Effects of resveratrol on DLD and NDUFB9 decrease in frozen semen of Mongolian sheep.

Mongolian sheep are a breed of sheep in China known for their excellent cold and drought resistance. Sperm from Mongolian sheep are often cryopreserved to improve breeding outcomes. However, cryopreservation of sperm often results in issues such as reduced vitality and altered morphology. Therefore, the objective of this study was to investigate the impact of the cryoprotectant resveratrol on frozen sperm from Mongolian sheep, specifically examining its effects on key proteins during cryopreservation. In this study, sperm samples were obtained from three adult Mongolian rams and processed through semen centrifugation. The sperm motility parameters of Fresh Sperm Group (FR), Resveratrol added before freezing group (FF-Res), Resveratrol-free frozen sperm group (FT), and Resveratrol added after freeze-thawing group (FA-Res) were determined. The tandem mass tags (TMT) peptide labeling combined with LC-MS/MS was used for proteomic analysis of the total proteins in FR and FT groups. A total of 2651 proteins were identified, among which 41 proteins were upregulated and 48 proteins were downregulated after freezing. In-depth bioinformatics analysis of differentially abundant proteins (DAPs) revealed their close association with the tricarboxylic acid cycle (TCA) and oxidative phosphorylation pathway. The energy-related protein dihydrolipoamide dehydrogenase (DLD) and the reactive oxygen species (ROS)-related protein NADH dehydrogenase 1 beta subcomplex subunit 9 (NDUFB9) exhibited significant decreases, indicating their potential role as key proteins contributing to reduced sperm vitality. The study demonstrated that the addition of resveratrol (RES) to semen could elevate the expression levels of DLD and NDUFB9 proteins. This study represents the pioneering proteomic analysis of Mongolian ram sperm before and after cryopreservation, establishing the significance of DLD and NDUFB9 as key proteins influencing the decline in vitality following cryopreservation of Mongolian ram sperm. These findings clarify that resveratrol can enhance the levels of DLD and NDUFB9 proteins in cryopreserved Mongolian ram sperm, consequently enhancing their vitality.

Role of white adipose tissue browning in cold seasonal acclimation in grazing Mongolian sheep (Ovisaries).

Mongolian sheep are characteristically cold-tolerant and thus can survive well and maintain genetic stability in the extremely cold environment of the Mongolian Plateau. However, the adaptive mechanism of Mongolian sheep during the cold season in the plateau environment remains unknown. Browning of white adipose tissues (WAT) can trigger nonshivering thermogenesis as a potential strategy to promote an animal's tolerance to cold environments. Thus, a comparative analysis of the genes and proteins of uncoupling protein 1 (UCP1)-dependent and UCP1-independent browning pathways, mitochondrial biogenesis, lipogenic and lipolytic processes of WAT from grazing Mongolian sheep in the cold and warm seasons was conducted. We found seasonal browning of both retroperitoneal WAT and perirenal WAT, and the signalling of the process was mainly transduced by the UCP1- dependent pathway, primarily reflected in the upregulated gene levels of UCP1 and peroxisome proliferative activated receptor gamma coactivator 1 alpha (PGC-1α). In addition, the mean adipocyte diameter and mRNA expression of lipogenic genes in both interscapular WAT and subcutaneous WAT were significantly elevated during the cold season. The findings of this study demonstrate that grazing Mongolian sheep could depend on seasonal browning of both retroperitoneal WAT and perirenal WAT together with the expansion of both interscapular WAT and subcutaneous WAT to acclimate to cold environments of the Mongolian Plateau.

Relationships between rumen microbes, short-chain fatty acids, and markers of white adipose tissue browning during the cold season in grazing Mongolian sheep (Ovisaries).

Mongolian sheep are characteristically cold-tolerant and they partially depend on seasonal browning of white adipose tissue (WAT) to acclimate to cold environments. The present work aimed to examine the rumen microbes, rumen fermentation profile, and relationships between the rumen microbiota, short-chain fatty acids (SCFAs), and markers of WAT browning and are thus conducive to exploring the plateau environment adaptability of Mongolian sheep in the cold season. A comparative analysis of the rumen microbes and SCFAs in the cold and warm seasons was conducted. Rumen microbes were analyzed using Illumina sequencing of the 16S rRNA gene. Ruminal SCFAs were determined by gas chromatography. Spearman's correlation test was used to determine the relationships between the rumen microbiota, SCFAs, and markers of WAT browning. Microbial 16S rRNA sequencing revealed a marked shift in rumen microbiota composition between the two seasons, and the bacteria were characterized by increased levels of the Actinobacteria and genera Christensenellaceae R-7 group, Ruminococcaceae UCG-011, Rikenellaceae RC9 gut group, Papillibacter, and Butyrivibrio 2 and reduced levels of Prevotella 1 and Ruminococcaceae UCG-014 in the cold season (P<0.05). Furthermore, the concentrations of SCFAs such as acetate and butyrate were significantly increased in the cold season (P<0.001 and P<0.05, respectively). Correlation analysis demonstrated that the relative abundances of the Actinobacteria and the genera Christensenellaceae R-7 group, Butyrivibrio 2, Ruminococcaceae UCG-002, and Ruminococcaceae UCG-011, identified as members of the Christensenellaceae, Lachnospiraceae, and Ruminococcaceae families (all within Firmicutes), were positively correlated with markers of browning in either retroperitoneal WAT or perirenal WAT, and acetate was positively correlated with Ruminococcaceae UCG-011 and Butyrivibrio 2 and markers of browning in either retroperitoneal WAT or perirenal WAT. Overall, there are distinct relationships between the rumen microbiota, ruminal SCFAs and markers of WAT browning during the cold season in grazing Mongolian sheep.

GLIS1, a potential candidate gene affect fat deposition in sheep tail.

Fat deposition in sheep tails is as a result of a complicated mechanism. Mongolian sheep (MG) and Small Tail Han sheep (STH) are two fat-tailed Chinese indigenous sheep breeds while DairyMeade and East Friesian (DS) are two thin-tailed dairy sheep breeds recently introduced to China. In this study, population genomics analysis was applied to identify candidate genes associated with sheep tails based on an in-depth whole-genome sequencing of MG, STH and DS. The selective signature analysis demonstrated that GLIS1, LOC101117953, PDGFD and T were in the significant divergent regions between DS and STH-MG. A nonsynonymous point mutation (g.27807636G>T) was found within GLIS1 in STH-MG and resulted in a Pro to Thr substitution. As a pro-adipogenic factor, GLIS1 may play critical roles in the mesodermal cell differentiation during fetal development affecting fat deposition in sheep tails. This study gives a new insight into the genetic basis of species-specific traits of sheep tails.

Contribution of the mutation T865G in TPH1 gene to the genetic potentiality of housed Mongolian sheep to year-round breeding.

Seasonal breeding is widespread in sheep and significantly affects the development of the housed sheep industry. To improve and balance the reproduction performance of sheep, year-round breeding has the goal of modern sheep farming. The tryptophan hydroxylase (TPH), which initiates and regulates biosynthesis of melatonin, is an important player in the formation of mammalian year-round breeding. However, little is known about its role in regulation of sheep seasonal breeding. In this study, a missense mutation, T865G in TPH1 gene was detected in 328 individuals of six Mongolian sheep groups. It was positively selected among Mongolian sheep. This mutation may appear between 13,683 and 350,973 years ago and only exist in Hu sheep now. In Hu sheep, the frequency of allele T was 89.66%, and that of allele G was 10.34%. The TPH1 protein structure and property analysis suggested that this mutation from T to G affect the three-dimensional structure and reduce the hydropathicity of catalytic core. When the allele is T, the protein activity is twice that of the allele G, and their difference was significant (p < .05). In conclusion, T865G is an ancient mutation of TPH1 gene and affects the function of TPH protein, which may contribute to the genetic potentiality of Mongolian sheep to year-round breeding.

Understanding molecular mechanisms of vertebral number of variations on Mongolian sheep using candidate genes analysis.

Objective: This study aimed to investigate the genetic link between variations in vertebral number and meat production traits, such as body weight and body measurements (body length, body height, heart girth, and shin width) in Mongolian (Bayantsagaan) sheep. Additionally, we examined the association of single-nucleotide polymorphisms (SNPs) in candidate genes, particularly Vertnin (VRTN), Nuclear receptor subfamily 6, group A, member 1 (NR6A1), and synapse differentiation-inducing 1-like (SYNDIG1L), with vertebral number variations and their potential impact on meat production traits. Materials and Methods: The study involved 220 Bayantsagaan sheep from Bayantsagaan soum, Tov province, Mongolia, including 104 sheep with extra vertebrae group and 116 individuals with typical vertebral number as the control group. Morphological data, including body weight and body measurements, were collected, and genetic samples were obtained. The impact of vertebral number on morphological traits was estimated using a general linear model. SNPs in the VRTN, NR6A1, and SYNDIG1L genes were sequenced, and their association with vertebral number was analyzed using one-way ANOVA. Results: Bayantsagaan sheep with extra vertebrae were, on average, 4.45 kg heavier and exhibited higher variability in body size traits compared to the control group. Four polymorphic sites were identified at the VRTN gene, with one polymorphic locus (VRTN1716) showing a significant association with vertebrae number and body size. Sheep with C/C genotype at VRTN1716 locus, had more vertebrae and larger body size compared to other genotypes. Conclusion: The findings suggest that variations in vertebral number and VRTN gene polymorphisms are linked to favorable meat production traits in Bayantsagaan sheep. The identified SNP (VRTN1716) associated with vertebral number and body size offers the potential for marker-assisted selection in breeding programs. These results provide valuable insights into the genetic basis of meat production traits in Bayantsagaan sheep and may contribute to the development of more efficient breeding strategies.

Phylogenetic analysis of small ruminant lentiviruses in Mongolian sheep supports an ancient east-west split for the genotype A.

The ovine maedi-visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV) are small ruminant lentiviruses (SRLVs) with striking genetic and structural similarities. The presence of SRLV in Mongolian sheep and goats was serologically demonstrated more than a decade ago; however, the viral genotype remains unknown. In total, 329 blood samples were collected from two sheep breeds (i.e., Khalkha and Sumber) in Tov, Govisumber, Arkhangay, Dornogovi, Zavkhan, and Sukhbaatar provinces, Mongolia. Serological and phylogenetic analyses were performed regardless of any apparent clinical signs, although most of the animals appeared healthy. All sheep in three of the six provinces were seronegative, whereas the seroprevalence in the Tov, Govisumber, and Zavkhan provinces averaged 7.9%. Genomic DNA from seropositive animals was tested using hemi-nested polymerase chain reaction, and sub-genomic SRLV sequences were determined from nine samples. Mongolian SRLV sequences clustered within the divergent subtype A22, which was previously found only in Fertile Crescent regions, including Lebanon, Jordan, and Iran, where the first sheep-domestication (Ovis aries) occurred. According to the phylogenetic analysis, genotype A has two ancestors from the ancient Fertile Crescent: (1) Turkish strains and (2) Iranian, Jordanian, and Lebanese strains. The first ancestor spread westward, whereas the second spread eastward, ultimately reaching Mongolia.

A Single-Cell Atlas of an Early Mongolian Sheep Embryo.

Cell types have been established during organogenesis based on early mouse embryos. However, our understanding of cell types and molecular mechanisms in the early embryo development of Mongolian sheep has been hampered. This study presents the first comprehensive single-cell transcriptomic characterization at E16 in Ujumqin sheep and Hulunbuir short-tailed sheep. Thirteen major cell types were identified at E16 in Ujumqin sheep, and eight major cell types were identified at E16 in Hulunbuir short-tailed sheep. Function enrichment analysis showed that several pathways were significantly enriched in the TGF-beta signaling pathway, the Hippo signaling pathway, the platelet activation pathway, the riboflavin metabolism pathway, the Wnt signaling pathway, regulation of the actin cytoskeleton, and the insulin signaling pathway in the notochord cluster. Glutathione metabolism, glyoxylate, and dicarboxylate metabolism, the citrate cycle, thyroid hormone synthesis, pyruvate metabolism, cysteine and methionine metabolism, thermogenesis, and the VEGF signaling pathway were significantly enriched in the spinal cord cluster. Steroid biosynthesis, riboflavin metabolism, the cell cycle, the Hippo signaling pathway, the Hedgehog signaling pathway, the FoxO signaling pathway, the JAK-STAT signaling pathway, and the Wnt signaling pathway were significantly enriched in the paraxial mesoderm cluster. The notochord cluster, spinal cord cluster, and paraxial mesoderm cluster were found to be highly associated with tail development. Pseudo-time analysis demonstrated that the mesenchyme can translate to the notochord in Ujumqin sheep. Molecular assays revealed that the Hippo signaling pathway was enriched in Ujumqin sheep. This comprehensive single-cell map revealed previously unrecognized signaling pathways that will further our understanding of the mechanism of short-tailed sheep formation.

Lipid transformation during postmortem chilled aging in Mongolian sheep using lipidomics.

This present study aimed to investigate the effects of postmortem chilled aging on lipid and molecular transformation mechanisms in Mongolian sheep. UPLC-ESI-MS/MS was used to identify 812 lipids in Mongolian sheep within four days of chilled aging. Using multivariate analysis, 95 significant differential lipids were identified. The levels of phosphatidylcholine, phosphatidylinositol, and certain free fatty acids were observed to rise significantly, while other phospholipids and acylcarnitines showed a downward trend. In addition, levels of most of the lysophospholipids increased significantly in the early postmortem period; however, their levels did not alter significantly after 48 h. The Kyoto encyclopedia of genes and genomes pathway analysis revealed that these differential lipids are primarily involved in glycerophospholipid metabolism, linoleic acid metabolism, arachidonic acid metabolism, and thermogenesis. Our results have important implications in terms of understanding lipid transformation and changes in meat quality during postmortem chilled aging in Mongolian sheep.

Effects of physical exercise on muscle metabolism and meat quality characteristics of Mongolian sheep.

The objective of this study was to investigate the effects of exercise training on muscle metabolism, fatty acid composition, carcass traits, and meat quality characteristics of Mongolian sheep. Fourteen Mongolian sheep were randomly divided into two groups (7 sheep in each) and placed in two adjacent livestock pens. One group of sheep was kept in the pen (Control [C] group) and the other group of sheep (Training [T] group) were driven away in a field to walk twice a day. The results showed a reduction in pH measured 45 min post mortem, L*, a*, and b* value, intramuscular fat, and carcass length, and an increase in the ultimate pH value and shear force in the meat of T group in comparison with that of C group (p < .050). Also, exercise training moderately affected the fatty acid composition of LT muscle. Compared with C group, the concentrations of myristoleic acid (C14:1) and stearic acid (C18:0) were increased (p < .050), while the concentrations of C20:3 n-6, neurolic acid (C24:1), and n-3 polyunsaturated fatty acid (PUFA) were decreased in T group (p < .050). Transcriptome analysis highlighted 621 genes differentially expressed in two groups, including 385 were up-regulated (e.g., GLUT4 and PGC-1α) and 236 were down-regulated (e.g., PLIN1 and ACSL3) in T with respect to C group. Besides, considering these genes, a number of enrichment pathways related to muscle metabolic processes, involving carbohydrate metabolism, lipid metabolism, oxidation reduction process, and muscle tissue development, were highlighted. In conclusion, these results contributed to a better understanding of the possible biological and molecular processes underlying the effects of exercise training on muscle metabolism and meat quality in Mongolian sheep, and provide useful information for contributing to understand the phenotypic and functional differences in meat quality of sheep.

Diet selection of sheep shifted from quality to quantity characteristics of forages as sward availability decreased.

Understanding the dynamic inter-relationship between grazing animals and the pasture sward is critical for sustainable grazing management. A field study was conducted to investigate the relationships between the quantity and quality characteristics of forages and diet selection of 30-kg Mongolian ewes in different seasons in Bromus inermis improved meadow steppe. Using a residual herbage mass method (1 000 kg/ha in spring, 800 kg/ha in summer and 600 kg/ha in autumn) to adjust stocking rate, three seasonal rest grazing strategies (spring rest, summer rest and autumn rest) combined with continuous grazing were studied. In each season, diet selection of sheep, quantified by diet composition and selectivity index, was estimated using a plant wax marker technique. Quantity (dry weight proportion, coverage, frequency, density and height) and quality (CP, NDF, ADF and DM digestibility) characteristics of forages consumed by sheep were determined simultaneously. Our results showed that in spring with the highest sward availability (herbage mass), diet selection of sheep could be predicted by the positive linear relationship between quality characteristics of forages and their selectivity index. In summer, the diet selection could be predicted by both positive linear relationship between quality characteristics and selectivity index, and the relationship between quantity characteristics and diet composition. While in autumn with the lowest sward availability, it could be predicted by the positive linear relationship between quantity characteristics and diet composition. During the whole grazing season, the diet composition of B. inermis (40.4%) and Leymus chinensis (35.1%) were larger than that of Potentilla bifurca (9.5%) and Carex (15.1%) and the selectivity index of P. bifurca was the highest (0.62), followed by Carex (0.17) and B. inermis (0.05), and L. chinensis (-0.29) was the lowest. The two parameters of diet selection above were positively related to their quantity and quality characteristics, respectively. These results suggested that the influence of quality characteristics on diet selection of sheep decreased gradually as a result of the decline in sward availability, while the influence of quantity characteristics increased. This study provides a comprehensive understanding of diet selection of sheep examining the trade-offs between quantity and quality characteristics of forages. The knowledge of diet selection of sheep and the corresponding prediction regressions acquired in this study could give the basis for designing appropriate grazing management strategies.

Physiological, Immune Response, Antioxidant Capacity and Lipid Metabolism Changes in Grazing Sheep during the Cold Season.

Mongolian sheep are characteristically cold-tolerant. However, their cold adaptive processes, such as the physiological feedback adjustments that occur during the cold season, remain unexplored. Therefore, the present study aimed to evaluate the physiological adaptations of Mongolian sheep in cold plateau environments. A comparative analysis of the serum biochemical parameters, immune response, antioxidant capacity, and glucose and lipid metabolism of grazing Mongolian sheep in the cold and warm seasons was conducted. The results showed that in the cold season, the glucose and lipid metabolism and thermogenesis of the grazing Mongolian sheep were notably enhanced. Moreover, the immune responses were stimulated by increased levels of cytokines, such as IL-2, IL-1ß, and IL-6, during the cold season. However, the antioxidant defense system was damaged; this damage was mainly characterized by decreased activity of antioxidant enzymes and an increased level of MDA during the cold season. Overall, glucose metabolism, lipid metabolism, thermogenesis, and immune responses were stimulated to meet the requirements of organismal metabolic regulation to enable grazing Mongolian sheep to physiologically adapt to cold climatic conditions.

Impact of feeding regimens on the composition of gut microbiota and metabolite profiles of plasma and feces from Mongolian sheep.

Mongolian sheep are an indigenous ruminant raised for wool and meat production in China. The gut microbial community plays an important role in animal performance and metabolism. The objective of this study was to investigate the effects of two feeding regimens on the diversity and composition of gut microbiota and metabolite profiles of feces and plasma from Mongolian sheep. A total of 20 Mongolian sheep were assigned to one of two feeding regimens: free grazing (FG) and barn confinement (BC). When samples were collected, the average live weights of the sheep were 31.28 ± 1.56 kg and 34.18 ± 1.87 kg for the FG and BC groups, respectively. At the genus level, the FG group showed higher levels of Bacteroides, RC9_gut_group, Alistipes, Phocaeicola, Barnesiella, and Oscillibacter, and lower levels of Succinivibrio, Treponema, and Prevotella, compared to the BC group. The butyric acid content in feces was lower in the FG group (P > 0.05). Higher levels of palmitic acid, oleic acid, alpha-linolenic acid, L-carnitine, L-citrulline, and L-histidine, and lower levels of L-tyrosine, L-phenylalanine, and L-kynurenine were found in the plasma of the FG sheep. Moreover, there were substantial associations between several gut microbiota genera and alterations in feces and plasma metabolites especially those involved in the metabolism of butyric acid, linolenic acid, and L-tyrosine. Feeding regimens can not only influence the composition of gut microbiota, but also alter metabolic homeaostasis in sheep.

Isolation, culture, and induced multiple differentiation of Mongolian sheep adipose-derived mesenchymal stem cells.

Adipose-derived mesenchymal stem cells (ADSC) are adult pluripotent cells and important resources for cell-based therapies of animals. There are presently different kinds of somatic cells used as donor cells for clone successfully. However, studies on somatic cell nuclear transplantation (SCNT) using ADSC as donor cells from Mongolian sheep have not been reported up to now. This study tested optimal methods of isolating, purifying, and proliferating Mongolian sheep ADSC, and determine their multiple differentiation potentiality. Adipose tissue was removed from approximately 2-year-old sheep and ADSC were harvested by pancreatic enzyme decomposition and adherent culture method. The growth curves of the Passages 1, 5, and 10 cultures were plotted and the exponential growth was determined as a population doubling time of 34.1 h. The expression of OCT4, SOX2, and NANOG genes were increased at Passage 3 (P3) as seen by reverse transcription polymerase chain reaction (RT-PCR) analysis. ADSC from Passage 3 were induced to undergo neurogenesis and form cardiomyocytes and pancreatic islet-like cells under inductive environments in vitro. The differentiation properties of cardiomyocytes and islet-like cells were confirmed by histological staining with toluidine blue, periodic acid-Schiff, and dithizone. The expression of specific genes in these cells were also detected by RT-PCR. Our study results confirm that isolated cells were indeed ADSC and may provide valuable materials for somatic cell clone and transgenic research.

Study on the correlation between BMPR1B protein in sheep blood and reproductive performance.

Sheep reproductive performance is one of the important economic traits in sheep farming. The bone morphogenetic protein receptor 1B (BMPR1B) gene and protein may play an important role in sheep fertility. This study was to investigate the association of blood BMPR1B protein expression with reproductive performance in sheep. Mongolian sheep with single and twin births and polytocous Small Tail Han sheep were selected due to differences in birth numbers. The BMPR1B mRNA in sheep blood was measured by a reverse transcription-polymerase chain reaction as well as the BMPR1B protein was measured by enzyme-linked immunosorbent assay in blood samples of Mongolian and Small Tail Han sheep. The results demonstrated that blood BMPR1B concentration in Mongolian sheep with twin birth was higher (P < 0.05) than Small Tail Han sheep and Mongolian sheep with single birth. The protein concentration in the anestrus season was higher (P < 0.045) than those in the estrus season for both Mongolian and Small Tail Han sheep. Moreover, BMPR1B concentration in Mongolian sheep increased (P < 0.05) at the age of 6 to 12 mo and that in Small Tail Han sheep increased (P < 0.05) at the age of 3 to 6 mo. The result indicates that the increase in BMPR1B protein concentrations in the blood of Mongolian ewes and Small Tail Han ewes may be beneficial to follicular development, but too high or too low of this blood protein concentration in Mongolian and Small Tail Han sheep is not conducive to ovulation.

The potential application of plant wax markers from alfalfa for estimating the total feed intake of sheep.

Estimating the feed intake of grazing herbivores is critical for determining their nutrition, overall productivity and utilization of grassland resources. A 17-day indoor feeding experiment was conducted to evaluate the potential use of Medicago sativa as a natural supplement for estimating the total feed intake of sheep. A total of 16 sheep were randomly assigned to four diets (four sheep per diet) containing a known amount of M. sativa together with up to seven forages common to typical steppes. The diets were: diet 1, M. sativa + Leymus chinensis + Puccinellia distans; diet 2, species in diet 1 + Phragmites australis; diet 3, species in diet 2 + Chenopodium album + Elymus sibiricus; and diet 4, species in diet 3 + Artemisia scoparia + Artemisia tanacetifolia. After faecal marker concentrations were corrected by individual sheep recovery, treatment mean recovery or overall recovery, the proportions of M. sativa and other dietary forages were estimated from a combination of alkanes and long-chain alcohols using a least-square procedure. Total intake was the ratio of the known intake of M. sativa to its estimated dietary proportion. Each dietary component intake was obtained using total intake and the corresponding dietary proportions. The estimated values were compared with actual values to assess the estimation accuracy. The results showed that M. sativa exhibited a distinguishable marker pattern in comparison to the other dietary forage species. The accuracy of the dietary composition estimates was significantly (P < 0.001) affected by both diet diversity and the faecal recovery method. The proportion of M. sativa and total intake across all diets could be accurately estimated using the individual sheep or the treatment mean recovery methods. The largest differences between the estimated and observed total intake were 2.6 g and 19.2 g, respectively, representing only 0.4% and 2.6% of the total intake. However, they were significantly (P < 0.05) biased for most diets when using the overall recovery method. Due to the difficulty in obtaining individual sheep recovery under field conditions, treatment mean recovery is recommended. This study suggests that M. sativa, a natural roughage instead of a labelled concentrate, can be utilized as a dietary supplement to accurately estimate the total feed intake of sheep indoors and further indicates that it has potential to be used in steppe grassland of northern China, where the marker patterns of M. sativa differ markedly from commonly occurring plant species.

Herbivore-Diet Analysis Based on Illumina MiSeq Sequencing: The Potential Use of an ITS2-Barcoding Approach to Establish Qualitative and Quantitative Predictions of Diet Composition of Mongolian Sheep.

DNA-barcoding approaches to estimate the diet compositions of grazing animals have received significant attention, and particularly when combined with next-generation sequencing, these techniques have substantially improved in recent years. In this study, the identity and species composition of plant material ingested by Mongolian sheep were estimated through the use of 350 bp ITS2 gene sequences of the vegetation found in fecal samples. Four diets were formulated using varying amounts of eight plant species that are common in the grasslands of northern China. Sixteen Mongolian sheep were taken from pastures and randomly assigned to four groups, and each group received one of four diets. Each sheep was randomly assigned to one of 16 confinement pens and fed its respective diet for 12 consecutive days. Fecal samples were removed from each pen from days 7-12, preserved, and composited for each pen. All herbage species included in the daily diets were detected in each fecal sample, with the exception of Phragmites australis. Moreover, 12 additional different plant species were retrieved from feces of the experimental sheep. The obtained data provided preliminary support for the use of the ITS2 barcode to determine which plants were consumed. Moreover, the proportions of the herbage DNA sequences recovered from sheep feces and those of the herbage masses in the daily diets did not completely match. These results indicate that the non-Gramineae DNA sequences amplified with ITS2 primers (including those of Chenopodium album, Artemisia scoparia, Artemisia tanacetifolia, and Medicago sativa) far exceeded those of the Gramineae species (including Leymus chinensis and Puccinellia distans), which constitute the largest share of the experimental diets. A significant positive correlation (Spearman's ρ = 0.376, P = 0.003) between the actual herbage mass proportions in the experimental diets and the herbage-DNA-sequence proportions provided sufficiently favorable support for the further investigation of DNA barcoding for the quantification of plants in feces. A significant regression coefficient was found between the relative DNA-sequence proportions of L. chinensis ( R2 = 0.82, P < 0.0001), P. distans ( R2 = 0.64, P = 0.0017), and C. album ( R2 = 0.98, P < 0.0001) and their respective herbage mass proportions. The quantitative relationship can be expressed by the linear-regression equations y = 0.90 x - 0.22, y = 0.98 x - 0.03, and y = 5.00 x - 0.25, respectively. Thus, these results demonstrate that dietary-DNA-barcoding methods exhibited potential in providing valuable quantitative information regarding food-item components. However, it should be noted that this explorative data needs to be further improved by using additional genes and by creating a sophisticated reference database, thus enhancing both quality and accuracy of the obtained results.

Microbial community compositions in the gastrointestinal tract of Chinese Mongolian sheep using Illumina MiSeq sequencing revealed high microbial diversity.

Chinese Mongolian sheep are an important ruminant raised for wool and meat production. However, little is known about the microbiota of the gastrointestinal tract (GIT) of Chinese Mongolian sheep. To increase our understanding of the microbial community composition in the GIT of Chinese Mongolian sheep, microbiota of five sheep is investigate for the first time using the Illumina MiSeq platform. High microbial diversity was obtained from the GIT, and the microbiota exhibited a higher biodiversity in the stomach and large intestine than in the small intestine. Firmicutes (44.62%), Bacteroidetes (38.49%), and Proteobacteria (4.11%) were the three most abundant phyla present in the GIT of the sheep. The present study also revealed the core genera of Prevotella, Bacteroides, Ruminococcus, Oscillospira, Treponema, and Desulfovibrio in the GIT. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States indicated that the metabolic pathway related to carbohydrate metabolism was the richest in the sheep GIT. In addition, a series of metabolic pathways related to plant secondary metabolism was most abundant in the stomach and large intestine than in the small intestine. Overall, the present study provides insight into the microbial community composition in GIT of the Chinese Mongolian sheep which is highly diverse and needs to be studied further to exploit the complex interactions with the host.