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Yak (Bos grunniens) is a large ruminant endemic to the Tibetan plateau. The addition of enzyme complexes to feed can significantly improve their growth performance. Therefore, studying the effects of ruminant compound enzyme preparations dosage on yak rumen microorganisms and production performance is crucial to promoting the development of the yak industry. This study aimed to determine the effects of feeding yaks with different doses of ruminant enzyme compounds on the performance, meat quality, and rumen microorganisms of yaks. Three kinds of experimental diets with doses of 0.5 g/kg (LE group), 1 g/kg (ME group), and 2 g/kg (HE group) were selected to determine the growth index, meat quality, serum biochemical indexes, rumen fluid pH and other indexes of the three experimental groups. Metagenomics studies were used to investigate the differences in rumen microbial composition and function among yak groups, and transcriptome sequencing of the longest dorsal muscle was performed to reveal the expression of differential genes among different groups. It was determined that the levels of dietary enzyme complexes significantly affected growth performance, rumen fluid pH, and serum biochemical indices. At the phylum level, the dominant phylum in all three treatment groups was Bacteroidota, Bacillota, Kiritimatiellota, and Pseudomonadota. At the genus level, Prevotella, Methanobrevibacter, Oscillibacter. Fibrobacter showed statistically significant differences in abundance (p

This study aimed to investigate the effect of prickly ash seeds (PAS) on the microbial community found in rumen microbes of Hu sheep by adding different percentages of prickly ash seeds and to carry out research on the relation between rumen flora and production performance. Twenty-seven male lambs of Hu sheep were classified into three groups based on the content of prickly ash seeds (PAS) fed for 90 days, i.e., 0%, 3%, and 6%. At the end of the feeding trial, rumen fluid samples were collected from six sheep in each group for 16S amplicon sequencing. The results showed that the addition of prickly ash seeds significantly increased both Chao1 and ACE indices (P < 0.05), and the differences between groups were greater than those within groups. The relative content of Bacteriodota decreased, and the relative content of Fusobacteriota, Proteobacteria, Acidobacteriota, and Euryarchaeota increased. The relative content of Papillibacter and Saccharofermentans was increased at the genus level, and the relative content of Bacteroides and Ruminococcus was decreased. The test group given 3% of prickly ash seeds was superior to the test group given 6% of prickly ash seeds. In addition, the addition of 3% of prickly ash seeds improved the metabolism or immunity of sheep. Fusobacteriota and Acidobacteriota were positively correlated with total weight, dressing percentage, and average daily gain (ADG) and negatively correlated with average daily feed intake (ADFI), feed-to-gain ratio (F/G), and lightness (L*). Methanobrevibacter and Saccharofermentans were positively correlated with ADG and negatively correlated with ADFI and L*. In conclusion, under the present experimental conditions, the addition of prickly ash seeds increased the abundance and diversity of rumen microorganisms in Hu sheep and changed the relative abundance of some genera. However, the addition of 6% prickly ash seedsmay negatively affect the digestive and immune functions in sheep rumen. [ABSTRACT FROM AUTHOR]

Simple Summary: Simple Summary: The rumen is the most important digestive organ of ruminants, and its rich microbial co−evolved with the host to play a crucial role in immunomodulation, defense, digestive metabolism, animal productivity, and meat quality control. It also forms a complex regulatory network with host genes and phenotypes that are involved in maintaining organismal homeostasis. In this study, we compared the differences in rumen microorganisms between crossbred progeny of sheep and Hu sheep, identified key biomarkers, and analyzed their relationship with rumen function. The results showed that microbial diversity was significantly higher in crossbred progeny, which was able to alter the microbial community structure to regulate rumen fermentation, promote carbon homeostasis, and enhance amino acid metabolism. In addition, there were significant correlations between biomarkers and key traits. Therefore, we suggest that there is a link between sheep rumen microbes and differential traits in crossbred progeny, which provides new insights for sheep trait improvement. This experiment was conducted to investigate the effect of three–way hybrid sheep and Hu sheep on serum indicators, rumen fermentation, rumen enzyme activity, and microorganisms in sheep. Healthy and similar birth weights from three groups (Hu, n = 11; Charolais × Australian White × Hu, CAH, n = 11; Charolais × Dorper × Hu, CDH, n = 11) were selected to be fed by the ewes until 45 days of age. Subsequently, they were weaned intensively and underwent short–term fattening for 3 months along with selected male lambs fed intensively. During this period, they were fed and watered ad libitum. Blood and rumen fluid were collected and analyzed for serum indicators and rumen fluid microorganisms, enzyme activity, and VFA, respectively, at the end of the fattening period. Compared with Hu lamb, the offspring of the three–way hybrid lamb showed significant improvements in body weight, serum lactate dehydrogenase, and creatinine content. However, there was no significant effect on serum immunity and antioxidant indices. In addition, the rumen fluid volatile fatty acid (VFA) molar concentration and microcrystalline cellulose and lipase content were significantly lower in the three–way hybrid lamb compared to Hu lamb, but β–glucosidase, amylase, pepsin, and VFA molar ratio were not significantly affected. Subsequently, 16S rRNA sequencing diversity analysis revealed that three–way hybrid lamb significantly increased rumen microbial ACE and Chao1 indices compared to Hu lamb. Meanwhile, the abundance of Verrucomicrobiota and Synergistota significantly increased at the phylum level. Correlation analysis showed that Prevotella had the highest proportion, while Rikenellaceae_RC9_gut_group correlated most closely with others genus. The microbial communities isovaleric acid molar concentration and proportion were strongly correlated. In addition, there were significant differences in correlations between microbial communities and isobutyric acid, butyric acid and valeric acid content, and their molar proportion, but they were not significantly correlated with digestive enzymes. From the functional enrichment analysis, it was found that hybrid progeny were mainly enriched in the pyruvate metabolism, microbial metabolism in diverse environments, carbon metabolism, and quorum sensing pathways. In contrast, the Hu sheep were primarily enriched in the cysteine and methionine, amino sugar and nucleotide sugar, and biosynthesis of secondary metabolite pathways. These results suggest that hybridization can play a role in regulating organismal metabolism and improve animal production performance by influencing the structure and characteristics of microbial communities. [ABSTRACT FROM AUTHOR]

Bovine reproduction, including male fertility traits like semen quality, are influenced by a variety of different factors like breed, nutrition, environment, and feeding management. Diet in a crucial determinant, and in this regard although corn silage is generally considered to be a favorable roughage for fattening meat type breeds, it tends to have a negative impact on semen quality. In the current study, alfalfa hay was substituted by corn silage as a roughage source in the diet of bulls to investigate its effects on the fertility of breeding bulls. A feeding trail spanning 140 days was conducted, with semen collection occurring twice a week commencing 60 days after the start of trial. Semen quality parameters, serum antioxidant indexes, sex hormone content in semen, rumen microflora, and sperm transcriptome were characterized. Feeding corn silage enhanced host antioxidant capacity, significantly decreased spermatozoal motility and increased sperm deformity rate in bulls. Furthermore, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) content in semen were significantly decreased (P < 0.05), and the inhibin B (INHB) content was significantly increased (P < 0.01). Feeding corn silage led to significant changes in the diversity of rumen microbiota of cattle at the phylum and genus levels, some of which were significantly correlated with semen quality. Subsequent RNA sequencing indicated that DHH and PITHD1, two genes related to sperm and reproductive development, were differentially expressed, and enrichment analysis also identified several pathways and biological functions relevant to sperm development and reproduction. These results indicate that feeding corn silage modulates semen quality via different pathways. Firstly, corn silage metabolites likely affect the secretion of INHB through the testicular capillaries, which affects semen quality by regulating genes involved in spermatogenesis. Secondly, low lignin content in silage corn appears to reduce abundance of rumen flora that are positively correlated with semen quality. Overall, results indicate that feeding bulls corn silage as the primary source of forage could negatively impact semen quality and may not be appropriate as the primary roughage of forage for breeding bulls. [ABSTRACT FROM AUTHOR]

Yak (Bos grunniens) is a unique large ruminant species in the Qinghai-Tibetan Plateau (QTP). Changing the energy levels of their rations can significantly improve their growth performance. Therefore, studying the effects of dietary energy levels on the rumen microflora and metabolites of yak is crucial for enhancing the development of the yak industry. Currently, there is a lack of understanding regarding the impact of feeding energy diets on rumen fermentation parameters, microbial functions, and metabolites. This study was designed to determine the appropriate energy level for feeding yak. Three test diets with metabolizable energy levels of 7.57 MJ/kg, 9.44 MJ/kg, and 11.9 MJ/kg were used and the concentration of volatile fatty acids (VFA) in rumen fluid was measured. The microbial communities, functions, and metabolites in yaks were studied by 16S rRNA sequencing, metagenome, and LC-MS non-targeted metabolomics to investigate the relationships among rumen fermentation parameters, microbial diversity, and metabolites. Ration energy levels significantly affect total VFA, acetate, propionate, butyrate, iso-valerate, valerate, and acetate/propionate (p < 0.05). At the phylum level, the dominant phyla in all three treatment groups were Bacteroidota, Firmicutes, and Actinobacteriota. At the genus level, the abundance of the unclassified_o__Bacteroidales, norank_f_Muribaculaceae, Lachnospiraceae_NK4A136_group, and Family _XIII_AD3011_group showed significant differences (p < 0.05) and were significantly correlated with differential metabolites screened for phosphatidylcholine [PC(16:0/0:0), PC(18:3/0:0)], uridine 30-monophosphate, and adenosine monophosphate, etc. CAZymes family analysis showed that GHs and CEs differed significantly among the three groups. In addition, differential metabolites were mainly enriched in the pathways of lipid metabolism, nucleotide metabolism, and biosynthesis of other secondary metabolites, and the concentrations of differential metabolites were correlated with microbial abundance. In summary, this study analyzed the effects of ration energy levels on rumen microorganisms and metabolites of yaks and their relationships. The results provided a scientific basis for the selection of dietary energy for yaks in the house feeding period in the future. [ABSTRACT FROM AUTHOR]

This experiment was conducted to investigate the effect of three–way hybrid sheep and Hu sheep on serum indicators, rumen fermentation, rumen enzyme activity, and microorganisms in sheep. Healthy and similar birth weights from three groups (Hu, n = 11; Charolais × Australian White × Hu, CAH, n = 11; Charolais × Dorper × Hu, CDH, n = 11) were selected to be fed by the ewes until 45 days of age. Subsequently, they were weaned intensively and underwent short–term fattening for 3 months along with selected male lambs fed intensively. During this period, they were fed and watered ad libitum. Blood and rumen fluid were collected and analyzed for serum indicators and rumen fluid microorganisms, enzyme activity, and VFA, respectively, at the end of the fattening period. Compared with Hu lamb, the offspring of the three–way hybrid lamb showed significant improvements in body weight, serum lactate dehydrogenase, and creatinine content. However, there was no significant effect on serum immunity and antioxidant indices. In addition, the rumen fluid volatile fatty acid (VFA) molar concentration and microcrystalline cellulose and lipase content were significantly lower in the three–way hybrid lamb compared to Hu lamb, but β–glucosidase, amylase, pepsin, and VFA molar ratio were not significantly affected. Subsequently, 16S rRNA sequencing diversity analysis revealed that three–way hybrid lamb significantly increased rumen microbial ACE and Chao1 indices compared to Hu lamb. Meanwhile, the abundance of Verrucomicrobiota and Synergistota significantly increased at the phylum level. Correlation analysis showed that Prevotella had the highest proportion, while Rikenellaceae_RC9_gut_group correlated most closely with others genus. The microbial communities isovaleric acid molar concentration and proportion were strongly correlated. In addition, there were significant differences in correlations between microbial communities and isobutyric acid, butyric acid and valeric acid content, and their molar proportion, but they were not significantly correlated with digestive enzymes. From the functional enrichment analysis, it was found that hybrid progeny were mainly enriched in the pyruvate metabolism, microbial metabolism in diverse environments, carbon metabolism, and quorum sensing pathways. In contrast, the Hu sheep were primarily enriched in the cysteine and methionine, amino sugar and nucleotide sugar, and biosynthesis of secondary metabolite pathways. These results suggest that hybridization can play a role in regulating organismal metabolism and improve animal production performance by influencing the structure and characteristics of microbial communities.

Introduction: The crude protein level in the diet will affect the fermentation parameters, microflora, and metabolites in the rumen of ruminants. It is of great significance to study the effect of crude protein levels in supplementary diet on microbial community and metabolites for improving animal growth performance. At present, the effects of crude protein level in supplementary diet on rumen fermentation parameters, microbial community, and metabolites of Jersey-Yak (JY) are still unclear. Methods: The purpose of this experiment was to study the appropriate crude protein level in the diet of JY. The rumen fermentation indexes (volatile fatty acids and pH) were determined by supplementary diets with crude protein levels of 15.16 and 17.90%, respectively, and the microbial community and metabolites of JYs were analyzed by non-target metabonomics and metagenome sequencing technology, and the changes of rumen fermentation parameters, microbial flora, and metabolites in the three groups and their interactions were studied. Results and Discussion: The crude protein level in the supplementary diet had significant effects on pH, valeric acid, and the ratio of acetic acid to propionic acid (p < 0.05). The protein level had no significant effect on the dominant microflora at the phylum level (p > 0.05), and all three groups were Bacteroides and Firmicutes. The results of metabolite analysis showed that the crude protein level of supplementary diet significantly affected the metabolic pathways such as Bile secretion and styrene degradation (p < 0.05), and there were different metabolites between the LP group and HP group, and these different metabolites were related to the dominant microbial to some extent. To sum up, in this experiment, the effects of crude protein level in supplementary diet on rumen microorganisms and metabolites of JY and their relationship were studied, which provided the theoretical basis for formulating a more scientific and reasonable supplementary diet in the future. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the effect of prickly ash seeds (PAS) on the microbial community found in rumen microbes of Hu sheep by adding different percentages of prickly ash seeds and to carry out research on the relation between rumen flora and production performance. Twenty-seven male lambs of Hu sheep were classified into three groups based on the content of prickly ash seeds (PAS) fed for 90 days, i.e., 0%, 3%, and 6%. At the end of the feeding trial, rumen fluid samples were collected from six sheep in each group for 16S amplicon sequencing. The results showed that the addition of prickly ash seeds significantly increased both Chao1 and ACE indices ( P < 0.05), and the differences between groups were greater than those within groups. The relative content of Bacteriodota decreased, and the relative content of Fusobacteriota, Proteobacteria, Acidobacteriota , and Euryarchaeota increased. The relative content of Papillibacter and Saccharofermentans was increased at the genus level, and the relative content of Bacteroides and Ruminococcus was decreased. The test group given 3% of prickly ash seeds was superior to the test group given 6% of prickly ash seeds. In addition, the addition of 3% of prickly ash seeds improved the metabolism or immunity of sheep. Fusobacteriota and Acidobacteriota were positively correlated with total weight, dressing percentage, and average daily gain (ADG) and negatively correlated with average daily feed intake (ADFI), feed-to-gain ratio (F/G), and lightness (L * ). Methanobrevibacter and Saccharofermentans were positively correlated with ADG and negatively correlated with ADFI and L * . In conclusion, under the present experimental conditions, the addition of prickly ash seeds increased the abundance and diversity of rumen microorganisms in Hu sheep and changed the relative abundance of some genera. However, the addition of 6% prickly ash seeds may negatively affect the digestive and immune functions in sheep rumen., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Li, Li, Ma, Wang, Wang, Wang, Liu, Li and Ma.)

Yak ( Bos grunniens ) is a unique large ruminant species in the Qinghai-Tibetan Plateau (QTP). Changing the energy levels of their rations can significantly improve their growth performance. Therefore, studying the effects of dietary energy levels on the rumen microflora and metabolites of yak is crucial for enhancing the development of the yak industry. Currently, there is a lack of understanding regarding the impact of feeding energy diets on rumen fermentation parameters, microbial functions, and metabolites. This study was designed to determine the appropriate energy level for feeding yak. Three test diets with metabolizable energy levels of 7.57 MJ/kg, 9.44 MJ/kg, and 11.9 MJ/kg were used and the concentration of volatile fatty acids (VFA) in rumen fluid was measured. The microbial communities, functions, and metabolites in yaks were studied by 16S rRNA sequencing, metagenome, and LC-MS non-targeted metabolomics to investigate the relationships among rumen fermentation parameters, microbial diversity, and metabolites. Ration energy levels significantly affect total VFA, acetate, propionate, butyrate, iso -valerate, valerate, and acetate/propionate ( p < 0.05). At the phylum level, the dominant phyla in all three treatment groups were Bacteroidota, Firmicutes, and Actinobacteriota. At the genus level, the abundance of the unclassified&#95;o&#95;&#95;Bacteroidales , norank&#95;f&#95;Muribaculaceae , Lachnospiraceae&#95;NK4A136&#95;group , and Family &#95;XIII&#95;AD3011&#95;group showed significant differences ( p < 0.05) and were significantly correlated with differential metabolites screened for phosphatidylcholine [PC(16:0/0:0), PC(18:3/0:0)], uridine 3'-monophosphate, and adenosine monophosphate, etc. CAZymes family analysis showed that GHs and CEs differed significantly among the three groups. In addition, differential metabolites were mainly enriched in the pathways of lipid metabolism, nucleotide metabolism, and biosynthesis of other secondary metabolites, and the concentrations of differential metabolites were correlated with microbial abundance. In summary, this study analyzed the effects of ration energy levels on rumen microorganisms and metabolites of yaks and their relationships. The results provided a scientific basis for the selection of dietary energy for yaks in the house feeding period in the future., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ma, La, Yang, Dai, Zhang, Zhang, Jin, Ma, Guo, Chu, Yan, Zhang and Liang.)

We present draft genome sequences of 12 Enterobacter hormaechei strains from feces isolated from cow rectums in the North West province, South Africa. The genome sizes ranged from 4.43 to 5.02Mb, with G + C contents of 55.5-56%, and contained 16-262 contigs. These data will contribute to our knowledge of the ecology and diversity of this species in South Africa., Competing Interests: The authors declare no conflict of interest.

Mulberry leaves have been used as a protein source in replacing concentrates of domestic animals, however, little is known about the relationship between supplementation level and the development of rumen epithelium. This experiment aimed to investigate the effects of different proportions of mulberry leaf powder (MLP) in dietary concentrate on rumen fermentation and rumen epithelium morphology in fattening Hu sheep. Forty three-month-old male Hu sheep with an initial body weight of 16.5 ± 0.6 kg (BW ± SD) were chosen and randomly divided into five treatments: 0% (control), 15% (T15), 30% (T30), 45% (T45) and 60% (T60) of MLP in concentrate, respectively. The results showed that the dry matter intake (DMI) and average daily gain (ADG) in treatments T15 and T30 have no significant difference with respect to the control treatment, but DMI and ADG in treatments T45 and T60 were lower than the control treatment (p < 0.05). The apparent digestibility of organic matter (OM) and neutral detergent fiber (NDF) increased linearly and quadraticly as MLP supplementation increased (p < 0.05). The concentration of ammonia (NH3-N) trended to decrease linearly with the increase of MLP supplementation (p < 0.1), whereas the microbial protein (MCP) concentration increased linearly as MLP supplementation increased (p < 0.05). In the results of rumen epithelium morphology, the width of stratum corneum was reduced, whereas the width of ruminal papillae increased (p < 0.05), and the width of stratum granulosum and stratum basale also increased as MLP increased. In summary, MLP supplementation could improve nutrient digestibility, the development of rumen papillae and stratum basale. However, high content MLP (45%−60%) supplementation decreased the growth and food intake performance of fattening Hu sheep. Therefore, 30% MLP is recommended to supplement in concentrate for fattening Hu sheep.

This study was conducted to evaluate the effects of Lactiplantibacillus plantarum BX62 with high-antioxidant activity on the dynamics of antioxidant capacity, rumen fermentation, methane (CH 4) emission, and ruminal bacterial diversity during incubation of alfalfa substrate by in vitro fermentation. The rumen inocula from sheep were incubated in batch cultures in three groups: (1) control group without inoculation, (2) L. plantarum MTD/1 (MTD) inoculated-group, and (3) L. plantarum BX62 (BX) inoculated-group. The incubations were maintained for 3, 6, 12, 24 and 36 h, with 3 replicates per group and incubation time point. Results showed that the groups × incubation time interactions (P < 0.05) were significant for gas production (GP), CH 4 production, and dry matter digestibility (DMD). The BX-inoculated group increased GP (P < 0.05) and DMD (P = 0.001) at 3 h and 6 h of fermentation versus control, whereas decreased the CH 4 production (P < 0.05) at 24 h and 36 h of fermentation. In addition, compared to control, two inoculation groups increased total volatile fatty acid (P = 0.028) and acetate (P = 0.034) concentrations at 3 h of fermentation. Total antioxidant capacity during fermentation, except after 6 h, was higher (P < 0.001) for both inoculated groups than control. Higher (P < 0.05) superoxide dismutase activity during fermentation was also found with both inoculated groups than control. BX- and MTD-inoculated groups notably decreased NAD+ content (P < 0.05) and NAD+ to NADH ratio (P < 0.05) versus control at 3 h and 6 h of fermentation. Bacteroidetes abundance increased (P < 0.05) while Firmicutes decreased (P < 0.05) for both inoculated groups compared to control. The relative abundances of fibrolytic bacteria such as Prevotella_1 and Lachnospiraceae , and of H 2 -incorporating bacteria, such as Succinivibrio , were higher (P < 0.05) for BX- and MTD-inoculated groups than control. In conclusion, the BX strain with high-antioxidant activity could be used as a candidate strain to improve the in vitro antioxidant status of ruminants and might have the potential to mitigate CH 4 emissions by competing for hydrogen utilization with methanogens, but it had little difference with the MTD strain. • Influences of antioxidant lactic acid bacteria on rumen fluid were first studied. • Lactiplantibacillus plantarum BX62 improved antioxidant capacity of rumen in vitro. • Inoculation of L. plantarum BX62 might mitigate methane emissions. • L. plantarum BX62 could improve in vitro ruminal fermentation characteristics. [ABSTRACT FROM AUTHOR]

Introduction: The crude protein level in the diet will affect the fermentation parameters, microflora, and metabolites in the rumen of ruminants. It is of great significance to study the effect of crude protein levels in supplementary diet on microbial community and metabolites for improving animal growth performance. At present, the effects of crude protein level in supplementary diet on rumen fermentation parameters, microbial community, and metabolites of Jersey-Yak (JY) are still unclear., Methods: The purpose of this experiment was to study the appropriate crude protein level in the diet of JY. The rumen fermentation indexes (volatile fatty acids and pH) were determined by supplementary diets with crude protein levels of 15.16 and 17.90%, respectively, and the microbial community and metabolites of JYs were analyzed by non-target metabonomics and metagenome sequencing technology, and the changes of rumen fermentation parameters, microbial flora, and metabolites in the three groups and their interactions were studied., Results and Discussion: The crude protein level in the supplementary diet had significant effects on pH, valeric acid, and the ratio of acetic acid to propionic acid ( p < 0.05). The protein level had no significant effect on the dominant microflora at the phylum level ( p > 0.05), and all three groups were Bacteroides and Firmicutes. The results of metabolite analysis showed that the crude protein level of supplementary diet significantly affected the metabolic pathways such as Bile secretion and styrene degradation ( p < 0.05), and there were different metabolites between the LP group and HP group, and these different metabolites were related to the dominant microbial to some extent. To sum up, in this experiment, the effects of crude protein level in supplementary diet on rumen microorganisms and metabolites of JY and their relationship were studied, which provided the theoretical basis for formulating a more scientific and reasonable supplementary diet in the future., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Dai, Ma, Dingkao, Huang, La, Li, Ma, Wu, Chu, Guo, Pei, Yan and Liang.)

Simple Summary: Rumen is the center of nutrient digestion, absorption and metabolism of ruminants. Ruminal microbes such as cellulolytic bacteria degrade the feed nutrients into smaller molecules, which are absorbed into the body mainly through rumen epithelium. Our study found that mulberry leaf powder supplementation improved the development of rumen epithelium, especially stratum basale, the most important layer of rumen epithelium for the energy metabolism. Our study suggests that supplementing ruminants with mulberry leaf powder may be one of the nutritional strategies that can improve the digestion and absorption performance of ruminants. Mulberry leaves have been used as a protein source in replacing concentrates of domestic animals, however, little is known about the relationship between supplementation level and the development of rumen epithelium. This experiment aimed to investigate the effects of different proportions of mulberry leaf powder (MLP) in dietary concentrate on rumen fermentation and rumen epithelium morphology in fattening Hu sheep. Forty three-month-old male Hu sheep with an initial body weight of 16.5 ± 0.6 kg (BW ± SD) were chosen and randomly divided into five treatments: 0% (control), 15% (T15), 30% (T30), 45% (T45) and 60% (T60) of MLP in concentrate, respectively. The results showed that the dry matter intake (DMI) and average daily gain (ADG) in treatments T15 and T30 have no significant difference with respect to the control treatment, but DMI and ADG in treatments T45 and T60 were lower than the control treatment (p < 0.05). The apparent digestibility of organic matter (OM) and neutral detergent fiber (NDF) increased linearly and quadraticly as MLP supplementation increased (p < 0.05). The concentration of ammonia (NH3-N) trended to decrease linearly with the increase of MLP supplementation (p < 0.1), whereas the microbial protein (MCP) concentration increased linearly as MLP supplementation increased (p < 0.05). In the results of rumen epithelium morphology, the width of stratum corneum was reduced, whereas the width of ruminal papillae increased (p < 0.05), and the width of stratum granulosum and stratum basale also increased as MLP increased. In summary, MLP supplementation could improve nutrient digestibility, the development of rumen papillae and stratum basale. However, high content MLP (45%–60%) supplementation decreased the growth and food intake performance of fattening Hu sheep. Therefore, 30% MLP is recommended to supplement in concentrate for fattening Hu sheep. [ABSTRACT FROM AUTHOR]