Xiang-Dong Zi

Reproduction in female yaks (Bos grunniens) and opportunities for improvement.

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.

Different preferences of IVF and SCNT bovine embryos for culture media

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.

Zebularine and Scriptaid Significantly Improve Epigenetic Reprogramming of Yak Fibroblasts and Cloning Efficiency

Abnormal epigenetic reprogramming of the donor nucleus after somatic cell nuclear transfer (SCNT) is thought to be the main cause of low cloning efficiency. Following SCNT, the donor nucleus often fails to express early embryonic genes and establish a normal embryonic pattern of chromatin modification. Therefore, in this study, we have attempted to improve epigenetic reprogramming of the donor nucleus and cloned embryos with Zebularine and Scriptaid. Yak fibroblasts were treated with 20 μM Zebularine alone or 20 μM Zebularine plus 0.5 μM Scriptaid for 24 h, whereas yak cloned embryos were treated exclusively with 0.5 μM Scriptaid for 12 h. There was no effect on cellular viability and proliferation after drug treatment. The treatment of fibroblasts with Zebularine or Zebularine plus Scriptaid increased histone acetylation of histone 3 lysine 9 (H3K9), but decreased the level of DNA methylation of Oct-4 and Sox-2 promoter regions. When donor cells were used after Zebularine plus Scriptaid treatment to reconstruct cloned embryos and then treated with Scriptaid, the developmental competence and cryosurvival of embryos were improved significantly. In addition, the relative expression of Oct-4 and Sox-2 were increased significantly. The expression levels of Dnmt-1 and Hdac-1 were significantly decreased when fibroblasts and cloned embryos were treated with Zebularine or Scriptaid. This work provides functional evidence that treatment with Zebularine and Scriptaid modifies the epigenetic status of yak fibroblasts, subsequently enhancing in vitro developmental potential and the quality of yak cloned embryos.

Developmental competence of frozen-thawed yak (Bos grunniens) oocytes followed by in vitro maturation and fertilization

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.

Epigenetic reprogramming of Yak iSCNT embryos after donor cell pre-treatment with oocyte extracts

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.

Cloning and mRNA expression levels of GDF9, BMP15, and BMPR1B genes in prolific and non-prolific goat breeds.

Recent findings indicate that the bonemorphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) are key regulators of follicular development in many mammalian species (Otsuka et al., 2011), but there is little information about them in goats. The present study, for the first time, investigated the nucleotide sequences and mRNA expression of BMP15, GDF9, and BMP receptors types 1B (BMPR1B) genes in Lezhi black goats, a local Chinese breed famous for its high fecundity (producing 3--5 kids per kidding), and Tibetan goats (Capra hircus), a single-birth breed characterized by adapting to cold, hypoxic ecological conditions in the Qinghai-Tibet Plateau. Ovaries, anterior pituitaries, and endometria were recovered from adult female Lezhi black goats (n1⁄4 6) and Tibetan goats (n1⁄4 6) at 12--24hr after onset of estrus. Follicles were also dissected from ovaries for isolated samples. We obtained sequences for goat GDF9 (1,362 bp), BMP15 (1,185 bp), and BMPRIB (1,527 bp) by reverse-transcriptase polymerase chain reaction (RT-PCR). GDF9 contained two exons with the lengths of 397 bp and 965 bp. All sequences were deposited in the GenBank database (accession numbers, JN049449, JN100107--JN100109, JF824148, JF824149). Comparison of the nucleotide sequences between the two breeds showed that the sequence identities of GDF9, BMP15, and BMPRIB were respectively 99.78%, 100%, and 99.55%. In contrast to previous comparative study in prolific Boer goats and non-prolific Yunling goats (Cui et al., 2009), the present study showed that a GDF9 base change at nucleotide 149 (T to C) resulted in amino acid substitution (L toP), but thebase changesat nucleotide 118 (C to T) produced no amino acid substitution.BMPR1B base changes at nucleotides 637 (G to A), 649 (G to A), 715 (G to C), 781 (C to T), and 827 (A to G) result in amino acid substitutions (G to S, E to K, A to P, L to F, and Y to C). There were no base differences in the BMP15 gene of the two goat breeds (Fig. 1A and Supplementary Fig. 1). Mutations in theBMP15,GDF9, and BMPR1B genes have been found to increase the ovulation rate and infertility in mice, sheep, and humans (Hanrahan et al., 2004;Otsukaet al., 2011). Therefore, thebasechanges inGDF9 and BMPR1B resulting in amino acid substitutions may be an important molecular mechanism that regulates the differential fecundity of these goat breeds. Real-time PCR analysis in the present study revealed broad tissue expression of the target mRNAs. The expression level of BMP15 mRNA in follicle of Lezhi black goat was 5.72-fold greater than that in Tibetan goat (P< 0.05). In contrast, expression levels of follicularGDF9 (Fig. 1B) and total ovarianBMPR1B were not different between these two breeds (Fig. 1C). This observation is similar to the previous study comparing Yunling and Boer goat breeds (Cui et al., 2009). We conclude that BMP15 gene expression and structural differences in GDF9 andBMPRIBmaybeassociatedwith the reproductive difference between Lezhi black and Tibetan goats.

Identification and comparative analysis of the ovarian microRNAs of prolific and non-prolific goats during the follicular phase using high-throughput sequencing

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.

Comparative messenger RNA expression of FSHβ, LHβ, FSHR, LHR, and ERβ in high and low prolific goat breeds.

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 ERβ 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.

Cloning of cDNAs for H1F0, TOP1, CLTA and CDK1 and the effects of cryopreservation on the expression of their mRNA transcripts in yak (Bos grunniens) oocytes ☆ ☆☆

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.

The effect of retroviral vector on uptake of human lactoferrin DNA by yak (Bos Grunniens) spermatozoa and their fertilizability in vitro.

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.