Determination of the Effects of Different Levels of Prosopis farcta Fruit Supplementation to Low-Quality Roughages on Organic Matter Digestibility and Methane Formation Using the In Vitro Digestion Method

Determination of the Effects of Different Levels of Prosopis farcta Fruit Supplementation to Low-Quality Roughages on Organic Matter Digestibility and Methane Formation Using the In Vitro Digestion Method | 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 Determination of the Effects of Different Levels of Prosopis farcta Fruit Supplementation to Low-Quality Roughages on Organic Matter Digestibility and Methane Formation Using the In Vitro Digestion Method Oktay Kaplan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7180785/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In arid regions and areas facing forage scarcity, Prosopis species have long been used in ruminant nutrition, yet their full potential remains inadequately assessed. Domesticated ruminants contribute approximately 15% of global methane emissions. Previously, antibiotics were routinely added to ruminant diets to reduce methane-associated energy losses and improve feed efficiency; however, sustainable alternative strategies are now required. In this study, widely used but nutritionally limited wheat straw and maize silage were supplemented with varying levels (2%, 4%, 6%, 8%, and 10%) of Prosopis farcta fruit. Using the in vitro gas production technique and cattle rumen fluid, parameters including total gas production, methane formation, ammonia nitrogen concentration, in vitro organic matter digestibility, and metabolizable energy were evaluated. Each treatment was conducted in quadruplicate under standard laboratory conditions. The lowest methane production was observed in wheat straw at control and 8% inclusion levels, and in maize silage at control, 8%, and 100% supplementation groups. The highest methane production occurred in wheat straw at 2%, 4%, 6%, 10%, and 100% inclusion levels, and in maize silage at 6% and 10% supplementation. In wheat straw, 2% and 6% inclusion levels significantly increased metabolizable energy, although methane production also rose considerably. Conversely, 8% supplementation did not increase metabolizable energy but maintained methane production close to control levels (14,56% vs. 13,87%). In maize silage, 10% inclusion maximised metabolizable energy but caused excessive methane emissions. Inclusion levels of 4–6% in maize silage offer productivity benefits but pose environmental risks, whereas 8% supplementation provides a more sustainable balance. feed additives in vitro digestibility metabolic energy methane emission Prosopis farcta Introduction Prosopis farcta ( P. farcta ) is a perennial shrub of the Fabaceae family, typically growing 0,3 to 1 metre in height and producing one to two dark brown pods per cluster (Pasiecznik et al. 2004 ). The genus Prosopis includes 44 species, such as P. juliflora , P. velutina , P. glandulosa , P. laevigata , P. pallida , and P. cineraria (García-Andrade et al. 2013). The nutritional composition of Prosopis pods has been well-documented, with crude protein ranging from 7–22%, fibre from 11–35%, fat from 1–6%, ash from 3–6%, and carbohydrates from 30–75% (Sawal et al. 2004 ; Choge et al. 2007 ). Notably, P. juliflora pods are richer in protein than leaves and contain most essential amino acids (Eldaw 2016 ). Furthermore, Prosopis seeds offer amino acids such as alanine, arginine, glutamic acid, lysine, methionine, and trace amounts of tryptophan, along with fatty acids like palmitic, oleic, and linoleic acid (Robertson et al. 2011 ). The utilisation of Prosopis species in ruminant diets is attributed to their high carbohydrate and protein contents (Khobondo et al. 2019 ). Their energy, mineral, and protein richness makes them a preferred feed component for goats, sheep, camels, and cattle (Mohamed et al. 2014 ; Pasiecznik et al. 2001 ). However, the shortage of quality forage remains a critical issue in livestock production (Paul et al. 2020 ). Although maize silage has been widely adopted, low-quality forages like wheat straw continue to dominate ruminant diets. Straw-based rations, however, are associated with lower dietary nitrogen and higher enteric methane emissions (Blümmel et al. 2005 ), which may be mitigated through the inclusion of natural plant additives (Yurtseven et al. 2009). Ruminants are significant contributors to global methane emissions, with the rumen accounting for roughly one quarter (Thorpe 2009 ). Over the past 250 years, methane emissions have increased by approximately 149%, exacerbating the effects of climate change. Simultaneously, arid and semi-arid lands now represent about 41% of the Earth’s surface and sustain over a third of the global population (Gutiérrez et al. 2018 ). Reducing methane emissions from livestock by 50% has thus become a priority for mitigating climate change (Ocko et al. 2021 ). Prosopis species are rich in bioactive compounds such as saponins, alkaloids, tannins, and oxalates, which may modulate rumen fermentation and reduce methane production (Anhwange et al. 2020 ; Shilwant et al. 2023 ). In particular, P. farcta contains high levels of palmitic acid methyl ester (~ 32,61%), a compound with known pharmaceutical value (Al-Waheeb 2021 ). The species also exhibits anti-inflammatory, antimicrobial, and antidiabetic properties (Sharifi-Rad et al. 2019) and is recognised for its high flavonoid content (Omidi et al. 2013 ). Flavonoids such as apigenin, quercetin, and daidzein (Amarowicz and Pegg 2008 ), and specifically luteolin, myricetin, and quercetin in Prosopis (Young et al. 2017 ), contribute to its antioxidant activity (Jahromi et al. 2018 ; Cotelle 2001 ). C-glycosyl flavonoids like schaftoside and vitexin have also been associated with biological activity (Sharifi-Rad et al. 2019). Furthermore, alkaloids from Prosopis seeds display antibacterial activity (Rahman et al. 2011 ), and the wood’s high tannin content (up to 9%) enhances antimicrobial efficacy (Prabha et al. 2014 ). In the context of rising antibiotic resistance, these natural antimicrobials are of growing interest (Henciya et al. 2017 ). Tannins, particularly condensed tannins, can influence rumen microbial populations and reduce protein degradation by forming stable protein complexes (Piluzza et al. 2014 ; Ali et al. 2012 ). Hydrolysable tannins, while more absorbable and potentially toxic, also offer protective effects in vitro (Getachew et al. 2008 ). Essential oils and saponins have been shown to reduce ammonia levels and support ruminant performance by inhibiting proteolytic microbes (Golbotteh et al. 2022 ; Alsubait et al. 2023 ; Yanza et al. 2024 ). This study aims to assess the feed value of P. farcta , a largely underutilised species, and evaluate the effects of its bioactive compounds on methane production in ruminants. Given the ongoing decline in feed and water resources due to climate change, alternative feedstuffs like P. farcta have become increasingly important. In this context, the fruit of P. farcta was added to wheat straw and maize silage at 2%, 4%, 6%, 8%, and 10% inclusion levels, with a 100% P. farcta group included to evaluate its viability as a sole feed component and observe the effects at higher inclusion levels. The study employed the in vitro gas production technique to assess total gas, methane, ammonia nitrogen, in vitro organic matter digestibility (IVOMD), and metabolisable energy (ME). Materials and Methods Wheat straw and maize silage were obtained from the local market. P. farcta fruits were collected from wild plants naturally growing in the arid, hot climate of Şanlıurfa. Once fully ripe, the fruits were harvested, shade-dried, and ground whole (including seeds) using a 1 mm mesh sieve (Simsek Laborteknik Ltd. Sti). Comprehensive chemical analyses were performed to determine the nutritional composition of P. farcta fruit, wheat straw, and maize silage, following AOAC official methods (AOAC 2005). Dry matter (DM) was measured by drying samples at 105°C in a laboratory oven (Nuve FN 500) to constant weight. Crude ash was determined by incineration at 600°C in a muffle furnace (Elektro-Mag N1), and organic matter (OM) was calculated by subtracting ash from DM. Crude protein (CP) content was determined by the Kjeldahl method, involving digestion, distillation, and titration steps (Simsek Laborteknik Ltd. Sti). Fibre fractions, including neutral detergent fibre (NDF) and acid detergent fibre (ADF), were analysed using the detergent system developed by Van Soest et al. ( 1991 ), employing Gooch crucibles with porosity grade 1. These analyses provided insights into cell wall composition and digestibility potential. As rumen fluid was collected from slaughtered animals at the abattoir, ethical approval was not required. The chemical compositions of the feed samples are shown in Table 1 . Table 1 Crude nutritional content (%DM) of the forages used and P. farcta fruit. %DM %OM %CP %ADF %NDF %Ash Wheat Straw 95,17 85,20 4,65 50,83 80,12 9,97 Corn Silage 93,35 88,85 6,74 9,61 54,47 4,85 PFF 95,96 91,97 9,87 33,93 41,11 3,99 DM: Dry matter, OM: Organic matter, CP: Crude protein, ADF: Acid detergent fibre, NDF: Neutral detergent fibre, PFF: P. farcta fruit. A total of 400 g of ground P. farcta fruit was extracted with 800 mL of 85% ethanol, homogenised at 10,000 rpm for 30 seconds (Isolab heavy duty homogeniser), and incubated in a 25°C shaking water bath for 24 hours. The extract was then centrifuged at 5,000 rpm for 15 minutes (M4815 PR), filtered (Whatman No. 1), and concentrated using a rotary evaporator at 40°C for 30 minutes (RE-2010). This process was repeated three times for maximum extraction. The final extract was stored at 4°C for further analysis (Sharifi-Rad et al. 2021). Determination of total phenolic content (TPC) TPC was measured using the Folin–Ciocalteu method. Diluted extracts were mixed with 150 µL Folin–Ciocalteu reagent and 450 µL sodium carbonate, vortexed, and kept in the dark for 30 minutes. Absorbance was read at 765 nm (Perkin Elmer Lambda 45 UV-Vis), and results were expressed as mg gallic acid equivalents (GAE)/g DM (Meyers et al. 2003 ). Determination of total flavonoid content (TFC) TFC was determined by the aluminium chloride method (Chang et al. 2002 ). A reaction mixture containing extract, methanol, aluminium chloride, potassium acetate, and water was incubated for 40 minutes, and absorbance was read at 415 nm. Results were given as mg quercetin equivalents (QE)/g DM. Antioxidant activity DPPH radical scavenging activity was assessed by mixing 0,1 mL extract with 2,9 mL of 0,1 µM DPPH solution and incubating for 30 minutes in the dark. Absorbance was measured at 517 nm (Kulisic et al. 2004 ). The extract showed 35,98 mg GAE/g TPC, 27,89 mg QE/g TFC, and 33,78% DPPH inhibition. Preparation of feed mixtures and formation of groups Experimental groups were created by supplementing wheat straw and maize silage with P. farcta fruit at 0% (control), 2%, 4%, 6%, 8%, and 10% inclusion levels (e.g., 10% group: 90 g wheat straw + 10 g P. farcta powder). A 100% P. farcta group ( P. farcta Control) was added to evaluate its potential as a sole feed or to clarify outcomes in case of unclear results from the mixed groups. In total, seven treatment groups were established. For each, approximately 0,2 g was placed in in vitro gas production syringes in quadruplicate, and exact weights were recorded. In vitro gas production was conducted according to Menke et al. ( 1988 ). Processing of rumen fluid and in vitro digestion Rumen fluid was freshly collected from healthy cattle post-slaughter at a local abattoir. Carbon dioxide was bubbled through the fluid, which was kept at 39°C in thermos flasks and transported to the lab. There, it was filtered through four layers of cheesecloth under carbon dioxide flow. Then, 500 mL of rumen fluid was mixed with 1000 mL of laboratory-prepared artificial saliva, and carbon dioxide was continuously bubbled through the mixture for about 15 minutes. All procedures were conducted at 38°C. In vitro ıncubation and gas measurement Approximately 0,2 g of each ground feed sample (1 mm sieve) was placed into glass syringes (200–220 mg capacity) and pre-incubated at 39°C. Then, 30 mL of the rumen fluid–artificial saliva mixture was added to each syringe. After removing air bubbles and recording the initial gas volume, syringes were incubated in a custom water bath at 39°C for 24 hours. Gas volumes were recorded at the end of incubation. All treatments were tested in quadruplicate (n = 4). The results were used to calculate ME and IVOMD. Methane and carbon dioxide levels in the fermentation gas were measured in real time with a methane analyser (Sensors Analysentechnik GmbH & Co. KG, Berlin, Germany) connected to a networked computer. After incubation, syringe contents were filtered through four layers of cheesecloth, and pH was measured (WTW 7310). The filtered fluid was frozen at − 20°C for ammonia nitrogen analysis, performed using the Markham distillation method (Broderick and Kang, 1980 ). Calculation of IVOMD and ME Gas production data were used to calculate IVOMD and ME using the following equations: $$\:IVOMD\:\left(\%\right)\:=\:14,88\:+\:0,889GP\:+\:0,45CP\:+\:0,0651CA$$ $$\:ME\:(MJ/kg\:DM)\:=\:\text{2,20}\:+\:0,136GP\:+\:0,057CP$$ Where: GP = gas production (mL) at 24 h; CP = crude protein (% DM); CA = crude ash (% DM). The effects of P. farcta fruit supplementation on in vitro gas production, IVOMD, ME, carbon dioxide, methane, pH, and ammonia nitrogen are summarised in Tables 2 and 3 . Data were analysed using one-way ANOVA in SPSS (SPSS 2010 ). Where significant effects were found, group means were compared using Duncan’s multiple range test (Duncan 1955 ). Differences were considered at P < 0,05. This statistical approach ensured robust evaluation of dietary treatment effects. Results and Discussion The P. farcta fruit used in this study showed a TPC of 35,98 mg GAE/g, TFC of 27,89 mg QE/g, and antioxidant activity of 33,78% inhibition. These values were lower than those reported by Salari et al. ( 2019 ), (TPC: 366,21 mg GAE/g, TFC: 283,33 mg QE/g), but higher than the values found in P. farcta leaves by Sharifi-Rad et al . (2021), (TPC: 16,47 mg GAE/g, TFC: 0,21 mg QE/g). Similarly, Jahromi et al. ( 2018 ), reported phenolic and flavonoid contents of 61,55 mg GAE/g and 17 mg QE/g, respectively, in ethanolic fruit extracts of P. farcta fruit. Flavonoids are common polyphenolic secondary metabolites in plants (Panche et al. 2016 ), and phenolic compounds represent one of the most widespread metabolite groups (Prabha et al. 2014 ). Cardozo et al. ( 2010 ), also identified substantial levels of anti-nutritional factors in Prosopis pods, including saponins (317 mg/100 g), total phenols (640 mg/100 g), tannins (860 mg/100 g), and phytic acid (181 mg/100 g). The chemical composition of the forage materials and P. farcta fruit is presented in Table 1 . Since wheat straw is harvested after completing its vegetative phase, it showed the highest levels of acid detergent fibre, neutral detergent fibre and ash. In contrast, P. farcta fruit had the highest OM (92,13%) and CP content (9,87%). In this study, the effects of adding different levels of P. farcta fruit to wheat straw on total gas, methane, carbon dioxide, ammonia nitrogen, pH, IVOMD, and ME are presented in Table 2 . Adding P. farcta fruit to wheat straw had no significant effect on pH ( P > 0,05), but it significantly influenced total gas and CO₂ production ( P < 0,01), as well as methane, ammonia nitrogen, IVOMD, and ME values ( P < 0,001). Both the control and 8% inclusion groups showed similarly low levels of gas, methane, and CO₂, while 2% and 6% levels resulted in the highest gas production—suggesting that low doses may help overcome wheat straw’s nutritional limitations. The reduced gas and methane at 8% are likely due to phenolic compounds and tannins inhibiting microbial fermentation. Although P. farcta supplementation generally improved ME and IVOMD in wheat straw and maize silage, it also led to higher methane emissions at most inclusion levels. In wheat straw, 2% and 6% supplementation significantly increased ME (7,79 and 7,52 MJ/kg DM vs. 6,58 in control), but nearly doubled methane output. The 8% level produced methane close to control (14,56% vs. 13,87%), though ME was relatively low (6,04 MJ/kg DM). These results highlight a trade-off: 4–6% inclusion may maximise energy, while 8% offers a more sustainable option by limiting methane emissions. Saponins are natural compounds known to regulate rumen fermentation and feed digestibility in ruminants (Baah et al. 2007 ). They can reduce methane production by suppressing methanogenic microorganisms (Zúñiga-Serrano et al . 2022) and exert antimicrobial effects on bacteria, protozoa, and methanogens (Cieslak et al. 2013 ). In the rumen, they also cause defaunation by disrupting protozoal membranes (Patra and Saxena 2009 ). Extracts from P. farcta seeds and pods, particularly methanolic and ethanolic fruit pod extracts, have shown high phenolic content and significant antibacterial activity (Poudineh et al. 2015 ). Jahromi et al. ( 2018 ) identified 27 different compounds in P. farcta fruit oil, which together accounted for 97,3% of the total oil content. They also reported that the oil exhibited strong antimicrobial activity, with a minimum inhibitory concentration of 16 µg/ml. Regarding ammonia nitrogen levels, the highest values were observed in the 6% and 10% P. farcta fruit groups. In contrast, the lowest level was recorded in the 100% group, which consisted solely of P. farcta fruit powder. This reduction is likely due to the inhibitory effects of tannins, saponins, and essential oils on protein degradation. Table 2 The effect of adding different levels of P. farcta fruit to wheat straw on total gas (ml/g DM), methane (%),ammonia nitrogen (mg/dl), pH, IVOMD in vitro (%DM), ME (MJ/kg DM) and carbon dioxide formation (ml/g DM) in vitro. Parameters Control ± SE %2PFF ± SE %4PFF ± SE %6PFF ± SE %8PFF ± SE %10PFF ± SE %100PFF ± SE SEM P Total gas ml/g DM 152,17 bc ± 1,48 196,28 a ± 12,29 164,67 ab ± 12,19 185,39 a ± 0,39 130,37 c ± 12,55 180,95 ab ± 10,97 167,19 ab ± 11,76 8,80 * % Methane 13,87 b ± 0,37 28,19 a ± 2,40 22,41 a ± 3,04 25,83 a ± 0,66 14,56 b ± 2,80 26,57 a ± 2,03 23,29 a ± 2,31 1,94 ** Carbon dioxide ml/ g DM 87,88 bc ± 1,09 118,45 a ± 10,10 92,96 ab ± 9,14 109,65 ab ± 0,48 65,94 c ± 9,88 104,62 ab ± 8,82 93,83 ab ± 9,67 0,24 * Ammonia nitrogenmg/dl 9,43 d ± 0,72 19,34 b ± 0,24 20,34 b ± 0,36 22,88 a ± 1,32 15,10 c ± 0,20 24,38 a ± 0,57 5,85 e ± 0,02 0,49 ** pH 6,94 ± 0,04 6,96 ± 0,01 6,97 ± 0,01 6,96 ± 0,01 6,97 ± 0,03 6,95 ± 0,02 6,99 ± 0,01 0,02 - IVOMD % DM 44,35 bc ± 0,26 52,26 a ± 2,19 46,69 ab ± 2,17 50,44 a ± 0,07 40,72 c ± 2,23 49,77 ab ± 1,95 50,05 ab ± 2,09 1,57 ** ME MJ/kg DM 6,58 bc ± 0,04 7,79 a ± 0,33 6,95 ab ± 0,33 7,52 a ± 0,01 6,04 c ± 0,34 7,43 ab ± 0,30 7,79 a ± 0,32 0,24 ** PFF: P. farcta fruit, DM: Dry Matter, IVOMD: In vitro organic matter digestibility, ME: Metabolisable energy, SE: Standard error, SEM: Standard error of means. a,b,c for each line, mean values with different letters are significantly different (*: P < 0,01, **: P < 0,001, -: Non significant). Table 3 The effect of adding different levels of P. farcta fruit to corn silage on total gas (ml/g DM), methane (%), ammonia nitrogen (mg/dl), pH, IVOMD in vitro (%DM), ME (MJ/kg DM) and carbon dioxide formation (ml/g DM) in vitro . Parameters Control ± SE %2PFF ± SE %4PFF ± SE %6PFF ± SE %8PFF ± SE %10PFF ± SE %100PFF ± SE SEM P Total gas ml/g DM 229,96 c ± 7,78 280,79 b ± 7,74 234,83 c ± 18,32 293,97 ab ± 13,40 214,47 c ± 17,97 332,51 a ± 22,65 167,19 d ± 11,76 14,23 ** % Methane 25,85d e ± 0,96 45,31 bc ± 1,97 36,83 cd ± 4,45 48,30 ab ± 3,43 30,20 de ± 5,27 58,50 a ± 4,91 23,29 e ± 2,31 3,33 ** Carbon dioxide ml/ g DM 153,15 cd ± 7,72 184,53 bc ± 5,67 147,19 d ± 14,02 194,53 ab ± 10,44 133,79 d ± 13,10 223,51 a ± 17,62 93,83 e ± 9,67 11,18 ** Ammonia nitrogen mg/dl 7,30 d ± 0,34 19,94 c ± 0,66 20,78 bc ± 0,06 21,38 b ± 0,29 21,32 b ± 0,02 24,40 a ± 0,30 5,85 e ± 0,02 0,24 ** pH 6,84 d ± 0,01 6,96 ab ± 0,02 6,90 c ± 0,02 6,91 c ± 0,01 6,92 bc ± 0,01 6,91 c ± 0,01 6,99 a ± 0,01 0,01 ** IVOMD % DM 59,23 c ± 1,38 68,31 b ± 1,38 60,17 c ± 3,26 70,73 ab ± 2,38 56,63 cd ± 3,20 77,67 a ± 4,03 50,05 d ± 2,09 2,53 ** ME MJ/kg DM 8,97 cd ± 0,21 10,36 b ± 0,21 9,12 c ± 0,50 10,73 ab ± 0,36 8,58 cd ± 0,49 11,80 a ± 0,62 7,79 d ± 0,32 0,39 ** PFF: P. farcta fruit, DM: Dry Matter, IVOMD: In vitro organic matter digestibility, ME: Metabolisable energy, SE: Standard error, SEM: Standard error of means. a,b,c,d,e for each line, mean values with different letters are significantly different (**: P < 0,001). The control group showed the fourth lowest ammonia nitrogen level, which can be attributed to the low protein content of wheat straw. The 2% and 4% P. farcta fruit groups displayed the second lowest levels, indicating that the ammonia-reducing effects of compounds in P. farcta fruit begin to take effect at these supplementation levels. Tannins form complexes with proteins, reducing ruminal degradation and increasing protein flow to the intestines (Patra and Saxena 2011 ). Hydrolyzable tannins limit rumen proteolysis by binding to bacteria and proteins (Zhao et al. 2023 ), and suppress protozoal growth, lowering ammonia nitrogen levels (Santoso et al. 2007 ). Essential oils can also reduce nitrous oxide and ammonia nitrogen emissions in dairy cattle (Carrazco et al. 2020 ). Güler et al. ( 2019 ) found that the addition of B. lactis to wheat straw reduced total gas production, methane formation, carbon dioxide levels, and IVOMD, whereas supplementation with S. boulardii increased methane production levels. Yucca schidigera extract (25–50 g/day) reduced ruminal volatyl fatty acids levels (Guyader et al. 2017 ) and saponins—whether from plants or extracts—were shown to lower ammonia nitrogen concentrations (Hu et al. 2005 ) and nitrogen excretion (Jayanegara et al. 2019 ). In lambs, Prosopis laevigata pods increased ammonia nitrogen at 250–500 g/kg levels (Pena-Avelino et al . 2016), suggesting safe use up to 500 g/kg. Yanza et al. ( 2024 ) reported that saponins up to 40 g/kg DM had no negative effect on intake or palatability. The effects of P. farcta fruit supplementation to corn silage on in vitro total gas (ml/g DM), methane (%), ammonia nitrogen (%), pH, IVOMD (% DM), ME (MJ/kg DM), and carbon dioxide (ml/g DM) are presented in Table 3 . In this study, significant differences in pH values were observed among the maize silage groups, unlike the wheat straw groups ( P < 0,001). The highest pH (6,99) occurred in the group with 100% P. farcta fruit, likely due to fermentation-inhibiting compounds in the fruit. The naturally acidic nature of maize silage may have also contributed to this variation. P. farcta inclusion significantly affected total gas, methane, carbon dioxide, ammonia nitrogen, pH, IVOMD, and ME values ( P < 0,001). As shown in Table 3 , total gas production was lower in the control, 4%, 8%, and 100% groups, while it increased at 2% and 6%, peaking at 10%. These results suggest that maize silage deficiencies started to be addressed at 2% and were best corrected at 10%. However, the low gas output in the 100% group may reflect inhibition of microbial fermentation by excess bioactive compounds. Therefore, using P. farcta fruit at 6–8% inclusion appears to offer a more balanced and practical supplementation approach than using it as a sole feed source. Soltan et al. ( 2012 ) suggested that the methane-reducing effect of P. juliflora leaves may result not only from tannins but also other bioactive compounds. Khan et al. ( 2010 ) identified moderate levels of indole alkaloids in the leaves, potentially responsible for reduced gas output. Despite low tannin levels (1,0 g/kg DM), P. juliflora leaves were associated with lower methane emissions ( P < 0,05), indicating the presence of additional inhibitory metabolites. Dos Santos et al. ( 2013 ) demonstrated that chloroformic extracts of P. juliflora pods, rich in juliprosopine, prosoflorine and juliprosine, suppressed gas production similarly to monensin after 36 h. Melesse et al. ( 2019 ) reported higher ME and IVOMD in P. juliflora pods than in other legumes, despite elevated gas production. Saad et al. ( 2017 ) observed moderate antimicrobial activity in P. farcta aerial parts, particularly in n-hexane and methylene chloride extracts. Güler et al. ( 2019 ) found that probiotic supplementation (including Lactobacillus rhamnosus , Bifidobacterium lactis , Saccharomyces boulardii ) to maize silage had no significant effect on methane or carbon dioxide. Saponin-rich diets have been linked to improved fermentation, health, and reduced methane emissions (Baheg et al. 2017 ). Meena et al. ( 2017 ) noted that increasing concentrates in Prosopis cineraria -based rations elevated ammonia nitrogen, VFAs, and protozoa populations ( P < 0,05). In the present study, ammonia nitrogen levels peaked at 10% P. farcta fruit supplementation, decreasing progressively in 6%, 8%, and 100% groups, suggesting that its bioactive compounds may inhibit ammonia nitrogen formation and promote bypass protein. Gül et al. ( 2017 ) found that black cumin and its oil increased gas production ( P < 0,05) without affecting methane, Carbon dioxide, ammonia nitrogen or pH, whereas thyme and thyme oil significantly influenced all gas parameters. According to Table 3 , the highest IVOMD and ME values were observed in the group supplemented with 10% P. farcta fruit ( P < 0,001), indicating that nutritional deficiencies in maize silage were partially addressed at 2–6% and optimally corrected at 10%. In contrast, the 100% P. farcta group showed the lowest values, likely due to the inhibitory effects of saponins and tannins on microbial activity. Its similarity to the control group highlights the poor nutritional value of unsupplemented silage. These results suggest that P. farcta is most effective at 8–10% inclusion. While the 10% group achieved the highest ME (11,80 MJ/kg DM) and IVOMD (77,67%), it also had the highest methane production (58,50%). The 8% group produced lower ME (8,58 MJ/kg DM) but maintained methane levels close to the control (30,20% vs. 25,85%). Despite low methane, the 100% group offered limited nutritional benefit, indicating it is unsuitable as a sole feed. Overall, 6–8% inclusion appears optimal for improving efficiency while minimising methane emissions. Gül et al. ( 2017 ) reported that supplementing corn silage with black cumin and its oil affected IVOMD and ME values ( P < 0,05), with reductions at 0,92% black cumin and increases at 0,3% black cumin oil, likely due to enhanced microbial activity. Similarly, thyme and thyme oil influenced IVOMD and ME, with the lowest and highest values observed at 8,6% and 0,15%, respectively. Meena et al. ( 2017 ) found that increasing concentrate levels elevated gas, methane, and ME ( P < 0,05), while high-tannin diets reduced methane and improved IVOMD. These results help to explain why the nutrients present in P. farcta fruit support microbial activity and gas production in the rumen environment up to a certain level, as they compensate for the nutritional deficiencies inherent in maize silage. According to IPCC ( 2022 ) 81% of agricultural nitrous oxide emissions arise from the sector itself, with 46% linked to ruminant excreta. Phytochemicals may reduce such emissions by accumulating natural nitrification inhibitors in urine (Totty et al. 2013 ). Effective feeding strategies can modulate nitrogen metabolism, decreasing both urinary nitrogen and atmospheric nitrous oxide (Stewart et al. 2019 ). While P. farcta fruit supplementation shows potential for improving rumen fermentation and reducing emissions, limitations must be acknowledged. As the study was conducted in vitro , results may not reflect in vivo rumen dynamics (Baah et al. 2007 ; Jahromi et al. 2018 ) and practical application should be approached with caution. Additionally, the phytochemical composition of P. farcta may vary seasonally and environmentally (Salari et al. 2019 ; Sharifi-Rad et al. 2021) affecting consistency. Nevertheless, its nutrient profile and emission-reducing properties (Sharifi-Rad et al. 2021) support its use as a potential feedstuff (Sawal et al. 2004 ). Future in vivo studies are needed to evaluate animal performance, health, and environmental outcomes. Moreover, synergistic effects with other additives or probiotics warrant investigation (Güler et al. 2019 ). Conclusion Existing literature shows that different fractions of Prosopis species can affect digestibility, performance, rumen fermentation, blood metabolites, and nitrogen utilisation in ruminants, with effects depending on species, dose, and origin. Owing to their rich bioactive content, Prosopis species may modulate rumen fermentation and improve metabolic responses. In wheat straw groups, 2% and 6% P. farcta supplementation significantly increased ME but also raised methane emissions. The 8% level did not enhance ME but kept methane close to control levels (14,56% vs 13,87%), suggesting it may offer a more sustainable balance than 4–6%, which, while efficient, pose environmental concerns. The 100% P. farcta group had higher IVOMD and ME than control, indicating strong nutritional potential compared to wheat straw. In maize silage, 10% supplementation maximised ME but also methane output. Although 8% reduced methane to near-control levels, it failed to improve ME. Thus, while 4–6% may be optimal for energy, methane remains a concern. The 100% P. farcta group showed low methane and moderate digestibility, but its low energy value limits its use as a sole feed. Declarations Ethics approval: As rumen fluid was collected from slaughtered animals at the abattoir, ethical approval was not required. Consent to participate: The participant provided both oral and written consent after being informed about all aspects of the experimental study. Conflict of interest: The authors declare that there are no financial or other conflicts of interest. References Ali A, Tudsri S, Rungmekarat S, Kaewtrakulpong K (2012) Effect of feeding Prosopis juliflora pods and leaves on performance and carcass characteristics of Afar sheep. Agric Nat Resour 46(6):871–881 Alsubait IS, Alhidary IA, Al-Haidary AA (2023) Effects of different levels of yucca supplementation on growth rates, metabolic profiles, fecal odor emissions, and carcass traits of growing lambs. Animals 13(4):755. 10.3390/ani13040755 Al-Waheeb AN (2021) Chemical composition of Prosopis farcta (Banks & Soland) Macbride (Leguminosae or Fabaceae) fruits. Iranian Journal of Ichthyology , 8:120–126. 10.22034/iji.v8i0.652 Amarowicz R, Pegg RB (2008) Legumes as a source of natural antioxidants. 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The genus \u003cem\u003eProsopis\u003c/em\u003e includes 44 species, such as \u003cem\u003eP. juliflora\u003c/em\u003e, \u003cem\u003eP. velutina\u003c/em\u003e, \u003cem\u003eP. glandulosa\u003c/em\u003e, \u003cem\u003eP. laevigata\u003c/em\u003e, \u003cem\u003eP. pallida\u003c/em\u003e, and \u003cem\u003eP. cineraria\u003c/em\u003e (Garc\u0026iacute;a-Andrade et al. 2013). The nutritional composition of \u003cem\u003eProsopis\u003c/em\u003e pods has been well-documented, with crude protein ranging from 7\u0026ndash;22%, fibre from 11\u0026ndash;35%, fat from 1\u0026ndash;6%, ash from 3\u0026ndash;6%, and carbohydrates from 30\u0026ndash;75% (Sawal et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Choge et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Notably, \u003cem\u003eP. juliflora\u003c/em\u003e pods are richer in protein than leaves and contain most essential amino acids (Eldaw \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Furthermore, \u003cem\u003eProsopis\u003c/em\u003e seeds offer amino acids such as alanine, arginine, glutamic acid, lysine, methionine, and trace amounts of tryptophan, along with fatty acids like palmitic, oleic, and linoleic acid (Robertson et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe utilisation of Prosopis species in ruminant diets is attributed to their high carbohydrate and protein contents (Khobondo et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Their energy, mineral, and protein richness makes them a preferred feed component for goats, sheep, camels, and cattle (Mohamed et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Pasiecznik et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). However, the shortage of quality forage remains a critical issue in livestock production (Paul et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Although maize silage has been widely adopted, low-quality forages like wheat straw continue to dominate ruminant diets. Straw-based rations, however, are associated with lower dietary nitrogen and higher enteric methane emissions (Bl\u0026uuml;mmel et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), which may be mitigated through the inclusion of natural plant additives (Yurtseven et al. 2009). Ruminants are significant contributors to global methane emissions, with the rumen accounting for roughly one quarter (Thorpe \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Over the past 250 years, methane emissions have increased by approximately 149%, exacerbating the effects of climate change. Simultaneously, arid and semi-arid lands now represent about 41% of the Earth\u0026rsquo;s surface and sustain over a third of the global population (Guti\u0026eacute;rrez et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Reducing methane emissions from livestock by 50% has thus become a priority for mitigating climate change (Ocko et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eProsopis species\u003c/em\u003e are rich in bioactive compounds such as saponins, alkaloids, tannins, and oxalates, which may modulate rumen fermentation and reduce methane production (Anhwange et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Shilwant et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In particular, \u003cem\u003eP. farcta\u003c/em\u003e contains high levels of palmitic acid methyl ester (~\u0026thinsp;32,61%), a compound with known pharmaceutical value (Al-Waheeb \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The species also exhibits anti-inflammatory, antimicrobial, and antidiabetic properties (Sharifi-Rad et al. 2019) and is recognised for its high flavonoid content (Omidi et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Flavonoids such as apigenin, quercetin, and daidzein (Amarowicz and Pegg \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), and specifically luteolin, myricetin, and quercetin in \u003cem\u003eProsopis\u003c/em\u003e (Young et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), contribute to its antioxidant activity (Jahromi et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Cotelle \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). C-glycosyl flavonoids like schaftoside and vitexin have also been associated with biological activity (Sharifi-Rad et al. 2019).\u003c/p\u003e\u003cp\u003eFurthermore, alkaloids from \u003cem\u003eProsopis\u003c/em\u003e seeds display antibacterial activity (Rahman et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), and the wood\u0026rsquo;s high tannin content (up to 9%) enhances antimicrobial efficacy (Prabha et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In the context of rising antibiotic resistance, these natural antimicrobials are of growing interest (Henciya et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Tannins, particularly condensed tannins, can influence rumen microbial populations and reduce protein degradation by forming stable protein complexes (Piluzza et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Ali et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Hydrolysable tannins, while more absorbable and potentially toxic, also offer protective effects \u003cem\u003ein vitro\u003c/em\u003e (Getachew et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Essential oils and saponins have been shown to reduce ammonia levels and support ruminant performance by inhibiting proteolytic microbes (Golbotteh et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Alsubait et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Yanza et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThis study aims to assess the feed value of \u003cem\u003eP. farcta\u003c/em\u003e, a largely underutilised species, and evaluate the effects of its bioactive compounds on methane production in ruminants. Given the ongoing decline in feed and water resources due to climate change, alternative feedstuffs like \u003cem\u003eP. farcta\u003c/em\u003e have become increasingly important. In this context, the fruit of \u003cem\u003eP. farcta\u003c/em\u003e was added to wheat straw and maize silage at 2%, 4%, 6%, 8%, and 10% inclusion levels, with a 100% \u003cem\u003eP. farcta\u003c/em\u003e group included to evaluate its viability as a sole feed component and observe the effects at higher inclusion levels. The study employed the \u003cem\u003ein vitro\u003c/em\u003e gas production technique to assess total gas, methane, ammonia nitrogen, \u003cem\u003ein vitro\u003c/em\u003e organic matter digestibility (IVOMD), and metabolisable energy (ME).\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eWheat straw and maize silage were obtained from the local market. \u003cem\u003eP. farcta\u003c/em\u003e fruits were collected from wild plants naturally growing in the arid, hot climate of Şanlıurfa. Once fully ripe, the fruits were harvested, shade-dried, and ground whole (including seeds) using a 1 mm mesh sieve (Simsek Laborteknik Ltd. Sti). Comprehensive chemical analyses were performed to determine the nutritional composition of \u003cem\u003eP. farcta\u003c/em\u003e fruit, wheat straw, and maize silage, following AOAC official methods (AOAC 2005). Dry matter (DM) was measured by drying samples at 105\u0026deg;C in a laboratory oven (Nuve FN 500) to constant weight. Crude ash was determined by incineration at 600\u0026deg;C in a muffle furnace (Elektro-Mag N1), and organic matter (OM) was calculated by subtracting ash from DM. Crude protein (CP) content was determined by the Kjeldahl method, involving digestion, distillation, and titration steps (Simsek Laborteknik Ltd. Sti). Fibre fractions, including neutral detergent fibre (NDF) and acid detergent fibre (ADF), were analysed using the detergent system developed by Van Soest et al. (\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e1991\u003c/span\u003e), employing Gooch crucibles with porosity grade 1. These analyses provided insights into cell wall composition and digestibility potential. As rumen fluid was collected from slaughtered animals at the abattoir, ethical approval was not required. The chemical compositions of the feed samples are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\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\u003eCrude nutritional content (%DM) of the forages used and \u003cem\u003eP. farcta\u003c/em\u003e fruit.\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=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\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\u003e%DM\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e%OM\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e%CP\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e%ADF\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e%NDF\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e%Ash\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eWheat Straw\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e95,17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e85,20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4,65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e50,83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e80,12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e9,97\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCorn Silage\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e93,35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e88,85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e6,74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e9,61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e54,47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e4,85\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePFF\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e95,96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e91,97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e9,87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e33,93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e41,11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e3,99\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eDM: Dry matter, OM: Organic matter, CP: Crude protein, ADF: Acid detergent fibre, NDF: Neutral detergent fibre, PFF: \u003cem\u003eP. farcta\u003c/em\u003e fruit.\u003c/p\u003e\u003cp\u003eA total of 400 g of ground \u003cem\u003eP. farcta\u003c/em\u003e fruit was extracted with 800 mL of 85% ethanol, homogenised at 10,000 rpm for 30 seconds (Isolab heavy duty homogeniser), and incubated in a 25\u0026deg;C shaking water bath for 24 hours. The extract was then centrifuged at 5,000 rpm for 15 minutes (M4815 PR), filtered (Whatman No. 1), and concentrated using a rotary evaporator at 40\u0026deg;C for 30 minutes (RE-2010). This process was repeated three times for maximum extraction. The final extract was stored at 4\u0026deg;C for further analysis (Sharifi-Rad et al. 2021).\u003c/p\u003e\u003cp\u003e\u003cb\u003eDetermination of total phenolic content (TPC)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTPC was measured using the Folin\u0026ndash;Ciocalteu method. Diluted extracts were mixed with 150 \u0026micro;L Folin\u0026ndash;Ciocalteu reagent and 450 \u0026micro;L sodium carbonate, vortexed, and kept in the dark for 30 minutes. Absorbance was read at 765 nm (Perkin Elmer Lambda 45 UV-Vis), and results were expressed as mg gallic acid equivalents (GAE)/g DM (Meyers et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eDetermination of total flavonoid content (TFC)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTFC was determined by the aluminium chloride method (Chang et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). A reaction mixture containing extract, methanol, aluminium chloride, potassium acetate, and water was incubated for 40 minutes, and absorbance was read at 415 nm. Results were given as mg quercetin equivalents (QE)/g DM.\u003c/p\u003e\u003cp\u003eAntioxidant activity\u003c/p\u003e\u003cp\u003eDPPH radical scavenging activity was assessed by mixing 0,1 mL extract with 2,9 mL of 0,1 \u0026micro;M DPPH solution and incubating for 30 minutes in the dark. Absorbance was measured at 517 nm (Kulisic et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). The extract showed 35,98 mg GAE/g TPC, 27,89 mg QE/g TFC, and 33,78% DPPH inhibition.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePreparation of feed mixtures and formation of groups\u003c/b\u003e\u003c/p\u003e\u003cp\u003eExperimental groups were created by supplementing wheat straw and maize silage with \u003cem\u003eP. farcta\u003c/em\u003e fruit at 0% (control), 2%, 4%, 6%, 8%, and 10% inclusion levels (e.g., 10% group: 90 g wheat straw\u0026thinsp;+\u0026thinsp;10 g \u003cem\u003eP. farcta\u003c/em\u003e powder). A 100% \u003cem\u003eP. farcta\u003c/em\u003e group (\u003cem\u003eP. farcta\u003c/em\u003e Control) was added to evaluate its potential as a sole feed or to clarify outcomes in case of unclear results from the mixed groups. In total, seven treatment groups were established. For each, approximately 0,2 g was placed in \u003cem\u003ein vitro\u003c/em\u003e gas production syringes in quadruplicate, and exact weights were recorded. \u003cem\u003eIn vitro\u003c/em\u003e gas production was conducted according to Menke et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1988\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eProcessing of rumen fluid and\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003edigestion\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRumen fluid was freshly collected from healthy cattle post-slaughter at a local abattoir. Carbon dioxide was bubbled through the fluid, which was kept at 39\u0026deg;C in thermos flasks and transported to the lab. There, it was filtered through four layers of cheesecloth under carbon dioxide flow. Then, 500 mL of rumen fluid was mixed with 1000 mL of laboratory-prepared artificial saliva, and carbon dioxide was continuously bubbled through the mixture for about 15 minutes. All procedures were conducted at 38\u0026deg;C.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eıncubation and gas measurement\u003c/b\u003e\u003c/p\u003e\u003cp\u003eApproximately 0,2 g of each ground feed sample (1 mm sieve) was placed into glass syringes (200\u0026ndash;220 mg capacity) and pre-incubated at 39\u0026deg;C. Then, 30 mL of the rumen fluid\u0026ndash;artificial saliva mixture was added to each syringe. After removing air bubbles and recording the initial gas volume, syringes were incubated in a custom water bath at 39\u0026deg;C for 24 hours. Gas volumes were recorded at the end of incubation. All treatments were tested in quadruplicate (n\u0026thinsp;=\u0026thinsp;4). The results were used to calculate ME and IVOMD. Methane and carbon dioxide levels in the fermentation gas were measured in real time with a methane analyser (Sensors Analysentechnik GmbH \u0026amp; Co. KG, Berlin, Germany) connected to a networked computer. After incubation, syringe contents were filtered through four layers of cheesecloth, and pH was measured (WTW 7310). The filtered fluid was frozen at \u0026minus;\u0026thinsp;20\u0026deg;C for ammonia nitrogen analysis, performed using the Markham distillation method (Broderick and Kang, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1980\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eCalculation of IVOMD and ME\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGas production data were used to calculate IVOMD and ME using the following equations:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:IVOMD\\:\\left(\\%\\right)\\:=\\:14,88\\:+\\:0,889GP\\:+\\:0,45CP\\:+\\:0,0651CA$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:ME\\:(MJ/kg\\:DM)\\:=\\:\\text{2,20}\\:+\\:0,136GP\\:+\\:0,057CP$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eWhere:\u003c/p\u003e\u003cp\u003eGP\u0026thinsp;=\u0026thinsp;gas production (mL) at 24 h;\u003c/p\u003e\u003cp\u003eCP\u0026thinsp;=\u0026thinsp;crude protein (% DM);\u003c/p\u003e\u003cp\u003eCA\u0026thinsp;=\u0026thinsp;crude ash (% DM).\u003c/p\u003e\u003cp\u003eThe effects of \u003cem\u003eP. farcta\u003c/em\u003e fruit supplementation on \u003cem\u003ein vitro\u003c/em\u003e gas production, IVOMD, ME, carbon dioxide, methane, pH, and ammonia nitrogen are summarised in Tables\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Data were analysed using one-way ANOVA in SPSS (SPSS \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Where significant effects were found, group means were compared using Duncan\u0026rsquo;s multiple range test (Duncan \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1955\u003c/span\u003e). Differences were considered at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0,05. This statistical approach ensured robust evaluation of dietary treatment effects.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eThe \u003cem\u003eP. farcta\u003c/em\u003e fruit used in this study showed a TPC of 35,98 mg GAE/g, TFC of 27,89 mg QE/g, and antioxidant activity of 33,78% inhibition. These values were lower than those reported by Salari et al. (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), (TPC: 366,21 mg GAE/g, TFC: 283,33 mg QE/g), but higher than the values found in \u003cem\u003eP. farcta\u003c/em\u003e leaves by Sharifi-Rad \u003cem\u003eet al\u003c/em\u003e. (2021), (TPC: 16,47 mg GAE/g, TFC: 0,21 mg QE/g). Similarly, Jahromi et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), reported phenolic and flavonoid contents of 61,55 mg GAE/g and 17 mg QE/g, respectively, in ethanolic fruit extracts of \u003cem\u003eP. farcta\u003c/em\u003e fruit. Flavonoids are common polyphenolic secondary metabolites in plants (Panche et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), and phenolic compounds represent one of the most widespread metabolite groups (Prabha et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Cardozo et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), also identified substantial levels of anti-nutritional factors in \u003cem\u003eProsopis\u003c/em\u003e pods, including saponins (317 mg/100 g), total phenols (640 mg/100 g), tannins (860 mg/100 g), and phytic acid (181 mg/100 g).\u003c/p\u003e\u003cp\u003eThe chemical composition of the forage materials and \u003cem\u003eP. farcta\u003c/em\u003e fruit is presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Since wheat straw is harvested after completing its vegetative phase, it showed the highest levels of acid detergent fibre, neutral detergent fibre and ash. In contrast, \u003cem\u003eP. farcta\u003c/em\u003e fruit had the highest OM (92,13%) and CP content (9,87%).\u003c/p\u003e\u003cp\u003eIn this study, the effects of adding different levels of \u003cem\u003eP. farcta\u003c/em\u003e fruit to wheat straw on total gas, methane, carbon dioxide, ammonia nitrogen, pH, IVOMD, and ME are presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eAdding \u003cem\u003eP. farcta\u003c/em\u003e fruit to wheat straw had no significant effect on pH (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0,05), but it significantly influenced total gas and CO₂ production (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0,01), as well as methane, ammonia nitrogen, IVOMD, and ME values (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0,001). Both the control and 8% inclusion groups showed similarly low levels of gas, methane, and CO₂, while 2% and 6% levels resulted in the highest gas production\u0026mdash;suggesting that low doses may help overcome wheat straw\u0026rsquo;s nutritional limitations. The reduced gas and methane at 8% are likely due to phenolic compounds and tannins inhibiting microbial fermentation. Although \u003cem\u003eP. farcta\u003c/em\u003e supplementation generally improved ME and IVOMD in wheat straw and maize silage, it also led to higher methane emissions at most inclusion levels. In wheat straw, 2% and 6% supplementation significantly increased ME (7,79 and 7,52 MJ/kg DM vs. 6,58 in control), but nearly doubled methane output. The 8% level produced methane close to control (14,56% vs. 13,87%), though ME was relatively low (6,04 MJ/kg DM). These results highlight a trade-off: 4\u0026ndash;6% inclusion may maximise energy, while 8% offers a more sustainable option by limiting methane emissions. Saponins are natural compounds known to regulate rumen fermentation and feed digestibility in ruminants (Baah et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). They can reduce methane production by suppressing methanogenic microorganisms (Z\u0026uacute;\u0026ntilde;iga-Serrano \u003cem\u003eet al\u003c/em\u003e. 2022) and exert antimicrobial effects on bacteria, protozoa, and methanogens (Cieslak et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In the rumen, they also cause defaunation by disrupting protozoal membranes (Patra and Saxena \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Extracts from \u003cem\u003eP. farcta\u003c/em\u003e seeds and pods, particularly methanolic and ethanolic fruit pod extracts, have shown high phenolic content and significant antibacterial activity (Poudineh et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Jahromi et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) identified 27 different compounds in \u003cem\u003eP. farcta\u003c/em\u003e fruit oil, which together accounted for 97,3% of the total oil content. They also reported that the oil exhibited strong antimicrobial activity, with a minimum inhibitory concentration of 16 \u0026micro;g/ml. Regarding ammonia nitrogen levels, the highest values were observed in the 6% and 10% \u003cem\u003eP. farcta\u003c/em\u003e fruit groups. In contrast, the lowest level was recorded in the 100% group, which consisted solely of \u003cem\u003eP. farcta\u003c/em\u003e fruit powder. This reduction is likely due to the inhibitory effects of tannins, saponins, and essential oils on protein degradation.\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\u003eThe effect of adding different levels of \u003cem\u003eP. farcta\u003c/em\u003e fruit to wheat straw on total gas (ml/g DM), methane (%),ammonia nitrogen (mg/dl), pH, IVOMD \u003cem\u003ein vitro\u003c/em\u003e (%DM), ME (MJ/kg DM) and carbon dioxide formation (ml/g DM) in vitro.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"10\"\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\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParameters\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl\u0026thinsp;\u0026plusmn;\u0026thinsp;SE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e%2PFF\u0026thinsp;\u0026plusmn;\u0026thinsp;SE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e%4PFF\u0026thinsp;\u0026plusmn;\u0026thinsp;SE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e%6PFF\u0026thinsp;\u0026plusmn;\u0026thinsp;SE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e%8PFF\u0026thinsp;\u0026plusmn;\u0026thinsp;SE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e%10PFF\u0026thinsp;\u0026plusmn;\u0026thinsp;SE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003e%100PFF\u0026thinsp;\u0026plusmn;\u0026thinsp;SE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eSEM\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\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\u003e\u003cb\u003eTotal gas ml/g DM\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e152,17\u003csup\u003ebc\u003c/sup\u003e \u0026plusmn; 1,48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e196,28\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;12,29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e164,67\u003csup\u003eab\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;12,19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e185,39\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0,39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e130,37\u003csup\u003ec\u003c/sup\u003e \u0026plusmn; 12,55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e180,95\u003csup\u003eab\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;10,97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e167,19\u003csup\u003eab\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;11,76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e8,80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e% Methane\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e13,87\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0,37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e28,19\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;2,40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e22,41\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;3,04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e25,83\u003csup\u003ea\u003c/sup\u003e \u0026plusmn; 0,66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e14,56\u003csup\u003eb\u003c/sup\u003e \u0026plusmn; 2,80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e26,57\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;2,03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e23,29\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;2,31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1,94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCarbon dioxide\u003c/b\u003e \u003cb\u003eml/ g DM\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e87,88\u003csup\u003ebc\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1,09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e118,45\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;10,10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e92,96\u003csup\u003eab\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;9,14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e109,65\u003csup\u003eab\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0,48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e65,94\u003csup\u003ec\u003c/sup\u003e \u0026plusmn; 9,88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e104,62\u003csup\u003eab\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;8,82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e93,83\u003csup\u003eab\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;9,67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0,24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAmmonia nitrogenmg/dl\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9,43\u003csup\u003ed\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0,72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e19,34\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0,24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20,34\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0,36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e22,88\u003csup\u003ea\u003c/sup\u003e \u0026plusmn; 1,32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e15,10\u003csup\u003ec\u003c/sup\u003e \u0026plusmn; 0,20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e24,38\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0,57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e5,85\u003csup\u003ee\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0,02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0,49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003epH\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6,94\u0026thinsp;\u0026plusmn;\u0026thinsp;0,04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6,96\u0026thinsp;\u0026plusmn;\u0026thinsp;0,01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6,97\u0026thinsp;\u0026plusmn;\u0026thinsp;0,01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6,96\u0026thinsp;\u0026plusmn;\u0026thinsp;0,01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e6,97\u0026thinsp;\u0026plusmn;\u0026thinsp;0,03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e6,95\u0026thinsp;\u0026plusmn;\u0026thinsp;0,02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e6,99\u0026thinsp;\u0026plusmn;\u0026thinsp;0,01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0,02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cem\u003e-\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIVOMD % DM\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e44,35\u003csup\u003ebc\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0,26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e52,26\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;2,19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e46,69\u003csup\u003eab\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;2,17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e50,44\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0,07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e40,72\u003csup\u003ec\u003c/sup\u003e \u0026plusmn; 2,23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e49,77\u003csup\u003eab\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1,95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e50,05\u003csup\u003eab\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;2,09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1,57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eME MJ/kg DM\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6,58\u003csup\u003ebc\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0,04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7,79\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0,33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6,95\u003csup\u003eab\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0,33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e7,52\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0,01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e6,04\u003csup\u003ec\u003c/sup\u003e \u0026plusmn; 0,34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e7,43\u003csup\u003eab\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0,30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e7,79\u003csup\u003ea\u003c/sup\u003e \u0026plusmn; 0,32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0,24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ePFF: \u003cem\u003eP. farcta\u003c/em\u003e fruit, DM: Dry Matter, IVOMD: \u003cem\u003eIn vitro\u003c/em\u003e organic matter digestibility, ME: Metabolisable energy, SE: Standard error, SEM: Standard error of means. a,b,c for each line, mean values with different letters are significantly different (*: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0,01, **: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0,001, -: Non significant).\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\u003eThe effect of adding different levels of \u003cem\u003eP. farcta\u003c/em\u003e fruit to corn silage on total gas (ml/g DM), methane (%), ammonia nitrogen (mg/dl), pH, IVOMD \u003cem\u003ein vitro\u003c/em\u003e (%DM), ME (MJ/kg DM) and carbon dioxide formation (ml/g DM) \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"10\"\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\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParameters\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl\u0026thinsp;\u0026plusmn;\u0026thinsp;SE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e%2PFF\u0026thinsp;\u0026plusmn;\u0026thinsp;SE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e%4PFF\u0026thinsp;\u0026plusmn;\u0026thinsp;SE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e%6PFF\u0026thinsp;\u0026plusmn;\u0026thinsp;SE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e%8PFF\u0026thinsp;\u0026plusmn;\u0026thinsp;SE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e%10PFF\u0026thinsp;\u0026plusmn;\u0026thinsp;SE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003e%100PFF\u0026thinsp;\u0026plusmn;\u0026thinsp;SE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eSEM\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\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\u003e\u003cb\u003eTotal gas ml/g DM\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e229,96\u003csup\u003ec\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;7,78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e280,79\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;7,74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e234,83\u003csup\u003ec\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;18,32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e293,97\u003csup\u003eab\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;13,40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e214,47\u003csup\u003ec\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;17,97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e332,51\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;22,65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e167,19\u003csup\u003ed\u003c/sup\u003e \u0026plusmn; 11,76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e14,23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e% Methane\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e25,85d\u003csup\u003ee\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0,96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e45,31\u003csup\u003ebc\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1,97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e36,83\u003csup\u003ecd\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;4,45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e48,30\u003csup\u003eab\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;3,43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e30,20\u003csup\u003ede\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;5,27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e58,50\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;4,91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e23,29\u003csup\u003ee\u003c/sup\u003e \u0026plusmn; 2,31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e3,33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCarbon dioxide ml/ g DM\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e153,15\u003csup\u003ecd\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;7,72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e184,53\u003csup\u003ebc\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;5,67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e147,19\u003csup\u003ed\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;14,02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e194,53\u003csup\u003eab\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;10,44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e133,79\u003csup\u003ed\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;13,10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e223,51\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;17,62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e93,83\u003csup\u003ee\u003c/sup\u003e \u0026plusmn; 9,67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e11,18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAmmonia nitrogen mg/dl\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7,30\u003csup\u003ed\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0,34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e19,94\u003csup\u003ec\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0,66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20,78\u003csup\u003ebc\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0,06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e21,38\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0,29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e21,32\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0,02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e24,40\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0,30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e5,85\u003csup\u003ee\u003c/sup\u003e \u0026plusmn; 0,02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0,24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003epH\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6,84\u003csup\u003ed\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0,01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6,96\u003csup\u003eab\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0,02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6,90\u003csup\u003ec\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0,02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6,91\u003csup\u003ec\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0,01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e6,92\u003csup\u003ebc\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0,01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e6,91\u003csup\u003ec\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0,01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e6,99\u003csup\u003ea\u003c/sup\u003e \u0026plusmn; 0,01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0,01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIVOMD % DM\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e59,23\u003csup\u003ec\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1,38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e68,31\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1,38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e60,17\u003csup\u003ec\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;3,26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e70,73\u003csup\u003eab\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;2,38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e56,63\u003csup\u003ecd\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;3,20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e77,67\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;4,03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e50,05\u003csup\u003ed\u003c/sup\u003e \u0026plusmn; 2,09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2,53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eME MJ/kg DM\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8,97\u003csup\u003ecd\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0,21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10,36\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0,21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9,12\u003csup\u003ec\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0,50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10,73\u003csup\u003eab\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0,36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8,58\u003csup\u003ecd\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0,49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e11,80\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0,62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e7,79\u003csup\u003ed\u003c/sup\u003e \u0026plusmn; 0,32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0,39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ePFF: \u003cem\u003eP. farcta\u003c/em\u003e fruit, DM: Dry Matter, IVOMD: \u003cem\u003eIn vitro\u003c/em\u003e organic matter digestibility, ME: Metabolisable energy, SE: Standard error, SEM: Standard error of means. a,b,c,d,e for each line, mean values with different letters are significantly different (**: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0,001).\u003c/p\u003e\u003cp\u003eThe control group showed the fourth lowest ammonia nitrogen level, which can be attributed to the low protein content of wheat straw. The 2% and 4% \u003cem\u003eP. farcta\u003c/em\u003e fruit groups displayed the second lowest levels, indicating that the ammonia-reducing effects of compounds in \u003cem\u003eP. farcta\u003c/em\u003e fruit begin to take effect at these supplementation levels. Tannins form complexes with proteins, reducing ruminal degradation and increasing protein flow to the intestines (Patra and Saxena \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Hydrolyzable tannins limit rumen proteolysis by binding to bacteria and proteins (Zhao et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and suppress protozoal growth, lowering ammonia nitrogen levels (Santoso et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Essential oils can also reduce nitrous oxide and ammonia nitrogen emissions in dairy cattle (Carrazco et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). G\u0026uuml;ler et al. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) found that the addition of \u003cem\u003eB. lactis\u003c/em\u003e to wheat straw reduced total gas production, methane formation, carbon dioxide levels, and IVOMD, whereas supplementation with \u003cem\u003eS. boulardii\u003c/em\u003e increased methane production levels. \u003cem\u003eYucca schidigera\u003c/em\u003e extract (25\u0026ndash;50 g/day) reduced ruminal volatyl fatty acids levels (Guyader et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and saponins\u0026mdash;whether from plants or extracts\u0026mdash;were shown to lower ammonia nitrogen concentrations (Hu et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) and nitrogen excretion (Jayanegara et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In lambs, \u003cem\u003eProsopis laevigata\u003c/em\u003e pods increased ammonia nitrogen at 250\u0026ndash;500 g/kg levels (Pena-Avelino \u003cem\u003eet al\u003c/em\u003e. 2016), suggesting safe use up to 500 g/kg. Yanza et al. (\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) reported that saponins up to 40 g/kg DM had no negative effect on intake or palatability.\u003c/p\u003e\u003cp\u003eThe effects of \u003cem\u003eP. farcta\u003c/em\u003e fruit supplementation to corn silage on \u003cem\u003ein vitro\u003c/em\u003e total gas (ml/g DM), methane (%), ammonia nitrogen (%), pH, IVOMD (% DM), ME (MJ/kg DM), and carbon dioxide (ml/g DM) are presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. In this study, significant differences in pH values were observed among the maize silage groups, unlike the wheat straw groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0,001). The highest pH (6,99) occurred in the group with 100% \u003cem\u003eP. farcta\u003c/em\u003e fruit, likely due to fermentation-inhibiting compounds in the fruit. The naturally acidic nature of maize silage may have also contributed to this variation. \u003cem\u003eP. farcta\u003c/em\u003e inclusion significantly affected total gas, methane, carbon dioxide, ammonia nitrogen, pH, IVOMD, and ME values (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0,001). As shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, total gas production was lower in the control, 4%, 8%, and 100% groups, while it increased at 2% and 6%, peaking at 10%. These results suggest that maize silage deficiencies started to be addressed at 2% and were best corrected at 10%. However, the low gas output in the 100% group may reflect inhibition of microbial fermentation by excess bioactive compounds. Therefore, using \u003cem\u003eP. farcta\u003c/em\u003e fruit at 6\u0026ndash;8% inclusion appears to offer a more balanced and practical supplementation approach than using it as a sole feed source.\u003c/p\u003e\u003cp\u003eSoltan et al. (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) suggested that the methane-reducing effect of \u003cem\u003eP. juliflora\u003c/em\u003e leaves may result not only from tannins but also other bioactive compounds. Khan et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) identified moderate levels of indole alkaloids in the leaves, potentially responsible for reduced gas output. Despite low tannin levels (1,0 g/kg DM), \u003cem\u003eP. juliflora\u003c/em\u003e leaves were associated with lower methane emissions (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0,05), indicating the presence of additional inhibitory metabolites. Dos Santos et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) demonstrated that chloroformic extracts of \u003cem\u003eP. juliflora\u003c/em\u003e pods, rich in juliprosopine, prosoflorine and juliprosine, suppressed gas production similarly to monensin after 36 h. Melesse et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) reported higher ME and IVOMD in \u003cem\u003eP. juliflora\u003c/em\u003e pods than in other legumes, despite elevated gas production. Saad et al. (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) observed moderate antimicrobial activity in \u003cem\u003eP. farcta\u003c/em\u003e aerial parts, particularly in n-hexane and methylene chloride extracts. G\u0026uuml;ler et al. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) found that probiotic supplementation (including \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e, \u003cem\u003eBifidobacterium lactis\u003c/em\u003e, \u003cem\u003eSaccharomyces boulardii\u003c/em\u003e) to maize silage had no significant effect on methane or carbon dioxide. Saponin-rich diets have been linked to improved fermentation, health, and reduced methane emissions (Baheg et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Meena et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) noted that increasing concentrates in \u003cem\u003eProsopis cineraria\u003c/em\u003e-based rations elevated ammonia nitrogen, VFAs, and protozoa populations (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0,05). In the present study, ammonia nitrogen levels peaked at 10% \u003cem\u003eP. farcta\u003c/em\u003e fruit supplementation, decreasing progressively in 6%, 8%, and 100% groups, suggesting that its bioactive compounds may inhibit ammonia nitrogen formation and promote bypass protein. G\u0026uuml;l et al. (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) found that black cumin and its oil increased gas production (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0,05) without affecting methane, Carbon dioxide, ammonia nitrogen or pH, whereas thyme and thyme oil significantly influenced all gas parameters.\u003c/p\u003e\u003cp\u003eAccording to Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the highest IVOMD and ME values were observed in the group supplemented with 10% \u003cem\u003eP. farcta\u003c/em\u003e fruit (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0,001), indicating that nutritional deficiencies in maize silage were partially addressed at 2\u0026ndash;6% and optimally corrected at 10%. In contrast, the 100% \u003cem\u003eP. farcta\u003c/em\u003e group showed the lowest values, likely due to the inhibitory effects of saponins and tannins on microbial activity. Its similarity to the control group highlights the poor nutritional value of unsupplemented silage. These results suggest that \u003cem\u003eP. farcta\u003c/em\u003e is most effective at 8\u0026ndash;10% inclusion. While the 10% group achieved the highest ME (11,80 MJ/kg DM) and IVOMD (77,67%), it also had the highest methane production (58,50%). The 8% group produced lower ME (8,58 MJ/kg DM) but maintained methane levels close to the control (30,20% vs. 25,85%). Despite low methane, the 100% group offered limited nutritional benefit, indicating it is unsuitable as a sole feed. Overall, 6\u0026ndash;8% inclusion appears optimal for improving efficiency while minimising methane emissions. G\u0026uuml;l et al. (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) reported that supplementing corn silage with black cumin and its oil affected IVOMD and ME values (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0,05), with reductions at 0,92% black cumin and increases at 0,3% black cumin oil, likely due to enhanced microbial activity. Similarly, thyme and thyme oil influenced IVOMD and ME, with the lowest and highest values observed at 8,6% and 0,15%, respectively. Meena et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) found that increasing concentrate levels elevated gas, methane, and ME (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0,05), while high-tannin diets reduced methane and improved IVOMD. These results help to explain why the nutrients present in \u003cem\u003eP. farcta\u003c/em\u003e fruit support microbial activity and gas production in the rumen environment up to a certain level, as they compensate for the nutritional deficiencies inherent in maize silage. According to IPCC (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) 81% of agricultural nitrous oxide emissions arise from the sector itself, with 46% linked to ruminant excreta. Phytochemicals may reduce such emissions by accumulating natural nitrification inhibitors in urine (Totty et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Effective feeding strategies can modulate nitrogen metabolism, decreasing both urinary nitrogen and atmospheric nitrous oxide (Stewart et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWhile \u003cem\u003eP. farcta\u003c/em\u003e fruit supplementation shows potential for improving rumen fermentation and reducing emissions, limitations must be acknowledged. As the study was conducted \u003cem\u003ein vitro\u003c/em\u003e, results may not reflect \u003cem\u003ein vivo\u003c/em\u003e rumen dynamics (Baah et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Jahromi et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and practical application should be approached with caution. Additionally, the phytochemical composition of \u003cem\u003eP. farcta\u003c/em\u003e may vary seasonally and environmentally (Salari et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sharifi-Rad \u003cem\u003eet al.\u003c/em\u003e 2021) affecting consistency. Nevertheless, its nutrient profile and emission-reducing properties (Sharifi-Rad \u003cem\u003eet al.\u003c/em\u003e 2021) support its use as a potential feedstuff (Sawal et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Future \u003cem\u003ein vivo\u003c/em\u003e studies are needed to evaluate animal performance, health, and environmental outcomes. Moreover, synergistic effects with other additives or probiotics warrant investigation (G\u0026uuml;ler et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eExisting literature shows that different fractions of \u003cem\u003eProsopis species\u003c/em\u003e can affect digestibility, performance, rumen fermentation, blood metabolites, and nitrogen utilisation in ruminants, with effects depending on species, dose, and origin. Owing to their rich bioactive content, \u003cem\u003eProsopis species\u003c/em\u003e may modulate rumen fermentation and improve metabolic responses. In wheat straw groups, 2% and 6% \u003cem\u003eP. farcta\u003c/em\u003e supplementation significantly increased ME but also raised methane emissions. The 8% level did not enhance ME but kept methane close to control levels (14,56% vs 13,87%), suggesting it may offer a more sustainable balance than 4\u0026ndash;6%, which, while efficient, pose environmental concerns. The 100% \u003cem\u003eP. farcta\u003c/em\u003e group had higher IVOMD and ME than control, indicating strong nutritional potential compared to wheat straw. In maize silage, 10% supplementation maximised ME but also methane output. Although 8% reduced methane to near-control levels, it failed to improve ME. Thus, while 4\u0026ndash;6% may be optimal for energy, methane remains a concern. The 100% \u003cem\u003eP. farcta\u003c/em\u003e group showed low methane and moderate digestibility, but its low energy value limits its use as a sole feed.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs rumen fluid was collected from slaughtered animals at the abattoir, ethical approval was not required.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe participant provided both oral and written consent after being informed about all aspects of the experimental study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no financial or other conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAli A, Tudsri S, Rungmekarat S, Kaewtrakulpong K (2012) Effect of feeding \u003cem\u003eProsopis juliflora\u003c/em\u003e pods and leaves on performance and carcass characteristics of Afar sheep. 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Agriculture 12(8):1198. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/agriculture12081198\u003c/span\u003e\u003cspan address=\"10.3390/agriculture12081198\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"feed additives, in vitro digestibility, metabolic energy, methane emission, Prosopis farcta","lastPublishedDoi":"10.21203/rs.3.rs-7180785/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7180785/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn arid regions and areas facing forage scarcity, \u003cem\u003eProsopis species\u003c/em\u003e have long been used in ruminant nutrition, yet their full potential remains inadequately assessed. Domesticated ruminants contribute approximately 15% of global methane emissions. Previously, antibiotics were routinely added to ruminant diets to reduce methane-associated energy losses and improve feed efficiency; however, sustainable alternative strategies are now required. In this study, widely used but nutritionally limited wheat straw and maize silage were supplemented with varying levels (2%, 4%, 6%, 8%, and 10%) of \u003cem\u003eProsopis farcta\u003c/em\u003e fruit. Using the \u003cem\u003ein vitro\u003c/em\u003e gas production technique and cattle rumen fluid, parameters including total gas production, methane formation, ammonia nitrogen concentration, \u003cem\u003ein vitro\u003c/em\u003e organic matter digestibility, and metabolizable energy were evaluated. Each treatment was conducted in quadruplicate under standard laboratory conditions. The lowest methane production was observed in wheat straw at control and 8% inclusion levels, and in maize silage at control, 8%, and 100% supplementation groups. The highest methane production occurred in wheat straw at 2%, 4%, 6%, 10%, and 100% inclusion levels, and in maize silage at 6% and 10% supplementation. In wheat straw, 2% and 6% inclusion levels significantly increased metabolizable energy, although methane production also rose considerably. Conversely, 8% supplementation did not increase metabolizable energy but maintained methane production close to control levels (14,56% vs. 13,87%). In maize silage, 10% inclusion maximised metabolizable energy but caused excessive methane emissions. Inclusion levels of 4\u0026ndash;6% in maize silage offer productivity benefits but pose environmental risks, whereas 8% supplementation provides a more sustainable balance.\u003c/p\u003e","manuscriptTitle":"Determination of the Effects of Different Levels of Prosopis farcta Fruit Supplementation to Low-Quality Roughages on Organic Matter Digestibility and Methane Formation Using the In Vitro Digestion Method","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-29 03:14:10","doi":"10.21203/rs.3.rs-7180785/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8a186671-584b-423a-8e0e-f578afed44fd","owner":[],"postedDate":"July 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-09T22:36:58+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-29 03:14:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7180785","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7180785","identity":"rs-7180785","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}