Rumen microbiota helps Tibetan sheep obtain energy more efficiently to survive in the extreme environment of the Qinghai-Tibet Plateau.

Introduction: T-sheep and H-sheep exhibit different environmental adaptability and production performance. The rumen microbiome has co-evolved with hosts and plays a vital role in nutrient digestion and energy metabolism. In our previous study, we found that T-sheep have a higher efficiency in energy metabolism than H-sheep, but the rumen microbial community remains unclear. Methods: In this study, we determined the rumen bacterial profile and rumen fermentation parameters to reveal the bacterial profiles and predictive functions among breeds and diets with four different energy levels, as well as the correlation between bacterial profiles and rumen fermentation characteristics. Results: The results showed that the rumen total volatile fatty acids (VFAs), acetate, butyrate, total branched-chain VFAs, iso-butyrate, and iso-valerate were higher in T-sheep than H-sheep. The alpha diversity of ruminal bacteria is not affected by dietary energy, but it shows a distinction between the sheep breeds. Specifically, T-sheep rumen bacteria exhibit higher alpha diversity than H-sheep. The beta diversity of ruminal bacteria is not influenced by dietary energy or sheep breeds, indicating similar communities of ruminal bacteria between different diets and sheep breeds. The phyla of Bacteroidetes and Firmicutes predominate in the rumen, with a higher relative abundance of Firmicutes observed in T-sheep than H-sheep. The two most abundant genera in the rumen were Prevotella 1 and Rikenellaceae RC9 gut group. Prevotella 1 is the predominant bacterial genus in the rumen of H-sheep, while the Rikenellaceae RC9 gut group dominates in the rumen of T-sheep. Microbial co-occurrence network analysis reveals that variations in rumen fermentation characteristics result from differences in module abundance, with a higher abundance of VFA-producing modules observed in the rumen of T-sheep. Microbial function prediction analysis showed that dietary energy rarely alters the functional composition of rumen bacteria. However, there were differences in the functions of rumen bacteria between sheep breeds, with T-sheep showing a greater emphasis on energy metabolism-related functions, while H-sheep showed a greater emphasis on protein metabolism-related functions. Discussion: These findings provide evidence of the special rumen microbial community that helps T-sheep efficiently obtain energy from low-protein and low-energy diets, enabling them to survive in the extreme environment of the Qinghai-Tibet Plateau.

The comparison of meat yield, quality, and flavor between small-tailed Han sheep and two crossbred sheep and the verification of related candidate genes.

Introduction: In Northeast China, Dorper and Australian White rams are commonly crossbred with small-tailed Han (STH) ewes to improve the offspring's meat yield and quality. However, the differences in traits and the flavor between the crossbred sheep and STH sheep remain unclear. In addition, the candidate genes potentially influencing the meat quality in the three sheep breeds require further verification. Methods: A total of 18 2-month-old healthy rams were raised over a period of 5 months, which included 6 STH, 6 Dorper and small-tailed Han crossbred (Do × STH), and 6 Australian white and small-tailed Han crossbred (Au × STH) offspring. The differences in slaughter, meat quality traits, fatty acid and amino acid composition in the muscular longissimus dorsi (MLD), and volatile compounds in the semitendinosus muscle were compared between the sheep breeds. The candidate genes related to intramuscular fat (IMF) content and fatty acids were validated. Results: The results of this study revealed that the crossbred sheep had higher body weight, carcass weight, bone weight, net meat weight, and IMF content than the STH sheep (p < 0.05). The Do × STH offspring had a higher pH value (24 h), moisture content, and cooking percentage; they also had redder and brighter meat color. The content of myristate, palmitic, and margaric acids in the crossbred sheep was higher than that in the STH sheep (p < 0.05). The Do × STH offspring had the highest saturated fatty acid content (p < 0.05). The Au × STH offspring had the highest protein content (p < 0.05). The arachidonic acid and amino acid (Asp, Ala, Ile, Leu, Lys, Thr, and essential amino acid) contents were higher in the STH sheep than in the crossbred sheep (p < 0.05). The odor activity value (OAV) analysis showed that most of the aldehydes in the Au × STH offspring had higher values. The PDK4 gene expression was positively associated with the IMF content and was negatively correlated with the linoleic acid content in the Do × STH sheep (p < 0.05). The TMEM273 gene expression was positively associated with linoleic and arachidonic acid contents and was negatively correlated with oleic and palmitic acid contents in the Do × STH sheep (p < 0.05). Discussion: The results showed the differences between the crossbred sheep and STH sheep and provided the candidate genes related to meat quality in sheep.

Regulation of the growth performance and the gastrointestinal microbiota community by the addition of defective pear fermentation to feed of small-tailed Han sheep.

This study investigated the effects of defective pear fermentation (DPF) diets on growth performance and gastrointestinal microbial communities in 60 healthy male small-tailed Han sheep, aged 90 days. The sheep were randomly divided into four groups, each consisting of three replicates with five sheep per replicate. Initially, all groups received a basal diet for seven days during the adaptation stage. Subsequently, for 60 days, group C (control) was fed a basal diet, group X received a basal diet with 2% DPF, group Y had a basal diet with 4% DPF, and group Z was fed a basal diet with 6% DPF. The results indicated that group Y experienced a significant increase in average daily gain (ADG) and average daily feed intake (ADFI). The addition of DPF significantly elevated the levels of GSH-Px and notably reduced MDA content compared to group C. Analysis of gastrointestinal microbiota showed that groups receiving DPF had increased relative abundances of Lachnospiraceae_NK3A20_group, norank_f p-2534-18B5_gut_group, Acetitomaculum, Actinobacteriota, Bacteroidota and Ruminococcus_gauvreauii_group, and decreased abundances of Proteobacteria, Prevotella, Staphylococcus, and Psychrobacter compared to group C. Group X exhibited the highest relative abundance of Olsenella, while group Y showed a significant increase in unclassified_f Lachnospiraceae compared to the other groups. Bacterial function prediction indicated that pathways related to energy metabolism were more prevalent in group X and Y. This study preliminarily confirms the feasibility of using DPF as feed additives, providing a foundation for further research and evaluation of DPF's application in animal production.

Temporary sensory separation of lamb groups from ewes affects behaviors and serum levels of stress-related indicators of small-tailed Han lambs.

Under the current meat sheep breeding system, newborn lambs usually live with their mothers until weaning, and in daily management, they often need to be separated from their ewes for a short period due to dehorning, disease treatment, etc. Such short-term separation was considered to be a high-intensity stress for the lambs. This study aimed to explore the effects of 1 h sensory separations on behaviors and the concentration of stress-related indicators of small-tailed Han lambs. Lambs were assigned to four groups: auditory, visual, and tactile separation (AVT) group; visual and tactile separation (VT) group; tactile separation (T) group; and control (C) group. Then they were separated from their mothers for one hour on postnatal days 14, 21, 28, 35 and 42. Results showed the separated lambs (AVT, VT, and T groups) spent less time lying down relaxing and more time looking around, exploring, vocalizing, and attempting to escape (P < 0.05). Lambs separated by lack of tactile contact only exhibited the most escaping and moving behavior. Twin-born lambs showed less moving, escaping, and vocalizing than single-born lambs (P < 0.05). The separation also led to a rise in serum globulin levels and a decrease in tetraiodothyronine. In conclusion, this study showed that temporary 1 h ewe-lamb separations could affect behaviors and the serum levels of stress indicators of lambs. The behavioral responses were more obvious when lambs were separated by lack of tactile contact only, and in single-born lambs. It can conclude that indicated that when lambs need to be temporarily separated from ewes in daily management production, it would be better to let them stay together with their littermates, and make them avoid hearing or seeing the ewes, such management may partially reduce the separation stress, thereby improving the welfare and breeding efficiency of sheep.

Regulatory Effects of FGF9 on Dermal Papilla Cell Proliferation in Small-Tailed Han Sheep.

Fibroblast growth factor 9 (FGF9) is crucial for the growth and development of hair follicles (HFs); however, its role in sheep wool growth is unknown. Here, we clarified the role of FGF9 in HF growth in the small-tailed Han sheep by quantifying FGF9 expression in skin tissue sections collected at different periods. Moreover, we evaluated the effects of FGF9 protein supplementation on hair shaft growth in vitro and FGF9 knockdown on cultured dermal papilla cells (DPCs). The relationship between FGF9 and the Wnt/ß-catenin signaling pathway was examined, and the underlying mechanisms of FGF9-mediated DPC proliferation were investigated. The results show that FGF9 expression varies throughout the HF cycle and participates in wool growth. The proliferation rate and cell cycle of FGF9-treated DPCs substantially increase compared to that of the control group, and the mRNA and protein expression of CTNNB1, a Wnt/ß-catenin signaling pathway marker gene, is considerably lower than that in the control group. The opposite occurs in FGF9-knockdown DPCs. Moreover, other signaling pathways are enriched in the FGF9-treated group. In conclusion, FGF9 accelerates the proliferation and cell cycle of DPCs and may regulate HF growth and development through the Wnt/ß-catenin signaling pathway.

Effects of alfalfa saponins on the production performance, serum biochemical factors, and immune factors in Small-Tailed Han sheep.

This study aimed to determine the potential effects of alfalfa saponins on the production performance, serum biochemical factors, and immune factors in sheep. Twenty Small-Tailed Han sheep were equally and randomly divided into Groups 1-4, fed with diets containing 0, 5, 10, and 20 g alfalfa saponins per kg, respectively, for 40 consecutive days. During the treatments, the body weight change was recorded for each sheep. Before, during, and after the treatments of alfalfa saponins, serum was collected from each group to compare the levels of biochemical and immune factors. All sheep were killed after the treatments, and the longissimus dorsi muscle was collected to compare the meat quality. The results validated the effects of alfalfa saponins on the growth performance and meat quality in Small-Tailed Han sheep, and the supplementation level of 10 g/kg was the best. Alfalfa saponins also had effects on the levels of biochemical factors in serum. However, both dose- and time-dependent effects were observed. After a shorter feeding period (14 days), the concentrations of cholesterol (CHOL) and low-density lipoprotein (LDL) in Groups 2, 3, and 4 were all lower than those in the control group; however, when alfalfa saponins were continuously fed, this effect was not apparent or even gone. Supplying alfalfa saponins increased serum concentrations of IgA, IgG, IgE, IgM, IL-1, IFN-α, and IFN-ß. And this effect was distinctly observed in Groups 3 and 4. Based on the current results, the alfalfa saponins concentration of 10 g/kg (for 14 consecutive days) could be suggested as the optimum ratio for good health conditions of Small-Tailed Han sheep.

Transcriptome Analysis of the Liver and Muscle Tissues of Dorper and Small-Tailed Han Sheep.

It is well known that Dorper (DP) is a full-bodied, fast-growing and high dressing percentage breed, while the production performance of Small-tailed Han sheep (STH) is not so excellent, in contrast to DP. Therefore, in this study, a comparative transcriptomic analysis of liver and muscle tissues from DP and STH breeds was carried out to find differentially expressed genes (DEGs) that affect their growth and meat quality traits. The results showed that the total number of DEGs was 2,188 in the two tissues. There were 950, 160 up-regulated and 1,007, 71 down-regulated genes in the liver and muscle, respectively. Several DEGs such as TGFB1, TGFB3, FABP3, LPL may be associated with growth and development in DP. Also, several GO terms were found to be associated with muscle growth and development, such as developmental growth (GO:0048589), and myofibril (GO:0030016). Further validation of eight genes (6 up-regulated, and 2 down-regulated) was performed using quantitative RT-PCR. These findings will provide valuable information for studying growth and development as well as meat quality traits in sheep.

Hypothalamic regulation of energy homoeostasis when consuming diets of different energy concentrations: comparison between Tibetan and Small-tailed Han sheep.

Seasonal energy intake of Tibetan sheep on the harsh Qinghai-Tibetan Plateau (QTP) fluctuates greatly and is often well below maintenance requirements. The aim of this study was to gain insight into how the hypothalamus regulates energy homoeostasis in Tibetan sheep. We compared Tibetan and Small-tailed Han sheep (n 24 of each breed), which were each allocated randomly into four groups and offered one of four diets that differed in digestible energy densities: 8·21, 9·33, 10·45 and 11·57 MJ/kg DM. Sheep were weighed every 2 weeks, and it was assumed that the change in body weight (BW) reflected the change in energy balance. The arcuate nucleus of the hypothalamus in Tibetan sheep had greater protein expressions of neuropeptide Y (NPY) and agouti-related peptide (AgRP) when in negative energy balance, but lesser protein expressions of proopiomelanocortin (POMC) and cocaine and amphetamine-regulated transcript (CART) when in positive energy balance than Small-tailed Han sheep. As a result, Tibetan sheep had a lesser BW loss when in negative energy balance and stored more energy and gained more BW when in positive energy balance than Small-tailed Han sheep with the same dietary intake. Moreover, in the hypothalamic adenosine monophosphate-activated protein kinase (AMPK) regulation pathway, Tibetan sheep had greater adenosine monophosphate-activated protein kinase-α 2 protein expression than Small-tailed Han sheep, which supported the premise of a better ability to regulate energy homoeostasis and better growth performance. These differences in the hypothalamic NPY/AgRP, POMC/CART and AMPK pathways between breeds conferred an advantage to the Tibetan over Small-tailed Han sheep to cope with low energy intake on the harsh QTP.

Small RNA deep sequencing reveals the expressions of microRNAs in ovine mammary gland development at peak-lactation and during the non-lactating period.

MicroRNAs (miRNAs) are small non-coding RNAs that are involved in mammary gland development and lactation in livestock. Little is known about the roles of miRNAs in ovine mammary gland development, hence in this study the expression profiles of miRNAs of the mammary gland tissues of ewes at peak-lactation and during the non-lactating period were investigated using RNA sequencing. A total of 147 mature miRNAs were expressed in the two periods. Compared with peak-lactation, eight miRNAs in the non-lactating ewe mammary gland were significantly up-regulated, whereas fifteen miRNAs were down-regulated. A KEGG analysis revealed that the target genes of the up-regulated miRNAs were significantly enriched in lysosome, Wnt and MAPK signaling pathways, while the target genes of down-regulated miRNAs were significantly enriched in the PI3K-Akt signaling pathway, protein processing in endoplasmic reticulum and axon guidance. These results suggest that further study of the differentially expressed miRNAs could provide a better understanding of the molecular mechanisms of mammary development and lactation in sheep.

Comparison of the Transcriptome of the Ovine Mammary Gland in Lactating and Non-lactating Small-Tailed Han Sheep.

Small-Tailed Han (STH) sheep are known for their high fecundity, but the survival of lambs is compromised and influences the commercial return from farming these sheep, with this being attributed in part to starvation from insufficient milk production by the ewes. In this study, the transcriptome profiles of the mammary gland of lactating and non-lactating STH ewes were investigated using paired-end RNA sequencing (RNA-Seq). An average of 14,447 genes were found to be expressed at peak-lactation in the STH sheep, while 15,146 genes were expressed in non-lactating ewes. A total of 4,003 differentially expressed genes (DEGs) were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that the DEGs were associated with a wide range of cellular components, biological processes and metabolic pathways, including binding activities, signaling pathways, cellular structures, and immune responses. The most highly expressed genes at peak-lactation included CSN2, LGB, LALBA, CSN1S1, CSN1S2, and CSN3, and the 10 most highly expressed genes accounted for 61.37% of the total Reads Per Kilobase of transcript, per Million mapped reads (RPKM). The most highly expressed genes in the mammary gland of non-lactating ewes included IgG, THYMB4X, EEF1A1, IgA, and APOE, and the 10 most highly expressed genes accounted for only 12.97% of the total gene RPKM values. This suggests that the sheep mammary gland undergoes a substantial development in milk protein synthesis infrastructure and promotion of protein transportation during lactation.

Comparison between Tibetan and Small-tailed Han sheep in adipocyte phenotype, lipid metabolism and energy homoeostasis regulation of adipose tissues when consuming diets of different energy levels.

This study aimed to gain insight into how adipose tissue of Tibetan sheep regulates energy homoeostasis to cope with low energy intake under the harsh environment of the Qinghai-Tibetan Plateau (QTP). We compared Tibetan and Small-tailed Han sheep (n 24 of each breed), all wethers and 1·5 years of age, which were each divided randomly into four groups and offered diets of different digestible energy (DE) densities: 8·21, 9·33, 10·45 and 11·57 MJ DE/kg DM. When the sheep lost body mass and were assumed to be in negative energy balance: (1) adipocyte diameter in subcutaneous adipose tissue was smaller and decreased to a greater extent in Tibetan than in Small-tailed Han sheep, but the opposite occurred in the visceral adipose tissue; (2) Tibetan sheep showed higher insulin receptor mRNA expression and lower concentrations of catabolic hormones than Small-tailed Han sheep and (3) Tibetan sheep had lower capacity for glucose and fatty acid uptake than Small-tailed Han sheep. Moreover, Tibetan sheep had lower AMPKα mRNA expression but higher mammalian target of rapamycin mRNA expression in the adipocytes than Small-tailed Han sheep. We concluded that Tibetan sheep had lower catabolism but higher anabolism in adipose tissue and reduced the capacity for glucose and fatty acid uptake to a greater extent than Small-tailed Han sheep to maintain energy homoeostasis when in negative energy balance. These responses provide Tibetan sheep with a high ability to cope with low energy intake and with the harsh environment of the QTP.

Exploring the roles of fecundity-related long non-coding RNAs and mRNAs in the adrenal glands of small-tailed Han Sheep.

BACKGROUND: Long non-coding RNAs (lncRNAs) can play important roles in uterine and ovarian functions. However, little researches have been done on the role of lncRNAs in the adrenal gland of sheep. Herein, RNA sequencing was used to compare and analyze gene expressions in adrenal tissues between follicular phases and luteal phases in FecBBB (MM) and FecB++ (WW) sheep, respectively, and differentially expressed lncRNAs and genes associated with reproduction were identified. RESULTS: In MM sheep, 38 lncRNAs and 545 mRNAs were differentially expressed in the adrenal gland between the luteal and follicular phases; In WW sheep, 513 differentially expressed lncRNAs and 2481 mRNAs were identified. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses indicated that differentially expressed lncRNAs and their target genes are mainly involved in the circadian rhythm, the mitogen activated protein kinase, thyroid, ovarian steroidogenesis and transforming growth factor beta signaling pathways. Differentially expressed lncRNAs can regulate reproduction by modulating genes involved in these signaling pathways and biological processes. Specifically, XLOC_254761, XLOC_357966, 105,614,839 and XLOC_212877 targeting CREB1, PER3, SMAD1 and TGFBR2, respectively, appear to play key regulatory roles. CONCLUSION: These results broaden our understanding of lncRNAs in adrenal gland of sheep and provide new insights into the molecular mechanisms underlying sheep reproduction.

Carcass parameters and meat quality of Tibetan sheep and Small-tailed Han sheep consuming diets of low-protein content and different energy yields.

Today, consumers are very health conscious and are more aware of the nutritional value of food, especially of meat, than they were in the past. The aim of this study was to evaluate the carcass parameters and meat quality of Tibetan sheep and Small-tailed Han sheep when consuming a diet of low-protein (~7%) and different energy yields (digestible energy, 8.21, 9.33, 10.45 and 11.57 MJ/kg) in the cold season. Twelve sheep of each breed were divided randomly into four treatments of different diets with three replicates per treatment per breed. Crude protein of the meat decreased linearly (p < .05), whereas energy increased linearly (p < .05) with an increase in energy level. Tibetan sheep tended to have a higher (p < .1) dressing percentage and rib eye area, while live body weight and hot carcass weight did not differ between breeds but increased linearly (p < .01) with an increase in energy level. Water holding capacity, as indicated by pressing loss and drip loss, did not differ between breeds and was not affected by dietary energy. The concentration of n-3 polyunsaturated fatty acids (PUFAs) was greater in Tibetan sheep meat but saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs) and n-6 PUFA did not differ between breeds. With an increase in energy content of the diet, SFA decreased (p < .05), whereas MUFA increased (p < .05). The n-6:n-3 PUFA ratio was lower (p < .001) in Tibetan sheep meat, while the atherogenic index did not differ between breeds, but tended to decrease (p < .1) with an increase in dietary energy content. The essential amino acid (EAA) content and ratio of EAA:NEAA (non-essential amino acid) were close to the world standards for healthy meat. In summary, (a) Tibetan sheep meat was preferable to Small-tailed Han sheep meat, although differences between breeds were small; and (b) some carcass parameters and meat quality were improved with an increase in dietary energy level when a low-protein diet was offered.

Tibetan sheep have a high capacity to absorb and to regulate metabolism of SCFA in the rumen epithelium to adapt to low energy intake.

The nutritional intake of Tibetan sheep on the harsh Qinghai-Tibetan Plateau is often under maintenance requirements, especially during the long, cold winter. However, they have adapted well and even thrive under these conditions. The aim of the present study was to gain insight into how the rumen epithelium of Tibetan sheep has adapted to the consumption of low-energy-level diets. For this purpose, we compared Tibetan and small-tailed Han sheep (n 24 of each breed, all wethers and 1·5 years of age), which were divided randomly into one of four groups and offered ad libitum diets of different digestible energy (DE) densities: 8·21, 9·33, 10·45 and 11·57 MJ DE/kg DM. The Tibetan sheep had higher rumen concentrations of total SCFA, acetate, butyrate and iso-acids but lower concentrations of propionate than small-tailed Han sheep. The Tibetan sheep had higher absorption capability of SCFA due to the greater absorption surface area and higher mRNA expression of the SCFA absorption relative genes than small-tailed Han sheep. For the metabolism of SCFA in the rumen epithelium, the small-tailed Han sheep showed higher utilisation of the ketogenesis pathway than Tibetan sheep; however, Tibetan sheep had greater regulation capacity in SCFA metabolism pathways. These differences between breeds allowed the Tibetan sheep to have greater capability of absorbing SCFA and better capacity to regulate the metabolism of SCFA, which would allow them to cope with low energy intake better than small-tailed Han sheep.

Comparative analysis on genome-wide DNA methylation in longissimus dorsi muscle between Small Tailed Han and Dorper×Small Tailed Han crossbred sheep.

OBJECTIVE: The objective of this study was to compare the DNA methylation profile in the longissimus dorsi muscle between Small Tailed Han and Dorper×Small Tailed Han crossbred sheep which were known to exhibit significant difference in meat-production. METHODS: Six samples (three in each group) were subjected to the methylated DNA immunoprecipitation sequencing (MeDIP-seq) and subsequent bioinformatics analyses to detect differentially methylated regions (DMRs) between the two groups. RESULTS: 23.08 Gb clean data from six samples were generated and 808 DMRs were identified in gene body or their neighboring up/downstream regions. Compared with Small Tailed Han sheep, we observed a tendency toward a global loss of DNA methylation in these DMRs in the crossbred group. Gene ontology enrichment analysis found several gene sets which were hypo-methylated in gene-body region, including nucleoside binding, motor activity, phospholipid binding and cell junction. Numerous genes were found to be differentially methylated between the two groups with several genes significantly differentially methylated, including transforming growth factor beta 3 (TGFB3), acyl-CoA synthetase long chain family member 1 (ACSL1), ryanodine receptor 1 (RYR1), acyl-CoA oxidase 2 (ACOX2), peroxisome proliferator activated receptor-gamma2 (PPARG2), netrin 1 (NTN1), ras and rab interactor 2 (RIN2), microtubule associated protein RP/EB family member 1 (MAPRE1), ADAM metallopeptidase with thrombospondin type 1 motif 2 (ADAMTS2), myomesin 1 (MYOM1), zinc finger, DHHC type containing 13 (ZDHHC13), and SH3 and PX domains 2B (SH3PXD2B). The real-time quantitative polymerase chain reaction validation showed that the 12 genes are differentially expressed between the two groups. CONCLUSION: In the current study, a tendency to a global loss of DNA methylation in these DMRs in the crossbred group was found. Twelve genes, TGFB3, ACSL1, RYR1, ACOX2, PPARG2, NTN1, RIN2, MAPRE1, ADAMTS2, MYOM1, ZDHHC13, and SH3PXD2B, were found to be differentially methylated between the two groups by gene ontology enrichment analysis. There are differences in the expression of 12 genes, of which ACSL1, RIN2, and ADAMTS2 have a negative correlation with methylation levels and the data suggest that DNA methylation levels in DMRs of the 3 genes may have an influence on the expression. These results will serve as a valuable resource for DNA methylation investigations on screening candidate genes which might be related to meat production in sheep.

Cryopreservation and multipotential characteristics evaluation of a novel type of mesenchymal stem cells derived from Small Tailed Han Sheep fetal lung tissue.

Lung mesenchymal stem cells (L-MSCs) characterized by plasticity, reduced relative immune privilege and high anti-fibrosis characteristics play the crucial role in lung tissue regenerative processes. However, up to date, the multi-differentiation potentials and application values of L-MSCs are still uncertain. In the current study, the Small Tailed Han Sheep embryo L-MSCs line from 12 samples, stocking 124 cryogenically-preserved vials, was successfully established by using primary culture and cell cryopreservation techniques. Isolated L-MSCs were morphologically consistent with fibroblasts, could be passaged for at least 18 passages and more than 91.8% of cells were diploid (2n = 54) analyze by G-banding. The majority of cells were in the G0/G1 phase (70.5-91.2%), and the growth curves were all typically sigmoidal. Moreover, L-MSCs were found to express pluripotent genes Oct4, Nanog and MSCs-associated genes ß-integrin, CD29, CD44, CD71, CD73 and CD90, while the expressions of hematopoietic cell markers CD34 and CD45 were negative. In addtion, the L-MSCs could be differentiated into cells of three layers with induction medium in vitro, which confirmed their multilineage differentiation potential. The secretion of urea and ALB showed the differentiated hepatocytes still possessed the detoxification function. These results indicated that the isolated L-MSCs displayed typical characteristics of mesenchymal stem cells and that the culture conditions were suitable for their maintenance of stemness and their proliferation in vitro.

Three slow skeletal muscle troponin genes in small-tailed Han sheep (Ovis aries): molecular cloning, characterization and expression analysis.

To explore the basic characteristics and expressing profile of the three slow skeletal muscle troponin genes TNNC1 (Troponin C type 1), TNNI1 (troponin I type 1) and TNNT1 (troponin T type 1). Three purebred Dorper sheep and another three purebred small-tailed Han sheep were selected. The sequence of the genes from the small-tailed Han sheep was cloned using rapid amplification of cDNA ends and reverse transcription-polymerase chain reaction; The characteristics of the predicted amino acids sequences were analyzed using bioinformatics analysis software; Gene expression analyses were performed using quantitative reverse transcription PCR. The full-length cDNA sequences of the genes were 707, 898, and 1001 bp, respectively, and were submitted to GenBank under accession numbers KR153938, KT218688 and KT218690. The three predicted proteins were predicted to be hydrophilic, non-secretory proteins and contain several phosphorylation sites. Multiple alignments and phylogenetic tree analyses showed that the predicted proteins were relatively conserved in mammals. The expression results of the three genes in eight tissues of Dorper and small-tailed Han sheep revealed that the three genes had a similar mRNA expression pattern, whereas distinct differences were observed among the eight tissues of the two sheep species. We cloned the full-length cDNA of the three genes, analyzed the amino acid sequences, and determined the expression levels of the genes. These results might play important roles in facilitating the future research of the three genes.

Polymorphisms of the Ovine BMPR-IB, BMP-15 and FSHR and Their Associations with Litter Size in Two Chinese Indigenous Sheep Breeds.

The Small Tailed Han sheep and Hu sheep are two prolific local sheep in China. In this study, the polymorphisms of BMPR-IB (Bone morphogenetic protein receptor IB), BMP-15 (Bone morphogenetic protein 15) and FSHR (follicle stimulating hormone receptor) were investigated to check whether they are associated with litter size in Small Tailed Han sheep and Hu sheep. Consequently, three polymorphisms, FecB mutation in BMPR-IB (c.746A>G), FecG mutation in BMP-15 (c.718C>T) and the mutation (g. 47C>T) in FSHR were found in the above two sheep breeds with a total number of 1630 individuals. The single marker association analysis showed that the three mutations were significantly associated with litter size. The ewes with genotype FecBB/FecBB and FecBB/FecB+ had 0.78 and 0.58 more lambs (p < 0.01) than those with genotype FecB+/FecB+, respectively. The heterozygous Han and Hu ewes with FecXG/FecX+ genotype showed 0.30 (p = 0.05) more lambs than those with the FecX+/FecX+ genotype. For FSHR gene, the ewes with genotype CC had 0.52 (p < 0.01) and 0.75 (p < 0.01) more lambs than those with genotypes TC and TT, respectively. Combined effect analyses indicated an extremely significant interaction (p < 0.01) between the random combinations of BMPR-IB, BMP-15 and FSHR genes on litter size. In addition, the Han and Hu ewes with BB/G+/CC genotype harbor the highest litter size among ewes analyzed in current study. In conclusion, BMPR-IB, BMP-15 and FSHR polymorphisms could be used as genetic markers in multi-gene pyramiding for improving litter size in sheep husbandry.