Evaluating sustainable ingredients including black soldier fly larvae (Hermetia illucens) in laying hens through performance, physiology and nutrient metabolism

Evaluating sustainable ingredients including black soldier fly larvae (Hermetia illucens) in laying hens through performance, physiology and nutrient metabolism | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluating sustainable ingredients including black soldier fly larvae (Hermetia illucens) in laying hens through performance, physiology and nutrient metabolism Yosra ZNAZEN, Marwa Gaddes, Raja Chalghoumi, Geert P.J. Janssens, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8450877/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract To increase the sustainability of laying hen diets in subtropical rural conditions, corn and soybean meal were partially substituted with locally produced ingredients, topped or not with black soldier fly ( Hermetia illucens , BSF) larvae, simulating rural practices. To understand the underlying metabolic causes of potentially altered performance, blood biochemical parameters, including acylcarnitines, were evaluated. From 30 to 40 weeks of age, 150 Lohman White laying hens were allocated to three diets: standard corn–soybean meal diet (CONTROL), an alternative diet with triticale, faba beans and rapeseed meal (ALTER), and an ALTER diet supplemented with 5% BSF-dried larvae (ALTER + BSF). Laying performance, organ traits, selected carnitine esters and serum biochemical parameters were assessed. The CONTROL and ALTER diets resulted in comparable laying performance, whereas ALTER + BSF decreased the laying rate by 2% ( p < 0.001 ) and increased egg weight by 2 grams ( p < 0.001 ). Multivariate analysis revealed coordinated remodeling of the global acylcarnitine profile (p = 0.002 , R 2 = 0.74), which was associated with BSF supplementation and driven by free carnitine (C0), ϐ-oxidation (3-hydroxybutyrylcarnitine, C4-OH) and amino acid degradation (propionylcarnitine, C3) markers. The ALTER diet increased alanine aminotransferase (ALT) levels, which were normalized by BSF feeding ( p = 0.01 ). No differences were observed in organ weights. In conclusion, the substitution of alternative ingredients for soybean and corn effectively maintained laying performance. The BSF larvae acted as a functional nutrient, favoring egg weight and stimulating fat oxidation. These findings support the use of alternative ingredients for sustainable poultry production in subtropical rural areas. Laying hens Hermetia illucens alternative ingredients performance metabolism Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The sustainability and economic stability of poultry production are intrinsically linked to feed formulation. For decades, the industry has relied on a foundational diet of corn and soybean meals, a combination recognized for its high digestibility, excellent amino acid profile and consistent support of hen productivity. However, the reliance on these commodities poses a significant sustainability challenge, particularly for smallholder farmers in subtropical areas, as their prices are highly sensitive to fluctuations in the global market. Recent disruptions to international supply chains during the COVID-19 pandemic have further highlighted the vulnerability of this centralized model (Rahimi et al., 2022) and the urgent need to develop resilient, sustainable and locally adapted feed strategies. With Tunisia as a case, where poultry production heavily depends on imported feed ingredients, developing locally sourced and sustainable alternatives is crucial to improve feed autonomy and production resilience. In this context, locally produced ingredients such as triticale, faba beans and rapeseed meals offer greater availability and sustainability while reducing the economic and environmental costs associated with transportation. Triticale, a high-yielding cereal grain, provides approximately 14.08 MJ/kg of metabolizable energy (McNab et Shannon, 1975). A study conducted by Fernandez et al. ( 1973 ) demonstrated that replacing up to 85% of corn with triticale in laying hens did not have any adverse effects on egg production. Furthermore, Aggoor et al. ( 2000 ) reported similar results with substitution levels up to 75%. In terms of fiber composition, triticale is rich in insoluble fibers such as cellulose and lignin (Zhu et Fan, 2018), which promote digesta passage without inducing excessive gut viscosity or hindering enzymatic nutrient access (Ferreira et al. 2017). Moreover, non-starch polysaccharides (NSPs), including arabinoxylan and ϐ-glucan, in some modern triticale cultivars generally lack long-chain structures capable of binding water and increasing intestinal viscosity in the monogastric gut (Rkha et al. 2011). Faba beans constitute a protein-rich legume suitable for the partial replacement of soybean meals. They typically contain approximately 30% crude protein (CP) with high apparent digestibility of up to 90% in low-tannin, low-vicine and high-convicine cultivars (Crépon et al., 2010 ). Faba beans are particularly rich in lysine (20 g/kg), with high ileal digestibility values reaching 90% in selected cultivars. An inclusion rate of 50 g/kg of faba beans has shown no adverse effects on laying performance or livability (Koivunen et al.,2014) (Algawany et al., 2019). For some cultivars low in vicine and convicine, faba beans can even safely reach 200 g/kg in the diet (Perez-Maldonado et al.,1999). Although NSPs in faba beans may modestly reduce the availability of the apparent metabolizable energy in the gut, this limitation can be offset by increased cecal fermentation, leading to increased short-chain fatty acid (SCFA) production (Drażbo et al., 2018 ). Given their low methionine and cysteine contents, which are crucial for egg production, faba beans can be effectively complemented by rapeseed meals, a byproduct rich in both methionine and cysteine (Cheng et al., 2022 ). The crude protein content in rapeseed meals ranges between 30% and 50%, depending on the agronomic and processing conditions. However, its protein and amino acid contents are typically lower than those of soybean meal because of its high fiber level, thermal processing and interactions with antinutritional compounds such as tannins and phytic acid (Gołębiewska et al., 2022). In laying hens, inclusion rates below 117 g/kg of low glucosinolate rapeseed meal have been shown to maintain performance (Zhu et al., 2018 ), whereas higher levels may reduce egg weight by approximately 10% in Hy-Line and ISA Brown strains (Kamińska, 2003 ). In rural farming conditions, details on the nutritive value of in situ growing ingredients are often unknown. Additionally, the successful adoption of these alternative feedstuffs requires careful formulation to overcome potential limitations associated with antinutritional factors and fiber content. To address these challenges, the incorporation of black soldier fly ( Hermetia illucens; BSF) larvae may ensure sufficient energy and protein intake. They possess the unique ability to be produced sustainably on agricultural side streams and organic waste (Odongo et al., 2024 ). A key advantage for smallholders is their ease of use, as the larvae can be provided whole without requiring complex processing. Previous studies have demonstrated that BSF larvae can effectively replace soybean meals in various forms, such as alive (Tahamtani et al., 2021 ), whole dried (Bejaei et Cheng, 2023) or meal (Romero et al., 2024 ), without any negative effects on laying performance. The crude protein content of BSF larvae varies with life stage, substrate, rearing conditions and processing method (Salam et al., 2022 ), ranging from 39 to 48% on a dry matter basis for dried larvae. Although their protein content is lower than that of conventional soybean and fish meals, full-fat BSF larvae are rich in essential amino acids such as lysine (23–68 g/kg), leucine (27–78 g/kg) and valine (28–67 g/kg), which are crucial for albumen synthesis, energy metabolism and muscle maintenance (Lu et al., 2022 ). Owing to their high lipid content, which can range between 11% (when reared in ensiled mussels) and 57% on a dry matter basis (when reared in bread), BSF larvae can serve as a valuable energy source. They also possess functional properties that can increase nutrient utilization and overall feed value. The chitin in their exoskeletons has prebiotic potential (Muslykhah et al.,2024), and their fat fraction is uniquely characterized by a predominance of lauric acid, which has antimicrobial properties (Suryati et al., 2023 ). The purpose of the present study was, therefore, to simulate poultry diets under rural farming conditions by substituting a conventional corn–soybean meal diet with locally produced ingredients, with or without topping-up with dried whole BSF larvae. A comprehensive evaluation was conducted to assess the effects on laying performance as well as the overall physiological and metabolic responses of laying hens to these dietary modifications. Materials and methods Animals and housing The experiment was conducted at the experimental poultry farm of the Higher Institute of Agronomy of Chott Mariem (35°55'7.26" N, 10°33'49.72" E). A total of 28-week-old Lohmann White laying hens (n = 150) were acquired from a commercial farm and randomly assigned to 15 floor pens (1.9 m × 1.12 m × 6 m H × W × L) such that the average initial live weight was consistent across the three dietary treatments (1452 g). The pens were each equipped with two nest boxes (840 cm² per nest), 2.25 meters of perches, a 10-liter drinker and a suspended circular feeder. The internal area, covering two-thirds of the pen's surface, was lined with wood shavings, with fresh layers added weekly. The hens were allowed two weeks to acclimate. The lighting schedule consisted of a combination of daylight and artificial light, totaling 16 hours of light and 8 hours of darkness, maintained until the end of the study, as recommended by the Lohman LSL-CLASSIC layers guide (Lohmann breeders 2021). During the trial conducted from March to June, the environmental temperature fluctuated between 14.5°C and 33.0°C. The pens were visually separated to avoid group interaction. All hens were individually labeled with leg rings at the beginning of the experimental phase. Diet design and chemical analysis At 30 weeks of age, each pen was randomly assigned to one of the three experimental dietary treatments (Table 1 ), each with five replicates of ten birds as follows: Table 1 Ingredients (%) and nutrient composition of the basal feeds used in the study CONTROL ALTER (%) Corn 62.6 35.2 Soybean meal 26.2 17.3 Triticale 0 20 Faba beans 0 10 Rapeseed meal 0 5 Soybean oil 0 1.35 Salt 0.35 0.37 Dicalcium phosphate 1.5 1.39 Limestone 8.97 9 Methionine 0.08 0.09 PREMIX 1 0.3 0.3 BSF dried larvae 0 0 Calculated analysis AME (MJ/kg) 11.44 11.44 CP 17.01 17.07 Methionine 0.39 0.38 Methionine + Cysteine 0.69 0.69 Lysine 0.91 0.9 Tryptophan 0.19 0.19 Threonine 0.66 0.64 Available P 0.34 0.35 Na 0.15 0.16 Cl 0.25 0.27 Vitamin K 0.76 0.76 Vitamin A 7.2 7.2 Vitamin D 1.98 1.98 Vitamin E 6.57 6.57 Laboratory analysis (%) DM 2 89.1 89.2 OM 3 88.3 89.1 Starch 42.6 41 Crude fat 2.3 2.3 NDF 4 7.09 7.6 ADF 5 3.07 2.22 1 PREMIX : vitamin and mineral premix contained the following: crude ash: 63.75%, calcium: 18.89%, sulfur: 3.87%, chlorine: 2.90%, magnesium: 0.35%, methionine: 16.96%, methionine + cysteine: 16.96%, manganese: 34.392 g/kg, zinc: 27.7 g/kg, iron: 24.628 g/kg, copper: 2.754 g/kg, iodine: 640 mg/kg, cobalt: 133.40 mg/kg, selenium: 104.04 mg/kg, choline: 114.8 g/kg, vitamin PP (Niacin): 5.4 g/kg, vitamin B5: 2700 mg/kg, vitamin B2: 1350 mg/kg, vitamin B6: 675 mg/kg, Vitamin B1: 448.34 mg/kg, vitamin K3: 360.07 mg/kg, folic acid: 90.9 mg/kg, vitamin B12: 2.7 mg/kg, Vitamin A: 3.599 k UI, vitamin E: 3284.10 UI, vitamin D3: 989.89 UI. 2 DM: Dry matter, 3 OM: organic matter, 4 NDF: Neutral detergent fiber, 5 ADF acid detergent fiber. CONTROL: A commercial pelleted concentrate for adult laying hens was fed with corn and soybean meal as the main components, providing 11.44 MJ/kg apparent metabolizable energy (AME) and 17.01% crude protein (CP). ALTER: Fed alternative pelleted concentrate for adult laying hens in which corn and soybean meal were partially substituted by local feed crops, i.e., faba beans, triticale and rapeseed meal designed to match the control’s energy (11.44 MJ/kg) and crude protein (17.07%) contents. ALTER + BSF: 95% ALTER diet supplemented with a daily portion of whole dried BSF larvae, adjusted to 5% of the expected daily dry matter intake, which was set at 6 g of larvae per hen per day during the study. The concentrate was provided daily at 9 am, and the hens had continuous access to both feed and water for ad libitum consumption. For the ALTER + BSF replicates, the dried BSF larvae were weighed, placed on two plates and provided as a top-up to the hens two hours after concentrate distribution. The proximate composition of the BSF dried larvae used in this study was as follows: 5.7% moisture, 43.58% crude protein, 30.54% ether extract, 6.04% total ash and 2.34% chitin on a dry matter basis. The gross energy (GE) of BSF larvae was estimated via the regression model developed by the Institut National de la Recherche Agronomique (INRA), as detailed in Sauvant et al. ( 2004 ): GE (MJ/kg) = 17.3 + 0.0617 CP + 0.2193 EE + 0.0387 CF − 0.1867 Ash + Δ where GE is the gross energy (MJ/kg), CP is the crude protein (g/kg), EE is the ether extract (g/kg), Δ is a correction factor equal to 0. With this equation, the GE of BSF larvae was calculated to be 25.88 MJ/kg on a dry matter basis, corresponding to 24.41 MJ/kg on a crude matter basis. Thus, an apparent total tract retention coefficient (ATTRC) for gross energy equal to 0.729, as mentioned in Mahmoud et al. ( 2023 ), was adopted to estimate the apparent metabolizable energy. The authors conducted an in vivo digestive balance experiment on broiler chickens using a full fat BSF meal containing 26.6 MJ/kg GE on a crude matter basis, 48.6% CP, 32.4% EE and 5.3% ash on a dry matter basis. The GE content was determined via the bomb calorimetry method. On the basis of this retention coefficient, the AME of the BSF larvae used in the present study was estimated to be 17.79 MJ/kg on a crude matter basis. The ALTER + BSF diet had an estimated AME content of 11.75 MJ/kg and a CP content of 17.35% on a crude matter basis. Chemical analysis Representative samples of each feed type were collected, marked and ground to pass through a 1 mm sieve via a laboratory mill prior to analysis. The samples were analyzed following the standard procedures outlined by the Association of Official Analytical Chemists (AOAC, 2016). For dry matter determination, 2 to 3 grams of each sample were oven-dried for 16 hours at 105°C. After cooling in a desiccator, the samples were weighed, and the weight loss was recorded as the moisture content. The crude protein content was determined via the Khjeldal method, in which total nitrogen was measured, and a coefficient of 6.25 was used to estimate the protein content. Ether extraction was determined via the Soxhlet extraction method. A dried ground sample was weighed and placed in a Soxhlet apparatus, where lipids were extracted over 6 hours of repeated cycles. Afterwards, the samples were oven-dried, cooled, and the total fat content was determined via the gravimetric method. The total ash content was determined by placing pre weighed samples in a ceramic crucible, incinerating them in a muffle furnace at 550°C for 6 hours, and then cooling and weighing the residue. The two concentrates used were analyzed for neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents via a Tecator Fibertec™. The starch in the feeds was analyzed via polarimetry. The chitin present in the BSF larvae was extracted via a cascade process to eliminate proteins, oils, minerals and pigments, resulting in a purified chitin fraction, following the method described by Khayrova et al. (2019) in triplicate. Briefly, subsamples were dried at 95°C for 24 hours, treated in a 1 L beaker with 500 mL of 10% (w/w) NaOH and left in a 50°C water bath for 12 hours with occasional stirring. The resulting broth was filtered, washed, added to a 1 L beaker containing 5% HCl, and left at 20°C for 6 hours. The residue was filtered, washed with distilled water, oven-dried at 95°C and weighed. A second deproteinization was applied using 2% NaOH for 2 hours, followed by a second demineralization using 2% HCl for 2 hours. After drying, the remaining solid fraction was treated with 50 mL of bleach at 1% for 2 hours for depigmentation. Postmortem assessment At the end of the experiment (40 weeks), a total of 30 hens (2 hens per pen) were marked, weighed, and slaughtered. Hens were dissected, and the following traits were measured: intestine weight, liver weight, filled and empty gizzard weight, heart weight, abdominal fat weight, ovary and oviduct weight, small intestine length and rectum length. All digestive organ weights were measured via an electronic balance with an accuracy of 0.1 g. The lengths were measured with a measuring tape to the nearest millimeter. The relative weights of the previous organs were calculated as the weight of the specific organ divided by the live body weight (Huang et al., 2022 ). Performance For each hen, body weight was recorded at the beginning (week 30) and at the end of the experimental period (week 40). Mortality and health status were monitored daily. The feed residue weight and water intake were recorded daily by replication. All eggs produced were collected daily, counted and individually weighed via an analytical balance (CRYSTAL 300 CE*, precision ± 0,0001 g). Water consumption, average daily feed intake (ADFI), the number of eggs laid, egg mass, the egg laying rate, the water-to-feed ratio, and the feed conversion ratios expressed per mass (FCRmass) and per dozen (FCRdozen) were determined weekly. Serum biochemical analysis and acylcarnitine profiling On day 69 of the trial, fresh blood samples were collected from the wing vein of 10 birds per treatment group (2 hens per pen). A volume of 2.5 mL was placed in a K3EDTA vacutainer tube and centrifuged at 3000 rpm for 15 minutes. The serum obtained was collected and stored at − 20°C and then analyzed for biochemical indices, including glucose, total cholesterol, triglycerides, creatinine, uric acid, urea, total protein and serum electrolytes, including sodium (Na), chloride (Cl), potassium (K) and alkaline reserves. Additionally, liver function was evaluated through analysis of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma-glutamyl transferase (GGT) levels via specific commercial kits (Marono et al., 2017 ). For acylcarnitine profiling, a 50 µL aliquot of each EDTA-treated blood sample was spotted onto circular specimen collection papers (Whatman Protein Saver cards, 903™, UK), dried, and then sent to Ghent University Hospital (laboratory for clinical chemistry) for acylcarnitine profiling via ultraperformance liquid chromatography (UPLC)-tandem mass spectrometry, as described in Gucciardi et al. ( 2015 ). In brief, acylcarnitine from 3.2 mm dried blood spots was extracted with 100 µL of methanol containing labeled internal standards. The extract was then dried under nitrogen at 60°C and derivatized to form butyl esters by adding 100 µL of freshly prepared butanol with 5% acetyl chloride, followed by heating at 60°C for 20 minutes. Chromatographic separation was performed on a UPLC system equipped with an ethylene-bridged hybrid C (18) column. Detection was carried out on a Waters Micromass Quattro Ultima tandem quadrupole mass spectrometer. Acylcarnitine concentrations were determined by integrating peak areas and using a calibration curve with a correlation coefficient ranging from 0.990 to 0.999. Statistical analysis The data were organized and validated in Microsoft Excel. Statistical analyses were performed via IBM SPSS Statistics for Windows (version 31.0.0.0), whereas multivariate analyses (PERMANOVA, PERMADISP, PCA and sPLS-DA) were conducted in R (version 4.4.0) via appropriate packages. Every replicate was treated as the experimental unit, and statistical significance was defined as p < 0.05 . For laying performance, a linear mixed-effects model was used. The diet group and week were specified as fixed effects, whereas the pen was included as a random effect. Pairwise comparisons among diets were conducted via the Bonferroni-adjusted method. For single time-point variables, normality and homogeneity of variance were evaluated via the Shapiro‒Wilk test and Levene’s test. When the assumptions were met, one-way ANOVA followed by Tukey’s HSD was applied. A permutational multivariate analysis of variance (PERMANOVA) test based on a Euclidean distance matrix (999 permutations) was applied in R via the adonis2 function in the vegan package to evaluate differences in 33 acylcarnitine metabolites among the three dietary groups. The homogeneity of multivariate dispersions PERMADISP was run via the betadisper function. Significant global effects were followed by pairwise PERMANOVA comparisons. Principal component analysis (PCA) was performed via the FactoMineR package to explore major sources of variation in the acylcarnitine profiles. Eigenvalues were used to determine the percentage of variance and metabolite correlations, which were assessed via the Pearson correlation coefficient (r) with two-tailed correlation significance testing ( p ). The first two principal components were used to generate the PCA biplot. To identify metabolites contributing to group separation, a supervised sPLS-DA model was performed via the mixOmics package. Discriminant metabolites were ranked according to their variable importance in projection (VIP) score. Individual metabolites (n = 33) were further analyzed via the Kruskal‒Wallis test, and p values were adjusted for multiple testing via the Benjamini‒Hochberg false discovery rate (FDR) method. The derived sums and metabolic ratios were evaluated via the Kruskal‒Wallis test, and the data are presented as medians (P25, P75) and box‒whisker plots. Results Growth and laying performance Hens’ body weights were similar across all dietary treatments, with no significant differences at any time point ( p > 0.05 ). The birds started with an average initial body weight of 1452 g and reached a final body weight of 1599 g, resulting in a homogeneous body weight gain of 149 g ( p = 0.31 ), indicating that dietary modifications did not affect overall growth performance. The effects of dietary treatments on laying performance throughout the trial period are presented in Table 2 . The hens in the ALTER + BSF group presented a 2% lower laying rate ( p < 0.001 ) than did those in the CONTROL and ALTER groups, which maintained similar rates. In contrast, the ALTER + BSF group laid heavier eggs (66 g) than did the ALTER and CONTROL groups, which presented similar egg weights (65 g, p 0.05 ). The feed conversion ratio per mass (FCRmass) was identical for hens fed the CONTROL and ALTER diets (1.69), whereas it decreased significantly for the hens fed dried BSF larvae (1.65, p = 0.01 ). The remaining parameters, such as ADFI, feed conversion ratio per dozen (FCRdozen), drinking and water-to-feed ratio, were not affected by diet. No mortalities or pathological signs were observed throughout the trial. Table 2 Laying performance (mean ± SD) of Lohman white hens fed a conventional diet (CONTROL), a diet with alternative ingredients without (ALTER) or with 5% black soldier fly (ALTER + BSF) (n = 5) DIET GROUP CONTROL ALTER ALTER + BSF p ADFI 1 (g DM/hen/day) 107.97 ± 7.97 108.65 ± 7.75 105.67 ± 9.00 0.16 Drinking (L/hen/day) 0.29 ± 0.08 0.30 ± 0.10 0.29 ± 0.09 0.72 Laying rate (%) 98.25 ± 1.89 a 98.54 ± 1.54 a 96.28 ± 3.22 b < 0.001 Egg weight (g) 65.03 ± 1.54 b 65.12 ± 0.68 b 66.27 ± 1.10 a < 0.001 Egg mass (g/day) 63.91 ± 2.13 64.17 ± 1.88 63.81 ± 2.32 0.67 FCRmass 2 (kgDM/kg) 1.69 ± 0.08 a 1.69 ± 0.08 a 1.65 ± 0.08 b 0.01 FCRdozen 3 (gDM/dozen) 1.31 ± 0.05 1.32 ± 0.05 1.31 ± 0.06 0.8 Water:feed (g/g) 2.48 ± 0.72 2.53 ± 0.80 2.57 ± 0.77 0.32 1 ADFI: average daily feed intake, 2 FCRdozen: feed conversion ratio per dozen, 3 FCRmass: feed conversion ratio per mass. P values report the overall group comparison performed using linear mixed-effects model with Bonferroni correction. a-b: Means with different superscript letters within each main effect are significantly different ( p < 0.05 ). Weekly performance parameters are presented in Fig. 1 . The three dietary groups presented consistent feed intake, except for the ALTER + BSF group, which declined significantly during the last week of the trial, reaching its lowest level (100 g DM/hen/day, p = 0.04 ). Laying rates were comparable among the three dietary groups at the beginning of the trial. A progressive decrease in laying rate was observed in the ALTER + BSF group, reaching a significant decrease of 3% in the fourth week ( p = 0.04 ), after which laying performance recovered and remained comparable to that of the other groups until the end of the trial. Additionally, hens fed the ALTER + BSF diet consistently produced numerically heavier eggs, with this difference reaching statistical significance in the ninth week. Postmortem results Table 3 presents the effects of the three dietary treatments on live body weight, relative organ weights, and relative intestinal lengths in hens. No significant differences were observed for any of the measured parameters ( p > 0.05 ). The live body weights ranged from 1594 g to 1609 g. The relative organ weights, including those of the digestive (gizzard, liver, and intestine) and reproductive (ovary and oviduct) organs, did not differ significantly among the treatments ( p > 0.05 ). Similarly, small intestine length and rectum length were not influenced by diet ( p > 0.05 ), indicating that dietary inclusion of BSF larvae did not affect organ development or gross intestinal morphology. Table 3 Live body weight (g), relative organ weight (%) and relative intestinal length (%) (mean ± SD) of Lohman white hens fed a conventional diet (CONTROL), a diet with alternative ingredients without (ALTER) or with 5% BSF (ALTER + BSF) (n = 5) CONTROL ALTER ALTER + BSF p Live body weight (g) 1594.36 ± 32.35 1609.1 ± 23.25 1596.42 ± 27.84 0.92 Relative weight (%) Intestine 5.1 ± 0.21 4.73 ± 0.12 5.05 ± 0.17 0.27 Filled gizzard 1.93 ± 0.16 1.86 ± 0.16 1.70 ± 0.08 0.49 Empty gizzard 1.33 ± 0.09 1.33 ± 0.09 1.24 ± 0.04 0.6 Liver 2.39 ± 0.08 2.19 ± 0.09 2.22 ± 0.12 0.3 Abdominal fat 1.90 ± 0.22 2.35 ± 0.21 2.57 ± 0.21 0.09 Ovary & oviduct 8.97 ± 0.44 8.51 ± 0.62 9.55 ± 0.35 0.32 Relative length (%) Small intestine 8.07 ± 1.84 8.26 ± 1.77 8.69 ± 1.15 0.42 Rectum 1.84 ± 0.27 1.77 ± 0.25 1.93 ± 0.32 0.46 Acylcarnitine profiling The global PERMANOVA revealed a highly significant effect of diet on the overall acylcarnitine profile ( p = 0.002 ), with dietary treatment explaining 74.7% of the total variance (R 2 = 0.74). The test for homogeneity of dispersion PERMADISP did not reveal a significant difference in variance between the groups ( p = 0.29 ). Pairwise PERMANOVAs revealed that the ALTER diet significantly altered the metabolic profile compared with the CONTROL diet ( p = 0.054 ). In addition, significant differences were observed between the ALTER and ALTER + BSF groups ( p = 0.005 ), with BSF inclusion accounting for 74.6% of the explained variance. No significant differences were detected between the CONTROL and ALTER + BSF groups ( p = 0.29 ). The PCA revealed ten components with eigenvalues greater than 1 (Fig. 2 ). The first two principal components (Fig. 3 . A) collectively explained 45.3% of the total variance in the dataset (Components 1: 28% and 2: 17.1%). The positive axis of component 1 was driven mainly by long-chain carnitine forms such as myristoylcarnitine (C14), which was positively correlated with tetradecenoylcarnitine (C14:1) (r = 0.9, p = 0.001 ), tetradecadienoylcarnitine (C14:2) (r = 0.81, p < 0.001 ), palmitoylcarnitine (C16) (r = 0.8, p = 0.002 ). The positive axis of component 2 was driven mainly by acetylcarnitine (C2), which was positively correlated with propionylcarnitine (C3) (r = 0.65, p = 0.02 ) and most of ϐ-oxidation forms such as 3-Hydroxybutyrylcarnitine (C4-OH) (r = 0.76, p = 0.004 ), 3-Hydroxyisovalerylcarnitine (C5-OH) (r = 0.64, p = 0.023 ) and 3-Hydroxypalmitoleylcarnitine (C16:1-OH) (r = 0.73, p = 0.006 ), which was positively correlated with 3-Hydroxylorylcarnitine (3OH-C12) (r = 0.65, p = 0.03 ). The negative axis of component 2 was strongly driven by linoylcarnitine (C18:2), which was negatively correlated with lauroylcarnitine (C12) (r = − 0.62, p = 0.03 ) and glutarylcarnitine (C5DC) (r = − 0.67, p = 0.015 ). Free carnitine (C0) was positively correlated with C3 (r = 0.72, p = 0.006 ), isovalerylcarnitine (C5) (r = 0.85, p < 0.001 ) and C4-OH (r = 0.29, p = 0.048 ). The results of Pearson correlation matrix are presented in the Supplementary Materials (Table S1 ). The CONTROL group presented a compact elongated cluster primarily located in the lower-left quadrant, indicating a relatively homogeneous metabolic profile with minimal metabolic disturbance. The ALTER group formed a tight cluster that was mostly positioned in the upper-left quadrant between the negative axis of component 1 and the positive axis of component 2, displaying moderate heterogeneity and showing a metabolic shift from CONTROL without extreme divergence. The ALTER + BSF group exhibited the widest cluster, which was clearly separated from the other groups and positioned along the positive axes of components 1 and 2, covering most metabolites contributing to the global variance. The sPLS-DA model (Fig. 3 . B) selected nine unique metabolites with the highest VIP scores (Table 4 ) as optimal discriminators among the three dietary groups, with 18% explained variance for component 1, which was driven by C0, C3, C5, C4-OH and C10, and 13% explained variance for component 2, which was driven by C5DC, adipoylcarnitine (C6DC), stearoylcarnitine (C18), C12 and C4-OH. Univariate Kruskal‒Wallis tests revealed that none of the metabolites were significantly different after FDR correction was applied (Table 4 ). Likewise, no significant effects of diet were observed for the derived acylcarnitine sums and ratios presented in Fig. 4 . Table 4 Acylcarnitine profile (mean ± SD) in dried blood spot from hens fed a conventional diet (CONTROL), a diet with alternative ingredients without (ALTER) or with 5% BSF (ALTER + BSF). Metabolite Metabolite name CONTROL ALTER ALTER + BSF p VIP (µmol/L) C0 Free carnitine 44.21 ± 0.17 37.13 ± 2.76 50.03 ± 3.26 0.17 3.26 C3 Propionylcarnitine 0.3 ± 0.05 0.33 ± 0.02 0.57 ± 0.09 0.17 2.49 C5 Isovalerylcarnitine 0.1 ± 0.01 0.07 ± 0.01 0.12 ± 0.02 0.17 1.70 C4-OH Hydroxybutyrylcarnitine 0.1 ± 0.03 0.13 ± 0.01 0.2 ± 0.03 0.17 0.91 C10 Decanoylcarnitine 0.03 ± 0.006 0.02 ± 0.003 0.04 ± 0.018 0.17 0.19 C5DC Glutarylcarnitine 0.02 ± 0.004 0.03 ± 0.006 0.03 ± 0.003 0.18 2.13 C6DC Adipoylcarnitine 0.01 ± 0.004 0.02 ± 0.005 0.02 ± 0.005 0.49 2.09 C18:2 Linoylcarnitine 0.03 ± 0.007 0.02 ± 0.006 0.02 ± 0.003 0.49 1.48 C12 Lauroylcarnitine 0.02 ± 0.004 0.03 ± 0.01 0.03 ± 0.005 0.49 1.16 C2 Acetylcarnitine 20.00 ± 1.85 20.88 ± 3.19 25.65 ± 3.63 0.49 - C14:2 Tetradecadienoylcarnitine 0.015 ± 0.00 0.02 ± 0.004 0.06 ± 0.086 0.49 - C14 Myristoylcarnitine 0.04 ± 0.017 0.04 ± 0.007 0.06 ± 0.02 0.49 - C16:1 Palmitoleylcarnitine 0.03 ± 0.007 0.02 ± 0.009 0.03 ± 0.009 0.49 - C16:1-OH Hydroxypalmitoleylcarnitine 0.03 ± 0.006 0.03 ± 0.01 0.04 ± 0.025 0.49 - C16-OH Hydroxypalmitoylcarnitine 0.03 ± 0.003 0.03 ± 0.007 0.04 ± 0.01 0.49 - C6 Hexanoylcarnitine 0.09 ± 0.014 0.07 ± 0.015 0.08 ± 0.043 0.50 - C14:1 Tetradecenoylcarnitine 0.02 ± 0.003 0.02 ± 0.008 0.03 ± 0.018 0.50 - C18-OH Hydroxystearoylcarnitine 0.08 ± 0.007 0.08 ± 0.02 0.10 ± 0.02 0.50 - C8 Octanoylcarnitine 0.07 ± 0.008 0.07 ± 0.006 0.08 ± 0.015 0.53 - C4 Butyrylcarnitine 0.52 ± 0.23 0.34 ± 0.12 0.53 ± 0.23 0.77 - C5-OH Hydroxyisovalerylcarnitine 0.13 ± 0.007 0.14 ± 0.03 0.15 ± 0.04 0.77 - C3DC Malonylcarnitine 0.04 ± 0.01 0.05 ± 0.02 0.06 ± 0.02 0.77 - 3OH-C12 3-Hydroxylauroylcarnitine 0.04 ± 0.007 0.05 ± 0.016 0.05 ± 0.014 0.77 - C14-OH Hydroxymyristoylcarnitine 0.02 ± 0.01 0.02 ± 0.01 0.02 ± 0.003 0.77 - C16 Palmitoylcarnitine 0.07 ± 0.023 0.07 ± 0.016 0.1 ± 0.04 0.77 - C18 Stearoylcarnitine 0.05 ± 0.016 0.04 ± 0.012 0.04 ± 0.005 0.77 - C18:1-OH Hydroxyoleylcarnitine 0.03 ± 0.004 0.03 ± 0.005 0.03 ± 0.013 0.77 - C14:1-OH Hydroxymyristoleylcarnitine 0.02 ± 0.009 0.02 ± 0.006 0.02 ± 0.002 0.83 - C10:2 Decadienoylcarnitine 0.01 ± 0.005 0.01 ± 0.003 0.01 ± 0.004 0.94 - C10:1 Decenoylcarnitine 0.02 ± 0.005 0.02 ± 0.005 0.02 ± 0.007 0.94 - C4DC Succinylcarnitine 0.05 ± 0.012 0.05 ± 0.013 0.06 ± 0.01 0.94 - C18:1 Oleylcarnitine 0.08 ± 0.014 0.08 ± 0.02 0.08 ± 0.035 0.94 - C5:1 Tiglylcarnitine 0.07 ± 0.033 0.07 ± 0.036 0.06 ± 0.015 0.99 - p values were adjusted for multiple testing using the False Discovery Rate (FDR) method, VIP: Variable importance in projection score. Serum biochemistry Table 5 presents the biochemical parameters of the blood serum. There were no significant differences observed among the dietary groups for glucose, total cholesterol, or triglyceride levels ( p > 0.05 ). The urea concentration was lower in the alternative diet group than in the CONTROL group ( p = 0.002 ). Conversely, the alkaline reserve increased significantly with the ALTER diet (16.9 mmol/L) ( p = 0.01 ), whereas the ALTER + BSF diet slightly decreased it to 15 mmol/L. No significant differences were detected for the liver enzymes AST and GGT ( p > 0.05 ). In contrast, feeding the ALTER diet increased ALT activity to 14.40 UI/L, while feeding the ALTER + BSF diet significantly reduced ALTER activity to 11.1 UI/L, which was statistically similar to that of the CONTROL group (10.38 UI/L) ( p = 0.01 ). Table 5 Serum biochemical parameters (mean ± SD) in laying hens fed a conventional diet (CONTROL), a diet with alternative ingredients without (ALTER) or with 5% BSF (ALTER + BSF) (n = 8). CONTROL ALTER ALTER + BSF p Energy & lipid metabolism (g/L) Glucose 2.91 ± 0.27 2.7 ± 0.21 2.85 ± 0.55 0.65 Total cholesterol 1.58 ± 0.41 1.42 ± 0.23 1.49 ± 0.49 0.82 Triglycerides 0.25 ± 0.06 0.33 ± 0.05 0.37 ± 0.14 0.15 Protein metabolism & kidney function (mg/L) Creatinine 2.32 ± 0.59 2.34 ± 0.46 2.72 ± 0.62 0.47 Uric acid 41.80 ± 12.54 42.19 ± 5.69 43.91 ± 14.86 0.95 Urea 40 ± 8 a 30 ± 6 b 20 ± 6 b 0.002 Total protein (g/L) 39 ± 8 40 ± 5 43 ± 11 0.77 Electrolytes & acid‒base balance (mmol/L) Sodium 84.6 ± 6.61 110 ± 25.17 113.8 ± 20.86 0.07 Chloride 67.4 ± 4.87 81.9 ± 19.34 85.9 ± 15.77 0.15 Potassium 4.12 ± 0.67 4.1 ± 0.86 4.61 ± 1 0.59 Alkaline reserve 11.9 ± 1.08 b 16.9 ± 2.3 a 15 ± 2.62 ab 0.01 Liver function (UI/L) AST 1 349.2 ± 74.72 339.7 ± 40.97 311.1 ± 119.44 0.76 ALT 2 10.38 ± 0.75 b 14.4 ± 2.33 a 11.1 ± 1.52 b 0.01 GGT 3 28.8 ± 4.04 29.9 ± 8.09 30.1 ± 2.61 0.92 ALT/AST 0.03 ± 0.009 0.04 ± 0.009 0.04 ± 0.016 0.25 1 Aspartate Aminotransferase; 2 Alanine aminotransferase; 3 Gamma-glutamyl transferase. a-b: Means with different superscript letters within each main effect are significantly different ( p < 0.05 ). Discussion This study revealed that the ALTER diet was effectively utilized by the layers to maintain performance comparable to that of the standard CONTROL diet. Even with dietary complexity (Triticale, faba beans and rapeseed meal), it seems that the plant-based alternative diet did not disturb the energy-to-protein balance during the ten weeks of the trial. These findings align with those of Mills et al. ( 2024 ), who reported that commercial strains of laying hens are well adapted to diets rich in structural materials with insoluble fiber without compromising laying performance. Among the three dietary groups, only ALTER + BSF resulted in significantly different performance, characterized by a slower laying rate but heavier eggs. Despite the reduced laying frequency observed in the group fed BSF, the three diet groups were similar in terms of the mass of egg produced. These findings suggest that hens fed a diet supplemented with 5% dried BSF larvae did not exhibit reduced productivity but rather underwent significant physiological changes. This biological response could be explained by the feeding strategy itself. The ALTER + BSF hens received highly palatable and nutrient-rich BSF larvae on top-up. This likely resulted in a sudden influx of easily digestible fats and amino acids (Alafif et al., 2025 ), which may have slowed the frequency of ovulation and led the hens’ metabolism to invest more resources in each egg. In fact, consuming fat-rich BSF larvae (23% CF) two hours after receiving high-fiber meal may cause abnormal gastrointestinal motility (Rodriguez-Sinovas, 1997). The high fat content likely triggered the release of satiety hormones, mainly cholecystokinin (CCK) (Denbow et Cline, 2015), reducing the meal size and increasing the inter-meal interval (Stengel et Tache, 2011). These effects align with the numerically reduced ADFI observed in the ALTER + BSF group throughout the trial, particularly during the last week. Shneider et al. (2004) extensively discussed how the body’s energy balance influences the hormonal regulation of the reproductive system. Following the sudden increase in energy available from the BSF, the hypothalamus may have temporarily suppressed the reproductive cascade. This response could inhibit the nocturnal luteinizing hormone (LH) preovulatory surge, disrupting the hormonal signals related to circadian and follicular growth rhythms and leading to a skipped ovulation the next morning (Johnson, 2014 ). Once the daily BSF signal is sufficient to push the total stress level above the threshold, the LH surge is inhibited, and the day is skipped. Continuous nutrient intake combined with reduced reproductive energy expenditure creates a significant surplus of protein and energy. This surplus is reallocated by the hen’s body to the next egg in the sequence, resulting in heavier eggs (Cucco et al., 2017 ; Nilson & Svensson, 1993). This aligns perfectly with the decreased laying rate and increased egg weight observed in the ALTER + BSF group. Despite the daily occurrence of sudden BSF meal, the hens exhibited physiological adaptations to this disruptive event following a threshold response. The weekly egg laying and egg weight curves clearly illustrate this physiological process. The decline in laying frequency observed during the third week of the trial likely reflects the initial impact of the top-up BSF meal. The stress induced may have pushed many hens beyond their tolerance threshold, resulting in a significant drop in egg production. By the fourth week, the laying rate of the ALTER + BSF group had rebounded, and egg weight tended to increase. These findings suggest that the endocrine and metabolic systems adapted to the new daily rhythm, establishing a new physiological equilibrium capable of accommodating the BSF-supplemented diet (Nilson et Svensson, 1993). Thereafter, egg production recovered and remained comparable to that of the other groups until the end of the trial. This study demonstrated a pronounced effect of supplementation with BSF larvae on the global acylcarnitine profile, reflecting coordinated remodeling in multiple biochemical pathways rather than changes in individual metabolites. Multivariate analyses confirmed this integrated metabolic response, which was driven mainly by nine discriminating metabolites with the most pronounced difference observed between the ALTER and ALTER + BSF groups. The PCA results revealed a stable and homogeneous metabolic profile in CONTROL hens, whereas the ALTER group presented moderate metabolic shifts. In contrast, the ALTER + BSF group displayed the greatest within-group variability, highlighting a strong metabolic response to BSF supplementation. Component 1 is driven by markers associated with lipid metabolism and long-chain fatty acid (LCFA) oxidation. These results are consistent with those of Aprianto et al. ( 2023 ), who reported that BSF larval oil suppresses the expression of fat-synthesis genes (acetyl-CoA carboxylase and fatty acid synthase) while upregulating the expression of genes involved in β-oxidation pathways. However, the unchanged 3OHLC:LC ratio, along with the C4-OH, C10 and C12 discriminators, which tended to be high in BSF-fed hens, suggest that the ketogenic state observed in the ALTER + BSF group was driven mainly by medium-chain fatty acids (MCFAs) rather than LCFAs. This could be explained by the fact that unlike LCFAs, MCFAs such as lauric acid, which is abundant in BSF larvae (Shu et al.,2025), diffuse directly into the mitochondria, bypassing the body’s main regulatory system (carnitine palmitoyltransferase 1: CPT1) for fat burning (Pereyra et al., 2023 ), as reflected by the unaffected levels of malonylcarnitine (C3DC). Anas et al., ( 2024 ) reported that hens fed a diet containing 2% calcium salt of BSF larvae oil, which lauric acid is the main component, produced eggs with increased yolk, albumen and eggshell weights. Furthermore, the elevated propionylcarnitine observed in ALTER + BSF hens indicates the degradation of branched-chain amino acids (leucine, isoleucine, valine), likely reflecting the detoxification of excess nitrogen derived from the BSF protein supply rich in those amino acids (Fuso et al., 2021 ; Lu et al. 2022 ). Consistently, free carnitine was positively correlated with short-chain acylcarnitines and 3-xydroxybutyrylcarnitine and it showed elevated levels in the ALTER + BSF group and the highest VIP score. This suggests that BSF supplementation prevented functional carnitine deficiency associated with the ketogenic state. Therefore, the elevated free carnitine levels likely resulted from direct dietary intake of the BSF larvae, which is consistent with findings by Kuppusamy et al. ( 2020 ), who demonstrated that BSF larvae can accumulate carnitines at levels substantially greater than those present in their substrate. However, the three dietary groups presented comparable overall functional metabolic indices, suggesting that 5% BSF supplementation induced coordinated remodeling of fatty acid oxidation without evident metabolic dysfunction, revealing a fuel-switching adaptation in response to the BSF fat and protein supply. Analysis of serum biochemical indicators revealed a clear pattern of physiological stress and subsequent mitigation. A primary finding was that hens fed the ALTER diet experienced a state of hepatocellular stress, as evidenced by a significant increase in the liver-specific ALT enzyme. The concurrent stability of AST levels, and therefore the ALT:AST ratio, suggests that mild-to-moderate injury is characterized by increased cell membrane permeability, rather than severe necrosis. Importantly, supplementation with BSF larvae effectively attenuated these elevations, restoring ALT levels to those of the CONTROL group. Since the direct effect of BSF on liver enzymes has not been previously reported, the protective mechanism observed may be linked to improved gut health. Dabbou et al. ( 2021 ) reported that modified BSF larval fat can improve gut health without inducing any adverse histopathological alterations. The abundant lauric acid in BSF has been shown to have antimicrobial proprieties, and chitin can act as a prebiotic compound (Shu et al.,2025; Bonomini et al., 2024 ). Together, these compounds may promote a healthier gut microbiome, reducing the metabolic and detoxification load on the hen liver. However, other metabolic indicators revealed that general metabolic processes and renal functions remained stable. The levels of glucose, total protein, and uric acid remained unaffected across all groups, suggesting a stable overall metabolic status. Since urea in birds is the primary byproduct of arginine catabolism (Ali et al., 2025 ), the significantly higher concentration of urea found in the blood of CONTROL hens is most likely due to the higher content of soybean meal, which is rich in arginine (Oviedo-Rondón et al., 2024 ). This study also demonstrated successful adaptation to changes in the dietary acid-base balance. Despite the balanced formulation of diets in terms of potassium, sodium and chloride, a significant increase in the alkaline reserve observed in the ALTER group indicates a net alkalizing effect of certain plant ingredients. The kidney may compensate by retaining more bicarbonate ions to stabilize the blood pH. The fact that the serum levels of sodium, chloride and potassium remained stable across all the groups is proof of a successful regulatory renal response. The relative weights of major reproductive, metabolic, and digestive organs were not affected by dietary treatment, confirming that their effects did not progress to structural alterations. The hens fed BSF larvae produced heavier eggs without a corresponding increase in the relative weights of their reproductive organs, indicating an efficient redistribution of energy and nutrients toward egg formation. Furthermore, the stability of live body weight, abdominal fat content, and intestinal length and weight across groups demonstrated that the digestive challenges posed by the alternative ingredients did not result in wasting, fat accumulation, or global intestinal morphological remodeling. These findings suggest that the observed effects reflect a physiological adaptation to the nutritional characteristics of alternative diets rather than pathological responses. Conclusions In conclusion, the partial substitution of corn and soybean meals with alternative plant-based feedstuffs maintained laying performance. However, this substitution entails a subtle metabolic cost. Careful consideration should be given to the antinutritional factors that could pose a potential risk to long-term health and productivity. The inclusion of 5% BSF dried larvae elicited a distinct physiological response. The BSF larvae acted as a functional ingredient, exhibiting a potential hepatoprotective effect that attenuated the liver stress signs observed in the hens fed alternative plant-based diets. Furthermore, the altered metabolic pathways shown in hens fed BSF larvae may explain the observed shift in reproduction physiology and, consequently, in performance, where the nutrients were channeled toward slowing laying frequency and producing heavier eggs. Overall, the use of plant-based alternative ingredients did not compromise laying performance, while BSF larvae supplemented with 5% BSF acted as a functional nutrient, favoring heavier eggs and stimulating fat oxidation. These findings support the use of alternative ingredients for sustainable poultry production in subtropical rural areas. Declarations Acknowledgements: The author used OpenAI’s Curie service to help improve wording and correct spelling and grammar. Funding: This study was supported by the PRIMA program (European Union) under grant agreement No. 2015, project SUSTAvianFEED. Conflicts of interest: The authors declare that they have no competing interests. Author Contributions: Yosra Znazen: data collection and treatment, writing the first draft of the manuscript, and participation in all aspects related to this manuscript; Marwa Gaddes: Installing the laying performance trial and collecting data; Raja Chalghoumi: reviewing the final draft; Geert P.J. Janssens: performing acylcarnitine analysis, interpreting results and reviewing final manuscript draft; Madiha Hadj Ayed: conceptualization, supervision and project administration. All the authors commented on previous versions of the manuscript and approved the final version. Data availability: Any further data related to the manuscript will be available upon request. Ethical approval: The experimental protocol (94-CEEA-ENMV/25) was approved by the Animal Ethics Committee of the National School of Veterinary Medicine, Sidi Thabet (Tunisia). References Aggoor, F., T. Tag El-Din, A. Abd El-Ghani, et Neven Heneish. « THE USE OF TRITICALE AS ALTERNATIVE CEREAL GRAIN TO YELLOW CORN IN LAYING HENS DIET ». Journal of Animal and Poultry Production 25, n o 2 (2000): 771‑85. https://doi.org/10.21608/jappmu.2000.258593. Alafif, Mohammad S., Louwrens C. Hoffman, Daniel Cozzolino, et al. « Assessment of Apparent Metabolizable Energy, and Ileal Amino Acid Digestibility of Full-Fat Black Soldier Fly Larvae ( Hermetia illucens ) in Broiler Chickens ». Poultry Science 104, n o 10 (2025): 105506. https://doi.org/10.1016/j.psj.2025.105506. Anas, M.A, M.A. Aprianto, M. Muhlisin, et al. « Improving Egg Quality in Laying Hens Using Calcium Salt of Black Soldier Fly Larvae Oil ». Journal of Insects as Food and Feed 11, no 5 (2024): 907‑19. https://doi.org/10.1163/23524588-00001300. Alagawany, Mahmoud, Mohamed E. Abd El-Hack, Elwy A. Ashour, et al. « Raw Faba Bean (Vicia Faba) as an Alternative Protein Source in Laying Hen Diets». Journal of Applied Poultry Research 28, nᵒ 4 (2019): 808‑17. https://doi.org/10.3382/japr/pfz037. Ali, Awais, Xuan Liu, Mebratu Melaku, et al. « Effect of Arginine Supplementation on Liver and Pectoral Muscle: Tissue-Specific Lipid Metabolism in Broilers ». Poultry Science 104, n o 10 (2025): 105601. https://doi.org/10.1016/j.psj.2025.105601. AOAC International. (2016). Official Methods of Analysis of AOAC International (18th ed.). Gaithersburg, MD, USA: AOAC International. Aprianto, Muhammad Anang, Muhlisin, Asih Kurniawati, Chusnul Hanim, Bambang Ariyadi, et Muhsin Al Anas. « Effect Supplementation of Black Soldier Fly Larvae Oil ( Hermetia Illucens L. ) Calcium Salt on Performance, Blood Biochemical Profile, Carcass Characteristic, Meat Quality, and Gene Expression in Fat Metabolism Broilers ». Poultry Science 102, no 10 (2023): 102984. https://doi.org/10.1016/j.psj.2023.102984. Bejaei, Masoumeh, et Kimberly M. Cheng. « Inclusion of Dried Black Soldier Fly Larvae in Free-Range Laying Hen Diets: Effects on Production Efficiency, Feed Safety, Blood Metabolites, and Hen Health ». Agriculture 14, n o 1 (2023): 31. https://doi.org/10.3390/agriculture14010031. Bonomini, M.G., S. Verstringe, G. Bruggeman, R. Vandercruyssen, H. Carmans, et A. Caligiani. « Characterization, Antibacterial Activity, and Prebiotic Potential of Dried Hermetia illucens L. Larvae and of Their Fractions ». Journal of Insects as Food and Feed 11, n o 2 (2024): 273‑88. https://doi.org/10.1163/23524588-00001152. Cheng, Hao, Xiang Liu, Qingrui Xiao, et al. « Rapeseed Meal and Its Application in Pig Diet: A Review ». Agriculture 12, nᵒ 6 (2022): 849. https://doi.org/10.3390/agriculture12060849. Crépon, Katell, Pascal Marget, Corinne Peyronnet, Benoit Carrouée, Paolo Arese, et Gérard Duc. « Nutritional Value of Faba Bean (Vicia Faba L.) Seeds for Feed and Food ». Field Crops Research 115, no 3 (2010): 329‑39. https://doi.org/10.1016/j.fcr.2009.09.016. Cucco, Marco, Marco Grenna, et Irene Pellegrino. « Egg Characteristics in Relation to Skipped Days of Laying in the Gray Partridge ». Avian Biology Research 10, n o 4 (2017): 231‑40. https://doi.org/10.3184/175815617X15036738758853. Dabbou, Sihem, Angelo Lauwaerts, Ilario Ferrocino, et al. « Modified Black Soldier Fly Larva Fat in Broiler Diet: Effects on Performance, Carcass Traits, Blood Parameters, Histomorphological Features and Gut Microbiota ». Animals 11, n o 6 (2021): 1837. https://doi.org/10.3390/ani11061837. Denbow, D. Michael, et Mark A. Cline. « Food Intake Regulation ». In Sturkie’s Avian Physiology . Elsevier, 2015. https://doi.org/10.1016/B978-0-12-407160-5.00021-X. Drażbo, A., D. Mikulski, J. Jankowski, et Z. Zduńczyk. « The Effect of Diets Containing Raw and Fermented Faba Beans on Gut Functioning and Growth Performance in Young Turkeys ». Journal of Animal and Feed Sciences 27, n o 1 (2018): 65‑73. https://doi.org/10.22358/jafs/82779/2018. Fernandez, R., S.M. Kim, J.L. Buenrostro, et J. McGinnis. « Triticale and Rye as Main Ingredients in Diets for Laying Hens ». Poultry Science 52, n o 6 (1973): 2244‑52. https://doi.org/10.3382/ps.0522244. Ferreira, João Paulo, Frank George Guimarães Cruz, Oliveira Filho, Pedro Alves de, et Ramon Duque. Fibra alimentar em dietas para aves – Uma revisão . 3, n o 2 (2017). Fuso, Andrea, Silvia Barbi, Laura Ioana Macavei, et al. « Effect of the Rearing Substrate on Total Protein and Amino Acid Composition in Black Soldier Fly ». Foods 10, n o 8 (2021): 1773. https://doi.org/10.3390/foods10081773. Gołębiewska, Kinga, Anna Fraś, et Damian Gołębiewski. « Rapeseed Meal as a Feed Component in Monogastric Animal Nutrition – a Review ». Annals of Animal Science 22, nᵒ 4 (2022): 1163‑83. https://doi.org/10.2478/aoas-2022-0020. Gucciardi, Antonina, Patrizia Zaramella, Irene Costa, et al. « Analysis and Interpretation of Acylcarnitine Profiles in Dried Blood Spot and Plasma of Preterm and Full-Term Newborns ». Pediatric Research 77, no 1 (2015): 36‑47. https://doi.org/10.1038/pr.2014.142. Huang, Qiang, Chaoliang Wen, Wei Yan, et al. « Comparative Analysis of the Characteristics of Digestive Organs in Broiler Chickens with Different Feed Efficiencies». Poultry Science 101, n o 12 (2022): 102184. https://doi.org/10.1016/j.psj.2022.102184. Johnson, Alan L. « The Avian Ovary and Follicle Development: Some Comparative and Practical Insights ». TURKISH JOURNAL OF VETERINARY AND ANIMAL SCIENCES 38 (2014): 660‑69. https://doi.org/10.3906/vet-1405-6. Kamińska, B. « Substitution of Soyabean Meal with Rapeseed meal or Its High-Protein Fraction in the Nutrition of Hens Laying Brown-Shelled Eggs ». Journal of Animal and Feed Sciences 12, nᵒ 1 (2003): 111‑19. https://doi.org/10.22358/jafs/67665/2003. Khayrova, Adelya, Sergey Lopatin, et Valery Varlamov. « Black Soldier Fly Hermetia illucens as a Novel Source of Chitin and Chitosan ». International Journal of Sciences 8, n o 04 (2019): 81‑86. https://doi.org/10.18483/ijSci.2015. Koivunen, Erja, Petra Tuunainen, Eija Valkonen, Laila Rossow, et Jarmo Valaja. « Use of Faba Beans (Vicia Faba L.) in Diets of Laying Hens ». Agricultural and Food Science 23, no 3 (2014): 165‑72. https://doi.org/10.23986/afsci.41329. Kuppusamy, Giva, Chee Kei Kong, Ganeswaran Chandra Segaran, et al. « Hummingbird-Leaves-Reared Black Soldier Fly Prepupae: Assessment of Nutritional and Heavy Metal Compositions ». Biology 9, n o 9 (2020): 274. https://doi.org/10.3390/biology9090274. Lohman Breeders. Guide d'élevage: LOHMANN LSL-CLASSIC, Production en cage. Cuxhaven, Germany: Lohman Breeders GmbH, 2021. Lu, Shengyong, Nittaya Taethaisong, Weerada Meethip, et al. « Nutritional Composition of Black Soldier Fly Larvae ( Hermetia illucens L.) and Its Potential Uses as Alternative Protein Sources in Animal Diets: A Review ». Insects 13, n o 9 (2022): 831. https://doi.org/10.3390/insects13090831. Mahmoud, A. E., P. C. H. Morel, M. A. Potter, et V. Ravindran. « The Apparent Metabolisable Energy and Ileal Amino Digestibility of Black Soldier Fly ( Hermetia illucens ) Larvae Meal for Broiler Chickens ». British Poultry Science 64, no 3 (2023): 377‑83. https://doi.org/10.1080/00071668.2022.2161873. Marono, S., R. Loponte, P. Lombardi, et al. « Productive Performance and Blood Profiles of Laying Hens Fed Hermetia illucens Larvae Meal as Total Replacement of Soybean Meal from 24 to 45 Weeks of Age ». Poultry Science 96, n o 6 (2017): 1783‑90. https://doi.org/10.3382/ps/pew461. McNab, J. M., et D. W. F. Shannon. « The Nutritive Value of Triticale and Rye for the Laying Hen ». British Poultry Science 16, n o 1 (1975): 9‑15. https://doi.org/10.1080/00071667508416153. Mills, Alisha, Anderson N. Maina, et Elijah G. Kiarie. « Comparative Responses of Lohmann Select Leghorn-Lite and Shaver Heritage White Leghorn Hens When Fed Diets Containing Fine and Coarse Oat Hulls ». Canadian Journal of Animal Science 104, n o 3 (2024): 346‑55. https://doi.org/10.1139/cjas-2024-0005. Muslykhah, Uswatun, Srisan Phupaboon, Chaichana Suriyapha, Maharach Matra, et Metha Wanapat. « Encapsulation of Protein-Based Bioactive from Black Soldier Fly for Ruminant Feeding ». Journal of Agriculture and Food Research 18 (décembre 2024): 101325. https://doi.org/10.1016/j.jafr.2024.101325. Nilsson, Jan-Ake, et Erik Svensson. « The Frequency and Timing of Laying Gaps ». Ornis Scandinavica 24, n o 2 (1993): 122. https://doi.org/10.2307/3676361. Odongo, Ernest Emmanuel, Wilfred Kisaakye Bbosa, et Priscilla Kagoro Kahunde. « Black Soldier Fly (BSF): A Sustainable Solution for Protein, Waste Management, and a Circular Bio-Economy ». European Journal of Theoretical and Applied Sciences 2, n o 3 (2024): 822‑34. https://doi.org/10.59324/ejtas.2024.2(3).64. Oviedo-Rondón, E.O., A. Toscan, N.S. Fagundes, J.K. Vidal, J. Barbi, et P. Thiery. « Soybean Meal Nutrient Composition, Amino Acid Digestibility, and Energy Content According to the Country of Origin and Year of Harvest Evaluated via NIRS ». Journal of Applied Poultry Research 33, n o 3 (2024): 100448. https://doi.org/10.1016/j.japr.2024.100448. Pereyra, Andrea S., Kelsey L. McLaughlin, Katherine A. Buddo, et Jessica M. Ellis. « Medium-Chain Fatty Acid Oxidation Is Independent of l -Carnitine in Liver and Kidney but Not in Heart and Skeletal Muscle ». American Journal of Physiology-Gastrointestinal and Liver Physiology 325, n o 4 (2023): G287‑94. https://doi.org/10.1152/ajpgi.00105.2023. Perez-Maldonado, R. A., P.F. Mannion, et D.J. Farrell. « Optimum Inclusion of Field Peas, Faba Beans, Chickpeas and Sweet Lupins in Poultry Diets. I. Chemical Composition and Layer Experiments ». British Poultry Science 40, n o 5 (1999): 667‑73. https://doi.org/10.1080/00071669987061. Rahimi, Parastoo, Md Saiful Islam, Phelipe Magalhães Duarte, et al. « Impact of the COVID-19 Pandemic on Food Production and Animal Health ». Trends in Food Science & Technology 121 (mars 2022): 105‑13. https://doi.org/10.1016/j.tifs.2021.12.003. Rakha, A., P. Åman, et R. Andersson. « Dietary Fiber in Triticale Grain: Variation in Content, Composition, and Molecular Weight Distribution of Extractable Components ». Journal of Cereal Science 54, n o 3 (2011): 324‑31. https://doi.org/10.1016/j.jcs.2011.06.010. Rodriguez-Sinovas, A., E. Fernandez, X. Manteca, A. G. Fernandez, et E. Gonalons. « CCK Is Involved in Both Peripheral and Central Mechanisms Controlling Food Intake in Chickens ». American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 272, n o 1 (1997): R334‑40. https://doi.org/10.1152/ajpregu.1997.272.1.R334. Romero, Carlos, Juan Carlos Cenalmor, Susana Chamorro, et César Redondo. « Effect of Different Dietary Doses of Black Soldier Fly Meal on Performance and Egg Quality in Free-Range Reared Laying Hens ». Animals 14, n o 22 (2024): 3340. https://doi.org/10.3390/ani14223340. Salam, Muhammad, Amina Shahzadi, Huaili Zheng, et al. « Effect of Different Environmental Conditions on the Growth and Development of Black Soldier Fly Larvae and Its Utilization in Solid Waste Management and Pollution Mitigation ». Environnemental Technology & Innovation 28 (novembre 2022): 102649. https://doi.org/10.1016/j.eti.2022.102649. Sauvant, D., Perez, J. M., &Tran, G. (Eds.). (2004). Tables of composition and nutritional value of feed materials. Wageningen Academic Publishers/INRA editions. Schneider, Jill E. « Energy Balance and Reproduction ». Physiology & Behavior 81, n o 2 (2004): 289‑317. https://doi.org/10.1016/j.physbeh.2004.02.007. Shu, Mung Kwan, Cheuk Ming Li, William Eduardo Furtado, Qianjun Huang, Sophie St-Hilaire, et Ákos Kenéz. « Antibacterial Properties of Oil Extracts of Black Soldier Fly Larvae Reared on Bread Waste ». Animal Production Science 64, n o 8 (2024). https://doi.org/10.1071/AN23394. Stengel, Andreas, et Yvette Tache. « Interaction between Gastric and Upper Small Intestinal Hormones in the Regulation of Hunger and Satiety: Ghrelin and Cholecystokinin Take the Central Stage ». Current Protein & Peptide Science 12, n o 4 (2011): 293‑304. https://doi.org/10.2174/138920311795906673. Suryati, Tuti, Euis Julaeha, Kindi Farabi, Hanies Ambarsari, et Ace Tatang Hidayat. « Lauric Acid from the Black Soldier Fly ( Hermetia illucens ) and Its Potential Applications ». Sustainability 15, n o 13 (2023): 10383. https://doi.org/10.3390/su151310383. Tahamtani, Fernanda M., Emma Ivarsson, Viktoria Wiklicky, et al. « Feeding Live Black Soldier Fly Larvae ( Hermetia illucens ) to Laying Hens: Effects on Feed Consumption, Hen Health, Hen Behavior, and Egg Quality ». Poultry Science 100, n o 10 (2021): 101400. https://doi.org/10.1016/j.psj.2021.101400. Zhu, Fan. « Triticale: Nutritional Composition and Food Uses ». Food Chemistry 241 (2018): 468‑79. https://doi.org/10.1016/j.foodchem.2017.09.009. Zhu, L.P., J.P. Wang, X.M. Ding, et al. « Effects of Dietary Rapeseed Meal on Laying Performance, Egg Quality, Apparent Metabolic Energy, and Nutrient Digestibility in Laying Hens ». Livestock Science 214 (2018): 265‑71. https://doi.org/10.1016/j.livsci.2018.06.007. Zulkifli, Nor Fatin Najihah Mohamad, Annita Yong Seok-Kian, Lim Leong Seng, Saleem Mustafa, Yang-Su Kim, et Rossita Shapawi. « Nutritional Value of Black Soldier Fly ( Hermetia illucens ) Larvae Processed by Different Methods ». PLOS ONE 17, no 2 (2022): e0263924. https://doi.org/10.1371/journal.pone.0263924. Supplementary Files Supplementarymaterials.pdf Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 28 Jan, 2026 Reviewers invited by journal 28 Jan, 2026 Editor assigned by journal 28 Dec, 2025 First submitted to journal 25 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8450877","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":581597114,"identity":"ca4a3d66-ea90-4c4c-a1ff-3437b8fa1760","order_by":0,"name":"Yosra ZNAZEN","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIie2QsQrCMBCGEw7qEp0rCPERIp0ExVepOLjUd1AK7ZI66+RbdG65waU4C3ETnBzqJtLB6KSDUTfBfMPljvzfcEeIxfLrZKUuTu0Lg+aLmwJfKIDs/ryJiXV8KM/VjvM4ybB3SXkDCC1PgUEpCm8p2aEzkxsfJ3PViYBAc5kalG0AhLlIQzcQOJGKasWBulEZ72klcBDxo8CuVIMPFN8D5uNQukwgOavhW6Wpd4FWhqMFC0SeTNUoAhoad2ms4z09VthfxYWnT6d0E+blyaC0s8eJRvc6fZ3X8Ofvyhi2WCyWP+UKvttVIiVOBu4AAAAASUVORK5CYII=","orcid":"https://orcid.org/0009-0009-6091-5265","institution":"Higher Institute of Agricultural Sciences of Chott Mariem: Institut Superieur Agronomique de Chott Mariem","correspondingAuthor":true,"prefix":"","firstName":"Yosra","middleName":"","lastName":"ZNAZEN","suffix":""},{"id":581597115,"identity":"1170f518-959e-4717-aae0-1c23f67ab23a","order_by":1,"name":"Marwa Gaddes","email":"","orcid":"","institution":"Higher Institute of Agricultural Sciences of Chott Mariem: Institut Superieur Agronomique de Chott Mariem","correspondingAuthor":false,"prefix":"","firstName":"Marwa","middleName":"","lastName":"Gaddes","suffix":""},{"id":581597116,"identity":"dcb4c9be-a978-436e-b500-7752f2ac42f9","order_by":2,"name":"Raja Chalghoumi","email":"","orcid":"","institution":"Higher School of Agriculture Mateur: Ecole Superieure d'Agriculture Mateur","correspondingAuthor":false,"prefix":"","firstName":"Raja","middleName":"","lastName":"Chalghoumi","suffix":""},{"id":581597117,"identity":"2bfea8dc-115d-4c54-b30e-b7c5bde786e4","order_by":3,"name":"Geert P.J. Janssens","email":"","orcid":"","institution":"UGent: Universiteit Gent","correspondingAuthor":false,"prefix":"","firstName":"Geert","middleName":"P.J.","lastName":"Janssens","suffix":""},{"id":581597118,"identity":"a3f167f7-4ff4-4b3e-b3b7-112517dd199a","order_by":4,"name":"Madiha Hadj Ayed","email":"","orcid":"","institution":"Higher Institute of Agricultural Sciences of Chott Mariem: Institut Superieur Agronomique de Chott Mariem","correspondingAuthor":false,"prefix":"","firstName":"Madiha","middleName":"Hadj","lastName":"Ayed","suffix":""}],"badges":[],"createdAt":"2025-12-25 20:23:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8450877/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8450877/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101480643,"identity":"591dcc9d-f84b-4382-8419-8ff64a9b8aa3","added_by":"auto","created_at":"2026-01-30 08:03:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":111929,"visible":true,"origin":"","legend":"\u003cp\u003eWeekly laying performance (mean±SE) of Lohman white hens fed a conventional diet (CONTROL), an alternative diet without (ALTER) or with 5% BSF supplementation (ALTER+BSF) (n=5). ADFI: average daily feed intake; FCRdozen: feed conversion ratio per dozen eggs; FCRmass: feed conversion ratio per kilogram. a-b: Means with different superscripts within each main effect are significantly different (p \u0026lt; 0.05). *: p \u0026lt; 0.05, ** p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8450877/v1/6845cb1eb6229887d148141d.png"},{"id":101480646,"identity":"1b4ec6bd-043c-480c-95c0-007c31960ff7","added_by":"auto","created_at":"2026-01-30 08:03:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":54081,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of variance explained by extracted principal components with eigenvalues \u0026gt; 1 from principal components analysis of hens fed a conventional (CONTROL), alternative without (ALTER) or with 5% BSF supplementation (ALTER+BSF) diets.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8450877/v1/d3e2e1fcc2a156d4ffe664cd.png"},{"id":101480644,"identity":"42718d1b-0ba4-45d8-a46a-5a166a464eca","added_by":"auto","created_at":"2026-01-30 08:03:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":121931,"visible":true,"origin":"","legend":"\u003cp\u003eMultivariate analysis of acylcarnitine profiles in hens fed a conventional diet (CONTROL), a diet with alternative ingredients without (ALTER) or with 5% BSF (ALTER+BSF). (A) Principal component analysis (PCA) biplot. Colored ellipses represent the 95% confidence intervals for each dietary group cluster. The arrows indicate the contributions and correlations of all 33 metabolites with the two principal components; their length and direction reflect the magnitude and orientation of each metabolite’s contribution to the total variance. (B) Sparse PLS-DA (sPLS-DA) biplot. This panel shows the optimal discrimination space achieved by the metabolites selected by the model. Dots represent samples projected onto the component axes. The length and direction of the arrows indicate each metabolite’s contribution to group separation.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8450877/v1/27bae42c9e0a5a3aa11a108d.png"},{"id":101480645,"identity":"5618d5be-29ac-47ea-a677-7f38c7c6701d","added_by":"auto","created_at":"2026-01-30 08:03:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":102782,"visible":true,"origin":"","legend":"\u003cp\u003eBox-and-whisker plots of summed acylcarnitine metabolites and metabolic ratios from dried blood spots of laying hens fed a standard diet (CONTROL), an alternative diet without (ALTER) and with 5% BSF (ALTER+BSF) diets. AC:C0: Acylcarnitine to free carnitine ratio; C3:C2: propionylcarnitine to acetylcarnitine ratio; C3DC:C2: malonylcarnitine to acetylcarnitine ratio; C4DC:C3: Succinylcarnitine to propionylcarnitine ratio; LCFA: Sum of long-chain acylcarnitines; SUM AC: Sum of total esterified carnitines; 3OHC4:C2: Hydroxybutyrylcarnitine to acetylcarnitine ratio; 3OHLC: sum of hydroxy long-chain acylcarnitine; 3OHLC:LC: hydroxy long-chain acylcarnitine to long chain acylcarnitine ratio. P values represent overall group comparisons performed via the Kruskal–Wallis test.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8450877/v1/11eaa95fb7d600e2e1c321b5.png"},{"id":101751484,"identity":"a49c040c-8091-43b8-bb0e-20efea47cd05","added_by":"auto","created_at":"2026-02-03 10:20:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1404312,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8450877/v1/0ddb14ea-4dbd-4826-a7a0-bc3b51aa4afd.pdf"},{"id":101480647,"identity":"8140b97a-2e3a-45e3-9aec-061a0fe66374","added_by":"auto","created_at":"2026-01-30 08:03:38","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":42043,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8450877/v1/46c76e8bfabc271745d92e3b.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eEvaluating sustainable ingredients including black soldier fly larvae (Hermetia illucens) in laying hens through performance, physiology and nutrient metabolism\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe sustainability and economic stability of poultry production are intrinsically linked to feed formulation. For decades, the industry has relied on a foundational diet of corn and soybean meals, a combination recognized for its high digestibility, excellent amino acid profile and consistent support of hen productivity. However, the reliance on these commodities poses a significant sustainability challenge, particularly for smallholder farmers in subtropical areas, as their prices are highly sensitive to fluctuations in the global market. Recent disruptions to international supply chains during the COVID-19 pandemic have further highlighted the vulnerability of this centralized model (Rahimi et al., 2022) and the urgent need to develop resilient, sustainable and locally adapted feed strategies.\u003c/p\u003e\u003cp\u003e With Tunisia as a case, where poultry production heavily depends on imported feed ingredients, developing locally sourced and sustainable alternatives is crucial to improve feed autonomy and production resilience. In this context, locally produced ingredients such as triticale, faba beans and rapeseed meals offer greater availability and sustainability while reducing the economic and environmental costs associated with transportation. Triticale, a high-yielding cereal grain, provides approximately 14.08 MJ/kg of metabolizable energy (McNab et Shannon, 1975). A study conducted by Fernandez et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1973\u003c/span\u003e) demonstrated that replacing up to 85% of corn with triticale in laying hens did not have any adverse effects on egg production. Furthermore, Aggoor et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) reported similar results with substitution levels up to 75%. In terms of fiber composition, triticale is rich in insoluble fibers such as cellulose and lignin (Zhu et Fan, 2018), which promote digesta passage without inducing excessive gut viscosity or hindering enzymatic nutrient access (Ferreira et al. 2017). Moreover, non-starch polysaccharides (NSPs), including arabinoxylan and ϐ-glucan, in some modern triticale cultivars generally lack long-chain structures capable of binding water and increasing intestinal viscosity in the monogastric gut (Rkha et al. 2011).\u003c/p\u003e\u003cp\u003eFaba beans constitute a protein-rich legume suitable for the partial replacement of soybean meals. They typically contain approximately 30% crude protein (CP) with high apparent digestibility of up to 90% in low-tannin, low-vicine and high-convicine cultivars (Cr\u0026eacute;pon et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Faba beans are particularly rich in lysine (20 g/kg), with high ileal digestibility values reaching 90% in selected cultivars. An inclusion rate of 50 g/kg of faba beans has shown no adverse effects on laying performance or livability (Koivunen et al.,2014) (Algawany et al., 2019). For some cultivars low in vicine and convicine, faba beans can even safely reach 200 g/kg in the diet (Perez-Maldonado et al.,1999). Although NSPs in faba beans may modestly reduce the availability of the apparent metabolizable energy in the gut, this limitation can be offset by increased cecal fermentation, leading to increased short-chain fatty acid (SCFA) production (Drażbo et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Given their low methionine and cysteine contents, which are crucial for egg production, faba beans can be effectively complemented by rapeseed meals, a byproduct rich in both methionine and cysteine (Cheng et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The crude protein content in rapeseed meals ranges between 30% and 50%, depending on the agronomic and processing conditions. However, its protein and amino acid contents are typically lower than those of soybean meal because of its high fiber level, thermal processing and interactions with antinutritional compounds such as tannins and phytic acid (Gołębiewska et al., 2022). In laying hens, inclusion rates below 117 g/kg of low glucosinolate rapeseed meal have been shown to maintain performance (Zhu et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), whereas higher levels may reduce egg weight by approximately 10% in Hy-Line and ISA Brown strains (Kamińska, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn rural farming conditions, details on the nutritive value of \u003cem\u003ein situ\u003c/em\u003e growing ingredients are often unknown. Additionally, the successful adoption of these alternative feedstuffs requires careful formulation to overcome potential limitations associated with antinutritional factors and fiber content. To address these challenges, the incorporation of black soldier fly (\u003cem\u003eHermetia illucens;\u003c/em\u003e BSF) larvae may ensure sufficient energy and protein intake. They possess the unique ability to be produced sustainably on agricultural side streams and organic waste (Odongo et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). A key advantage for smallholders is their ease of use, as the larvae can be provided whole without requiring complex processing. Previous studies have demonstrated that BSF larvae can effectively replace soybean meals in various forms, such as alive (Tahamtani et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), whole dried (Bejaei et Cheng, 2023) or meal (Romero et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), without any negative effects on laying performance. The crude protein content of BSF larvae varies with life stage, substrate, rearing conditions and processing method (Salam et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), ranging from 39 to 48% on a dry matter basis for dried larvae. Although their protein content is lower than that of conventional soybean and fish meals, full-fat BSF larvae are rich in essential amino acids such as lysine (23\u0026ndash;68 g/kg), leucine (27\u0026ndash;78 g/kg) and valine (28\u0026ndash;67 g/kg), which are crucial for albumen synthesis, energy metabolism and muscle maintenance (Lu et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Owing to their high lipid content, which can range between 11% (when reared in ensiled mussels) and 57% on a dry matter basis (when reared in bread), BSF larvae can serve as a valuable energy source. They also possess functional properties that can increase nutrient utilization and overall feed value. The chitin in their exoskeletons has prebiotic potential (Muslykhah et al.,2024), and their fat fraction is uniquely characterized by a predominance of lauric acid, which has antimicrobial properties (Suryati et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e The purpose of the present study was, therefore, to simulate poultry diets under rural farming conditions by substituting a conventional corn\u0026ndash;soybean meal diet with locally produced ingredients, with or without topping-up with dried whole BSF larvae. A comprehensive evaluation was conducted to assess the effects on laying performance as well as the overall physiological and metabolic responses of laying hens to these dietary modifications.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and housing\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe experiment was conducted at the experimental poultry farm of the Higher Institute of Agronomy of Chott Mariem (35\u0026deg;55'7.26\" N, 10\u0026deg;33'49.72\" E). A total of 28-week-old Lohmann White laying hens (n\u0026thinsp;=\u0026thinsp;150) were acquired from a commercial farm and randomly assigned to 15 floor pens (1.9 m \u0026times; 1.12 m \u0026times; 6 m H \u0026times; W \u0026times; L) such that the average initial live weight was consistent across the three dietary treatments (1452 g). The pens were each equipped with two nest boxes (840 cm\u0026sup2; per nest), 2.25 meters of perches, a 10-liter drinker and a suspended circular feeder. The internal area, covering two-thirds of the pen's surface, was lined with wood shavings, with fresh layers added weekly. The hens were allowed two weeks to acclimate. The lighting schedule consisted of a combination of daylight and artificial light, totaling 16 hours of light and 8 hours of darkness, maintained until the end of the study, as recommended by the Lohman LSL-CLASSIC layers guide (Lohmann breeders 2021). During the trial conducted from March to June, the environmental temperature fluctuated between 14.5\u0026deg;C and 33.0\u0026deg;C. The pens were visually separated to avoid group interaction. All hens were individually labeled with leg rings at the beginning of the experimental phase.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDiet design and chemical analysis\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAt 30 weeks of age, each pen was randomly assigned to one of the three experimental dietary treatments (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), each with five replicates of ten birds as follows:\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIngredients (%) and nutrient composition of the basal feeds used in the study\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCONTROL\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eALTER\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCorn\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e62.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoybean meal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTriticale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFaba beans\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRapeseed meal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoybean oil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSalt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.37\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDicalcium phosphate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.39\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLimestone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethionine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePREMIX\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBSF dried larvae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCalculated analysis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAME (MJ/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.44\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethionine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethionine\u0026thinsp;+\u0026thinsp;Cysteine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.69\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLysine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTryptophan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThreonine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAvailable P\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVitamin K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.76\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVitamin A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVitamin D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.98\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVitamin E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.57\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLaboratory analysis (%)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDM\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e89.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e89.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOM\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e88.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e89.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStarch\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCrude fat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNDF\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eADF\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e\u003csup\u003e1\u003c/sup\u003e PREMIX : vitamin and mineral premix contained the following: crude ash: 63.75%, calcium: 18.89%, sulfur: 3.87%, chlorine: 2.90%, magnesium: 0.35%, methionine: 16.96%, methionine\u0026thinsp;+\u0026thinsp;cysteine: 16.96%, manganese: 34.392 g/kg, zinc: 27.7 g/kg, iron: 24.628 g/kg, copper: 2.754 g/kg, iodine: 640 mg/kg, cobalt: 133.40 mg/kg, selenium: 104.04 mg/kg, choline: 114.8 g/kg, vitamin PP (Niacin): 5.4 g/kg, vitamin B5: 2700 mg/kg, vitamin B2: 1350 mg/kg, vitamin B6: 675 mg/kg, Vitamin B1: 448.34 mg/kg, vitamin K3: 360.07 mg/kg, folic acid: 90.9 mg/kg, vitamin B12: 2.7 mg/kg, Vitamin A: 3.599 k UI, vitamin E: 3284.10 UI, vitamin D3: 989.89 UI. \u003csup\u003e2\u003c/sup\u003e DM: Dry matter, \u003csup\u003e3\u003c/sup\u003e OM: organic matter, \u003csup\u003e4\u003c/sup\u003e NDF: Neutral detergent fiber, \u003csup\u003e5\u003c/sup\u003e ADF acid detergent fiber.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eCONTROL: A commercial pelleted concentrate for adult laying hens was fed with corn and soybean meal as the main components, providing 11.44 MJ/kg apparent metabolizable energy (AME) and 17.01% crude protein (CP).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e ALTER: Fed alternative pelleted concentrate for adult laying hens in which corn and soybean meal were partially substituted by local feed crops, i.e., faba beans, triticale and rapeseed meal designed to match the control\u0026rsquo;s energy (11.44 MJ/kg) and crude protein (17.07%) contents.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eALTER\u0026thinsp;+\u0026thinsp;BSF: 95% ALTER diet supplemented with a daily portion of whole dried BSF larvae, adjusted to 5% of the expected daily dry matter intake, which was set at 6 g of larvae per hen per day during the study.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe concentrate was provided daily at 9 am, and the hens had continuous access to both feed and water for \u003cem\u003ead libitum\u003c/em\u003e consumption. For the ALTER\u0026thinsp;+\u0026thinsp;BSF replicates, the dried BSF larvae were weighed, placed on two plates and provided as a top-up to the hens two hours after concentrate distribution. The proximate composition of the BSF dried larvae used in this study was as follows: 5.7% moisture, 43.58% crude protein, 30.54% ether extract, 6.04% total ash and 2.34% chitin on a dry matter basis. The gross energy (GE) of BSF larvae was estimated via the regression model developed by the Institut National de la Recherche Agronomique (INRA), as detailed in Sauvant et al. (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2004\u003c/span\u003e):\u003c/p\u003e\u003cp\u003eGE (MJ/kg)\u0026thinsp;=\u0026thinsp;17.3\u0026thinsp;+\u0026thinsp;0.0617 CP\u0026thinsp;+\u0026thinsp;0.2193 EE\u0026thinsp;+\u0026thinsp;0.0387 CF \u0026minus;\u0026thinsp;0.1867 Ash\u0026thinsp;+\u0026thinsp;Δ\u003c/p\u003e\u003cp\u003ewhere\u003c/p\u003e\u003cp\u003eGE is the gross energy (MJ/kg),\u003c/p\u003e\u003cp\u003eCP is the crude protein (g/kg),\u003c/p\u003e\u003cp\u003eEE is the ether extract (g/kg),\u003c/p\u003e\u003cp\u003eΔ is a correction factor equal to 0.\u003c/p\u003e\u003cp\u003eWith this equation, the GE of BSF larvae was calculated to be 25.88 MJ/kg on a dry matter basis, corresponding to 24.41 MJ/kg on a crude matter basis. Thus, an apparent total tract retention coefficient (ATTRC) for gross energy equal to 0.729, as mentioned in Mahmoud et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), was adopted to estimate the apparent metabolizable energy. The authors conducted an \u003cem\u003ein vivo\u003c/em\u003e digestive balance experiment on broiler chickens using a full fat BSF meal containing 26.6 MJ/kg GE on a crude matter basis, 48.6% CP, 32.4% EE and 5.3% ash on a dry matter basis. The GE content was determined via the bomb calorimetry method. On the basis of this retention coefficient, the AME of the BSF larvae used in the present study was estimated to be 17.79 MJ/kg on a crude matter basis. The ALTER\u0026thinsp;+\u0026thinsp;BSF diet had an estimated AME content of 11.75 MJ/kg and a CP content of 17.35% on a crude matter basis.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eChemical analysis\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eRepresentative samples of each feed type were collected, marked and ground to pass through a 1 mm sieve via a laboratory mill prior to analysis. The samples were analyzed following the standard procedures outlined by the Association of Official Analytical Chemists (AOAC, 2016). For dry matter determination, 2 to 3 grams of each sample were oven-dried for 16 hours at 105\u0026deg;C. After cooling in a desiccator, the samples were weighed, and the weight loss was recorded as the moisture content. The crude protein content was determined via the Khjeldal method, in which total nitrogen was measured, and a coefficient of 6.25 was used to estimate the protein content. Ether extraction was determined via the Soxhlet extraction method. A dried ground sample was weighed and placed in a Soxhlet apparatus, where lipids were extracted over 6 hours of repeated cycles. Afterwards, the samples were oven-dried, cooled, and the total fat content was determined via the gravimetric method. The total ash content was determined by placing pre weighed samples in a ceramic crucible, incinerating them in a muffle furnace at 550\u0026deg;C for 6 hours, and then cooling and weighing the residue. The two concentrates used were analyzed for neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents via a Tecator Fibertec\u0026trade;. The starch in the feeds was analyzed via polarimetry. The chitin present in the BSF larvae was extracted via a cascade process to eliminate proteins, oils, minerals and pigments, resulting in a purified chitin fraction, following the method described by Khayrova et al. (2019) in triplicate. Briefly, subsamples were dried at 95\u0026deg;C for 24 hours, treated in a 1 L beaker with 500 mL of 10% (w/w) NaOH and left in a 50\u0026deg;C water bath for 12 hours with occasional stirring. The resulting broth was filtered, washed, added to a 1 L beaker containing 5% HCl, and left at 20\u0026deg;C for 6 hours. The residue was filtered, washed with distilled water, oven-dried at 95\u0026deg;C and weighed. A second deproteinization was applied using 2% NaOH for 2 hours, followed by a second demineralization using 2% HCl for 2 hours. After drying, the remaining solid fraction was treated with 50 mL of bleach at 1% for 2 hours for depigmentation.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003ePostmortem assessment\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAt the end of the experiment (40 weeks), a total of 30 hens (2 hens per pen) were marked, weighed, and slaughtered. Hens were dissected, and the following traits were measured: intestine weight, liver weight, filled and empty gizzard weight, heart weight, abdominal fat weight, ovary and oviduct weight, small intestine length and rectum length. All digestive organ weights were measured via an electronic balance with an accuracy of 0.1 g. The lengths were measured with a measuring tape to the nearest millimeter. The relative weights of the previous organs were calculated as the weight of the specific organ divided by the live body weight (Huang et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003ePerformance\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFor each hen, body weight was recorded at the beginning (week 30) and at the end of the experimental period (week 40). Mortality and health status were monitored daily. The feed residue weight and water intake were recorded daily by replication. All eggs produced were collected daily, counted and individually weighed via an analytical balance (CRYSTAL 300 CE*, precision\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0001 g). Water consumption, average daily feed intake (ADFI), the number of eggs laid, egg mass, the egg laying rate, the water-to-feed ratio, and the feed conversion ratios expressed per mass (FCRmass) and per dozen (FCRdozen) were determined weekly.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSerum biochemical analysis and acylcarnitine profiling\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eOn day 69 of the trial, fresh blood samples were collected from the wing vein of 10 birds per treatment group (2 hens per pen). A volume of 2.5 mL was placed in a K3EDTA vacutainer tube and centrifuged at 3000 rpm for 15 minutes. The serum obtained was collected and stored at \u0026minus;\u0026thinsp;20\u0026deg;C and then analyzed for biochemical indices, including glucose, total cholesterol, triglycerides, creatinine, uric acid, urea, total protein and serum electrolytes, including sodium (Na), chloride (Cl), potassium (K) and alkaline reserves. Additionally, liver function was evaluated through analysis of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma-glutamyl transferase (GGT) levels via specific commercial kits (Marono et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). For acylcarnitine profiling, a 50 \u0026micro;L aliquot of each EDTA-treated blood sample was spotted onto circular specimen collection papers (Whatman Protein Saver cards, 903\u0026trade;, UK), dried, and then sent to Ghent University Hospital (laboratory for clinical chemistry) for acylcarnitine profiling via ultraperformance liquid chromatography (UPLC)-tandem mass spectrometry, as described in Gucciardi et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In brief, acylcarnitine from 3.2 mm dried blood spots was extracted with 100 \u0026micro;L of methanol containing labeled internal standards. The extract was then dried under nitrogen at 60\u0026deg;C and derivatized to form butyl esters by adding 100 \u0026micro;L of freshly prepared butanol with 5% acetyl chloride, followed by heating at 60\u0026deg;C for 20 minutes. Chromatographic separation was performed on a UPLC system equipped with an ethylene-bridged hybrid C (18) column. Detection was carried out on a Waters Micromass Quattro Ultima tandem quadrupole mass spectrometer. Acylcarnitine concentrations were determined by integrating peak areas and using a calibration curve with a correlation coefficient ranging from 0.990 to 0.999.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe data were organized and validated in Microsoft Excel. Statistical analyses were performed via IBM SPSS Statistics for Windows (version 31.0.0.0), whereas multivariate analyses (PERMANOVA, PERMADISP, PCA and sPLS-DA) were conducted in R (version 4.4.0) via appropriate packages. Every replicate was treated as the experimental unit, and statistical significance was defined as \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e. For laying performance, a linear mixed-effects model was used. The diet group and week were specified as fixed effects, whereas the pen was included as a random effect. Pairwise comparisons among diets were conducted via the Bonferroni-adjusted method. For single time-point variables, normality and homogeneity of variance were evaluated via the Shapiro‒Wilk test and Levene\u0026rsquo;s test. When the assumptions were met, one-way ANOVA followed by Tukey\u0026rsquo;s HSD was applied. A permutational multivariate analysis of variance (PERMANOVA) test based on a Euclidean distance matrix (999 permutations) was applied in R via the adonis2 function in the vegan package to evaluate differences in 33 acylcarnitine metabolites among the three dietary groups. The homogeneity of multivariate dispersions PERMADISP was run via the betadisper function. Significant global effects were followed by pairwise PERMANOVA comparisons. Principal component analysis (PCA) was performed via the FactoMineR package to explore major sources of variation in the acylcarnitine profiles. Eigenvalues were used to determine the percentage of variance and metabolite correlations, which were assessed via the Pearson correlation coefficient (r) with two-tailed correlation significance testing (\u003cem\u003ep\u003c/em\u003e). The first two principal components were used to generate the PCA biplot. To identify metabolites contributing to group separation, a supervised sPLS-DA model was performed via the mixOmics package. Discriminant metabolites were ranked according to their variable importance in projection (VIP) score. Individual metabolites (n\u0026thinsp;=\u0026thinsp;33) were further analyzed via the Kruskal‒Wallis test, and p values were adjusted for multiple testing via the Benjamini‒Hochberg false discovery rate (FDR) method. The derived sums and metabolic ratios were evaluated via the Kruskal‒Wallis test, and the data are presented as medians (P25, P75) and box‒whisker plots.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGrowth and laying performance\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eHens\u0026rsquo; body weights were similar across all dietary treatments, with no significant differences at any time point (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e). The birds started with an average initial body weight of 1452 g and reached a final body weight of 1599 g, resulting in a homogeneous body weight gain of 149 g (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.31\u003c/em\u003e), indicating that dietary modifications did not affect overall growth performance.\u003c/p\u003e \u003cp\u003eThe effects of dietary treatments on laying performance throughout the trial period are presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The hens in the ALTER\u0026thinsp;+\u0026thinsp;BSF group presented a 2% lower laying rate (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) than did those in the CONTROL and ALTER groups, which maintained similar rates. In contrast, the ALTER\u0026thinsp;+\u0026thinsp;BSF group laid heavier eggs (66 g) than did the ALTER and CONTROL groups, which presented similar egg weights (65 g, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e), resulting in a stable egg mass throughout the trial (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e). The feed conversion ratio per mass (FCRmass) was identical for hens fed the CONTROL and ALTER diets (1.69), whereas it decreased significantly for the hens fed dried BSF larvae (1.65, \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.01\u003c/em\u003e). The remaining parameters, such as ADFI, feed conversion ratio per dozen (FCRdozen), drinking and water-to-feed ratio, were not affected by diet. No mortalities or pathological signs were observed throughout the trial.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLaying performance (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) of Lohman white hens fed a conventional diet (CONTROL), a diet with alternative ingredients without (ALTER) or with 5% black soldier fly (ALTER\u0026thinsp;+\u0026thinsp;BSF) (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDIET GROUP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCONTROL\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eALTER\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eALTER\u0026thinsp;+\u0026thinsp;BSF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eADFI\u003csup\u003e1\u003c/sup\u003e (g DM/hen/day)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e107.97\u0026thinsp;\u0026plusmn;\u0026thinsp;7.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e108.65\u0026thinsp;\u0026plusmn;\u0026thinsp;7.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e105.67\u0026thinsp;\u0026plusmn;\u0026thinsp;9.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDrinking (L/hen/day)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.72\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLaying rate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e98.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.89 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e98.54\u0026thinsp;\u0026plusmn;\u0026thinsp;1.54 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e96.28\u0026thinsp;\u0026plusmn;\u0026thinsp;3.22 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEgg weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e65.03\u0026thinsp;\u0026plusmn;\u0026thinsp;1.54 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e65.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e66.27\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEgg mass (g/day)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e63.91\u0026thinsp;\u0026plusmn;\u0026thinsp;2.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64.17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e63.81\u0026thinsp;\u0026plusmn;\u0026thinsp;2.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFCRmass\u003csup\u003e2\u003c/sup\u003e (kgDM/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFCRdozen\u003csup\u003e3\u003c/sup\u003e (gDM/dozen)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWater:feed (g/g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e1\u003c/sup\u003e ADFI: average daily feed intake, \u003csup\u003e2\u003c/sup\u003eFCRdozen: feed conversion ratio per dozen, \u003csup\u003e3\u003c/sup\u003eFCRmass: feed conversion ratio per mass. P values report the overall group comparison performed using linear mixed-effects model with Bonferroni correction. a-b: Means with different superscript letters within each main effect are significantly different (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWeekly performance parameters are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The three dietary groups presented consistent feed intake, except for the ALTER\u0026thinsp;+\u0026thinsp;BSF group, which declined significantly during the last week of the trial, reaching its lowest level (100 g DM/hen/day, \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.04\u003c/em\u003e). Laying rates were comparable among the three dietary groups at the beginning of the trial. A progressive decrease in laying rate was observed in the ALTER\u0026thinsp;+\u0026thinsp;BSF group, reaching a significant decrease of 3% in the fourth week (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.04\u003c/em\u003e), after which laying performance recovered and remained comparable to that of the other groups until the end of the trial. Additionally, hens fed the ALTER\u0026thinsp;+\u0026thinsp;BSF diet consistently produced numerically heavier eggs, with this difference reaching statistical significance in the ninth week.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePostmortem results\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e presents the effects of the three dietary treatments on live body weight, relative organ weights, and relative intestinal lengths in hens. No significant differences were observed for any of the measured parameters (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e). The live body weights ranged from 1594 g to 1609 g. The relative organ weights, including those of the digestive (gizzard, liver, and intestine) and reproductive (ovary and oviduct) organs, did not differ significantly among the treatments (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e). Similarly, small intestine length and rectum length were not influenced by diet (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e), indicating that dietary inclusion of BSF larvae did not affect organ development or gross intestinal morphology.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLive body weight (g), relative organ weight (%) and relative intestinal length (%) (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) of Lohman white hens fed a conventional diet (CONTROL), a diet with alternative ingredients without (ALTER) or with 5% BSF (ALTER\u0026thinsp;+\u0026thinsp;BSF) (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCONTROL\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eALTER\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eALTER\u0026thinsp;+\u0026thinsp;BSF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLive body weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1594.36\u0026thinsp;\u0026plusmn;\u0026thinsp;32.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1609.1\u0026thinsp;\u0026plusmn;\u0026thinsp;23.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1596.42\u0026thinsp;\u0026plusmn;\u0026thinsp;27.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.92\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eRelative weight (%)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIntestine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFilled gizzard\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEmpty gizzard\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLiver\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAbdominal fat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOvary \u0026amp; oviduct\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eRelative length (%)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSmall intestine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.07\u0026thinsp;\u0026plusmn;\u0026thinsp;1.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.26\u0026thinsp;\u0026plusmn;\u0026thinsp;1.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.69\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRectum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.46\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAcylcarnitine profiling\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe global PERMANOVA revealed a highly significant effect of diet on the overall acylcarnitine profile (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.002\u003c/em\u003e), with dietary treatment explaining 74.7% of the total variance (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.74). The test for homogeneity of dispersion PERMADISP did not reveal a significant difference in variance between the groups (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.29\u003c/em\u003e). Pairwise PERMANOVAs revealed that the ALTER diet significantly altered the metabolic profile compared with the CONTROL diet (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.054\u003c/em\u003e). In addition, significant differences were observed between the ALTER and ALTER\u0026thinsp;+\u0026thinsp;BSF groups (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.005\u003c/em\u003e), with BSF inclusion accounting for 74.6% of the explained variance. No significant differences were detected between the CONTROL and ALTER\u0026thinsp;+\u0026thinsp;BSF groups (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.29\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eThe PCA revealed ten components with eigenvalues greater than 1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The first two principal components (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. A) collectively explained 45.3% of the total variance in the dataset (Components 1: 28% and 2: 17.1%). The positive axis of component 1 was driven mainly by long-chain carnitine forms such as myristoylcarnitine (C14), which was positively correlated with tetradecenoylcarnitine (C14:1) (r\u0026thinsp;=\u0026thinsp;0.9, \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.001\u003c/em\u003e), tetradecadienoylcarnitine (C14:2) (r\u0026thinsp;=\u0026thinsp;0.81, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e), palmitoylcarnitine (C16) (r\u0026thinsp;=\u0026thinsp;0.8, \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.002\u003c/em\u003e). The positive axis of component 2 was driven mainly by acetylcarnitine (C2), which was positively correlated with propionylcarnitine (C3) (r\u0026thinsp;=\u0026thinsp;0.65, \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.02\u003c/em\u003e) and most of ϐ-oxidation forms such as 3-Hydroxybutyrylcarnitine (C4-OH) (r\u0026thinsp;=\u0026thinsp;0.76, \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.004\u003c/em\u003e), 3-Hydroxyisovalerylcarnitine (C5-OH) (r\u0026thinsp;=\u0026thinsp;0.64, \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.023\u003c/em\u003e) and 3-Hydroxypalmitoleylcarnitine (C16:1-OH) (r\u0026thinsp;=\u0026thinsp;0.73, \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.006\u003c/em\u003e), which was positively correlated with 3-Hydroxylorylcarnitine (3OH-C12) (r\u0026thinsp;=\u0026thinsp;0.65, \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.03\u003c/em\u003e). The negative axis of component 2 was strongly driven by linoylcarnitine (C18:2), which was negatively correlated with lauroylcarnitine (C12) (r\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.62, \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.03\u003c/em\u003e) and glutarylcarnitine (C5DC) (r\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.67, \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.015\u003c/em\u003e). Free carnitine (C0) was positively correlated with C3 (r\u0026thinsp;=\u0026thinsp;0.72, \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.006\u003c/em\u003e), isovalerylcarnitine (C5) (r\u0026thinsp;=\u0026thinsp;0.85, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) and C4-OH (r\u0026thinsp;=\u0026thinsp;0.29, \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.048\u003c/em\u003e). The results of Pearson correlation matrix are presented in the Supplementary Materials (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The CONTROL group presented a compact elongated cluster primarily located in the lower-left quadrant, indicating a relatively homogeneous metabolic profile with minimal metabolic disturbance. The ALTER group formed a tight cluster that was mostly positioned in the upper-left quadrant between the negative axis of component 1 and the positive axis of component 2, displaying moderate heterogeneity and showing a metabolic shift from CONTROL without extreme divergence. The ALTER\u0026thinsp;+\u0026thinsp;BSF group exhibited the widest cluster, which was clearly separated from the other groups and positioned along the positive axes of components 1 and 2, covering most metabolites contributing to the global variance.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe sPLS-DA model (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. B) selected nine unique metabolites with the highest VIP scores (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) as optimal discriminators among the three dietary groups, with 18% explained variance for component 1, which was driven by C0, C3, C5, C4-OH and C10, and 13% explained variance for component 2, which was driven by C5DC, adipoylcarnitine (C6DC), stearoylcarnitine (C18), C12 and C4-OH. Univariate Kruskal‒Wallis tests revealed that none of the metabolites were significantly different after FDR correction was applied (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Likewise, no significant effects of diet were observed for the derived acylcarnitine sums and ratios presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAcylcarnitine profile (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) in dried blood spot from hens fed a conventional diet (CONTROL), a diet with alternative ingredients without (ALTER) or with 5% BSF (ALTER\u0026thinsp;+\u0026thinsp;BSF).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMetabolite\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMetabolite name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCONTROL\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eALTER\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eALTER\u0026thinsp;+\u0026thinsp;BSF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eVIP\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003e(\u0026micro;mol/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFree carnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e44.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e37.13\u0026thinsp;\u0026plusmn;\u0026thinsp;2.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e50.03\u0026thinsp;\u0026plusmn;\u0026thinsp;3.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePropionylcarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIsovalerylcarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC4-OH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHydroxybutyrylcarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.91\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDecanoylcarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC5DC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGlutarylcarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC6DC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAdipoylcarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC18:2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLinoylcarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.48\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLauroylcarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAcetylcarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.88\u0026thinsp;\u0026plusmn;\u0026thinsp;3.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25.65\u0026thinsp;\u0026plusmn;\u0026thinsp;3.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC14:2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTetradecadienoylcarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.015\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.086\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMyristoylcarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.017\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC16:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePalmitoleylcarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC16:1-OH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHydroxypalmitoleylcarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.025\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC16-OH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHydroxypalmitoylcarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHexanoylcarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.043\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC14:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTetradecenoylcarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC18-OH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHydroxystearoylcarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOctanoylcarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eButyrylcarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC5-OH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHydroxyisovalerylcarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC3DC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMalonylcarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3OH-C12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3-Hydroxylauroylcarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC14-OH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHydroxymyristoylcarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePalmitoylcarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStearoylcarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC18:1-OH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHydroxyoleylcarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC14:1-OH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHydroxymyristoleylcarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC10:2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDecadienoylcarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC10:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDecenoylcarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC4DC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSuccinylcarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC18:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOleylcarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.035\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC5:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTiglylcarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.033\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.036\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003ep values were adjusted for multiple testing using the False Discovery Rate (FDR) method, VIP: Variable importance in projection score.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSerum biochemistry\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e presents the biochemical parameters of the blood serum. There were no significant differences observed among the dietary groups for glucose, total cholesterol, or triglyceride levels (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e). The urea concentration was lower in the alternative diet group than in the CONTROL group (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.002\u003c/em\u003e). Conversely, the alkaline reserve increased significantly with the ALTER diet (16.9 mmol/L) (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.01\u003c/em\u003e), whereas the ALTER\u0026thinsp;+\u0026thinsp;BSF diet slightly decreased it to 15 mmol/L. No significant differences were detected for the liver enzymes AST and GGT (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e). In contrast, feeding the ALTER diet increased ALT activity to 14.40 UI/L, while feeding the ALTER\u0026thinsp;+\u0026thinsp;BSF diet significantly reduced ALTER activity to 11.1 UI/L, which was statistically similar to that of the CONTROL group (10.38 UI/L) (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.01\u003c/em\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSerum biochemical parameters (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) in laying hens fed a conventional diet (CONTROL), a diet with alternative ingredients without (ALTER) or with 5% BSF (ALTER\u0026thinsp;+\u0026thinsp;BSF) (n\u0026thinsp;=\u0026thinsp;8).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCONTROL\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eALTER\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eALTER\u0026thinsp;+\u0026thinsp;BSF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eEnergy \u0026amp; lipid metabolism (g/L)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlucose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal cholesterol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTriglycerides\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eProtein metabolism \u0026amp; kidney function (mg/L)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCreatinine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.47\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUric acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e41.80\u0026thinsp;\u0026plusmn;\u0026thinsp;12.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e42.19\u0026thinsp;\u0026plusmn;\u0026thinsp;5.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e43.91\u0026thinsp;\u0026plusmn;\u0026thinsp;14.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.95\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40\u0026thinsp;\u0026plusmn;\u0026thinsp;8 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30\u0026thinsp;\u0026plusmn;\u0026thinsp;6 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20\u0026thinsp;\u0026plusmn;\u0026thinsp;6 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal protein (g/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e39\u0026thinsp;\u0026plusmn;\u0026thinsp;8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e43\u0026thinsp;\u0026plusmn;\u0026thinsp;11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.77\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eElectrolytes \u0026amp; acid‒base balance (mmol/L)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSodium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e84.6\u0026thinsp;\u0026plusmn;\u0026thinsp;6.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e110\u0026thinsp;\u0026plusmn;\u0026thinsp;25.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e113.8\u0026thinsp;\u0026plusmn;\u0026thinsp;20.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChloride\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e67.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e81.9\u0026thinsp;\u0026plusmn;\u0026thinsp;19.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e85.9\u0026thinsp;\u0026plusmn;\u0026thinsp;15.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePotassium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.61\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.59\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlkaline reserve\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15\u0026thinsp;\u0026plusmn;\u0026thinsp;2.62 ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eLiver function (UI/L)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAST\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e349.2\u0026thinsp;\u0026plusmn;\u0026thinsp;74.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e339.7\u0026thinsp;\u0026plusmn;\u0026thinsp;40.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e311.1\u0026thinsp;\u0026plusmn;\u0026thinsp;119.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.76\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALT\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.33 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.52 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGGT\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.9\u0026thinsp;\u0026plusmn;\u0026thinsp;8.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.92\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALT/AST\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003e1\u003c/sup\u003e Aspartate Aminotransferase; \u003csup\u003e2\u003c/sup\u003e Alanine aminotransferase; \u003csup\u003e3\u003c/sup\u003e Gamma-glutamyl transferase. a-b: Means with different superscript letters within each main effect are significantly different (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study revealed that the ALTER diet was effectively utilized by the layers to maintain performance comparable to that of the standard CONTROL diet. Even with dietary complexity (Triticale, faba beans and rapeseed meal), it seems that the plant-based alternative diet did not disturb the energy-to-protein balance during the ten weeks of the trial. These findings align with those of Mills et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), who reported that commercial strains of laying hens are well adapted to diets rich in structural materials with insoluble fiber without compromising laying performance. Among the three dietary groups, only ALTER\u0026thinsp;+\u0026thinsp;BSF resulted in significantly different performance, characterized by a slower laying rate but heavier eggs. Despite the reduced laying frequency observed in the group fed BSF, the three diet groups were similar in terms of the mass of egg produced. These findings suggest that hens fed a diet supplemented with 5% dried BSF larvae did not exhibit reduced productivity but rather underwent significant physiological changes. This biological response could be explained by the feeding strategy itself. The ALTER\u0026thinsp;+\u0026thinsp;BSF hens received highly palatable and nutrient-rich BSF larvae on top-up. This likely resulted in a sudden influx of easily digestible fats and amino acids (Alafif et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), which may have slowed the frequency of ovulation and led the hens\u0026rsquo; metabolism to invest more resources in each egg. In fact, consuming fat-rich BSF larvae (23% CF) two hours after receiving high-fiber meal may cause abnormal gastrointestinal motility (Rodriguez-Sinovas, 1997). The high fat content likely triggered the release of satiety hormones, mainly cholecystokinin (CCK) (Denbow et Cline, 2015), reducing the meal size and increasing the inter-meal interval (Stengel et Tache, 2011). These effects align with the numerically reduced ADFI observed in the ALTER\u0026thinsp;+\u0026thinsp;BSF group throughout the trial, particularly during the last week. Shneider et al. (2004) extensively discussed how the body\u0026rsquo;s energy balance influences the hormonal regulation of the reproductive system. Following the sudden increase in energy available from the BSF, the hypothalamus may have temporarily suppressed the reproductive cascade. This response could inhibit the nocturnal luteinizing hormone (LH) preovulatory surge, disrupting the hormonal signals related to circadian and follicular growth rhythms and leading to a skipped ovulation the next morning (Johnson, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Once the daily BSF signal is sufficient to push the total stress level above the threshold, the LH surge is inhibited, and the day is skipped. Continuous nutrient intake combined with reduced reproductive energy expenditure creates a significant surplus of protein and energy. This surplus is reallocated by the hen\u0026rsquo;s body to the next egg in the sequence, resulting in heavier eggs (Cucco et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Nilson \u0026amp; Svensson, 1993). This aligns perfectly with the decreased laying rate and increased egg weight observed in the ALTER\u0026thinsp;+\u0026thinsp;BSF group.\u003c/p\u003e \u003cp\u003eDespite the daily occurrence of sudden BSF meal, the hens exhibited physiological adaptations to this disruptive event following a threshold response. The weekly egg laying and egg weight curves clearly illustrate this physiological process. The decline in laying frequency observed during the third week of the trial likely reflects the initial impact of the top-up BSF meal. The stress induced may have pushed many hens beyond their tolerance threshold, resulting in a significant drop in egg production. By the fourth week, the laying rate of the ALTER\u0026thinsp;+\u0026thinsp;BSF group had rebounded, and egg weight tended to increase. These findings suggest that the endocrine and metabolic systems adapted to the new daily rhythm, establishing a new physiological equilibrium capable of accommodating the BSF-supplemented diet (Nilson et Svensson, 1993). Thereafter, egg production recovered and remained comparable to that of the other groups until the end of the trial.\u003c/p\u003e \u003cp\u003eThis study demonstrated a pronounced effect of supplementation with BSF larvae on the global acylcarnitine profile, reflecting coordinated remodeling in multiple biochemical pathways rather than changes in individual metabolites. Multivariate analyses confirmed this integrated metabolic response, which was driven mainly by nine discriminating metabolites with the most pronounced difference observed between the ALTER and ALTER\u0026thinsp;+\u0026thinsp;BSF groups. The PCA results revealed a stable and homogeneous metabolic profile in CONTROL hens, whereas the ALTER group presented moderate metabolic shifts. In contrast, the ALTER\u0026thinsp;+\u0026thinsp;BSF group displayed the greatest within-group variability, highlighting a strong metabolic response to BSF supplementation. Component 1 is driven by markers associated with lipid metabolism and long-chain fatty acid (LCFA) oxidation. These results are consistent with those of Aprianto et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), who reported that BSF larval oil suppresses the expression of fat-synthesis genes (acetyl-CoA carboxylase and fatty acid synthase) while upregulating the expression of genes involved in β-oxidation pathways. However, the unchanged 3OHLC:LC ratio, along with the C4-OH, C10 and C12 discriminators, which tended to be high in BSF-fed hens, suggest that the ketogenic state observed in the ALTER\u0026thinsp;+\u0026thinsp;BSF group was driven mainly by medium-chain fatty acids (MCFAs) rather than LCFAs. This could be explained by the fact that unlike LCFAs, MCFAs such as lauric acid, which is abundant in BSF larvae (Shu et al.,2025), diffuse directly into the mitochondria, bypassing the body\u0026rsquo;s main regulatory system (carnitine palmitoyltransferase 1: CPT1) for fat burning (Pereyra et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), as reflected by the unaffected levels of malonylcarnitine (C3DC). Anas et al., (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) reported that hens fed a diet containing 2% calcium salt of BSF larvae oil, which lauric acid is the main component, produced eggs with increased yolk, albumen and eggshell weights.\u003c/p\u003e \u003cp\u003eFurthermore, the elevated propionylcarnitine observed in ALTER\u0026thinsp;+\u0026thinsp;BSF hens indicates the degradation of branched-chain amino acids (leucine, isoleucine, valine), likely reflecting the detoxification of excess nitrogen derived from the BSF protein supply rich in those amino acids (Fuso et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Lu et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Consistently, free carnitine was positively correlated with short-chain acylcarnitines and 3-xydroxybutyrylcarnitine and it showed elevated levels in the ALTER\u0026thinsp;+\u0026thinsp;BSF group and the highest VIP score. This suggests that BSF supplementation prevented functional carnitine deficiency associated with the ketogenic state. Therefore, the elevated free carnitine levels likely resulted from direct dietary intake of the BSF larvae, which is consistent with findings by Kuppusamy et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), who demonstrated that BSF larvae can accumulate carnitines at levels substantially greater than those present in their substrate. However, the three dietary groups presented comparable overall functional metabolic indices, suggesting that 5% BSF supplementation induced coordinated remodeling of fatty acid oxidation without evident metabolic dysfunction, revealing a fuel-switching adaptation in response to the BSF fat and protein supply.\u003c/p\u003e \u003cp\u003eAnalysis of serum biochemical indicators revealed a clear pattern of physiological stress and subsequent mitigation. A primary finding was that hens fed the ALTER diet experienced a state of hepatocellular stress, as evidenced by a significant increase in the liver-specific ALT enzyme. The concurrent stability of AST levels, and therefore the ALT:AST ratio, suggests that mild-to-moderate injury is characterized by increased cell membrane permeability, rather than severe necrosis. Importantly, supplementation with BSF larvae effectively attenuated these elevations, restoring ALT levels to those of the CONTROL group. Since the direct effect of BSF on liver enzymes has not been previously reported, the protective mechanism observed may be linked to improved gut health. Dabbou et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) reported that modified BSF larval fat can improve gut health without inducing any adverse histopathological alterations. The abundant lauric acid in BSF has been shown to have antimicrobial proprieties, and chitin can act as a prebiotic compound (Shu et al.,2025; Bonomini et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Together, these compounds may promote a healthier gut microbiome, reducing the metabolic and detoxification load on the hen liver.\u003c/p\u003e \u003cp\u003eHowever, other metabolic indicators revealed that general metabolic processes and renal functions remained stable. The levels of glucose, total protein, and uric acid remained unaffected across all groups, suggesting a stable overall metabolic status. Since urea in birds is the primary byproduct of arginine catabolism (Ali et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), the significantly higher concentration of urea found in the blood of CONTROL hens is most likely due to the higher content of soybean meal, which is rich in arginine (Oviedo-Rond\u0026oacute;n et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This study also demonstrated successful adaptation to changes in the dietary acid-base balance. Despite the balanced formulation of diets in terms of potassium, sodium and chloride, a significant increase in the alkaline reserve observed in the ALTER group indicates a net alkalizing effect of certain plant ingredients. The kidney may compensate by retaining more bicarbonate ions to stabilize the blood pH. The fact that the serum levels of sodium, chloride and potassium remained stable across all the groups is proof of a successful regulatory renal response.\u003c/p\u003e \u003cp\u003eThe relative weights of major reproductive, metabolic, and digestive organs were not affected by dietary treatment, confirming that their effects did not progress to structural alterations. The hens fed BSF larvae produced heavier eggs without a corresponding increase in the relative weights of their reproductive organs, indicating an efficient redistribution of energy and nutrients toward egg formation. Furthermore, the stability of live body weight, abdominal fat content, and intestinal length and weight across groups demonstrated that the digestive challenges posed by the alternative ingredients did not result in wasting, fat accumulation, or global intestinal morphological remodeling. These findings suggest that the observed effects reflect a physiological adaptation to the nutritional characteristics of alternative diets rather than pathological responses.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIn conclusion, the partial substitution of corn and soybean meals with alternative plant-based feedstuffs maintained laying performance. However, this substitution entails a subtle metabolic cost. Careful consideration should be given to the antinutritional factors that could pose a potential risk to long-term health and productivity. The inclusion of 5% BSF dried larvae elicited a distinct physiological response. The BSF larvae acted as a functional ingredient, exhibiting a potential hepatoprotective effect that attenuated the liver stress signs observed in the hens fed alternative plant-based diets. Furthermore, the altered metabolic pathways shown in hens fed BSF larvae may explain the observed shift in reproduction physiology and, consequently, in performance, where the nutrients were channeled toward slowing laying frequency and producing heavier eggs.\u003c/p\u003e \u003cp\u003eOverall, the use of plant-based alternative ingredients did not compromise laying performance, while BSF larvae supplemented with 5% BSF acted as a functional nutrient, favoring heavier eggs and stimulating fat oxidation. These findings support the use of alternative ingredients for sustainable poultry production in subtropical rural areas.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003eThe author used OpenAI’s Curie service to help improve wording and correct spelling and grammar.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This study was supported by the PRIMA program (European Union) under grant agreement No. 2015, project SUSTAvianFEED.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest:\u003c/strong\u003e The authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e Yosra Znazen: data collection and treatment, writing the first draft of the manuscript, and participation in all aspects related to this manuscript; Marwa Gaddes: Installing the laying performance trial and collecting data; Raja Chalghoumi: reviewing the final draft; Geert P.J. Janssens: performing acylcarnitine analysis, interpreting results and reviewing final manuscript draft; Madiha Hadj Ayed: conceptualization, supervision and project administration. All the authors commented on previous versions of the manuscript and approved the final version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u003c/strong\u003eAny further data related to the manuscript will be available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval:\u003c/strong\u003e The experimental protocol (94-CEEA-ENMV/25) was approved by the Animal Ethics Committee of the National School of Veterinary Medicine, Sidi Thabet (Tunisia).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAggoor, F., T. Tag El-Din, A. Abd El-Ghani, et Neven Heneish. \u0026laquo; THE USE OF TRITICALE AS ALTERNATIVE CEREAL GRAIN TO YELLOW CORN IN LAYING HENS DIET \u0026raquo;. \u003cem\u003eJournal of Animal and Poultry Production\u003c/em\u003e 25, n\u003csup\u003eo\u003c/sup\u003e 2 (2000): 771‑85. https://doi.org/10.21608/jappmu.2000.258593.\u003c/li\u003e\n\u003cli\u003eAlafif, Mohammad S., Louwrens C. Hoffman, Daniel Cozzolino, et al. \u0026laquo; Assessment of Apparent Metabolizable Energy, and Ileal Amino Acid Digestibility of Full-Fat Black Soldier Fly Larvae (\u003cem\u003eHermetia illucens\u003c/em\u003e) in Broiler Chickens \u0026raquo;. \u003cem\u003ePoultry Science\u003c/em\u003e 104, n\u003csup\u003eo\u003c/sup\u003e 10 (2025): 105506. https://doi.org/10.1016/j.psj.2025.105506.\u003c/li\u003e\n\u003cli\u003eAnas, M.A, M.A. Aprianto, M. Muhlisin, et al. \u0026laquo; Improving Egg Quality in Laying Hens Using Calcium Salt of Black Soldier Fly Larvae Oil \u0026raquo;. Journal of Insects as Food and Feed 11, no 5 (2024): 907‑19. https://doi.org/10.1163/23524588-00001300.\u003c/li\u003e\n\u003cli\u003eAlagawany, Mahmoud, Mohamed E. Abd El-Hack, Elwy A. Ashour, et al. \u0026laquo; Raw Faba Bean (Vicia Faba) as an Alternative Protein Source in Laying Hen Diets\u0026raquo;. Journal of Applied Poultry Research 28, nᵒ 4 (2019): 808‑17. https://doi.org/10.3382/japr/pfz037.\u003c/li\u003e\n\u003cli\u003eAli, Awais, Xuan Liu, Mebratu Melaku, et al. \u0026laquo; Effect of Arginine Supplementation on Liver and Pectoral Muscle: Tissue-Specific Lipid Metabolism in Broilers \u0026raquo;. \u003cem\u003ePoultry Science\u003c/em\u003e 104, n\u003csup\u003eo\u003c/sup\u003e 10 (2025): 105601. https://doi.org/10.1016/j.psj.2025.105601.\u003c/li\u003e\n\u003cli\u003eAOAC International. (2016). \u003cem\u003eOfficial Methods of Analysis of AOAC International\u003c/em\u003e (18th ed.). Gaithersburg, MD, USA: AOAC International.\u003c/li\u003e\n\u003cli\u003eAprianto, Muhammad Anang, Muhlisin, Asih Kurniawati, Chusnul Hanim, Bambang Ariyadi, et Muhsin Al Anas. \u0026laquo; Effect Supplementation of Black Soldier Fly Larvae Oil (\u003cem\u003eHermetia Illucens L.\u003c/em\u003e) Calcium Salt on Performance, Blood Biochemical Profile, Carcass Characteristic, Meat Quality, and Gene Expression in Fat Metabolism Broilers \u0026raquo;. Poultry Science 102, no 10 (2023): 102984. https://doi.org/10.1016/j.psj.2023.102984.\u003c/li\u003e\n\u003cli\u003eBejaei, Masoumeh, et Kimberly M. Cheng. \u0026laquo; Inclusion of Dried Black Soldier Fly Larvae in Free-Range Laying Hen Diets: Effects on Production Efficiency, Feed Safety, Blood Metabolites, and Hen Health \u0026raquo;. \u003cem\u003eAgriculture\u003c/em\u003e 14, n\u003csup\u003eo\u003c/sup\u003e 1 (2023): 31. https://doi.org/10.3390/agriculture14010031.\u003c/li\u003e\n\u003cli\u003eBonomini, M.G., S. Verstringe, G. Bruggeman, R. Vandercruyssen, H. Carmans, et A. Caligiani. \u0026laquo; Characterization, Antibacterial Activity, and Prebiotic Potential of Dried \u003cem\u003eHermetia illucens\u003c/em\u003e L. Larvae and of Their Fractions \u0026raquo;. \u003cem\u003eJournal of Insects as Food and Feed\u003c/em\u003e 11, n\u003csup\u003eo\u003c/sup\u003e 2 (2024): 273‑88. https://doi.org/10.1163/23524588-00001152.\u003c/li\u003e\n\u003cli\u003eCheng, Hao, Xiang Liu, Qingrui Xiao, et al. \u0026laquo; Rapeseed Meal and Its Application in Pig Diet: A Review \u0026raquo;. Agriculture 12, nᵒ 6 (2022): 849. https://doi.org/10.3390/agriculture12060849.\u003c/li\u003e\n\u003cli\u003eCr\u0026eacute;pon, Katell, Pascal Marget, Corinne Peyronnet, Benoit Carrou\u0026eacute;e, Paolo Arese, et G\u0026eacute;rard Duc. \u0026laquo; Nutritional Value of Faba Bean (Vicia Faba L.) Seeds for Feed and Food \u0026raquo;. Field Crops Research 115, no 3 (2010): 329‑39. https://doi.org/10.1016/j.fcr.2009.09.016.\u003c/li\u003e\n\u003cli\u003eCucco, Marco, Marco Grenna, et Irene Pellegrino. \u0026laquo; Egg Characteristics in Relation to Skipped Days of Laying in the Gray Partridge \u0026raquo;. \u003cem\u003eAvian Biology Research\u003c/em\u003e 10, n\u003csup\u003eo\u003c/sup\u003e 4 (2017): 231‑40. https://doi.org/10.3184/175815617X15036738758853.\u003c/li\u003e\n\u003cli\u003eDabbou, Sihem, Angelo Lauwaerts, Ilario Ferrocino, et al. \u0026laquo; Modified Black Soldier Fly Larva Fat in Broiler Diet: Effects on Performance, Carcass Traits, Blood Parameters, Histomorphological Features and Gut Microbiota \u0026raquo;. \u003cem\u003eAnimals\u003c/em\u003e 11, n\u003csup\u003eo\u003c/sup\u003e 6 (2021): 1837. https://doi.org/10.3390/ani11061837.\u003c/li\u003e\n\u003cli\u003eDenbow, D. Michael, et Mark A. Cline. \u0026laquo; Food Intake Regulation \u0026raquo;. In \u003cem\u003eSturkie\u0026rsquo;s Avian Physiology\u003c/em\u003e. Elsevier, 2015. https://doi.org/10.1016/B978-0-12-407160-5.00021-X.\u003c/li\u003e\n\u003cli\u003eDrażbo, A., D. Mikulski, J. Jankowski, et Z. Zduńczyk. \u0026laquo; The Effect of Diets Containing Raw and Fermented Faba Beans on Gut Functioning and Growth Performance in Young Turkeys \u0026raquo;. \u003cem\u003eJournal of Animal and Feed Sciences\u003c/em\u003e 27, n\u003csup\u003eo\u003c/sup\u003e 1 (2018): 65‑73. https://doi.org/10.22358/jafs/82779/2018.\u003c/li\u003e\n\u003cli\u003eFernandez, R., S.M. Kim, J.L. Buenrostro, et J. McGinnis. \u0026laquo; Triticale and Rye as Main Ingredients in Diets for Laying Hens \u0026raquo;. \u003cem\u003ePoultry Science\u003c/em\u003e 52, n\u003csup\u003eo\u003c/sup\u003e 6 (1973): 2244‑52. https://doi.org/10.3382/ps.0522244.\u003c/li\u003e\n\u003cli\u003eFerreira, Jo\u0026atilde;o Paulo, Frank George Guimar\u0026atilde;es Cruz, Oliveira Filho, Pedro Alves de, et Ramon Duque. \u003cem\u003eFibra alimentar em dietas para aves \u0026ndash; Uma revis\u0026atilde;o\u003c/em\u003e. 3, n\u003csup\u003eo\u003c/sup\u003e 2 (2017).\u003c/li\u003e\n\u003cli\u003eFuso, Andrea, Silvia Barbi, Laura Ioana Macavei, et al. \u0026laquo; Effect of the Rearing Substrate on Total Protein and Amino Acid Composition in Black Soldier Fly \u0026raquo;. \u003cem\u003eFoods\u003c/em\u003e 10, n\u003csup\u003eo\u003c/sup\u003e 8 (2021): 1773. https://doi.org/10.3390/foods10081773.\u003c/li\u003e\n\u003cli\u003eGołębiewska, Kinga, Anna Fraś, et Damian Gołębiewski. \u0026laquo; Rapeseed Meal as a Feed Component in Monogastric Animal Nutrition \u0026ndash; a Review \u0026raquo;. Annals of Animal Science 22, nᵒ 4 (2022): 1163‑83. https://doi.org/10.2478/aoas-2022-0020.\u003c/li\u003e\n\u003cli\u003eGucciardi, Antonina, Patrizia Zaramella, Irene Costa, et al. \u0026laquo; Analysis and Interpretation of Acylcarnitine Profiles in Dried Blood Spot and Plasma of Preterm and Full-Term Newborns \u0026raquo;. Pediatric Research 77, no 1 (2015): 36‑47. https://doi.org/10.1038/pr.2014.142.\u003c/li\u003e\n\u003cli\u003eHuang, Qiang, Chaoliang Wen, Wei Yan, et al. \u0026laquo; Comparative Analysis of the Characteristics of Digestive Organs in Broiler Chickens with Different Feed Efficiencies\u0026raquo;. \u003cem\u003ePoultry Science\u003c/em\u003e 101, n\u003csup\u003eo\u003c/sup\u003e 12 (2022): 102184. https://doi.org/10.1016/j.psj.2022.102184.\u003c/li\u003e\n\u003cli\u003eJohnson, Alan L. \u0026laquo; The Avian Ovary and Follicle Development: Some Comparative and Practical Insights \u0026raquo;. \u003cem\u003eTURKISH JOURNAL OF VETERINARY AND ANIMAL SCIENCES\u003c/em\u003e 38 (2014): 660‑69. https://doi.org/10.3906/vet-1405-6.\u003c/li\u003e\n\u003cli\u003eKamińska, B. \u0026laquo; Substitution of Soyabean Meal with Rapeseed meal or Its High-Protein Fraction in the Nutrition of Hens Laying Brown-Shelled Eggs \u0026raquo;. Journal of Animal and Feed Sciences 12, nᵒ 1 (2003): 111‑19. https://doi.org/10.22358/jafs/67665/2003.\u003c/li\u003e\n\u003cli\u003eKhayrova, Adelya, Sergey Lopatin, et Valery Varlamov. \u0026laquo; Black Soldier Fly \u003cem\u003eHermetia illucens\u003c/em\u003e as a Novel Source of Chitin and Chitosan \u0026raquo;. \u003cem\u003eInternational Journal of Sciences\u003c/em\u003e 8, n\u003csup\u003eo\u003c/sup\u003e 04 (2019): 81‑86. https://doi.org/10.18483/ijSci.2015.\u003c/li\u003e\n\u003cli\u003eKoivunen, Erja, Petra Tuunainen, Eija Valkonen, Laila Rossow, et Jarmo Valaja. \u0026laquo; Use of Faba Beans (Vicia Faba L.) in Diets of Laying Hens \u0026raquo;. Agricultural and Food Science 23, no 3 (2014): 165‑72. https://doi.org/10.23986/afsci.41329.\u003c/li\u003e\n\u003cli\u003eKuppusamy, Giva, Chee Kei Kong, Ganeswaran Chandra Segaran, et al. \u0026laquo; Hummingbird-Leaves-Reared Black Soldier Fly Prepupae: Assessment of Nutritional and Heavy Metal Compositions \u0026raquo;. \u003cem\u003eBiology\u003c/em\u003e 9, n\u003csup\u003eo\u003c/sup\u003e 9 (2020): 274. https://doi.org/10.3390/biology9090274.\u003c/li\u003e\n\u003cli\u003eLohman Breeders. Guide d\u0026apos;\u0026eacute;levage: LOHMANN LSL-CLASSIC, Production en cage. Cuxhaven, Germany: Lohman Breeders GmbH, 2021.\u003c/li\u003e\n\u003cli\u003eLu, Shengyong, Nittaya Taethaisong, Weerada Meethip, et al. \u0026laquo; Nutritional Composition of Black Soldier Fly Larvae (\u003cem\u003eHermetia illucens\u003c/em\u003e L.) and Its Potential Uses as Alternative Protein Sources in Animal Diets: A Review \u0026raquo;. \u003cem\u003eInsects\u003c/em\u003e 13, n\u003csup\u003eo\u003c/sup\u003e 9 (2022): 831. https://doi.org/10.3390/insects13090831.\u003c/li\u003e\n\u003cli\u003eMahmoud, A. E., P. C. H. Morel, M. A. Potter, et V. Ravindran. \u0026laquo; The Apparent Metabolisable Energy and Ileal Amino Digestibility of Black Soldier Fly (\u003cem\u003eHermetia illucens\u003c/em\u003e) Larvae Meal for Broiler Chickens \u0026raquo;. British Poultry Science 64, no 3 (2023): 377‑83. https://doi.org/10.1080/00071668.2022.2161873.\u003c/li\u003e\n\u003cli\u003eMarono, S., R. Loponte, P. Lombardi, et al. \u0026laquo; Productive Performance and Blood Profiles of Laying Hens Fed \u003cem\u003eHermetia illucens\u003c/em\u003e Larvae Meal as Total Replacement of Soybean Meal from 24 to 45 Weeks of Age \u0026raquo;. \u003cem\u003ePoultry Science\u003c/em\u003e 96, n\u003csup\u003eo\u003c/sup\u003e 6 (2017): 1783‑90. https://doi.org/10.3382/ps/pew461.\u003c/li\u003e\n\u003cli\u003eMcNab, J. M., et D. W. F. Shannon. \u0026laquo; The Nutritive Value of Triticale and Rye for the Laying Hen \u0026raquo;. \u003cem\u003eBritish Poultry Science\u003c/em\u003e 16, n\u003csup\u003eo\u003c/sup\u003e 1 (1975): 9‑15. https://doi.org/10.1080/00071667508416153.\u003c/li\u003e\n\u003cli\u003eMills, Alisha, Anderson N. Maina, et Elijah G. Kiarie. \u0026laquo; Comparative Responses of Lohmann Select Leghorn-Lite and Shaver Heritage White Leghorn Hens When Fed Diets Containing Fine and Coarse Oat Hulls \u0026raquo;. \u003cem\u003eCanadian Journal of Animal Science\u003c/em\u003e 104, n\u003csup\u003eo\u003c/sup\u003e 3 (2024): 346‑55. https://doi.org/10.1139/cjas-2024-0005.\u003c/li\u003e\n\u003cli\u003eMuslykhah, Uswatun, Srisan Phupaboon, Chaichana Suriyapha, Maharach Matra, et Metha Wanapat. \u0026laquo; Encapsulation of Protein-Based Bioactive from Black Soldier Fly for Ruminant Feeding \u0026raquo;. \u003cem\u003eJournal of Agriculture and Food Research\u003c/em\u003e 18 (d\u0026eacute;cembre 2024): 101325. https://doi.org/10.1016/j.jafr.2024.101325.\u003c/li\u003e\n\u003cli\u003eNilsson, Jan-Ake, et Erik Svensson. \u0026laquo; The Frequency and Timing of Laying Gaps \u0026raquo;. \u003cem\u003eOrnis Scandinavica\u003c/em\u003e 24, n\u003csup\u003eo\u003c/sup\u003e 2 (1993): 122. https://doi.org/10.2307/3676361.\u003c/li\u003e\n\u003cli\u003eOdongo, Ernest Emmanuel, Wilfred Kisaakye Bbosa, et Priscilla Kagoro Kahunde. \u0026laquo; Black Soldier Fly (BSF): A Sustainable Solution for Protein, Waste Management, and a Circular Bio-Economy \u0026raquo;. \u003cem\u003eEuropean Journal of Theoretical and Applied Sciences\u003c/em\u003e 2, n\u003csup\u003eo\u003c/sup\u003e 3 (2024): 822‑34. https://doi.org/10.59324/ejtas.2024.2(3).64.\u003c/li\u003e\n\u003cli\u003eOviedo-Rond\u0026oacute;n, E.O., A. Toscan, N.S. Fagundes, J.K. Vidal, J. Barbi, et P. Thiery. \u0026laquo; Soybean Meal Nutrient Composition, Amino Acid Digestibility, and Energy Content According to the Country of Origin and Year of Harvest Evaluated via NIRS \u0026raquo;. \u003cem\u003eJournal of Applied Poultry Research\u003c/em\u003e 33, n\u003csup\u003eo\u003c/sup\u003e 3 (2024): 100448. https://doi.org/10.1016/j.japr.2024.100448.\u003c/li\u003e\n\u003cli\u003ePereyra, Andrea S., Kelsey L. McLaughlin, Katherine A. Buddo, et Jessica M. Ellis. \u0026laquo; Medium-Chain Fatty Acid Oxidation Is Independent of l -Carnitine in Liver and Kidney but Not in Heart and Skeletal Muscle \u0026raquo;. \u003cem\u003eAmerican Journal of Physiology-Gastrointestinal and Liver Physiology\u003c/em\u003e 325, n\u003csup\u003eo\u003c/sup\u003e 4 (2023): G287‑94. https://doi.org/10.1152/ajpgi.00105.2023.\u003c/li\u003e\n\u003cli\u003ePerez-Maldonado, R. A., P.F. Mannion, et D.J. Farrell. \u0026laquo; Optimum Inclusion of Field Peas, Faba Beans, Chickpeas and Sweet Lupins in Poultry Diets. I. Chemical Composition and Layer Experiments \u0026raquo;.\u003cem\u003eBritish Poultry Science\u003c/em\u003e 40, n\u003csup\u003eo\u003c/sup\u003e 5 (1999): 667‑73. https://doi.org/10.1080/00071669987061.\u003c/li\u003e\n\u003cli\u003eRahimi, Parastoo, Md Saiful Islam, Phelipe Magalh\u0026atilde;es Duarte, et al. \u0026laquo; Impact of the COVID-19 Pandemic on Food Production and Animal Health \u0026raquo;. \u003cem\u003eTrends in Food Science \u0026amp; Technology\u003c/em\u003e 121 (mars 2022): 105‑13. https://doi.org/10.1016/j.tifs.2021.12.003.\u003c/li\u003e\n\u003cli\u003eRakha, A., P. \u0026Aring;man, et R. Andersson. \u0026laquo; Dietary Fiber in Triticale Grain: Variation in Content, Composition, and Molecular Weight Distribution of Extractable Components \u0026raquo;. \u003cem\u003eJournal of Cereal Science\u003c/em\u003e 54, n\u003csup\u003eo\u003c/sup\u003e 3 (2011): 324‑31. https://doi.org/10.1016/j.jcs.2011.06.010.\u003c/li\u003e\n\u003cli\u003eRodriguez-Sinovas, A., E. Fernandez, X. Manteca, A. G. Fernandez, et E. Gonalons. \u0026laquo; CCK Is Involved in Both Peripheral and Central Mechanisms Controlling Food Intake in Chickens \u0026raquo;. \u003cem\u003eAmerican Journal of Physiology-Regulatory, Integrative and Comparative Physiology\u003c/em\u003e 272, n\u003csup\u003eo\u003c/sup\u003e 1 (1997): R334‑40. https://doi.org/10.1152/ajpregu.1997.272.1.R334.\u003c/li\u003e\n\u003cli\u003eRomero, Carlos, Juan Carlos Cenalmor, Susana Chamorro, et C\u0026eacute;sar Redondo. \u0026laquo; Effect of Different Dietary Doses of Black Soldier Fly Meal on Performance and Egg Quality in Free-Range Reared Laying Hens \u0026raquo;. \u003cem\u003eAnimals\u003c/em\u003e 14, n\u003csup\u003eo\u003c/sup\u003e 22 (2024): 3340. https://doi.org/10.3390/ani14223340.\u003c/li\u003e\n\u003cli\u003eSalam, Muhammad, Amina Shahzadi, Huaili Zheng, et al. \u0026laquo; Effect of Different Environmental Conditions on the Growth and Development of Black Soldier Fly Larvae and Its Utilization in Solid Waste Management and Pollution Mitigation \u0026raquo;. Environnemental Technology \u0026amp; Innovation 28 (novembre 2022): 102649. https://doi.org/10.1016/j.eti.2022.102649.\u003c/li\u003e\n\u003cli\u003eSauvant, D., Perez, J. M., \u0026amp;Tran, G. (Eds.). (2004). Tables of composition and nutritional value of feed materials. Wageningen Academic Publishers/INRA editions.\u003c/li\u003e\n\u003cli\u003eSchneider, Jill E. \u0026laquo; Energy Balance and Reproduction \u0026raquo;. \u003cem\u003ePhysiology \u0026amp; Behavior\u003c/em\u003e 81, n\u003csup\u003eo\u003c/sup\u003e 2 (2004): 289‑317. https://doi.org/10.1016/j.physbeh.2004.02.007.\u003c/li\u003e\n\u003cli\u003eShu, Mung Kwan, Cheuk Ming Li, William Eduardo Furtado, Qianjun Huang, Sophie St-Hilaire, et \u0026Aacute;kos Ken\u0026eacute;z. \u0026laquo; Antibacterial Properties of Oil Extracts of Black Soldier Fly Larvae Reared on Bread Waste \u0026raquo;. \u003cem\u003eAnimal Production Science\u003c/em\u003e 64, n\u003csup\u003eo\u003c/sup\u003e 8 (2024). https://doi.org/10.1071/AN23394.\u003c/li\u003e\n\u003cli\u003eStengel, Andreas, et Yvette Tache. \u0026laquo; Interaction between Gastric and Upper Small Intestinal Hormones in the Regulation of Hunger and Satiety: Ghrelin and Cholecystokinin Take the Central Stage \u0026raquo;. \u003cem\u003eCurrent Protein \u0026amp; Peptide Science\u003c/em\u003e 12, n\u003csup\u003eo\u003c/sup\u003e 4 (2011): 293‑304. https://doi.org/10.2174/138920311795906673.\u003c/li\u003e\n\u003cli\u003eSuryati, Tuti, Euis Julaeha, Kindi Farabi, Hanies Ambarsari, et Ace Tatang Hidayat. \u0026laquo; Lauric Acid from the Black Soldier Fly (\u003cem\u003eHermetia illucens\u003c/em\u003e) and Its Potential Applications \u0026raquo;. \u003cem\u003eSustainability\u003c/em\u003e 15, n\u003csup\u003eo\u003c/sup\u003e 13 (2023): 10383. https://doi.org/10.3390/su151310383.\u003c/li\u003e\n\u003cli\u003eTahamtani, Fernanda M., Emma Ivarsson, Viktoria Wiklicky, et al. \u0026laquo; Feeding Live Black Soldier Fly Larvae (\u003cem\u003eHermetia illucens\u003c/em\u003e) to Laying Hens: Effects on Feed Consumption, Hen Health, Hen Behavior, and Egg Quality \u0026raquo;. \u003cem\u003ePoultry Science\u003c/em\u003e 100, n\u003csup\u003eo\u003c/sup\u003e 10 (2021): 101400. https://doi.org/10.1016/j.psj.2021.101400.\u003c/li\u003e\n\u003cli\u003eZhu, Fan. \u0026laquo; Triticale: Nutritional Composition and Food Uses \u0026raquo;. \u003cem\u003eFood Chemistry\u003c/em\u003e 241 (2018): 468‑79. https://doi.org/10.1016/j.foodchem.2017.09.009.\u003c/li\u003e\n\u003cli\u003eZhu, L.P., J.P. Wang, X.M. Ding, et al. \u0026laquo; Effects of Dietary Rapeseed Meal on Laying Performance, Egg Quality, Apparent Metabolic Energy, and Nutrient Digestibility in Laying Hens \u0026raquo;. \u003cem\u003eLivestock Science\u003c/em\u003e 214 (2018): 265‑71. https://doi.org/10.1016/j.livsci.2018.06.007.\u003c/li\u003e\n\u003cli\u003eZulkifli, Nor Fatin Najihah Mohamad, Annita Yong Seok-Kian, Lim Leong Seng, Saleem Mustafa, Yang-Su Kim, et Rossita Shapawi. \u0026laquo; Nutritional Value of Black Soldier Fly (\u003cem\u003eHermetia illucens\u003c/em\u003e) Larvae Processed by Different Methods \u0026raquo;. PLOS ONE 17, no 2 (2022): e0263924. https://doi.org/10.1371/journal.pone.0263924.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"tropical-animal-health-and-production","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trop","sideBox":"Learn more about [Tropical Animal Health and Production](https://www.springer.com/journal/11250)","snPcode":"11250","submissionUrl":"https://submission.nature.com/new-submission/11250/3","title":"Tropical Animal Health and Production","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Laying hens, Hermetia illucens, alternative ingredients, performance, metabolism","lastPublishedDoi":"10.21203/rs.3.rs-8450877/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8450877/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTo increase the sustainability of laying hen diets in subtropical rural conditions, corn and soybean meal were partially substituted with locally produced ingredients, topped or not with black soldier fly (\u003cem\u003eHermetia illucens\u003c/em\u003e, BSF) larvae, simulating rural practices. To understand the underlying metabolic causes of potentially altered performance, blood biochemical parameters, including acylcarnitines, were evaluated. From 30 to 40 weeks of age, 150 Lohman White laying hens were allocated to three diets: standard corn\u0026ndash;soybean meal diet (CONTROL), an alternative diet with triticale, faba beans and rapeseed meal (ALTER), and an ALTER diet supplemented with 5% BSF-dried larvae (ALTER\u0026thinsp;+\u0026thinsp;BSF). Laying performance, organ traits, selected carnitine esters and serum biochemical parameters were assessed.\u003c/p\u003e \u003cp\u003eThe CONTROL and ALTER diets resulted in comparable laying performance, whereas ALTER\u0026thinsp;+\u0026thinsp;BSF decreased the laying rate by 2% (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) and increased egg weight by 2 grams (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e). Multivariate analysis revealed coordinated remodeling of the global acylcarnitine profile (p\u0026thinsp;=\u0026thinsp;\u003cem\u003e0.002\u003c/em\u003e, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.74), which was associated with BSF supplementation and driven by free carnitine (C0), ϐ-oxidation (3-hydroxybutyrylcarnitine, C4-OH) and amino acid degradation (propionylcarnitine, C3) markers. The ALTER diet increased alanine aminotransferase (ALT) levels, which were normalized by BSF feeding (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.01\u003c/em\u003e). No differences were observed in organ weights.\u003c/p\u003e \u003cp\u003eIn conclusion, the substitution of alternative ingredients for soybean and corn effectively maintained laying performance. The BSF larvae acted as a functional nutrient, favoring egg weight and stimulating fat oxidation. These findings support the use of alternative ingredients for sustainable poultry production in subtropical rural areas.\u003c/p\u003e","manuscriptTitle":"Evaluating sustainable ingredients including black soldier fly larvae (Hermetia illucens) in laying hens through performance, physiology and nutrient metabolism","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-30 08:03:31","doi":"10.21203/rs.3.rs-8450877/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-01-28T09:04:12+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-28T07:32:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-29T02:56:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"Tropical Animal Health and Production","date":"2025-12-26T02:59:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"tropical-animal-health-and-production","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trop","sideBox":"Learn more about [Tropical Animal Health and Production](https://www.springer.com/journal/11250)","snPcode":"11250","submissionUrl":"https://submission.nature.com/new-submission/11250/3","title":"Tropical Animal Health and Production","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b44dcc05-2ed5-4696-9e92-48b3f1689696","owner":[],"postedDate":"January 30th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-29T14:57:42+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-30 08:03:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8450877","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8450877","identity":"rs-8450877","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}