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Contents Small RNA represents several unique non-coding RNA classes that have important function in a wide range of biological processes including development of germ cells and early embryonic, cell differentiation, cell proliferation and apoptosis in diverse organisms. However, little is known about their expression profiles and effects in yak oocytes maturation and early development. To investigate the function of small RNAs in the maturation process of yak oocyte and early development, two small RNA libraries of oocytes were constructed from germinal vesicle stage ( GV) and maturation in vitro to metaphase II-arrested stage (M II) and then sequenced using small RNA high-throughput sequencing technology. A total of 9,742,592 and 12,168,523 clean reads were obtained from GV and M II oocytes, respectively. In total, 801 and 1,018 known mi RNAs were acquired from GV and M II oocytes, and 75 mi RNAs were found to be significantly differentially expressed: 47 mi RNAs were upregulated and 28 mi RNAs were downregulated in the M II oocytes compared to the GV stage. Among the upregulated mi RNAs, miR-342 has the largest fold change (9.25-fold). Six highly expressed mi RNAs (let-7i, miR-10b, miR-10c, miR-143, miR-146b and miR-148) were validated by real-time quantitative PCR ( RT- qPCR) and consistent with the sequencing results. Furthermore, the expression patterns of two mi RNAs and their potential targets were analysed in different developmental stages of oocytes and early embryos. This study provides the first mi RNA profile in the mature process of yak oocyte. Seventy-five mi RNAs are expressed differentially in GV and M II oocytes as well as among different development stages of early embryos, suggesting mi RNAs involved in regulating oocyte maturation and early development of yak. These results showed specific mi RNAs in yak oocytes had dynamic changes during meiosis. Further functional and mechanistic studies on the mi RNAs during meiosis may beneficial to understanding the role of mi RNAs on meiotic division. [ABSTRACT FROM AUTHOR]

TG interaction factor 1 (TGIF1) plays a major role in transcriptional inhibition and suppression of TGF-β signaling, but its functional roles in granulosa cells (GCs) have not been elucidated; in particular, there is no information about the yak (Bos grunniens) TGIF1 gene. Therefore, the objectives of this study were to clone yak TGIF1 and investigate TGIF1 functions in yak GCs. RT‒PCR results showed that the coding region of yak TGIF1 is 759 bp and encodes 252 amino acids. Its nucleotide sequence showed 85.24-99.74% similarity to mouse, human, pig, goat and cattle homologous genes. To explore the functional roles of TGIF1, we studied proliferation, apoptosis, cell cycle progression, steroidogenesis and the expression levels of related genes in yak GCs transfected with small interfering RNA specific to TGIF1. The results showed that TGIF1 knockdown promoted proliferation and cell cycle progression and inhibited apoptosis and estradiol (E 2 ) and progesterone (P 4 ) production in cultured yak GCs. Conversely, TGIF1 overexpression inhibited proliferation and cell cycle progression and stimulated apoptosis and E 2 and P 4 production. In addition, these functional changes in yak GCs were observed parallel to the expression changes in genes involved in the cell cycle (PCNA, CDK2, CCND1, CCNE1, CDK4 and P53), apoptosis (BCL2, BAX and CASPASE3), and steroidogenesis (CYP11A1, 3β-HSD and StAR). In conclusion, TGIF1 was relatively conserved in the course of animal evolution. TGIF1 inhibited GC viability and stimulated apoptosis and the secretion of E 2 and P 4 by yak GCs. Our results will help to reveal the mechanism underlying yak follicular development and improve the reproductive efficiency of female yaks., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The objectives of the study were to investigate changes in the mRNA expression levels of five genes during antral follicle development and to assess the efficacy of four timed-artificial insemination (TAI) protocols in female yaks ( Bos grunniens ). RT-qPCR analysis revealed that expression levels were greater for follicle-stimulating hormone receptor and bone morphogenic protein 15 in the small follicle, luteinizing hormone receptor, and kit ligand in the large follicle, and growth differentiation factor 9 in the medium follicle ( p  < 0.05). Non-suckling yaks were treated as a 7-d CIDR, and PGF 2α + eCG at CIDR withdrawal and TAI with frozen yak semen at 56-58 h after PGF 2α (PPe-7d); either a 7-d CIDR (PPG-7d) or a 5-d CIDR (PPG-5d), and PGF 2α at CIDR withdrawal and TAI + GnRH at 70-72 h after PGF 2α ; and GnRH treatment on Day 0, followed by PGF 2α on Day 7 and TAI + GnRH on Day 9 (GPG-7d). The results showed that the pregnancy rate (P/AI) was greater in PPG-5d than in GPG-7d ( p  < 0.05), but the P/AI was not different among the other TAI protocols. In conclusion, the expression levels of these genes in follicles are dynamically changed during antral follicle development in yaks. The PPG-5d protocol achieved a greater P/AI.

Vascular endothelial growth factor (VEGF) is crucial for follicle development through the regulation of granulosa cell (GC) function in some mammals, but its mechanism is unclear in yak (Bos grunniens). Therefore, the objectives of this study were to investigate the effects of VEGF on the viability, apoptosis and steroidogenesis of yak GCs. First, we investigated the localization of VEGF and its receptor (VEGFR2) in yak ovaries by immunohistochemistry analysis and evaluated the effect of culture medium containing different VEGF concentrations and culture times on the viability of yak GCs by Cell Counting Kit-8. Then, optimal treatment with 20 ng/mL VEGF for 24 h was selected to analyze the effects of this compound on intracellular reactive oxygen species levels by DCFH-DA kit, cell cycle and apoptosis by flow cytometry, steroidogenesis by ELISA kit and the expression of the related genes by RT‒qPCR. The results showed that VEGF and VEGFR2 were highly coexpressed in GCs and theca cells. GCs cultured in medium containing 20 ng/mL VEGF for 24 h significantly improved cell viability, decreased ROS production, promoted the transition from G1 phase to S phase (P < 0.05), increased the expression of the CCND1 (P < 0.05), CCNE1, CDK2, CDK4, and PCNA genes (P < 0.01) and decreased the expression of the P53 gene (P < 0.05). This treatment significantly reduced GC apoptosis (P < 0.05) by promoting the expression of BCL2 and GDF9 (P < 0.01) and inhibiting the expression of BAX and CASPASE3 (P < 0.05). VEGF promoted progesterone secretion (P < 0.05) accompanied by increased expression of HSD3B, StAR and CYP11A1 (P < 0.05). Taken together, our findings highlight the beneficial influence exerted by VEGF in improving GC viability and reducing ROS production and the apoptosis rate through the modulation of related gene expression., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Supplementation with acetyl-l-carnitine (ALC) during in vitro maturation significantly improves the rates of oocyte cleavage and morula and blastocyst formation in sheep and buffalo; however, the mode of action of ALC in improving oocyte competence is not completely understood. Therefore, the aim of this study was to investigate the effects of ALC on proliferation, antioxidant properties, lipid droplet accumulation and steroid hormone secretion in yak (Bos grunniens) granulosa cells (GCs). Yak GCs were identified using FSHR immunofluorescence. The cells were treated with different concentrations of ALC, cell proliferation was detected by cell counting kit-8, and the optimal concentration and treatment time were determined for subsequent experiments. Then, reactive oxygen species (ROS) were detected by a DCFH-DA probe, and lipid droplet accumulation was observed by oil red O staining. Estradiol (E2) and progesterone (P4) in the medium were detected by ELISA, and the expression of genes related to cell proliferation, apoptosis, the cell cycle, antioxidants and steroid synthesis was determined by RT‒qPCR. The results showed that 1 mM ALC treatment for 48 h was the optimum treatment. It significantly increased cell viability (P < 0.05), significantly decreased the amount of ROS and lipid droplet content, and promoted P4 and E2 secretion (P < 0.05) of yak GCs. RT‒qPCR results verified that GCs treated with 1 mM ALC for 48 h significantly increased the expression of genes related to anti-apoptosis and the cell cycle (BCL-2, PCNA, CCND1 and CCNB1), antioxidants (CAT, SOD2 and GPX1), and E2 and P4 secretion (StAR, CYP19A1 and HSD3B1) (P < 0.05), but it significantly decreased the expression of apoptosis genes (BAX and P53) (P < 0.05). In conclusion, ALC increased the viability of yak GCs, reduced the amount of ROS and lipid droplets, increased P4 and E2 synthesis and affected the expression of related genes in yak GCs., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Endoplasmic reticulum stress (ERS) plays an important role in regulating the reproductive process of female mammals, mainly involved in follicular atresia and corpus luteum regression. DNA damage induced transcript 3 (DDIT3) is a marker gene of ERS. The objectives of the present study were to clone and analyze the sequence and tissue expression characteristics of DDIT3 gene in female yaks. By reverse transcriptase-polymerase chain reaction (RT-PCR) strategy, we obtained full-length 507-bp DDIT3-cDNA, encoding for 168-aa protein. Yak DDIT3 exhibited highest and least identity with that of bison and horse, respectively. Real-time PCR analyses revealed that the expression level of DDIT3 gene in ovary was higher than that in heart, liver, kidney, spleen, lung, uterus and oviduct ( p  < 0.05). DDIT3 expression level in ovary and uterus during pregnancy was higher than that in follicular phase, luteal phase and fetus stage. DDIT3 was highly expressed in metaphase II oocytes and granulosa cells than that in germinal vesicle and metaphase I oocytes ( p  < 0.05), respectively. This is the first molecular characterization and expression patterns of DDIT3 gene in female yaks. These results indicated that the DDIT3 gene possibly plays an important role in regulating ovary function and pregnancy maintenance in yaks.

Hypoxia-inducible factors (HIFs) are oxygen-dependent transcriptional activators, but there is little information about their role in yak ( Bos grunniens ) reproduction. The present study, for the first time, investigated the adaptive mechanism of yak reproduction to high-altitude hypoxic stress by comparing the expression of HIF mRNAs between female yaks at high-altitude and cattle at low-altitude. Hypothalamus, anterior pituitary, oviduct, ovary and uterus tissue samples were collected from five adult female yaks and cattle. mRNA expression was determined by the quantitative real-time polymerase chain reaction. Both HIF-1α and HIF-2α were expressed in all five tissues examined from both species, albeit at different levels. In yaks, the highest mRNA levels of HIF-1α and HIF-2α occurred in the oviduct and anterior pituitary, respectively. Both HIF-1α and HIF-2α mRNA levels were higher in yaks than in cattle ( p  < 0.01). These data provide evidence that adaptation of reproduction to hypoxic conditions is associated with a greater expression of HIF-1α and HIF-2α in the reproductive axis of female yaks than cattle.

This study investigated the variations of the nucleotide sequences and ovarian expression levels of genes related to follicular development and atresia in prolific Jintang black goats and nonprolific Tibetan goats. Eight genes, FSHB, LHB, FSHR, LHCGR, ESR2, B4GANT2, BCL2 and BAX, were examined using reverse transcription-polymerase chain reaction and quantitative real-time PCR. The results showed that the nucleotide and deduced amino acid sequences of the LHB and BAX genes were not different, but there was one base change in the FSHR genes between the two breeds. There was one base change in the FSHB gene, which resulted in one amino acid substitution; there were nine base changes in the LHCGR gene, which resulted in five amino acid substitutions; and there were six base changes in the B4GANT2 gene, which resulted in four amino acid substitutions. The expression levels of the FSHR, LHCGR, ESR2, B4GANT2, BCL2 and BAX genes in the ovaries were not different between the two breeds. The plasma concentrations of FSH were not different, but the plasma concentrations of LH, P 4 and E 2 were lower in prolific Jintang black goats than in nonprolific Tibetan goats (P ˂ 0.05) at 40 hr after removal of the Controlled Internal Drug Release Devices. These results provide some foundations elucidating the endocrine and molecular mechanisms controlling ovulation rate in goats, but these need to be further verified., (© 2019 The Authors. Veterinary Medicine and Science Published by John Wiley & Sons Ltd.)

Follicle-stimulating hormone receptor (FSHR) plays a central role in promoting follicle maturation through the follicle-stimulating hormone (FSH)-mediated cAMP pathway in animals. The objectives of the present study were to clone the FSHR gene of yaks (Bos grunniens) and compare differences in FSHR mRNA expression in the reproductive axis between yaks and cattle. Hypothalamus, anterior pituitary, oviduct, ovary, and uterus tissue samples were collected from adult female yaks (n = 5) and cattle (n = 5) during the follicular phase. Using reverse transcriptase-polymerase chain reaction (RT-PCR), we found that the FSHR coding region of the yak is 2088 bp and encodes 695 amino acids. Its amino acid sequence showed 99.38%-72.22% similarity to the homologous genes of cattle, goats, sheep, cats, donkeys, horses, humans, chickens, monkeys, mice, rats, and wild boar. Real-time PCR analysis revealed that the FSHR gene was expressed in all tissues examined. Expression of the FSHR gene in the yak was higher in the uterus than other tissues (P < 0.05) but, in cattle, was higher in the ovary than other tissues (P < 0.05). The FSHR gene expression level in the cattle ovary was significantly higher than that in the yak ovary (P < 0.01). These results indicate that the FSHR gene is relatively conserved in the course of animal evolution. The variation in sequence and expression level of FSHR between the two species might be associated with the difference in their reproduction., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The development of an accurate and noninvasive preselection process for competent oocytes is essential to achieve a highly efficient in vitro production (IVP) of embryos. Cumulus cells (CCs) have important functions in oocyte growth, development, maturation, and fertilization. It, therefore, is important to know if the quality of oocytes can be ascertained by assessment of gene expression of the surrounding CCs or not. The aim of this study was to identify differentially expressed genes in yak CCs from oocytes with varying developmental competences as possible biomarkers for distinguishing oocyte competence. The isolated CCs were pooled into immature and mature groups in accordance with the maturation outcome of oocytes. A total of 9516 genes were differentially expressed in the two CC categories (P <  0.05). With a minimum change of 2.5-fold, 45 up-regulated and 79 down-regulated genes were observed in CCs belonging to the mature group compared with those in the immature group (P <  0.01). These genes were primarily enriched for the cell cycle, meiosis, cell signaling, metabolism, and apoptosis. The selected candidate genes (CCND1, BMP15, GDF9, H19, KLF4, GPC1, SYCP3, and CTSB) were validated using quantitative real-time polymerase chain reaction (RT-qPCR) and there were expression patterns similar to those detected with transcriptome analysis. The CCs from fertilized oocytes arrested at the 2-cell (2-cell group), or 8-cell (8-cell group) stages or that developed into blastocysts (the blastocyst group) had a 1.5-, 1.8-, and 2.3-fold increase, respectively, in mRNA relative abundance of CCND1 compared with CCs from unfertilized oocytes (P <  0.05). The results with the RT-qPCR analysis confirmed that the relative abundance of CCND1 mRNA in CCs was associated with oocyte developmental competence. In conclusion, RNA-Seq is useful in extracting transcriptomes and selecting markers associated with oocyte developmental competence. Furthermore, the expression of the CCND1 gene in yak CCs can be used to preselect oocytes for IVP efficiency., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Functional reprogramming of a differentiated cell toward pluripotent cell may have long-term applications in numerous aspects, especially in regenerative medicine. Evidences accumulating from recent studies suggest that cellular extracts from stem cells or pluripotent cells can induce epigenetic reprogramming and facilitate pluripotency in otherwise highly differentiated cell types. Epigenetic reprogramming using cellular extracts has gained increasing attention and applied to recognize the functional factors, acquire the target cell types, and explain the mechanism of reprogramming. Now, more and more researches have proved that cellular extract treatment is an important strategy of cellular reprogramming. Thus, this review mainly focused on the progresses and potential mechanisms in epigenetic reprogramming using cellular extracts.

During mammalian pre-implantation embryonic development, dramatic and orchestrated changes occur in gene transcription. Pregnancy rates were low when yak females were crossbred with cattle breeds, but few studies exist to describe the unique molecular network regulation behind the pre-implantation development of these embryos. We determined the transcriptomes of crossbred embryos derived from yak oocytes in vitro fertilized with Jersey sperm using Illumina RNA-seq for the first time in this study. Embryos were sampled at the 2-, 4-, and 8-cell, morula and blastocyst stages. The results showed that in total, 291.9 million short reads were generated from the five libraries of 2-, 4-, and 8-cell, morula and blastocyst stages, with 276.2 million high-quality reads selected for further analysis. Eighty to 91% of the clean reads were aligned against the yak reference genome. A total of 19,072 transcripts were identified in five libraries, of which 7,785 transcripts were co-expressed in each stage and 2,013 transcripts were stage-specific. When a |log 2 ratio| ≥1 and q-value ≤ 0.05 were set as thresholds for identifying differentially expressed genes (DEGs), we detected a total of 3,690 to 10,298 DEGs between any two consecutive stages. Based on the results of GO and KEGG enrichment, some of these DEGs potentially play an important role in regulating pre-implantation development, but they are most likely stage-specific. There were 2,960, 7,287, 6,420, 7,724 and 10,417 DEGs in 2-, 4-, 8-cell, morula and blastocyst stages between the crossbred embryos and purebred embryos of the yak, respectively, leading to a large difference in GO terms and pathways. In conclusion, we sequenced transcriptomes of in vitro-produced crossbred embryos of yak and cattle during pre-implantation and provided comprehensive examinations of gene activities. These will be helpful for development of assisted reproductive technology and better understanding the early maternal-fetal or maternal-embryonic dialog in inter-species crossbreeding.

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 |log 2 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., (© 2018 Blackwell Verlag GmbH.)

To increase the current understanding of the gene expression in the pre-ovulatory ovary and identify the key genes involved in the regulation of ovulation rate, we compared the transcriptomes of ovaries from the prolific Jintang black goat (JTG) and the non-prolific Tibetan goat (TBG) during the follicular phase using the Illumina RNA-Seq method. Three ovarian libraries were constructed for each breed. On average, we obtained approximately 49.2 and 45.9 million reads for each individual ovary of TBGs and JTGs, respectively, of which 79.76% and 78.67% reads were covered in the genome database. A total of 407 differentially expressed genes (DEG) were detected between these two breeds, in which 316 were upregulated, and 91 were downregulated in the ovaries of JTGs versus TBGs. Based on the results of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, some of these DEGs potentially play an important role in controlling the development of ovarian follicles. SRD5A2, MSMB, STAR and 3BHSD, etc. were the most significantly differentially expressed between these two distinct breeds. In addition, each ovary expressed 1,066 versus 989 novel transcripts, and 171,829 versus 140,529 putative SNPs in TBGs versus JTBs, respectively. All data sets (GEO and dbSNP) were available via public repositories. Our study provides insight into the transcriptional regulation of the ovaries of two distinct breeds of goats that might serve as a key resource for understanding goat fecundity. SRD5A2, MSMB, STAR and 3BHSD may be associated with the high fecundity of JTGs., (© 2017 Blackwell Verlag GmbH.)

The kidding rate is one of the most important economic traits for goat production, but the genetic mechanism that is associated with ovulation rate is poorly understood. Recently, increasing evidence has suggested that microRNAs (miRNAs) influence ovarian biological processes. The present study provides the first comparison of the ovarian miRNAs of prolific Jintang black goats (JTGs) and non-prolific Tibetan goats (TBGs) during the follicular phase using RNA-Seq technology. We generated 11.19 million (M) and 11.34 M clean reads from the TBG and JTG libraries, respectively, from which a total of 389 known miRNAs were identified and 142 novel miRNAs were predicted. A total of 191 miRNAs were differentially expressed between the two breeds. Among the 10 most abundant miRNAs, miR-21-5p was defined as differentially expressed miRNA with a higher level in the JTG library than in the TBG library, but the other miRNAs were not different between the breeds. The predicted miRNA-targeted genes were further analyzed by Gene Ontology and KEGG pathway analyses. The results revealed that miR-21, miR-99a, miRNA-143, let-7f, miR-493 and miR-200b may affect follicular development. These findings will increase the current understanding of the role of ovarian miRNAs in the regulation of ovulation rate in goats.

Introduction: We cloned and sequenced four pivotal cDNAs involved in DNA structural maintenance (H1F0 and TOP1) and the cell cycle (CLTA and CDK1) from yak oocytes. In addition, we studied the consequences of freezing-thawing (F/T) processes on the expression of their mRNA transcripts in yak immature and in vitro matured (MII) oocytes., Material and Methods: H1F0, TOP1, CLTA and CDK1 cDNAs were cloned from yak oocytes by reverse transcriptase-polymerase chain reaction (RT-PCR) strategy. The expression of their mRNA transcript analyses were performed upon fresh and frozen-thawed immature germinal vesicle (GV) and MII yak oocytes following normalization of transcripts with GAPDH by real-time PCR., Results: The yak H1F0, TOP1, CLTA and CDK1 cDNA sequences were found to consist of CDK1 585, 2539, 740, and 894 bp, respectively. Their coding regions encoded 195, 768, 244, and 298 amino acids, respectively. The homology with that of cattle was very high (95.2%, 98.8%, 93.6%, and 89.5%, respectively nucleotide sequence level, and 94.3%, 98.2%, 87.7%, and 90.9%, respectively at the deduced amino acid level). The overall mRNA expression levels of these four transcripts were reduced by F/T process, albeit at different levels. TOP1 in GV-oocytes, and H1F0 and CDK1 in MII-oocytes of the yak were significantly down-regulated (P<0.05)., Conclusions: This is the first isolation and characterization of H1F0, TOP1, CLTA, and CDK1 cDNAs from yak oocytes. The lower fertility and developmental ability of yak oocytes following fertilization after cryopreservation may be explained by the alterations to their gene expression profiles., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The functional reprogramming of a differentiated cell to a pluripotent state presents potential beneficial applications in disease mechanisms and regenerative medicine. Epigenetic modifications enable differentiated cells to perpetuate molecular memory to retain their identity. Therefore, the aim of this study was to investigate the reprogramming modification of yak fibroblast cells that were permeabilized and incubated in the extracts of mesenchymal stem cells derived from mice adipose tissue [adipose-derived stem cells (ADSCs)]. According to the results, the treatment of ADSC extracts promoted colony formation. Moreover, pluripotent gene expression was associated with the loss of repressive histone modifications and increased global demethylation. The genes Col1a1 and Col1a2, which are typically found in differentiated cells only, demonstrated decreased expression and increased methylation in the 5'-flanking regulatory regions. Moreover, yak fibroblast cells that were exposed to ADSC extracts resulted in significantly different eight-cell and blastocyst formation rates of cloned embryos compared with their untreated counterparts. This investigation provides the first evidence that nuclear reprogramming of yak fibroblast cells is modified after the ADSC extract treatment. This research also presents a methodology for studying the dedifferentiation of somatic cells that can potentially lead to an efficient way of reprogramming somatic cells toward a pluripotent state without genetic alteration.

Background: The competence for embryonic development after IVF is low in the yak, therefore, we investigated the effects of supplementation of FSH, LH and the proteasome inhibitor MG132 in IVM media on yak oocyte competence for development after IVF., Methods: In Experiment 1, yak cumulus-oocyte complexes (COCs) were in vitro matured (IVM) in TCM-199 with 20% fetal calf serum (FCS), 1 microg/mL estradiol-17beta, and different combinations of LH (50 or 100 IU/mL) and FSH (0, 1, 5, 10 microg/mL) at 38.6 degrees C, 5% CO2 in air for 24 h. Matured oocytes were exposed to frozen-thawed, heparin-capacitated yak sperm. Presumptive zygotes were cultured in SOF medium containing 6 mg/ml BSA, 0.5 mg/mL myoinositol, 3% (v/v) essential amino acids, 1% nonessential amino acids and 100 μg/mL L-glutamine (48 h, 38.5 degrees C, 5% CO2, 5% O2, and 90% N2). In Experiment 2, cumulus cells were collected at the end of IVM to determine FSHR and LHR mRNA expression by real-time PCR. In Experiment 3 and 4, COCs were cultured in the presence or absence of the proteasomal inhibitor MG132 from either 0-6 h or 18-24 h after initiation of maturation., Results: The optimum concentration of FSH and LH in IVM media was 5 microg/mL FSH and 50 IU/mL LH which resulted in the greatest cleavage (79.1%) and blastocyst rates (16.1%). Both FSHR and LHR mRNA were detected in yak cumulus cells after IVM. Treatment with MG132 early in maturation reduced (P<0.05) cleavage and blastocyst rates. Conversely, treatment with MG132 late in maturation improved (P<0.05) blastocyst rate. Optimal results with MG132 were achieved at a concentration of 10 microM., Conclusions: An optimum concentration of FSH and LH in IVM medium, and treatment with MG132 late in maturation can improve yak oocytes competence for development after IVF.

The preference of fertilized (IVF) and somatic cell nuclear transfer (SCNT) presumptive zygotes for different media when cultured in vitro to the blastocyst stage was evaluated in this study. The experiment comprised two zygote production methods (IVF and SCNT) × two culture media (mSOF and G1.5/G2.5) factorial design in which culture droplets that contained approximate 30 presumptive zygotes formed the experimental plots for the assessment of cleavage and blastocyst development. There were 15 to 20 replicates (culture droplets) per treatment combination. Sub-samples 30 to 41 of the blastocysts produced were assessed for cell number and cell apoptosis. A further 10 blastocysts per treatment combination were used for quantitative real-time polymerase chain reaction (RT-PCR) to evaluate the relative abundance of Hsp70 and Bax mRNA. Presumptive zygotes produced by IVF were developmentally more competent than SCNT zygotes in terms of cleavage rate (66.9 vs. 57.0%; P < 0.05) and blastocyst development rates (blastocysts of presumptive zygotes 29.7 vs. 24.8%; blastocysts of cleaved zygotes 44.4 vs. 36.6%; P < 0.05). Over both zygote production systems, however, the results were similar whether culture was in mSOF or in G1.5/G2.5 media for cleavage rate (63.2 vs. 62.4%; P > 0.05) and blastocyst development rate (blastocysts of presumptive zygotes 26.4 vs. 25.7%; P > 0.05; blastocysts of cleaved zygotes 41.8 vs. 41.2%; P > 0.05). There was, however, a significant interaction between the method of zygote production and culture medium for the apoptotic index of blastocysts. The interaction was such that IVF-produced zygotes cultured in mSOF had a lower apoptotic index compared with those cultured in G1.5/G2.5 (4.7 ± 1.2% vs. 9.8 ± 0.9%; P < 0.05) whereas SCNT zygotes had a higher apoptotic index when cultured in mSOF compared with those cultured in G1.5/G2.5 (11.9 ± 1.5% vs. 4.5 ± 1.2%; P < 0.05). Moreover, RT-PCR analysis showed that embryos from IVF-produced zygotes cultured in mSOF had a lower expression level of stress-related and apoptosis genes (Hsp70 and Bax) than those cells cultured in G1.5/G2.5 medium, while SCNT-derived embryos cultured in mSOF had a higher expression level of these genes than those embryos cultured in G1.5/G2.5 medium. The results of this study show that bovine IVF- and SCNT-produced presumptive zygotes have different nutrient requirements for in vitro culture to the blastocyst stage of development. IVF-derived zygotes have a preference for mSOF as the culture medium whereas the G1.5/G2.5 medium is more suitable for the culture of bovine SCNT-derived zygotes.

In the present study, we examined the ability of immature germinal vesicle (GV) and subjected to in vitro matured (MII) yak oocytes to survive after cryopreservation as well as their subsequent development following in vitro maturation and fertilization. Both GV and MII oocytes were cryopreserved by using two different vitrification solutions (VS); VS-I contained 10% ethylene glycol (EG) and 10% dimethylsulfoxide (DMSO) in TCM-199 + 20% (v/v) fetal calf serum (FCS) whereas VS-II contained 40% EG + 18% Ficoll + 0.5 M sucrose in TCM-199 + 20% FCS. The percentage of oocytes found to be morphologically normal was greater (P < 0.01) in VS-I group than in VS-II group. Rates of cleavage (30.6-42.2%) and blastocyst formation (2.9-8.9%) did not differ among groups, but were lower than in unfrozen control (55.7% and 25.4%, P < 0.01). These results show that a combination of EG and DMSO or EG, Ficoll and sucrose can be used to cryopreserve yak oocytes in French straws., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Growth hormone (GH), insulin-like growth factor-I (IGF-I), and II (IGF-II) play a key role in the development of preantral to preovulatory follicles in some species. To better understand the role of these genes in controlling follicular development and fecundity in goats, in the present study, we first cloned the cDNA encoding GH, IGF-I and IGF-II from prolific Lezhi black goat and non-prolific Tibetan goat (Capra hircus), and their mRNA expression between the two breeds were compared. By reverse transcriptase-polymerase chain reaction (RT-PCR) strategy, we obtained full-length 688-bp GH, 493-bp IGF-I, and 566-bp IGF-II cDNAs, encoding for 217 amino acid (aa) GH, 154 aa IGF-I, and 179 aa IGF-II putative proteins. Analysis of their nucleotide and amino acid sequences revealed a high degree of identity between the two breeds, although one base change in GH resulted in one amino acid substitution in the translated proteins. However, two base changes in IGF-I and IGF-II did not lead to any amino acid changes. Real-time PCR analyses revealed that in the middle of estrus, GH, IGF-I and IGF-II genes were expressed, albeit at different levels, in all three tissues (anterior pituitary, endometrium and ovary) examined. GH was most highly expressed in ovary (P<0.01) and its expression was greater in all three tissues examined in Lezhi black goat than in Tibetan goat (P<0.05). IGF-I and IGF-II genes were expressed at a higher (P<0.05) level in anterior pituitary of Lezhi black goat than that in Tibetan goat, but they had a similar expression pattern in endometrium and ovary. These results provide the foundation of information required for future studies of these gene effects on goat fecundity., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) have a central role in follicle growth and maturation, but no clear differences between breeds with different ovulation rates have been found. Therefore, this study investigated mRNA expression of FSHβ, LHβ, FSH receptor (FSHR), LH receptor (LHR), and estrogen receptor-β (ERβ) genes in prolific Lezhi black (LB) goats and nonprolific Tibetan (TB) goats by real-time PCR. Follicles and pituitaries were recovered from goats at 12-24 h after onset of estrus. Real-time PCR analysis revealed that the expression levels of FSHβ and LHβ mRNA were significantly higher (p < 0.01) in pituitary of LB than in TB does, but the expression levels of FSHR and LHR mRNA in follicle of TB were greater (p < 0.05). Expression level of follicular ER β was not different between the two breeds. Data provide evidence that the greater ovulation rate in the LB goat as compared to the TB breed is associated with a greater gonadotropin expression during follicular phase.

The treatment of donor cells with oocyte extracts before inter-species somatic cell nuclear transfer (iSCNT) is a novel method for cellular reprogramming. This study aims to evaluate the effect of pre-treatment donor cell with oocyte extracts on the early developmental competence of yak iSCNT embryos. Yak fibroblasts were reversibly permeabilized with streptolysin O, and then treated with yak oocyte extracts (YOE) or bovine oocyte extracts (BOE) prior to iSCNT. The 8-cell and blastocyst formation increased significantly compared with the control group (P<0.05) when donor cells pre-treated with YOE or BOE. The relative expression level of embryo-specific genes TBP1 and Mash2 were also up-regulated both in the blastocysts of the YOE and BOE groups. In addition, the methylation level of pluripotency-specific genes (Oct4 and Nanog) in the blastocysts of the YOE and BOE groups were similar to that of its IVF counterpart (53.1%, 48.8% vs. 40.1%; 24.8%, 26.5% vs. 35.9%). Our results suggested that pre-treatment of donor cells with oocyte extracts can improve nuclear-cytoplasmic reprogramming; thus representing a novel way to improve the efficiency of yak iSCNT., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

Prolactin (PRL) plays central roles in a wide range of body functions in mammals, and the actions are mediated by the specific cell surface receptor, the prolactin receptor (PRLR). To better understand the role of PRL in the yak (Bos grunniens), in the present study, we first cloned yak PRLR cDNA, and compared its mRNA expression in several tissues with cattle (Bos taurus). By reverse transcriptase-polymerase chain reaction (RT-PCR) strategy, we obtained full-length of yak PRLR cDNA sequence comprised of an open reading frame of 1746bp encoding a 581 amino acid protein, and contained a signal sequence and a transmembrane region. The intracellular domain had two pairs of cysteine residues and a WSXWS motif. The cytoplasmic domain comprised 323 residues and contained box 1 sequence. The yak PRLR shared 66.0-98.5% protein sequence identity with mammalian homologs. Real-time PCR analysis revealed that PRLR mRNA was higher in mammary tissue than in ovary and endometrium (P<0.01). During pregnancy, the ovary and mammary PRLR mRNA expression increased by 33- and 2.9-fold in yak, respectively, and increased by 46- and 3.8-fold in cattle, respectively. PRLR mRNA expression was higher (P<0.05) in mammary tissue and ovary of pregnant cow than that of pregnant yak. It is proposed that the increased ovarian and mammary PRLR mRNA expression during pregnancy may be associated with corpus luteum function for maintenance of pregnancy and mammary development for subsequent lactation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The purpose of this study was to investigate fertilization ability and embryo development to the blastocyst stage after reciprocal in vitro fertilization (IVF) between yak and cattle in an attempt to clarify the problem of low conception rate after mating yak females with cattle bulls. In vitro-matured (IVM) cattle and yak oocytes were inseminated with either Holstein or yak spermatozoa, and after an 18-h of coincubation period, a proportion of the oocytes was fixed and examined for sperm penetration, polyspermy and male pronuclear formation. The remaining oocytes were cultured in vitro and evaluated for cleavage and blastocyst formation rates. The percentage of IVM oocytes penetrated by spermatozoa ranged from 78.5 to 90.5%, and the formation of one or two pronuclei and the incidence of polyspermy did not differ among the different combinations. The cleavage and blastocyst rates were not affected by the species of the sperm, but they were affected by the species of the oocytes (P<0.05), with cattle oocytes having a higher (P<0.05) cleavage and blastocyst rates (69.9 and 31.3%) than yak oocytes (62.7 and 11.5%). The blastocyst formation rate was calculated from the cleaved zygotes. The interaction between sire and oocytes species (P<0.05) influenced blastocyst formation rate, with the highest blastocyst rate occurring in cattle oocytes fertilized with yak spermatozoa (36.5%) and the lowest rate occurring in yak oocytes fertilized with yak spermatozoa (9.4%). The effect of heterosis was apparent at the blastocyst stage, but there was a large reciprocal difference in blastocyst production between crosses. It was concluded that the low conception rate that results from crossing yaks with cattle is not due to either a species-specific block of fertilization or the developmental competence of the early stage embryo.

The objectives of this study were to evaluate the transfection effectiveness of retroviral vector PLNCX2 in yak sperm-mediated gene transfer (SMGT) and the effect of SMGT on sperm motility and fertilizability. Human lactoferrin (hLF) DNA was ligated into PLNCX2 to construct recombinant plasmid PLNCX2-hLF, then, using PLNCX2-hLF+FuGene 6 to generate SMGT yak spermatozoa for fertilizing bovine oocytes. The result showed that DNA-binding rate increased with the extension of incubation period and DNA treatment did not decrease sperm motility. Oocytes inseminated with SMGT-spermatozoa had a lower (P < 0.05) cleavage rate (57.7%) than the control (73.4%), but development up to blastocyst stage was not different (26.8 vs. 31.7%). It appears that PLNCX2 is useful for generating transgenic yaks by SMGT.

The objectives of this study were to investigate differences in fertilization and development of embryos after in vitro fertilization of Bos taurus (cow) oocytes with sperm from either yaks (Bos grunniens) or Holstein bulls. Frozen-thawed spermatozoa (Holstein n=5 sires; yak n=5 sires) were evaluated for motility (forward progression) and acrosomal status immediately post-thaw and then 1, 2, 3, and 8h later. In vitro-matured cow oocytes (n=1652) were inseminated with either Holstein bull or yak spermatozoa and after an 18-h co-incubation period, a proportion of the oocytes were fixed and examined for sperm penetration, polyspermy, and male pronuclear formation. The remaining oocytes were cultured in vitro and evaluated for cleavage and blastocyst production rates. Overall, there were species differences (P<0.05) and an effect of time (P<0.01) in sperm motility and acrosome integrity. An effect (P<0.01) of a species-by-time interaction was detected for motility, but not for acrosome integrity. The percentage of oocytes penetrated and the formation of two pronuclei when cow oocytes were inseminated with yak spermatozoa (97.4% and 81.6%, respectively) were greater (P<0.01) than that achieved with Holstein bull spermatozoa (77.8% and 65.9%, respectively), but the incidence of polyspermy (>2 pronuclei) was similar (P>0.05; 10.8% vs. 15.8%). The yak male symbolxcow combination gave a higher cleavage rate than the Holstein male symbolxcow combination (P<0.05; 76.3% vs. 63.3%), but there was no difference in the blastocyst rate (17.9% vs. 14.5%). It is concluded that yak spermatozoa could successfully fertilize cattle oocytes and their hybrid embryos had normal competence to develop to blastocysts.

The objectives of the present study were to evaluate the induction of estrus and fertility in yak cows treated with Co-Synch regimens or progesterone (P(4)). In Experiment 1, postpartum suckled yaks were assigned to three treatments: (1) A (n=28), insertion of an intravaginal device containing P(4) (CIDR) on Day 0, PGF(2alpha) (i.m.) on Day 6 and PMSG (i.m.) at the time of CIDR removal on Day 7 (P(4)-PGF(2alpha)-PMSG); (2) B (n=21), PGF(2alpha) (i.m.) on Day 6 and PMSG on Day 7; (3) C (n=26), control group. Seven yak bulls were grazed with the cows for natural breeding. Rate of estrus within 96h of the end of treatment was greater (P<0.05) in A (100.0%) than in B (28.6%) or C (0.0%). First service conception rate (CR) determined by serum P(4) on Day 21 after breeding was greater (P<0.05) in A (78.6%) than in B (22.2%). Also, pregnancy rate (PR) during the breeding season was greater (P<0.05) in A (82.1%) than in B (19.0%) and C (7.7%). In Experiment 2, non-suckled yaks that calved in previous years but not in the current year were assigned to three treatments: (1) A (n=31), GnRH (i.m.) on Day 0, followed by PGF(2alpha) on Day 7 and timed artificial insemination (TAI) concurrently with GnRH treatment on Day 9 (Co-Synch regimen); (2) B (n=50), a CIDR device for 7 days plus PGF(2alpha) and PMSG at the time of CIDR withdrawal on Day 7 and TAI on Day 9 (P(4)-PGF(2alpha)-PMSG); (3) C (n=50), yak cows were artificially inseminated at spontaneous estrus. Frozen semen of Holstein and Jersey were used for insemination in Experiment 2. The CR assessed by rectal palpation 35 days after TAI was not different in A (22.6%), B (30.0%) and C (33.3%), but PR was greater in A and B than in C, when based on those cows presented for estrous synchronization programs. It is concluded that P(4)-PGF(2alpha)-PMSG protocol could efficiently induce estrus and result in an acceptable pregnancy rate in postpartum suckled yak cows. This technique and Co-Synch regimen can be applied successfully for TAI of non-suckled yak cows.

This paper reviews seasonal breeding, puberty, postpartum anestrus, embryonic loss and calf survival and their constraints in female yaks. Methods for improving fertility in postpartum yak cows are also considered. Yaks are seasonal breeders with mating and conception restricted in the warm season. Puberty generally occurs in the 2nd to the 4th warm season following birth, i.e. between 13 and 36 months of age. The cows usually have a long postpartum anestrus period; only a small proportion of the cows return to estrus in the 1st breeding season after calving, most come into estrus in the 2nd and 3rd years. Nutritional status is the most important determinant of reproduction in female yaks. Reproductive success is a direct result of the availability of pasture determined by climate, season, and management practices. Milking delays puberty by reducing milk intake (restricted suckling) and growth rate for the calf. Milking interferes with grazing and prolongs the duration of postpartum acyclicity in cows. Calves born early in the season have a longer suckling season than those born later in the season before the onset of winter. Thus, they can have their first cycle in the breeding season of the following year, while those born late in the season may not have their first estrus until 25 or 26 months of age. Cows calving early in the season are more likely to return to estrus in the year of calving because they have a longer period to recover from the demand on body reserves before the onset of winter. Inbreeding in smallholder yak farms is also discussed and minimizing inbreeding by exchanging bulls among different herds is suggested. Reproductive efficiency can be improved by nutritional supplementation during the winter, however, the most cost-effective and practical strategy for this needs to be determined. Early weaning or restricted suckling may shorten the duration of postpartum acyclicity, however, it is impractical due to reduced growth rates and increased winter mortality of early weaned calves. A single treatment with either GnRH, or PGF(2alpha)+GnRH can successfully induce estrus in yak cows that calved in previous years (with or without calf) but did not calve in the current year, however, it has little effect in cows nursing a calf born in the current year. The effects of administration of exogenous progestogens plus GnRH on the fertility of yak cows are worthy of further study.