The antioxidant effects of butylated hydroxytoluene on cryopreserved goat sperm from a proteomic perspective.

At present, there are few reports about the proteomics changes provoked by butylated hydroxytoluene (BHT) supplementation on cryopreserved semen in mammals. Thus, we aimed to evaluate the effects of different concentrations of BHT on goat sperm and to investigate the proteomics changes of adding BHT to cryopreserved goat (Capra hircus) sperm. Firstly, semen samples were collected from four goats, and frozen in the basic extenders containing different concentrations of BHT (0.5 mM, 1.0 mM, 2.0 mM) and a control without BHT, respectively. After thawing, the protective effects of dose-dependent replenished BHT to the freezing medium on post-thaw sperm motility, integrities of plasma membrane and acrosome, reactive oxygen species levels were confirmed, with 0.5 mM BHT being the best (B group) as compared to the control (without BHT, C group). Afterwards, TMT-based quantitative proteomic technique was performed to profile proteome of the goat sperm between C group and B group. Parallel reaction monitoring was used to confirm reliability of the data. Overall, 2,476 proteins were identified and quantified via this approach. Comparing the C and B groups directly (C vs. B), there were 17 differentially abundant proteins (DAPs) po-tentially associated with sperm characteristics and functions were identified, wherein three were upregulated and 14 were downregulated, respectively. GO annotation analysis demonstrated the potential involvement of the identified DAPs in metabolic process, multi-organism process, reproduction, reproductive process, and cellular process. KEGG enrichment analysis further indicated their potential roles in renin-angiotensin system and glutathione metabolism pathways. Together, this novel study clearly shows that BHT can effectively improve quality parameters and fertility potential of post-thawed goat sperm at the optimal concentration, and its cryoprotection may be realized through regulation of sperm metabolism and antioxidative capability from the perspective of sperm proteomic modification.

Association of litter size with the ruminal microbiome structure and metabolomic profile in goats.

The Yunshang black goat is a renowned mutton specialist breed mainly originating from China that has excellent breeding ability with varying litter sizes. Litter size is an important factor in the economics of goat farming. However, ruminal microbiome structure might be directly or indirectly regulated by pregnancy-associated factors, including litter sizes. Therefore, the current experiment aimed to evaluate the association of different litter sizes (low versus high) with ruminal microbiome structure by 16S rRNA gene sequencing and metabolomic profiling of Yunshang black does. A total of twenty does of the Yunshang Black breed, approximately aged between 3 and 4 years, were grouped (n = 10 goats/group) into low (D-l) and high (D-h) litter groups according to their litter size (the lower group has ≤ 2 kids/litter and the high group has ≧ 3 kids/litter, respectively). All goats were sacrificed, and collected ruminal fluid samples were subjected to 16S rRNA sequencing and LC-MS/MC Analysis for ruminal microbiome and metabolomic profiling respectively. According to PCoA analysis, the ruminal microbiota was not significantly changed by the litter sizes among the groups. The Firmicutes and Bacteroidetes were the most dominant phyla, with an abundance of 55.34% and 39.62%, respectively. However, Ruminococcaceae_UCG-009, Sediminispirochaeta, and Paraprevotella were significantly increased in the D-h group, whereas Ruminococcaceae_UCG-010 and Howardella were found to be significantly decreased in the D-l group. The metabolic profiling analysis revealed that litter size impacts metabolites as 29 and 50 metabolites in positive and negative ionic modes respectively had significant differences in their regulation. From them, 16 and 24 metabolites of the D-h group were significantly down-regulated in the positive ionic mode, while 26 metabolites were up-regulated in the negative ionic mode for the same group. The most vibrant identified metabolites, including methyl linoleate, acetylursolic acid, O-desmethyl venlafaxine glucuronide, melanostatin, and arginyl-hydroxyproline, are involved in multiple biochemical processes relevant to rumen roles. The identified differential metabolites were significantly enriched in 12 different pathways including protein digestion and absorption, glycerophospholipid metabolism, regulation of lipolysis in adipocytes, and the mTOR signaling pathway. Spearman's correlation coefficient analysis indicated that metabolites and microbial communities were tightly correlated and had significant differences between the D-l and D-h groups. Based on the results, the present study provides novel insights into the regulation mechanisms of the rumen microbiota and metabolomic profiles leading to different fertility in goats, which can give breeders some enlightenments to further improve the fertility of Yunshang Black goats.

Calcium Requirement of Yunnan Semi-fine Wool Rams (Ovis aries) Based on Growth Performance, Calcium Utilization, and Selected Serum Biochemical Indexes.

Calcium (Ca) is required for the growth and development of sheep, but the requirement of Yunnan semi-fine wool (YSW) rams remains uncovered. The current study aims to estimate the Ca requirement of growing YSW rams based on their growth performance, Ca utilization, and serum biochemical indexes. Forty-five YSW rams (10-month-olds) were randomly allocated to five dietary treatments with varying Ca levels of 0.50% (D1), 0.68% (D2), 0.73% (D3), 0.89% (D4), and 0.98% (D5). A higher value for average daily gain and a lower value for the feed conversion ratio were observed in the D3 group compared to the D5 group (p < 0.05). The dry matter intake amount changed quadratically with the increased Ca levels (p < 0.05). The levels of Ca intake, fecal Ca, and excreted Ca were significantly higher in the D5 group than those in the D1 group (p < 0.05). The apparent Ca digestibility rate and the Ca retention rate were significantly higher in the D4 group than in the D1 group (p < 0.05). The serum Ca concentration increased linearly with the incremental levels of dietary Ca (p < 0.05). The activity of alkaline phosphatase was significantly higher in the D1 group than in the D2 group (p < 0.05). The serum levels of hydroxyproline, osteocalcin, and calcitonin decreased from the D1 group to the D2 group, and then significantly ascended (p < 0.05) with the dietary Ca levels from the D3 group to the D5 group. The serum parathyroid hormone content was elevated from the D1 group to the D3 group and then decreased from the D4 group to the D5 group. After calculation, the daily net Ca requirement for the maintenance of YSW rams was 0.073 g/kg of BW0.75, and the daily total Ca requirement was 0.676 g/kg of BW0.75. To optimize the growth performance and the Ca utilization of YSW rams, the recommended dietary Ca level ranges from 0.73% to 0.89% based on this study.

An updated review on the application of proteomics to explore sperm cryoinjury mechanisms in livestock animals.

This comprehensive review critically examines the application of proteomics in understanding sperm cryoinjury mechanisms in livestock animals, in the context of the widespread use of semen cryopreservation for genetic conservation. Despite its global adoption, cryopreservation often detrimentally affects sperm quality and fertility due to cryoinjuries. These injuries primarily arise from ice crystal formation, osmotic shifts, oxidative stress, and the reorganization of membrane proteins and lipids during freezing and thawing, leading to premature capacitation-like changes. Moreover, the cryopreservation process induces proteome remodeling in mammalian sperm. Although there have been technological advances in semen cryopreservation, the precise mechanisms of mammalian sperm cryoinjury remain elusive. This review offers an in-depth exploration of how recent advancements in proteomic technologies have enabled a detailed investigation into these molecular disruptions. It presents an analysis of protein-level alterations post-thaw and their impact on sperm viability and functionality. Additionally, it discusses the role of proteomics in refining cryopreservation techniques to mitigate cryoinjury and enhance reproductive outcomes in livestock. This work synthesizes current knowledge, highlights gaps, and suggests directions for future research in animal reproductive science and biotechnology.

Transcriptome analysis of porcine embryos derived from oocytes vitrified at the germinal vesicle stage.

Vitrification of porcine immature oocytes at the germinal vesicle (GV) stage reduces subsequent embryo yield and changes at the molecular level may occur during embryonic development. Therefore, the present study used porcine parthenogenetic embryos as a model to investigate the effect of GV oocyte vitrification on the transcriptional profiles of the resultant embryos at the 4-cell and blastocyst stages using the Smart-seq2 RNA-seq technique. We identified 743 (420 up-regulated and 323 down-regulated) and 994 (554 up-regulated and 440 down-regulated) differentially expressed genes (DEGs) from 4-cell embryos and blastocysts derived from vitrified GV oocytes, respectively. Functional enrichment analysis of DEGs in 4-cell embryos showed that vitrification of GV oocytes influenced regulatory mechanisms related to transcription regulation, apoptotic process, metabolism and key pathways such as the MAPK signaling pathway. Moreover, DEGs in blastocysts produced from vitrified GV oocytes were enriched in critical biological functions including cell adhesion, cell migration, AMPK signaling pathway, GnRH signaling pathway and so on. In addition, the transcriptomic analysis and quantitative real-time PCR results were consistent. In summary, the present study revealed that the vitrification of porcine GV oocytes could alter gene expression patterns during subsequent embryonic developmental stages, potentially affecting their developmental competence.

Dietary protein levels modulate the gut microbiome composition through fecal samples derived from lactating ewes.

In ruminants, the digestion and utilization of dietary proteins are closely linked to the bacterial populations that are present in the gastrointestinal tract. In the present study, 16S rDNA sequencing, together with a metagenomic strategy was used to characterize the fecal bacteria of ewes in the early lactation stage after feeding with three levels of dietary proteins 8.58%, 10.34%, and 13.93%, in three different groups (H_1), (H_m) and (H_h), respectively. A total of 376,278,516 clean data-points were obtained by metagenomic sequencing. Firmicutes and Bacteroidetes were the dominant phyla, regardless of the dietary protein levels. In the H_h group, the phyla Proteobacteria, Caldiserica, and Candidatus_Cryosericota were less abundant than those in the H_I group. In contrast, Lentisphaerae, Chlamydiae, and Planctomycetes were significantly more abundant in the H_h group. Some genera, such as Prevotella, Roseburia, and Firmicutes_unclassified, were less abundant in the H_h group than those in the H_I group. In contrast, Ruminococcus, Ruminococcaceae_noname, Anaerotruncus, Thermotalae, Lentisphaerae_noname, and Paraprevotella were enriched in the H_h group. The acquired microbial genes were mainly clustered into biological processes; molecular functions; cytosol; cellular components; cytoplasm; structural constituents of ribosomes; plasma membranes; translation; and catalytic activities. 205987 genes were significantly enriched in the H_h group. In contrast, 108129 genes were more abundant in the H_I group. Our findings reveal that dynamic changes in fecal bacteria and their genes are strongly influenced by the levels of dietary proteins. We discovered that differentially expressed genes mainly regulate metabolic activity and KEGG demonstrated the primary involvement of these genes in the metabolism of carbohydrates, amino acids, nucleotides, and vitamins. Additionally, genes responsible for metabolism were more abundant in the H_h group. Investigating fecal bacterial characteristics may help researchers develop a dietary formula for lactating ewes to optimize the growth and health of ewes and lambs.

Effects of graded levels of dietary protein supplementation on milk yield, body weight gain, blood biochemical parameters, and gut microbiota in lactating ewes.

Diet-associated characteristics such as dietary protein levels can modulate the composition and diversity of the gut microbiota, leading to effects on the productive performance and overall health of animals. The objective of this study was to see how changes in dietary protein levels affect milk yield, body weight gain, blood biochemical parameters, and gut microbiota in lactating ewes. In a completely randomized design, eighteen ewes were randomly assigned to three groups (n = 6 ewes/group), and each group was assigned to one of three dietary treatments with different protein contents. The ewes' groups were fed on 8.38% (S-I), 10.42% (S-m), and 13.93% (S-h) dietary protein levels on a dry basis. The body weight gain and milk yield were greater (p < 0.05) in ewes fed the S-h dietary treatment than in those fed the S-m and S-1 diets, respectively. However, milk protein contents were similar (p > 0.05) across the treatments. The blood glucose, total protein, cholesterol, triglycerides, high-density lipoprotein, low-density lipoprotein, lactate, creatinine, and C-reactive protein contents of lactating ewes were not influenced (p > 0.05) by different dietary protein levels. The alanine transaminase, aminotransferase, and lactate dehydrogenase activities were also not changed (p > 0.05) across the groups. However, blood urea nitrogen and albumin contents of lactating ewes were changed (p < 0.05) with increasing levels of dietary protein, and these metabolite concentrations were higher (p < 0.05) for S-h than the rest of the treatments. In the different treatment groups, Firmicutes and Bacteroidetes were found to be the most dominant phyla. However, the abundance of Lachnospiraceae species decreased as dietary protein levels increased. Within the Bacteroidetes phylum, Rikenellaceae were more abundant, followed by Prevotellaceae, in ewes fed the S-m diet compared to those fed the other diets. Based on the results, feeding at an optimal protein level improved milk yield and body weight gain through modifying the digestive tract's beneficial bacterial communities. The results of blood metabolites suggested that feeding higher-protein diets has no negative impact on health.

Genetic variation in the OPN gene affects milk composition in Chinese Holstein cows.

The aim of this study was to investigate the association between genotypes and haplotypes of OPN, and milk composition in dairy cows. A total of 317 Chinese Holstein cows were genotyped via DNA sequencing in this study. Three single nucleotide polymorphisms (SNPs), g.2916G > A, g.58675C > T and g.58899C > A, and eight haplotypes were identified. Of the eight possible haplotypes, four haplotypes i.e., Hap2 (ACC; 55.30%), Hap6 (GCC, 15.6%), Hap1 (ACA, 13.6%) and Hap4 (ATC, 5.70%), were considered to be major with a cumulative estimated frequency of >90%. Single markers (g.2916G > A and g.58899C > A) and Haplotype Hap6/4 were found to be associated with an increase in butter-fat percentage (p < 0.05). Taken together, our results provided evidence that polymorphisms in OPN are associated with milk composition, and could potentially be used for marker-assisted selection in Chinese Holstein cows.

The Proteomic Modification of Buck Ejaculated Sperm Induced by the Cryopreservation Process.

Using two-dimensional electrophoresis along with mass spectroscopy, we have investigated how the cryopreservation process affected the protein profile of goat ejaculated sperm. In this study, five bucks were used for semen collection. After removal of seminal plasma, the Tris-based extender containing glycerol and egg yolk was used to freeze semen. The results indicated that the post-thaw sperm quality showed a significant reduction compared with fresh sperm. The numbers of protein spots acquired in fresh and post-thaw sperm were 2926 ± 57 and 3061 ± 81, respectively. Twenty-two different abundant proteins (DAPs) were identified between fresh sperm and frozen-thawed sperm (≥3.0-folds, p < 0.05). The abundances of 19 proteins were significantly higher in the fresh sperm than the post-thaw sperm. The results of the gene ontology annotation showed the primary location of the DAPs on sperm cytoskeleton, protein complex, cytoplasm, and mitochondria. In addition, these proteins were mainly involved in ion binding, small molecular metabolic processes, structure molecule activity, guanosine triphosphatase activity, oxidoreductase activity, and protein complex assembly. The interaction networks among these DAPs demonstrated that they may play roles in oxidoreductase activity, structure, acrosomal function, and motility of sperm. Collectively, the proteome of goat sperm was altered during the cryopreservation process, demonstrating that protein modification induced by cryopreservation may be associated with the reduced quality of goat sperm after thawing.

Fertility results after exocervical insemination using goat semen cryopreserved with extenders based on egg yolk, skim milk, or soybean lecithin.

To evaluate the effects of four extenders on the post-thaw quality and fertility of goat semen, six Yunshang Black bucks' semen was collected, pooled, diluted with Andromed® (Andr®), Optidyl® (Opt®), P3644 Sigma l-phosphatidylcholine (l-α SL), and skim milk-based (Milk) extenders, and then cryopreserved. The sperm motilities, abnormalities, membrane and acrosome integrity, mitochondrial activity, apoptosis, and reactive oxygen species (ROS) production were evaluated after thawing. After exocervical insemination with the thawed semen, the pregnancy, lambing, and twinning rates were recorded and compared. The results showed that sperm motilities, membrane integrity, acrosome integrity, mitochondrial activity, and viable spermatozoa were significantly higher in the Andr® and Opt® groups than those in the l-α SL and Milk groups (p < .05). Furthermore, there was no difference between Andr® and Opt® (p > .05). The sperm abnormality was lower in semen frozen with the Andr® or Opt® extenders, as compared to the l-α SL or Milk extender (p < .05). Regarding, the viable cells with low ROS production, the optimal results were obtained in the semen frozen with Andr® and Opt® extenders. Following exocervical insemination, the pregnancy and lambing rates in the Milk group were significantly lower than those in the other groups (p < .05). No difference was found in the pregnancy and lambing rates between Andr®, Opt®, and l-α SL (p > .05). Furthermore, the twinning rates were similar between these four groups (p > .05). In conclusion, egg yolk or skim milk can be substituted by soybean lecithin during cryopreservation of goat semen.

Effects of Dietary Protein Levels on Sheep Gut Metabolite Profiles during the Lactating Stage.

Diet-associated characteristics such as dietary protein levels can modulate the gut's primary or secondary metabolites, leading to effects on the productive performance and overall health of animals. Whereas fecal metabolite changes are closely associated with gut metabolome, this study aimed to see changes in the rumen metabolite profile of lactating ewes fed different dietary protein levels. For this, eighteen lactating ewes (approximately 2 years old, averaging 38.52 ± 1.57 kg in their initial body weight) were divided into three groups (n = 6 ewes/group) by following the complete randomized design, and each group was assigned to one of three low-protein (D_I), medium-protein (D_m), and high-protein (D_h) diets containing 8.58%, 10.34%, and 13.93% crude protein contents on a dry basis, respectively. The fecal samples were subjected to untargeted metabolomics using ultra-performance liquid chromatography (UPLC). The metabolomes of the sheep fed to the high-protein-diet group were distinguished as per principal-component analysis from the medium- and low-protein diets. Fecal metabolite concentrations as well as their patterns were changed by feeding different dietary protein levels. The discriminating metabolites between groups of nursing sheep fed different protein levels were identified using partial least-squares discriminant analysis. The pathway enrichment revealed that dietary protein levels mainly influenced the metabolism-associated pathways (n = 63 and 39 in positive as well as negative ionic modes, respectively) followed by protein (n = 15 and 8 in positive as well as negative ionic modes, respectively) and amino-acid (n = 14 and 7 in positive as well as negative ionic modes, respectively) synthesis. Multivariate and univariate analyses showed comparative changes in the fecal concentrations of metabolites in both positive and negative ionic modes. Major changes were observed in protein metabolism, organic-acid biosynthesis, and fatty-acid oxidation. Pairwise analysis and PCA reveal a higher degree of aggregation within the D-h group than all other pairs. In both the PCA and PLS-DA plots, the comparative separation among the D_h/D_m, D_h/D_I, and D_m/D_I groups was superior in positive as well as negative ionic modes, which indicated that sheep fed higher protein levels had alterations in the levels of the metabolites. These metabolic findings provide insights into potentiated biomarker changes in the metabolism influenced by dietary protein levels. The target identification may further increase our knowledge of sheep gut metabolome, particularly regarding how dietary protein levels influence the molecular mechanisms of nutritional metabolism, growth performance, and milk synthesis of sheep.

Interaction of MyoD and MyoG with Myoz2 gene in bovine myoblast differentiation.

This study aims to explore the functional role of Myoz2 in myoblast differentiation, and elucidate the potential factors interact with Myoz2 in promoter transcriptional regulation. The temporal-spatial expression results showed that the bovine Myoz2 gene was highest expressed in longissimus dorsi, and in individual growth stages and myoblast differentiation stages. Knockdown of Myoz2 inhibited the differentiation of myoblast, and negative effect of MyoD, MyoG, MyH and MEF2A expression on mRNA levels. Subsequently, the promoter region of bovine Myoz2 gene with 1.7 Kb sequence was extracted, and then it was set as eight series of deleted fragments, which were ligated into pGL3-basic to detect core promoter regions of Myoz2 gene in myoblasts and myotubes. Transcription factors MyoD and MyoG were identified as important cis-acting elements in the core promoter region (-159/+1). Also, it was highly conserved in different species based on dual-luciferase analysis and multiple sequence alignment analysis, respectively. Furthermore, a chromatin immunoprecipitation (ChIP) analysis combined with site-directed mutation and siRNA interference and overexpression confirmed that the combination of MyoD and MyoG occurred in region -159/+1, and played an important role in the regulation of bovine Myoz2 gene. These findings explored the regulatory network mechanism of Myoz2 gene during the development of bovine skeletal muscle.

The establishment of goat semen protein profile using a tandem mass tag-based proteomics approach.

In this study, the complete proteome of goat ejaculated semen including spermatozoa and seminal plasma was established, applying a tandem mass tag (TMT) labeling together with liquid chromatography-tandem mass spectrometry (LC-MS/MS). In seminal plasma, 2299 proteins were identified and 2098 proteins were quantified. The GO analysis demonstrated that 32% proteins were involved in metabolic activities. 46% proteins are located at intracellular region, intracellular organelle, and membrane-bounded organelle. Regarding molecular function, 40% proteins are engaged on protein binding, hydrolase activity, and ion binding. The KEGG analysis indicated a primary involvement of the identified proteins in protein processing in endoplasmic reticulum, lysosome, and proteome. In spermatozoa, 2491 proteins were identified and quantified. 39% proteins are involved in metabolic activities. 48% proteins are located at intracellular region, intracellular organelle, and membrane-bounded organelle. 38% proteins are engaged on protein binding, hydrolase activity, and ion binding. The KEGG analysis demonstrated their roles derived from the identified proteins in proteasome, glycolysis, pyruvate metabolism, and citrate cycle. Additionally, 1312 proteins were simultaneously presented in spermatozoa and seminal plasma. The involvement of 42% proteins in metabolic activities were observed. 47% proteins are located at intracellular region, intracellular organelle, and membrane-bounded organelle. The common proteins are mainly engaged on protein processing in endoplasmic reticulum, proteome, glycolysis, lysosome, and citrate cycle. Collectively, this study established the protein database of goat semen. More studies should be used to elucidate functionality of these identified proteins.

Ultrastructural Modification of Ram Sperm Frozen with Cyclohexanediol and Trehalose.

In this study, the effects of trehalose and 1, 3-cyclohexanediol (1, 3-CHD) on the ultrastructure of frozen-thawed ram sperm were assessed and compared. In the control group, sperm were frozen without trehalose and 1, 3-CHD. In the trehalose group, 100 mM trehalose was used for sperm cryopreservation. In the cyclohexanediol group, the freezing extender contained 100 mM 1, 3-CHD. The transmission electron microscope (TEM) was used to observe the ultrastructural alterations of sperm. For verification of the TEM results, the plasma membrane and acrosome integrity of ram frozen sperm was assessed. Three fertility-proven rams were used in this study. Semen collection was repeated 6 times. The collected semen was pooled to preclude the individual difference each time. The sperm collected from a representative ram were used for ultrastructural observation. The TEM results indicated extensive and severe cryoinjuries on the main organelles of ram frozen sperm. Some alterations in plasma membrane, including detachment, rupture, dilation, or loss, appeared in post-thaw sperm. The bending shape and leakage of genetic materials were also observed in the nucleus. In addition, the outer acrosome membrane in some frozen sperm was broken or partly lost. Further, leakage of the inner contents of acrosomes also occurred. Sperm mitochondria was negatively influenced by cryopreservation. With 1, 3-CHD or trehalose, the percentage of sperm with normal acrosomes was 62% or 64%, and it was significantly higher than that of the control (41.51%, p < 0.05). However, different from trehalose, 1, 3-CHD cannot efficiently protect the post-thaw integrity of the plasma membrane (48.09% vs. 26.92%, p < 0.05). The TEM results were consistent with the quality assessment of frozen-thawed sperm. Collectively, trehalose and 1, 3-CHD can mitigate cryoinjuries on sperm ultrastructure. The cryoprotective effects of trehalose on sperm plasma membrane are superior to 1, 3-CHD. Sperm plasma membrane is the most sensitive to cryoinjuries, followed by acrosomes, mitochondria, and nuclei.

Identification and validation of ram sperm proteins associated with cryoinjuries caused by the cryopreservation process.

The change of sperm protein profile after the cryopreservation process may influence fertilization and early embryonic development. The purpose of the present study was to identify ram sperm proteomic modifications induced by the cryopreservation process using the isobaric tags for relative and absolute quantification labeling technology (iTRAQ) coupled with the parallel reaction monitoring (PRM) technology. Semen samples were collected from five Yunnan semi-fine wool rams using an electroejaculator. Sperm motility (CASA), plasma membrane (HOST test), and acrosome integrity (FITC-PSA) were evaluated after freeze-thawing. The total proteins of fresh and frozen-thawed sperm were extracted and purified, followed by identifying ram sperm proteomic modifications using the isobaric tags for relative and absolute quantification labeling technique (iTRAQ) coupled with the parallel reaction monitoring (PRM) technology. The results showed a significant reduction (P < 0.05) in all sperm parameters after thawing. 126 differentially abundant proteins (DAPs) were identified through comparison of the proteomes between fresh and frozen-thawed sperm. Among them, 90 proteins were down-regulated after the cryopreservation process. The remaining 36 proteins were up-regulated in frozen-thawed sperm. The results of functional annotation demonstrated the potential relationship of 10 DAPs with oxidoreductase activity. 18 and 15 DAPs may be involved in the stress and carbohydrate metabolic process, respectively. Furthermore, 8 DAPs may be functionally associated with reproduction. The Kyoto Encyclopedia of Genes and Genomes (KEGG) results demonstrated the primary enrichment of these identified DAPs in metabolic activities, disease, and oxidative phosphorylation. In order to confirm the reliability of the iTRAQ results, the changing trends of 10 proteins analyzed by PRM were similar to those of the corresponding proteins identified by iTRAQ. In conclusion, the cryopreservation process modifies the proteome of ram sperm, possibly leading to compromised fertility of post-thaw sperm. Additionally, the identified DAPs in this study may function as potential biomarkers for assessing the post-thaw quality of ram semen.

Sequencing Reveals Population Structure and Selection Signatures for Reproductive Traits in Yunnan Semi-Fine Wool Sheep (Ovis aries).

Yunnan semi-fine wool sheep are among the most important cultivated sheep breeds in China. However, their population structure, genetic characteristics and traits of interest are poorly studied. In this study, we systematically studied the population characteristics and selection signatures of 40 Yunnan semi-fine wool sheep using SNPs obtained from whole-genome resequencing data. A total of 1393 Gb of clean data were acquired. The mapping rate against the reference genome was 91.23% on average (86.01%-92.26%), and the average sequence depth was 9.51X. After filtering, 28,593,198 SNPs and 4,725,259 indels with high quality were obtained. The heterozygosity rate, inbreeding coefficient and effective population size of the sheep were calculated to preliminarily explore their genetic characteristics. The average heterozygosity rate was 0.264, the average inbreeding coefficient was 0.0099, and the effective population size estimated from the heterozygote excess (HE) was 242.9. Based on the Tajima's D and integrated haplotype score (iHS) approaches, 562 windows and 11,356 core SNPs showed selection signatures in the Yunnan semi-fine wool sheep population. After genome annotation and gene enrichment analysis, we found traces of early domestication in sensory organs, behavioural activity and the nervous system as well as adaptive changes in reproductive and wool traits under selection in this population. Some selected genes related to litter size, including FSHR, BMPR1B and OXT, were identified as being under selection. Specific missense mutations of the FSHR gene that differed from the reference genome were also identified in the population, and we found some SNP variations that may affect litter size. Our findings provide a theoretical basis for the conservation and utilization of Yunnan semi-fine wool sheep. Furthermore, our results reveal some changes common to sheep after domestication and provide a new opportunity to investigate the genetic variation influencing fecundity within a population evolving under artificial selection.

The Assessment of Various Factors Affecting the Postwarming Viability and Developmental Capability of Goat Metaphase II Oocytes Vitrified by Cryotop.

The effects of the equilibration time, the vitrification procedure, and the warming procedure on the quality of goat oocytes vitrified by Cryotop were assessed. In the first part of the study, oocytes were exposed to 10% dimethyl sulfoxide (DMSO) and 10% ethylene glycol (EG) for 1, 3, 5, or 10 minutes, respectively, followed by vitrification. In the second part, after equilibration in 7.5% DMSO +7.5% EG for 3 minutes, 10% DMSO +10% EG for 3 minutes, or 4% EG for 10 minutes, oocytes were equilibrated in 15% DMSO +15% EG, 20% DMSO +20% EG, or 35% EG for 30 seconds before vitrification. The vitrification procedures were designated as first vitrification procedure (VPI), second vitrification procedure (VPII), and third vitrification procedure (VPIII), respectively. In the third part, oocytes vitrified using VPIII were warmed by the three procedures (first warming procedure [TPI], second warming procedure [TPII], or third warming procedure [TPIII]) containing different concentrations of trehalose. The results showed that after equilibration for 1 or 3 minutes in 10% DMSO and 10% EG, the viability and developmental capability of vitrified oocytes were significantly superior to the groups after equilibration for over 5 minutes (p < 0.05). With the VPIII procedure, the frequencies with normal morphology, cleavage, and blastocyst formation of vitrified oocytes were 91.87% ± 4.14%, 76.51% ± 4.37%, and 39.84% ± 2.91%, respectively, demonstrating a significant increase compared to the VPI or VPII group (p < 0.05). The rates of vitrified oocytes with normal morphology and cleavage in the TPI group were higher than the TPII or TPIII group (p < 0.05). In conclusion, equilibration in 10% DMSO and 10% EG for <3 minutes benefits the viability of vitrified oocytes. EG may be more efficient for vitrification of goat oocytes compared to DMSO. Higher concentrations (more than 1 M) of trehalose enhance cryosurvival of goat oocytes when warming.

Genetic Diversity Relationship in Azakheli Buffalo Inferred from mtDNA and MC1R Sequences Comparison.

Azakheli is relatively smaller riverine breed with a very peculiar characteristics kept under unique traditional husbandry practices in comparison with rest of the Pakistani buffalo breeds; however, milk production is comparable. The present study was conducted to evaluate the genetic diversity of Azakheli breed. A total of sixty-six blood samples were collected for the amplification of mtDNA D-loop region and MC1R gene sequencing analysis. Median-joining network analysis of 191 mtDNA D-loop sequences of Azakheli and eight Indian riverine buffalo breeds clustered into three clades. Ancient Azakheli Region 1 clade was the oldest with the highest mutation steps and was present close to the root of UPGMA phylogenetic tree. There was 5 mutated lines distance between Pakistan buffalo and Indian riverine buffaloes. The populations of neighboring countries did not share any haplotypes with Azakheli buffalo of Pakistan. Possibly, residing for so long in the cold atmosphere and high elevation regions caused the mutation in mtDNA D-loop, though these conditions did not affect the overall performance of Azakheli as milch buffalo breed of Pakistan. MC1R analyses showed high mutations in Azakheli of Albino phenotype and all the black phenotype individuals of Azakheli buffalo share haplotypes with dominant Chinese and Indian black phenotypes buffaloes in MC1R median-joining network, indicating the reason of black coat color is due to MC1R gene. The haplotype diversity and nucleotide diversity was (H. 0.923, Pi: 0.00895) in Azakheli. Current results illustrated Asian ancestry for Azakheli buffalo, and mtDNA and MC1R analyses provided further evidence. Additional genetic analyses and archeological studies may provide further insight into the domestication period and history of Azakheli buffalo breed. The further studies are required on different coat colors with different genes on Azakheli buffalo to understand the phenotype variation.

CEBPß binding directly to the promoter region drives CEBPɑ transcription and improves FABP4 transcriptional activity in adipose tissue of yak (Bos grunniens).

Fatty acid binding protein 4 (FABP4) was crucial to fatty acid uptake and intracellular transport. However, the mechanisms regulating yak (Bos grunniens) FABP4 transcription were not determined. In the current study, predominant expression levels of yak FABP4 were identified in subcutaneous fat and longissimus dorsi muscles by quantitative real-time polymerase chain reactions (qPCR). The CCAAT/enhancer binding protein alpha (CEBPα) and myocyte enhancer factor 2A (MEF2A), as transcriptional activator or repressor in the promoter region of FABP4, were confirmed by both site-directed mutagenesis experiment and chromatin immunoprecipitation assay. Additionally, molecular mechanisms of CEBPɑ regulation were analyzed to explore the transcriptional regulatory property of FABP4, which indicated that transcriptional activity of CEBPɑ depended on CCAAT/ enhancer binding protein beta (CEBPß) transcription factor. Our results demonstrated that CEBPß binding directly to the promoter region drove CEBPɑ transcription, improving yak FABP4 transcriptional activity in adipocytes. This mechanism expanded the information on the transcriptional regulatory network of adipogenesis.