Obesity has become a global epidemic with major implications for fertility. In particular, obesity can trigger follicular atresia by initiating the apoptosis of granulosa cells (GCs). Emerging evidence suggests that this process may be closely linked to the dysregulation of cellular autophagy. Metformin has been shown to restore autophagic flux and mitigate obesity-related cellular dysfunction in mice; however, the ability of metformin to alleviate lipid overload-induced damage in goat granulosa cells has yet to be investigated. Analyses showed that 400 μM palmitic acid (PA) significantly increased lipid accumulation and reduced cell viability (P < 0.05) in goat granulosa cells. Furthermore, PA impaired mitochondrial function, associated with a significant increase in the populations of both early and late apoptotic cells (P < 0.05). However, treatment with 5 μM metformin (MET) under PA exposure significantly enhanced the viability of GCs and reduced the expression levels of pro-apoptotic BAX (P < 0.05). Next, we evaluated the effect of MET on cellular autophagy and found that MET treatment significantly downregulated the expression levels of phosphorylated mTORC1 (Ser2448), LC3B, and P62 while upregulating the expression levels of ULK1 in PA-treated GCs (P < 0.05). Our findings indicate that metformin improved palmitate-induced granulosa cell dysfunction by activating ULK1-mediated autophagy. Our findings will advance our understanding of reproductive dysfunction in obese ruminants, and provide a theoretical foundation for improving fertility in obese mammals.

This study examined the effects of different concentrations of roscovitine (ROSC) on cytoplasmic maturation in bovine oocytes and subsequent embryo development. Oocytes were prematured with ROSC at concentrations of 0, 25, 37.5, 50 and 75 µM for 6 h. A significant increase in the number of oocytes at GV stage was observed in the treatment groups compared to the control group, indicating an inhibitory effect of ROSC on meiosis. After continued culture in a maturation solution without ROSC for 18 h, oocytes did not exhibit significant difference in maturation rates between the treatment and control groups, suggesting that the inhibitory effect of ROSC is reversible. Further analysis revealed that a 37.5 µM ROSC treatment significantly enhanced homogeneous mitochondrial distribution, mitochondrial membrane potential, and glutathione levels in mature oocytes, while reducing reactive oxygen radical content. IVF blastocysts from oocytes treated with 37.5 μM ROSC had a significant increase in the total cell number, and inner cell mass/total cell number ratio, and a reduction in apoptosis rate, despite no significant differences in cleavage or blastocyst rates compared to controls. These findings suggested that prematuration with 37.5 µM ROSC promoted cytoplasmic maturation in bovine oocytes and improved embryo quality. This study analyzed comprehensively the impacts of meiosis arrest induced by ROSC on cytoplasmic maturation in oocyte and embryo quality from various perspectives for the first time.

Negative energy balance (NEB) typically occurs in dairy cows after delivery. Cows with a high yield are more likely to experience significant NEB. This type of metabolic imbalance could cause ketosis, which is often accompanied by a decline in reproductive performance. However, the molecular mechanisms underlying NEB have yet to be fully elucidated. During excessive NEB, the body fat is extensively broken down, resulting in the abnormal accumulation of non-esterified fatty acids (NEFAs), represented by palmitic acid (PA), within the uterus. Such an abnormal accumulation has the potential to damage bovine endometrial epithelial cells (BEECs), while the molecular mechanisms underlying its involvement in the PA-induced injury of BEECs remains poorly understood. Melatonin (MT) is recognized for its regulatory role in maintaining the homeostasis of mitochondrial reactive oxygen species (mitoROS). However, little is known as to whether MT could ameliorate the damage incurred by BEECs in response to PA and the molecular mechanism involved. Analysis showed that 0.2 mmol/L PA stress increased the level of cellular and mitochondrial oxidative stress, as indicated by increased reactive oxygen species (ROS) level. In addition, we observed mitochondrial dysfunction, including abnormal mitochondrial structure and respiratory function, along with a reduction in mitochondrial membrane potential and mitochondrial copy number, and the induction of apoptosis. Notably, we also observed the upregulation of autophagy proteins (PINK, Parkin, LC3B and Ubiquitin), however, the P62 protein was also increased. As we expected, 100 μmol/L of MT pre-treatment attenuated PA-induced mitochondrial ROS and restored mitochondrial respiratory function. Meanwhile, MT pretreatment reversed the upregulation of P62 induced by PA and activated the AMPK-mTOR-Beclin-1 pathway, contributing to an increase of autophagy and decline apoptosis. Our findings indicate that PA can induce mitochondrial dysfunction and enhance autophagy in BEECs. In addition, MT is proved to not only reduce mitochondrial oxidative stress but also facilitate the clearance of damaged mitochondria by upregulating autophagy pathways, thereby safeguarding the mitochondrial pool and promoting cellular viability. Our study provides a better understanding of the molecular mechanisms underlying the effect of an excess of NEB on the fertility outcomes of high yielding dairy cows.

Fertilization proteins JUNO and CD9 play vital roles in sperm-egg fusion, but little is known about their expression patterns during in vitro maturation (IVM) and their function during in vitro fertilization (IVF) of bovine oocytes. In this study, qRT-PCR and immunofluorescence staining were used to detect the mRNA and protein expression levels of JUNO and CD9 genes in bovine oocytes and cumulus cells. Then, fertilization rate of MII oocytes treated with (i) JUNO antibody (1, 5 and 25 mu g/ml) or (ii) CD9 antibody (1, 5 and 25 mu g/ml) or (iii) CD9 antibody (5 mu g/ml) + JUNO antibody (5 mu g/ml) were recorded. Our results showed that the mRNA and protein expression levels of JUNO and CD9 genes significantly increased from bovine GV oocytes to MII oocytes, and similar mRNA expression patterns of JUNO and CD9 were also detected in cumulus cells. All groups of oocytes treated with CD9 antibody or JUNO antibody showed significantly decreased fertilization rates (p < .05). Particularly, the fertilization ability of oocytes treated with CD9 antibody (5 mu g/ml) + JUNO antibody (5 mu g/ml) sharply decreased to 3.48 +/- 0.11%. In conclusion, our study revealed the expression levels of JUNO and CD9 genes in oocytes and cumulus cells increased during IVM of bovine oocytes, with JUNO protein playing a major role in the fertilization of bovine oocytes.

Dairy products are an important source of high quality protein supplement in people’s lives, with high nutritional value and many health benefits. The increasing consumer demand for dairy products has brought opportunities and challenges to dairy production, and improving the productivity of dairy cows has become a hot research topic. However, modern dairy production faces many dilemmas, such as heat stress in dairy cows due to high summer temperatures, resulting in reduced fertility. Heat stress in dairy cows has adverse effects on embryo quality, including embryonic cell cycle progression and cell survival, resulting in lower embryo quality and reduced productivity. In addition, abnormal epigenetic modifications are another mechanism by which heat stress in cows affects embryo quality, mainly including abnormal histone modifications and abnormal DNA methylation. Numerous studies have shown that restoration of cellular mitochondrial function and the use of melatonin can effectively mitigate the adverse effects of heat stress on embryos. Therefore, this paper outlines the mechanisms by which heat stress leads to abnormal epigenetic modifications in embryos and thus decreases embryo quality, and two solutions to mitigate heat stress by mitochondrial transplantation and melatonin treatment to improve embryo quality, thus contributing to improve the productivity of dairy cows, with the aim of providing a reference for reducing the losses caused by heat stress in animal husbandry.

随着牛奶生产的强化,奶牛生产效率大幅提高,牛奶产量不断增加,高产奶牛的繁殖力却在不断下降,这与奶牛产后能量不平衡相关.当奶牛产后所需能量超过摄入能量时,会出现能量负平衡.研究表明,能量负平衡通过多种机制影响奶牛的繁殖力,其中包括能量负平衡引起奶牛内分泌和代谢机制的改变.因此,本文概述了能量负平衡对奶牛繁殖力的影响,以及能量负平衡导致奶牛繁殖力下降的内分泌和代谢机制,旨在为制定缓解能量负平衡的方法提供参考,以期降低能量负平衡对畜牧业造成的损失.

牛胚胎基因组选择(embryonic genome selection,EGS)是指活检牛早期胚胎中部分滋养层细胞,通过微量胚胎细胞全基因组扩增,进行遗传育种值评估,筛选出优质早期胚胎.该项技术的基本步骤是:对第6天左右的早期牛囊胚进行活检取样,利用显微切割系统切取微量滋养层细胞,随后通过微量细胞全基因组扩增(whole genomic amplification),经单核苷酸多态性(single-nucleotide polymorphism)分析后,对早期胚胎进行生产性能预测,从而筛选出基因型优良的胚胎进行移植.本文总结了 EGS技术中常用的活检方法、扩增方法,并概述了该技术在牛上的应用现状与该项技术当前存在的一些问题,以期为未来EGS技术的全面发展提供参考.

水牛是我国产奶畜种之一,其适应能力强,耐高温高湿、耐粗饲,其奶营养丰富,经济价值高,被誉为"奶中之王".然而,奶水牛性成熟晚、发情症状不明显,且其发情易受环境因素影响,导致其繁殖效率低下,从而在生产中造成人力浪费和经济损失.因此,掌握有效的奶水牛发情鉴定技术进而确定最佳配种时间对奶水牛生产具有重要意义.本文参考国内外相关报道,简要总结了奶水牛各发情阶段的人工和自动化鉴定方法及其优缺点,回顾了通过血液、尿液、唾液、宫颈阴道液(cervical-vaginal fluid,CVF)以及粪便等物质鉴定奶水牛发情的相关研究,探讨了利用组学技术开发新的奶水牛发情鉴定方法的可能性,旨在为开发奶水牛发情鉴定新方法提供一定参考.

乳房炎是奶牛产后乳腺组织发生的病变,是奶牛产后最常见的疾病之一,可分为临床乳房炎和亚临床乳房炎.乳房炎不仅严重影响奶牛泌乳性能,增加泌乳奶牛被动淘汰率,而且也对奶牛繁殖性能产生不利的影响.研究表明,奶牛产后泌乳期配种前和(或)妊娠期间患乳房炎可降低人工授精受胎率、增加早期胚胎死亡(妊娠损失)和流产率、增加空怀天数和产犊间隔,从而影响奶牛养殖经济效益.乳房炎主要是通过影响奶牛生殖内分泌、体液免疫和发热等途径影响母牛卵巢机能、子宫机能和子宫内环境,从而影响母牛发情周期和发情表现、胚胎和胎儿发育而影响奶牛繁殖性能.本文综述了乳房炎对奶牛繁殖性能影响及其机制的研究最新进展,以期为提高我国奶牛繁殖性能提供参考.

卵母细胞和胚胎冷冻技术可与超数排卵和胚胎移植等繁殖技术相结合,打破生理、地区和时间对母畜繁殖潜力的限制,对国际种源流通、优良家畜扩繁和濒危动物种质资源保存具有重要意义.然而,卵母细胞和胚胎中丰富的脂质在降温过程中容易引起膜结构损伤、内质网和线粒体损伤、脂质过氧化,降低其解冻后发育能力,这极大地限制了冷冻卵母细胞和胚胎的应用.众多研究表明,在冷冻前降低卵母细胞和胚胎的脂质含量有利于提高其解冻后的存活率、囊胚率和妊娠率.目前,常用的降脂方法包括3种,即离心极化脂滴后通过显微操作去除脂滴、添加化学物质促进脂质代谢以及通过调控脂质代谢相关基因的表达降低脂质含量.作者简要阐述了过高的脂质含量引起卵母细胞和胚胎冷冻损伤的机制,总结了离心去脂法与化学去脂法的原理、应用效果以及局限性,并探讨了通过调控脂质代谢相关基因的表达从而去脂的可能性,以期为开发更稳定的卵母细胞和胚胎冷冻技术提供一定的参考.

Oocyte vitrification is crucial for livestock reproduction, germplasm conservation, and human-assisted reproduction, but the overabundance of lipids is highly detrimental to oocyte development. It is necessary to reduce the lipid droplet content of oocytes before cryopreservation. This study analyzed the impact of β-nicotinamide mononucleotide (NMN), berberine (BER), or cordycepin (COR) on various aspects of bovine oocytes, including lipid droplet content and the expression levels of genes related to lipid synthesis in bovine oocytes, development ability, reactive oxygen species (ROS), apoptosis, and the expression levels of genes associated with endoplasmic reticulum (ER) stress, and mitochondrial function in vitrified bovine oocytes. The results of our study indicated that 1 μM NMN, 2.5 μM BER, and 1 μM COR were effective in reducing the lipid droplet content and suppressing the expression levels of genes involved in lipid synthesis in bovine oocytes. Our findings showed that the vitrified bovine oocytes treated with 1 μM of NMN had a significantly higher survival rate and better development ability compared to the other vitrified groups. Additionally, 1 μM NMN, 2.5 μM BER, and 1 μM COR decreased the levels of ROS and apoptosis, decreased the mRNA expression levels of genes involved in ER stress and mitochondrial fission but increased the mRNA expression levels of genes associated with mitochondrial fusion in the vitrified bovine oocytes. Our study results suggested that 1 μM NMN, 2.5 μM BER, and 1 μM COR effectively decreased the lipid droplet content and enhanced the development ability of vitrified bovine oocytes by lowering ROS levels, reducing ER stress, regulating mitochondrial function, and inhibiting apoptosis. Furthermore, the results showed that 1 μM NMN was more effective than 2.5 μM BER and 1 μM COR.

Oocyte maturation is a critical step in the completion of female gametogenesis in the ovary; thus, for subsequent fertilization and embryogenesis. Vitrification of embryo also has been shown to be closely associated with oocyte maturation. To improve the quality and developmental potential of bovine oocytes derived from in vitro maturation (IVM), Pre-IVM with C-type natriuretic peptide (CNP), melatonin (MT) and in combination, IGF1, FGF2, LIF (FLI) were supplemented in the IVM medium. In this current study, we cultured bovine oocytes in Pre-IVM with CNP for 6 h before transferring them to the IVM medium supplemented with MT and FLI. The developmental potential of bovine oocytes was then investigated by measuring the reactive oxygen species (ROS), the intracellular glutathione (GSH) and ATP levels, the transzonal projections (TZP), the mitochondrial membrane potential (ΔΨm), cacline-AM, and the expression of related genes (cumulus cells (CCs), oocytes, blastocysts). The results revealed that oocytes treated with a combination of CNP, MT, and FLI had dramatically improved the percentage of oocytes developed to blastocyst, ATP content, GSH levels, TZP intensity, the ΔΨm, cacline-AM fluorescence intensity, and considerably reduced ROS levels of oocytes. Furthermore, the survival rate and the hatched rate after vitrification of the CNP+MT+FLI group were significantly higher than those other groups. Thus, we speculated that CNP+MT+FLI increases the IVM of bovine oocytes. In conclusion, our findings deepen our understanding and provide new perspectives on targeting the combination of CNP, MT and FLI to enhance the quality and developmental potential of bovine oocytes.

牦牛广泛分布于海拔3 000～5 000 m的高原地区,是当地畜牧业经济发展的重要畜种.经过长期适应性进化,牦牛在生理结构以及遗传分子方面表现出高原适应性特征.随着组学技术的广泛应用,牦牛高原适应机制得到进一步揭示.本文参考国内外相关报道,对牦牛高原低氧适应的肺、心生理结构和分子机制,高寒环境适应的被毛周期性调控及脂肪沉积代谢的分子机制,抗病分子基础,雌雄牦牛繁殖的生理基础和分子机制相关研究进展进行综述和展望.

Vitrification of oocytes is crucial for embryo biotechnologies, germplasm cryopreservation of endangered and excellent female animals, and the fertility of humans. However, vitrification significantly impairs the fertilization ability of oocytes, which significantly limits its widely used application. JUNO protein, a receptor for Izumo1, is involved in sperm-oocyte fusion and is an indispensable protein for mammalian fertilization, and its abundance is susceptible to vitrification. However, it is still unclear how vitrification reduces the fertilization capacity of bovine oocytes by affecting JUNO protein. This study was designed to investigate the effect of vitrification on the abundance and post-translational modifications of JUNO protein in bovine oocytes. Our results showed that vitrification did not alter the amino acid sequence of JUNO protein in bovine oocytes. Furthermore, the liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis results showed that vitrification significantly reduced the number and changed the location of disulfide bonds, and increased the number of both phosphorylation and glycosylation sites of JUNO protein in bovine oocytes. Finally, the fertilization capacity and development ability of vitrified oocytes treated with 200 pg JUNO mRNA microinjection and cholesterol-loaded methyl-β-cyclodextrin (CLC/MβCD) were similar to those of fresh oocytes. In conclusion, our results showed that vitrification of bovine oocytes did not alter the protein sequence of JUNO, but induced post-translational modifications and changed protein abundance. Moreover, the fertilization and development ability of vitrified bovine oocytes were improved by the combination treatment of JUNO mRNA microinjection and CLC/MβCD.

Recent years, techniques for in vitro embryo production（IVP） have advanced significantly, especially the ovum pick-up（OPU） technology grows widely. OPU is a new technology developed in the 1990 s that can obtain oocytes from excellent breeding heifers, which has a broad application prospect in the research of the mechanism of animal embryo production and development and in the clinical practice of human assisted reproductive technology. It can improve the resource protection efficiency of breeding females and precious wild animals, and the genetic potential of excellent females, and provide the necessary supporting technology for the development of cattle biotechnology. The quality of oocytes is the key to the success of the IVP system, and the in vitro maturation（IVM） process of the oocytes is critical for successful fertilization and embryo development. Oocyte IVM completes the cytoplasmic and nuclear maturation of the oocyte, preparing all necessary components for development to the activation of the embryonic genome. Developmental ability of oocytes matured in vitro is lower than that of oocytes matured in vivo, non-synchronization of oocyte meiosis and nucleocytoplasmic maturation, oxidative stress damage, et al are important for successful fertilization and embryo development. Therefore, it is necessary to further improve the in vitro maturation ability of oocytes to improve the bovine IVP system. In this paper, the problems of in vitro maturation of oocytes such as non-synchronization of nucleocytoplasmic maturation and oxidative stress were summarized, as well as the improvement effects of C-type natriuretic peptide, melatonin, FLI and other substances on oocytes IVM were discussed in order to improve the bovine IVP system.

[目的]为了评估胚胎质量和发育阶段对奶牛胚胎移植妊娠率的影响.[方法]使用63 头青年奶牛作为供体进行超数排卵,评估回收胚胎质量和发育阶段.选择334 头青年奶牛作为受体鲜胚移植不同质量和发育阶段胚胎.对胚胎质量分布、发育阶段分布、不同质量胚胎和不同发育阶段胚胎移植30d妊娠率进行统计分析.[结果]可用胚胎中A级胚胎比例(60.78%)显著高于B级和C级胚胎比例(36.70%和2.52%)(P<0.05);致密桑椹胚比例(54.36%)显著高于早期囊胚,囊胚和扩张囊胚比例(18.35%,25.0%和 2.29%)(P<0.05).A级和B级胚胎移植30d妊娠率(63.55%和64.35%)显著高于C级胚胎移植30d妊娠率(44.44%)(P<0.05);致密桑椹胚、早期囊胚、囊胚和扩张囊胚移植 30d妊娠率差异不显著(P<0.05),早期囊胚、囊胚移植30d妊娠率高于致密桑椹胚、扩张囊胚移植30d妊娠率(P<0.05).[结论]选择不同发育阶段的A级和B级胚胎能够获得较高胚胎移植妊娠率,增加早期囊胚和囊胚阶段胚胎移植数量能够提高胚胎移植妊娠率.

奶牛养殖是我国畜牧业重要的组成部分,在促进乡村发展和振兴中起重要作用.近年来,奶牛养殖规模化、集约化、现代化程度越来越高,人民对牛奶及奶制品需求与日俱增.但随着养牛规模扩大,常见传染性疾病的发病率居高不下,部分传染病甚至出现"抬头"趋势.传染性疾病不仅对奶牛机体健康和泌乳性能造成巨大影响,还会显著降低奶牛的繁殖性能,给奶牛养殖业造成巨大的经济损失.本文详细介绍布氏杆菌病、传染性鼻气管炎、牛病毒性腹泻病等牧场常见传染性疾病对奶牛繁殖性能的影响及其调控机制的最新研究进展,为提高我国奶牛繁殖效率及牧场经济效益提供参考.

[目的]研究性控精液对奶牛体内胚胎质量、胚胎发育和胚胎移植妊娠率的影响.[方法]将144头青年奶牛随机分为对照组(63头)和试验组(81头),使用促卵泡激素(FSH,260 mg/头)进行超排处理.对照组和试验组分别使用常规精液和性控精液输精,并对获得的体内性控胚胎进行移植,对胚胎生产、胚胎质量、胚胎发育和胚胎移植妊娠情况进行统计.[结果]试验组供体获得的平均可用胚胎数(5.67枚)显著(P<0.05)低于对照组(6.92枚);试验组供体获得的可用胚胎中A级胚胎比例(62.53％)、B级胚胎比例(35.29％)与对照组(A级胚胎比例66.51％、B级胚胎比例30.97％)相比差异均不显著(P>0.05);试验组供体获得的可用胚胎中桑葚胚比例(84.10％)显著(P<0.05)高于对照组(61.24％),囊胚比例(15.90％)显著(P<0.05)低于对照组(38.76％);试验组的鲜胚移植妊娠率(52.41％)显著(P<0.05)低于对照组(66.13％).[结论]与常规精液相比,使用性控精液生产奶牛体内性控胚胎并移植后,平均可用胚胎数、可用胚胎中囊胚比例和胚胎移植妊娠率降低,可用胚胎质量未明显降低;优化性控精液使用方案和胚胎移植技术能够提高体内性控胚胎生产和胚胎移植效率.

Sulforaphane (SFN), a bioactive phytocompound extracted from cruciferous plants, has received increasing attention due to its vital cytoprotective role in eliminating oxidative free radical through activation of nuclear factor erythroid 2-related factor (Nrf2)-mediated signal transduction pathway. This study aims at a better insight into the protective benefit of SFN in attenuating paraquat (PQ)-caused impairment in bovine in vitro-matured oocytes and the possible mechanisms involved therein. Results showed that addition of 1 μM SFN during oocyte maturation obtained higher proportions of matured oocytes and in vitro-fertilized embryos. SFN application attenuated the toxicological effects of PQ on bovine oocytes, as manifested by enhanced extending capability of cumulus cell and increased extrusion proportion of first polar body. Following incubation with SFN, oocytes exposed to PQ exhibited reduced intracellular ROS and lipid accumulation levels, and elevated T-SOD and GSH contents. SFN also effectively inhibited PQ-mediated increase in BAX and CASPASE-3 protein expressions. Besides, SFN promoted the transcription of NRF2 and its downstream antioxidative-related genes GCLC, GCLM, HO-1, NQO-1, and TXN1 in a PQ-exposed environment, indicating that SFN prevents PQ-caused cytotoxicity through activation of Nrf2 signal transduction pathway. The mechanisms underlying the role of SFN against PQ-induced injury included the inhibition of TXNIP protein and restoration of the global O-GlcNAc level. Collectively, these findings provide novel evidence for the protective role of SFN in alleviating PQ-caused injury, and suggest that SFN application may be an efficacious intervention strategy against PQ cytotoxicity.

Correct reprogramming of the DLK1-DIO3 imprinted region is critical for the development of cloned animals. However, in pigs, the imprinting and regulation of the DLK1-DIO3 region has not been systematically analyzed. The objective of this study was to investigate the imprinting status and methylation regulation of the DLK1-DIO3 region in wild-type and cloned neonatal pigs. We mapped the imprinting control region, IG-DMR, by homologous alignment and validated it in sperm, oocytes, fibroblasts, and parthenogenetic embryos. Subsequently, single nucleotide polymorphism-based sequencing and bisulfite sequencing polymerase chain reaction were conducted to analyze imprinting and methylation in different types of fibroblasts, as well as wild-type and cloned neonatal pigs. The results showed that Somatic cell nuclear transfer (SCNT) resulted in hypermethylation of the IG-DMR and aberrant gene expression in the DLK1-DIO3 region. Similar to wild-type pigs, imprinted expression and methylation were observed in the surviving cloned pigs, whereas in dead cloned pigs, the IG-DMR was hypermethylated and the expression of GTL2 was nearly undetectable. Our study reveals that abnormal imprinting of the DLK1-DIO3 region occurs in cloned pigs, which provides a theoretical basis for improving the cloning efficiency by gene editing to correct abnormal imprinting.