Relation between Physical Properties of the Zona Pellucida and Viability of Bovine Embryos after Slow-freezing and Vitrification

In vitro-produced bovine morulae/blastocyst embryos (n = 119) were slow-frozen and vitrified and the physical alterations of the zona pellucida (ZP) was observed by scanning electron microscopy (SEM) to find an explanation for the loss of developmental capacity of the embryos after freezing/thawing. A control group was provided, in which embryos (n = 38) were neither frozen nor vitrified. Embryos were in vitro-cultured in a standard CO2 Heraeus incubator and their viability was assessed 24 and 48 h after the start of culture, evaluating their morphological aspect. After 24 h of culture, embryo survival rate for slow-freezing/thawed (n = 23), vitrified/thawed (n = 20) and control embryos (n = 20) was 39, 27 and 90%, and 35, 14 and 65% after 48 h of culture, respectively. For evaluation of physical changes occurring in ZP, 20 embryos were slow-frozen, 18 were vitrified and 18 were used as control. All embryos were fixed, dried and examined under an SEM. Embryo's diameter, as well as the number of pores and their diameter was measured in squares of 6.4 microm width. We observed that, on average, the diameter of the embryos (92.26 +/- 10.15 microm) did not differ significantly among all embryos. As far as the diameter of the pores in the outer surface of the ZP is concerned, the results revealed a significant difference (P < 0.05) between control (0.48 +/- 0.0025 microm), slow-frozen (0.34 +/- 0.0007 microm) and vitrified (0.27 +/- 0.0006 microm) embryos. For the number of pores, statistical differences (p < 0.05) were observed between control and vitrified embryos (45.4 +/- 7.3 vs 38.2 +/- 8.2). It is possible that ZP functions as a barrier which is positive when dealing with pathogens, but is harmful when nutrients were supplied from the outside, especially at 48 h of culture. Results indicate that the steps of cryopreservation cause alterations in ZP, with irreversible damage on the further developmental competence of bovine embryos.     

Three-dimensional live imaging of bovine embryos by optical coherence tomography

While embryo transfer (ET) is widely practiced, many of the transferred embryos fail to develop in cattle. To establish a more effective method for selecting bovine embryos for ET, here we quantified morphological parameters of living embryos using three-dimensional (3D) images non-invasively captured by optical coherence tomography (OCT). Seven Japanese Black embryos produced by in vitro fertilization that had reached the expanded blastocyst stage after 7 days of culture were transferred after imaged by OCT. Twenty-two parameters, including thickness and volumes of the inner cell mass, trophectoderm, and zona pellucida, and volumes of blastocoel and whole embryo, were quantified from 3D images. Four of the seven recipients became pregnant. We suggest that these 22 parameters can be potentially employed to evaluate the quality of bovine embryos before ET.     

腺病毒介导的高效生产水牛转基因胚胎条件的摸索

试验旨在探究腺病毒感染水牛颗粒细胞和胚胎的最佳条件,以提高转基因水牛胚胎的生产效率。以水牛颗粒细胞和体外发育的胚胎为研究对象,分别用0、1×10~0、1×10^-1、1×10^-2、1×10^-3、1×10^-4、1×10^-5 GFU/mL腺病毒感染水牛颗粒细胞,获得最佳感染浓度后,在最佳浓度条件下分别感染24、48、72、96 h,摸索最佳感染时间,用倒置荧光显微镜观察试验结果。利用腺病毒感染水牛颗粒细胞的最佳浓度和时间分别感染2细胞和4细胞期胚胎,对胚胎的最佳感染条件进行摸索,分析胚胎分裂率、囊胚率和转染囊胚率。结果显示,1×10^-2 GFU/mL浓度和48 h感染时间可获得水牛颗粒细胞的最佳腺病毒感染效率。腺病毒感染2细胞和4细胞期无透明带和非完整透明带胚胎后胚胎发绿光,而感染完整透明带胚胎后不发光,2细胞期胚胎感染后停止发育,4细胞期开始感染的非完整透明带胚胎可继续发育至囊胚。于4细胞期分别感染完整透明带组、非完整透明带组和无透明带组水牛胚胎,结果显示,非完整透明带转染组在囊胚率和囊胚转染效率上均优于其他组。综上,非完整透明带,1×10^-2 GFU/mL和48 h为感染浓度和时间,4细胞期为感染起始期的腺病毒介导的水牛转基因胚胎生产方法能够实现目标基因在水牛胚胎中的高效表达,从而达到提高转基因水牛胚胎生产效率的目的。     

Morphology and size of embryos recovered from superovulated cows.

A total of 691 normal embryos were recovered from 183 superovulated donor cows on the 5th and 6th days after the first insemination, and were examined for their morphology and size in relation to their developmental stage. There was no significant difference in the thickness of the zona pellucida, the diameter of the cell mass, and the overall diameter of the embryos among zygotes, 2-, 4-, 8- and 16-cell embryos, and morulae. In the blastocyst stage, however, the diameter of the cell mass and the overall embryo diameter were significantly greater and the zona pellucida was significantly thinner than in the earlier-stage embryos. The volume of the blastomere significantly decreased from zygote to morula in proportion to the increase in the number of blastomeres. The volume of the cell mass of 2-cell embryos was decreased by about 30% compared with that of zygotes and no increase in the volume of the cell mass was observed during the progression from 2-cell stage to morula. The diameter of the cell mass and the overall diameter of morulae recovered on the 6th day after the first insemination were significantly greater than those of morulae recovered on the 5th day.     

Comparison of persistence of seven bovine viruses on bovine embryos following in vitro exposure

The ability of seven cytopathic strains of bovine viruses to adhere to the zona pellucida of six-to-eight day-old bovine embryos were compared. Embryos were exposed to virus by placing them either in virus suspensions or by culturing them on infected bovine turbinate cultures for 18-24 h. After exposure to bovine virus diarrhea virus (BVDV), infectious bovine rhinotracheitis virus (IBV), bluetongue virus (BTV), pseudorabies virus (PRV), vesicular stomatitis virus (VSV), parainfluenza 3 virus (PI3), or bovine enterovirus virus (BEV), the embryos were tested for virus by culture in bovine turbinate cells and by morphological examination using electron microscopy (EM). A special technique to minimize loss of embryos processed for EM was developed. More embryos had viral particles on the surface of the zona pellucida after exposure to 18-24 hour infected cell cultures than did embryos exposed to viral culture suspensions. The most dramatic finding was that BTV adhered in large numbers to the surface of the zona pellucida of exposed embryos. IBRV, PRV, and VSV comprised an intermediate group, with virions occasionally detected on the surface of exposed embryos after 5 washes. Therefore, extensive washing is required. The PI3 and BEV were easily removed from embryo-exposed virus by washing. BVD was difficult to identify morphologically, making assessment by EM unreliable. There was no evidence that any one of the seven viruses penetrated the intact zona pellucida. Using a micromanipulator, 42 embryos were also directly inoculated through the zona pellucida with +/- 50 picoliters of virus inoculum or medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Adherence of bovine viral diarrhea virus to bovine oocytes and embryos with a hardened zona pellucida cultured in vitro

The purpose of this study was to investigate the adherence of bovine viral diarrhea virus (BVDV) to bovine mature, or immature, cumulus-free oocytes and to in vitro fertilized embryos, maintained in vitro in a ligated bovine oviduct to allow for the hardening of the zona pellucida. Incubation of the oocytes and embryos in the oviduct for 5 h caused hardening of the zona pellucida as measured by resistance to pronase digestion (which increased from approximately 3 min to 7 h; P >0.001). However, there was no difference between the number of infected oocytes and embryos (n = 965 in 193 samples) following experimental exposure to BVDV regardless of whether or not they were previously incubated in the oviduct (P > 0.05). It was concluded that the modification of the proteolytic resistance properties of the zona pellucida during in vitro oviductal incubation did not influence the adherence of BVDV to zona pellucida of oocytes or in vitro fertilized embryos.     

Correlation between the Cell Number and Diameter in Bovine Embryos Produced In Vitro

This study was designed to examine the effects of age and developmental stage of in vitro‐produced bovine embryos on the cell number of the embryos and to investigate the correlation between the cell number and diameter in the embryos. The diameter and cell number in blastocysts and expanded blastocysts collected on days 7–9 after in vitro fertilization (IVF) were examined. Although the diameters of the blastocysts collected on days 7 and 8 after IVF were smaller than those of the expanded blastocysts collected on day 9, the cell number in both types of embryos was similar. The cell numbers of the blastocysts and expanded blastocysts decreased with increasing embryo age. There were positive correlations between the cell number and diameter in bovine embryos at each stage collected on each day after IVF. However, the value of the correlation coefficient in the day‐9 expanded blastocyst group tended to be higher than that in the other groups. These results indicate that the cell number of in vitro‐produced embryos is affected by the embryonic stage and age. The diameter of the embryo may be potentially used for the viability testing of the expanded blastocysts collected on day 9 after IVF.     

Protein A gold identification of ureaplasmas on the bovine zona pellucida.

The object of this study was to develop a prefixation protein A gold labelling technique for Ureaplasma diversum and to apply this to bovine embryos. Sixteen hour cultures of Ureaplasma diversum strain 2312 were incubated with either specific antiserum or nonimmune serum, followed by exposure to protein A gold and negative staining. The ureaplasmas which were incubated with specific antiserum were labelled with gold particles while those ureaplasmas which were incubated with nonimmune serum were not labelled. Twenty-three unhatched, day 7 bovine embryos were then incubated in either embryo culture medium (ECM) alone, ECM with sterile ureaplasma broth added or ECM with 1.7 X 10(6) colony forming units of Ureaplasma diversum strain 2312 per embryo. After 16 hours, the embryos were washed twice and incubated with either specific antiserum or nonimmune serum. The embryos were then incubated with medium containing protein A gold and examined by electron microscopy. No ureaplasmas were identified on the zona pellucida of the control embryos. Ureaplasmas were identified on the outer surface of the zona pellucida of 13 of the 17 embryos which had been exposed to the organism. Of these, the embryos which were incubated with specific antiserum had labelled ureaplasmas while the embryos which were incubated with nonimmune serum had unlabelled ureaplasmas on the zona pellucida. It was concluded that the protein A gold method was a suitable technique for the identification of ureaplasmas in EM preparations. The presence of ureaplasmas on the outer surface of the bovine zona pellucida following in vitro exposure to the organism was confirmed.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Susceptibility of bovine naked 2- and 4-cell embryos and hatched blastocysts produced in vitro to infection with noncytopathogenic bovine viral diarrhea virus.

To investigate the susceptibility of early bovine embryos to noncytopathogenic bovine viral diarrhea virus (NCP BVDV), 2- and 4-cell embryos produced in vitro from which zona pellucida had been removed by pronase treatment, and hatched blastocysts were exposed to 10(6) TCID50/m/ of NCP BVDV No. 12 strain. The virus was detected in all embryo samples immediately prior to cultivation but not in the medium. After 24-hr culture, the virus was isolated from four media and two embryo samples in four experiments in the blastocyst group, and the viral antigen was demonstrated in the cytoplasm of the embryo cells by the immunofluorescent technique. By contrast, no virus was recovered from, or viral antigen detected in samples from the 2- and 4-cell embryo group in any of the experiments, even though they were exposed to the virus after removal of the zona pellucida. These findings suggest that 2- and 4-cell embryos are unlikely to be susceptible to NCP BVDV, but that blastocysts are capable of being infected with the virus. hatched blastocyst, noncytopathogenic bovine viral diarrhea virus.     

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.

     

Development of interspecies cloned monkey embryos reconstructed with bovine enucleated oocytes

This study was carried out to determine whether culture media reconstructed with bovine enucleated oocytes and the expression pattern of Oct-4 could support dedifferentiaton of monkey fibroblasts in interspecies cloned monkey embryos. In this study, monkey and bovine skin fibroblasts were used as donor cells for reconstruction with bovine enucleated oocytes. The reconstructed monkey interspecies somatic cell nuclear transfer (iSCNT) embryos were then cultured under six different culture conditions with modifications of the embryo culture media and normal bovine and monkey specifications. The Oct-4 expression patterns of the embryos were examined at the two-cell to blastocyst stages using immunocytochemistry. The monkey iSCNT embryos showed similar cleavage rates to those of bovine SCNT and bovine parthenogenetic activation (PA). However, the monkey iSCNT embryos were not able to develop beyond the 16-cell stage under any of the culture conditions. In monkey and bovine SCNT embryos, Oct-4 could be detected from the two-cell to blastocyst stage, and in bovine PA embryos, Oct-4 was detectable from the morula to blastocyst stage. These results suggested that bovine ooplasm could support dedifferentiation of monkey somatic cell nuclei but could not support embryo development to either the compact morula or blastocyst stage. In conclusion, we found that the culture conditions that tend to enhance monkey iSCNT embryo development and the expression pattern of Oct-4 in cloned embryos (monkey iSCNT and bovine SCNT) are different than in bovine PA embryos.     

The effect of experimental infection of mouse preimplantation embryos with paramyxovirus Sendai.

Zona-intact and zona-free mouse embryos at the morula stage were exposed to Sendai virus in vitro, and then transferred to the uteri of recipient foster mothers. Both embryos developed into expanded blastocyst stage after 48 hr of culture. Zona-intact embryos were resistant to infection and subsequent transfer resulted in the development of fetuses, indicating that the zona pellucida plays a role of a barrier to virus infection. On the other hand, zona-free embryos were susceptible to infection and only one fetus out of 64 transfers developed to term. Implantation sites were scarcely observed in the uteri of the foster mothers that received zona-free embryos, suggesting that most of the embryos did not develop after embryo transfer. Sendai virus was shed in the culture fluid of the zona-free embryos indicating viral replication in the embryonic cells. By immunofluorescence assay, viral antigens were detected in the embryos, tissues of the fetus and implantation site derived from the zona-free embryos. These findings indicate that replication of Sendai virus in the embryonic cells interfere with early embryonic development and fetal growth of the embryo.     

Embryonic migration relative to maternal recognition of pregnancy in sheep

Twenty-five crossbred ewes were utilized to examine the timing of embryonic migration relative to maternal recognition of pregnancy. These ewes also were utilized to examine whether ovine embryos synthesized estradiol-17 beta in association with embryonic elongation and intrauterine migration. Embryos were flushed on d 11 through 15 from hemiovariectomized ewes. Recovery of embryos from the uterine horn contralateral to the remaining ovary indicated that migration had occurred. Ewes subsequently were returned with rams to determine their interestrous interval. Recovered embryos were classified morphologically, their length determined and individually incubated. Changes in estradiol within the medium were determined after a 6-h incubation. Embryo migration began on d 14 (P less than .05); on consecutive days from 11 through 15, 0, 0, 0, 60 and 100% of ewes examined, respectively, had an embryo in the contralateral horn. Extended estrous cycles (greater than 20 d) were observed in 0, 0, 40, 80 and 100% of ewes examined (P less than .05) following removal of embryos on d 11 through 15 of the cycle. Ovine embryos were longer (P less than .05) on d 14 (4.8 +/- 1.1 cm length, mean +/- SE) compared with d 13 (.2 +/- .1 cm) and increased further (P less than .05) on d 15 (7.8 +/- 1.1 cm). Incidence of intrauterine migration was correlated with embryonic length (r = .83; P less than .01) and estradiol synthesis (r = .77; P less than .01).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.