Delipidating in vitro-produced bovine zygotes: effect on further development and consequences for freezability

To study the effect of partial removal of intracytoplasmatic lipids from bovine zygotes on their in vitro and in vivo survival, presumptive zygotes were delipidated by micromanipulation and cocultured with Vero cells in B2+10% FCS. Blastocyst rates of delipidated (n=960), sham (centrifuged but not delipidated, n=830) and control embryos (n=950) were 42.1, 42.3 and 39.9% respectively (P > 0.05). Day 7 blastocysts derived from delipidated zygotes had a mean of 123.9 +/-45.6 nuclei compared to 137.5+/-32.9 for control blastocysts (P > 0.05). The full-term development of delipidated blastocysts after single transfer to recipients was similar to that of control IVF blastocysts (41.2% vs 45.4% respectively). To assess the effect of delipidation on the embryo tolerance to freezing/thawing, delipidated (n=73), control (n=67) and sham (n=50) Day 7 blastocysts were frozen in 1.36 M glycerol + 0.25 M sucrose in PBS. After thawing, embryos were cocultured for 72 h with Vero cells in B2+10% FCS. Survival rates at 24 h were not significantly different between groups. However, in the delipidated group, the survival rate after 48 h in culture was significantly higher than in the control group (56.2 vs 39.8, P < 0.02), resulting in a higher hatching rate after 3 days in culture (45.2 vs 22.4, P < 0.02). Pregnancy rates for delipidated and control frozen/thawed embryos were respectively 10.5 and 22.2% (P > 0.05). Electron microscopic observations showed much fewer lipid droplets (and smaller) in delipated blastocysts than in controls. Taken together, our data show that delipidation of one cell stage bovine embryos is compatible with their normal development to term and has a beneficial effect on their tolerance to freezing and thawing at the blastocyst stage. This procedure, however, alters the developmental potential of such blastocysts, suggesting that maternally inherited lipid stores interfere with metabolic recovery after thawing.     

Treatment with chemical delipidation forskolin prior to cryopreservation improves the survival rates of swamp buffalo (Bubalus bubalis) and bovine (Bos indicus) in vitro produced embryos

The cryopreservation of embryos is a technology developed for long-term genetic preservation. However, high sensitivity to low temperatures due to a large number of intracellular lipids within ruminant embryos compromises the success of this technique. The aim of this study was to examine the effects of using of lipolytic chemical agent forskolin, during in vitro producing of buffalo and bovine embryos on lipid contents, cryotolerance and subsequent developmental competence of these embryos. Buffalo and bovine oocytes were collected by the aspiration technique from follicles and submitted for in vitro fertilisation; the embryos were later divided into four experiments. Experiment 1, buffalo and bovine embryos were pre-treated in the presence and absence of 10 μM forskolin for 24 h. Lipid contents were determined by Nile red staining and confocal microscopy. We found that 10 μM forskolin was capable to reduce lipid contents within developing embryos in both of species (P < 0.01). Lipid contents within Day 2 embryos exhibited greater fluorescence intensity than did Day 7 embryos in both animal species. The purpose of Experiment 2 was to investigate the adverse effects of 10 μM forskolin on embryo development. In Experiments 3 and 4, Day 2 (4- to 8-cell stage) and Day 7 (blastocyst stage) embryos were pre-treated with 10 μM forskolin for 24 h and further cryopreserved with a controlled-rate freezing technique. The successful cryopreservation was determined by post-thawed embryonic development in vitro. The results showed that the blastocyst rate of the 4–8 cell stage in the forskolin-treated group had increased in both species, while the hatching and hatched blastocyst rates of forskolin-treated day 7 bovine embryos were significantly higher than those of the non-treated group (52.1% vs. 39.4%; P < 0.05). However, delipidation with forskolin did not affect the developmental rate of the day 7 buffalo embryos (P = 0.73). Our studies showed that delipidation by forskolin treatment increased the survival rate of cryopreservation in buffalo and bovine in vitro produced embryos.     

The ovine uterus as a host for in vitro-produced bovine embryos

A series of experiments were conducted to determine whether bovine blastocysts would develop beyond the blastocyst stage in the ovine uterine environment. In Experiment 1, in vitro matured, fertilized and cultured (IVM/IVF/IVC) expanded bovine blastocysts were transferred into uteri of ewes on Day 7 or 9 of the estrous cycle and collected on Day 14 or 15 to determine if the bovine blastocysts would elongate and form an embryonic disk. Springtime trials with ewes that were synchronized with a medroxyprogesterone acetate (MAP) sponge resulted in a 78% blastocyst recovery rate, and 68% of the recovered spherical or elongated embryos had embryonic disks. In Experiment 2, transfer of 4-cell bovine embryos to the oviducts of ewes at Day 3 resulted in a lower recovery (47 vs 80%) than the transfer of blastocysts at Day 7 when embryos were recovered at Day 14. However, the percentage of embryos containing embryonic disks was higher for embryos transferred at the 4-cell stage (71%) than for embryos transferred as blastocysts (50%). In Experiment 3, IVF embryos from super-ovulated cows or Day 8 in vitro produced embryos transferred to cows were collected at Day 14 and were found to be similar in size to those produced by transfer to ewes in Experiment 2. In Experiment 4, the transfer of bovine blastocysts to ewes did not prolong the ovine estrous cycle. In Experiment 5, extension of the ovine estrous cycle by administration of a MAP releasing intravaginal device allowed bovine embryos to elongate extensively and to become filamentous. In Experiment 6, uterine flushings on Day 14 or Day 16 contained elevated levels of interferon-tau when bovine blastocyst were transferred on Day 7. Transfer of bovine embryos to the reproductive tract of a ewe allows some embryos to develop normally to advanced perimplantation stages and may be a useful tool for studying critical stages of embryo development and the developmental capacity of experimental embryos.     

The effects of time of first cleavage, developmental stage, and delipidation of nuclear-transferred porcine blastocysts on survival following vitrification

The effect of removing cytoplasmic lipid droplets (delipidation) at the 2-cell and developmental stages on the survival of porcine somatic cell nuclear-transferred blastocysts developed from the enucleated oocytes receiving somatic cells from kidney of an adult female after cryopreservation was examined. Vitrification was performed using the Cryoloop method with a small volume of medium (0.5 μl). To select 2-cell embryos with a high potential to develop into blastocysts, the relationship between the timing of the first cleavage and the developmental potential was examined. The potential of nuclear-transferred oocytes to develop into blastocysts in the intermediate-cleavage group (20–24 h after activation, 25%) was slightly or significantly (P < 0.05) higher than that in fast-cleavage (<20 h after activation, 13%) and slow-cleavage groups (>24 h after activation, 5%). Most non-delipidated blastocysts did not survive after thawing (0% for early-stage and 9% for advanced-stage blastocysts), but the survival rate of delipidated blastocysts 48 h after culture (54% and 72%, respectively) was not significantly different from that of non-vitrified blastocysts (80% and 92%, respectively). The survival rate of advanced-stage blastocysts after vitrification was slightly higher than that of early-stage blastocysts. The present study demonstrates that somatic cell nuclear-transferred porcine blastocysts developed from embryos selected at the 2-cell stage can be preserved by vitrification with a small volume of medium if the lipid droplets of the embryos are first removed.     

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.     

In vitro development and post-thaw survival of blastocysts derived from delipidated zygotes from domestic cats

The ability to cryopreserve in vitro-produced feline embryos was investigated. To improve the survival rate of cryopreserved embryos, first the developmental ability of in vitro fertilized feline zygotes (after removal of intracellular lipids) was determined, followed by the post-thaw survival of cryopreserved blastocysts derived from delipidated zygotes. More than 67% of the delipidated zygotes cleaved and 36% of them developed to the morula stage. The developmental ability of delipidated zygotes to the blastocyst stage (26%) was similar to that of sham-operated (30.5%) or control embryos (31.3%). Although the survival rate of delipidated blastocysts (81.8%) after freezing and thawing tended to be higher than that of control embryos without delipidation (60.6%), rates were not significantly different between the both groups. In conclusion, in vitro-produced feline blastocysts were successfully frozen, removal of the cytoplasmic lipid content in feline zygotes did not impair their in vitro developmental competence (up to the blastocyst stage), and reduction of cytoplasmic lipids by aspiration had no apparent effects on the survival of in vitro-derived blastocysts after cryopreservation.     

Cryopreservation of in vitro produced bovine embryos: effects of lipid segregation and post-thaw laser assisted hatching

The objective was to determine if lipid segregation, with or without post-thaw laser assisted hatching (LAH) of in vitro produced (IVP) bovine embryos, would enhance in vitro survivability and development 24 h post-thaw. On Day 6 of culture (Day 0 = IVF), in vitro produced bovine embryos were divided into three developmental stages: 32-cell (n = 78), compact morula (CM n = 223), and blastocyst (n =56). Embryos within each stage were allocated to the following treatments prior to cryopreservation in 1.5M ethylene glycol: no treatment (Control), 7.5 μg/mL Cytochalasin B for 20 min (CB), or CB with centrifugation (16,000 × g) for 20 min (CBCF). All CB treatments were extended to include embryo freezing. Immediately post-thaw, one-half of the CBCF and Control groups were subjected to zona pellucida drilling (LAH), using the XY Clone® system, creating groups CBCFLAH and LAH, respectively. All thawed embryos were cultured for 24 h and evaluated. No treatment differences were observed for either post-thaw survival or 24 h development. Within the CM stage, CBCFLAH and LAH exhibited a greater number of both total and live cells than Control (total: 69.4, 69.3, 53.0, live: 56.4, 54.7, 39.3 respectively; P < 0.05). In conclusion, LAH post-thaw alone or in combination with CBCF improved embryo viability following cryopreservation.     

Birth of piglets from in vitro-produced, zona-intact porcine embryos vitrified in a closed system

As the importance of swine models in biomedical research increases, it is essential to develop low-cost, high-throughput systems to cryopreserve swine germplasm for maintenance of these models. However, porcine embryos are exceedingly sensitive to low temperature and successful cryopreservation is generally limited to the use of vitrification in open systems that allow direct contact of the embryos with liquid nitrogen (LN₂). This creates a high risk of pathogen transmission. Therefore, cryopreservation of porcine embryos in a “closed” system is of very high importance. In this study, in vitro-produced (IVP) porcine embryos were used to investigate cryosurvival and developmental potential of embryos cryopreserved in a closed system. Optimal centrifugal forces to completely disassociate intracellular lipids from blastomeres were investigated using Day-4 embryos. Cryosurvival of delipidated embryos was investigated by vitrifying the embryos immediately after centrifugation, or after development to blastocysts. In this study, centrifugation for 30 min at 13,000 g was adequate to completely delipidate the embryos; furthermore, these embryos were able to survive cryopreservation at a rate comparable to those centrifuged for only 12 min. When delipidated embryos were vitrified at the blastocyst stage, there was no difference in survival between embryos vitrified using OPS and 0.25 mL straws. Some embryos vitrified by each method developed to term. These experiments demonstrated that porcine embryos can be cryopreserved in a closed system after externalizing their intracellular lipids. This has important implications for banking swine models of human health and disease.     

Survival of frozen-thawed sheep embryos cryopreserved at cleavage stages

This study evaluated the effect of freezing-thawing procedures on the viability of sheep embryos cryopreserved at various developmental stages. The survival rates of frozen-thawed embryos were compared with non-frozen counterparts. Embryos were recovered from the oviduct and uterus, at different days of the early luteal phase, and were classified at six different developmental stages: 2- to 4-cell (n = 72), 5- to 8-cell (n = 73), 9- to 12-cell (n = 70), early morulae (n = 42), morulae (n = 41), and blastocyst (n = 70). For each early cleavage stage and blastocysts, approximately half of the embryos, were frozen immediately by slow freezing with an ethylene glycol-based solution. The remaining embryos were cultured to the hatched blastocyst stage. All morulae and compact morulae were frozen after recovery with the same protocol. Cryoprotectants were removed using 1M sucrose solution, and then warmed the embryos were cultured to the hatched stage in a standardized in vitro culture. Embryo developmental stage had a significant effect on the ability to hatch following freezing (P<0.0001). The cryotolerance of the embryos fitted a regression (r2 = 0.908), increasing linearly from 2- to 4-cell embryos (17.1%) to morula stage (46.3%) and in a quadratic regression from the morula to the blastocyst stage (83.7%). Frozen early cleavage stage embryos had a significantly lower viability than their fresh counterparts (23.1 vs 83.1%; P<0.0001), with a similar rate of viability between fresh or frozen blastocysts (92.5 vs 83.7%). In conclusion, early sheep embryos are very sensitive to freezing per se and the survival rates following conventional freezing improve as embryo developmental stage progresses.     

Morphological features of lipid droplet transition during porcine oocyte fertilisation and early embryonic development to blastocyst in vivo and in vitro

Lipid content in mammalian oocytes or embryos differs among species, with bovine and porcine oocytes and embryos showing large cytoplasmic droplets. These droplets are considered to play important roles in energy metabolism during oocyte maturation, fertilisation and early embryonic development, and also in the freezing ability of oocytes or embryos; however, their detailed distribution or function is not well understood. In the present study, changes in the distribution and morphology of porcine lipid droplets during in vivo and in vitro fertilisation, in contrast to parthenogenetic oocyte activation, as well as during their development to blastocyst stage, were evaluated by transmission electron microscopy (TEM). The analysis of semi-thin and ultra-thin sections by TEM showed conspicuous, large, electron-dense lipid droplets, sometimes associated with mitochondrial aggregates in the oocytes, irrespective of whether the oocytes had been matured in vivo or in vitro. Immediately after sperm penetration, the electron density of the lipid droplets was lost in both the in vivo and in vitro oocytes, the reduction being most evident in the oocytes developed in vitro. Density was restored in the pronculear oocytes, fully in the in vivo specimens but only partially in the in vitro ones. The number and size of the droplets seemed, however, to have decreased. At 2- to 4-cell and blastocyst stages, the features of the lipid droplets were almost the same as those of pronuclear oocytes, showing a homogeneous or saturated density in the in vivo embryos but a marbled or partially saturated appearance in the in vitro embryos. In vitro matured oocytes undergoing parthenogenesis had lipid droplets that resembled those of fertilised oocytes until the pronuclear stage. Overall, results indicate variations in both the morphology and amount of cytoplasmic lipid droplets during porcine oocyte maturation, fertilisation and early embryo development as well as differences between in vivo and in vitro development, suggesting both different energy status during preimplantation development in pigs and substantial differences between in vitro and in vivo development.     

Oviduct Epithelial Cell Co-culture of early Porcine Embryos

One- to 16-cell porcine embryos were cultured in either Whittens medium supplemented with bovine serum albumin and fetal calf serum (WM) or in the same medium with porcine oviduct epithelial cell co-culture (WM-Poec). All stages of embryos cultured in WM-POEC had higher cell counts after 144–168 h of development than did embryos in WM. There was however, no significant difference in blastocyst formation rate of embryos cultured in WM-POEC over those cultured in WM. A high proportion of the embryos entering culture at the 1-2-cell were able to pass the 4-cell block stage in both WM and WM-POEC, 81% and 77%, respectively. In both media, most of the 1-2-cell embryos arrested their development at the compacted morula stage and failed to blastulate while embryos initiating culture at the 4-and 8-16-cell embryos formed blastocysts in culture at a rate of 80–90%.     

Development of centrifuged cow zygotes cultured in rabbit oviducts

Zygotes from superovulated cows were centrifuged and pronuclei were detected by differential interference-contrast microscopy in 73% of 106 zygotes. Zygotes were then transferred to ligated oviducts of follicular-phase, 1-day pseudopregnant or 7-day pseudopregnant rabbits and recovered 5 days later. Their development did not differ from that of uncentrifuged zygotes transferred to the opposite oviduct: 41% of the embryos recovered from rabbit oviducts contained 17-32 nuclei and an additional 5% contained greater than 32 nuclei. In another experiment, 399 ova from unmated cows were transferred to rabbit oviducts to determine whether centrifugation induced parthenogenetic development. After 7 days, 257 ova were recovered; 16% of the recovered ova had developed parthenogenetically and contained 2-30 nuclei. Neither centrifugation of the ova nor reproductive status of the rabbits influenced the proportion of parthenogenotes found. Parthenogenetic development was also observed in 14 of 71 ova (20%) recovered on Day 7 from uninseminated superovulated cows. In an attempt to increase the probability of detecting treatment differences, centrifuged and control cow zygotes were incubated for 7 (rather than 5) days in opposite oviducts of fourteen 1-day pseudopregnant rabbits. Development was unaffected by centrifugation: 61% of the zygotes recovered had developed beyond the 16-cell stage, with 23, 24 and 15% containing 17-32, 33-64, and greater than 64 nuclei, respectively. Taking into account the percentage of zygotes in which pronuclei can be seen, the recovery rate from rabbit oviducts, and the proportion of embryos that develop to the morula stage or beyond, 26% of the original group of zygotes would be candidates for transfer into recipient cows.     

The effects of cryopreservation and transfer on embryonic development in the common marmoset monkey, Callithrix jacchus

Embryos were collected at the 4-10-cell stage from the oviducts (Day 4; Day 1 = ovulation) or as morulae (Day 7) from the uterus of marmosets and frozen in 1.5 M-DMSO (Days 4 and 7) or 1.0 M-glycerol (Day 4 only), using a slow freezing and thawing technique. Of 22 Day-4 embryos frozen in DMSO, 18 were recovered and 16 of these were transferred to 10 synchronized recipients; 7 recipients became pregnant compared with all 7 control recipients receiving 10 unfrozen embryos. Fifteen frozen-thawed morulae were transferred to 9 Day-6 recipients; the pregnancy rate (55.6%) was lower than for control embryos (85.7%). Embryos frozen in glycerol suffered severe osmotic stress during glycerol addition and removal. Of 8 recipients, 3 (37.5%) became pregnant but only one fetus was carried to term. These results on embryo collection, freezing and transfer in the marmoset have important implications for developing improved methods for freezing human embryos and the breeding of endangered primates.     

Survival rates and sex ratio of bovine IVE embryos frozen at different developmental stages on day 7

Two experiments were designed to determine the effects of stage of development on Day 7 of in vitro-produced bovine embryos on survival after deep freezing and on sex ratio. Bovine IVF embryos and bovine oviductal epithelial cells (BOEC) were co-cultured in TCM-199 and, on Day 7 after insemination (Day 0), were morphologically evaluated and divided into groups by developmental stage. In Experiment 1, embryos classified as early blastocysts, blastocysts and full-expanding blastocysts were randomly subdivided into 2 groups by replicate: 50% of the embryos were placed immediately in a new BOEC co-culture (fresh group), while the other 50% were frozen, thawed and placed in a new BOEC co-culture (frozen/thawed group). Embryos were frozen in 1.5 M glycerol using a standard slow cooling technique. Fresh and frozen/thawed embryos were compared for survival rate (embryos hatching/hatched) in BOEC co-culture over the following 3 d (i.e., Days 7 to 10). The overall survival of the 425 embryos (early to full-expanding blastocysts) was 33% and was not different between fresh (35%) and frozen/thawed (30%) embryos. Survival of embryos cultured fresh or after freezing/thawing was higher for full-expanding blastocysts than for early blastocysts or for blastocysts, both of which were not different. In Experiment 2, all frozen/thawed embryos used in Experiment 1 plus all morulae and hatched blastocysts collected and frozen on Day 7 without regard to survival were sexed utilizing the polymerase chain reaction (PCR) technique. Sex of the embryos, by stage of development on Day 7, was determined in order to compare the rate of development in BOEC co-culture with the sex ratio (percentage of males). A total of 235 embryos was sex-determined with an overall percentage of males of 51%, which was not different from the expected 1:1 sex ratio. Both full-expanding blastocysts and hatched blastocysts had a significantly higher (P < 0.05) proportion of males (68 and 100%, respectively), while morulae had a significantly lower proportion of males (24%). Early blastocysts and blastocysts did not differ from a 1:1 sex ratio. The results indicate that male embryos develop faster in vitro than female embryos. The higher survival rate of full-expanding blastocysts after freezing/thawing, and the production of a higher number of males than females among embryos of this developmental stage suggest that a greater number of male fetuses may result from the successful freezing and transfer of in vitro-produced bovine embryos.     

In vitro development of reconstructed bovine embryos and fate of donor mitochondria following nuclear injection of cumulus cells

In this study we examined the developmental potential of reconstructed embryos and the fate of donor mitochondria during preimplantation development after nuclear transfer in cattle. Isolated cumulus cells were used as donor cells in nuclear transfer. Cumulus cells labelled with MitoTracker Green FM fluorochrome were injected into enucleated bovine MII oocytes and cultured in vitro. MitoTracker labelling on donor cells did not have a detrimental effect on blastocyst formation following nuclear transfer. Cleavage rate was about 69% (56/81) and blastocyst formation rate was 6.2% (5/81) at 7 days after nuclear transfer. The labelled mitochondria dispersed to the cytoplasm and became distributed between blastomeres and could be identified up to the 8- to 15-cell stage. Small patches of mitochondria were detected in some 8- to 15-cell stage embryos (5/20). However, donor mitochondria were not detected in embryos at the 16-cell stage and subsequent developmental stages. In the control group, mitochondria could be identified in arrested 1-cell embryos up to 7 days after nuclear transfer. These results suggest that disappearance of the labelled donor mitochondria in nuclear transfer bovine embryos is not due to fading of the fluorochrome marker, but is rather an as yet undefined cytoplasmic event.     

Effects of quality and developmental stage on the survival of IVF-derived bovine blastocysts cultured in vitro after freezing and thawing

Factors affecting viability of IVF-derived bovine blastocysts after freezing and thawing were investigated. A total of 1,101 ova matured and fertilized in vitro were cultured under 2 different conditions, 1) in TCM-199 on granulosa cell monolayers at 5% CO(2) in air and 2) in synthetic oviduct fluid (SOF) medium without somatic cell support at 5% CO(2), 5% O(2), 90% N(2). All blastocysts that developed from the 2 different culture systems were individually classified into 4 grades of embryo quality and were then frozen by conventional slow freezing. Developmental rates of the IVF-derived ova to blastocysts and the survival rates of the frozen-thawed blastocysts were not different between the SOF medium (16 and 49%) and the co-culture system (13 and 61%, respectively). Survival of frozen-thawed blastocysts was affected by embryo quality in both the SOF and co-culture systems (P<0.001). Blastocysts produced in vitro were also individually classified into 3 developmental stages and were then cultured for 3 d in the co-culture system with granulosa cells after freezing and thawing. There was a difference in the survival rate of frozen-thawed embryos between blastocyst developmental stages (early vs mid, P<0.05; mid vs expanded, P<0.01; early vs expanded, P<0.001). The post-thawing survival rate of blastocysts frozen at Day 7 (62%) of culture was higher compared with that of Day 8 (45%), but there was no difference in survival rate between Day 7 and 8 of culture. The results indicate that the quality and developmental stage of blastocysts are important factors influencing their survival after freezing and thawing.     

Tolerance to vitrification of rat embryos at various developmental stages

Numerous genetically engineered rat strains have been produced via genome editing. Although freezing of embryos is helpful for the production and storage of these valuable strains, the tolerance to freezing of embryos varies at each developmental stage of the embryo. This study examined the tolerance to freezing of rat embryos at various developmental stages, particularly at the pronuclear stage. Embryos that had developed to the pronuclear, 2-cell, and morula stages were frozen via vitrification using ethylene glycol- and propylene glycol-based solutions. More than 90% of the embryos at all developmental stages survived after warming. The developmental rates to offspring of thawed embryos at the pronuclear, 2-cell, and morula stages were 19%, 41%, and 52%, respectively. Pronuclear stage embryos between the early and late developmental stages were then vitrified. The developmental rates to offspring of the thawed pronuclear stage embryos collected at 24, 28, and 31 h after the induction of ovulation were 17%, 21%, and 23%, respectively. These results indicated that the tolerance to vitrification of rat embryos increased with the development of embryos. The establishment of vitrification method of rat embryos at various developmental stages is helpful for improving the production and storage of valuable rat strains used for biomedical science.     

Nuclear transplantation in the bovine embryo: assessment of donor nuclei and recipient oocyte

Blastomeres from 2- to 32-cell bovine embryos were transferred to enucleated oocytes matured either in vivo or in vitro by micromanipulation and electrofusion. The percentage of donor cells fusing with the recipient oocytes was dependent on relative cell size or stage of development. Therefore, when smaller donor karyoplasts (17- to 32-cell vs. 2- to 8-cell) were transferred, the rate of fusion was significantly less (p less than 0.01). After fusion, nuclear transfer embryos were cultured either in vitro or in vivo (in a ligated ovine oviduct). Nuclear transfer embryos cultured in vitro developed to the 4- to 6-cell stage after 72 h (4-cell, 71%; 8-cell, 33%, 16-cell, 33%; p less than 0.30), whereas nuclear transfer embryos cultured in vivo developed to the morula or blastocyst stage (2- to 8-cell, 11.7%; 9- to 16-cell, 16.0%; 17- to 32-cell, 8.3%; p greater than 0.30) after 4 or 5 days. Freshly ovulated oocytes (collected 36 h after the onset of estrus), when used as recipients, resulted in morula/blastocyst-stage embryos more often than in vitro-matured oocytes or in vivo-matured oocytes collected 48 h after the onset of estrus (20% vs. 7.8% and 6.7%, respectively; p less than 0.02). After in vivo culture, nuclear transfer embryos were mounted and fixed or transferred nonsurgically to the uteri of 6- to 8-day postestrus heifers. Seven pregnancies resulted from the transfer of 19 embryos into 13 heifers; 2 heifers completed pregnancy with the birth of live calves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Piglets born from centrifuged and vitrified early and peri-hatching blastocysts

Cryopreservation of zona-intact porcine embryos has been relatively unsuccessful to date, although some success has been obtained with lipid reduced morulae and early blastocysts. This study adapted some vitrification protocols used successfully with late blastocysts for use with early zona-intact blastocysts, using actin depolymerization, centrifugation, and open-pulled (OPS) straws. Initially, Day 6 peri-hatching blastocysts were collected, cultured for 40 min in 7.5 microg/ml cytochalasin B and vitrified in 6.5 M glycerol and 6% BSA (VS1) in either heat-sealed (HS) or open straws (OS). The post-thaw survival of those stored in HS was 15.4% after 24 and 48 h in vitro; storage in OS significantly improved survival (58.8% for both 24 and 48 h). When similar stage blastocysts were cultured in cytochalasin B and vitrified with 8 M ethylene glycol and 7% polyvinylpyrrolidone (PVP; VS2) in OS, survival was 44.4 and 33.3% for 24 and 48 h, respectively. Day 5 late morulae and early blastocysts were collected, cultured with cytochalasin B, and centrifuged or left intact (control), then vitrified with VS1 in HS or OS, or vitrified in VS2 in OS only. None of the intact control embryos survived thawing and 48 h culture in vitro. Centrifuged early blastocysts vitrified with VS1 showed good post-thaw survival in culture when stored in HS (62.8 and 60.5% for 24 and 48 h, respectively), or OS (75 and 63.6%). When vitrified with VS2 in OS, survival improved (80 and 76.7%). Peri-hatching blastocysts were vitrified in VS1, and early blastocysts were vitrified with VS1 and VS2. All blastocysts were stored in OS. The embryos were recovered and transferred to Day 4 and 5 pseudopregnant recipients (for Day 5 and 6 blastocysts, respectively). Of the five recipients receiving peri-hatching blastocysts, two became pregnant and delivered a total of eight piglets. All three recipients of early blastocysts vitrified in VS1 had a delayed return to estrus; while of the four receiving embryos vitrified with VS2, two were delayed in returning to estrus, and one was confirmed pregnant after 45 days. A litter of five piglets, one male and four female, was produced at 116 days of gestation. To our knowledge, this is the first litter of piglets produced from early blastocysts vitrified without micromanipulation to remove polarized lipid droplets.