山羊超数排卵与胚胎移植技术的研究与应用

本研究于 2 0 0 0年 9月～ 2 0 0 3年 11月分别对 883只 (次 )波尔山羊、95只黑山羊、5 2只努比羊和 37只波黑杂交羊采用孕激素 FSH法进行超排处理 ,对 1190 0只受体黑山羊埋置CIDR或孕酮海绵栓并分别注射Folligon的方法进行同期发情处理 ,通过对不同品种、季节、年龄和重复超排对山羊超排反应的影响 ,胚胎分割、胚龄和黄体状况对移植妊娠率和产羔率的影响及不同孕激素制剂对同期发情效果的影响等因素进行分析 ,为山羊胚胎移植产业化提供理论和技术依据     

奶牛胚胎移植中GnRH、PGF2α同期发情处理效果的比较

同期发情处理是牛胚胎移植中一项重要的技术,不同的方法因不同的环境条件产生不同的效果。本文就同期发情的不同方法进行了比较,以寻找适合当地条件的曲 情方案。１９３头健康,经产的黑白花奶牛分为３个组,用３种不同的方法进行同期发情处理和胚胎移植。第１组;注射１次Ｌｕｔａｌｙｓｅ;第２组;注射２次Ｌｕｔａｌｙｓｅ;第３组：注射Ｆｅｒｔａｇｙｌ－Ｌｕｔａｌｙｓｅ－Ｆｅｒｔａｇｙｌ。     

天祝白牦牛胚胎移植实验研究

选用健康成年天祝母白牦牛(Poephagus grunniens)(供体)10头、黑牦牛(受体)37头进行实验. 供体牛(组1)用CIDR-B+FSH＋PG进行同期发情和超数排卵; 受体牛(组2和组3)分别采用CIDR-B+PG和单次注射PG法进行同期发情. 结果表明, 平均黄体数、卵泡数、可移植胚胎数和退化胚胎数分别为4.75±2.19, 1.13±0.83, 2.50±1.31和1.38±0.92. 在3组中, 组1的同期发情率最高, 为80%, 组2和3分别为60%和45.5%. 处理后不同组别的发情开始时间不同, 组1的大部分白牦牛在处理结束后的25~75 h发情, 而组2和3的受体黑牦牛分别在73~96和49~72 h发情. 平均胚胎回收率55.6%. 共收集18枚可用胚胎, 将其中12枚移植到10头同期发情的受体牦牛, 最终妊娠率50%, 分娩率40%.     

母畜同期发情激素使用的探讨

针对高中生物学教学中同期发情激素使用的问题,对家畜的自然发情机理以及在此基础之上衍生的同期发情机理给予了介绍,说明了进行母畜同期发情使用的激素是孕激素或前列腺素;而促性腺激素的配合使用能促使母畜发情有较好的准确性和同期性。     

云南省奶牛胚胎移植试验

实验用12头黑白花奶牛（3-9岁）,PGF[2α]作同步发情处理后,周期第10-12天,总剂量36 mg的FSH-P按递减方式每日肌肉注射2次,间隔12小时,共注射4天,在第3天加注PGF[2α]35 mg,进行超数排卵处理。12头供体牛共得胚135枚,可用胚数118枚,为总胚数的87.47%。平均每头得胚11.25枚。胚胎分别移植给20头同步发情处理的黑白花奶牛和20头黄牛受体（有3头为自然发情）,每头移植1枚胚胎。结果,奶牛受孕率为35%（受孕7头,产犊7头）;黄牛受孕率为40%（受孕8头,流产2头,产犊6头）。奶牛和黄牛总受孕率为37.5%。     

胚胎工程中激素选择的生理学解析

从生理学的角度解析了家畜发情机制及胚胎工程中用孕激素进行同期发情处理和用促性腺激素进行超数排卵的原因.     

不同日龄小鼠的超排、体外授精及二细胞胚胎移植的比较研究(简报)

小鼠是现代生物医学中应用最为广泛的实验动物,特别是随着基因工程动物的大量开发,对小鼠卵母细胞和小鼠胚胎的需求量将日趋增多。但是如果单纯依靠动物的自然排卵,一方面所得到的卵母细胞数或胚胎数较少,一方面又消耗大量的时间和浪费较多的动物。因此,研究者期望使用较少的动物但仍能得到较多的、正常的卵母细胞或胚胎来满足生命科学研究的需要。Onadera,M等的研究表明,小鼠卵母细胞的发育与年龄有关,同一品系小鼠不同日龄间的自然排卵数差异不显著,出现异常卵的比例有显著性差异。但对不同日龄、不同品系小鼠的超排的效果尚     

小鼠超数排卵优化试验方案研究

探讨建立一种适合贵州地区、高效、稳定的小鼠超数排卵优化方案。在饲养环境相同的基础上,对激素(PMSG, hCG)不同的剂量组合、注射间隔时间、小鼠周龄等影响因素进行了相关研究。试验结果表明:(1)平均采胚数量组间、平均异常胚组间与平均可用胚组间差异显著(p<0.05),注射10 IU的激素剂量组合获得受精卵最多,且异常胚最少,效果最佳。(2)第1、第2、第3组平均采胚数量组间差异显著(p<0.05),第1组与第2组平均可用胚组间差异不显著(p>0.05),但第1、2组与第3组差异显著(p<0.05),异常胚组间差异不显著(p>0.05),选择4周龄超排效果最佳。(3)第1、第2、第3组平均采胚数量、平均可用胚组间差异显著(p<0.05),平均异常胚组间差异不显著(p>0.05),PMSG,hCG间隔注射时间为48 h为最佳。     

孕马血清诱导猕猴超数排卵试验

1930年H.H.Cole等发现马妊娠期某一阶段血清内含有促性腺激素,其中激素之一就是促卵泡生成素。至今孕马血清在畜牧业上已被广泛地用于诱导排卵和增加多胎率。     

不同日龄小鼠超排、体外授精及二细胞胚胎移植的比较研究（摘报）

The development of oocytes, superovulated at 28, 56 and 112 days in three mice strains (ICR, B6C3F1, and C57BL/6J) with PMSG and HCG, were examined using the techniques of in vitro fertilization, culture, and transfer of these two-cell embryos to pseudopregnant recipients. The highest numbers of oocytes were obtained from superovulated 28-day-old mice in three strains. Approximately 90% of oocytes developed to the two-cell stage after in vitro fertilization, and about 50% became pregnant through the recipients. These results suggested that donor age at 28 days had prominent discrepancy with 56 and 112-day-old mice (P < 0.01) in oocytes superovulation, and no influence on the rate of insemination and pregnancy.     

Comparison of progesterone-based protocols with gonadotropin-releasing hormone or estradiol benzoate for timed artificial insemination or embryo transfer in lactating dairy cows

The objective was to compare two protocols for synchronizing ovulation in lactating Holstein cows submitted to timed AI (TAI) or timed ET (TET). Within each farm (n = 8), cows (n = 883; mean ± SEM 166.24 ± 3.27 d postpartum, yielding 36.8 ± 0.34 kg of milk/d) were randomly assigned to receive either: 1) an intravaginal progesterone insert (CIDR^®) with 1.9 g of progesterone + GnRH on Day -10, CIDR^® withdrawal + PGF2α on Day -3, and 1 mg estradiol cypionate on Day -2 (treatment GP-P-E; n_TAI = 180; n_TET = 260); or 2) a CIDR^® insert + 2 mg estradiol benzoate on Day -10, PGF2α on Day -3, CIDR^® withdrawal + 1 mg estradiol cypionate on Day -2 (treatment EP-P-E; n_TAI = 174; n_TET = 269). Cows were subsequently randomly assigned to receive either TAI on Day 0 or TET on Day 7. Serum progesterone concentration on Day -3 was greater in GP-P-E than in EP-P-E (2.89 ± 0.15 vs 2.29 ± 0.15 ng/mL; P < 0.01), with no significant effect of group on serum progesterone on Day 7. Compared to cows submitted to TAI, those submitted to TET had greater pregnancy rates on Day 28 (44.0% [233/529] vs 29.7% [105/354]; P < 0.001) and on Day 60 (37.6% [199/529] vs 26.5 [94/354]; P < 0.001). However, there were no effects of treatments (GP-P-E vs EP-P-E; P > 0.10) on synchronization (87.0% [383/440] vs 85.3% [378/443]), conception (TAI: 35.3% [55/156] vs 33.8% [50/148]; TET: 50.7% [115/227] vs 51.3% [118/230]) and pregnancy rates on Days 28 (TAI: 30.5% [55/180] vs 28.7% [50/174]; TET: 44.2% [115/260] vs 43.9% [118/269]) and 60 (TAI: 27.2% [49/80] vs 25.9% [45/174]; TET: 38.8% [101/260] vs 36.4% [98/269]). In conclusion, GP-P-E increased serum progesterone concentrations on Day -3, but rates of synchronization, conception, and pregnancy were not significantly different between cows submitted to GP-P-E and EP-P-E protocols, regardless of whether they were inseminated or received an embryo.     

胚胎冷冻技术应用于抗菌肽转基因小鼠的保种传代

目的研究胚胎冷冻在抗菌肽转基因FVB小鼠保种传代中的应用。方法对6~8周正常雌性FVB小鼠进行超排分别与雄性杂合子抗菌肽转基因FVB小鼠交配,收集2-cell胚胎,进行胚胎冷冻。1周后进行胚胎复苏移植,通过PCR方法对仔代鉴定。结果冻存胚胎140枚,复苏获得存活胚胎98枚,移植85枚,产仔38只,获得阳性后代12只。结论通过胚胎冷冻技术保种及复苏移植技术可对抗菌肽转基因小鼠进行传代。     

Forty years of embryo transfer in cattle: A review focusing on the journal Theriogenology, the growth of the industry in North America,and personal reminisces

After the first successful transfer of mammalian embryos in 1890, it was approximately 60 years before significant progress was reported in the basic technology of embryo transfer (ET) in cattle. Starting in the early 1970s, technology had progressed sufficiently to support the founding of commercial ET programs in several countries. Today, well-established and reliable techniques involving superovulation, embryo recovery and transfer, cryopreservation, and IVF are utilized worldwide in hundreds, if not thousands, of commercial businesses located in many countries. The mean number of embryos produced via superovulation has changed little in 40 years, but there have been improvements in synchrony and hormonal protocols. Cryopreservation of in vivo-derived embryos is a reliable procedure, but improvements are needed for biopsied and in vitro-derived embryos. High pregnancy rates are achieved when good quality embryos are transferred into suitable recipients and low pregnancy rates are often owing to problems in recipient management and not technology per se. In the future, unanticipated disease outbreaks and the ever-changing economics of cattle and milk prices will continue to influence the ET industry. The issue of abnormal pregnancies involving in vitro embryos has not been satisfactorily resolved and the involvement of abnormal epigenetics associate with this technology merits continued research. Last, genomic testing of bovine embryos is likely to be available in the foreseeable future. This may markedly decrease the number of embryos that are actually transferred and stimulate the evolution of more sophisticated ET businesses.     

中国的动物胚胎技术研究进展

胚胎移植技术最早是由英国的Walter Heape率先研究的,随后经过了胚胎学家几十年的努力才在上个世纪来期逐渐开始广泛应用于医学生殖,畜牧业,动物保种等领域。本文集中论述中国的胚胎移植技术发展以及它在畜牧业领域的应用。在中国商业性的动物胚胎移植正在蓬勃发展,其成功率已经和国外同行相差不大。通过胚胎移植工作者的努力,一些从事商业性胚胎移植的公司已经能够做到鲜胚移植后奶牛的妊娠率55％～70％,冷冻胚胎的妊娠率达到45％～60％。不仅如此,在各个省还建立了自己的专门的冷冻胚胎中心,为本省的畜牧产业提供胚胎或者相关的服务。在动物保种方面,胚胎技术给人们提供了一种全新的思路：通过胚胎技术将活的胚胎冷冻在液氮罐以实现永久保存。例如中国科学院动物所陈大元先生提出通过胚胎移植技术实现对大熊猫的物种保存,并取得了一定的成果。随着经济和社会的进一步发展,人民生活水平的提高对于肉和奶的需求会进一步提高。依靠常规的选育技术来改良牲畜是无法满足人民对于畜牧产品急切要求,要实现畜牧业的跨越式发展,就必须依靠科技的力量来实现畜牧业的大发展,满足人民生活需要。胚胎技术等新技术会为我国的畜牧业的产业提高提供了一个新的机遇,可以相信,在不远的将来,动物胚胎技术必将在中国有一个较大发展。     

Induction of superovulation in South American camelids

The development of assisted reproductive technologies such as embryo transfer (ET), artificial insemination (AI) and in vitro fertilization (IVF) in South American camelids is considerably behind that of other livestock species. Poor success of the embryo transfer technique has been related to a lack of an effective superstimulatory treatment, low embryo recovery rate, and the recovery of hatched blastocysts that are not conducive to the cryopreservation process. Superstimulation has been attempted using equine chorionic gonadotropin (eCG) and follicle stimulating hormone (FSH) during the luteal, or the sexually receptive phase, sometimes given at follicular wave emergence. The rationale for inducing a luteal phase prior to or during superstimulation in camelids is not clearly understood, but it may simply reflect an empirical bias to conventional methods used in other ruminants. The number of ovulations or CL varies widely among studies, ranging from 2 to more than 15 per animal, with the number of transferable embryos ranging from 0 to 4 per animal. The control of follicular growth combined with superstimulatory protocols has resulted in a more consistent ovarian response and a greater number of follicles available for aspiration and oocyte collection. Recent studies in llamas have demonstrated that the use of ovulation inducing treatments or follicle ablation can synchronize follicular wave emergence allowing the initiation of gonadotropin treatment in the absence of a dominant follicle resulting in a more consistent ovulatory response. Few studies in alpacas have been reported, but it appears from recent field studies that the ovarian response is more variable and that there is a greater number of poor responders than in llamas. A review of superstimulation protocols that have been used in llamas and alpacas in the last 15 years is provided, including a discussion of the potential of protocols designed to initiate treatment at specific stages of follicular growth.     

Efficiency of superovulation and in vivo embryo production in eFSH-treated donor mares after estrus synchronization with progesterone and estradiol-17β

Reliable methods of regulating estrus and stimulating superovulations in equine embryo transfer programs are desirable. Our objectives were to investigate the efficacy of a progesterone and estradiol-17β (P&E) estrus synchronization regimen in mares with and without subsequent equine follicle-stimulating hormone (eFSH) treatment and to examine the effects of eFSH on folliculogenesis and embryo production. Cycling mares were treated with P&E daily for 10 d. On the final P&E treatment day, prostaglandin F_2α was administered, and mares were randomly assigned to one of two treatment groups (n = 20 mares/group). In both groups, mares were examined daily by transrectal ultrasonography. In the eFSH group, twice-daily eFSH treatments were initiated at follicle diameter 20 to 25 mm and ceased at follicle ≥35 mm; human chorionic gonadotrophin (hCG) was administered after 36 h. In the control group, eFSH treatments were not given, but hCG was administered at follicle ≥35 mm. Mares were inseminated with fresh semen, and embryo recovery attempts were performed 8 d postovulation. Synchrony of ovulations within each group appeared to be similar. Six mares in the eFSH group failed to ovulate. The eFSH treatment resulted in higher (P < 0.05) numbers of preovulatory follicles and ovulations; however, embryo recovery rate did not increase (eFSH 1.0 ± 0.4 vs. control 0.95 ± 0.1 embryos/recovery attempt), and embryo per ovulation rate was significantly lower (36% vs. 73%). The eFSH-treated mares had significantly higher frequency of nonovulatory follicles (28% vs. 0) and higher periovulatory serum concentrations of estradiol-17β. Based on our findings, combined P&E and eFSH regimens cannot be recommended for cycling donor mares.     

The cryoprotective effect of trehalose supplementation on boar spermatozoa quality

The objective was to compare the reproductive performances associated with the first (Cycle-1), second (Cycle-2), and mid-season (MS-Cycle) ovulations of the breeding season in donor mares that were treated with equine-FSH (eFSH) in the early vernal transition. Mares (n = 15) kept under ambient light were examined ultrasonographically per-rectum starting January 30. When an ovarian follicle ≥25 mm in diameter was detected, twice daily eFSH treatments were initiated. The eFSH treatments ceased when a follicle ≥35 mm was detected, and 36 h later hCG was administered. Thereafter, mares were artificially inseminated every 48 h until ovulation (Day 0). Trans-cervical embryo recovery attempts were performed on Day 8, and subsequently PGF2α was administered. Equine FSH was not administered in the subsequent estrous cycles. In Cycle-2 and in the MS-Cycle, hCG was administered when a follicle ≥35 mm was detected; breeding, embryo recovery, and PGF2α administration, were similar to Cycle-1. Mares had an untreated estrous cycle (no treatment or breeding) between Cycle-2 and the MS-Cycle. All mares developed follicle(s) ≥35 mm after 4.9 ± 0.6 days of eFSH treatment, and subsequently ovulations occurred; mean (95% CI) interval from treatment initiation to ovulation was 7.9 (6.5–9.3) days. The number of preovulatory follicles (≥30 mm) at the time of hCG administration (Cycle-1: 2.2 ± 0.3 compared with Cycle-2: 1.0 ± 0 compared with MS-Cycle: 1.1 ± 0.1 follicles), and the number of ovulations (2.5 ± 0.4 compared with 1.0 ± 0 compared with 1.1 ± 0.1 ovulations) were greater (p < 0.05) in Cycle-1. Nevertheless, mean embryo numbers did not differ among cycles (0.8 ± 0.2 compared with 0.5 ± 0.1 compared with 0.5 ± 0.1 embryo/mare). On average, embryo morphology grade was less (p < 0.05) in Cycle-1 as compared to non-eFSH cycles (combined Cycle-2 and MS-Cycle). This impaired embryo quality could be due to a seasonal effect, or negative effect of the eFSH treatment, which was possibly related to alterations in the hormonal environment (estradiol-17β and progesterone). A prolonged IOI (>21 days) was recorded in 7 of 15 mares following the Cycle-1 ovulation, but not subsequently. In conclusion, eFSH treatment of vernal transitional donor mares stimulated ovulation within only few days of treatment, and the following embryo recovery rate was at least as good as in the subsequent estrous cycles; however, on average, embryos were morphologically impaired. In subsequent estrous cycles in the breeding season, ovulations, embryo recovery rates, and embryo variables did not appear to be negatively affected; however, the first inter-ovulatory interval of the breeding season was prolonged in approximately half of the mares.