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Yak has strong adaptability to plateau hypoxia environment. However, the endothelin‐1 (ET‐1) and endothelial nitric oxide synthase (eNOS) are important regulators in blood oxygen transportation. Yak testes: newborn (3 days), young (1 years), adult (4 years) and old (9 years) were collected for microscopic analyses using haematoxylin and eosin staining (H&E), immunohistochemistry and immunofluorescence, as well as Western blot to compare the expression of ET‐1 and eNOS. Furthermore, the levels of ET‐1 mRNA and eNOS mRNA was detected by real‐time quantitative polymerase chain reaction (RT‐qPCR). The results showed that ET‐1 mRNA and eNOS mRNA in old yaks were higher than other developmental stages (p <.01). And the levels of ET‐1 and eNOS protein increased with age. Immunohistochemistry and immunofluorescence showed that ET‐1 and eNOS were mainly localized in gonocytes and spermatogenic membrane of newborn yaks. These two factors were expressed in both Leydig cells of young yaks and endothelial cells of adult yaks. In old yaks, ET‐1 was mainly expressed in Sertoli cells, while eNOS was obviously positive in capillaries and Leydig cells. Therefore, the positive results of ET‐1 and eNOS in gonocyte and spermatogenic basement were closely related to the development of testes. The expression of Leydig and Sertoli cells indicated that they played an important role in testes function. The expression in endothelial cells or interstitial capillaries, suggesting that they are involved in the regulation of microcirculation in yak testes. This study could provide clues for further revealing the regulation of yak testicular blood vessels in alpine cold and hypoxic environments. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the distribution and expression of matrix metalloproteinase‐2 (MMP‐2) and tissue inhibitor of matrix metalloproteinase‐2 (TIMP‐2) in yak testes. The testes of healthy yaks at different ages: newborn [3 days], young [1 year], adult [4 years], and old [9 years] were collected for microscopic analyses using hematoxylin and eosin staining, immunohistochemistry and immunofluorescence, as well as western blot to compare the expression of MMP‐2 and TIMP‐2. Furthermore, the levels of MMP‐2mRNA and TIMP‐2mRNA was detected by real‐time quantitative polymerase chain reaction (qPCR). The results of immunohistochemistry and immunofluorescence demonstrated that MMP‐2 and TIMP‐2 were mainly located in gonocytes of newborn, Sertoli cells of young, spermatozoa of adult and Leydig cells of old. The protein levels of MMP‐2 and TIMP‐2 exhibited a downward from newborn to adult, but increased again in old yaks. The analysis of qPCR showed that MMP‐2 was higher in young compared with newborn or adult(**p <.01), but a lower expression was detected in adult compared with old yak testicular tissues (*p <.05). Compared with adults, TIMP‐2 was significantly higher in newborn and young yaks (**p <.01), and slightly higher in old yaks (*p <.05). Hence, The location of MMP‐2 and TIMP‐2 in gonocytes were associated with the development of newborn yak testes. The expression of MMP‐2 and TIMP‐2 in Sertoli cells at young and adult yaks suggested that they provided a clue for the regulation of spermatogenesis. The positive labeling of MMP‐2 and TIMP‐2 in Leydig cells in old yaks suggested that both may be involved in the interstitial metabolism of the testes during this period. This study revealed the possible role of MMP‐2 and TIMP‐2 in testicular functionality of yaks at different ages. [ABSTRACT FROM AUTHOR]

Cattle‐yak, the hybrid offspring of yak (Bos grunniens) and cattle (Bos taurus), serves as a unique model to dissect the molecular mechanisms underlying reproductive isolation. While female cattle‐yaks are fertile, the males are completely sterile due to spermatogenic arrest at the meiosis stage and massive germ cell apoptosis. Interestingly, meiotic defects are partially rescued in the testes of backcrossed offspring. The genetic basis of meiotic defects in male cattle‐yak remains unclear. Structure‐specific endonuclease subunit (SLX4) participates in meiotic double‐strand break (DSB) formation in mice, and its deletion results in defects in spermatogenesis. In the present study, we examined the expression patterns of SLX4 in the testes of yak, cattle‐yak, and backcrossed offspring to investigate its potential roles in hybrid sterility. The results showed that the relative abundances of SLX4 mRNA and protein were significantly reduced in the testis of cattle‐yak. The results of immunohistochemistry revealed that SLX4 was predominately expressed in spermatogonia and spermatocytes. Chromosome spreading experiments showed that SLX4 was significantly decreased in the pachytene spermatocytes of cattle‐yak compared with yak and backcrossed offspring. These findings suggest that SLX4 expression was dysregulated in the testis of cattle‐yak, potentially resulting in the failure of crossover formation and collapses of meiosis in hybrid males. [ABSTRACT FROM AUTHOR]

Cumulus granulosa cells (CGCs) are a type of important ovarian somatic cells that carries out various functions related to oogenesis, follicular development and embryogenesis. The study on the development and function of CGCs facilitates the understanding of reproductive regulation in female animals. Sirtuin 7 (SIRT7) is a member of the sirtuin family of NAD+‐dependent deacetylases mediating numerous biological processes. In this study, we detected the localization of SIRT7 in yak ovaries as well as explored the function of SIRT7 in yak CGCs. The results revealed that the SIRT7 protein was mainly localized in the cytoplasm of oocytes, granulosa cells and theca cells. The knockdown of SIRT7 in yak CGCs repressed cell proliferation and impacted the expressions of several apoptosis‐related genes. Furthermore, oestrogen synthesis was also inhibited in SIRT7‐deficient yak CGCs. The expressions of several sterogenesis‐related genes decreased significantly following SIRT7 knockdown. In addition, the lack of SIRT7 in yak CGCs resulted in decreased levels of the TGFB/SMAD family members TGFB1, TGFBR1 SMAD2 and SMAD3. Moreover, the activation of the TGFB/SMAD pathway by adding TGFB/SMAD pathway activator SRI‐011381 partially rescued the level of oestrogen secreted by SIRT7‐deficient yak CGCs, as well as the expressions of steroidogenesis‐related genes NR5A1 and CYP19A1. This research is the first to focus on the role of SIRT7 in yak ovary, and the outcomes offer new insights into the mechanism governing yak reproduction. [ABSTRACT FROM AUTHOR]

The aims of this study were to analyse the protein phosphatase 1 regulatory subunit 11 (PPP1R11) expression and cellular localization in yak follicles and investigate its effects on cell proliferation, apoptosis and oestrogen secretion in granulosa cells (GCs). Ten healthy and non‐pregnant female yaks (4‐year‐old) were used as experimental animals. The mRNA relative expression level of PPP1R11 in GCs from small (<3.0 mm), medium (3.0–5.9 mm) and large (6.0–9.0 mm) follicles was detected by RT‐qPCR, and the cellular localization of PPP1R11 protein was detected by immunohistochemistry staining (IHC). After isolation, culture and identification of yak GCs in vitro, si‐PPP1R11 and si‐NC (negative control) were transfected into GCs. RT‐qPCR and immunofluorescence staining were used to evaluate the interference efficiency, and ELISA was performed to detect oestrogen concentration. Then, EdU staining and TUNEL staining were conducted to analyse cell proliferation and apoptosis. In addition, the oestrogen synthesis, proliferation‐ and apoptosis‐related genes were detected by RT‐qPCR after knockdown PPP1R11. The results showed that PPP1R11 is mainly located in ovarian GCs, and the expression levels of PPP1R11 in GCs from large follicles were significantly higher than that from medium and small follicles. Transfection of si‐PPP1R11 into GCs could significantly inhibit the expression of PPP1R11. Interestingly, the oestrogen secretion ability and the expression level of oestrogen pathway‐related genes (STAR, CYP11A1, CYP19A1 and HSD17B1) were also significantly downregulated. Moreover, the proportion of positive cells was decreased, and cellular proliferation‐related genes (PCNA, CCNB1 and CDC25A) were significantly downregulated after knockdown PPP1R11. However, the proportion of apoptotic cells was increased, and apoptosis‐related genes (BAX, CASP3 and P53) were significantly upregulated. Taken together, this study was the first revealed the expression and cellular localization of PPP1R11 in yak follicles. Interference PPP1R11 could reduce oestrogen secretion, inhibit proliferation and promote apoptosis in GCs, which provided a basis for further studies on the regulatory mechanism of PPP1R11 in follicle development. [ABSTRACT FROM AUTHOR]

We investigated the structural features of arterial blood vessels in yak uterine caruncle and the effects of the expression of vascular regulation‐related factors on angiogenesis in pregnant and non‐pregnant yak uterus. Three‐dimensional specimens of the uterine artery of non‐pregnant and pregnant yaks were produced to observe and measure the distribution characteristics and number of arterial vessels in the uterus and caruncle in the two periods. The uterine caruncle structure was observed and analysed by haematoxylin–eosin staining. The expression features of vascular endothelial growth factor (VEGF) and angiopoietin‐1 (Ang‐1) in the uterine caruncle were detected with immunohistochemistry, quantitative real‐time PCR (qRT‐PCR) and western blotting. The length and number of blood vessels in the caruncle were increased, the degree of curvature was decreased, and the folding was more complicated during pregnancy as compared with that during non‐pregnancy. The immunohistochemical results demonstrated that VEGF and Ang‐1 were mainly expressed strongly in the mucosal epithelial cytoplasm. The glandular lumen of the uterine gland, lymphocytes and the media and adventitia of blood vessels are widely distributed, and they are all positive. VEGF and Ang‐1 mRNA and protein levels were highest in pregnancy, followed by that in the luteal phase and in the follicular phase, and three stages were significantly different (p <.05). These findings provide an anatomical reference and theoretical basis for improving the diagnosis and treatment of yak reproductive disorders and other diseases in high‐altitude and low‐oxygen environments. [ABSTRACT FROM AUTHOR]

The balance between proliferation, differentiation and apoptosis is well‐coordinated in spermatogenesis for the timely production of appropriate numbers of sperm in animals. Disruption or decrease in sperm production is due to many conditions, including changes in testicular cell fate balance. Interspecies hybridization of domestic yaks and cattle results in sterility in males because of spermatogenic arrest; however, the underlying mechanisms involved in sterility are still unclear. In the present study, we investigated the proliferation and apoptosis status during the development of yaks and crossbred cattle‐yaks using immunohistochemistry of proliferating cell nuclear antigen and terminal deoxynucleotidyl transferase‐mediated dUTP nick end labelling assays. Testicular tissues from yaks (immature: 1 year old, mature: 2–3 years old) and backcrossed hybrids (2 year old) were collected and used to investigate the expression of each parameter in testicular cells. During the maturation of yak testes, proliferation and apoptosis became active only in spermatogenic cells, and not in other somatic cells, such as Sertoli cells, myoid cells and Leydig cells. Furthermore, hybrid cattle‐yak testes maintained proliferation ability but less apoptotic ability in spermatogenic cells when compared to yaks of the same age, suggesting that normal spermatogenic cell fate control is disrupted by changes in the balance between proliferation and apoptosis. In addition, Leydig cell proliferation rate was higher than apoptosis rate in the cattle‐yak testes, indicating an increased number of Leydig cells, which may affect spermatogenesis through changes in steroidogenesis. Although epigenetic changes may be involved in cattle‐yak testes, further studies are needed to clarify the modulation of proliferation and apoptosis to elucidate the mechanisms of infertility in hybrid cattle‐yak males. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) play vital roles in the development of oocytes and ovarian follicles. We have previously shown differential expression of miR‐342‐3p during yak oocyte maturation. In this study, we investigated the role of miR‐342‐3p in meiotic maturation of yak oocytes and the underlying mechanism. The profile of ovarian DNA methyltransferase 1 (DNMT1) expression was investigated in yak by RT‐qPCR and western blot analyses. The pattern of Dnmt1 expression in various meiotic stages (GV stage, MI stage and MII stage) of yak oocyte maturation was then measured by immunofluorescence staining. The interaction between Dnmt1 and miR‐342‐3p was verified by dual‐luciferase reporter assay. Finally, miR‐342‐3p inhibitors were microinjected into yak cumulus‐oocyte complex to evaluate the effects on oocyte maturation. MiR‐342‐3p expression was upregulated in oocytes during meiotic maturation, with significantly higher levels in the MII stage compared with the GV‐ and MI stages (p <.05), whereas the opposite pattern of Dnmt1 expression was detected. In the period to sexual maturity (3‐year‐old), DNMT1 showed an age‐related pattern of ovarian expression at both the gene and protein levels. Immunohistochemistry analysis also indicated maturation‐stage‐related differences in DNMT1 expression in the ovarian follicles and corpus luteum, with expression predominantly detected in cumulus cells and oocytes. MiR‐342‐3p inhibitors effectively upregulated Dnmt1 expression and significantly inhibited oocyte meiotic maturation. Taken together, our results indicate that miR‐342‐3p plays a vital role in the meiotic maturation of yak oocytes by targeting the 3'‐untranslated regions (UTR) of Dnmt1 and provide a new perspective on the mechanism of this process. [ABSTRACT FROM AUTHOR]

Our goal was to investigate heparin‐induced capacitation of frozen‐thawed yak sperm and to assess the effects of caffeine or ouabain supplementation with heparin on sperm capacitation. Sperm were incubated with varying heparin concentrations, namely 0, 12.5, 25, 50 and 100 μg/ml, for 0, 15, 30 and 60 min. In every treatment, sperm capacitation was assessed using microscopic examination of the sperm acrosomal status and western blot analysis of the levels of tyrosine phosphorylation (Tyr‐P). Based on our results, the optimal condition for frozen‐thawed yak sperm capacitation was a 30‐min exposure to 50‐μg/ml heparin. Next, we incubated frozen‐thawed yak sperm with 50‐μg/ml heparin, along with varying concentrations of caffeine supplementation, namely 0, 2.5, 5 and 10 mM for 30 min. Interestingly, caffeine significantly increased yak sperm acrosome reaction (AR) and Tyr‐P (p <.05). The optimal caffeine concentration was 5 mM, followed by 2.5 and 10 mM, with the lowest AR and Tyr‐P found in sperm cells that did not receive any caffeine. To examine the effects of ouabain on sperm capacitation, we next incubated frozen‐thawed yak sperm with 50‐μg/ml heparin, along with varying concentrations of ouabain, namely 0, 25, 50 and 100 µM for 30 min. We demonstrated that ouabain supplementation did not alter yak sperm AR or Tyr‐P in sperm cells, relative to the control (p >.05). In summary, our findings suggested that caffeine acts synergistically with heparin to increase yak sperm capacitation, but ouabain does not synergize with heparin to promote yak sperm capacitation. [ABSTRACT FROM AUTHOR]

The gas‐phase environment of in vitro culture system plays an important role in the development of oocytes, and oxygen concentration is one of the important factors. In the present study, we aimed to explore the effect of different oxygen concentrations (20%, 10%, 5% or 1% O2) in yak oocyte maturation and to detect the expression of hypoxia‐inducible factor 1α (HIF‐1α), vascular endothelial growth factor (VEGF) and cell apoptosis in yak COCs. First, the maturation rate of oocytes, cleavage rate and blastocysts rate following parthenogenetic activation in the group with 5% oxygen concentration were significantly higher (p <.05) than the other groups. Then, TUNEL analysis showed that the 5% oxygen concentration group significantly inhibited apoptosis of cumulus–oocyte complexes (COCs) compared to the other group, and the transcription and protein levels of pro‐apoptotic factor Bax, HIF‐1α and VEGF in yak COCs significantly reduced, while anti‐apoptotic factor Bcl‐2 significantly increased. Furthermore, immunohistochemical staining results indicated that HIF‐1α protein was mainly located in theca follicle interna, mural follicular stratum granulosum, corona radiata and ovarian stroma in the follicular ovarian tissue, while VEGF protein was mainly located in the granulosa and theca cell layers. In summary, our findings demonstrate that 5% oxygen concentration may promote maturation and inhibit apoptosis of oocytes through HIF‐1α‐mediated VEGF expression. [ABSTRACT FROM AUTHOR]

Although the interspecies hybridization of bovids, such as cattle–yak (Bos taurus × Bos grunniens), has heterosis benefits, the infertility of hybrid males affects the maintenance of dominant traits in subsequent generations. To achieve reproductive capacity, male germ cell development requires coordinated changes in gene expression, including DNA methylation and generalized histone modifications. Although gene expression‐related mechanisms underlying hybrid male sterility have been investigated recently, information on the cell types and stage‐specific controls remains limited. Here, we used immunohistochemistry and image analyses to evaluate the 5‐methylcytosine (5MC) and acetyl‐histone H3 Lys9 (AcK9) expression in all spermatogonia and testicular somatic cell types to determine their roles in cattle–yak spermatogenesis. Testicular tissues from yak (1–3 years old) and backcrossed hybrids (2 years old) were used. In yak, the AcK9 expression levels increased in all cell types during maturation, but the 5MC expression levels did not change until reaching 3 years when they increased in all testicular cell types, except spermatogonia. Cattle–yak hybrids showed higher 5MC expression levels and different AcK9 expression levels in all cell types compared to the same‐aged yak. These results suggested that both gene modulation by AcK9 and constant levels of DNA methylation are required for spermatogenesis during maturation in yak. Therefore, inappropriate expression levels of both AcK9 and DNA methylation might be the major factors for disruption of normal germ cell development in cattle–yak. Additionally, various modulations occurred depending on the cell type. Further experiments are needed to identify the stage‐specific gene expression modulations in each cell type in yak and cattle–yak to potentially solve the infertility issue in crossbreeding. [ABSTRACT FROM AUTHOR]

Post‐partum ovarian cycle arrest is the main factor affecting yak reproductive efficiency. There are few reports regarding the molecular regulatory mechanism of post‐partum oestrus at transcriptome and proteome levels in yaks. Our previous studies focussed on the ovaries of yaks with post‐partum ovarian cycle arrest and post‐partum oestrus yaks. In this study, RNA sequencing transcriptomic study was combined with quantitative proteomic analyses to identify post‐partum ovarian cycle‐related genes and proteins. Consequently, 1,149 genes and 24 proteins were found to be up‐ or downregulated during post‐partum oestrus. The analysis of differentially regulated genes identified three gene or protein pairs that were synchronously upregulated and no gene or protein pairs that were synchronously downregulated, suggesting that these upregulated genes may regulate the post‐partum ovarian cycle. The functional classification of these differentially expressed genes and proteins indicated their connection with the oocyte meiosis, the oestrogen signalling pathway, the progesterone‐mediated oocyte maturation and the gonadotrophin‐releasing hormone (GnRH) signalling pathway. In this study, a total of six genes and two proteins involved in the oocyte meiosis, the oestrogen signalling pathway, the progesterone‐mediated oocyte maturation and the GnRH signalling pathway were identified. The CSNK1A1, M91_09723, M91_11326, M91_21439, M91_19073, SHC2, Atf6b, M91_03062, HSPCA and calmodulin could regulate oestrus, respectively, in the post‐partum so as to control the anoestrus status. [ABSTRACT FROM AUTHOR]

Luteinizing hormone receptor (LHR), prolactin receptor (PRLR), growth hormone (GH) and insulin‐like growth factor 1 (IGF1) have been shown to be key regulators of germ cell development. However, the role of LHR, PRLR, GH and IGF1 in the development of yak testis remains unclear. In this study, we aimed to describe and compare gene expression and protein localization of LHR, PRLR, GH and IGF1 in the development of yak testes. Testes were collected from 6, 24, 36 and 72 months yak, and the kidney, liver, testicular, lung, skeletal muscle, heart and spleen tissues were collected from 36 months yak. The quantitative real‐time PCR (qRT‐PCR) results showed that the expression of these four genes was widely expressed in kidney, liver, testicular, lung, skeletal muscle, heart and spleen, while the LHR and PRLR were highly expressed in the kidney, skeletal muscle and testis, and higher levels of GH and IGF were expressed in spleen and testis. Moreover, the mRNA expression of these genes in adults was higher than in pre‐pubertal yak. In the testis, the LHR‐, PRLR‐, GH‐ and IGF1‐positive signals were detected in the Leydig cells of the 6 months, while the intense positive signals were discovered in Leydig cells, spermatogonia and spermatocytes of the 36 and 72 months. Thus, LHR, PRLR, GH and IGF1 may be involved in the development of spermatids and spermatocytes, and in the regulation of spermatogonia proliferation and Leydig cell function. [ABSTRACT FROM AUTHOR]

Cattleyaks (CY) are interspecific hybrids between cattle (Bos taurus) and yak (Bos gruniens, YK) exhibiting the same prominent adaptability and higher performances than YK. The main problem of this crossbreeding is that the males are sterile. Different series of events of spermatogenesis coordinate to regulate gene expressing, involving microRNAs (miRNAs). As non‐coding ribonucleic acids (ncRNAs), miRNAs predominantly facilitate the regulation of gene expression at post‐transcriptional stages and play important roles in the acquisition and maintenance of male fertility in reproduction. The function of miRNA in the male reproductive system extends from the testis into the epididymis, regulating gene expression and contributing to regional gene expression variations. RNA sequencing on biological replicates, we described differentially expressed miRNAs profiles for tissue from epididymis of YK and CY. In the present study, High‐throughput sequencing analysis showed that 55 differentially expressed (DE) miRNAs were identified in the epididymis of YK and CY. Among these, 43 DE miRNAs were upregulated while the remaining 12 DE miRNAs were downregulated between epididymis of YK and CY. In addition, we identified that the top most important DE miRNAs, bta‐miR‐449c, bta‐miR‐539, bta‐ miR‐136, bta‐miR‐504, bta‐miR‐31 and bta‐miR‐222 were involved in the process of sperm maturation in epididymis CY. It was identified that the bta‐miR‐449c and bta‐miR‐222 may play major roles in the process of sperm maturation, sperm quality, sperm count, sperm production and male infertility of CY. Furthermore, GO and KEGG analyses were performed to classify the functions of target genes for DE miRNAs. In addition, RT‐qPCR validation of the DE miRNAs and its targeted genes revealed that putative miRNAs are involved in the male CY infertility by altering the gene expression. Present findings may not only increase our understanding of the molecular mechanisms regulated by the miRNAs in epididymis, but also provide a valuable information to understand the male infertility mechanism of CY. [ABSTRACT FROM AUTHOR]

The yak is an important source for the people living and ecological environment in the Qinghai–Tibet Plateau. In every winter, many domestic yaks will lose bodyweight or dead under cold and food scarcity. Moving the plateau yaks to farm in the plain is a useful approach to reduce their environmental stress and gain more production. In this study, we measured growth, slaughter and beef quality traits every month and sequenced mRNA expression levels of muscles of two groups yaks living in plateau and plain respectively. We found there is significant difference (p‐value <0.01) in the third (60 days), fourth (90 days), fifth (120 days) and sixth (150 days) weights between subpopulations in the plateau and plain. We identified 540 different expressed genes (DEGs) including 123 known genes and 417 unknown genes. Using the weighted correlation network analysis (WGCNA) to build a co‐express network, the modules were strong relative to weight traits. The findings highlighted the underlying way and a relative network to yield a new view about gene expression between the yaks living plateau and plain. [ABSTRACT FROM AUTHOR]

We examined the changes in body weight (BW), back‐fat thickness (BFT) and blood metabolites in relation to postpartum (PP) ovarian activity status in twenty female yaks raised under semi‐intensive system. BFT and ovarian activities, like follicle development, ovulation (OV) and corpus luteum (CL) development, were monitored from 4 to 15 weeks (wk) PP using ultrasonography. Resumption of ovarian activity was confirmed with ovulation of dominant follicle (DF) and subsequent CL development, and >1 ng/ml progesterone concentration in blood plasma sample after 1week of ovulation. Yaks were further classified as cyclic (with CL), acyclic (without CL), and cystic (with >25 mm follicular cyst; FC). Within 20 weeks PP, 60% yaks resumed cyclic ovarian activity, while 25% failed to initiate cycling activity, and 15% developed follicular cysts. In all categories of yak, BW gradually decreased (p <.05) till nadir; however, nadir reached earlier (p <.05) in acyclic yaks. BFT differed (p <.05) among the yak groups, but it tended to be higher in cyclic yaks as compared to acyclic and cystic. No difference (p >.05) in non‐esterified fatty acids (NEFA) values was found among the different categories of yaks, whereas, beta‐hydroxy butyrate (BHB) levels were higher in cystic animals as compared to acyclic and cyclic. Blood glucose levels decreased in all yaks during initial 2 weeks after calving. Our findings suggest that yaks with low BW, BFT and glucose levels, and higher BHB values were at risk of delayed resumption of ovarian activity and concomitant development of follicular cysts. [ABSTRACT FROM AUTHOR]

The goal of this study was to characterize and evaluate the main markers of macrophages, T lymphocytes, B lymphocytes and plasmocytes in the testis of juvenile and adult yaks by quantitative real‐time polymerase chain reaction and immunohistochemistry. Within the same age group, the mRNA expression of CD68 was always highest, followed by that of CD3ε, CD79α, IgG and IgA. Moreover, CD68, CD3, CD79α, IgA and IgG positive cells were all located in the testicular interstitial tissues of juvenile and adult yaks. In the same age group, the frequency of CD68 positive macrophages was higher than that of CD3 positive T lymphocytes, which was followed by that of CD79α positive B lymphocytes and IgA and IgG positive plasmocytes. No significant difference was observed between the B lymphocyte and plasmocyte frequencies in yak testes. Furthermore, CD68, CD3ε, CD79α, IgA and IgG mRNA expression levels and the frequencies of CD68, CD3, CD79α, IgA and IgG positive cells increased from juveniles to adults. Similarly, the frequencies of CD68, CD3, CD79α, IgA and IgG positive cells also increased with age. These results suggest that in the yak testis, the immune defence system against pathogens might primarily comprise macrophages and T lymphocytes in the testicular interstitial tissue. Moreover, the testicular immune environment may mature and expand to a fully functional state in adult yaks. The low frequencies of B lymphocyte and plasmocyte in yaks, differing from those in rodents and humans, might be related to the fact that yaks live in low‐oxygen plateaus. [ABSTRACT FROM AUTHOR]

Known as the 'ship of the plateau', through thousands of years evolution and cruelty environments selection containing low oxygen and strong ultraviolet radiation, yaks have adapted plateau environments and supplied important goods and materials for the people in the Qinghai‐Tibet Plateau. This study aimed to identify differentially expressed (DE) genes and novel long non‐coding RNAs (lncRNAs) of yaks for the Plateau adaptation and their underlying co‐expression and regulatory network. We carried out RNA‐seq analysis for cerebral and cerebellar tissue specimens of Bos taurus, Bos grunniens × Bos Taurus and B. grunniens. Furthermore, 12,072 pseudo lncRNAs were predicted using three software. In total, 4,257 significant DE transcripts were identified using the Ballgown R package (p <.01), of which 1,021 were protein‐coding genes, 14 were known lncRNAs, and 661 were novel lncRNAs. Using WGCNA, a co‐expression network of DE mRNAs and lncRNAs comprising 5 modules was generated to determine functional associations clusters. This study reveals a valuable sub‐network comprising 8 hub genes, one known lncRNA and 5 novel lncRNAs in the major module. These hub genes are associated with blood pressure regulation, generation of reactive oxygen species and metabolism. The analysis of co‐expressed genes thus provides a basis for the regulatory mechanisms in PA in Yaks and for the detection of additional genes between cross‐breed and parent populations. [ABSTRACT FROM AUTHOR]

The yak (Bos grunniens) is the most important livestock animal in high‐altitude regions owing to its prominent adaptability to cold conditions, nutritional deficiencies and hypoxia. The reproductive organs exhibit different histological appearances and physiological processes at different reproductive stages. Hypoxia‐inducible factor‐1 alpha (HIF‐1α) is the regulatory subunit of HIF‐1 that crucially regulates the response to hypoxia in mammalian organisms. The goal of our study was to investigate the expression and distribution of HIF‐1α in the primary yak reproductive organs at different reproductive stages. Samples of the ovary, oviduct and uterus of 15 adult female yaks were collected and used in the experiment. The expression and localization of HIF‐1α proteins and mRNA were investigated using quantitative real‐time polymerase chain reaction (qRT‐PCR), Western blot (WB) and immunohistochemistry (IHC). The results indicated that the expression of HIF‐1α protein in the ovary was higher during the luteal phase than during the follicular phase and gestation period (p <.05). In the oviduct, HIF‐1α protein was also more highly expressed during the luteal phase than during the follicular phase and gestation period (p <.01). However, in the uterus, the HIF‐1α protein had stronger expression during the gestation period than during the follicular phase (p <.01) and luteal phase (p <.05). The expression of HIF‐1α mRNA was similar to that of its protein. Immunohistochemical analysis revealed intense immunostaining of HIF‐1α proteins in the follicular granulosa cells, granular luteal cells, villous epithelial cells of the oviduct, endometrial glandular epithelium and luminal epithelium, foetal villous trophoblast, and epithelia of caruncular crypts. This study showed that the expression of HIF‐1α in the ovary, oviduct and uterus varies according to the stage of the reproductive cycle. This implies that HIF‐1α plays an important role in regulating the stage‐specific physiological function of yak reproductive organs under hypoxic environments. [ABSTRACT FROM AUTHOR]

Cattleyak, which are interspecific hybrids between cattle and yak, display much higher growth performances than yak. However, F1 male cattleyak are infertile due to defective testicular development. Sirtuin 1 (SIRT1) is a histone deacetylase that is essential for various biological processes, while the roles of testicular SIRT1 in yak and cattleyak are still poorly understood. Here, we found that SIRT1 was localized in various kinds of yak testicular cells except elongated spermatids while it was deficient in cattleyak testis. Further studies indicated that cattleyak testis exhibited decreased histone acetylation levels on H3 and H4. One of SIRT1 co‐factors, steroidogenic factor‐1 (SF‐1), was lost in cattleyak testis at protein level. Expressions of several SF‐1 target genes responsible for Sertoli cell development and steroidogenesis, including STAR, CYP11A1, CYP26B1, FDX1 and HSD3B, decreased significantly in cattleyak testis. In addition, SIRT1‐mediated P53 acetylation was not responsible for the cell apoptosis in cattleyak testis. Taken together, our results suggested the deficiency of SIRT1 in yak testis caused inactivation of SF‐1 and the impairment of testicular development. This research provides theoretical bases for understanding the mechanism of cattleyak sterility and gives new insights in revealing the roles of SIRT1 in regulating yak testicular development. [ABSTRACT FROM AUTHOR]

In Mongolia, yak (Bos grunniens) are able to live in alpine areas and their products greatly influence the lives of the local people. Increased vigour in hybridized yak and cattle can offer benefits for livestock farmers. However, male hybrids show reproductive defects resulting from spermatogenesis arrest, affecting the conservation and maintenance of dominant traits in the next generation. The underlying mechanisms involved in hybrid cattle–yak infertility have recently been investigated; however, the genetic cause is still unclear. Androgens and androgen receptor (AR) signalling are required for spermatogenesis. We, therefore, evaluated the expression of AR, 3β‐hydroxysteroid dehydrogenase (3βHSD) and 5α‐reductase 2 (SRD5A2) in Leydig cells to investigate their function in cattle–yak spermatogenesis. Testicular tissues from yaks (1–3 years old) and hybrids (F1–F3, 2 years old) were collected and subjected to immunohistochemistry and image analyses to investigate the expression of each parameter in the Leydig cells. After maturation at 2 years, the expression levels of AR increased and the levels of 3βHSD decreased, but the SRD5A2 levels remained constant in yak. However, the cattle–yak hybrid F2 showed immature testicular development and significantly different expression levels of AR and 3βHSD compared with mature yak. These results suggest that the decreased expression of AR and increased expression of 3βHSD in the Leydig cells of cattle–yak hybrid testes may represent one of the causes of infertility. Our study might help in solving the problem of infertility in crossbreeding. [ABSTRACT FROM AUTHOR]

Contents: Morphological changes of the uterus and alterations in its secretory activity under the influence of steroid hormones been well documented. The oestrous cycle is also associated with significant changes in plasma follicle‐stimulating hormone (FSH), whose effects are mediated through its receptor (FSHR). Reports showed that in many mammals, FSHR was expressed in gonadal and extragonadal tissues including cervix, female reproductive tract, and pituitary gland. Follicle‐stimulating hormone (FSH) signals through endothelial FSHR directly stimulate angiogenesis and involved in the timing of birth in human, and the presence of FSHR in the placenta is essential for normal pregnancy in mice. But little is known about FSHR expression in the yak uterus. The main objective of the present study was to determine the expression and localization of FSHR in the yak uterus during different phases of the oestrous cycle. Results showed that FSHR protein was localized in the surface and glandular epithelial cells, stroma cells, myometrial smooth muscle cells and blood vessel endothelial cells. The expression of FSHR protein peaked at oestrus, significantly decreased at dioestrus (p < 0.05) and increased again at proestrus. FSHR mRNA was highly expressed at both proestrus and oestrus, and decreased at metestrus with the lowest values at dioestrus (p < 0.05). In conclusion, FSHR expression in the yak uterus changed with the stage of the oestrous cycle suggesting that FSHR plays an essential role in regulating the endometrial and myometrial functions during the oestrus cycle in the yak. [ABSTRACT FROM AUTHOR]

Contents: The objective of this study was to investigate the mechanism that regulates pre‐implantation development of the yak (Bos grunniens). We determined the transcriptomes of in vitro‐produced yak embryos at two‐cell, four‐cell, eight‐cell stages, and morula and blastocyst using the Illumina RNA‐seq for the first time. We obtained 47.36–50.86 million clean reads for each stage, of which, 85.65%–90.02% reads were covered in the reference genome. A total of 17,368 genes were expressed during the two‐cell stage to blastocyst of the yak, of which 7,236 genes were co‐expressed at all stages, whereas 10,132 genes were stage‐specific expression. Transcripts from 9,827 to 14,893 different genes were detected in various developmental stages. When |log2 ratio| ≥ 1 and q‐value <0.05 were set as thresholds for identifying differentially expressed genes (DEGs), we detected a total of 6,922–10,555 DEGs between any two consecutive stages. The GO distributions of these DEGs were classified into three categories: biological processes (23 terms), cellular components (22 terms) and molecular functions (22 terms). Pathway analysis revealed 310 pathways of the DEGs that were operative in early pre‐implantation yak development, of which 32 were the significantly enriched pathways. In conclusion, this is the first report to investigate the mechanism that regulates yak embryonic development using high‐throughput sequencing, which provides a comprehensive framework of transcriptome landscapes of yak pre‐implantation embryos. [ABSTRACT FROM AUTHOR]

Contents The objective of our present study was to determine the effects of insulin-like growth factor I ( IGF-I) on the development of yak ( Bos grunniens) embryos after cumulus-oocyte complex ( COC) vitrification and warming followed by in vitro fertilization ( IVF). In Experiment 1, the yak COCs underwent vitrification and then IVF. Embryos were incubated in synthetic oviductal fluid ( SOF) supplemented with four concentrations (0, 50, 100 and 200 ng/ml) of IGF-I, while the yak COCs without vitrification or IGF-I supplementation acted as the control group; the BAX, BCL-2, AQP3 mRNA and aquaporin 3 ( AQP3) protein expression levels in the five groups of blastocysts were evaluated using quantitative real-time PCR and immunofluorescence analyses. In Experiment 2, the groups described above were fertilized and incubated. The cleavage rate, blastocyst rate, total cell count per blastocyst and the rate of growth of the inner cell mass ( ICM) and trophectoderm ( TE) were evaluated. The results were as follows: (1) the AQP3 gene expression and protein expression in the control and 100 ng/ml IGF-I treatment groups were the highest. (2) The BAX gene expression was the lowest and the BCL-2 gene expression was the highest in the control and 100 ng/ml IGF-I treatment groups. (3) The rates of cleavage and blastocysts in the control and 100 ng/ml IGF-I groups were higher than those in the other three groups. The total cell count per blastocyst in the vitrified and warmed 100 ng/ml IGF-I group (106.7 ± 4.9) and the control group (107.3 ± 4.2) was higher than that in the vitrified and warmed 0 ng/ml IGF-I (91.2 ± 3.1), 50 ng/ml IGF-I (92.3 ± 3.7) and 200 ng/ml IGF-I (92.4 ± 3.7) groups. Therefore, we conclude that IGF-I can improve yak blastocyst developmental ability, cytomembrane permeability and formation of the blastocyst cavity after COC vitrification by improving the BAX, BCL-2 and AQP3 expression levels. [ABSTRACT FROM AUTHOR]

Growth factors play critical role in cell proliferation, regulate tissue differentiation and modulate organogenesis. Several growth factors have been identified in the testes of various mammalian species in last few years. In present investigation, the objective was to determine the expression of epidermal growth factor (EGF) and the epidermal growth factor receptor (EGFR) in yak testicular tissue by relative quantitative real time polymerase chain reaction (RT-PCR), Western blot (WB) and immunohistochemistry (IHC) from mRNA and protein levels. The testicular tissues were collected from male yak at 6 and 24 months old. Results of RT-PCR and WB showed that the expression quantity of EGF and EGFR at 24 months of age was higher than at 6 months, and the increase rate of EGFR on mRNA and protein levels was higher than the increase rate EGF during post-natal testes development. Positive staining for EGF and EGFR was very low and mainly localized to Leydig cells testes at 6 months of age with immunohistochemistry, and seminiferous tubules were not observed. At 24 month of age, both the EGF and EGFR could be detected in Leydig cells, peritubular myoid cells, sertoli cells and germ cells of the yak testes. However, EGF and EGFR were localized to preferential adluminal compartment and basal compartment in the seminiferous tubules, respectively. In conclusion, the findings in present studies suggest that EGF and EGFR as important paracrine and/or autocrine regulators in yak testes development and spermatogenesis. [ABSTRACT FROM AUTHOR]

Contents With an aim to improve the efficiency of yak-bovine interspecies somatic cell nuclear transfer (i SCNT), this study investigated the effect of different culture systems on the development, quality and gene expression profile of yak-bovine i SCNT embryo. Reconstructed embryos were cultured in modified synthetic oviductal fluid (m SOF) or relative defined culture medium ( RDCM) with 5% or 20% oxygen tension. Relative m RNA abundance of Oct-4, IFNT, IGF-2, Bax, GPX-1, SOD-1, CAT and GSS was analysed in blastocysts with q RT-PCR. The blastocyst formation rate in RDCM under 5% oxygen tension was significantly higher than that under 20% oxygen tension ( P < 0.05). The total cell number of blastocyst derived from RDCM with 20% oxygen tension was lower than that of other groups, whereas the group of RDCM with 5% oxygen tension showed a beneficial effect on apoptosis index and tolerance to cryopreservation ( P < 0.05). However, under the same oxygen tension, the m RNA abundance of IFNT of RDCM groups was higher than that of the m SOF groups. In addition, high oxygen tension during in vitro culture ( IVC) with RDCM significantly increases the m RNA expression of oxidative stress-related genes ( GPX-1, SOD-1, CAT and GSS) ( P < 0.05). 3, 4-Dihydroxyflavone ( DHF) during high oxygen tension was able to improve the cloned blastocyst formation rate in RDCM ( P < 0.05). These results for the first time showed that low oxygen tension and RDCM could improve the developmental competence and quality and alleviate the oxidative stress for yak-bovine i SCNT embryo during IVC. [ABSTRACT FROM AUTHOR]

Contents Synaptonemal complex protein 3 (SYCP3) is a DNA-binding protein located on the lateral elements of the synaptonemal complex. This protein plays an important role in homologous chromosome pairing and is necessary for male meiosis of spermatogenesis. To further understand the SYCP3 gene function and its relationship to cattle-yak male sterility, we investigated the characteristics of the bovine SYCP3 ( bSYCP3) gene as well as its transcription level and epigenetic modification status. The bSYCP3 gene encodes a 225-amino acid protein with the Cor1 motif and two coiled-coil-forming regions, homologous with other mammals (59-77% identity overall). Real-time PCR analysis indicated that the expression level of bSYCP3 mRNA in yak testes was significantly higher than that in cattle-yak (p < 0.05). The methylation level of the bSYCP3 promoter (mainly 7th, 13th and 17th CpG sites) in cattle-yaks (40%) was significantly higher than that in yaks (0%) (p < 0.05). This suggests that bSYCP3 plays an important role in meiosis of bovine spermatogenesis; further, interspecific hybridization between yak and cattle might influence bSYCP3 gene expression in cattle-yak testes, which might be influenced by bSYCP3 gene promoter methylation. [ABSTRACT FROM AUTHOR]

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Contents An attempt was undertaken to investigate the efficacy of Ovsynch protocol for timed artificial insemination (TAI) with or without Norprolac (antiprolactin) treatment during non-breeding season (winter months) in yaks (n = 25). During non-breeding season, plasma prolactin profile has been reported high due to cold and nutritional stress. The Norprolac dose of i.m. administration was standardized for prolactin suppression. Three different doses viz. 2.5, 5.0 and 7.5 mg were attempted and the dose of 7.5 mg Norprolac i.m. per animal was found to be suitable for suppression of prolactin levels up to 30 h. Ovsynch treatment with Norprolac induced more number of oestrous symptoms per animal (4.8 vs 2.1), higher LH peak concentration (24.01 vs 16.16 ng/ml), longer duration of LH surge (6.8 vs 5.2 h) and higher conception rate (70 vs 30%) in Ovsynch plus Norprolac treated animals compared with animals treated with Ovsynch alone. Therefore, this study clearly indicates the opportunity for practical application of the Ovsynch plus Norprolac protocol for TAI in yaks during non-breeding seasons. [ABSTRACT FROM AUTHOR]

Contents An attempt was made to determine plasma concentrations of, 13, 14-dihydro-15-keto-prostaglandin F2α (PGFM), cortisol and progesterone during periparturient period in yak. Plasma PGFM level showed an increasing trend beginning day 5 prior to parturition (0.48 ± 0.14 ng/ml) and increased steeply thereafter to reach a peak level (17.16 ± 1.31 ng/ml) on the day of parturition. The levels, then, declined sharply on day 1 postpartum to reach 1.20 ± 0.40 ng/ml and thereafter declined gradually over the days to reach 0.28 ± 0.09 ng/ml on day 20 postpartum and this level was maintained with fluctuation within narrow limits thereafter till conclusion of the blood sampling on day 90 post-calving. The plasma progesterone concentration on days 7 and 6 before parturition was high (2.10 ± 0.10 and 2.12 ± 0.10 ng/ml, respectively). The level then decreased gradually and abrupt fall was observed 1–2 days before parturition and became basal on day of parturition (0.24 ± 0.04 ng/ml). This basal level was maintained till the end of the blood sampling on day 90 postpartum. Plasma cortisol level showed an increasing trend beginning day 2 prior to parturition (2.36 ± 0.65 ng/ml) and increased steeply thereafter to reach a peak level (26.65 ± 5.28 ng/ml) on the day of parturition. The levels, then, declined gradually over the days and touched 2.36 ± 0.25 ng/ml on day 3 postpartum and this level was maintained with fluctuation within narrow limits thereafter till day 7 post-calving. [ABSTRACT FROM AUTHOR]

Contents Yak and mithun are two domesticated herbivores of high economic importance to the farming community living in highlands. Improved yak and mithun production could significantly enhance the living standards of these highlanders. Over the years, their dwindling numbers have been a cause of serious concern. In view of the lack of knowledge on the reproductive physiology of these ruminants, studies have been undertaken to investigate their reproductive endocrinology in recent years. This paper attempts to present the latest information on the endocrine changes associated with their various reproductive processes viz. growth and puberty, oestrous cyclicity and oestrous behaviour, ovulation, and, pregnancy and parturition. The paper also provides the recent developments on research done towards the enhancement of yak and mithun reproductive efficiencies through endocrine and embryo biotechniques. [ABSTRACT FROM AUTHOR]