Effect of replacement of berseem clover by almond hulls from Palestine or Tunisia with <i>Pichia manshurica</i> yeast as feed additives in mixed diet on rumen fermentation, methane emissions, and protozoa counts, in vitro

Halimeh Zoabi; Yosra A. Soltan; Amr S. Morsy; Hani M. El-Zaiat; Ahmed E. Kholif; Soha Ghzayel; Bassam AbuAziz; Raouia Ben Rhouma; Jihen Jemaï; Sawsan Hassan; Hajer Ammar; Halimeh Zoabi; Yosra A. Soltan; Amr S. Morsy; Hani M. El-Zaiat; Ahmed E. Kholif; Soha Ghzayel; Bassam AbuAziz; Raouia Ben Rhouma; Jihen Jemaï; Sawsan Hassan; Hajer Ammar

doi:10.3934/agrfood.2026048

AIMS Agriculture and Food

2026, Volume 11, Issue 4: 974-995. doi: 10.3934/agrfood.2026048

Previous Article Next Article

Research article Topical Sections

Effect of replacement of berseem clover by almond hulls from Palestine or Tunisia with Pichia manshurica yeast as feed additives in mixed diet on rumen fermentation, methane emissions, and protozoa counts, in vitro

1.
High School of Agriculture of Le Kef, University of Jendouba, Jendouba 8100, Tunisia
2.
Laboratory of Agricultural Production System and Sustainable Development (SPADD), High School of Agriculture of Mograne, University of Carthage, Zaghouan 1121, Tunisia
3.
Veterinary Services and Animal Health Department, Jenin 009704, Palestine
4.
Department of Animal and Fish Production, Faculty of Agriculture, University of Alexandria, Aflaton St., El-Shatby, P.O. Box 21545, Alexandria 21526, Egypt
5.
Livestock Research Department, Arid Lands Cultivation Research Institute, City of Scientific Research and Technological Applications, New Borg El-Arab, P.O. Box 21934, Alexandria 21934, Egypt
6.
Department of Animal and Veterinary Sciences, College of Agricultural and Marine Sciences, Qaboos University, P.O. Box 34, Al-Khod 123, Oman
7.
Department of Animal Sciences, North Carolina Agricultural and Technical State University, Greensboro, NC 27411, USA
8.
Dairy Science Department, National Research Centre, 33 Bohouth St., Dokki, Giza 12622, Egypt
9.
Biotechnology Directorate, National Agricultural Research Center (NARC), Ministry of Agriculture, Jenin 009704, Palestine
10.
International Center for Agricultural Research in the Dry Areas (ICARDA), Resilient Agri-Food Systems Program, Tunis, CP 1004, Tunisia

Received: 21 July 2026 Revised: 10 September 2026 Accepted: 30 September 2026 Published: 04 November 2026

This study was conducted to evaluate the effects of combined supplementation of two rumen-isolated Pichia manshurica yeast strains (FFNLYFC1and FFNLYFC2), which were combined 1:1 and dosed at 1.4 × 10⁸ CFU/g dry matter (DM) from each strain on in vitro ruminal fermentation of diets in which almond hulls from Palestine (AHP) or Tunisia (AHT) replaced berseem clover (Trifolium alexandrinum) as the primary forage source. Five experimental treatments were evaluated at a 60:40 forage-to-concentrate ratio: a control diet (berseem clover hay); AHP without yeast (AHP–); AHP with yeast (AHP+); AHT without yeast (AHT–); and AHT with yeast (AHT+). Three independent biological runs were performed using rumen fluid from three water buffalo males. The asymptotic gas production potential (b) was highest in the control and AHP+ (149.3 and 149.2 mL/g DM, respectively; P = 0.016) and significantly lower in AHT diets (140.4–141.1 mL/g DM). True dry matter digestibility was highest in AHP+ (81.4%) and lowest in AHT– (77.9%). The AHP+ diet yielded the highest total volatile fatty acid (VFA) concentration (129.6 mmol/L), representing a 37.6% increase over the control (94.2 mmol/L), alongside the greatest propionate proportion (35.4% vs. 21.1% in the control; P = 0.001). Microbial biomass production was highest in AHP+ (523.3 mg/g DM; P = 0.003). True digestible organic matter was significantly greater in AHT– and AHT+ (812.9 and 813.8 g/kg DM, respectively) than in the control (786.4 g/kg DM), reflecting lower lignification in Tunisian hulls. The true neutral detergent fiber degradability (TNDF) was markedly higher in the control (539.1 g/kg DM NDF than in all almond hull diets (357.4–421.5 g/kg NDF; P < 0.001), though yeast supplementation improved TNDF in AHP diets by 17.9%. Methane production per gram of DM was highest in AHP+ (32.6 mL/g DM; P = 0.039), though methane (CH₄) per unit of truly digested organic matter did not differ significantly among treatments (P = 0.059), indicating that the elevated absolute CH₄ in AHP+ reflects greater total fermentation activity rather than enhanced methanogenesis per se. Ruminal pH and protozoal counts were not significantly different among treatments. These findings indicated that P. manshurica yeast supplementation substantially enhanced fermentation efficiency and VFA output in Palestinian almond hull diets and exerted a meaningful but more modest benefit in Tunisian hull diets, with the geographic origin of the almond hulls appearing to modulate the fermentation response to yeast inclusion. P. manshurica-supplemented Palestinian almond hull diets (AHP+) produced the best combined fermentation outcome among the almond hull treatments evaluated.
- P. manshurica,
- almond hulls,
- in vitro rumen fermentation,
- volatile fatty acids,
- methane
Citation: Halimeh Zoabi, Yosra A. Soltan, Amr S. Morsy, Hani M. El-Zaiat, Ahmed E. Kholif, Soha Ghzayel, Bassam AbuAziz, Raouia Ben Rhouma, Jihen Jemaï, Sawsan Hassan, Hajer Ammar. Effect of replacement of berseem clover by almond hulls from Palestine or Tunisia with Pichia manshurica yeast as feed additives in mixed diet on rumen fermentation, methane emissions, and protozoa counts, in vitro[J]. AIMS Agriculture and Food, 2026, 11(4): 974-995. doi: 10.3934/agrfood.2026048

Related Papers:

Abstract

This study was conducted to evaluate the effects of combined supplementation of two rumen-isolated Pichia manshurica yeast strains (FFNLYFC1and FFNLYFC2), which were combined 1:1 and dosed at 1.4 × 10⁸ CFU/g dry matter (DM) from each strain on in vitro ruminal fermentation of diets in which almond hulls from Palestine (AHP) or Tunisia (AHT) replaced berseem clover (Trifolium alexandrinum) as the primary forage source. Five experimental treatments were evaluated at a 60:40 forage-to-concentrate ratio: a control diet (berseem clover hay); AHP without yeast (AHP–); AHP with yeast (AHP+); AHT without yeast (AHT–); and AHT with yeast (AHT+). Three independent biological runs were performed using rumen fluid from three water buffalo males. The asymptotic gas production potential (b) was highest in the control and AHP+ (149.3 and 149.2 mL/g DM, respectively; P = 0.016) and significantly lower in AHT diets (140.4–141.1 mL/g DM). True dry matter digestibility was highest in AHP+ (81.4%) and lowest in AHT– (77.9%). The AHP+ diet yielded the highest total volatile fatty acid (VFA) concentration (129.6 mmol/L), representing a 37.6% increase over the control (94.2 mmol/L), alongside the greatest propionate proportion (35.4% vs. 21.1% in the control; P = 0.001). Microbial biomass production was highest in AHP+ (523.3 mg/g DM; P = 0.003). True digestible organic matter was significantly greater in AHT– and AHT+ (812.9 and 813.8 g/kg DM, respectively) than in the control (786.4 g/kg DM), reflecting lower lignification in Tunisian hulls. The true neutral detergent fiber degradability (TNDF) was markedly higher in the control (539.1 g/kg DM NDF than in all almond hull diets (357.4–421.5 g/kg NDF; P < 0.001), though yeast supplementation improved TNDF in AHP diets by 17.9%. Methane production per gram of DM was highest in AHP+ (32.6 mL/g DM; P = 0.039), though methane (CH₄) per unit of truly digested organic matter did not differ significantly among treatments (P = 0.059), indicating that the elevated absolute CH₄ in AHP+ reflects greater total fermentation activity rather than enhanced methanogenesis per se. Ruminal pH and protozoal counts were not significantly different among treatments. These findings indicated that P. manshurica yeast supplementation substantially enhanced fermentation efficiency and VFA output in Palestinian almond hull diets and exerted a meaningful but more modest benefit in Tunisian hull diets, with the geographic origin of the almond hulls appearing to modulate the fermentation response to yeast inclusion. P. manshurica-supplemented Palestinian almond hull diets (AHP+) produced the best combined fermentation outcome among the almond hull treatments evaluated.

References

[1]	Zoabi H, Ammar H, Ghzayel S, et al. (2024) Nutritional characteristics of almond hulls treated with sodium hydroxide and urea or supplemented with polyethylene glycol as an alternative feed resource for ruminant nutrition in Mediterranean area: In vitro study. Cogent Food Agric 10: 2422534. https://doi.org/10.1080/23311932.2024.2422534 doi: 10.1080/23311932.2024.2422534
[2]	Rad MI, Rouzbehan Y, Rezaei J (2016) Effect of dietary replacement of alfalfa with urea-treated almond hulls on intake, growth, digestibility, microbial nitrogen, nitrogen retention, ruminal fermentation, and blood parameters in fattening lambs. J Anim Sci 94: 349–358. https://doi.org/10.2527/jas.2015-9437 doi: 10.2527/jas.2015-9437
[3]	Recalde A, Evan T de, Fernández C, et al. (2024) Chemical composition and nutritive value of almond hulls from two almond varieties and influence of including almond hulls in the diet on in vitro ruminal fermentation and methane production. Vet Sci 11: 242. https://doi.org/10.3390/vetsci11060242 doi: 10.3390/vetsci11060242
[4]	Zoabi H, Ammar H, Ghzayel S, et al. (2025) Feeding sodium hydroxide-treated almond hulls to Assaf sheep: Effects on chemical composition, nutrient digestibility, and zootechnical performance. Agriculture 15: 1000. https://doi.org/10.3390/agriculture15091000 doi: 10.3390/agriculture15091000
[5]	Elahi MY, Kargar H, Dindarlou MS, et al. (2017) The chemical composition and in vitro digestibility evaluation of almond tree (Prunus dulcis D. A. Webb syn. Prunus amygdalus; var. Shokoufeh) leaves versus hulls and green versus dry leaves as feed for ruminants. Agroforestry Systems 91: 773–780. https://doi.org/10.1007/s10457-016-9964-5 doi: 10.1007/s10457-016-9964-5
[6]	Kholif AE, Anele A, Anele UY (2024) Microbial feed additives in ruminant feeding. AIMS Microbiol 10: 542–571. https://doi.org/10.3934/microbiol.2024026 doi: 10.3934/microbiol.2024026
[7]	Wang H, Liu G, Zhou A, et al. (2024) Effects of yeast culture on in vitro ruminal fermentation and microbial community of high concentrate diet in sheep. AMB Express 14: 37. https://doi.org/10.1186/s13568-024-01692-6 doi: 10.1186/s13568-024-01692-6
[8]	Baker LM, Kraft J, Karnezos TP, et al. (2022) Review: The effects of dietary yeast and yeast-derived extracts on rumen microbiota and their function. Anim Feed Sci Technol 294: 115476. https://doi.org/10.1016/j.anifeedsci.2022.115476 doi: 10.1016/j.anifeedsci.2022.115476
[9]	Chaucheyras-Durand F, Walker ND, Bach A (2008) Effects of active dry yeasts on the rumen microbial ecosystem: Past, present and future. Anim Feed Sci Technol 145: 5–26. https://doi.org/10.1016/j.anifeedsci.2007.04.019 doi: 10.1016/j.anifeedsci.2007.04.019
[10]	Amin AB, Mao S (2021) Influence of yeast on rumen fermentation, growth performance and quality of products in ruminants: A review. Anim Nutr 7: 31–41. https://doi.org/10.1016/j.aninu.2020.10.005 doi: 10.1016/j.aninu.2020.10.005
[11]	Elmhadi ME, Ali DK, Khogali MK, et al. (2022) Subacute ruminal acidosis in dairy herds: Microbiological and nutritional causes, consequences, and prevention strategies. Anim Nutr 10: 148–155. https://doi.org/10.1016/j.aninu.2021.12.008 doi: 10.1016/j.aninu.2021.12.008
[12]	Mao J, Wang L (2025) Rumen acidosis in ruminants: A review of the effects of high-concentrate diets and the potential modulatory role of rumen foam. Front Vet Sci 12: 1595615. https://doi.org/10.3389/fvets.2025.1595615 doi: 10.3389/fvets.2025.1595615
[13]	Soltan Y, Morsy A, Elazab M, et al. (2024) Effects of Pichia manshurica yeast supplementation on ruminal fermentation, nutrient degradability, and greenhouse gas emissions in aflatoxin B1 contaminated diets. Trop Anim Health Prod 56: 367. https://doi.org/10.1007/s11250-024-04184-8 doi: 10.1007/s11250-024-04184-8
[14]	Liang X, Li J, Liu P, et al. (2026) Rumen ecological distribution of Pichia yeasts and their effects on rumen fermentation and microbial community. BMC Microbiol 26: 725. https://doi.org/10.1186/s12866-026-05281-4 doi: 10.1186/s12866-026-05281-4
[15]	Bueno ICS, Cabral Filho SLS, Gobbo SP, et al. (2005) Influence of inoculum source in a gas production method. Anim Feed Sci Technol 123–124: 95–105. https://doi.org/10.1016/j.anifeedsci.2005.05.003 doi: 10.1016/j.anifeedsci.2005.05.003
[16]	AOAC (2023) Official Methods of Analysis of AOAC International, Oxford University Press, Oxford.
[17]	Van Soest PJ, Robertson JB, Lewis BA (1991) Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J Dairy Sci 74: 3583–3597. https://doi.org/10.3168/jds.S0022-0302(91)78551-2 doi: 10.3168/jds.S0022-0302(91)78551-2
[18]	NRC (2021) Nutrient Requirements of Dairy Cattle, Washington, D. C., National Academies Press.
[19]	Menke KH, Steingass H (1988) Estimation of the energetic feed value obtained from chemical analysis and in vitro gas production using rumen fluid. Anim Res Dev 28: 7–55.
[20]	France J, Dijkstra J, Dhanoa MS, et al. (2000) Estimating the extent of degradation of ruminant feeds from a description of their gas production profiles observed in vitro: Derivation of models and other mathematical considerations. Br J Nutr 83: 143–150. https://doi.org/10.1017/S0007114500000180 doi: 10.1017/S0007114500000180
[21]	Menke KH, Raab L, Salewski A, et al. (1979) The estimation of the digestibility and metabolizable energy content of ruminant feedingstuffs from the gas production when they are incubated with rumen liquor in vitro. J Agric Sci 93: 217–222. https://doi.org/10.1017/S0021859600086305 doi: 10.1017/S0021859600086305
[22]	Blümmel M, Makkar HPS, Becker K (1997) In vitro gas production: a technique revisited. J Anim Physiol Anim Nutr (Berl) 77: 24–34. https://doi.org/10.1111/j.1439-0396.1997.tb00734.x doi: 10.1111/j.1439-0396.1997.tb00734.x
[23]	Kholif AE, Gouda GA, Morsy TA, et al. (2022) Dietary date palm leaves ensiled with fibrolytic enzymes decreased methane production, and improved feed degradability and fermentation kinetics in a ruminal in vitro system. Waste Biomass Valorization 13: 3475–3488.
[24]	Dehority BA, Tirabasso PA, Grifo AP (1989) Most-probable-number procedures for enumerating ruminal bacteria, including the simultaneous estimation of total and cellulolytic numbers in one medium. Appl Environ Microbiol 55: 2789–2792. https://doi.org/10.1128/aem.55.11.2789-2792.1989 doi: 10.1128/aem.55.11.2789-2792.1989
[25]	De Baere S, Eeckhaut V, Steppe M, et al. (2013) Development of a HPLC–UV method for the quantitative determination of four short-chain fatty acids and lactic acid produced by intestinal bacteria during in vitro fermentation. J Pharm Biomed Anal 80: 107–115. https://doi.org/10.1016/j.jpba.2013.02.032 doi: 10.1016/j.jpba.2013.02.032
[26]	Phesatcha K, Phesatcha B, Wanapat M, et al. (2020) Roughage to concentrate ratio and Saccharomyces cerevisiae inclusion could modulate feed digestion and in vitro ruminal fermentation. Vet Sci 7: 1–13. https://doi.org/10.3390/vetsci7040151 doi: 10.3390/vetsci7040151
[27]	Dijkstra J, Kebreab E, Bannink A, et al. (2005) Application of the gas production technique to feed evaluation systems for ruminants. Anim Feed Sci Technol 123-124: 561–578. https://doi.org/10.1016/j.anifeedsci.2005.04.048 doi: 10.1016/j.anifeedsci.2005.04.048
[28]	Liu H, Li F, Ma Z, et al. (2024) Effects of active dry yeast supplementation in in vitro and in vivo nutrient digestibility, rumen fermentation, and bacterial community. Animals 14: 2916. https://doi.org/10.3390/ani14192916 doi: 10.3390/ani14192916
[29]	Storm E, Ørskov ER (1983) The nutritive value of rumen micro-organisms in ruminants. Br J Nutr 50: 463–470. https://doi.org/10.1079/BJN19830114 doi: 10.1079/BJN19830114
[30]	Nueraihemaiti G, Huo X, Zhang H, et al. (2025) Effect of diet supplementation with two yeast cultures on rumen fermentation parameters and microbiota of fattening sheep in vitro. Microorganisms 13: 550. https://doi.org/10.3390/microorganisms13030550 doi: 10.3390/microorganisms13030550
[31]	Pang R, Xiao X, Mao T, et al. (2023) The molecular mechanism of propionate-regulating gluconeogenesis in bovine hepatocytes. Anim Biosci 36: 1693–1699. https://doi.org/10.5713/ab.23.0061 doi: 10.5713/ab.23.0061
[32]	Malyugina S, Holik S, Horky P (2025) Mitigation strategies for methane emissions in ruminant livestock: a comprehensive review of current approaches and future perspectives. Front Anim Sci 6: 1610376. https://doi.org/10.3389/fanim.2025.1610376 doi: 10.3389/fanim.2025.1610376
[33]	Zhang X, Dong X, Wanapat M, et al. (2022) Ruminal pH pattern, fermentation characteristics and related bacteria in response to dietary live yeast (Saccharomyces cerevisiae) supplementation in beef cattle. Anim Biosci 35: 184–195. https://doi.org/10.5713/ab.21.0200 doi: 10.5713/ab.21.0200
[34]	Jouany JP (1996) Effect of rumen protozoa on nitrogen utilization by ruminants. J Nutr 126: 1335–1346. https://doi.org/10.1093/jn/126.suppl_4.1335S doi: 10.1093/jn/126.suppl_4.1335S
[35]	Majewska MP, Miltko R, Bełżecki G, et al. (2021) Population of protozoa and carbohydrate-digesting enzymes in the rumen of sheep fed a diet supplemented with yeast Saccharomyces cerevisiae. Small Ruminant Res 205, 106544. https://doi.org/10.1016/j.smallrumres.2021.106544 doi: 10.1016/j.smallrumres.2021.106544
[36]	Lila ZA, Mohammed N, Yasui T, et al. (2004) Effects of a twin strain of Saccharomyces cerevisiae live cells on mixed ruminal microorganism fermentation in vitro. J Anim Sci 82: 1847–1854. https://doi.org/10.2527/2004.8261847x doi: 10.2527/2004.8261847x

Reader Comments

Your name:*

Email:*
© 2026 the Author(s), licensee AIMS Press. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0)