This study examined the impact of cyclicity (with or without cycle corpus luteum; CL) on oocyte quality and embryonic development in buffaloes. We collected oocytes from the ovaries of slaughtered buffaloes (N = 158 cyclic; n = 316 ovaries and N = 177 acyclic; n = 353 ovaries). Blood progesterone concentration and number of oocytes per ovary were higher in cyclic buffaloes. Cyclic buffalo ovaries produce higher oocytes with I + II and fewer III + IV grades. Oocytes from cyclic buffaloes had a higher maturation rate based on cumulus expansion, cleavage rate and embryo development to the 8-cell, morula and blastocyst stages than acyclic buffaloes. In conclusion, oocytes recovered from the ovaries of the cyclic buffaloes showed improved oocyte competence and subsequent in vitro blastocyst development.

Herein, we evaluated the effects of gonadotropin hormone-releasing hormone (GnRH) administration 84 h after medroxyprogesterone acetate (MAP) sponge removal on follicular growth, ovulation timing, and pregnancy per artificial insemination (AI) in cosynchronized postpartum Nili Ravi buffaloes. In this study, 58 Nili Ravi postpartum buffaloes (DIM = 103 +/- 1.64) were randomly divided into two treatment groups (n = 29/treatment): GnRH-TAI-84 and TAI-84. All buffaloes were administered a MAP sponge for seven days. Upon MAP sponge removal, all the subjects received prostaglandin F-2 alpha (PGF(2 alpha)) and Timed AI (TAI) was performed 84 h after sponge removal. In the GnRHTAI-84 group, the buffaloes received GnRH alongside insemination, whereas in the TAI-84 group, the buffaloes were inseminated without GnRH administration. Follicle diameter and blood estradiol levels were measured every 6 h from 72-108 h after MAP sponge removal. The animals were checked for pregnancy using ultrasonography 40 days after AI. Animals subjected to the GnRH-TAI-84 protocol had a higher follicular growth rate and preovulatory follicle size than those in the TAI-84 group. The follicular diameter was also larger in animals that received GnRH-TAI-84 than in those that received TAI-84 90 and 96 h after MAP sponge removal. Buffaloes in the GnRH-TAI-84 group had lower estradiol concentrations at 90, 96, 102, and 108 h than those in the TAI-84 group. Ovulation in GnRHTAI-84 buffaloes occurred 11 h earlier than that in buffaloes from the TAI-84 group. A shorter interval between AI and ovulation in GnRH-TAI-84 buffaloes (14 h vs. 25 h) led to greater pregnancies per AI (62% vs. 17%) compared to buffaloes from the TAI-84 group.

Leukemia inhibitory factor (LIF) is an important growth factor that supports the culture and maintenance of spermatogonial stem cells (SSCs) by suppressing spontaneous differentiation. Different LIF sequences may lead to differences in function. The protein sequences of buffalo LIF and mouse LIF differed by 65.5% according to MEGA software analysis. The PB-LIF-GFP-Puro vector was constructed, and the CHO-K1 cell line was established. The final LIF protein concentration in the CHO-K1 cell culture medium was approximately 4.268 ng/mL. Here, we report that buffalo LIF effectively maintains the self-renewal of buffalo spermatogonia during culture. Buffalo spermatogonia were cultured in conditioned medium containing no LIF (0 ng/mL), mouse LIF (1 ng/mL), mouse LIF (10 ng/mL), or buffalo LIF (1 ng/mL). Furthermore, the effects of mouse LIF and buffalo LIF culture on the maintenance of buffalo spermatogonia were determined by analyzing cell colony formation, quantitative real-time polymerase chain reaction, cell immunofluorescence, and cell counting. The buffalo LIF (1 ng/mL) group showed similar maintenance of the proliferation of buffalo spermatogonia to that in the mouse LIF (10 ng/mL) group. These results demonstrated that the proliferation of buffalo spermatogonia can be maintained in vitro by adding a low dose of buffalo LIF. This study provides a foundation for the further optimization of in vitro buffalo SSC culture systems.

Buffaloes are raised by small farm holders primarily as source of draft power owing to its resistance to hot climate, disease, and stress conditions. Over the years, transformation of these animals from draft to dairy was deliberately carried out through genetic improvement program leading to the development of buffalo-based enterprises. Buffalo production is now getting more attention and interest from buffalo raisers due to its socioeconomic impact as well as its contribution to propelling the livestock industry in many developing countries. Reproduction of buffaloes, however, is confronted with huge challenge and concern as being generally less efficient to reproduce compared with cattle due to both intrinsic and extrinsic factors such as poor estrus manifestation, silent heat, marked seasonal infertility, postpartum anestrus, long calving interval, delayed puberty, inherently low number of primordial follicles in their ovaries, high incidence of atresia, and apoptosis. Assisted reproductive technologies (ARTs) are major interventions for the efficient utilization of follicle reserve in buffaloes. The present review focuses on estrus and ovulation synchronization for fixed time artificial insemination, in vitro embryo production, intracytoplasmic sperm injection, cryopreservation of oocytes and embryos, somatic cell nuclear transfer, the factors affecting utilization in various ARTs, and future perspectives in buffaloes.

目前,水牛精原干细胞(spermatogonial stem cells,SSCs)体外培养系统因尚无法支持水牛(Bubalus bubalis)SSCs体外长久培养而限制了水牛雄性遗传资源的高效利用.通过添加合适小分子化合物优化水牛SSCs体外培养系统是一个可选方案.本研究推测GSK3β特异性抑制剂CHIR99021能促进水牛精原细胞的体外增殖和自我更新.为了验证此假说,本研究探索了添加不同浓度CHIR99021对水牛精原细胞体外培养效果的影响.分别将不同浓度CHIR99021(0,5,10,20 μmol/L)添加到水牛精原细胞体外培养细胞培养液中,设置4个试验组,体外培养7 d后收集细胞,进行实时定量PCR(real-time quantitative PCR,RT-qPCR)和免疫荧光染色细胞计数.RT-qPCR结果表明,添加10、20 μmol/L的CHIR99021显著提高了 SSCs特异性表达基因(PLZF,Nanos2)和增殖相关基因(CCND1,CCNE1)的表达水平;显著降低了分化相关基因(DMRT1,DAZL)和氧化相关基因(CAT,SOD3)的表达水平.通过免疫荧光染色进行DDX4+和PGP9.5+细胞计数,发现添加10 μmol/L CHIR99021显著增加了精原细胞数量.以上结果说明,在体外培养系统中添加CHIR99021能有效维持水牛精原细胞的SSCs分子特征,促进其体外增殖,显著改善了水牛精原细胞体外培养条件.本研究能为利用小分子化合物优化水牛SSCs体外培养系统提供重要参考.

[目的]探究罗汉果甜苷V(MV)缓解持续光照导致小鼠脂肪积累的作用,为罗汉果在畜禽健康养殖中的应用提供参考依据.[方法]以15周龄雄性C57BL/6小鼠为研究对象,随机分为对照组(12 h光照:12 h黑暗)、持续光照组(24 h光照)及持续光照添加MV处理组(每日灌胃100 mg/kg的MV),试验周期为5周.试验期间监测小鼠体质量和采食量,并检测其体脂率和能量代谢水平;试验结束后处死小鼠,采集腹股沟白色脂肪(iWAT)和附睾白色脂肪(eWAT)进行称重,通过苏木精—伊红(H-E)染色观察脂肪细胞形态学特征,并以实时荧光定量PCR检测脂肪组织中脂肪代谢及能量代谢相关基因的表达情况.[结果]在整个试验过程中,各处理组小鼠的体质量与采食量均无显著差异(P>0.05),但持续光照能导致小鼠体脂率极显著升高(P<0.01,下同),而氧气消耗量、二氧化碳排出量及产热量等基础代谢参数均极显著降低,iWAT和eWAT的百分比及细胞直径均极显著增大;iWAT和eWAT中的脂肪合成相关基因(Fabp4、Pparg2和Zfp423)相对表达量极显著上调,脂肪分解相关基因Lpl相对表达量极显著下调,能量代谢基因(Sirt1和Ppargc1a)相对表达量显著(P<0.05)或极显著下调,炎症反应相关基因Nfkb1相对表达量极显著上调.经MV处理后能使持续光照小鼠上述变化指标均恢复至正常水平,即MV能有效改善持续光照导致的小鼠能量代谢紊乱、缓解脂肪累积,以及缓解脂肪代谢、能量代谢及炎症反应相关基因的异常表达.[结论]持续光照会在不改变能量摄入的情况下导致机体脂肪积累,影响机体的正常代谢和健康.MV通过调控Sirt1、Ppargc1a等重要代谢相关基因表达,改善机体代谢紊乱,并通过促进脂肪分解和抑制脂肪合成相关基因表达,有效缓解持续光照导致的小鼠脂肪积累.

Although inducible pluripotent stem cells (iPSC) have been identified in poultry, the induction efficiency is low, because different culture media, feeder cells and feeder layer treatments affect the efficiency of somatic cell reprogramming. We investigated improvement of the feeder culture system for induction of chicken iPSC by comparing the effects of different types and treatments of feeder cells on the growth and proliferation of chicken iPSC. Mouse embryo fibroblasts (MEF), but not Sandoz inbred mouse-derived thioguanine-resistant and ouabain-buffalo rat cells, were suitable feeder cells that supported proliferation of chicken iPSC. Institute of Cancer Research (ICR) mice, but not Kunming mice, were suitable for preparing MEF that support cell proliferation. Also, MEF feeder cells that had been inactivated by mitomycin C were effective. Leukemia inhibitory factor was not required for chicken iPSC culture when MEF feeder cells were used. The optimal feeder culture system for growth and proliferation of chicken iPSC consisted of MEF feeder cells derived from ICR mice that were inactivated by mitomycin C combined with embryonic germ cell culture medium.

The microenvironment in the seminiferous tubules of buffalo changes with age, which affects the self-renewal and growth of spermatogonial stem cells (SSCs) and the process of spermatogenesis, but the mechanism remains to be elucidated. RNA-seq was performed to compare the transcript profiles of pre-pubertal buffalo (PUB) and adult buffalo (ADU) seminiferous tubules. In total, 17,299 genes from PUB and ADU seminiferous tubules identified through RNA-seq, among which 12,271 were expressed in PUB and ADU seminiferous tubules, 4,027 were expressed in only ADU seminiferous tubules, and 956 were expressed in only PUB seminiferous tubules. Of the 17,299 genes, we identified 13,714 genes that had significant differences in expression levels between PUB and ADU through GO enrichment analysis. Among these genes, 5,342 were significantly upregulated and possibly related to the formation or identity of the surface antigen on SSCs during self-renewal; 7,832 genes were significantly downregulated, indicating that genes in PUB seminiferous tubules do not participate in the biological processes of sperm differentiation or formation in this phase compared with those in ADU seminiferous tubules. Subsequently, through the combination with KEGG analysis, we detected enrichment in a number of genes related to the development of spermatogonial stem cells, providing a reference for study of the development mechanism of buffalo spermatogonial stem cells in the future. In conclusion, our data provide detailed information on the mRNA transcriptomes in PUB and ADU seminiferous tubules, revealing the crucial factors involved in maintaining the microenvironment and providing a reference for further in vitro cultivation of SSCs.

Spermatogonial stem cells (SSCs), a class of primitive germ cells located on the basement of the seminiferous tubules of the testes, are the only kind of adult stem cells that can pass their genetic material to offspring and are also capable of proliferation and differentiation, ensuring the efficiency of passing transgenes from parents to subsequent generations. In this research, to understand the growth characteristics of buffalo SSCs in vitro, methods for their isolation, enrichment, culture and preliminary identification were established. Testes from 3-to 6-month-old buffalos were digested with two-step enzymatic treatment to obtain isolated single cells and then enriched with a differential plating method. Isolated SSCs were cultured on STO feeder cells with SSCs culture medium (SSCM), which is serum-free. SSCs began to proliferate on the second day and quickly formed grape-like clusters that were consistent with the morphological features of SSCs. These cells were identified as positive by immunofluorescence staining. This study successfully isolated, enriched and identified buffalo SSCs and established an effective platform to explore the mechanisms of proliferation and differentiation of buffalo SSCs.

水牛睾丸支持细胞(Sertoli cells)是环绕在精原干细胞(spermatogonial stem cells,SSCs)周围的一类体细胞,为SSCs增殖提供物理支持及稳定的环境,同时参与血睾屏障形成.支持细胞可分泌FGF2,从而提高SSCs存活和增殖.至今为止,水牛SSCs培养体系仍然面临许多挑战,推测内源性的FGF2可提高SSCs在体外培养时的自我更新和增殖能力.为此,本研究探索了水牛支持细胞的分离纯化方法,对比了幼年和成年水牛支持细胞FGF2的表达差异,并构建了支持细胞FGF2过表达细胞系.结果表明,幼年水牛支持细胞作为饲养层更有利于维持SSCs的增殖.实时荧光定量PCR显示FGF2在过表达细胞系中的水平显著高于幼年水牛支持细胞对照组的水平.与幼年水牛支持细胞作为饲养层共培养的SSCs相比,FGF2过表达支持细胞系作为饲养层共培养SSCs显著提高PLZF(P＜0.05)、OCT4(P＜0.05)和GFRα1(P＜0.05)的表达水平,并且明显改善了SSCs的体外培养性能.本研究为优化水牛SSCs体外培养系统提供理论和实践基础.

Artificial light at night (ALAN) exposes us to prolonged illumination, that adversely affects female reproduction. However, it remains to be clarified how prolonged light exposure affects oocyte meiotic maturation and quality. To this end, we exposed female mice to a constant light (CL) of 250 lux for different durations. Our findings showed that CL exposure for 7 weeks reduced the oocyte maturation rate. Meanwhile, CL exposure caused greater abnormalities in spindle assembly and chromosome alignment and a higher rate of oocyte aneuploidy than the regular light dark cycle. CL exposure also induced oxidative stress and caused mitochondrial dysfunction, which resulted in oocyte apoptosis and autophagy. Notably, our results showed that CL exposure reduced the levels of α-tubulin acetylation, DNA methylation at 5 mC, RNA methylation at m6A and histone methylation at H3K4me2 but increased the levels of histone methylation at H3K27me2 in oocytes. In summary, our findings demonstrate that constant bright light exposure causes oocyte meiotic defects and reduces cytoplasmic quality. These results extend the current understanding of ALAN-mediated defects in female reproduction.

Oocyte in vitro maturation (IVM) plays a pivotal role in in vitro embryo production. However, the efficiency of IVM is still low and needs to be further improved. In the present study, we evaluated the beneficial effects of mogroside V, an extract derived from Siraitia grosvenorii, on oocyte IVM. Porcine cumulus-oocyte complexes were cultured in IVM medium supplemented or not supplemented with mogroside V for 40 h. We found that mogroside V supplementation increased the percentage of oocyte first polar body extrusion and improved subsequent blastocyst formation after parthenogenetic activation. Furthermore, mogroside V reduced the levels of reactive oxygen species (ROS) and increased the mRNA expression of oxidative stress-related genes (SOD, CAT and SIRT1). Moreover, mogroside V supplementation enhanced the mitochondrial content, mtDNA copy number, mitochondrial membrane potential (Delta Psi m), ATP generation, and the relative mRNA expression of mitochondria-related genes (PGC-1 alpha and TFAM). In summary, our findings demonstrate that mogroside V supplementation reduces intracellular ROS levels and enhances mitochondrial function to promote porcine oocyte IVM. (C) 2019 Elsevier Inc. All rights reserved.

鸡输卵管上皮细胞是卵清蛋白的主要分泌细胞,是研究输卵管特异表达蛋白调控的重要工具.在以往的研究中,多采用普通DMEM培养液对鸡输卵管上皮细胞进行分离与培养,容易造成其自身特性在体外培养过程中的改变.本研究我们优化了细胞分离方法,发现从输卵管漏斗部组织分离的输卵管上皮细胞增殖较快;用鸡输卵管上皮细胞培养基相比DMEM更适合促进细胞生长;与胰酶相比,用Accutase消化酶进行细胞传代,有利于输卵管上皮细胞特性维持.对所获得的输卵管上皮细胞鉴定发现,己烯雌酚能促进卵清蛋白的表达,说明分离培养的细胞保持了鸡输卵管上皮细胞特性.本研究建立的方法为输卵管特异表达蛋白调控以及家禽生物反应器的研究奠定了基础.

Buffalo is considered short-day breeder in tropical and subtropical part of the world and seasonality and photoperiodism impart major influence on its fertility. However, its impact on in vitro embryo production (IVEP) remains elusive. Therefore, this study investigated the effect of seasonal variations and photoperiodism on morphological and molecular parameters of IVEP in buffalo. For this purpose, we conducted two different experiments on the oocytes obtained by aspirating follicles from abattoir derived ovaries. In Exp. I, retrospective analysis was performed for oocyte recovery, blastocyst and hatching rate, during four consecutive seasonal periods (i.e. January-March, April-June, July-September and October-December). In Exp. II, oocytes from peak breeding and non-breeding seasons were subjected to 24 hr in vitro maturation and evaluated for polar body extrusion to assess maturation rate. Results showed that embryo development was markedly low during second quarter (April-June) and maximum during fourth quarter (October-December) of the year; referred as non-breeding and breeding seasons, respectively. Comparative data analysis demonstrated that poor oocyte quality is major reason for lesser efficiency of embryo production during non-breeding season than peak breeding season as suggested by poor oocyte recovery (2.31 +/- 0.10 vs. 3.65 +/- 0.27) and maturation rate (33.32 +/- 2.1 vs. 63.15 +/- 7.31). Subsequently, comparative gene expression analysis of blastocysts during peak breeding season significantly upregulated pluripotency gene (OCT-4) and downregulated heat shock protein 90, as compared to non-breeding season. Therefore, it could be divulged from the present study that seasonal variations and photoperiodism have profound effect on oocyte quality and subsequent embryo development. It is recommended to find suitable additives for in vitro maturation that could mitigate seasonal effects.

本研究的目的是通过低氧处理体外培养的鸡原始生殖细胞(Primordial germ cell)优化培养系统,降低细胞凋亡率,提高细胞活力,为高效生产转基因鸡提供科学依据.研究采用低氧气体(1％O2)分别处理鸡PGCs 6 h、12h、24 h,随后用q-PCR检测凋亡相关基因Caspase3的表达.与常氧组(21％ O2)相比,1％ O2-24h低氧组Caspase3显著下调(p＜0.05).进一步检测1％ O2-24 h低氧组p53和Bcl2的表达情况,结果显示p53表达显著下调(p＜0.05),Bcl2表达无显著差异.采用流式细胞术检测细胞凋亡比例,低氧组((13.5±0.8)％)凋亡比例低于常氧组((21.8±2.1)％).采用q-PCR,免疫荧光染色,细胞迁移鉴定低氧处理后的鸡PGCs生物学特性,发现其仍保持生殖细胞的生物学特性.本研究表明,在体外培养条件下,1％ O2低氧处理鸡PGCs 24 h能抑制细胞凋亡,且不改变其生物学特性.

The presence of serum in embryo culture medium has been implicated for increased embryo's sensitivity to cryopreservation, compromised viability, abnormal embryo and fetal development. Hence, designing a serum free culture system is indispensable. The present study aims to compare the efficiency of the serum and granulosa cells monolayer free commercial culture system (SFCS) with the conventional serum supplemented co-culture system (SSCS) and optimized culture system (OCS). Generally, SFCS is designed explicitly for bovine oocyte maturation and embryo culture (SF-IVM and SF-IVC), and SSCS (based on M199, SS-IVM, and SS-IVC) is utilized for buffalo in vitro embryo production. However, OCS is a newly designed culture system in which oocyte maturation is performed in serum supplemented maturation medium, and the subsequent embryos are co-cultured with granulosa cells in serum free culture medium. To evaluate the effect of serum on buffalo embryo production, buffalo oocytes, and their subsequent embryos were cultured in SSCS, SFCS, and OCS, simultaneously. The percentage of cleaved embryos cultured in SSCS and OCS was approximately 4% higher as compared to SFCS. However, OCS significantly showed the maximum proportion of embryos that developed to the blastocyst stage (7d) and hatched (6d) as compared to the SFCS and SSCS. Additionally, OCS promoted the expression of developmentally important genes (BCL2-L1 and VEGF-A), cell number, and cryo-survival ability of blastocysts in comparison with SSCS. Taken together, OCS is more suitable for the oocyte maturation and culture of buffalo embryos. However, to design the serum free culture system, it is recommended to find suitable serum alternatives for in vitro oocyte maturation.

Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor cloned from mouse brain tissue by Lin et al. (1993) and can also be secreted by Sertoli cells. GDNF has a variety of physiological functions and plays important roles in the nervous system and in the reproduction of spermatogonial stem cells (SSCs). To date, commercialized murine and human GDNF have typically been used for the in vitro culture of SSCs from domestic animals. However, whether endogenous GDNF is superior to xenogenic GDNF in SSC culture in vitro remains unclear. To answer this question, the buffalo GDNF gene was cloned and integrated into the PiggyBac eukaryotic expression vector, which was stably expressed in Sertoli cells, thus establishing a PB-GDNF-Sertoli cell line. Quantitative real-time polymerase chain reaction (qRT-PCR) results showed that the GDNF gene expression in the PB-GDNF-Sertoli cell line was significantly increased compared with that in the control Sertoli cell line (p<0.01). Enzyme-linked immunosorbent assay (ELISA) results showed that GDNF gene secretion was also significantly increased in PB-GDNF-Sertoli cells compared with that in control Sertoli cells (p<0.05). We further compared two types of spermatogonia cultured on either PB-GDNF-Sertoli cells or control Sertoli cells, revealing that the use of PB-GDNF-Sertoli cells as a feeder layer could significantly increase the expression levels of DDX4 (p<0.01), PLZF (p<0.01) and NANOS2 (p<0.05) in buffalo spermatogonia in vitro. These results suggested that overexpression of GDNF could effectively maintain the pluripotency of buffalo spermatogonia in vitro, laying the foundation for improving the in vitro culture of buffalo spermatogonia.

Postovulatory ageing compromises oocyte quality and subsequent development in various manners. We aimed to assay the protective effects of mogroside V on porcine oocyte quality during in vitro ageing and explore the related causes. We observed that mogroside V can effectively maintain normal oocyte morphology and early embryo development competence after prolonged culture for 24 h. Moreover, mogroside V can markedly reduce reactive oxygen species (ROS) levels, alleviate spindle formation and chromosome alignment abnormalities, improve mitochondrial contents, adenosine triphosphate (ATP) levels and the membrane potential (ΔΨm), and reduce early apoptosis in aged oocytes. We examined the molecular changes and found that SIRT1 expression was decreased in in vitro aged oocytes but was maintained by exposure to mogroside V. However, when SIRT1 was successfully inhibited by the specific inhibitor EX-527, mogroside V could not reduce ROS levels or alleviate abnormal spindle organization and chromosome misalignment. In summary, our results demonstrated that mogroside V can alleviate the deterioration of oocyte quality during in vitro ageing, possibly by reducing oxidative stress through SIRT1 upregulation.