Juvenile in vitro embryo transfer can shorten generation intervals, but its application in goats is constrained by the quantity-quality trade-off in oocytes from prepubertal donors. We established an age-specific workflow for Yudong black goats by separately optimizing ovarian superstimulation in juvenile and adult donors, evaluating anti-Müllerian hormone (AMH) as an exploratory indicator of ovarian response, improving in vitro maturation (IVM), and optimizing somatic cell nuclear transfer (SCNT). Under the selected juvenile protocol (1-2 months, 220 IU FSH plus 330 IU PMSG, collection 12 h after the final FSH injection), donors yielded 113.4 ± 10.9 follicles and 73.5 ± 6.3 cumulus-oocyte complexes, with 35.9 ± 0.9% Grade A/B oocytes. Pre-stimulation serum AMH was positively associated with follicle number; an exploratory cutoff of 4.54 ng/mL predicted ≥ 90 follicles (AUC 0.906; sensitivity 87.5%; specificity 80.0%). Supplementing IVM medium with 20% adult follicular fluid increased juvenile oocyte maturation from 53.7 ± 0.8% to 62.2 ± 0.8% and reduced intracellular reactive oxygen species. Optimal electrofusion voltages were 18 V for juvenile and 21 V for adult cytoplasts. Adult-derived parthenogenetic embryos had higher blastocyst rates than juvenile-derived embryos. SCNT embryos were transferred at the 2-4-cell stage; therefore, no in vitro SCNT blastocyst endpoint was generated. Preliminary Day-45 pregnancy establishment was 1/6 recipients for each cytoplast source. These findings support an age-specific, conservation-oriented workflow while emphasizing the need for independent AMH validation, minimally invasive oocyte recovery, and larger full-term SCNT studies.

Lycopene (LYC) is a plant-derived antioxidant that ameliorates oxidative and other stress-related damage in spermatozoa yet its inclusion in ovine freezing medium remains largely unexplored. In this study semen was collected from eight Oura-type Tibetan sheep with three ejaculates per male and cryopreserved in freezing medium supplemented with 0, 1, 2, 4, 8 and 16 μM LYC to identify the optimal concentration and evaluate its effects on structural integrity antioxidant status and fertilizing capacity. A dose of 2 μM LYC proved optimal markedly improving post-thaw survival and motion kinetics relative to the control (P < 0.01). At both 25 °C and 4 °C this concentration prolonged survival and increased the proportion of live cells at each observation point. It also enhanced acrosome (P < 0.01), head membrane (P < 0.05) and tail membrane (P < 0.01) integrity and elevated mitochondrial membrane potential (MMP) (P < 0.05). Compared with the control the 2 μM LYC group showed lower reactive oxygen species (ROS) and malondialdehyde (MDA) levels (P < 0.01) and higher superoxide dismutase (SOD) activity and total antioxidant capacity (T-AOC) (P < 0.05 and P < 0.01 respectively). In vitro fertilization (IVF) trials revealed that 2 μM LYC increased cleavage (P < 0.05) and blastocyst formation rates (P < 0.01) without affecting blastocyst cell number (P > 0.05). In a cervical artificial insemination (AI) programme pregnancy rates were 44 % for the 2 μM LYC group versus 28 % for the control. We conclude that adding 2 μM LYC to the freezing medium improves post-thaw quality counters oxidative stress and enhances the fertilizing capacity of Oura-type Tibetan sheep spermatozoa.

Sperm non-coding RNAs, including micro RNAs, transfer RNA-derived small RNAs, and long non-coding RNAs, are pivotal in cellular cytoskeletal remodeling, early embryonic development, and offspring phenotypes. Despite the identification of circular RNAs (circRNAs) in mammals, the roles of sperm-derived circRNAs in embryogenesis remain largely unexplored. This study identify circRNA-1572, a sperm-derived circRNA deliver into oocytes during fertilization, through whole-transcriptome sequencing of porcine metaphase II (MII) oocytes, purified mature sperm, and in vitro fertilized pronuclear (PN) embryos. Functional assays confirm circRNA-1572 competitively binds to bta-miR-2478-L-2 through a "sponge" mechanism, regulating the expression of the target gene cyclin B2 (CCNB2). Knockdown (KD) of circRNA-1572 or overexpression of bta-miR-2478-L-2 led to reduce levels of CCNB2 mRNA and protein, along with altered fibrous actin (F-actin) distribution and aberrant chromosomal organization, leading to increase developmental arrest and impair zygotic genome activation (ZGA) during early porcine embryogenesis. Importantly, these phenotypes are rescued upon supplementary mRNA of CCNB2. Moreover, SMART-seq analysis reveals KD of CCNB2 resulted in delayed degradation of maternal transcripts in 2-cell embryos and delayed initiation of ZGA in 4-cell. This study provides novel insights into the molecular regulatory functions of sperm-derived circRNAs in early mammalian embryogenesis and underscores the impact of paternal factors on embryonic development.

During the in vitro maturation process of oocytes, oxidative stress is commonly present, and excessive oxidative stress can affect oocyte maturation. Thus, adding antioxidants during maturation is an effective strategy for reducing oxidative stress. N-acetylcysteine (NAC), a derivative of cysteine, participates in glutathione (GSH) metabolism and stimulates glutathione synthesis. However, a clear understanding of the effect of NAC on sheep oocytes remains unknown. In this study, we investigated NAC's impact on the maturation of sheep oocytes, and the results revealed that the maturation rate, and subsequently the cleavage and blastocyst formation, were significantly enhanced by incubation with 1 mM NAC. The GSH and Ca2+ levels increased, and the cortical granules were significantly elevated, whereas the reactive oxygen species levels were significantly reduced in the 1 mM NAC-treated group. Additionally, the number of inner cell masses was significantly increased. The findings of this study support the hypothesis that NAC increases oocyte maturation rate by protecting them from oxidative stress damage. These discoveries provide a new approach for improving the efficiency of in vitro production of sheep embryos.

Apigenin (API), a naturally occurring flavonoid with potent antioxidant activity, has received limited attention regarding its protective effects on frozen-thawed sperm. Here, API was added to the freezing medium at 0-100 μM to determine the optimal concentration by assessing post-thaw survival and kinetic parameters. Dose-response screening identified 10 μM API as the most effective level, and this concentration was used to evaluate sperm quality, antioxidant capacity, fertilization potential and protein expression. Ten micromolar API significantly enhanced acrosomal and head membrane integrity and mitochondrial membrane potential, whereas tail membrane integrity and DNA integrity remained unchanged. Reactive oxygen species and malondialdehyde levels declined, superoxide dismutase activity and total antioxidant capacity increased; IVF cleavage rate improved, while blastocyst rate and blastocyst cell number remained unaffected, and AI pregnancy rate rose. iTRAQ profiling indicated that API primarily modulates proteins linked to energy metabolism, motility and mitochondrial function. API at 10 μM markedly improves the quality of frozen-thawed Oura-type Tibetan sheep sperm.

In vitro maturation (IVM) is a very important technology for the modernization of animal husbandry. Previous studies have demonstrated that the mitochondria-targeting antioxidant mitoquinone mesylate (MitoQ) was known for its protective role in a variety of tissues and cells; however, its function in Tibetan sheep oocytes is not fully understood. This study used a Tibetan sheep model to evaluate the effects of oocytes exposed to MitoQ on maturation and subsequent embryonic development. During IVM, cumulus-oocyte-complexes were exposed to 0-400 nM MitoQ to evaluate the viability of cumulus cells expansion and oocyte maturation, and determine the optimal concentration of 100 nM. Here, we discovered that 100 nM MitoQ addition to the medium improved the Tibetan sheep oocyte maturation rate (P < 0.05) and cumulus cells expansion rate (P > 0.05). In addition, immunostaining showed that decreased ROS levels (P < 0.01), increased GSH levels (P < 0.01). MitoQ-treated oocytes showed enhanced mitochondrial activity (P < 0.01) and mitochondrial membrane potential (P < 0.05). MitoQ increased Ca2 + levels (P < 0.01) and attenuated early apoptosis (P < 0.01). No differences were observed for cleavage rate (P > 0.05), and improved number of blastocyst cells and the blastocyst rate (P < 0.05) after in vitro fertilization. Moreover, various genes associated with oocyte oxidative stress (GCLC, SOD1) in mature oocytes were beneficially regulated in the MitoQ-treated oocytes. In conclusion, MitoQ can enhance the oocyte maturation rate, improve subsequent embryonic development in Tibetan sheep.

Myo-inositol (MYO) is an essential compound within the ovarian follicular microenvironment, which facilitates the transmission of follicle-stimulating hormone signals in oocytes, thereby aiding in their maturation. Metabolomics analysis of follicular fluid from Tibetan sheep and in vitro maturation experiments have revealed the presence of myo-inositol; however, it remains uncertain whether the administration of exogenous myo-inositol can effectively enhance the maturation of Tibetan sheep oocytes. Consequently, this study was designed to explore the effects of myo-inositol supplementation on oocyte maturation and early embryo development. The influence of myo-inositol on oocyte quality was measured through various parameters, including reactive oxygen species, glutathione levels, mitochondrial function, mitochondrial membrane potential, and calcium ion concentration. The findings showed that 25 mM MYO markedly boosted the maturation rate of Tibetan sheep oocytes, significantly lowered oxygen radical formation, and increased glutathione, calcium ion concentration, and endoplasmic reticulum activity. The addition of 25 mM MYO to the in vitro embryo culture medium also substantially enhanced blastocyst formation. In summary, this study demonstrated that myo-inositol supplementation can improve both the rate of maturation of Tibetan sheep oocytes and the development of early embryos. This research lays a foundational framework for enhancing the in vitro maturation system of Tibetan sheep oocytes and the overall efficiency of in vitro embryo production.

Butuo Black sheep (BBS), an ancient indigenous Chinese breed, has co-evolved with the Yi people's semi-nomadic lifestyle and demonstrated exceptional adaptability to high-altitude migrations. However, a high-quality reference genome for BBS is still lacking. In this study, we established a high-quality chromosome-level genome assembly of BBS using PacBio HiFi sequencing. The final assembled genome size was approximately 2.95 Gb, with a contig N50 of 71.45 Mb and a scaffold N50 of 92.26 Mb. The genome assembly achieved a high Benchmarking Universal Single-Copy Orthologs (BUSCO) score of 95.9%, indicating its high completeness and quality. The de novo genome prediction revealed that repetitive sequences accounted for 47.74% of the genome, with long interspersed nuclear elements (LINEs) being the most abundant. Additionally, we present 50 BBS shotgun genomes sequenced using the Illumina HiSeq 2000 platform, with a mean coverage of 10.36×. The study generated approximately 1.2 terabytes of raw data, with 99.9% clean reads mapping successfully to the sheep reference genome at 99.6% coverage. This extensive dataset provides a valuable resource for studying genetic diversity and evolutionary patterns in BBS.

Palmitic acid (PA) is known to be elevated in the follicular fluid of women with obesity, negatively affecting female fertility. However, the mechanism by which PA exposure reduces female fertility is not fully understood, and how it can be treated requires further investigation. We first established in vivo and in vitro models of mouse oocyte maturation at high concentrations of PA and determined the effects of treatment with agomelatine (Ago) which is a melatonin receptor agonist with antioxidant properties. We assessed oocyte maturation rates, spindle morphology and chromosome morphology, oxidative stress and apoptosis levels. Lastly, we examined energy levels, mitochondrial function, and mitochondrial synthesis-related protein expression levels. Our results showed that PA exposure disrupted spindle assembly and chromosome alignment, reduced microtubule stability, and impaired the meiotic maturation of oocytes. PA also disrupted mitochondrial function, leading to decreased ATP production, elevated Reactive Oxygen Species(ROS) levels, oxidative stress, and apoptosis. Remarkably, Ago supplementation promoted oocyte quality by restoring spindle/chromosome conformation, maintaining mitochondrial function, lowering ROS levels, and inhibiting apoptosis. This study establishes that Ago ameliorates metabolic stress-induced oocyte deterioration through mitochondrial functional restoration, providing mechanistic insights into obesity-associated infertility. Importantly, our study identifies a potentially favorable drug for combating obesity-induced female infertility.

Preimplantation embryos undergo a series of important biological events, including epigenetic reprogramming and lineage differentiation, and the key genes and specific mechanisms that regulate these events are critical to reproductive success. Ubiquitin-specific protease 7 (USP7) is a deubiquitinase involved in the regulation of a variety of cellular functions, yet its precise function and mechanism in preimplantation embryonic development remain unknown. Our results showed that RNAi-mediated silencing of USP7 in mouse embryos or treatment with P5091, a small molecule inhibitor of USP7, significantly reduced blastocyst rate and blastocyst quality, and decreased total and trophectoderm cell numbers per blastocyst, as well as destroyed normal lineage differentiation. The results of single-cell RNA-seq, reverse transcription-quantitative polymerase chain reaction, western blot, and immunofluorescence staining indicated that interference with USP7 caused failure of the morula-to-blastocyst transition and was accompanied by abnormal expression of key genes (Cdx2, Oct4, Nanog, Sox2) for lineage differentiation, decreased transcript levels, increased global DNA methylation, elevated repressive histone marks (H3K27me3), and decreased active histone marks (H3K4me3 and H3K27ac). Notably, USP7 may regulate the transition from the morula to blastocyst by stabilizing the target protein YAP through the ubiquitin-proteasome pathway. In conclusion, our results suggest that USP7 may play a crucial role in preimplantation embryonic development by regulating lineage differentiation and key epigenetic modifications.

肌肉生长抑制素(Myostatin,MSTN)基因突变时会导致肌纤维增粗,肌肉细胞增多,表现出双肌性状.为敲除欧拉藏绵羊MSTN基因以获得产肉性状更优的欧拉藏绵羊,利用胞嘧啶碱基编辑系统(CBE)在欧拉藏绵羊MSTN基因编码区提前引入终止密码子.在MSTN基因 3 个外显子区域上各设计 1 条sgRNA,分别连接至pEF1a-BE4max-NG-mU6-gRNA-blast质粒并转染欧拉藏绵羊耳成纤维细胞,通过Sanger测序和T-A克隆检测,成功筛选出 1 条靶向外显子I的符合预期的sgRNA,编辑效率 20％.本研究成功通过单碱基编辑技术在欧拉藏绵羊耳成纤维细胞MSTN基因编码区进行定点编辑,为通过分子育种培育产肉性状更佳的欧拉藏绵羊新品种奠定基础.

Microplastics (MPs) are an emerging pollutant that is becoming recognized as an increasingly serious environ-mental problem. The biological toxicity and resulting health risks of MPs have attracted much attention in the research community. While the effects of MPs on various mammalian organ systems have been described, their interactions with oocytes and the underlying mechanism of their activity within the reproductive system have remained ambiguous. Here, we discovered that oral administration of MPs to mice (40 mg/kg per day for 30 days) significantly reduced the oocyte maturation and fertilization rate, embryo development, and fertility. Ingestion of MPs significantly increased the ROS level in oocytes and embryos, leading to oxidative stress, mitochondrial dysfunction, and apoptosis. Moreover, mouse exposure to MPs caused DNA damage in oocytes, including spindle/chromosome morphology defects, and downregulation of actin and Juno expression in mouse oocytes. In addition, mice were also exposed to MPs (40 mg/kg per day) during gestation and lactation to determine trans-generational reproductive toxicity. The results showed that maternal exposure to MPs during pregnancy resulted in a decline in birth and postnatal body weight in offspring mice. Furthermore, MPs exposure of mothers markedly reduced oocyte maturation, fertilization rate, and embryonic development in their female offspring. This investigation provides new insights on the mechanism of MPs' reproductive toxicity and raises concerns for potential risks of MP pollution on the reproductive health of humans and animals.

In this study, a single base editing system was used to edit the FecB and GDF9 gene to achieve a targeted site mutation from A to G and from C to T in Ouler Tibetan sheep fibroblasts, and to test its editing efficiency. Firstly, we designed and synthesized sgRNA sequences targeting FecB and GDF9 genes of Ouler Tibetan sheep, followed by connection to epi-ABEmax and epi-BE4max plasmids to construct vectors and electrotransfer into Ouler Tibetan sheep fibroblasts. Finally, Sanger sequencing was performed to identify the target point mutation of FecB and GDF9 genes positive cells. T-A cloning was used to estimate the editing efficiency of the single base editing system. We obtained gRNA targeting FecB and GDF9 genes and constructed the vector aiming at mutating single base of FecB and GDF9 genes in Ouler Tibetan sheep. The editing efficiency for the target site of FecB gene was 39.13%, whereas the editing efficiency for the target sites (G260, G721 and G1184) of GDF9 gene were 10.52%, 26.67% and 8.00%, respectively. Achieving single base mutation in FecB and GDF9 genes may facilitate improving the reproduction traits of Ouler Tibetan sheep with multifetal lambs.

WDR43(WD Repeat Domain 43)是一种核糖体生成因子.本研究旨在分析牦牛WDR43 序列的结构功能及其在牦牛组织和早期胚胎不同阶段的表达情况,从而为研究WDR43 在牦牛早期胚胎发育过程中的作用机制提供实验依据.本实验选取 30～90 d胚龄左右的牦牛胎儿(n=2)和 3～4 岁健康的母牦牛(n=2)作为材料,克隆获得牦牛WDR43 的编码区,生物信息学分析预测WDR43 功能并通过qRT-PCR获得WDR43在牦牛不同组织的表达情况.结果表明,牦牛WDR43编码区长度为 2 046 bp,编码 681 个氨基酸,该序列有45.23％可能形成无规则卷曲,29.37％形成α螺旋,20.41％形成延伸链,4.99％形成β-转角.WDR43 总体带正电荷,属于不稳定蛋白,无跨膜结构及信号肽.WDR43 氨基酸序列高度保守,与牛、山羊、绵羊和野猪WDR43基因同源性较高,分别为 98.85％、98.53％、98.09％和 93.83％.WDR43 在卵巢的表达量显著高于其他组织,并且在早期胚胎过程中均有表达,说明WDR43 可能参与了牦牛的早期胚胎发育.本研究成功获得了牦牛WDR43基因编码区序列,分析其结构功能并明确其在牦牛不同组织的表达模式,为进一步研究WDR43基因在牦牛早期胚胎发育过程中的作用机制奠定了基础.

Zinc plays a crucial role in the growth and reproductive functions of animals. Despite the positive effects of zinc that have been reported in oocytes of cows, pigs, yaks, and other animals, the influence of zinc on sheep is little known. To investigate the effect of zinc on the in vitro maturation of sheep oocytes and subsequent parthenogenesis-activated embryonic development, we added different concentrations of zinc sulfate to the in vitro maturation (IVM) culture medium. The IVM culture medium with zinc improved the maturation of sheep oocytes and the subsequent blastocyst rate after parthenogenesis activation. Notably, it also enhanced the level of glutathione and mitochondrial activity while reducing levels of reactive oxygen species. Thus, zinc addition to the IVM medium improved the quality of oocytes with a positive effect on the subsequent development of oocytes and embryos.

CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) is widely used in the field of livestock breeding. However, its low efficiency, untargeted cutting and low safety have greatly hampered its use for introducing single base mutations in livestock breeding. Single base editing, as a new gene editing tool, can directly replace bases without introducing double strand breaks. Single base editing shows high efficiency and strong specificity, and provides a simpler and more effective method for precise gene modification in livestock breeding. This paper introduces the principle and development of single base editing technology and its application in livestock breeding.

Extensive epigenetic reprogramming occurs during preimplantation embryonic development. However, the impact of DNA methylation in plateau yak preimplantation embryos and how epigenetic reprogramming contributes to transcriptional regulatory networks are unclear. In this study, we quantified gene expression and DNA methylation in oocytes and a series of yak embryos at different developmental stages and at single- cell resolution using single-cell bisulfite-sequencing and RNAseq. We characterized embryonic genome activation and maternal transcript degradation and mapped epigenetic reprogramming events critical for embryonic development. Through cross-species transcriptome analysis, we identified 31 conserved maternal hub genes and 39 conserved zygotic hub genes, including SIN3A, PRC1, HDAC1/2, and HSPD1. Notably, by combining single-cell DNA methylation and transcriptome analysis, we identified 43 candidate methylation driver genes, such as AURKA, NUSAP1, CENPF, and PLK1, that may be associated with embryonic development. Finally, using functional approaches, we further determined that the epigenetic modifications associated with the histone deacetylases HDAC1/2 are essential for embryonic development and that the deubiquitinating enzyme USP7 may affect embryonic development by regulating DNA methylation. Our data represent an extensive resource on the transcriptional dynamics of yak embryonic development and DNA methylation remodeling, and provide new insights into strategies for the conservation of germplasm resources, as well as a better understanding of mammalian early embryonic development that can be applied to investigate the causes of early developmental disorders.

In mammals, the transcription of transposable elements (TEs) is important for maintaining early embryonic development. Here, we systematically analyzed the expression characteristics of TE-derived transcripts in early embryos by constructing a database of TEs and transcriptome data from goats and using it to study the function of endogenous retroviruses (ERVs) in regulating early embryo development. We found that ERV1 made up the highest proportion of TE sequences and exhibited a stage-specific expression pattern during early embryonic development. Among ERV elements, ERV1 had the potential to encode the Gag protein domain to form virus-like particles (VLPs) in early goat embryos. Knockdown of ERV1_1_574 significantly reduced the embryo development rate and the number of trophoblast cells ( P < 0.05). Transcriptome sequencing analysis of morula embryos showed that ERV1_1_574 mainly regulated the expression of genes related to embryo compaction and trophoblast cell differentiation, such as CX43 and CDX2. In summary, we found that ERV1 expression was essential for early embryonic development in goats through regulation of trophoblast cell differentiation.

探究双丁注射液和鱼腥草注射液对奶牛隐性乳房炎的治疗效果,为奶牛隐性乳房炎的防治提供依据和参考.对青海地区某规模化养殖奶牛分别进行中草药和抗生素的对比治疗,并综合评价了患病奶牛的治愈率、产奶量、体细胞数、血清理化性质、乳品质和乳酶活性.结果显示,中药组在治愈率、产奶量、体细胞数恢复上优于抗生素组;两种治疗方法对血清理化性质和肝代谢的影响总体无明显差异;在治疗6d后,中药组在乳糖率和乳蛋白率上优于抗生素组;在治疗3d后,中药组在N-乙酰基-β-葡萄糖苷酶、髓过氧化物酶和乳酸脱氢酶的活性恢复上优于抗生素组.结果表明,双丁注射液和鱼腥草注射液联合应用在治愈率、产奶量、体细胞、乳品质和乳酶活性均优于抗生素组,对奶牛隐性乳房炎有更好的治疗效果.