Transfer of Bisected Cattle Embryos within the Same Zona Pellucida*

Cattle embryos at the compact morula and blastocyst stages were bisected by two different methods. In some embryos the embryo bisection was performed outside of the zona pellucida and both halves were replaced into it. Other embryos were bisected within the zona pellucida and no pipetting of the halves was required. They were transferred to synchronized recipient cows and the pregnancy rates and twinning rates were evaluated.     

Laparoscopy vs. laparotomy for embryo transfer to produce transgenic goats (Capra hircus)

This study was performed to produce transgenic Korean native goat (Capra hircus) by laparoscopic embryo transfer (ET) to overcome the limitations of ET performed by laparotomy. Transgenic embryos were produced by DNA pronuclear microinjection of in vivo zygotes. The recipient goats were synchronized for estrus by using an introvaginal progesterone devices as a controlled internal drug-releasing insert (CIDR) for 13 days and injection of 400 IU PMSG 48 h before removal of the insert. Embryos were transferred on day 3 and 4 after removal of the insert. Recipient goats were deprived of feed for 48 h, then suspended in a laparotomy cradle at an angle of 45°. After obtaining a sufficient pneumoperitoneum, the laparoscope and forceps were inserted abdominally through 5 mm trocar sleeves. Examination of the ovaries and uterus was performed and then 213 embryos were transferred into the oviducts via the infundibula of 76 recipient goats. To compare pregnancy rates, ET was also performed by laparotomy in 82 recipient goats. The pregnancies in the recipient goats were diagnosed by ultrasound on day 30 after embryo transfer. The pregnancy rate with laparoscopic ET was significantly higher than with ET performed by laparotomy (46.1% vs. 28.6%, p < 0.05). In addition, the pregnancy rates were compared between ovulated and non-ovulated ovaries of the recipient goats in the laparoscopic ET group. No significant difference was observed between the pregnancy rates of ovulated and non-ovulated ovaries (41.3% vs. 33.3%, p < 0.05) suggesting that ET may also be possible in non-ovulated recipients through artificial rupture of Graafian follicles. These results suggest that laparoscopic ET is a highly efficient method for the transfer of goat embryos.     

Prospects for the use of embryos in the control of disease and the transport of genotypes

Transfer and low temperature storage of embryos are now proven techniques for a number of mammalian species. These techniques are useful in control of disease and in saving genotypes from infected animals. The place of embryos in the epidemiology of disease depends upon whether the causative organism can gain entry to the oocyte before or at fertilisation and on whether the young embryo can be invaded by organisms in the uterine environment. There is little evidence that important live-stock diseases are transmitted via gametes. The zona pellucida surrounding the embryo is an effective barrier against a number of important disease organisms; in some cases the embryo is susceptible once it has hatched from the zona pellucida. It is important therefore in considering the use of embryos in disease control, to ensure that virus is not attached to the surface of the zona pellucida from where it can infect the recipient and/or the embryo after hatching. Washing procedures have been devised together with the use of enzymes and antisera to remove virus from the surface of embryos. Some viruses enter pores and sperm tracks in the zona and removal of these may present a problem. African swine fever virus has been shown to resist removal by treatment with enzymes. There are no guidelines as to the likely interaction between a certain virus and embryos. Therefore each virus of interest must be tested to determine whether it can be transmitted via washed embryos. Nevertheless there are numerous instances of the use of embryo transfer to eradicate a specific disease or to save valuable genetic material from infected animals without transmitting disease.     

The effects of an advanced uterine environment on embryonic survival in the mare

Reasons for performing the study: During embryo transfer (ET) the equine embryo can tolerate a wide degree of negative asynchrony but positive asynchrony of >2 days usually results in embryonic death. There is still confusion over whether this is due to the inability of the embryo to induce luteostasis or to an inappropriate uterine environment. Objectives: To assess embryo survival and development in an advanced uterine environment. Hypothesis: Embryo–uterine asynchrony, not the embryo's inability to induce luteostasis, is responsible for embryonic death in recipient mares with a >2 days chronologically advanced uterus. Methods: Experiment 1: Thirteen Day 7 embryos were transferred to the uteri of recipient mares with luteal prolongation, occasioned by manual crushing of their own conceptus, such that donor–recipient asynchrony was between +13 and +49 days. Experiment 2: Day 7 embryos were transferred to recipient mares carrying their own conceptus at Days 18 (n = 2), 15 (n = 2), 14 (n = 4), 12 (n = 4) or 11 (n = 4) of gestation. In addition, Day 8 embryos were transferred to 4 pregnant recipient mares on Day 11 of gestation. Results: No pregnancies resulted following transfer of Day 7 embryos to recipients in prolonged dioestrus with asynchronies between +13 and +49 days. However, the use of early pregnant mares as recipients resulted in 5/20 (25%) twin pregnancies, 4 of which came from the transfer of a Day 8 embryo to a Day 11 recipient. All transferred embryos showed retarded growth, with death occurring in 4/5 (80%). Conclusions and potential relevance: The results emphasise the importance of an appropriate uterine environment for embryo growth and the inability of equine embryos to survive transfer to a uterus >2 days advanced even when luteostasis is achieved. It is possible that in normal, non‐ET equine pregnancy, embryo–uterine asynchrony may account for some cases of embryonic death.     

Pronuclear Embryo Yield in Canindé and Saanen Goats for DNA Microinjection

The objective of this study was to examine the effect of donor breed on pronuclear‐stage embryo yield to be used for DNA microinjection in a transgenesis goat program. Twelve Canindé and twelve Saanen goats were heat synchronized using a progestagen‐cloprostenol treatment. Forty‐eight hours before the sponge removal, superovulation was induced with a total administration of 4.4 mg/kg bodyweight NIH‐FSH‐P1, given twice daily in decreasing doses over 3 days. In addition, goats received 100 μg of GnRH and they were hand‐mated at 36 and 48 h after progestagen removal. Embryo recovery was performed by oviduct flushing at 72 h after sponge removal. Embryos were microinjected with a DNA construct and noticeable swelling of the nuclei was the criterion for successful microinjection. The total diameter, cytoplasm diameter, zona pellucida thickness and pronuclei diameter were measured for each microinjected embryo. A higher (p < 0.05) percentage of fertilized ova was observed in Canindé (89.9%) than Saanen (36.2%) goats. In addition, Canindé donors produced a higher percentage of pronuclear embryos when compared with Saanen: 72.5% vs 20.6% (p < 0.05), respectively. Successful microinjection was verified in 96.7% and 73.3% of times in Canindé and Saanen embryos, respectively (p < 0.05). Significant differences were observed for all morphometric parameters except for cytoplasm diameter. In conclusion, under our study experimental conditions, Canindé were more efficient than Saanen goats concerning the pronuclear embryo yield and manipulation. The use of Canindé goats in transgenesis programs could be increase the interest in their breeding and could be contribute to saving them from extinction.     

Practical Experience with the Treatment of Recipient Mares with a Non‐Steroidal Anti‐Inflammatory Drug in an Equine Embryo Transfer Programme

As part of a commercial embryo transfer programme, 20 embryos were transferred to spontaneously synchronous or synchronized recipient mares. In 14 cases, embryo recipients were treated with non‐steroidal anti‐inflammatory drugs (NSAID), receiving flunixin meglumine i.v. at the time of transfer and vedaprofen orally twice a day on the 3 days after embryo transfer, while six embryos were transferred to untreated mares that served as controls. Out of the 14 recipient mares treated with NSAID, 11 (79%) were pregnant at 6–8 days after transfer and in 10 mares, the pregnancy was continued. From the six untreated recipients, only one became pregnant but underwent early embryonic death between day 14 and 35 after ovulation. In conclusion, pregnancy rate in NSAID‐treated recipients is higher than that in untreated recipients and above reported average values, indicating that treatment of recipient mares with NSAID helps to increase pregnancy rates after transcervical transfer and can be recommended for equine embryo transfer.     

Secondary Corpora Lutea Induced by hCG Treatment Enhanced Demi‐Embryo Survival in Lactating High‐Yielding Dairy Cows

Using a novel in vivo model considering a low developmental competence embryo (demi‐embryo) and a subnormal fertility recipient (lactating high‐yielding dairy cow), this experiment evaluated the effect of human chorionic gonadotrophin (hCG) treatment at embryo transfer (ET) on embryonic size at implantation, embryonic survival and recipient plasma progesterone (P₄) and bovine pregnancy‐specific protein B (PSPB) concentrations until day 63 of pregnancy. Embryos were bisected and each pair of demi‐embryos was bilaterally transferred to recipients (n = 61) on day 7 of the oestrous cycle. At ET recipients were randomly assigned to treatment with 1500 IU hCG or to untreated controls. Higher (p < 0.01) pregnancy rates on days 25, 42 and 63, and embryo survival rate on day 63 were observed in hCG‐treated cows with secondary CL than in hCG‐treated cows without secondary CL and in untreated cows. Pregnancy rates and embryo survival rate were similar in hCG‐treated cows without secondary CL and untreated cows. Embryonic size on day 42 was not affected by treatment with hCG, presence of secondary CL and type of pregnancy (single vs twin). Presence of secondary CL increased (p < 0.05) plasma P₄ concentrations of pregnant cows on days 14, 19 and 25 but not thereafter and of non‐pregnant cows on days 14–21. Treatment with hCG and presence of secondary CL had no effect on plasma PSPB concentrations, which were higher (p < 0.05) in twin than in single pregnancies. In conclusion, secondary CL induced by hCG treatment at ET significantly increased plasma P₄ concentrations, the survival rate of demi‐embryos and the pregnancy rate of high‐yielding lactating dairy cows. Embryos were rescued beyond maternal recognition of pregnancy, but later embryonic survival, growth until implantation and placental PSPB secretion until day 63 of pregnancy were not affected by treatment or presence of secondary CL.     

Embryo transfer: a discussion on its potential for infectious disease control based on a review of studies on infection of gametes and early embryos by various agents

Studies on laboratory animals have shown that viruses vary as to whether or not they are transmissible by the gametes or are capable of passing through the zona pellucida and infecting the embryo.

Methods of studying early embryos for the presence of infectious agents include electron microscopy, immunocytochemistry and cell cultivation.

Determination that early bovine embryos do not become infected by certain agents might allow for easing of restrictions in the current import and export regulations for cattle embryos.

Embryo transfer could be used as a means of controlling or eliminating disease in a herd or flock if the causal agent does not infect the early embryo via the gametes or by penetrating the zona pellucida.

     

应用体细胞克隆技术和胚胎移植技术生产克隆水牛

为了解决克隆水牛技术在供核细胞、受体细胞中的选用问题,探索不同条件对移植受孕的影响,完善克隆技术的理论体系。试验以摩拉奶水牛的耳成纤维细胞作为供核细胞,本地水牛卵母细胞为受体胞质进行细胞克隆构建重组胚胎;将发育5～10 d的重组胚胎移植于本地水牛、杂交水牛的子宫内,观察不同发情方式、胚胎类型、胚龄及季节对移植受孕的影响。结果表明:水牛的克隆胚胎妊娠率较低,分别为12.05%、12.04%、13.95%。自然发情和同期发情对克隆胚胎移植受胎率的影响差异不显著;鲜胚胎移植186头,受胎22头,受胎率为11.83%(22/186);冻胚胎移植174头,受胎23头,受胎率为13.22%(23/174)。克隆水牛从出生至12月龄体尺增长明显高于本地水牛;水牛克隆胚胎移植受胎在秋季最好,秋季受胎数占全年的53.33%,显著高于其他季节。     

Embryo cloning in domestic animals]

The cloning of livestock is performed by the nuclear transfer of early embryonic stages into prepared oocytes in order to obtain a high number of genetic identical animals. As the most important technical steps there are maturation and enucleation of the oocytes, isolation of single blastomeres or karyoplasts of the donor embryo, transfer of the nucleus-containing membrane vesicle under the zona pellucida of the recipient cell, and fusion of the recipient cell and blastomere or karyoplast. Until now, the largest clone which is known exists of seven bulls. The efficiencies of the particular methodical steps have to be improved. More knowledge of the activation of oocytes, nucleus differentiation and availability of determined cell cycle-stages of mitosis is required. The combination of embryo cloning, cryopreservation of embryos and non-surgical embryo transfer is required for basic research and animal breeding.     

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.     

水牛活体采卵和无透明带胚胎培养体系的优化

本研究旨在探索哺乳动物体外受精（IVF）胚胎单独或少量培养时发育效率低、无透明带胚胎的体外发育潜能受阻的问题,以期建立提高水牛活体采卵（ovum-pick-up,OPU）和徒手克隆（handmade clone,HMC）胚胎发育潜能高效稳定的体外生产体系。研究首先比较了单个微滴内共培养的胚胎数量（1、3、5、10和20枚）对胚胎发育效果的影响;而后采用微穴体系（well-of-the-well,WOW）和辅助共培养体系（培养微滴中添加包埋IVF胚胎的琼脂糖小块）培养OPU-IVF胚胎,并用WOW体系培养无透明带的徒手克隆重构胚,与传统的微滴培养体系比较其体外发育效果。结果表明：单个微滴内培养的胚胎数量为1、3和5枚时,囊胚发育率极显著低于10枚和20枚组（P<0.01）;与微滴培养体系相比,辅助共培养和WOW体系均极显著提高OPU-IVF胚胎的囊胚率（P<0.01）,且WOW培养体系极显著促进HMC重构胚的卵裂率和囊胚率（P<0.01）。综上所述,胚胎群体培养有助于胚胎发育,在保证系谱明确的前提下琼脂糖包埋辅助胚胎共培养体系和WOW体系提高了OPU-IVF体外胚胎发育效率,且WOW体系还可用于无透明带胚胎的高效培养。     

Optimization of in vitro Culture System for Embryos Derived from Ovum-pick-up and Zona-free Embryos in Buffalos

The purpose of this study was to explore the problems of low development efficiency of small amounts in vitro fertilization (IVF) embryos and limited development potential of zona pellucida-free embryos when cultured in vitro of mammals,to establish an efficient and stable in vitro production system for improving the developmental potential of living ovum-pick-up (OPU) and handmade clone (HMC) embryos in buffalos.The study first compared the effect of the number of fertilized eggs (1,3,5,10 and 20) co-cultured within a single microdroplet on the embryonic development effect.Then,OPU-IVF embryos were cultured using the well-of-the-well (WOW) system and the auxiliary co-culture system (adding agarosaccharide fragments of embedded in in vitro fertilization (IVF) fertilized egg in the cultivation of microdroplets).Furthermore,the embryos without zona pellucida were cloned and reconstructed by using the WOW system and compared with the traditional microdroplet system in vitro.The results showed that the blastocyst development rates of the 10 and 20 embryos groups were significantly higher than that of the 1,3 and 5 embryos groups.Compared with the microdroplet system,the assisted co-culture system and the WOW system significantly improved the blastocyst rate of OPU-IVF embryos(P<0.01).Moreover,the WOW culture system significantly promoted the cleavage rate and blastocyst rate of HMC reconstructed embryos(P<0.01).To sum up,embryo mass culture contributed to embryo development,and under the premise of ensuring a clear pedigree,the agar-sugar embedding assisted embryo co-culture system and the WOW system improved the in vitro development efficiency of OPU-IVF embryos,the WOW system would also be applied to the high-efficient culture of zona pellucida free embryos.     

Effect of flunixin meglumine treatment during and after embryo transfer on the pregnancy rate in cattle

This study aimed to determine the effect of flunixin meglumine treatment during and after the transfer of in vivo produced embryos to Angus (cows) and Holstein (cows and heifers) breeds of cattle on pregnancy rate. Holstein cows were used as donors in the study. A double dose of prostaglandin F2α was administered to the recipient animals for synchronization. Uterine flushing was performed in donors on day 7 after artificial insemination. A total of 295 transferable embryos were obtained. These embryos were transferred to Angus cows (n = 85), Holstein heifers (n = 80) and Holstein cows (n = 130). After the transfer, these animals were divided into three subgroups. The first subgroup (TI) was administered flunixin meglumine during embryo transfer, and the second subgroup (TII) was administered flunixin meglumine both during embryo transfer and on days 8 and 9 after the transfer. The third subgroup (TIII) was not administered anything and it was considered the control group. Pregnancy examination of the recipients was performed on days 30–35 after the transfer using real-time ultrasonography. The pregnancy rates after embryo transfer were found to be 43.52% in Angus cows, 42.5% in Holstein heifers, and 24.61% in Holstein cows (p < .05). When the animals were not classified according to breed, the pregnancy rates in subgroups TI, TII and TIII were found to be 29.29%, 45.10% and 29.79%, respectively (p < .05). In addition, the pregnancy rates were higher in TII and TIII subgroups of Angus cows and Holstein heifers compared to that of Holstein cows (p < .05). As a result, the pregnancy rates obtained after embryo transfer in Angus cows and Holstein heifers were found to be higher than that in Holstein cows. In addition, it was concluded that the administration of flunixin meglumine during and during/after embryo transfer has a positive effect on pregnancy rates in Angus cows and Holstein heifers.     

Effects of Developmental Stage, Embryonic Interferon-τ Secretion and Recipient Synchrony on Pregnancy Rate after Transfer of in vitro Produced Bovine Blastocysts

Three separate trials of bovine embryo transfers were performed consisting of 32, 41 and 33 transfers, respectively, to examine the effects of (a) the developmental stage of in vitro‐derived blastocysts, (b) the amount of interferon‐τ (IFN‐τ) they secreted during culture and (c) the cyclic stage of the recipient at the time of transfer on the probability of establishment of pregnancy. One blastocyst was transferred into the ipsilateral uterine horn to the CL. At the time of transfer, blastocysts were classified into one of three developmental stages (early blastocyst, blastocyst and expanded blastocyst) and the cyclic stage of each cow was assessed (?12 h, on time, +12 h, +24 h, >24 h). Prior to the second and third trials, blastocysts were individually cultured for 24 h in 50 μl medium droplets and the IFN‐τ concentration in the droplet was determined. Logistic regression analyses revealed that expanded blastocysts had a significantly higher likelihood of establishing pregnancy (p = 0.009), and that there was a significant interaction with the cyclic stage of the recipient in this group with lower rates of pregnancy resulting from decreasing synchrony with the recipient (p = 0.033). IFN‐τ secretion during culture was significantly higher in expanded blastocysts than in the other two groups (p < 0.05). A significant effect of the pre‐transfer level of IFN‐τ secretion was found only in the ‘Blastocyst’ group where transfer of embryos with lower IFN‐τ production prior to transfer resulted in higher pregnancy rates (p = 0.047). These results demonstrate that IFN‐τ secretion may be a useful tool to predict pregnancy outcome, but only within certain developmental stages.     

Tiger,Bengal and Domestic Cat Embryos Produced by Homospecific and Interspecific Zona‐Free Nuclear Transfer

The aim of this study was to evaluate three different cloning strategies in the domestic cat (Felis silvestris) and to use the most efficient to generate wild felid embryos by interspecific cloning (iSCNT) using Bengal (a hybrid formed by the cross of Felis silvestris and Prionailurus bengalensis) and tiger (Panthera tigris) donor cells. In experiment 1, zona‐free (ZP‐free) cloning resulted in higher fusion and expanded blastocyst rates with respect to zona included cloning techniques that involved fusion or injection of the donor cell. In experiment 2, ZP‐free iSCNT and embryo aggregation (2X) were assessed. Division velocity and blastocyst rates were increased by embryo aggregation in the three species. Despite fewer tiger embryos than Bengal and cat embryos reached the blastocyst stage, Tiger 2X group increased the percentage of blastocysts with respect to Tiger 1X group (3.2% vs 12.1%, respectively). Moreover, blastocyst cell number was almost duplicated in aggregated embryos with respect to non‐aggregated ones within Bengal and tiger groups (278.3 ± 61.9 vs 516.8 ± 103.6 for Bengal 1X and Bengal 2X groups, respectively; 41 vs 220 ± 60 for Tiger 1X and Tiger 2X groups, respectively). OCT4 analysis also revealed that tiger blastocysts had higher proportion of OCT4‐positive cells with respect to Bengal blastocysts and cat intracytoplasmic sperm injection blastocysts. In conclusion, ZP‐free cloning has improved the quality of cat embryos with respect to the other cloning techniques evaluated and was successfully applied in iSCNT complemented with embryo aggregation.     

Transfer of bovine demi-embryos with and without the zona pellucida

Bisected bovine embryos with or without the zona pellucida were transferred to recipients nonsurgically in five field trials. Embryos were collected from superovulated donors 6.5 to 7.5 d after estrus; only embryos of good and excellent quality were bisected. Demi-embryos were transferred either within a zona pellucida, without a zona pellucida, without a zona pellucida, or in the third and fourth trials, without a zona but embedded in 7% gelatin. Pregnancies were diagnosed at 44 to 68 d of gestation. In a preliminary trial, 9/29 zona pellucida-intact demi-embryos developed into fetuses compared with 1/10 zona pellucida-free demi-embryos (P greater than .1). The proportion of zona-free demi-embryos developing to fetuses was not significantly different from the zona-intact group in the second trial either, 24/49 and 5/19, respectively. In trial 3, the proportion of zona pellucida-free demi-embryos developing was 8/25; of zona-enclosed embryos, 29/88; and of zona-free demi-embryos embedded in gelatin, 8/22 (P greater than .1). Similarly, in the fourth trial the rate of development of zona-free demi-embryos to fetuses was 5/12, that of zona-enclosed embryos was 32/81, and that of zona-free demi-embryos embedded in gelatin was 3/12 (P greater than .1). In trial 5, survival of zona-enclosed demi-embryos to fetuses was 40/105, and of zona-free demi-embryos, 46/109 (P greater than .1). Except for trial 2, half of the demi-embryos were twinned, one to each uterine horn; twinning did not significantly affect the proportion developing to fetuses for any of the demi-embryo groups. It is concluded that placing post-compaction demi-embryos into the zona pellucida for transfer does not improve pregnancy rates significantly.     

Effect of the size of zona pellucida opening on hatching in the common marmoset monkey (Callithrix jacchus) embryo

The use of the common marmoset monkey in biomedical research has increased recently, and further attention has been devoted to this model after the successful production of transgenic marmosets. To extend genetic engineering approaches to widespread biomedical research fields, efficient prolonged in vitro culturing of embryo development is necessary. We aimed to evaluate the effects of the size of the zona pellucida opening on promoting the hatching process in the marmoset embryo. Piezo‐microdrilling of a 6‐μm opening in eight embryos resulted in four partially hatched embryos and one hatched embryo after 5 days of culture. Piezo‐microdrilling a 20‐μm opening in 11 embryos resulted in nine partial hatchings and no hatched embryos. Piezo‐scraping an 80‐μm opening in six embryos resulted in no partially hatched embryos and five hatched embryos. These results suggest that an 80‐μm opening, rather than 6‐μm or 20‐μm openings, is suitable to complete the hatching process in the marmoset embryo.     

Developmental Kinetics of Pig Embryos by Parthenogenetic Activation or by Handmade Cloning

The developmental kinetics of pig embryos produced by parthenogenetic activation without (PAZF) or with (PAZI) zona pellucida or by handmade cloning (HMC) was compared by time‐lapse videography. After cumulus cell removal, the matured oocytes were either left zona intact (PAZI) or were made zona free by pronase digestion (PAZF) before they were activated (PA). Other matured oocytes were used for HMC based on foetal fibroblast cells. On Day 0 (day of PA or reconstruction), the embryos were cultured for 7 days in vitro in our time‐lapse system. Pictures were taken every 30 min, and afterwards, each cell cycle was identified for each embryo to be analysed. Results showed that the PA embryos (both PAZF and PAZI) had shorter first cell cycle compared with HMC (17.4. 17.8 vs 23.6 h), but had a longer time length from four cell to morula stages (57.9, 53.8 vs 44.9 h). However, at the second cell cycle, PAZF embryos needed shorter time, while PAZI embryos had similar time length as HMC embryos, and both were longer than PAZF (23.4, 24.8 vs 14.6 h). Both PAZF and PAZI embryos used similar time to reach the blastocyst stage, and this was later than HMC embryos. In addition, when all of these embryos were grouped into viable (developed to blastocysts) and non‐viable (not developed to blastocysts), the only difference in the time length was observed on the first cell cycle (18.6 vs 24.5 h), but not on the later cell cycles. In conclusion, our results not only give detailed information regarding the time schedule of in vitro‐handled pig embryos, but also indicate that the first cell cycle could be used as a selecting marker for embryo viability. However, to evaluate the effect of the produced techniques, the whole time schedule of the pre‐implantation developmental kinetics should be observed.     

Association between embryonic loss and damage to the zona pellucida by invasive micromanipulation during oviductal transfer of early-stage embryos in pigs

The aim of this study was to examine the impact of zona pellucida damage, which might arise during somatic cell nuclear transfer (SCNT), on the development and survival of transferred embryos. The zonae pellucidae of in vitro matured oocytes were either punctured with 8- to 10-microm square-ended nuclear injection pipettes and piezo pulses or slit with 35- to 40-microm enucleation pipettes. Intact oocytes were used as controls. These oocytes were electroactivated to induce parthenogenesis and transferred to the oviducts of estrus-synchronized recipient gilts. After 5 to 7 days, the recipient uteri were flushed to collect embryos, and embryonic development (morula-blastocyst stage embryos/collected embryos) and survival (viable embryos/collected embryos) were determined. In total, 221 zona-punctured, 129 zona-slitted and 57 intact embryos were transplanted into four, two and two gilts, respectively. The efficiency of embryo recovery was similar in all groups (64.3 to 79.1%). However, the zona-penetrated and incised embryos exhibited unstable development and survival compared with the controls; development and survival of the control embryos were 94.7 and 87.7%, whereas those of the zona-punctured embryos were 69.0 and 47.9% (P<0.01) and those of the zona-slit embryos were 64.7 and 50.0% (P<0.01). Cells with large foci that appeared to be macrophage giant cells were observed at the surface or inside the degenerated zona-damaged embryos. These results indicate that the recipient's immune response to damage to the zona pellucida may impair embryonic development after transplantation to the oviduct. This may be one of the factors causing the reduced efficiency of live progeny production by SCNT.