牛胚胎移植的生物安全措施

对胚胎的质量检查,目前只能凭其形态进行评估分级,而对胚胎的内在质量却无从判断,尤其是胚胎的生物安全问题往往被忽视。牛胚胎移植是从处于无菌状态的供体牛子宫内采取胚胎,在体外进行一系列操作,再移植到受体牛子宫内,其基本过程包括:供体牛和受体牛的选择,同期发情处理,供体牛的超排和配种,胚胎的回收与保存,检胚和移胚。在这一系列的过程中,如果被微生物污染,首先会影响胚胎的发育,造成胚胎早期死亡或者不受孕。即使胚胎能正常发育受孕,也可能造成传染性疾病的传播。因此,进行牛胚胎移植时,采取必须的生物安全措施,对提高胚胎的质量及其…     

如何提高牛超排效果

牛胚胎移植产业化势在必行 ,产业化的前提必须有充足的胚胎 ,但在体外胚胎生产技术、环境等因素尚未成熟前 ,体内胚胎生产 (即超数排卵 )技术仍是获得胚胎的重要来源。如何在减少供体母牛、资金、时间等投入的情况下提高超排效果 ,是目前体内生产胚胎面临的主要问题。其中 ,供体牛的选择至关重要。尽管有研究指出 ,牛个体间的超排反应差异是无法降低的 ,但我们完全可以通过对供体牛个体的多方面有效的选择来提高超排反应 ,从而相对降低牛个体间的超排差异。1　供体母牛子宫状况检查供体母牛子宫状况直接影响胚胎的质量。实践证明 ,子宫有较…     

牛胚胎移植的主要操作技术及其影响因素

1 胚胎移植的主要操作技术 主要包括供体和受体母牛的选择,供体母牛的超数排卵,供体与受体母牛的同期发情,胚胎的回收,胚胎质量的鉴别,胚胎的冷冻保存,胚胎移植,受体牛的妊娠诊断等.要提高牛胚胎移植的成功率,就要从以上各个环节着手,切实做好各项工作.     

影响南方地区母牛胚胎质量和数量主要因素分析

胚胎移植技术通过同期发情、超数排卵和人工技术在良种母牛(供体牛)体内生产多枚胚胎,并将早期的胚胎从供体牛取出来移植到另一头母牛(受体牛)胚胎移植技术是加快牛群改良和纯种快速繁育一种重要手段。胚胎质量和数量是影响胚胎移植技术效果的关键因素。     

娟姗牛与荷斯坦牛X-性控冷冻精液试验研究效果分析

采用娟姗牛与荷斯坦牛X-性控冷冻精液,给娟姗母牛、荷斯坦母牛及胚胎移植供体进行人工授精,以探索两种冷冻精液人工授精受胎效果及供体牛生产胚胎效果。研究结果表明,人工输精试验牛情期受胎率及供体母牛的平均可用胚胎数和可用胚胎率受公牛X-性控冷冻精液的影响;人工输精试验牛的受胎率青年牛优于经产牛,而荷斯坦牛的优于娟珊牛;生产胚胎的供体牛,综合可用胚胎数和可用胚胎率,在同一品种上青年牛的效果优于经产牛,在不同品种而同一年龄上的荷斯坦牛优于娟珊牛。     

牛胚胎移植技术(续)

<正>2牛胚胎移植技术的操作流程及其相关原理牛胚胎移植技术主要包括:供体牛的选择、供体牛的同期发情、超数排卵、人工授精、胚胎的收集、胚胎的     

青年牛不适宜做胚胎移植

有些人认为青年牛最适合用于胚胎移植,因为青年牛是不经产牛,所以繁殖疾病少,有利于受精和受孕。其实不然。下面就针对青年牛为什么不适合做供体牛和受体牛的原因分别进行阐述。1青年牛不适合做供体牛提供胚胎的牛称为供体牛,做胚胎移植时若用青年牛做供体牛易出现下列问题:对药物反应不强烈、输精和冲胚困难、胚胎质量差以及下次配种困难等。1.1对药物反应不强烈由于青年牛各部分器官刚刚发育成熟,内分泌调节尚未平衡,内分泌调节机能有待进一步完善加强。所以在超数排卵时青年牛不如经产牛对药物反应强烈。要使青年牛的超数排卵达到预期的…     

牛冷冻胚胎移植的探索与应用

用不同的冷冻方法对胚胎质量和移植成功率进行比较，常规冷冻法胚胎可用率高达８０．２６％；受体牛与供体牛发情同期化越接近，胚胎质量越好，移植成功率越高。３年来共移植冷冻胚胎１５２头牛，受胎６７头，冷冻胚胎移植成功率平均４４．０８％。     

提高娟姗奶牛体内胚胎生产效率的试验

试验选用10头娟姗牛作为供体牛,研究营养水平、FSH品牌、子宫内环境等对娟姗牛体内胚胎生产效率的影响,结果表明营养中等以上、进口FSH、输精前对子宫进行净化等可以提高供体牛体内胚胎生产效率。     

影响牛胚胎移植效果的因素分析

近年来,胚胎移植技术在我国发展迅速,并在生产中得到了一定的应用,但由于费用比较高,且移植成功率受到很多因素影响,使其推广受到了很大限制。本文就牛胚胎移植过程中影响其移植成功率的几个方面因素,包括供体和受体牛的选择、供体和受体牛的同期发情程度、胚胎质量、移植者的技术操作水平、受体牛的饲养管理以及移植季节等作一综述。     

小鼠胚胎冷冻保存及复苏的试验研究

为探讨提高人胚胎冷冻的操作技术水平及进行冷冻实验室质量控制的有效方法，采用慢速冷冻及快速融解复苏的方法对小鼠2－细胞胚胎进行冷冻，复苏后培养72h，通过胚胎的囊胚率来评价冷冻技术和进行质量控制。结果表明，本实验在人胚胎冷冻、复苏条件下，共冷冻小鼠胚胎140个，复苏存活率为89.29%，囊胚率为78.57%，获得较为理想的冷冻复苏效果。因此认为应用小鼠2－细胞胚胎进行程序冷冻及复苏是提高人胚胎冷冻技术水平及进行质量控制的良好方法。     

Reduced quality of bovine embryos cultured in media conditioned by exposure to an inflamed endometrium

OBJECTIVE: A suboptimal uterine environment contributes to the bovine repeat breeder syndrome. Subclinical endometritis is a component, so the mechanism by which inflammation affects embryo survival was investigated by assessing the effect of non-cellular products of an inflamed endometrium on embryo development to blastocyst. DESIGN: Endometrial fluid from a lactating dairy cow was collected by lavage using embryo culture medium. Aseptic inflammation was then induced by infusion of glycogen and lavage was repeated 6 h later. The recovered fluid was used to culture Day 5 in-vitro-derived embryos for 2 days. Embryo development and quality were compared for two treatment groups (culture media conditioned by inflamed or non-inflamed endometrium) and two controls (control or control + serum). RESULTS: Development to blastocyst was higher for conditioned media or media + serum (inflamed 42.2%; non-inflamed 49.3%; control + serum 50.9%; control 16.9%; P < 0.001). Blastocyst cell numbers were lower for the conditioned-inflamed group (inflamed, 83.1; non-inflamed, 99.8; control + serum, 100.6; control, 110.1; P < 0.001). Trophectoderm cell number, but not inner cell mass number, was reduced in the conditioned-inflamed group and the inner cell mass:trophectoderm ratio was increased (P < 0.001). CONCLUSION: Altering the embryo culture environment with the products of endometrial inflammation had a subtle effect on embryo quality. An increased inner cell mass:trophoblast ratio is likely to negatively affect embryonic survival. Altered embryo quality is a mechanism for early embryonic failure in cows with subclinical endometritis. Culture of embryos with normal endometrial fluid may improve in vitro embryo production.     

性控精液对奶牛体内胚胎质量、发育和移植妊娠率的影响

[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。     

牛胚胎移植技术的研究与应用

本文总结了牛胚胎移植技术在国内外的研究与发展概况,同时从供、受体母牛的选择与饲养管理、供体的超排、发情鉴定与配种、受体的同期发情与发情鉴定、胚胎的收集与质量鉴定、胚胎的移植等方面对牛胚胎移植技术的具体操作过程作了较详尽地论述,最后阐述了牛胚胎移植存在的问题与发展前景。     

西部农村发展奶牛小区的优势及注意问题

西部农村饲草资源充裕,发展奶牛养殖小区有利于减少草料运输费用,有利于减轻城郊养牛环境污染,有利于改善牛奶质量。本文提出小区建设应注意的五个问题:注重奶牛质量;采用胚胎移植技术,提高小区牛群的个体产奶量:种养结合,建立与奶牛养殖业配套的饲草生产基地;改进饲养管理,推行混合日粮饲养技术;实行行业准入制度,控制小区环境污染,提高牛奶质量,生产绿色奶品。     

超排过程中不同发情时间对胚胎生产效果的影响

在牛胚胎生产过程中,由于不同牛只存在个体差异（如超排月龄、体重、营养状况、卵巢发育情况及初情期月龄等）,其对外界环境的适应能力存在不同的差异;目的：如何有效的控制好供体母牛同期发情的发情时间,提升超排后胚胎的质量与数量。方法：笔者选用10-16月龄的纯种肉用型西门塔尔牛进行胚胎生产,通过对112头次10-16月龄牛只超排进行胚胎生产并对相关数据进行统计分析;结果：根据认真观察记录供体牛只拆栓后的发情期表现,出现部分牛只提前发情或延迟发情现象,其中大部分牛只处于拆栓后24h前后两h集中发情情况;结论：供体母牛在拆栓后24h前后两h集中发情的牛只,其头均生产胚胎的数量与质量均优于延迟发情和提前发情的牛只。     

Identification of key bacterial populations affecting early embryonic development in cattle uterus

Superovulation is an important animal breeding biotechnology, while the quality of embryos obtained from superovulation is unstable in cattle. The relationship between the microorganisms in the cattle uterus and embryo qualities was determined to identify the key bacterial populations affecting early embryonic development. A total of 10 Xia Nan cows underwent superovulation, we collected cervical mucus and flush samples to investigated by 16S rDNA sequencing. Results showed that there were abundant microorganisms in cervical mucus, but no obvious relationship with the quality of embryos. The clustering results of flush samples were consistent with the grouping of embryo quality. Proteobacteria accounted for more than 95% of the total bacterial community in group A with the best embryo quality (qualified embryo ratio above 0.8), and as embryo quality decreased, the Proteobacteria proportion also decreased. In contrast to the proportion of Proteobacteria, the proportions of Firmicutes and Bacteroidetes significantly increased as embryo quality decreased. For group C with the worst embryo quality, the proportions of Firmicutes and Bacteroidetes increased to 4.7 times and 12.3 times of group A, respectively. These results showed that the quantities and proportions of Firmicutes and Bacteroidetes may be related to early embryonic development in cattle.     

Beneficial Effect of Two Culture Systems with Small Groups of Embryos on the Development and Quality of In Vitro‐Produced Bovine Embryos

Currently, in vitro‐produced embryos derived by ovum pick up (OPU) and in vitro fertilization (IVF) technologies represent approximately one‐third of the embryos worldwide in cattle. Nevertheless, the culture of small groups of embryos from an individual egg donor is an issue that OPU‐IVF laboratories have to face. In this work, we tested whether the development and quality of the preimplantation embryos in vitro cultured in low numbers (five embryos) could be improved by the addition of epidermal growth factor, insulin, transferrin and selenium (EGF‐ITS) or by the WOW system. With this aim, immature oocytes recovered from slaughtered heifers were in vitro matured and in vitro fertilized. Presumptive zygotes were then randomly cultured in four culture conditions: one large group (LG) (50 embryos/500 μl medium) and three smaller groups [five embryos/50 μl medium without (control) or with EGF‐ITS (EGF‐ITS) and five embryos per microwell in the WOW system (WOW)]. Embryos cultured in LG showed a greater ability to develop to blastocyst stage than embryos cultured in smaller groups, while the blastocyst rate of WOW group was significantly higher than in control. The number of cells/blastocyst in LG was higher than control or WOW, whereas the apoptosis rate per blastocyst was lower. On the other hand, the addition of EGF‐ITS significantly improved both parameters compared to the control and resulted in similar embryo quality to LG. In conclusion, the WOW system improved embryo development, while the addition of EGF‐ITS improved the embryo quality when smaller groups of embryos were cultured.     

Effect of Embryo Density on In vitro Developmental Characteristics of Bovine Preimplantative Embryos with Respect to Micro and Macroenvironments

To overcome developmental problems as a consequence of single embryo culture, the Well of the Well (WOW) culture system has been developed. In this study, we aimed to examine the effect of embryo densities with respect to both microenvironment and macroenvironment on developmental rates and embryo quality to get a deeper insight into developmentally important mechanisms. WOW diameter and depth significantly affected developmental rates (p < 0.05). WOWs with diameter of 500 μm reached significantly higher blastocyst rates (32.5 vs 21.1% vs 20.3%) compared to embryos cultured in WOWs of 300 μm diameter or plain cultured controls. Embryos cultured in WOWs with 700 μm depth reached significant higher developmental rates compared with embryos cultured in WOWs of 300 μm depth and control embryos (30.6 vs 22.6% vs 20.3%). Correlation of the embryo per WOW volume with developmental rates was higher (r² = 0.92, p = 0.0004) than correlation of WOW diameter or WOW depth with developmental rates. However, the embryo per WOW volume did not affect differential cell counts. An embryo per culture dish volume of 1 : 30 μl was identified to be optimal when the embryo per WOW volume was 1 : 0.27 μl increasing developmental rates up to the level of mass embryo production. Giving the opportunity to track each embryo over the complete culture period while keeping high developmental rates with normal mitotic dynamics, the results of this work will provide benefit for the single culture of embryos in human assisted reproduction, mammalian embryos with high economic interest as well as for scientific purpose.     

Japanese Society for Animal Reproduction: award for outstanding research 2002. The development and prevalence of the transfer technique for frozen-thawed embryos of Japanese black beef cattle in Tochigi Prefecture

The conditions of embryo transfer by the stepwise method, in which frozen-thawed embryos are transferred on day 7 (day 0=onset of estrus), were investigated with the aim of increasing pregnancy rates in frozen-thawed embryo transfer. The use of a vaginal speculum to prevent bacterial infection when passing an embryo transfer gun through the vagina yielded a pregnancy rate equal to or higher than that with application of a sheath cover to the transfer gun. Administration of a sedative, xylazine, to recipient cattle for preventing movement at the time of embryo transfer improved the pregnancy rate. The influence of the time from thawing of frozen embryos to transfer and of the transportation of the recipient by truck upon pregnancy rate was investigated. Embryo transfer within 60 minutes after aspiration into a straw or transportation of the bovine recipient, 1.5 hours each way before and after transfer, had no influence on pregnancy rate. Relations of the embryonic developmental stage and morphological quality after thawing of frozen embryos to pregnancy rate were investigated in recipients of nulliparous Holstein heifers. The pregnancy rate increased as the embryonic developmental stage advanced from compacted morula, early blastocyst, and blastocyst in that order. The pregnancy rate obtained with blastocyst stage embryos was significantly (P<0.05) higher than that with compacted morula stage embryos, and there was no significant difference in pregnancy rates between excellent morphological quality and good morphological quality for compacted morula stage embryos. When correlation of luteal function and pregnancy rate was investigated in bovine recipients, pregnancy rate showed a tendency to increase with increasing blood progesterone (P) concentration on the day before (on day 6 after estrus) and the day of embryo transfer. The pregnancy rate in bovine recipients, which showed a blood P concentration of > or =2.5 ng/ml on the day before embryo transfer, was significantly (P<0.05) higher than that in those with a blood P concentration of <2.5 ng/ml. Pregnancy rate showed a tendency to increase with decreasing blood estradiol-17beta (E2) concentration on the day of embryo transfer. Activation of luteal function by administration of human chorionic gonadotropin (hCG) in cycling cattle was investigated for its effect on increasing pregnancy rate in bovine recipients. A follicle coexisting with cyclic CL ovulated and induced CL formed after injection of hCG 1,500 IU 5 days after ovulation. The blood P concentration was significantly (P<0.05) higher in the administration group than in the control group, and the blood E2 concentration rapidly decreased, showing a lower concentration than in the control group. These results suggest the possibility that the pregnancy rate could be improved by administration of hCG. Pregnancy rate following intramuscular injection hCG 1,500 IU was comparatively investigated in parous Japanese Black beef cattle receiving frozen-thawed embryos 7 days after estrus. Pregnancy rate was 67.5% in the group in which hCG was administered on day 6 after estrus, and was significantly (P<0.05) higher than that in the control group (45.0%) and the group in which hCG was administered on day 1 after estrus (42.5%), revealing that hCG administration facilitated pregnancy. Transfer of frozen-thawed embryos in the blastocyst stage within 60 minutes after the aspiration into a straw, with a vaginal speculum after administration of xylazine is suggested as a way of improving pregnancy rate in bovine recipients with favorable luteal function and in those with luteal function activated by administration of hCG on the day before embryo transfer.