Feed processing and macronutrient strategies to alleviate the effect of heat stress on the physiological and performance responses of lactating sows.

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Kwang Mun Kim, Habeeb Tajudeen, Jun Young Mun, Abdolreza Hosseindoust, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9194665/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 This study evaluated the effects of feed processing and dietary energy-protein strategies on physiological responses, performance, stress biomarkers, and fecal microbiota of lactating sows exposed to heat stress (HS). Fifty multiparous Landrace × Yorkshire sows were assigned to five dietary treatments as treatment a: mash diet with 3,320 kcal ME/kg, 18.0% crude protein (CP), without AA supplementation (CON). Treatment b: pellet diet with 3,320 kcal ME/kg, 18.0% CP without AA supplementation. Treatment c: pellet diet, 3,400 kcal ME/kg, 18.0% CP, no additional AA supplementation. Treatment d: pellet diet, 3,400 kcal ME/kg, 16.2% CP, no additional AA supplementation. Treatment e: pellet diet, 3,400 kcal ME/kg, 16.2% CP, with additional AA supplementation (10%+). Feed processing did not significantly affect rectal temperature, sow feed intake, body weight (BW) change, reproductive performance, stress biomarkers, or fecal microbial populations. Respiratory rate was generally unaffected by dietary treatment but increased (p < 0.05) during late lactation in sows fed the high-ME, low-CP pelleted diet with AA supplementation. Sow BW at weaning was higher (p < 0.05) in sows fed higher-CP diets, while piglet weaning weight and average daily gain were positively influenced by dietary ME and CP (p < 0.05). Inflammatory cytokines, antioxidant capacity, hair cortisol, and fecal microbiota were not altered by dietary treatments. However, malondialdehyde concentration increased (p < 0.05) with higher dietary ME. In conclusion, pelleted diets formulated with higher dietary ME (3,400 kcal/kg) and adequate CP (18.0%) supported superior piglet growth performance and improved sow BW at weaning under HS. Cortisol Homeostasis Lactation Lipid peroxidation Microbiota Figures Figure 1 Figure 2 Figure 3 1. INTRODUCTION Heat stress (HS) is a major limitation in modern pig production, particularly during lactation when nutrient demands are highest (Tajudeen et al. 2022 ). Elevated ambient temperature reduces voluntary feed intake, increases catabolic metabolism, alters endocrine and inflammatory responses, and ultimately compromises milk production, sow body reserves, and piglet growth (Mun et al. 2025 ). Nutritional interventions, including adjustment of dietary energy density, protein levels, amino acid (AA) balance, and feed processing technologies have been proposed to alleviate the adverse effects of HS (Min et al. 2019 ; He et al. 2018 ). Higher energy diets may compensate for reduced feed intake, while optimized AA ratios help maintain milk yield and tissue integrity (Bell et al. 2000 ; Cottrell et al. 2015 ). Feed processing techniques (e.g., pelletization vs. mash) may modify nutrient availability, digestibility, and metabolic heat production (Vukmirović et al. 2017 ). Pigs possess a simple, single-compartment stomach, which means their diets must consist of ingredients that are easy to digest and nutritionally efficient. One major factor influencing how well pigs utilize nutrients is the size of the feed particles (Vukmirović et al. 2017 ). When particles are ground more finely, their surface area increases, allowing the effective functions of digestive enzymes. As a result, fine grinding often enhances growth performance (Song et al. 2025 ). In modern pig production, pelleted diets are widely used due to their potential to improve feed conversion ratio (FCR) and offer handling and storage benefits compared to mash diets (Gaillard et al. 2020 ). During pelleting, however, feed particles undergo substantial additional size reduction. Although this can improve nutrient digestibility. An excess of very fine particles in either mash or pelleted feed can harm the gastrointestinal tract and is strongly associated with stomach ulcers and other forms of gastric damage, including keratinization and erosions (Vukmirović et al. 2017 ; Cybulski et al. 2024 ; Krepelková et al. 2024 ). Studies consistently indicate that reducing the amount of very fine feed particles is among the most effective ways to prevent these ulcers (Jadhav et al. 2025 ; Song et al. 2025 ). Particle size also shapes the microbial environment of the pig’s digestive system (Burrough et al. 2025 ). Coarsely ground mash diets slow down stomach emptying and produce a thicker, drier gastric content. These conditions promote stronger acidification, elevate organic acid and lactic acid-producing bacteria levels, and ultimately lower stomach pH (Moesseler et al. 2010). A lower pH helps suppress harmful bacteria, providing additional layer of protection for the gut. However, limited data exist on the combined effects of feed processing, energy level, protein density, and AA fortification in lactating sows under HS. Therefore, this study evaluated these factors on physiological responses, inflammatory and antioxidant markers, microbiota, and performance of lactating sows and their litters. 2. MATERIALS AND METHODS 2.1. Ethics approval The Kangwon National University Institutional Animal Care and Use Committee approved all protocols involving animal use, care and handling (protocol, KW-240722-4). 2.2. Animals, housing, treatments, and diet Fifty multiparous LY sows (Landrace × Yorkshire; parity 2–5; mean parity = X.X ± SD) were enrolled in the study. The experimental period covered 35 days and commenced in late gestation (approximately 3–5 days prior to expected farrowing), continued through lactation, and ended at the time of re-mating/post-weaning (35 days total). All sows were managed under commercial conditions in the same farrowing facility at Haman-gun, South Korea, and were exposed to naturally occurring heat-stress conditions during the experimental period. The sows were distributed across 5 treatments as treatment a: mash diet with 3,320 kcal ME/kg, 18.0% crude protein (CP), with no additional AA supplementation (CON). Treatment b: pellet diet with 3,320 kcal ME/kg, 18.0% CP, no additional AA supplementation. Treatment c: pellet diet, 3,400 kcal ME/kg, 18.0% CP, no additional AA supplementation. Treatment d: pellet diet, 3,400 kcal ME/kg, 16.2% CP, no additional AA supplementation. Treatment e: pellet diet, 3,400 kcal ME/kg, 16.2% CP, with additional AA supplementation (10%+). The data were analyzed using mixed-effects models. Sows were housed in standard farrowing crates (dimensions: 2.1 × 0.8 m) with access to a slatted floor and individual feeder and water nipple. Environmental temperature and relative humidity in the farrowing room were recorded continuously using data-loggers (one per room) set to record at 15-min intervals. Temperature-humidity index (THI) was calculated daily as described in previous literature (Tajudeen et al. 2022 ) (THI = T - [(0.55–0.55 × RH) × (T − 58)], with T in °F and RH as fraction) to quantify heat-stress severity; mean daily THI and peak THI during the study are reported. The ambient temperature and THI remained above thermal comfort thresholds for sows throughout the experiment (Fig. 1), indicating persistent HS during the experiment. 2.3. Experimental design and management This study used a longitudinal (within-subject) design with repeated measurements on each sow and litter over the 35-day period. Sows received the same basal lactation diet formulated to meet or exceed NRC (2012) nutrient recommendations for lactating sows (Table 1). Feed and water were provided ad libitum from farrowing onward with feed refusals recorded daily. Feed offered and refusals were recorded daily to calculate average daily feed intake (ADFI). Parity, body condition score at entry, and expected farrowing date were used to balance initial sow status across the herd. 2.4. Continuous environmental monitoring (ambient temperature, RH) throughout the study Physiological indices (respiratory rate, rectal temperature): at Days − 1, 0 (within 24 h of farrowing), 7, 14, 21 and at weaning. Measurements were taken twice daily (0700 h and 1500 h) to capture diurnal variation and peak heat load. 2.5. Sample collection and chemical analyses 2.5.1. Respiratory rate and Rectal temperature Respiratory rate (breaths·min⁻¹) was determined by counting flank movements for 60 s while sows were resting. Rectal temperature (°C) was measured using a digital veterinary thermometer inserted approximately 6 cm into the rectum. Measurements were taken at post-partum (within 24 h), mid-lactation, and weaning. 2.5.2. Sow performance Body weight (BW) was measured using a calibrated electronic scale at 24h post-partum and weaning (d 21), and BW change during lactation was calculated. Backfat thickness (BF) was measured at the P2 position (65 mm from the dorsal midline at the last rib) using an ultrasound backfat meter (Renco Lean-Meater®, Renco Corp., USA) at post-partum and weaning; backfat change was calculated as the difference. At d 0–21, average daily feed intake (ADFI) was calculated over the lactation period. Farrowing duration was defined as the time from the birth of the first piglet to the birth of the last piglet. Weaning-to-estrus interval (WEI) was recorded as days from weaning to first standing estrus. 2.5.3. Litter and piglet performance Litter data included total born, born alive, stillborn, number weaned, and litter survivability (%). Individual piglet BW was recorded at birth (within 24 h) and at weaning using a digital scale. Litter birth weight and weaning weight were calculated as the sum of individual piglet weights. Piglet average daily gain (ADG) was calculated as the difference between weaning and birth weights divided by lactation length. 2.5.4. Blood sampling and serum analyses At d 21, blood samples (10 mL) were collected via jugular venipuncture between 0800–1000 h to reduce circadian variability. Samples were centrifuged at 1,800 × g for 15 min at 4°C, and serum was stored at -80°C until analysis. Serum concentrations were quantified using porcine specific Commercial ELISA kits (MyBioSource, San Diego, CA, USA) of tumor necrosis factor-α (TNF-α) (MBS262753), interleukin-1β (IL-1β) (MBS260684), and IL-10 (MBS2513043). All assays were performed in accordance with the manufacturers’ instructions. Cytokine concentrations were quantified using standard curves generated independently for each experiment. Each assay was conducted in two independent runs, with all samples analyzed in duplicate. Total antioxidant capacity (TAC; Cayman Chemical, Ann Arbor, MI, USA; Cat. No. 709001), superoxide dismutase (SOD; Cayman Chemical; Cat. No. 706002), and malondialdehyde (MDA; thiobarbituric acid reactive substances assay; Cayman Chemical; Cat. No. 10009055) were measured using colorimetric assay kits. All samples were also analyzed in duplicate. 2.5.5. Hair cortisol analysis At d 21, hair samples (100 mg) were collected from the nuchal region and stored in labelled paper envelopes at room temperature. Samples were washed twice with isopropanol, air-dried, and finely cut. Cortisol was extracted by incubating hair in methanol for 24 h at room temperature. Extracts were evaporated, reconstituted in assay buffer, and cortisol concentration was determined using a cortisol ELISA kit (Salimetrics, State College, PA, USA; Cat. No. 1-3002). Hair cortisol concentrations were expressed as pg·mg⁻¹ hair. 2.5.6. Quantitative PCR Analysis of Fecal Microbiota Fecal samples were collected directly from the rectum of sows on day 21 of lactation, immediately placed on ice, and stored at − 80°C until analysis. Genomic DNA was extracted from approximately 200 mg of fecal material using a commercial stool DNA extraction kit (QIAamp Fast DNA Stool Mini Kit, Qiagen, Hilden, Germany; Cat. No. 51604), following the manufacturer’s instructions. DNA concentration and purity were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Quantitative real-time PCR (qPCR) was performed to quantify selected bacterial groups, including Lactobacillus spp ., Bifidobacterium spp ., Clostridium spp ., and Escherichia coli . ( E. Coli ). Universal bacterial 16S rRNA gene primers were used to quantify total bacteria (Nadkami et al. 2002; Rinttilä et al. 2004 ). Amplification reactions were conducted in a final volume of 20 µL containing SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA), forward and reverse primers (Table 2), nuclease-free water, and template DNA. All samples were analyzed in duplicate, and non-template controls were included in each run to confirm the absence of contamination. Quantitative PCR was performed using an ABI 7500 Fast Real-Time PCR System (Applied Biosystems). Thermal cycling conditions consisted of an initial denaturation step followed by 40 cycles of denaturation, annealing, and extension. Melting curve analysis was conducted at the end of each run to confirm amplification specificity. Relative abundance of each bacterial group was calculated using the comparative Ct (2⁻ΔCt) method, where ΔCt was defined as the difference between the Ct value of the target bacterial group and that of total bacteria within the same sample. Results were expressed as relative abundance (2⁻ΔCt × 100). Data were log-transformed when necessary to meet assumptions of normality prior to statistical analysis (Walter et al. 2001 ; Rinttila et al. 2004; Park et al. 2006 ; Mirhosseini et al. 2010 ) 2.6. Statistical analysis Data were analyzed using mixed-effects models (SAS 9.4; SAS Institute Inc., Cary, NC, USA). Feed processing (mash vs. pellet), metabolizable energy (ME) level, CP level, and additional AA supplementation was included as fixed effects, with the barn housing the sow and piglets considered the experimental unit. All response variables were analyzed at each time point separately. Preplanned orthogonal contrasts were used to compare specific treatment combinations. Data were checked for normality, and log transformation was applied when necessary. Differences were considered significant at p < 0.05, and results are presented as means ± SEM. 3. RESULTS 3.1. Respiratory Rate The effects of feed processing, dietary ME, CP, and additional AA supplementation on the respiratory rate of heat-stressed lactating sows across the 21-d lactation period is shown in Fig. 2. Overall, there was no significant difference in respiratory rate in all treatments from d1-d19. However, a significant difference among treatments was observed near the end of lactation (day 20; p < 0.05), where sows fed the pelleted HME (dietary energy level of 3,400kcal/kg ME, LCP diet (dietary crude protein level of 16.2%) with additional AA showed a higher respiratory rate compared with other treatments. 3.2. Rectal Temperature The effects of feed processing, dietary ME, CP, and additional AA supplementation on rectal temperature of HS lactating sows over the 21-d lactation period is shown in Fig. 3. There was no significant difference in the rectal temperature across all treatments and days. 3.3. Sow Performance The effects of feed processing, dietary ME, CP, and additional AA supplementation on sow performance during lactation is shown in Table 3. There was no significant difference in the BW during post-partum and no difference in the BW change in all treatments. However, the sows fed pelleted diet with higher CP showed higher (p < 0.05) BW at weaning compared with the lower-CP diet. There was no difference in the backfat thickness at 24 h postpartum, at weaning, and BF change during lactation. There was also no significant difference in the ADFI, farrowing duration, and WEI in all treatments. 3.4. Litter and Piglet Performance The effects of feed processing, dietary ME, CP, and additional AA supplementation on litter and piglet performance is shown in Table 4. Reproductive performance, including total born, born alive, stillborn, number weaned, and piglet survivability, was not affected by treatment, feed processing, dietary ME, CP level, or AA supplementation. Similarly, litter birth weight and litter weaning weight did not differ among treatments. Piglet birth weight was not influenced by treatment or any main effect. However, piglet weaning weight was affected by treatment (p < 0.05), with significant effects of dietary ME (p < 0.027) and dietary CP (p < 0.007). Piglet ADG was also affected by treatment (p < 0.05), with significant effects of dietary ME (p < 0.05) and dietary CP (p < 0.05), whereas feed processing and AA supplementation had no effect. 3.5. Inflammatory and Oxidative Stress-Related Biomarkers The effects of feed processing, dietary ME, CP, and additional AA supplementation on the inflammatory and oxidative stress-related biomarkers are shown in Table 5. There was no significant difference in the TNF-α, IL-10, IL-1β, TAC and SOD across all treatments. However, there was a significant difference (p < 0.05) in the MDA concentration for dietary ME. 3.6. Hair Cortisol and Fecal Microflora The effects of feed processing, dietary ME, CP, and additional AA supplementation on hair cortisol and fecal microflora quantification are shown in Table 6 and Table 7. There was no significant difference in the hair cortisol concentration, as well as the fecal microflora quantity. 4. DISCUSSION Heat stress remains one of the most critical challenges in modern sow production systems, particularly during lactation when metabolic heat production and nutrient demands peak. In the present study, the combined effects of feed processing, dietary ME, CP, and additional AA supplementation were evaluated in HS lactating sows, with emphasis on physiological responses, performance, inflammatory and oxidative status, and fecal microbiota. Respiratory rate and rectal temperature are reliable indicators of thermoregulatory strain in heat-stressed sows (Freitas et al. 2023 ). In this study, respiratory rate did not differ among treatments during most of lactation, indicating that all diets supported adequate thermoregulation during early and mid-lactation. However, the significantly higher respiratory rate observed on day 20 in sows fed the pelleted high-ME, low-CP diet with AA supplementation suggests an increase in metabolic heat production toward late lactation. This response may reflect the combined thermogenic effects of increased dietary energy density and AA oxidation when nutrient intake exceeds anabolic capacity, particularly under HS conditions (Lucy et al. 2017; Zhang et al. 2020 ). Despite this late-lactation increase in respiratory rate, rectal temperature remained unchanged across treatments throughout the experimental period. This finding suggests that sows were able to maintain core body temperature within physiological limits, likely through increased evaporative heat loss mechanisms, such as panting, consistent with previous reports in lactating sows exposed to moderate HS (Oh et al. 2022 ; Mun et al. 2025 ). Sow BW at weaning was higher in sows fed pelleted diets with higher CP compared with lower CP diets, while BW change during lactation was unaffected by treatment. This indicates that the 18% CP may support improved tissue retention or reduced body reserve mobilization during lactation, even under HS. This finding is consistent with the study by Zang et al. (2020). Their study reported that a CP diet close to 19% improved tissue retention. Protein adequacy is critical for milk protein synthesis and maternal tissue turnover, particularly when feed intake is constrained (Bell et al. 2000 ; Cottrell et al. 2015 ). The lack of treatment effects on backfat thickness, ADFI, farrowing duration, and weaning-to-estrus interval suggests that neither feed processing nor dietary ME and AA manipulation markedly altered energy partitioning or reproductive recovery under the conditions of this study. This aligns with previous findings showing that, under HS, voluntary feed intake often becomes the primary limiting factor, overriding moderate dietary adjustments (Cottrell et al. 2015 ; He et al. 2018 ; Serviento et al. 2020 ). Reproductive performance parameters were not influenced by dietary treatments, indicating that gestational outcomes and early neonatal survival were largely unaffected by dietary ME, CP, or feed form. However, piglet weaning weight and ADG were significantly influenced by dietary ME and CP, but not by feed processing or AA supplementation. These results highlight the importance of dietary energy and protein supply in supporting milk yield and milk nutrient output under HS, which directly affects piglet growth performance (Choi et al. 2017 ; Min et al. 2019 ; Kim et al. 2019 ). The absence of additional benefits from AA supplementation suggests that basal AA supply may have already met or exceeded requirements, or that HS limited the efficiency of AA utilization for milk synthesis (Baumgard and Rhoads, 2013 ; Zhang and Trottier, 2019 ). Similar findings have been reported where AA fortification did not further enhance piglet growth when energy intake remained the primary constraint (Cottrell et al. 2015 ). Inflammation and oxidative stress are hallmark responses to HS, often mediated by altered gut permeability, endotoxin translocation, and mitochondrial dysfunction (Tang et al. 2022 ; Xia et al. 2022 ; Chirivi et al. 2024). In the present study, TNF-α, IL-10, IL-1β, TAC, and SOD were not affected by feed processing, dietary ME, CP, or AA supplementation, suggesting that none of the dietary strategies markedly altered systemic inflammatory or antioxidant capacity. However, MDA, a marker of lipid peroxidation, was significantly influenced by dietary ME, indicating that higher energy intake may exacerbate oxidative stress under HS. This finding is consistent with reports showing that elevated metabolic flux increases reactive oxygen species production, particularly when mitochondrial efficiency is compromised by thermal stress (Christen et al. 2018; Zhao et al. 2019; Mun et al. 2025 ). Hair cortisol provides an integrated measure of chronic stress exposure (Wiechers et al. 2021 ; Tajudeen et al. 2022 ). The absence of treatment effects on hair cortisol suggests that dietary interventions did not substantially modify long-term hypothalamic-pituitary-adrenal axis activation in heat-stressed sows. Similarly, fecal microbial populations were unaffected by dietary treatments. This may reflect the relative stability of adult sow microbiota or insufficient dietary contrast to induce measurable microbial shifts. Previous studies have shown that in most cases, only a substantial change in fiber source, fermentability, or feeding duration is often required to significantly alter fecal microbial composition in mature sows (Liu et al. 2021 ; Pi et al. 2021 ; Luo et al. 2024 ; Burrough et al. 2025 ). 5. CONCLUSION In summary, pelleted diets formulated with higher dietary ME (3,400 kcal/kg) and adequate CP (18.0%) supported superior piglet growth performance and improved sow BW at weaning under HS. Overall, these findings indicate that while feed form did not independently elicit pronounced physiological or microbial changes, it should be considered an important contextual factor influencing how dietary energy and nutrient density are utilized under heat stress. Nutritional strategies for heat-stressed lactating sows should therefore prioritize balanced energy-protein provision, with careful consideration of feed processing to avoid excessive metabolic heat production, particularly during late lactation. Declarations Acknowledgments We appreciate the technical and physical support provided by the graduate students of Kangwon National University Teaching and Research Farm during the course of this project. Author Contributions Writing - original draft: H.T, K.M.K; Validation: J.Y. M, J.S.K; Investigation: A.H, S.S.L; Formal analysis: A. H, P.N.S; Data curation: H.T, J.Y.M; Supervision: K.M.K, J.S.K; Resources: H.T., J.S.K; Project administration: J.Y.M, H.T; Methodology: P.N.S, H.T; Conceptualization: S.S.L; Funding acquisition: J.S.K; All authors reviewed the manuscript. Statement of Animal Rights This experiment was approved and conducted according to the guidelines of the Institutional Animal Care and Use Committee of Kangwon National University (KW-240722-4) and complied with the ARRIVE guidelines. Competing Interest The authors affirm that they have no conflict of interests. Data Availability Statement Data are available from the corresponding author on reasonable request. References Baumgard LH, Rhoads Jr, R. P (2013) Effects of heat stress on postabsorptive metabolism and energetics. Annu Rev Anim Biosci 1: 311-337. https://doi.org/10.1146/annurev-animal-031412-103644 Bell AW, Burhans WS, Overton TR (2000) Protein nutrition in late pregnancy, maternal protein reserves and lactation performance in dairy cows. Proc Nutr Soc 59: 119-126. https://doi:10.1017/S0029665100000148 Burrough ER, Gabler NK, Thomson JR (2025) Digestive System. 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National Academies Press. Oh S, Hosseindoust A, Ha S, Moturi J, Mun J, Tajudeen H, Kim J (2022) Metabolic responses of dietary fiber during heat stress: effects on reproductive performance and stress level of gestating sows. Metabolites 12: 280. https://doi.org/10.3390/metabo12040280 Park YS, Lee SR, Kim YG (2006) Detection of Escherichia coli O157: H7, Salmonella spp., Staphylococcus aureus and Listeria monocytogenes in kimchi by multiplex polymerase chain reaction (mPCR). J Microbiol 44: 92-97. Pi Y, Hu J, Bai Y, Wang Z, Wu Y, Ye H, Wang J (2021) Effects of dietary fibers with different physicochemical properties on fermentation kinetics and microbial composition by fecal inoculum from lactating sows in vitro. J Sci Food Agric 101: 907-917. https://doi.org/10.1002/jsfa.10698 Rinttilä T, Kassinen A, Malinen E, Krogius L, Palva A (2004) Development of an extensive set of 16S rDNA‐targeted primers for quantification of pathogenic and indigenous bacteria in faecal samples by real‐time PCR. J Appl Microbiol 97: 1166-1177.https://doi.org/10.1111/j.1365-2672.2004.02409.x Serviento AM, Labussière E, Castex M, Renaudeau D (2020) Effect of heat stress and feeding management on growth performance and physiological responses of finishing pigs. J Anim Sci 98: skaa387. https://doi.org/10.1093/jas/skaa387 Song J, Heuer CH, Patterson R, Nyachoti CM (2025) Standardized Ileal Digestibility of Amino Acids in Hybrid Rye Ground to Two Particle Sizes and Fed With or Without Multienzyme Supplement to Young Growing Pigs. J Anim Physiol Anim Nutr 109: 411-422.https://doi.org/10.1111/jpn.14053 Tajudeen H, Moturi J, Hosseindoust A, Ha S, Mun J, Choi Y, Kim J (2022) Effects of various cooling methods and drinking water temperatures on reproductive performance and behavior in heat stressed sows. J Anim Sci Technol 64: 782. https://doi.org/10.5187/jast.2022.e33 Tang S, Xie J, Fang W, Wen X, Yin C, Meng Q, Zhang H (2022) Chronic heat stress induces the disorder of gut transport and immune function associated with endoplasmic reticulum stress in growing pigs. Anim Nutr 11: 228-241. https://doi.org/10.1016/j.aninu.2022.08.008 Vukmirović Đ, Čolović R, Rakita S, Brlek T, Đuragić O, Solà-Oriol D (2017) Importance of feed structure (particle size) and feed form (mash vs. pellets) in pig nutrition-A review. Anim Feed Sci and Technol 233: 133-144. https://doi.org/10.1016/j.anifeedsci.2017.06.016 Walter D, Knittel J, Schwartz K, Kroll J, Roof M (2001) Treatment and control of porcine proliferative enteropathy using different tiamulin delivery methods. JSHAP 9:109-113. https://dx.doi.org/10.54846/jshap/291 Wiechers DH, Brunner S, Herbrandt S, Kemper N, Fels M (2021) Analysis of hair cortisol as an indicator of chronic stress in pigs in two different farrowing systems. Front Vet Sci 8: 605078. https://doi.org/10.3389/fvets.2021.605078 Xia B, Wu W, Fang W, Wen X, Xie J, Zhang H (2022) Heat stress-induced mucosal barrier dysfunction is potentially associated with gut microbiota dysbiosis in pigs. Anim Nutr 8: 289-299. https://doi.org/10.1016/j.aninu.2021.05.012 Zhang S, Trottier NL (2019) Dietary protein reduction improves the energetic and amino acid efficiency in lactating sows. Anim Prod Sci 59: 1980-1990. https://doi.org/10.1071/AN19309 Zhang S, Johnson JS, Trottier NL (2020) Effect of dietary near ideal amino acid profile on heat production of lactating sows exposed to thermal neutral and heat stress conditions. J Anim Sci Biotechnol 11: 75. https://doi.org/10.1186/s40104-020-00483-w Zhao Y, Kim SW (2019) Oxidative stress status and reproductive performance of sows during gestation and lactation under different thermal environments. Asian-Australas J Anim Sci 33: 722. https://doi.org/10.5713/ajas.19.0334 Tables Tables 1 to 7 are available in the Supplementary Files section. Supplementary Files Tables.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 04 Apr, 2026 Reviewers invited by journal 02 Apr, 2026 Editor assigned by journal 30 Mar, 2026 First submitted to journal 24 Mar, 2026 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-9194665","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":616439670,"identity":"39bd6c48-d2d9-436c-9760-b3c67ba24cd9","order_by":0,"name":"Kwang Mun Kim","email":"","orcid":"","institution":"Kangwon National University","correspondingAuthor":false,"prefix":"","firstName":"Kwang","middleName":"Mun","lastName":"Kim","suffix":""},{"id":616439671,"identity":"b60387af-34b0-4d5f-9eb6-82647dda9a55","order_by":1,"name":"Habeeb Tajudeen","email":"","orcid":"","institution":"Kangwon National University","correspondingAuthor":false,"prefix":"","firstName":"Habeeb","middleName":"","lastName":"Tajudeen","suffix":""},{"id":616439672,"identity":"09eed578-2097-46d0-86ab-499b912bbd1b","order_by":2,"name":"Jun Young Mun","email":"","orcid":"","institution":"Kangwon National University","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"Young","lastName":"Mun","suffix":""},{"id":616439673,"identity":"ad45f961-eedd-43d6-b34d-3eff7b4a0127","order_by":3,"name":"Abdolreza Hosseindoust","email":"","orcid":"","institution":"Kangwon National University","correspondingAuthor":false,"prefix":"","firstName":"Abdolreza","middleName":"","lastName":"Hosseindoust","suffix":""},{"id":616439674,"identity":"a4614af1-cf6b-4190-b45e-6c60cd8c9a55","order_by":4,"name":"Sang Sik Lee","email":"","orcid":"","institution":"Kangwon National University","correspondingAuthor":false,"prefix":"","firstName":"Sang","middleName":"Sik","lastName":"Lee","suffix":""},{"id":616439675,"identity":"99e22eaa-dd4b-49c6-ac36-e45fffb115e4","order_by":5,"name":"Priscilla Neves Silvestre","email":"","orcid":"","institution":"Kangwon National University","correspondingAuthor":false,"prefix":"","firstName":"Priscilla","middleName":"Neves","lastName":"Silvestre","suffix":""},{"id":616439676,"identity":"8a580d42-ca91-479d-a39d-9fd6d4b81841","order_by":6,"name":"JinSoo Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIiWNgGAWjYBACCYYDjA8+VNgYIImxEdTCbDjjTBpJWhjYpHlbDpOgRbLx+GNj3obzxvyz269JfNzBIM/fwJb2AZ8WaYYzhg/n7rhtJnHnTJnkzDMMhjMOsB2egU+LHMMZZoO3Z27bMNzISbvN28bAuIGBvRmvw+QYjj+T4G07ZyMP1WJPUIs0wwEzSd62A2YGN9KPgbQkbmBgO4xXi2TDGWNgICcbG97IYf85s00iecZhtmS8WiRuHH8IjEo7w3k30h8bfGyzse1vbzPGq4VB4gCMxQOKG2A8MePXwMDA3wBjsT8gpHYUjIJRMApGKAAARN5OEWcn9AYAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-9518-7917","institution":"Kangwon National University","correspondingAuthor":true,"prefix":"","firstName":"JinSoo","middleName":"","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2026-03-23 02:36:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9194665/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9194665/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106453810,"identity":"4cfdbd68-4634-4b4a-b09e-de10ac8d4509","added_by":"auto","created_at":"2026-04-08 17:20:01","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":92898,"visible":true,"origin":"","legend":"\u003cp\u003eAmbient temperature (blue line) and temperature-humidity index (THI) (Orange line) during experimental period\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9194665/v1/6dea7f79629940d64321e94f.jpg"},{"id":106453812,"identity":"e36d44e3-79fb-4d9b-8ace-d2e0f02a3fff","added_by":"auto","created_at":"2026-04-08 17:20:01","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":85159,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of feed processing, dietary metabolizable energy (ME), crude protein (CP), and additional amino acid supplementation on respiratory rate of heat-stressed lactation sows. LME: dietary energy level of 3,320kcal/kg ME; HME: dietary energy level of 3,400kcal/kg ME; LCP: dietary crude protein level of 16.2%; HCP: dietary crude protein level of 18%; AA+, 10% additional Lys, Met, Thr, Trp in diet.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9194665/v1/f77d2b22ed66576199b89141.jpg"},{"id":106453813,"identity":"06615a1a-54f8-4a48-8483-03198edbb19b","added_by":"auto","created_at":"2026-04-08 17:20:01","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":67530,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of feed processing, dietary metabolizable energy(ME), crude protein (CP), and additional amino acid supplementation on rectal temperature of heat-stressed lactation sows. LME: dietary energy level of 3,320kcal/kg ME; HME: dietary energy level of 3,400kcal/kg ME; LCP: dietary crude protein level of 16.2%; HCP: dietary crude protein level of 18%; AA+, 10% additional Lys, Met, Thr, Trp in diet.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9194665/v1/0cdbd314a2437a09ac39c6c2.jpg"},{"id":106959814,"identity":"fbd0bd90-8deb-4bfc-aabb-10de26cd6167","added_by":"auto","created_at":"2026-04-15 09:15:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":989344,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9194665/v1/13382a0f-ef2a-4aec-b8d1-cc056c04a3e6.pdf"},{"id":106724205,"identity":"8147f8ab-210d-42d2-8ac8-b783280e2e18","added_by":"auto","created_at":"2026-04-12 18:26:40","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":69933,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-9194665/v1/05c1c1c0ccd2c15d071d81f9.docx"}],"financialInterests":"","formattedTitle":"Feed processing and macronutrient strategies to alleviate the effect of heat stress on the physiological and performance responses of lactating sows.","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eHeat stress (HS) is a major limitation in modern pig production, particularly during lactation when nutrient demands are highest (Tajudeen et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Elevated ambient temperature reduces voluntary feed intake, increases catabolic metabolism, alters endocrine and inflammatory responses, and ultimately compromises milk production, sow body reserves, and piglet growth (Mun et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Nutritional interventions, including adjustment of dietary energy density, protein levels, amino acid (AA) balance, and feed processing technologies have been proposed to alleviate the adverse effects of HS (Min et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; He et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Higher energy diets may compensate for reduced feed intake, while optimized AA ratios help maintain milk yield and tissue integrity (Bell et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Cottrell et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Feed processing techniques (e.g., pelletization vs. mash) may modify nutrient availability, digestibility, and metabolic heat production (Vukmirović et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Pigs possess a simple, single-compartment stomach, which means their diets must consist of ingredients that are easy to digest and nutritionally efficient. One major factor influencing how well pigs utilize nutrients is the size of the feed particles (Vukmirović et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). When particles are ground more finely, their surface area increases, allowing the effective functions of digestive enzymes. As a result, fine grinding often enhances growth performance (Song et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn modern pig production, pelleted diets are widely used due to their potential to improve feed conversion ratio (FCR) and offer handling and storage benefits compared to mash diets (Gaillard et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). During pelleting, however, feed particles undergo substantial additional size reduction. Although this can improve nutrient digestibility. An excess of very fine particles in either mash or pelleted feed can harm the gastrointestinal tract and is strongly associated with stomach ulcers and other forms of gastric damage, including keratinization and erosions (Vukmirović et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Cybulski et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Krepelkov\u0026aacute; et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Studies consistently indicate that reducing the amount of very fine feed particles is among the most effective ways to prevent these ulcers (Jadhav et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Song et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Particle size also shapes the microbial environment of the pig\u0026rsquo;s digestive system (Burrough et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Coarsely ground mash diets slow down stomach emptying and produce a thicker, drier gastric content. These conditions promote stronger acidification, elevate organic acid and lactic acid-producing bacteria levels, and ultimately lower stomach pH (Moesseler et al. 2010). A lower pH helps suppress harmful bacteria, providing additional layer of protection for the gut.\u003c/p\u003e \u003cp\u003eHowever, limited data exist on the combined effects of feed processing, energy level, protein density, and AA fortification in lactating sows under HS. Therefore, this study evaluated these factors on physiological responses, inflammatory and antioxidant markers, microbiota, and performance of lactating sows and their litters.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Ethics approval\u003c/h2\u003e \u003cp\u003e The Kangwon National University Institutional Animal Care and Use Committee approved all protocols involving animal use, care and handling (protocol, KW-240722-4).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Animals, housing, treatments, and diet\u003c/h2\u003e \u003cp\u003eFifty multiparous LY sows (Landrace \u0026times; Yorkshire; parity 2\u0026ndash;5; mean parity\u0026thinsp;=\u0026thinsp;X.X\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) were enrolled in the study. The experimental period covered 35 days and commenced in late gestation (approximately 3\u0026ndash;5 days prior to expected farrowing), continued through lactation, and ended at the time of re-mating/post-weaning (35 days total). All sows were managed under commercial conditions in the same farrowing facility at Haman-gun, South Korea, and were exposed to naturally occurring heat-stress conditions during the experimental period. The sows were distributed across 5 treatments as treatment a: mash diet with 3,320 kcal ME/kg, 18.0% crude protein (CP), with no additional AA supplementation (CON). Treatment b: pellet diet with 3,320 kcal ME/kg, 18.0% CP, no additional AA supplementation. Treatment c: pellet diet, 3,400 kcal ME/kg, 18.0% CP, no additional AA supplementation. Treatment d: pellet diet, 3,400 kcal ME/kg, 16.2% CP, no additional AA supplementation. Treatment e: pellet diet, 3,400 kcal ME/kg, 16.2% CP, with additional AA supplementation (10%+). The data were analyzed using mixed-effects models. Sows were housed in standard farrowing crates (dimensions: 2.1 \u0026times; 0.8 m) with access to a slatted floor and individual feeder and water nipple. Environmental temperature and relative humidity in the farrowing room were recorded continuously using data-loggers (one per room) set to record at 15-min intervals. Temperature-humidity index (THI) was calculated daily as described in previous literature (Tajudeen et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) (THI\u0026thinsp;=\u0026thinsp;T - [(0.55\u0026ndash;0.55 \u0026times; RH) \u0026times; (T\u0026thinsp;\u0026minus;\u0026thinsp;58)], with T in \u0026deg;F and RH as fraction) to quantify heat-stress severity; mean daily THI and peak THI during the study are reported. The ambient temperature and THI remained above thermal comfort thresholds for sows throughout the experiment (Fig.\u0026nbsp;1), indicating persistent HS during the experiment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Experimental design and management\u003c/h2\u003e \u003cp\u003eThis study used a longitudinal (within-subject) design with repeated measurements on each sow and litter over the 35-day period. Sows received the same basal lactation diet formulated to meet or exceed NRC (2012) nutrient recommendations for lactating sows (Table\u0026nbsp;1). Feed and water were provided ad libitum from farrowing onward with feed refusals recorded daily. Feed offered and refusals were recorded daily to calculate average daily feed intake (ADFI). Parity, body condition score at entry, and expected farrowing date were used to balance initial sow status across the herd.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Continuous environmental monitoring (ambient temperature, RH) throughout the study\u003c/h2\u003e \u003cp\u003ePhysiological indices (respiratory rate, rectal temperature): at Days\u0026thinsp;\u0026minus;\u0026thinsp;1, 0 (within 24 h of farrowing), 7, 14, 21 and at weaning. Measurements were taken twice daily (0700 h and 1500 h) to capture diurnal variation and peak heat load.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Sample collection and chemical analyses\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1. Respiratory rate and Rectal temperature\u003c/h2\u003e \u003cp\u003eRespiratory rate (breaths\u0026middot;min⁻\u0026sup1;) was determined by counting flank movements for 60 s while sows were resting. Rectal temperature (\u0026deg;C) was measured using a digital veterinary thermometer inserted approximately 6 cm into the rectum. Measurements were taken at post-partum (within 24 h), mid-lactation, and weaning.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2. Sow performance\u003c/h2\u003e \u003cp\u003eBody weight (BW) was measured using a calibrated electronic scale at 24h post-partum and weaning (d 21), and BW change during lactation was calculated. Backfat thickness (BF) was measured at the P2 position (65 mm from the dorsal midline at the last rib) using an ultrasound backfat meter (Renco Lean-Meater\u0026reg;, Renco Corp., USA) at post-partum and weaning; backfat change was calculated as the difference.\u003c/p\u003e \u003cp\u003eAt d 0\u0026ndash;21, average daily feed intake (ADFI) was calculated over the lactation period. Farrowing duration was defined as the time from the birth of the first piglet to the birth of the last piglet. Weaning-to-estrus interval (WEI) was recorded as days from weaning to first standing estrus.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.5.3. Litter and piglet performance\u003c/h2\u003e \u003cp\u003eLitter data included total born, born alive, stillborn, number weaned, and litter survivability (%). Individual piglet BW was recorded at birth (within 24 h) and at weaning using a digital scale. Litter birth weight and weaning weight were calculated as the sum of individual piglet weights. Piglet average daily gain (ADG) was calculated as the difference between weaning and birth weights divided by lactation length.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.5.4. Blood sampling and serum analyses\u003c/h2\u003e \u003cp\u003eAt d 21, blood samples (10 mL) were collected via jugular venipuncture between 0800\u0026ndash;1000 h to reduce circadian variability. Samples were centrifuged at 1,800 \u0026times; g for 15 min at 4\u0026deg;C, and serum was stored at -80\u0026deg;C until analysis. Serum concentrations were quantified using porcine specific Commercial ELISA kits (MyBioSource, San Diego, CA, USA) of tumor necrosis factor-α (TNF-α) (MBS262753), interleukin-1β (IL-1β) (MBS260684), and IL-10 (MBS2513043). All assays were performed in accordance with the manufacturers\u0026rsquo; instructions. Cytokine concentrations were quantified using standard curves generated independently for each experiment. Each assay was conducted in two independent runs, with all samples analyzed in duplicate. Total antioxidant capacity (TAC; Cayman Chemical, Ann Arbor, MI, USA; Cat. No. 709001), superoxide dismutase (SOD; Cayman Chemical; Cat. No. 706002), and malondialdehyde (MDA; thiobarbituric acid reactive substances assay; Cayman Chemical; Cat. No. 10009055) were measured using colorimetric assay kits. All samples were also analyzed in duplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.5.5. Hair cortisol analysis\u003c/h2\u003e \u003cp\u003eAt d 21, hair samples (100 mg) were collected from the nuchal region and stored in labelled paper envelopes at room temperature. Samples were washed twice with isopropanol, air-dried, and finely cut. Cortisol was extracted by incubating hair in methanol for 24 h at room temperature. Extracts were evaporated, reconstituted in assay buffer, and cortisol concentration was determined using a cortisol ELISA kit (Salimetrics, State College, PA, USA; Cat. No. 1-3002). Hair cortisol concentrations were expressed as pg\u0026middot;mg⁻\u0026sup1; hair.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.5.6. Quantitative PCR Analysis of Fecal Microbiota\u003c/h2\u003e \u003cp\u003eFecal samples were collected directly from the rectum of sows on day 21 of lactation, immediately placed on ice, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until analysis. Genomic DNA was extracted from approximately 200 mg of fecal material using a commercial stool DNA extraction kit (QIAamp Fast DNA Stool Mini Kit, Qiagen, Hilden, Germany; Cat. No. 51604), following the manufacturer\u0026rsquo;s instructions. DNA concentration and purity were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Quantitative real-time PCR (qPCR) was performed to quantify selected bacterial groups, including \u003cem\u003eLactobacillus spp\u003c/em\u003e., \u003cem\u003eBifidobacterium spp\u003c/em\u003e., \u003cem\u003eClostridium spp\u003c/em\u003e., and \u003cem\u003eEscherichia coli\u003c/em\u003e. (\u003cem\u003eE. Coli\u003c/em\u003e). Universal bacterial 16S rRNA gene primers were used to quantify total bacteria (Nadkami et al. 2002; Rinttil\u0026auml; et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Amplification reactions were conducted in a final volume of 20 \u0026micro;L containing SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA), forward and reverse primers (Table\u0026nbsp;2), nuclease-free water, and template DNA. All samples were analyzed in duplicate, and non-template controls were included in each run to confirm the absence of contamination. Quantitative PCR was performed using an ABI 7500 Fast Real-Time PCR System (Applied Biosystems). Thermal cycling conditions consisted of an initial denaturation step followed by 40 cycles of denaturation, annealing, and extension. Melting curve analysis was conducted at the end of each run to confirm amplification specificity. Relative abundance of each bacterial group was calculated using the comparative Ct (2⁻ΔCt) method, where ΔCt was defined as the difference between the Ct value of the target bacterial group and that of total bacteria within the same sample. Results were expressed as relative abundance (2⁻ΔCt \u0026times; 100). Data were log-transformed when necessary to meet assumptions of normality prior to statistical analysis (Walter et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Rinttila et al. 2004; Park et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Mirhosseini et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2010\u003c/span\u003e)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Statistical analysis\u003c/h2\u003e \u003cp\u003eData were analyzed using mixed-effects models (SAS 9.4; SAS Institute Inc., Cary, NC, USA). Feed processing (mash vs. pellet), metabolizable energy (ME) level, CP level, and additional AA supplementation was included as fixed effects, with the barn housing the sow and piglets considered the experimental unit. All response variables were analyzed at each time point separately. Preplanned orthogonal contrasts were used to compare specific treatment combinations. Data were checked for normality, and log transformation was applied when necessary. Differences were considered significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, and results are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Respiratory Rate\u003c/h2\u003e \u003cp\u003eThe effects of feed processing, dietary ME, CP, and additional AA supplementation on the respiratory rate of heat-stressed lactating sows across the 21-d lactation period is shown in Fig.\u0026nbsp;2. Overall, there was no significant difference in respiratory rate in all treatments from d1-d19. However, a significant difference among treatments was observed near the end of lactation (day 20; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), where sows fed the pelleted HME (dietary energy level of 3,400kcal/kg ME, LCP diet (dietary crude protein level of 16.2%) with additional AA showed a higher respiratory rate compared with other treatments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Rectal Temperature\u003c/h2\u003e \u003cp\u003eThe effects of feed processing, dietary ME, CP, and additional AA supplementation on rectal temperature of HS lactating sows over the 21-d lactation period is shown in Fig.\u0026nbsp;3. There was no significant difference in the rectal temperature across all treatments and days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Sow Performance\u003c/h2\u003e \u003cp\u003eThe effects of feed processing, dietary ME, CP, and additional AA supplementation on sow performance during lactation is shown in Table\u0026nbsp;3. There was no significant difference in the BW during post-partum and no difference in the BW change in all treatments. However, the sows fed pelleted diet with higher CP showed higher (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) BW at weaning compared with the lower-CP diet. There was no difference in the backfat thickness at 24 h postpartum, at weaning, and BF change during lactation. There was also no significant difference in the ADFI, farrowing duration, and WEI in all treatments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Litter and Piglet Performance\u003c/h2\u003e \u003cp\u003eThe effects of feed processing, dietary ME, CP, and additional AA supplementation on litter and piglet performance is shown in Table\u0026nbsp;4. Reproductive performance, including total born, born alive, stillborn, number weaned, and piglet survivability, was not affected by treatment, feed processing, dietary ME, CP level, or AA supplementation. Similarly, litter birth weight and litter weaning weight did not differ among treatments. Piglet birth weight was not influenced by treatment or any main effect. However, piglet weaning weight was affected by treatment (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with significant effects of dietary ME (p\u0026thinsp;\u0026lt;\u0026thinsp;0.027) and dietary CP (p\u0026thinsp;\u0026lt;\u0026thinsp;0.007). Piglet ADG was also affected by treatment (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with significant effects of dietary ME (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and dietary CP (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas feed processing and AA supplementation had no effect.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Inflammatory and Oxidative Stress-Related Biomarkers\u003c/h2\u003e \u003cp\u003eThe effects of feed processing, dietary ME, CP, and additional AA supplementation on the inflammatory and oxidative stress-related biomarkers are shown in Table\u0026nbsp;5. There was no significant difference in the TNF-α, IL-10, IL-1β, TAC and SOD across all treatments. However, there was a significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the MDA concentration for dietary ME.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Hair Cortisol and Fecal Microflora\u003c/h2\u003e \u003cp\u003eThe effects of feed processing, dietary ME, CP, and additional AA supplementation on hair cortisol and fecal microflora quantification are shown in Table\u0026nbsp;6 and Table\u0026nbsp;7. There was no significant difference in the hair cortisol concentration, as well as the fecal microflora quantity.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eHeat stress remains one of the most critical challenges in modern sow production systems, particularly during lactation when metabolic heat production and nutrient demands peak. In the present study, the combined effects of feed processing, dietary ME, CP, and additional AA supplementation were evaluated in HS lactating sows, with emphasis on physiological responses, performance, inflammatory and oxidative status, and fecal microbiota. Respiratory rate and rectal temperature are reliable indicators of thermoregulatory strain in heat-stressed sows (Freitas et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In this study, respiratory rate did not differ among treatments during most of lactation, indicating that all diets supported adequate thermoregulation during early and mid-lactation. However, the significantly higher respiratory rate observed on day 20 in sows fed the pelleted high-ME, low-CP diet with AA supplementation suggests an increase in metabolic heat production toward late lactation. This response may reflect the combined thermogenic effects of increased dietary energy density and AA oxidation when nutrient intake exceeds anabolic capacity, particularly under HS conditions (Lucy et al. 2017; Zhang et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Despite this late-lactation increase in respiratory rate, rectal temperature remained unchanged across treatments throughout the experimental period. This finding suggests that sows were able to maintain core body temperature within physiological limits, likely through increased evaporative heat loss mechanisms, such as panting, consistent with previous reports in lactating sows exposed to moderate HS (Oh et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Mun et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSow BW at weaning was higher in sows fed pelleted diets with higher CP compared with lower CP diets, while BW change during lactation was unaffected by treatment. This indicates that the 18% CP may support improved tissue retention or reduced body reserve mobilization during lactation, even under HS. This finding is consistent with the study by Zang et al. (2020). Their study reported that a CP diet close to 19% improved tissue retention. Protein adequacy is critical for milk protein synthesis and maternal tissue turnover, particularly when feed intake is constrained (Bell et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Cottrell et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The lack of treatment effects on backfat thickness, ADFI, farrowing duration, and weaning-to-estrus interval suggests that neither feed processing nor dietary ME and AA manipulation markedly altered energy partitioning or reproductive recovery under the conditions of this study. This aligns with previous findings showing that, under HS, voluntary feed intake often becomes the primary limiting factor, overriding moderate dietary adjustments (Cottrell et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; He et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Serviento et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eReproductive performance parameters were not influenced by dietary treatments, indicating that gestational outcomes and early neonatal survival were largely unaffected by dietary ME, CP, or feed form. However, piglet weaning weight and ADG were significantly influenced by dietary ME and CP, but not by feed processing or AA supplementation. These results highlight the importance of dietary energy and protein supply in supporting milk yield and milk nutrient output under HS, which directly affects piglet growth performance (Choi et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Min et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Kim et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The absence of additional benefits from AA supplementation suggests that basal AA supply may have already met or exceeded requirements, or that HS limited the efficiency of AA utilization for milk synthesis (Baumgard and Rhoads, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Zhang and Trottier, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Similar findings have been reported where AA fortification did not further enhance piglet growth when energy intake remained the primary constraint (Cottrell et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInflammation and oxidative stress are hallmark responses to HS, often mediated by altered gut permeability, endotoxin translocation, and mitochondrial dysfunction (Tang et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Xia et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Chirivi et al. 2024). In the present study, TNF-α, IL-10, IL-1β, TAC, and SOD were not affected by feed processing, dietary ME, CP, or AA supplementation, suggesting that none of the dietary strategies markedly altered systemic inflammatory or antioxidant capacity. However, MDA, a marker of lipid peroxidation, was significantly influenced by dietary ME, indicating that higher energy intake may exacerbate oxidative stress under HS. This finding is consistent with reports showing that elevated metabolic flux increases reactive oxygen species production, particularly when mitochondrial efficiency is compromised by thermal stress (Christen et al. 2018; Zhao et al. 2019; Mun et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHair cortisol provides an integrated measure of chronic stress exposure (Wiechers et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Tajudeen et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The absence of treatment effects on hair cortisol suggests that dietary interventions did not substantially modify long-term hypothalamic-pituitary-adrenal axis activation in heat-stressed sows. Similarly, fecal microbial populations were unaffected by dietary treatments. This may reflect the relative stability of adult sow microbiota or insufficient dietary contrast to induce measurable microbial shifts. Previous studies have shown that in most cases, only a substantial change in fiber source, fermentability, or feeding duration is often required to significantly alter fecal microbial composition in mature sows (Liu et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Pi et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Luo et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Burrough et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e"},{"header":"5. CONCLUSION","content":"\u003cp\u003eIn summary, pelleted diets formulated with higher dietary ME (3,400 kcal/kg) and adequate CP (18.0%) supported superior piglet growth performance and improved sow BW at weaning under HS. Overall, these findings indicate that while feed form did not independently elicit pronounced physiological or microbial changes, it should be considered an important contextual factor influencing how dietary energy and nutrient density are utilized under heat stress. Nutritional strategies for heat-stressed lactating sows should therefore prioritize balanced energy-protein provision, with careful consideration of feed processing to avoid excessive metabolic heat production, particularly during late lactation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe appreciate the technical and physical support provided by the graduate students of Kangwon National University Teaching and Research Farm during the course of this project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWriting - original draft: H.T, K.M.K; Validation: J.Y. M, J.S.K; Investigation: A.H, S.S.L; Formal analysis: A. H, P.N.S; Data curation: H.T, J.Y.M; Supervision: K.M.K, J.S.K; Resources: H.T., J.S.K; Project administration: J.Y.M, H.T; Methodology: P.N.S, H.T; Conceptualization: S.S.L; Funding acquisition: J.S.K; All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatement of Animal Rights\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis experiment was approved and conducted according to the guidelines of the Institutional Animal Care and Use Committee of Kangwon National University (KW-240722-4) and complied with the ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors affirm that they have no conflict of interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBaumgard LH, Rhoads Jr, R. 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J Anim Physiol Anim Nutr 109: 411-422.https://doi.org/10.1111/jpn.14053\u003c/li\u003e\n\u003cli\u003eTajudeen H, Moturi J, Hosseindoust A, Ha S, Mun J, Choi Y, Kim J (2022) Effects of various cooling methods and drinking water temperatures on reproductive performance and behavior in heat stressed sows. J Anim Sci Technol 64: 782. https://doi.org/10.5187/jast.2022.e33\u003c/li\u003e\n\u003cli\u003eTang S, Xie J, Fang W, Wen X, Yin C, Meng Q, Zhang H (2022) Chronic heat stress induces the disorder of gut transport and immune function associated with endoplasmic reticulum stress in growing pigs. Anim Nutr 11: 228-241. https://doi.org/10.1016/j.aninu.2022.08.008\u003c/li\u003e\n\u003cli\u003eVukmirović Đ, Čolović R, Rakita S, Brlek T, Đuragić O, Sol\u0026agrave;-Oriol D (2017) Importance of feed structure (particle size) and feed form (mash vs. pellets) in pig nutrition-A review. Anim Feed Sci and Technol 233: 133-144. https://doi.org/10.1016/j.anifeedsci.2017.06.016\u003c/li\u003e\n\u003cli\u003eWalter D, Knittel J, Schwartz K, Kroll J, Roof M (2001) Treatment and control of porcine proliferative enteropathy using different tiamulin delivery methods. JSHAP 9:109-113. https://dx.doi.org/10.54846/jshap/291\u003c/li\u003e\n\u003cli\u003eWiechers DH, Brunner S, Herbrandt S, Kemper N, Fels M (2021) Analysis of hair cortisol as an indicator of chronic stress in pigs in two different farrowing systems. Front Vet Sci 8: 605078. https://doi.org/10.3389/fvets.2021.605078\u003c/li\u003e\n\u003cli\u003eXia B, Wu W, Fang W, Wen X, Xie J, Zhang H (2022) Heat stress-induced mucosal barrier dysfunction is potentially associated with gut microbiota dysbiosis in pigs. Anim Nutr 8: 289-299. https://doi.org/10.1016/j.aninu.2021.05.012\u003c/li\u003e\n\u003cli\u003eZhang S, Trottier NL (2019) Dietary protein reduction improves the energetic and amino acid efficiency in lactating sows. Anim Prod Sci 59: 1980-1990. https://doi.org/10.1071/AN19309\u003c/li\u003e\n\u003cli\u003eZhang S, Johnson JS, Trottier NL (2020) Effect of dietary near ideal amino acid profile on heat production of lactating sows exposed to thermal neutral and heat stress conditions. J Anim Sci Biotechnol 11: 75. https://doi.org/10.1186/s40104-020-00483-w\u003c/li\u003e\n\u003cli\u003eZhao Y, Kim SW (2019) Oxidative stress status and reproductive performance of sows during gestation and lactation under different thermal environments. Asian-Australas J Anim Sci 33: 722. https://doi.org/10.5713/ajas.19.0334\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 7 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"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":"Cortisol, Homeostasis, Lactation, Lipid peroxidation, Microbiota","lastPublishedDoi":"10.21203/rs.3.rs-9194665/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9194665/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study evaluated the effects of feed processing and dietary energy-protein strategies on physiological responses, performance, stress biomarkers, and fecal microbiota of lactating sows exposed to heat stress (HS). Fifty multiparous Landrace \u0026times; Yorkshire sows were assigned to five dietary treatments as treatment a: mash diet with 3,320 kcal ME/kg, 18.0% crude protein (CP), without AA supplementation (CON). Treatment b: pellet diet with 3,320 kcal ME/kg, 18.0% CP without AA supplementation. Treatment c: pellet diet, 3,400 kcal ME/kg, 18.0% CP, no additional AA supplementation. Treatment d: pellet diet, 3,400 kcal ME/kg, 16.2% CP, no additional AA supplementation. Treatment e: pellet diet, 3,400 kcal ME/kg, 16.2% CP, with additional AA supplementation (10%+). Feed processing did not significantly affect rectal temperature, sow feed intake, body weight (BW) change, reproductive performance, stress biomarkers, or fecal microbial populations. Respiratory rate was generally unaffected by dietary treatment but increased (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) during late lactation in sows fed the high-ME, low-CP pelleted diet with AA supplementation. Sow BW at weaning was higher (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in sows fed higher-CP diets, while piglet weaning weight and average daily gain were positively influenced by dietary ME and CP (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Inflammatory cytokines, antioxidant capacity, hair cortisol, and fecal microbiota were not altered by dietary treatments. However, malondialdehyde concentration increased (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) with higher dietary ME. In conclusion, pelleted diets formulated with higher dietary ME (3,400 kcal/kg) and adequate CP (18.0%) supported superior piglet growth performance and improved sow BW at weaning under HS.\u003c/p\u003e","manuscriptTitle":"Feed processing and macronutrient strategies to alleviate the effect of heat stress on the physiological and performance responses of lactating sows.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-08 17:19:57","doi":"10.21203/rs.3.rs-9194665/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-04-04T05:57:52+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-02T08:23:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-30T04:47:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"Tropical Animal Health and Production","date":"2026-03-24T22:09:20+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":"d6970b75-61d2-4a74-9d8f-8339d086067f","owner":[],"postedDate":"April 8th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-08T17:19:57+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-08 17:19:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9194665","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9194665","identity":"rs-9194665","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}