重复手术法收集超数排卵后的家兔卵母细胞

采用促卵泡素(FSH)和FSH混合25%聚乙烯吡咯烷酮(PVP)两种方法进行超数排卵,以正常排卵母兔作对照,经过3次重复剖腹手术(每次间隔至少50天以上)收集家兔卵母细胞,旨在探索大量获取家兔成熟卵母细胞简单有效的方法。结果超数排卵的两组获取总卵母细胞数和可用卵数比对照组均高出3倍以上(P=0.001),两种超数排卵效果无显著差异(P>0.1)。3次连续手术均成活的家兔中,对照组和FSH组3次取卵效果无显著差异(P>0.05),FSH混合25%PVP组首次手术与第三次手术卵母细胞数出现显著差异(P<0.01)。本方法可有效获取大量家兔卵母细胞,并提高实验动物利用率。     

绵羊卵母细胞采集、体外成熟和胚胎体外培养对体外受精技术效率的影响

利用屠宰场采集的绵羊卵巢作为试验材料,研究了不同卵母细胞采集方法(卵泡冲洗法、剖切法、注射器抽吸法和真空泵抽吸法)、成熟液中添加不同来源激素(BIONICHE或宁波激素厂生产 FSH/LH)和血清(发情绵羊血清或胎牛血清),以及mSOF和mCR胚胎培养体系对绵羊体外受精各环节效率的影响。结果表明,卵泡冲洗法获得A、B两级卵母细胞比例为77.1%,显著高于其他3种方法(P＜0.05),添加BIONICHE FSH/LH+ESS成熟液中,卵母细胞成熟率显著高于其他添加方式(P＜0.05),mSOF和mCR胚胎培养体系在卵裂率上无显著差异(P＞0.05),但mSOF组中囊胚率和孵化率均显著高于mCR组(P＜0.05)。综上所述,本研究中卵泡冲洗法更适合绵羊卵母细胞采集,成熟液中添加BIONICHE FSH/LH和ESS可显著促进绵羊卵母细胞成熟;与mCR培养体系相比,mSOF培养体系更适合绵羊体外受精胚胎的发育。     

Effects of Repeated Embryo Collection on Embryo Recovery Rate in Fertile Mares

In mares, repeated embryo collection in successive oestrous cycles is necessary if a greater number of foals should be produced. We investigated effects of repeated embryo collection in fertile donor mares on embryo recovery rates. In addition, an influence of the individual mare and season on embryo recovery rates was studied. In nine mares, a total of 153 embryo collections were performed during 30 months (17 ± 2.2 embryo collections per mare). The overall embryo recovery rate was 64% and did not differ among mares. Between successive embryo collection procedures, recovery rate varied significantly; however, no increase or decrease in the embryo recovery rate with increasing number of successive embryo collections was seen. In three mares, ovulation ceased from November to February. In the remaining six mares, embryo production was successfully continued throughout winter and no influence of the month on embryo recovery rates was detected.     

In Vivo Embryo Recovery Rate by Laparoscopic Technique from Rabbit Does Selected for Growth Rate

Rabbit does from R line selected for growth rate present a low reproductive performance and this study aimed to evaluate both the recovery efficacy and viability of recovered embryos after vitrification and the reproductive performance of donor does subjected to in vivo recovery. Does were divided into three groups: 28 does without in vivo recovery (control), 25 does in which in vivo recovery was started in the nulliparous state (group 1) and 30 does with at least one litter before in vivo recovery (group 2). Does were superovulated with a single subcutaneous injection of 50 IU of equine chorionic gonadotropin (eCG) per female, and were then artificially inseminated 60 h later and immediately administered an intravenous dose of 75 IU of human chorionic gonadotropin (hCG) per female. Does from group 1 and 2 were recovered in vivo 76-80 h post-insemination by repeated laparoscopies at one to four times and permitted one or two parturitions between recoveries [in vivo (IV) recovery]. At the end of the experiment, about 16 does of all groups were recovered post-mortem (PM recovery). All normal embryos were vitrified, devitrified and then cultivated in vitro to evaluate the viability after thawing. A significant increase in the ovulation rate was found in does recovered PM than in those recovered IV in the nulliparous state. However, no significant differences were observed in the recovery rate, the donor rate, the number of normal embryos recovered with at least one normal embryo per doe and the viability after thawing between the PM and IV groups. A significant decrease in the fertility rate, total born, live born and weaned kids was found for does from group 1 in comparison with does from group 2. Results support the use of repeated laparoscopy to increase the number of recovered embryos per donor doe especially in such R line does, if they are permitted to produce at least one litter before the beginning of in vivo recovery.     

季节对杜泊羊体内胚胎生产与移植效果的影响

为了研究季节对杜泊羊体内胚胎生产效率及胚胎移植妊娠率的影响,于2016年选取内蒙古赛诺草原羊业有限公司种羊场经产纯种杜泊母羊作为供体,采用同期发情、超数排卵及腹腔镜人工输精等方法对供体羊进行处理后,利用手术冲胚的方法获得纯种杜泊羊胚胎,同时对受体羊进行同期发情处理和胚胎移植。经统计分析,2016年全年该公司共有放栓供体4 241只,超排处理4 180只,配种供体4 131只,冲胚供体3 987只,供体可用率为94.01%;冲出胚胎总数为23 516枚,可用胚胎18 002枚,胚胎可利用率为76.55%,平均每只供体能获得可用胚胎4.52枚。供体在10月、11月、12月冲胚所获得的平均冲胚数和平均可用胚数明显高于4月、5月、6月(P<0.05),其中10月的平均冲胚数和平均可用胚数最高(P<0.01),分别为7.04枚和5.62枚。将获得的部分胚胎用于胚胎移植,移植单胚怀孕受体数为4 446只,妊娠率为53.67%;移植双胚怀孕受体数805只,妊娠率65.02%。此外,受体在1月、3月、6月、12月受胎率较其他月份高(P<0.05)。该研究可为杜泊种羊超数排卵、胚胎移植和肉羊的产业化生产提供一定的数据参考。     

浅析牛体内胚胎生产的影响因素

体内胚胎生产是胚胎移植的重要组成部分。最近几年,体内胚胎生产结合胚胎移植技术在良种牛快速扩繁领域里发挥了重要作用,但在生产中,效果仍不稳定。国内外很多学者对提高牛体内胚胎生产的稳定性做了大量研究,本文就国内外专家学者对影响牛体内胚胎生产的外在和内在因素进行了分析总结。     

Effect of Leptin on In Vivo Goat Embryo Production

The aim of this study was to evaluate the effect of leptin administration during superovulation on in vivo goat embryo production. Ten mature does were superovulated with 133 mg follicle‐stimulating hormone (FSH) i.m. in six descending doses at 12‐h intervals. The goats received 4.8 μg/kg human recombinant leptin s.c. (leptin group, n = 5) or phosphate‐buffered saline (PBS) (control group, n = 5) with the first and second FSH doses. The does were mated and subjected to embryo collection by transcervical technique 6 days later. The total number of cells per embryo and the number of cells with fragmented DNA were assessed in selected blastocysts by combining Hoechst 33342 and terminal dUTP nick‐end labelling (TUNEL) staining. Plasma concentrations of oestradiol (E₂) and progesterone (P₄) were determined by electrochemiluminescence from the day of FSH treatment, on the day of superovulatory oestrus and on the day before embryo collection. Compared with the control group, the does that received leptin had a higher number of transferable embryos (p < 0.005), fewer embryos classified as degenerated (p < 0.001) and fewer TUNEL‐positive cells/blastocyst (p < 0.001). The number of transferable embryos was positively correlated with E₂ concentrations on day of oestrus (r = 0.562; p < 0.01) and P₄ concentrations on the day of embryo collection (r = 0.912; p < 0.001). We concluded that in vivo leptin administration during FSH treatment improved embryo quality and affected ovarian steroidogenesis in superovulated goats.     

Procedure for Maximizing Oocyte Harvest for In Vitro Embryo Production in Small Ruminants

Possible effects of repeated hormonal treatments and laparoscopic ovum pick-up (LOPU) on the efficiency of oocyte recovery rate and quality were determined in sheep and goats. In six adult Merino sheep and five Criolla goats, ovarian status was synchronized by a prostaglandin F(2 alpha) analogue and the insertion of an intravaginal sponge 48 h later. Follicle development was stimulated by a single dose of FSH (60 mg NIH-FSH-P1) plus a single dose of equine chorionic gonadotrophin (eCG; 300 UI). The first FSH/eCG doses were administered 48 h after the sponge insertion, being repeated every 4 days to complete a total of four treatments in sheep and three in goats. Follicles in both ovaries were categorized according to their diameter and follicular fluid was aspirated under laparoscopic observation without a vacuum pump. In sheep, during a 12-day-period, a total of 347 follicles were aspirated with a recovery rate of 46.9%. In goats, during an 8-day-period, 219 follicles were aspirated with a recovery rate of 45.6%. In both species, there were no significant differences in the number of aspirated follicles, oocyte recovery rate and good quality oocyte recovery rate. However, in sheep the oocyte recovery rate was higher for large follicles, whereas in goats no such effect was detected. In summary, current results indicate that retrieval of oocytes can be maximized, without affecting oocyte quality, by repeating 'oneshot' FSH/eCG regimes and LOPUs at intervals as short as 4 days.     

Nonsurgical Embryo Collection in Rabbits

Contents: Attempts were made to collect nonsurgically rabbit embyos. The experiments were carried out on 70 superovulated white New Zealand does. 54–55 hours after mating prostaglandins were administered. Ernbyos were expulsed into the vagina in 40–75% of the females. The percent of reacting females and the percent of embryos expulsed depended on the kind and dose of used PGF_2α. Enzaprost applicated in doses of 2–3 ml resulted in approximately 90% reacting females and approximately 40% flushed embyos. The collected embyos developed both in vitro and in vivo at a similar percent level as in the control group .
Inhalt: Unblutige Embryogewinnung beim Kaninchen
Versuche zur unblutigen Embryogewinnung wurden an 70 superovulierten Weiβen Neuseeländer-Häsinnen durchgeführt. 54 bis 55 Std. nach dem Belegen wurden Prostaglandin-F_2α-Präparate appliziert. Bei 40–75% der Häsinnen wurden Embyonen in die Vagina ausgestoβen. Der Anteil positiv reagierender Weibchen und der Prozentanteil ausgestoβener Embyonen war von der Dosis und dem verwendeten Prostaglandin-Praparat abhängig. Nach 2–3 ml Enzaprost reagierten etwa 90% der Häsinnen und 40% der Embyonen wurden ausgespült. Die Weiterentwicklung dieser Embyonen in vitro und in vivo war ahnlich wie jene von Kontrolltieren .     

Effect of Days Post-Partum, Breed and Ovum Pick-Up Scheme on Bovine Oocyte Recovery and Embryo Development

The objective of this study was to investigate (i) the effect of two different ovum pick-up (OPU) schemes (once vs twice weekly aspirations) on oocyte recovery rate, quality and subsequent in vitro embryo development, (ii) the influence of days post-partum on oocyte recovery and (iii) possible differences in OPU results from two different herds. In group A, OPU was performed twice weekly in two Holstein Friesian (HF) and three Danish Red and White (DRW) cows from a private herd. In the research herd, two groups of eight HF cows were investigated: group B (OPU once weekly) and group C (OPU twice weekly). The collected oocytes were subsequently submitted to in vitro embryo production. More oocytes were recovered from the private herd when compared with the research herd. In the research herd, the twice weekly scheme aspirated more oocytes than the once weekly scheme. The quality of the retrieved oocytes was significantly different between groups B and C but not between groups A and C, and HF cows yielded higher quality oocytes than DRW cows (p = 0.029). Oocytes from group C showed higher level of embryonic development than group B oocytes. No differences in blastocyst rates were observed between groups A and C. Session affected the number of retrieved oocytes and subsequent developmental rates, with these being lower in the first compared with the last sessions. Finally, there was no significant effect of days post-partum in the number and quality of the retrieved oocytes, likely because of the small group size and high variation between sessions.     

乙酰荧光素(FDA)在小鼠和猪卵母细胞活力评价上作用

以小鼠卵母细胞和猪卵母细胞为材料研究了FDA活力检测上的应用。结果表明：1)受精卵和卵母细胞均有积聚FDA(乙酰荧光素)的能力；2)猪卵巢在不同温度保存之后，其卵母细胞的积聚FDA(乙酰荧光素)的能力有明显改变。这些结果表明，可以用积聚FDA(乙酰荧光素)的能力检测卵母细胞的活力。     

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。     

家兔超数排卵与早期胚胎发育研究

为了提高家兔繁殖力,使用不同剂量的FSH对家兔进行了超数排卵研究,结果表明：采用高剂量FSH处理母兔的平均排卵点数与平均卵泡发育数显著（P〈0.05）高于低剂量FSH组,两种处理方法的平均卵泡囊肿数与平均卵泡数则差异不显著（P〉0.05）。家兔早期胚胎发育观察结果表明：母兔交配后24h采胚所获取的胚胎大多处于1细胞期,交配后30h胚胎多处于2细胞期,交配后35h胚胎多处于4细胞期。采用52IU FSH进行超数排卵处理,对家兔的胚胎发育无影响。     

家兔超数排卵与早期胚胎发育研究

为了提高家兔繁殖力,使用不同剂量的FSH对家兔进行了超数排卵研究,结果表明:采用高剂量FSH处理母兔的平均排卵点数与平均卵泡发育数显著(P＜0.05)高于低剂量FSH组,两种处理方法的平均卵泡囊肿数与平均卵泡数则差异不显著(P＞0.05).家兔早期胚胎发育观察结果表明:母兔交配后24 h采胚所获取的胚胎大多处于1细胞期...     

家畜卵母细胞和胚胎去脂方法研究进展

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。     

Dynamic Study on Nuclear Pore Complexes in Swine Oocyte Matured In Vivo and In Vitro Using Field-Emission Scanning Electron Microscopy

Veterinary Research Communications -     

Endoscopic Recovery of Early Preimplantation Bovine Embryos: Effect of Hormonal Stimulation,Embryo Kinetics and Repeated Collection

The collection of extra numbers of bovine embryos by superstimulation of donors underlies variation concerning yield of morulae and blastocysts. Our study aimed at establishing a correlation between hormonal treatment and embryo development during oviductal passage including repeated flushing. A transvaginal endoscopic procedure was used to flush the oviducts at six different time intervals (beginning at 24 h until 105 h) after artificial insemination. In total, 119 animals were superovulated using either FSH or eCG. The hormonal treatment resulted in the stimulation of 2076 follicles of which 77% (1590 CL) ovulated. The bilateral flushing resulted in the collection of 1411 complexes (collection rate: 89%), of which 78% (1098) were assessed as viable embryos. The use of FSH resulted in significantly more stimulated follicles and ovulation sites compared with eCG (p < 0.001). Generally, the embryo kinetics were similar among the FSH and eCG treated animals. However, the embryo cleavage of the eCG treated animals was ahead of that of the FSH group comparing the different collection time points. The overall proportions of non‐viable embryos in both groups were similar. Regarding the embryo collection intervals in the eCG group, this proportion significantly increased during 51–105 h compared to 24–50 h (p < 0.05), whereas FSH delivered constant results. It was shown that the repeated endoscopic collection of oviductal stage embryos had no negative influence on the collection parameters. It is concluded that the introduced transvaginal endoscopic technique could have main impact on further studies focusing on early embryo development.     

猪早期胚胎体内、外发育规律研究

系统地掌握猪早期胚胎体内外发育规律是进行猪胚胎冷冻、胚胎分割、鲜胚移值、长途运输引种以及基因转移等生物技术研究和应用的基础。本研究包括２个实验，实验１，利用８９头超排母猪和１９头自然发情母猪，于配种后第２、３、４、５、６、７、８、９和１０天手术采得胚胎１６１２枚，系统地观察猪早期胚胎体内发育规律，即：２日龄胚胎中原核胚占７７．３９％（３９７／５１３），３日龄胚胎中４和６细胞胚占７９．０５％（８３／１０５），４日龄胚胎中４～８细胞占４０％（３５／７６），５日龄胚胎多为桑椹和囊胚８３．１２％（６４／７７），６日龄胚多为膨胀囊胚４２．０９％（１８１／４３０）和孵化囊胚３４．１９％（１４７／４３０），７和８、９、１０日龄胚发育到孵化囊胚分别为７６．３４／ｏｏ（７１／９３）和１００％（７７／７７），而自然发情的５日龄胚胎中桑椹和囊胚仅占３２．５％（１３／４０），６日龄胚胎中膨胀囊胚仅占１１．１１％（１７／１５３），孵化胚仅占１９．６１％（３０／１５３）。各发育阶段的胚胎比例均明显低于５、６日龄超排供体（Ｐ＜０．０５）。实地２，将１３８枚囊胚和膨胀囊胚，分别体外培养２４和１２小时，囊胚发育阶段的胚胎５０％（４８／９６）?