Blastomere content of cultured/frozen bovine demiembryos.

The purpose of the study was to evaluate whether a period of co-culture with bovine oviduct epithelial cells (BOEC) could improve the tolerance of bisected bovine embryos to freezing and thawing. Day 6 embryos were bisected and the resulting demiembryos were stained with Hoechst 33342 and cell counts were made by counting intact blastomere nuclei. Of these, 11 were stained as freshly manufactured demiembryos, 25 after co-culture for 24 h with BOEC and 37 stained after 24 h co-culture and freezing and thawing. The staining revealed, that there was no significant difference in cell count of demiembryos that were stained immediately after bisection, compared to those, that were co-cultured for a 24 h period. Also, the co-cultured/frozen/thawed demiembryos had a significant decrease in cell numbers compared to the non-frozen demiembryos. We conclude, that a 24 h period of co-culture with BOEC does not result in appreciable cellular proliferation in demiembryos and therefore instead of improving the survival of frozen/thawed demiembryos by giving them opportunity to multiply their cell number and thus make them more resistant to cell damage, rather compromised the viability of cryopreserved demiembryos.     

In vitro development of OPU‐derived bovine embryos cultured either individually or in groups with the silk protein sericin and the viability of frozen‐thawed embryos after transfer

The optimization of single‐embryo culture conditions is very important, particularly in the in vitro production of bovine embryos using the ovum pick‐up (OPU) procedure. The purpose of this study was to examine the development of embryos derived from oocytes obtained by OPU that were cultured either individually or in groups in medium supplemented with or without sericin and to investigate the viability of the frozen‐thawed embryos after a direct transfer. When two‐cell‐stage embryos were cultured either individually or in groups for 7 days in CR1aa medium supplemented with or without 0.5% sericin, the rates of development to blastocysts and freezable blastocysts were significantly lower for the embryos cultured individually without sericin than for the embryos cultured in groups with or without sericin. Moreover, the rate of development to freezable blastocysts of the embryos cultured individually with sericin was significantly higher than that of the embryos cultured without sericin. When the frozen‐thawed embryos were transferred directly to recipients, the rates of pregnancy, abortion, stillbirth and normal calving in the recipients were similar among the groups, irrespective of the culture conditions and sericin supplementation. Our findings indicate that supplementation with sericin during embryo culture improves the quality of the embryos cultured individually but not the viability of the frozen‐thawed embryos after transfer to recipients.     

精浆对猪精子冷冻效果影响的研究

为优化猪精子冷冻技术,提高解冻后精子的活力和受精能力,本试验分别以含精浆浓度为10%、20%和30%的冷冻保护剂处理精子,以冷冻前、解冻后精子活力和质膜完整性,解冻后精子进行体外受精(IVF)的卵裂率和囊胚率等作为检测指标,同时以含有卵黄的Tris-柠檬酸－葡萄糖(Tris-citric acid-glucose,TCG)冷冻基础液作为对照研究精浆对猪冷冻精子的保护作用。结果显示,在含有10%精浆浓度的稀释液中,冷冻前质膜完整性,解冻后精子活率、质膜完整性、IVF囊胚率相对于对照组均显著提高(P<0.05);当含有10%精浆的冷冻精液解冻后用于人工授精时,与配母猪妊娠率、窝产仔数、窝产活仔数等仍显著低于鲜精授精组(P<0.05)。上述结果表明,含10%精浆的冷冻保护剂能提高精子的冷冻后活力和IVF胚胎发育率,但用于人工授精配种与鲜精相比还有一定差距。     

牛胚胎冷冻与移植受胎技术研究

本文进行了牛胚胎冷冻与移植受胎技术研究。摸索出了提高牛冷冻胚胎成活率和移植受胎率的综合配套技术。牛冷冻胚胎解冻成活率达８４．４％（５７／１２２）。其中１９９６年移植受胎率达到５５．６％（２０／３６）。Ａ级胚胎达以７７．８％（２１／２７），并在我国首次获得中国荷斯坦牛冷冻胚胎的二分胚的同孵孪生牛犊。且达到４２．９％（３／７）移植受胎率。     

非繁殖季节胚胎移植受体绵羊妊娠效果影响因素的研究

在非繁殖季节应用CIDR法和Progestagen Sponge法对受体绵羊进行同期发情处理后,进行冷冻胚胎移植,研究不同同期发情处理方法、卵巢状况、胚龄、移植方法和移植时间对冷冻胚胎移植妊娠率的影响,以期为当地实现绵羊胚胎移植产业化提供理论依据和技术参考。结果表明:经同期发情处理后,绵羊在去栓后24~72 h集中发情,在去栓后36~48 h发情最为集中;CIDR法和Progestagen Sponge法的同期发情率分别为91.11%和87.78%(P>0.05),同期发情绵羊移植利用率分别为82.93%和78.48%(P>0.05);单侧卵巢上含有1个和2个黄体其妊娠率分别为46.07%和48.78%(P>0.05);桑椹胚和囊胚的妊娠率分别为49.25%和42.11%(P>0.05);腹腔镜法和手术法的妊娠率分别为45.16%和48.52%(P>0.05);11月和12月进行冷冻胚胎移植的妊娠率分别为50.76%和43.08%(P>0.05),该试验的绵羊冻胚移植妊娠率为46.92%,基本接近国内冻胚移植水平,但低于目前国内羊鲜胚移植妊娠率(50%~60%)。由此可以得出,不同同期发情处理方法、卵巢状况、胚龄、移植方法和移植时间对胚胎移植妊娠率的影响均无显著性差异(P>0.05),大规模群体羊同期发情时,可选价格便宜的Progestagen Sponge法;只要受体绵羊黄体质量好即可移植胚胎;为了减轻受体绵羊的应激反应,降低术后子宫粘连的发病率,可广泛应用腹腔镜法进行胚胎移植;该试验的绵羊冻胚移植妊娠率低于目前国内羊鲜胚移植妊娠率,通过筛选高效低毒的抗冻保护剂,以及提高基层技术人员操作水平,则有望进一步提高绵羊冷冻胚胎移植妊娠率。     

影响绵羊冻胚移植妊娠率的因素分析

本研究对胚胎质量、胚胎移植数量、胚胎发育阶段、冷冻方法、移植方法及饲养管理水平等因素对移植产羔率的影响进行研究,以提高绵羊胚胎移植效率。结果表明:采用1.5mol/L乙二醇做冷冻保护剂对胚胎进行常规冷冻,冻前A级胚胎冷冻/解冻后可用胚率、存活率、降级率均高于B级,差异极显著(P<0.01)。移植1枚冷冻解冻后A级、B级或2枚(A+B)胚胎的受体移植产羔率分别为44.48%、46.84%和44.81%。经χ2检验分析,三者之间无显著差异(P>0.05)。移植双胚的受体双羔率为22(%53/241),以胚胎为单位计算,产羔率仅为33.40%。A级囊胚和桑椹胚的产羔率之间无显著差异(P>0.05)。1.5 mol/L乙二醇常规冷冻保存的体内胚胎移植产羔率高于EFS40玻璃化冷冻保存,但经统计学分析,二者无显著差异(P>0.05)。子宫手术移植和腹腔内窥镜法子宫移植之间产羔率不具统计学差异(P>0.05)。在牧场条件下大规模移植的平均产羔率为43.95%,范围在20%￣70%之间,受体饲养管理水平对绵羊移植产羔率有较大影响。     

Comparison of Different Cryopreservation Techniques: Higher Survival and Implantation Rate of Frozen–Thawed Mouse Pronuclear Embryos in the Presence of Beta‐Mercaptoethanol in Post‐Thaw Culture

The objective of this study was to investigate the effects of beta‐mercaptoethanol (β‐ME) on post‐thaw embryo developmental competence and implantation rate of mouse pronuclear (PN) embryos that were cryopreserved after slow freezing, solid surface vitrification (SSV) or open‐pulled straw (OPS) vitrification methods. Mouse PN embryos were cryopreserved by using slow freezing, SSV and OPS methods. After cryopreservation, freeze–thawed PN embryos were cultured up to blastocyst stage in a defined medium supplemented without or with 50 μm β‐ME. The blastocyst formation rate of embryos that were cryopreserved by slow freezing method (40.0%) or vitrified by OPS method (18.3%) were lower than those vitrified by SSV method (55.6%) and fresh embryos (61.9%) in the absence of 50 β‐ME in the culture media (p < 0.05). The blastocyst formation rate of embryos that were cryopreserved by slow freezing method (53.1%) or by OPS method (41.9%) were lower than those vitrified by SSV method (79.5%) and that of fresh (85.7%) in the presence of β‐ME in the culture media (p < 0.05). The embryos transfer results revealed that the implantation rate of blastocyst derived from mouse PN embryos vitrified by SSV method (31.9% vs 51.2%) was similar to that of the control (39.0% vs 52.5%), but higher than those cryopreserved by slow freezing (28.2% vs 52.0%) and by OPS method (0.0% vs 51.2%) (p < 0.05). In conclusion, supplementation of β‐ME in an in vitro culture medium was shown to increase survival of embryo development and implantation rate of frozen–thawed mouse PN embryos after different cryopreservation protocols.     

Forced Collapse of the Blastocoel Cavity Improves Developmental Potential in Cryopreserved Bovine Blastocysts by Slow‐Rate Freezing and Vitrification

This study was conducted to evaluate the effectiveness of forced collapse of the blastocoel before slow‐rate freezing and vitrification of bovine blastocysts. Cryopreservation of bovine blastocysts has been proposed as a tool to improve the feasibility of cattle production using the embryo transfer technique. However, the low efficiency of frozen–thawed embryos survival and further development is a crucial problem. In this study, bovine in vitro and in vivo blastocysts were slow‐rate frozen and vitrified after forced blastocoele collapse (FBC) of the blastocyst cavity by puncturing the blastocoele with a pulled Pasteur pipet. Differences in the developmental potential of frozen–thawed blastocysts derived from FBC and non‐FBC groups were found in both slow‐rate freezing and vitrification. Furthermore, we found that the total cell number of blastocysts in FBC groups was increased and the index of apoptosis in FBC groups was decreased. Consistent with these results, real‐time RT‐PCR analysis data showed that expression of the anti‐apoptotic Bcl‐XL gene was significantly increased by FBC groups, whereas expression of the pro‐apoptotic Bax gene was significantly decreased by FBC groups. Our results also showed that pregnancy outcomes in both slow‐rate frozen and vitrified bovine in vivo blastocysts could be improved by reducing the fluid content after FBC of the blastocyst cavity. Therefore, we suggest that FBC of the blastocyst cavity with a pulled Pasteur pipet is an effective pre‐treatment technique for both slow‐rate freezing and vitrification of bovine blastocysts.     

Effects of resveratrol treatment on mitochondria and subsequent embryonic development of bovine blastocysts cryopreserved by slow freezing

This study evaluated the effects of cryopreservation by slow freezing on the mitochondrial function, DNA integrity, and developmental ability of bovine embryos and examined whether resveratrol treatment of the frozen‐thawed blastocysts improved embryonic viability. In vitro produced bovine embryos were subjected to slow freezing. After thawing, the ATP content and mitochondrial DNA integrity (mtDNA), determined by real‐time PCR targeting short and long mitochondrial sequences, was found to be lower in frozen‐thawed embryos than in fresh embryos, and mtDNA copy number was significantly reduced during the 24‐hr incubation post warming. Furthermore, immunostaining against double‐strand DNA revealed DNA damage in frozen‐thawed embryos. When frozen‐thawed embryos were incubated in the medium containing 0.5 µM resveratrol, SIRT1 expression, and survival rate of the embryos significantly improved compared with the vehicle‐treated embryos. In addition, cell‐free mtDNA content in medium was higher in case of resveratrol‐treated embryos than of vehicle‐treated embryos. In conclusion, slow freezing affects mitochondrial integrity and function in the blastocysts. In the frozen‐thawed embryos, mitochondria were removed during post‐thawing incubation and resveratrol enhanced the process, resulting in improved survivability of the embryos.     

Incidence and avoidance of zona fracture in cryopreserved bovine ova and embryos

Ova (n=62), which were collected from slaughterhouse bovine ovaries, and embryos (n=26), which were non-surgically recovered from 11 superovulated crossbred donor cows, were frozen. The frozen ova and embryos were then thawed using two conventional thawing protocols, i.e. at 37 degrees C for 30 seconds in a water bath and at 25 degrees C for 2 minutes in air. Some 64.5% of the ova and 53.8% of the embryos thawed in the water bath and 16.1% of the ova and 7.7% of the embryos thawed in ambient air exhibited fractured zonae pellucidae. The slow thawing protocol had a lower incidence of zona damage in cryopreserved oval and embryos than the fast thawing protocol. A low pregnancy rate (12.5%) was recorded for embryos transferred with zona fracture while embryos transferred with intact zonae had a rate of 35.3%) indicating that embryos with zona damage are less viable.     

Incidence and avoidance of zona fracture in cryopreserved bovine ova and embryos

Ova (n=62), which were collected from slaughterhouse bovine ovaries, and embryos (n=26), which were non-surgically recovered from 11 superovulated crossbred donor cows, were frozen. The frozen ova and embryos were then thawed using two conventional thawing protocols, i.e. at 37 °C for 30 seconds in a water bath and at 25 °C for 2 minutes in air. Some 64.5% of the ova and 53.8% of the embryos thawed in the water bath and 16.1% of the ova and 7.7% of the embryos thawed in ambient air exhibited fractured z:onae pellucidae. The slow thawing protocol had a lower incidence of zona damage in cryopreserved oval and embryos than the fast thawing protocol. A low pregnancy rate (12.5%) was recorded for embryos transferred with zona fracture while embryos transferred with intact zonae had a rate of 35.3%) indicating that embryos with zona damage are less viable.     

Viability of OPS Vitrified Sheep Embryos After Direct Transfer

The aim of this study was to evaluate the viability in the effect of open pulled straw (OPS) vitrification procedure of sheep embryos after direct transference. Embryos were produced in vivo and cryopreserved in slow freezing or OPS vitrification. The survival rates of cryopreserved embryos were compared to non-frozen standard pattern. In a first set of experiments, embryos at morula and blastocyst stages were dived in ethylene glycol (1.5 M) and frozen in an automatic freezer. After being thawed, they were directly or indirectly transferred to ewes recipient. A second group of embryos were drawn into OPS and plunged into liquid nitrogen after being exposed at room temperature for 1 min and 45 s in 10% EG plus 10% dimethyl sulphoxide (DMSO), then again for 30 s in 20% EG + 20% DMSO + 0.5 M sucrose. After being warmed, embryos were also directly transferred using a French mini straw as the catheter for the transplantation process or after in vitro dilution of cryoprotectants (two-step-process). No significant difference was observed among fresh, frozen or vitrified embryos on pregnancy rate (50.0%, 38.6% and 55.8%). However, when we evaluated only the direct transference, the pregnancy rate of OPS vitrified embryos was higher than that of frozen embryos (57.1% vs 34.8%) (p = 0.07). In addition, vitrified morulae had a higher pregnancy rate than the one with frozen embryos (64.0% vs 38.9%) (p = 0.07). Finally, our results indicate that OPS vitrification technique in association with direct transference improves the viability of sheep embryos with potential applications to field conditions.     

The transfer of fresh and frozen embryos in an elite swamp buffalo herd

To investigate the development of fresh and frozen swamp buffalo embryos after transfer to synchronized recipients, 14 fresh embryos and 28 frozen embryos, collected from Thai swamp buffalo cows of an elite herd at the Surin Breeding Center, were transferred nonsurgically to 31 synchronized recipients buffalo cows. One fresh embryo was transferred to each of 14 recipients. Twenty eight frozen embryos were transferred to 17 recipients of which 7 cows received I embryo, 9 cows received 2 embryos and 1 cow received 3 embryos. Pregnancy was diagnosed by real time B-mode ultrasonography one month after transfer and confirmed by rectal palpation one and two months later. The pregnant cows were kept under observation until calving. The results of fresh embryo transfer showed that 5/14 (35.7%) were pregnant after 30 days, 4/14 (28.6%) remained pregnant until the 3rd month and 2/14 (14.3%) calved. With the frozen embryos, only one cow which received three embryos became pregnant and remained so for 3 months although the embryo did not survive to full term. The overall pregnancy rate using frozen embryos was 5.9% (1/17). The study demonstrated the possibility of performing embryo transfer in elite buffalo herds for genetic improvement, however the use of frozen embryos needed further investigation.     

Use of a rapid method of cryopreservation of bovine embryos for non-surgical transfer in transplantation practice

The embryos were frozen and thawed in Cassou minipaillette by a rapid method. Embryos with cryoprotective agent (glycerol, 1.5 M) were placed directly into the freezing medium at the temperature of -6 to -7 degrees C, frozen after seedling at the temperature decrease by 0.3 to 0.5 degrees C per minute to the temperature of -32 degrees C and then transferred directly into liquid nitrogen. They were thawed in a bath warm 20 to 37 degrees C. After thawed the cryoprotective agent was evacuated in 1.1 M sucrose. The best-quality embryos were selected for freezing. Out of these 366 thawed so far, with average survival of 74.31%. The total of the 268 thawed embryos were transferred ipsilaterally, by a non-surgical method, to 190 synchronised heifers, out of which 105 (55.26%) got in calf. Rapid freezing method based on 1.5 M of glycerol and thawing at the presence of 1.1 M sucrose proved effective and suitable for practice, as not only sufficient reviviscence of embryos and their survival in womb are guaranteed, but also a substantial shortening of the freezing as well as thawing process.     

Current Reproductive Technologies Impacting Equine Embryo Production

Numerous reproductive technologies have been developed in the past several decades, which have dramatically changed the way mares are bred. This review will focus on embryo recovery and transfer, cooled-shipped embryos, embryo freezing, oocyte freezing, oocyte collection and transfer, intracytoplasmic sperm injection (ICSI), and sexed semen. Embryo transfer procedures have been constant for many years and the costs have not changed. The major change has been the ability to store embryos at 5 C for 12–24 hours and transport them to recipient stations. Embryo freezing has become more common using the technique of vitrification of embryos >300 μm or deflating embryos >300 μm before freezing. Oocyte vitrification has resulted in poor pregnancy rates although the technique works well in women. The ability to collect oocytes from mares and fertilize them by sperm injection has revolutionized the veterinarian’s approach to infertility in the mare and/or stallion. A transvaginal approach can be used to collect oocytes from preovulatory follicles and unstimulated follicles 5–25 mm in size. Although traditional in vitro fertilization does not work well in the horse, ICSI can be used to produce blastocysts which, upon nonsurgical transfer into recipients, provide a pregnancy rate similar to fresh embryos collected from donor mares. Sorting sperm by flow cytometry into X- and Y-bearing spermatozoa has been shown to provide about a 50% pregnancy rate with freshly sorted sperm but only 12% with sorted, frozen/thawed stallion sperm. It is likely that more advanced reproductive techniques will be developed in the future. Their acceptance will depend on how well they work, perceived need, cost, and, to some extent, the breed associations.     

Cryosurvival and pregnancy rates: One‐step protocol for freezing–thawing Shangri‐la Yak (Bos grunniens) Embryos

The yak is one of the most important and economically useful animals for highlanders. The decline in the yak population requires effective measures for the conservation and multiplication of elite germplasm. A standardized protocol will simplify the freezing and warming of yak embryos in straw and facilitate embryo transfer. In this work, we investigated a one‐step protocol that uses a stable basal medium, which comprised a warming medium (1.08 M sucrose) and a freezing medium (EFS40). We also assessed the effects of the new transfer method on embryo survival. A total of 145 yak frozen embryos were thawed in a standard medium system. The one‐step protocol led to a high recovery percentage (84.93) of yak embryos that survived vitrification and warming. The in vitro survival rates of these embryos significantly different from those of embryos frozen–thawed via the conventional method. The 95 embryos frozen–thawed via our one‐step protocol were then implanted in selected recipients. Thirty‐six singleton pregnancies were established. In conclusion, the proposed one‐step method is a simple, safe, and standardized freezing–thawing protocol that ensures embryo survival and quality under field conditions. This study establishes new possibilities for the widespread use of embryo transfer in yaks.     

Cryopreservation and In Vitro culture of Preimplantation Embryos in Djungarian Hamster (Phodopus sungorus)

Although embryo cryobanking was applied to Syrian golden and to Campbell's hamsters, no attempt has been made at freezing embryos in Djungarian hamsters. Four‐cell stage embryos were flushed from the reproductive ducts of pregnant females before noon of the third‐day post coitum and frozen in 0.25‐ml straws according to standard procedures of slow cooling. A mixture of permeating (ethylene glycol) and non‐permeating (sucrose) cryoprotectants was used. The thawing was performed by incubating at RT for 40 s followed by 40 s in a water bath at 30.0°C. Most (66.7%) of the non‐frozen four‐cell embryos developed up to the morula stage in rat one‐cell embryo culture medium (R1ECM). The use of hamster embryo culture medium (HECM) yielded fewer morulas (18.2%) during the same 24‐h period of culture. The rate of embryo's surviving the freezing–thawing procedures, as estimated by light microscopy, was 60.7–68.8%. After 24‐h culturing in R1ECM, 64.7% of frozen–thawed four‐cell embryos developed and all of them reached the morula stage. Supplementation of R1ECM with GM‐CSF (2 ng/ml) improved the rate of Djungarian hamster frozen–thawed embryo development: 100% of the four‐cell stage embryos developed, 50% of them achieved the morula stage, and 50% developed even further and reached the blastocyst stage within 24 h of culturing. This study reports the world's first successful transfer of frozen–thawed Djungarian hamster embryos yielding term pups. Taken together, the results of this study demonstrate the possibility of applying some key reproductive technologies, that is, embryo freezing/cryopreservation and in vitro culture, to Djungarian hamsters.     

日本和牛胚胎移植的试验研究

[目的]为了在我国引进日本和牛这一高等级肉牛品种并迅速扩大纯种日本和牛的数量.[方法]分别采用CIDR+E2法和两次PGF2α法对胚胎移植受体牛做同期发情处理,采用一步细管法冷冻的日本和牛胚胎进行移植,观察妊娠率.[结果]表明,受体牛经CIDR+E2法和两次PGF2α法处理后12～48 h的发情率分别为82.7%和45.9%,两者之间差异显著(P＜0.05),对90枚日本和牛胚胎进行一步细管法管内解冻,然后给62头CIDR+E2法处理和28头两次PGF2α法处理的受体奶牛移植,受胎率分别为43.5%和42.5%,产犊率分别为40.3%和39.3%,两者之间差异不显著(P＞0.05).[结论]经同期发情处理的受体牛,只要其子宫环境及卵巢黄体发育良好,被移植日本和牛胚胎都能正常妊娠和产犊;用日本和牛胚胎对低产奶牛进行移植,经三代纯化即可获得纯种日本和牛.     

小鼠胚胎的玻璃化冷冻研究

用不同冷冻载体(玻璃管、塑料管和0.25 mL细管)及不同冷冻方法(程序化冷冻和玻璃化冷冻)对小鼠3.5 d~4 d桑椹胚和囊胚进行冷冻保存,并与不做任何冷冻保存处理直接培养进行对比。结果表明,使用玻璃管、塑料管和0.25 mL细管作为胚胎的承载材料进行玻璃化冷冻,效果差异不显著;采用程序化冷冻与OPS玻璃化冷冻法,对小鼠胚胎进行冷冻保存可以取得较好的结果。从而得出,用不同材质的冷冻载体进行玻璃化冷冻,可以获得与程序化冷冻相同的良好效果。     

不同胚胎移植方法对天祝白牦牛受胎率的影响

为提高胚胎移植技术在牦牛上的运用效果,选用51头甘肃省天祝县健康黑牦牛作为受体,以纯种天祝白牦牛作为供体生产胚胎,分别对同期发情处理和自然发情的受体牛进行鲜胚和冻胚移植试验。结果,同期发情处理的受体牛鲜胚移植的受胎率显著高于冻胚移植的受胎率(P0.05),分别为52%和38.5%;在自然发情受体牛的胚胎移植中也得到了相似结果,鲜胚和冻胚的移植受胎率分别为60%和50%,同期发情处理牛的平均妊娠率则低于自然发情受体牛的平均妊娠率(P0.05),二者分别为47.5%和54.5%。结论,牦牛鲜胚移植的受胎率明显高于冻胚移植的受胎率,而且自然发情受体牛的受胎率高于同期发情处理牛。