Commercial aspects of bovine embryo transfer

Superovulated beef cows and heifers were nonsurgically collected 6 to 8 days post estrus. Commercial production results for 1976 through 1978 were 4979 pregnancies from 7814 embryos transferred for an overall pregnancy rate of 63%. In 1978, 519 superovulation procedures averaged 9.95 +/- 8.4 (S.D.) ova collected, 8.2 +/- 7.55 ova fertilized, 5.96 +/- 5.37 embryos transferred and 3.63 +/- 5.37 pregnancies per procedure. Embryos were transferred to recipient cows in estrus 12 hr before the donor (-12) the same time (0) or 12 hr after the donor (+12). The +12 group had a significantly lower pregnancy rate (61%, P<.05) than the 0 group (67%) or -12 group (66%). Transfer of early morula stage embryos resulted in a lower pregnancy rate (61%, P<.05) than late morula (67%) early blastocyst (67%) or late blastocyst (71%) stage embryos. A higher pregnancy rate (P<.05) was obtained with embryos of good morphological quality (71%) than with embryos graded fair and poor (55%). The pregnancy rate for embryos transferred nonsurgically was lower (44%) than the pregnancy rate for embryos transferred surgically during the same time period (66%). Pregnancy rates for three operators performing the nonsurgical transfers were 48%, 53%, 28%. No difference in pregnancy rate was found between embryos cultured 24 hr in BMOC-3 at 37C (62%) and embryos transferred the same day as collection (60%). Pregnancy rates for cultured embryos transferred to recipient cows in estrus 12, 24 or 36 hr after the donor were 68%, 62% and 60%, respectively. Embryos recovered on days 6, 7 and 8 were frozen in 1.5M DMSO and stored in liquid nitrogen several days to several weeks. Of 68 embryos frozen, 34 were viable post thaw. Upon transfer to recipient cows, the 34 viable embryos produced 23 confirmed pregnancies.     

Approaches to biosecurity in bovine embryo transfer programs

Although transfer of bovine embryos is much less likely to result in transmission of pathogens than transport of postnatal cattle, the epidemiologic risk associated with bovine embryo transfer merits examination. Much research has validated the efficacy of internationally approved processing protocols to render bovine in vivo-derived embryos free of specified pathogens. The purpose of this review is to summarize current sanitary recommendations for bovine embryo transfer, while emphasizing recent research to develop and validate novel approaches to biosecurity. Continued research will enable the development and validation of novel embryo treatments and culture reagents to minimize requirements for testing of embryo or oocyte donors, and testing of embryo recipients.     

Sexing and multiple genotype analysis from a single cell of bovine embryo

We described a procedure for multiple genotype analysis (determination of sex and of three genetic markers) from a single cell derived from bovine preimplantation embryo. It consists of primer extension preamplification-polymerase chain reaction (PEP-PCR) and subsequent single assay or multiplex PCR. A single blastomere that was isolated by microaspiration from bovine embryos at the 16- to 32-cell stage then was lysed and was subjected to the PEP-PCR. When testing 75 embryos, efficiency of genotyping by standard PCR for kappa-casein, growth hormone (GH) and prolactin (PRL) polymorphic alleles was 91, 88 and 89%, respectively. Sexing efficiency in the multiplex PCR was 91%, based on the amplification of Y-specific locus using kappa-casein internal standard. The microaspiration of a single blastomere was shown not to be invasive for the embryos. It did not alter their development potential in vitro (P > 0.05), as was seen by obtaining a similar percentage of embryos developing further into the blastocyst stage in the group subjected to biopsy (44/75, 59%) and in the control group of embryos (30/50, 60%).     

Cloning of bovine embryos by multiple nuclear transfer

The in vitro development of multiple generation bovine nuclear transferred embryos to blastocysts and their survival ability after freezing and thawing were examined. Parent donor embryos which had 20 to 50 cells were recovered from superovulated cows. Follicular oocytes matured in vitro were used as recipient oocytes. The recipient oocytes enucleated at 22 to 24 h after the onset of maturation were preactivated at 33 h. Enucleated oocytes with a donor blastomere were fused 9 h after activation by an electric stimulus and the fused oocytes were cultured in vitro (first generation). Reconstituted oocytes that had developed to the 8- to 16-cell stage 3 to 4 d after fusion were used as donor embryos for the next generation. Recloning procedures were performed twice (second and third generations). The proportion of recipient oocytes successfully fused with a blastomere increased with the cycle of nuclear transfer. Eighty to 86% of fused oocytes developed to the 2-cell stage and there was no significant difference with the generation. The proportion of reconstituted embryos receiving blastomeres derived from first generation embryos had higher developmental ability in vitro, than those derived from other generations (43 vs 31% for 8 to 16-cell stage, 37 vs 20 and 21% for blastocyst stage). The number of cloned blastocysts increased with repeated nuclear transfer (once: 6.2 +/- 4.3, twice: 19.8 +/- 9.2 and three times: 30.0 +/- 14.7) but varied greatly with each parent donor embryo. The in vitro viability of cloned blastocysts after freezing and thawing (59%) was low but not significantly different from that obtained for in vitro fertilized blastocysts (72%). After transfer of either fresh or frozen-thawed cloned blastocysts to 21 recipients, 10 of them were pregnant on Day 60. Four and 3 offspring were produced from 20 fresh and 14 frozen-thawed blastocysts,respectively.     

Single embryo transfer in IVF to prevent multiple pregnancies.

As the success rates of IVF clinics improve, one of the adverse consequences is the increased incidence of twins, due largely to the number of embryos transferred. Even if the number of embryos transferred is restricted to two, the twinning rate can exceed 40% of the pregnancies. An obvious way to reduce this high twin rate would be to transfer only one embryo. This would require that cryopreservation of the supernumerary embryos be efficacious enough so that the chance of achieving an ongoing pregnancy is not diminished by transferring a single embryo in the stimulated cycle. Previous studies utilising embryos on day 2 and 3 of development have shown that the pregnancy rates can be acceptable (about 40%) and that the cumulative rate can be up to 60%. Most of these studies, however, do not include a comparison with the cumulative pregnancy rate with two embryos transferred in the stimulated cycle. Therefore, the efficacy has not been proven. We present clinical data from the past few years to illustrate the increase in success rates and the concomitant increase in twinning rates. The increased success in the cryopreservation program has enabled us to trial a single embryo transfer program and compare the results to the transfer of two embryos. The results strongly suggest that the transfer of a single embryo is the better clinical option.     

Fast-developing preimplantation embryo progeny from heterogametic females in mammals.

Karyotyping and cell number estimates in preimplantation embryos from heterogametic (XY*) and homogametic (XX) females of the field mouse Akodon azarae were studied to determine whether XX-XY-XY* differences exist in the rate of preimplantation development. At the morula stage, XY embryos from heterogametic mothers had twice the mean number of cells compared with XX embryos. However, this difference in cell numbers was not seen between XX and XY embryos from homogametic mothers. In this case, mean cell numbers were similar despite embryos being XX or XY. Furthermore, the mean cell number for XX and XY morulae from homogametic females was comparable to that for XX embryos from heterogametic females. It is concluded that XY* embryos (which will develop into heterogametic females) show an accelerated rate of preimplantation development.     

In vitro production of bovine embryos and their application for embryo transfer

This review introduces newly developed serum-free media (IVD101 and IVMD101), that are effective for producing high yields of transferable embryos of good quality from in vitro-matured and -fertilized oocytes. Both serum-free media produced better results than serum-containing medium, including increased rates of blastocyst formation, post-thaw embryo viability, and pregnancy after transfer. In addition, reduced risks of calf mortality and large calf syndrome were also observed for the serum-free-derived embryos. Serum-derived embryos contained a large number of lipid droplets and immature mitochondria in their cytoplasm that may account for the lower production of transferable embryos and poor embryo quality. A non-invasive technique using scanning electrochemical microscopy was successful in quantitatively measuring oxygen consumption of single embryos. This technique may prove to be reliable for predicting embryo viability and subsequent developmental ability.     

Glycolipid transfer protein from bovine brain

Glycolipid transfer protein from bovine brain has been purified partially by ammonium sulfate precipitation, CM-52 ion-exchange, and Sephadex G-75 column chromatography. Both pyrene-labeled and tritium-labeled glucocerebrosides have been used to study the kinetics of protein-mediated transfer between donor and acceptor vesicles. Protein accelerates glucocerebroside transfer but does not accelerate phospholipid transfer. In colyophilized small sonicated vesicles (10% glucocerebroside, 90% 1-palmitoyl-2-oleoyl-phosphatidylcholine) about two-thirds of the glycolipid is transferred in 2 h and the remaining one-third does not transfer (up to 5 h). For donor and acceptor vesicles made of dipalmitoylphosphatidylcholine or 1-palmitoyl-2-oleoyl-phosphatidylcholine, glucocerebroside (10% in donors) is transferred rapidly only when both the donor and acceptor matrix phospholipids are in the liquid-crystalline state. If either donor or acceptor vesicles are in the gel state, transfer protein mediated transfer is much reduced. The amount of transfer protein bound specifically to glucocerebroside-containing vesicles is nearly equal above and below the matrix phospholipid phase transition temperature. Bound protein transfers glucocerebroside upon addition of acceptor vesicles.     

The characterization of bovine embryos obtained from prepubertal calf oocytes and their viability after non surgical embryo transfer

We have previously shown that the addition of epidermal growth factor (EGF) during in vitro maturation was capable of stimulating the cytoplasmic maturation of cow and calf oocytes. The aim of the present study was to compare calf and cow blastocysts produced in the presence of EGF in terms of total cell number and cell distribution between trophectoderm (TE) and inner cell mass (ICM), pattern of protein synthesis, and ability to establish pregnancy after embryo transfer to recipients. For all experiment, embryos at Day 7 were obtained from IVM/IVF/IVC oocytes. No significant differences were noted in total cell number (cow= 138±46 vs CALF= 142±59; mean±SD) or ICM and TE cell number between calf (ICM= 35 ± 19, TE= 107± 52) and cow (ICM= 38± 21, TE= 99 ± 32) blastocysts, nor in the ICM/total cell number ratio (cow= 0.27± 11, CALF= 0.25 ± 12). No differences were noted in the constitutive and the neosynthetic protein profiles between cow and calf embryos obtained in vitro. The results of embryo transfer, showed that there was higher pregnancy loss following transfer of calf compared with cow embryos. After Day 35, the rate of pregnancy decreases, with only 22% of calf embryos maintaining pregnancy until calving compared with 39% for cow embryos. In conclusion, it would seem that embryos originating from calf oocytes are less capable of establishing pregnancies than embryos obtained from adult oocytes, althrough this difference was not significant. This low viability cannot be explained by differences in cell number or by the protein profiles identifed between these 2 groups of embryos.     

Offspring born from chimeras reconstructed from parthenogenetic and in vitro fertilized bovine embryos

Chimeric embryos were produced by aggregation of parthenogenetic (Japanese Red breed) and in vitro fertilized (Holstein breed) bovine embryos at the Yamaguchi Research Station in Japan and by aggregation of parthenogenetic (Red Angus breed) and in vitro fertilized (Holstein breed) embryos at the St. Gabriel Research Station in Louisiana. After embryo reconstruction, live offspring were produced at each station from transplanting these embryos. The objective of this joint study was to evaluate the developmental capacity of reconstructed parthenogenetic and in vitro fertilized bovine embryos. In experiment I, chimeric embryos were constructed: by aggregation of four 8‐cell (demi‐embryo) parthenogenetic and four 8‐cell stage (demi‐embryo) IVF‐derived blastomeres (method 1) and by aggregation of a whole parthenogenetic embryo (8‐cell stage) and a whole IVF‐derived embryo (8‐cell stage) (method 2). Similarly in experiment II, chimeric embryos were constructed by aggregating IVF‐derived blastomeres with parthenogenetic blsatomeres. In this experiment, three categories of chimeric embryos with different parthenogenetic IVF‐derived blastomere ratios (2:6; 4:4, and 6:2) were constructed from 8‐cell stage bovine embryos. In experiment III, chimeric embryos composed of four 8‐cell parthenogenetic and two 4‐cell IVF‐derived blastomeres or eight 16‐cell parthenogenetic and four 8‐cell IVF‐derived blastomeres were constructed. Parthenogenetic demi‐embryos were aggregated with sexed (male) IVF demi‐embryos to produce chimeric blastocysts (experiment IV). In the blastocyst stage, hatching and hatched embryos were karyotyped. In experiment V, chimeric embryos that developed to blastocysts (zona‐free) were cryopreserved in ethylene glycol (EG) plus trehalose (T) with different concentrations of polyvinylpyrrolidone (PVP; 5%, 7.5%, and 10%). In experiment I, the aggregation rate of the reconstructed demi‐embryos cultured in vitro without agar embedding was significantly lower than with agar embedding (53% for 0% agar, 93% for 1% agar, and 95% for 1.2% agar, respectively). The aggregation was also lower when the aggregation resulted from a whole parthenogenetic and IVF‐derived embryos cultured without agar than when cultured with agar (70% for 0% agar, 94% for 1% agar, and 93% for 1.2% agar, respectively). The development rate to blastocysts, however, was not different among the treatments. In experiment II, the developmental rates to the morula and blastocyst stages were 81%, 89%, and 28% for the chimeric embryos with parthenogenetic:IVF blastomere ratios of 2:6, 4:4, and 6:2, respectively. In experiment III, the developmental rate to the morula and blastocyst stages was 60% and 65% for the two 4‐cell and four 8‐cell chimeric embryos compared with 10% for intact 8‐cell parthenogenetic embryos and 15% for intact 16‐cell parthenogenetic embryos. To verify participation of parthenogenetic and the cells derived from the male IVF embryos in blastocyst formation, 51 embryos (hatching and hatched) were karyotyped, resulting in 27 embryos having both XX and XY chromosome plates in the same sample, 14 embryos with XY and 10 embryos with XX. The viability and the percentage of zona‐free chimeric embryos at 24 hr following cryopreservation in EG plus T with 10% PVP were significantly greater than those cryopreserved without PVP (89% vs. 56%). Pregnancies were diagnosed in both stations after the transfer of chimeric blastocysts. Twin male (stillbirths) and single chimeric calves were delivered at the Yamaguchi station, with each having both XX and XY chromosomes detected. Three pregnancies resulted from the transferred 40 chimeric embryos at the Louisiana station. Two pregnancies were lost prior to 4 months and one phenotypically‐ chimeric viable male calf was born. We conclude that the IVF‐derived blastomeres were able to stimulate the development of bovine parthenogenetic blastomeres and that the chimeric parthenogenetic bovine embryos were developmentally competent. Mol. Reprod. Dev. 53:159–170, 1999. © 1999 Wiley‐Liss, Inc.     

Monolayer culture of cells originating from apreimplantation bovine embryo

Summary The objective of this study was to establish a method by which trophectodermal cells originating from individual preimplantation bovine embryos could be perpetuated in monolayer culture. A single, Day-11 bovine embryo collected nonsurgically from a mixed-breed beef cow was cultured in Ham's F10 medium supplemented with fetal bovine serum, sodium pyruvate, insulin and epidermal growth factor. After 13 d in culture the embryo had adhered to the surface of the plastic culture vessel and a monolayer covering 0.3 cm² had developed in the manner of a tissue explant. The monolayer was successfully dispersed using trypsin-EDTA and the cells were passaged Expansion to a 25-cm² flask was achieved by the 4th passage. By passaging cultures at a dilution ratio of 1∶2, cells were maintained for 38 passages before growth slowed. Transfers beyond the 44th passage were unsuccessful. The cell line, designated BE-13, was successfully frozen and thawed at the 9th, 12th, 15th, and 20th passages. The cell line contains both mono- and binucleate cells with a prominent rough endoplasmic reticulum characteristic of ruminant trophoblast cells. Susceptibility to eight bovine viruses was demonstrated. Such cell lines may provide inexpensive systems for the study of trophoblast metabolism and for investigation of the role of the trophoblast in the pathogenesis of selected bovine abortifacient diseases. Because of their range of viral susceptibility, these cells might also be useful for diagnostic purposes. Published as publication no. 1891 College of Veterinary Medicine, Auburn University, Alabama 36849. This work was funded in part by an Auburn University Faculty Research Grant-in-aid. Preliminary results of the study were presented in abstract form at the 1987 Annual Conference of the International Embryo Transfer Society.     

Development of rabbit zygotes into blastocysts in defined protein-free medium and offspring born following culture and embryo transfer

We report here on an improved, completely defined culture system for producing embryos in vitro which mimics development in vivo. This system avoids the confounding effects of the many unknowns introduced by the multivariate components of the serum or by unknowns attached to bovine serum albumin (BSA). Zygotes were obtained from superovulated rabbits and cultured in modified defined RPMI 1640:Dulbecco's MEM, 1:1 (RD) medium. The effect of a novel and potentially ideal antioxidant, tempol, was tested (20 to 0.001 mM) but found to be either toxic or ineffective. In the presence of 20% O(2), 600 units of Superoxide dismutase or 2.5 mM of taurine increased embryo hatching after 72 h of culture in RD medium to 75 and 76%, respectively, compared with 46% in the control (P < 0.05). The need for antioxidants was reduced with 5% O(2). The beneficial effects of RD medium were demonstrated when 60 zygotes cultured for 48 h to the early blastocyst stage in this medium were transferred and resulted in 30 young (50%) compared with 35/60 (58%) young from uncultured control transfers. Only 12% of the young were obtained from slower developing morulae. Thus, high viability was established for rapidly growing embryos in culture, but fewer slow growing embryos survived after transfer. A further comparison of embryos cultured in RD medium with a high potassium simple, optimized, defined medium (KSOM), revealed that both yielded results approaching those of direct transfer without culture. Simple defined media may also be useful for the culture of embryos of other species.