An approach to successful freezing of demi-embryos derived from day-7 bovine embryos

The developmental capacity of frozen/thawed bisected embryos (n = 33) derived from day-7 bovine embryos was investigated and compared to ordinary embryos after freezing and thawing (n = 28) and to freshly bisected embryos (n = 19). The freezing and thawing protocol was identical for ordinary and demi-embryos. The percentage of intact embryos classified as excellent, good, or poor after thawing was 92.9 and 96.3% for ordinary and demi-embryos, respectively. Pregnancy rates of 53.8 (8 15 ), 46.2 (6 13 ), and 47.5% (9 19 ) were obtained when frozen/thawed ordinary embryos and frozen/thawed demi-embryos classified as excellent or good and sealed with an additional zona pellucida from hatched pig blastocysts or freshly bisected embryos were transferred. One pair of identical twins resulted from the transfer of frozen/thawed demi-embryos sealed with an additional zona pellucida. Transfer of four frozen/thawed demi-embryos without an additional zona pellucida led to one pregnancy. In contrast, demi-embryos derived from frozen/thawed ordinary embryos (n = 8) as well as frozen/thawed demi-embryos classified as poor (n = 6) did not result in any pregnancies although two halves were transferred per recipient. It is concluded that sealing the punctured zona pellucida improves the developmental capacity of frozen/thawed demi-embryos derived from day-7 bovine embryos, and freezing demi-embryos is more efficient compared to the splitting of frozen/thawed ordinary embryos.     

Survival of sheep demi-embryos in vivo and in vitro

Sheep embryos (morulae and blastocysts) were bisected either by microscalpel or by microneedle after dissolving the zona pellucida with acidified Tyrode's solution. Fourteen and 11 cryopreserved demi-embryos failed to develop when transferred to recipients or placed in culture, respectively. When fresh demi-embryos were cultured in Dulbecco's phosphate buffered saline (DPBS) plus fetal calf serum (FCS) or Whitten's medium, the survival rate was 26% compared to 68% for whole embryos (P<0.01), and there was a suggestion that the presence of a zona pellucida was beneficial to survival. When two demi-embryos each within a zona pellucida were transferred into each of 10 ewes, six of them lambed to produce a total of eight lambs, including two sets of identical twins. Of 10 ewes receiving two demi-embryos without zonae pellucidae, three lambed to produce a total of four lambs, including one set of identical twins. Of 10 ewes that each received two whole embryos, 10 lambed to produce a total of 16 lambs. There was a suggestion that the zona pellucida might enhance the survival of demi-morulae but not demi-blastocysts.     

High survival of rabbit morulae after vitrification in an ethylene glycol-based solution by a simple method.

Rabbit morulae were exposed to a vitrification solution-modified PBS [PB1] medium containing 40% ethylene glycol + 18% Ficoll + 0.3 M sucrose (EFS) for 2, 5, or 10 min at 20 degrees C and were vitrified in liquid nitrogen. When morulae were rapidly warmed, 96% had an intact zona pellucida. When embryos were cultured after removal of the mucin coat, high proportions of them formed blastocoel (79-100%), but the percentage of embryos developed to fully expanded blastocysts decreased with increased exposure time 87%, 40%, and 17%). The survival rate of morulae vitrified after removal of the mucin coat was lower than that of mucin-intact embryos. To assess the development potential in vivo, 131 embryos were vitrified after 2 min of exposure to EFS solution; all the embryos were recovered and 120 were transferred to recipients without removal of the mucin coat, resulting in 78 (65%) full-term fetuses or young. This simple method, which yields high survival both in vitro and in vivo, will be of practical use for vitrifying rabbit embryos.     

Birth of piglets after OPS vitrification and transfer of compacted morula stage embryos with intact zona pellucida

In swine, five to six days post-insemination, morulae and blastocysts are collected together after uterine flushing. The purpose of this study was to vitrify zona pellucida-intact morulae with Open Pulled Straw (OPS) technology and obtain piglets after transfer. Morulae (200) were vitrified after a two-step equilibration in ethylene glycol, dimethyl sulfoxide and sucrose in Hepes-buffered TCM199 + 20% NBCS medium (TCM). 2-6 morulae were loaded into OPS and plunged into liquid nitrogen. At embryo warming, a three-step dilution with decreasing concentrations of sucrose was applied. In each of 10 recipients, 20 morulae were transferred surgically. Day 25, gestation rate and the farrowing rate were 80% and 70%, respectively. The pregnant recipients farrowed from 1 to 8 piglets and the survival of total transferred embryos was 13%. Although survival rates are still compromised, OPS technology is therefore appropriate to cryopreserve porcine morulae with intact zona pellucida.     

Freezing of bovine embryos: Effects of a one-step addition of 1.4 M glycerol

The effects of a one-step addition of 1.4 M glycerol (method A) upon morphological appearance and developmental capacity of frozen/thawed day 7 bovine embryos were investigated and compared to a standard stepwise addition of 1.0 M glycerol (method B). With method A, the percentage of intact embryos (classified as excellent, good and poor) was 95.3% (61 out of 64) without differences between morulae (96.5%) and blastocysts (94.4%). With method B, the percentage of intact embryos was 83.0% (44 out of 53). The percentage was similar for blastocysts (89.2%) and significantly (p < 0.05) lower for morulae (68.8%) when compared to method A. The percentage of embryos with a damaged zona pellucida was considerably increased with method A (26.6%) when compared to method B (13.2%). The proportion of embryos with excluded blastomeres was similar in both methods (21.9% method A, 17.0% method B). With method A, pregnancy rates after nonsurgical transfer were 51.0% (25 out of 49) and were better than with method B (40.5%; 15 out of 37). Embryos with a damaged zona pellucida resulted in a high pregnancy rate of 66.7% (8 out of 12). A pregnancy rate of 52.9% (10 out of 17) was obtained with embryos showing some excluded blastomeres. Thus, a one-step addition of 1.4 M glycerol facilitates and accelerates the process of embryo cryopreservation and is compatible with high pregnancy rates. Damage of the zona pellucida does not impair further development of frozen/thawed bovine embryos provided blastomeres are intact.     

The survival of whole and bisected rabbit morulae after cryopreservation by the vitrification method

The survival of whole and bisected rabbit morulae cryopreserved by the vitrification method was investigated. The embryos were loaded in a column of vitrification solution (VS, a mixture of 25% glycerol and 25% 1, 2-propanediol in PBS+16% calf serum), which was located between two columns of 1 M sucrose solution in a plastic straw. The embryos were frozen by being plunged into liquid nitrogen and thawed in a water bath at 20 degrees C. Two methods of loading embryos into straws were used: the single and double column vitrification solution methods. The embryonic survival rates between these two methods were compared. Seventy-one (86.6%) out of 82 morulae vitrified in double column straws developed into the blastocyst stage in vitro. Eleven (18.3%) live fetuses were obtained after the transfer of 60 frozen-thawed morulae to four recipients. By contrast, the survival rate (36.5%, 27 74 ) of embryos vitrified in the single column straws was significantly lower (P<0.05). The vitrification solution of the single column straws became opaque, indicating ice-crystal formation, upon thawing in 5 of 11 straws, which was assumed to have damaged the embryos. More than 80% (29 36 ) of the bisected morulae frozen and thawed in the double column straws developed to the blastocyst stage in vitro when cryoprotectant was diluted stepwise with 1 M and 0.25 M sucrose solution. When the cryoprotectant was removed by one-step dilution with 1 M sucrose solution, swelling in blastomeres was observed and the development rate of the recovered embryos decreased (45.8%, 11 24 ). These results indicate that the vitrification method employed in our experiment is not only efficient for the cryopreservation of rabbit morulae, but it can also be used for the preservation of bisected rabbit morulae, which had not been successful using previous methods.     

Micromanipulation of sheep morulae to produce monozygotic twins

Donor ewes were treated with an intravaginal sponge containing 30 mg fluorogestone acetate (FGA) which was removed 12 days later. On the mornings of days 10, 11, and 12, each animal received 30 mg horse anterior pituitary (HAP) extract. Donors were mated twice daily during oestrus. Egg recovery was attemped seven days after progestagen with-drawal, when late morula/early blastocysts could be expected. The zona pellucida was opened with the aid of a micromanipulator, and the embryo was removed by gentle positive pressure from an evacuating pipette. After removal from the zona pellucida, the embryo was bisected using a fine glass needle; each demi-embryo was immediately placed either into the original zona pellucida or into one from an evacuated oocyte. The original opening was closed and both halves were transferred bilaterally into previously synchronized recipients. From 17 embryos split and transferred to 17 ewes, nine became pregnant and produced seven sets of monozygotic twins and two singletons.     

Chimerism in cattle through microsurgical aggregation of morulae

A cattle chimera was produced by combining four halves of two parent embryos of different breeds (Brown-Swiss x Braunvieh plus Holstein-Friesian x Holstein-Friesian) in one zona pellucida. Parent embryos in the 32-cell morula stage were recovered non-surgically, were bisected, and the combined four halves were transferred non-surgically to recipient heifers. Chimerism of coat colour was used as evidence. Combining of only two half embryos from different parents resulted in five pregnancies carried to term but none of the calves born was a chimera.     

Viability of bovine demi- and quarter-embryos after transfer

The viability of bovine demi- and quarter-embryos was investigated. Early compacting morulae were nonsurgically flushed from superovulated donor cows and were bisected by two microneedles. One of the halves was then split further into two quarters. Each demi- and quarter-embryo was placed in an evacuated zona pellucida. One demi- or two quarter-embryos were transferred non-surgically into cow or heifer recipients. Viability was measured by ultrasound scanning of the fetuses on Days 35, 48 and 60 of pregnancy. The pregnancy rates at Day 60 were 46.2% (6 13 ) for heifers and 33.3% (4 12 ) for cows after the transfer of a single demi-embryo. The transfer of two quarter-embryos resulted in a pregnancy rate of 61.5% (8 13 ) for heifers and 8.3% (1 12 ) for cows. Seven (53.8%) and four (33.3%) live fetuses were found on Day 60 following the transfer of demi-embryos into heifers and cows, respectively. The transfer of quarter-embryos resulted in 10 fetuses (38.5%) in the heifer recipients and only one fetus (4.2%) in the cow recipients. The results of this study suggest that heifers are more suitable than cows as recipients for quarter-embryos.     

The freezability of biopsied bovine embryos

A biopsy of 1-5 blastomeres was taken from 86 bovine compacted morulae, using a technique, that did minimal damage to the zona pellucida. As soon as possible after the micromanipulation the embryos were frozen, without sealing the penetrated zona pellucida. In order to evaluate the viability of the biopsied frozen-thawed embryos, half of them were transferred to syncronized recipients while the remaining half were co-cultured to evaluate the developmental capacity. A control group of 43 intact embryos was subjected to the same procedures. This study revealed a slight, but not significant, decrease in the pregnancy rate of the biopsied embryos after freezing and thawing, although the embryos by co-culture with bovine oviduct epithelial cells revealed normal morphology and developmental rate. It is concluded that the described biopsy technique did not compromise the freezability of 7-day-old bovine embryos.     

Sheep embryo cryopreservation by vitrification and conventional freezing

The survival of ovine embryos (morulae and blastocysts) either frozen by a conventional method or vitrified was investigated in culture. In Experiment I, embryos were vitrified using a solution containing 25% propylene glycol and 25% glycerol. A group of embryos (simulated control) was processed without freezing to evaluate the toxicity of the vitrification solution. In Experiment II, embryos were exposed to a solution of PBS containing 10% glycerol and 0.25 M sucrose placed horizontally in a programmable freezer. Automatic seeding was applied at -7 degrees C in 2 positions on straws and cooled at -0.3 degrees C/min to -25 degrees C and then stored in liquid nitrogen. In vitro development rates of vitrified embryos were 12% (morulae) and 19% (blastocysts). Simulated embryos showed a higher rate of survival than embryos cryopreserved by vitrification (67 and 63%, morulae and blastocysts respectively). In conventional cooling, the blastocysts showed the highest viability percentage (67%) of all the experimental groups but these values decreased significantly in morulae (31%). Differences in temperature between straws placed in distinct positions in the freezing chamber and thermic deviation were observed when automatic seeding was applied. Embryo viability differed from 51 to 75% according the relative position of the embryos within the chamber. Survival was higher when automatic seeding was applied on the meniscus of the embryo column versus the central point of this column (65 vs 21%). The damage of both cryopreservation methods on zona pellucida integrity (27 and 35% in vitrified and conventionally frozen embryos, respectively) had no effect on the in vitro survival.     

Sexing of half-embryos produced by microsurgical bisection of mouse morulae and production of chimeric mouse of defined sex composition by aggregating two sexed half-embryos

Using the halved morulae of mice obtained with microsurgical technique, the following two experiments were performed. 1) Sexing of half-embryos by chromosomal analysis and transfer of the half-embryos after determining the sex of the other monozygotic half. One half of the bisected embryo was cultured in Colcemid solution (0.04 micrograms/ml) to be ensured for chromosomal preparation. More than 50% (152/270) of the blastulated embryos from the halves could be sexed by direct sex chromosome analysis. Thirty-nine of the half-embryos of which the co-twin halves were sexed, were transplanted in to the uterine horns of 18 pseudopregnant mice, and twelve became pregnant. The autopsies of them on Day 18 to 20 of pregnancy, revealed the presence of 16 fetuses. The morphological sex of these fetuses thus obtained coincided completely with the previous judgement based on the chromosomal sexing. 2) Production of chimeras of defined sex composition by aggregating two half-morulae of defined sex. Out of 147 pairs of half-morulae of two different strains (ICR and C3H/He), which were replaced in pairs into empty zona pellucidae, 107 (72.8%) were aggregated successfully and developed in vitro into full expanding blastocysts of typical form. Among the 107 aggregate blastocysts, 31 were sexed for both component embryos by chromosomal analysis on the co-twin half-embryos. When these 31 blastocysts were transferred, 11 living offspring including 4 chimeras were obtained. Transfer of 12 male-male and 5 female-female aggregate blastocysts resulted in 8 males and 1 female, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sex-dependent loss of bisected bovine morulae after culture and freezing.

The objective of this study was to determine the post-transfer survival rate of bovine embryos cultured between the time of bisection and freezing. In this experiment 158 morulae were bisected and both portions were cultured for 24-44 h either in vivo after transfer to sheep oviducts (n = 80 morulae) or in vitro (n = 78 morulae) in Ham's F10 medium with 20% fetal calf serum, with bovine oviduct cells or in medium collected from oviduct cultures (conditioned medium). After culture, half of each morula was fixed for cytogenetic sex determination (n = 125) and the other half was frozen. The frozen halves were later thawed and transferred (n = 115) to recipients, who were, if pregnant, slaughtered to determine the sex of the fetus. The culture resulted in better pregnancy rates than those previously reported for embryos frozen immediately after bisection. The sex of 49 (33 males, 16 females) of the fixed demi-morulae was determined, and 38 of the transferred demi-morulae established pregnancies (23 males, 10 females and 5 fetuses that were not recovered). The male:female ratio in in vivo and in vitro culture groups was significantly different from the expected ratio of 1:1 and suggests that manipulation and culture of embryos results in a preferential loss of female embryos.     

Effect of biopsy and vitrification on in vitro survival of ovine embryos at different stages of development

The objective of the present study was to assess the in vitro viability of ovine embryos at different stages of development after combining cell sampling and vitrification. Precompacted morulae, compacted morulae and blastocysts were obtained from superovulated Sarda ewes at 4, 5 or 6 d following insemination. Embryo cell biopsy was carried out in a 100-microl drop of PBS + 10% fetal calf serum (FCS) with 10 micromol nocodazole and 7.5 microg/ml cytochalasin-b by aspiration (3-5 cells). Embryos were cryopreserved at room temperature after exposure of 2 solutions for 5 min, transferred into a vitrification solution, loaded into the center of 0.25-ml straws separated by air bubbles from 2 columns of sucrose 0.5 M and plunged immediately into liquid nitrogen. In Experiment 1, the in vitro viability of manipulated or vitrified embryos after in vitro co-culture in TCM 199 medium with 10% FCS and sheep oviductal epithelial cells (SOEC) in 5% CO2 humidified atmosphere in air at 39 degrees C was significantly lower (P < 0.05 and P < 0.01, respectively) at precompacted morula (60 and 30%) and compacted morula (62 and 39%) stages than intact embryos at the same stages (87 and 88%). No differences were found at the blastocyst stage. In Experiment 2, the in vitro survival rate of precompacted morulae which were manipulated and immediately vitrified was lower (P < 0.05) than in those manipulated and, after a temporary period of culture, vitrified at blastocyst stage (21 vs 48%); while no differences were found at compacted morula and blastocyst stages. The results show that 1) the stage of development influences the subsequent in vitro viability of manipulated and vitrified ovine embryos, 2) temporary culture after manipulation and before vitrification improves the in vitro viability of embryos, and 3) the hole in the zona pellucida resulting from biopsy does not affect blastocyst survival after subsequent vitrification.     

Survival of biopsied and sexed bovine demi-embryos

The viability of sex-diagnosed bovine demi-embryos was investigated after transfer. Day-7 morulae and blastocysts were subjected to splitting and biopsy in PBS + 4mg/ml polyvinylpyrrolidone + 200mM sucrose using a microblade. The biopsy (approximately 2 to 8 blastomeres) was transferred to a tube, and its presence in the tube was verified by examination under a stereomicroscope. After proteinase K treatment, repetetive male-specific DNA was amplified by the polymerase chain reaction (PCR). No autosomal control primers were used in the PCR. Instead, the absence of a characteristic Y-specific product together with the amplification of non-specific products was considered an indication of a female sample. The biopsied demi-embryos were transferred either singly or in pairs to synchronous heifer or cow recipients 6 to 10 h after flushing. Sex diagnosis was carried out within 6 to 7 h. Of 19 original embryos, 7 were diagnosed as males and 5 as females. The DNA of the biopsies of the remaining 7 embryos did not result in any amplification products. Since 5 of these samples were seen in the tubes prior to PCR, the corresponding embryos were considered "potential females." The sex of the last 2 samples could not be determined. Nine of 10 embryos were correctly sexed as revealed by calving data. Of the 38 transferred demi-embryos, 16 had developed to live fetuses as detected by ultrasonography on Day 65 of pregnancy. Eleven live calves and three stillborn calves were delivered. After bisection, biopsy and single transfer, 6 live calves were born from 7 original embryos (86%). After transfer of both halves into the same recipient, only 5 live calves from 12 original embryos were produced (42%). None of the 4 manipulated Grade-2 embryos survived to term, nor did any of the 4 manipulated blastocysts. Of the 14 original Grade-1 morulae manipulated and transferred, 15 were live fetuses at Day 65, and 11 live calves were born.     

Quick-splitting of bovine embryos

Described is a simplified method of bovine embryo bisection amenable to on-farm embryo transfer. Using a microblade operated by a hand-held micromanipulator, Day 7 bovine embryos were bisected while in the zona pellucida. With a vertical motion, the embryo was pinned between the blade and the bottom of a plastic petri dish and bisected. Demi-embryos were transferred nonsurgically (without zonae pellucidae) into synchronized recipients. Pregnancy rates were normal with 5 13 (38%) and 9 20 (45%) of recipients confirmed pregnant 70 to 80 d after receiving either twin or single half embryos, respectively. This compared to 12 28 (43%) of recipients becoming pregnant from transfer of whole embryos. These data confirm that bovine demi-embryos do not need zonae pellucidae on Day 7 and that simplified field methods of bisection give normal pregnancy results.     

Production of bovine identical twins via transfer of demi-embryos without zonae pellucidae

Embryos were collected nonsurgically from n?turally-cycling or superovulated donors 7 d after estrus. Forty-four morulae, early blastocysts and blastocysts classified as good to excellent were bisected using a fine glass needle to produce forty-four identical demi-embryos. The bisected demi-embryos, without zonae pellucidae, were nonsurgically transferred, either by twin or single transfer. An additional forty-eight embryos were collected from the same donors and transferred as a control. Among the twin transfers, 8 of the 13 recipients became pregnant (61.5%). Seven of them conceived twin fetuses (87%) and one a single fetus. However, only two sets of normal identical twin calves were obtained. Among the single transfers, 72.6% (45/62) of bisected embryos without zonae pellucidae resulted in pregnancy, of which 48.4% (30/62) were identical twins, and 24.2% (15/62) were singletons. Another 27.4% (17/62) of the recipients did not became pregnant. The pregnancy rate for whole embryos with zonae pellucidae was 72.9% (35/48). These data show that there was no significant difference between the pregnancy rates of bisected embryos without zonae pellucidae and whole embryos with zonae pellucidae transferred 7 d after estrus. Bisection of bovine embryos was simplified and even morula stage embryos were transferred without zonae pellucidae.     

Commercial splitting of bovine embryos

Independent studies were undertaken at Alberta Livestock Transplants (ALT) and at Select Embryos, Inc. (SEI) to develop procedures for splitting bovine embryos. At both locations embryos were recovered seven days after the onset of estrus and superovulation. Initially, survival after splitting was evaluated by culture $\text{in}$$\text{vitro}$ for 18 to 24 hours. Culturing half or demi-embryos without a zona pellucida at ALT resulted in 15% survival compared to 35% survival when both halves were in separate zonae. Culturing demi-embryos on a monolayer of luteal cells at SEI did not improve survival $\text{in}$$\text{vitro}$. In fertility trials, best results were obtained at ALT when both demi-embryos within separate zonae were nonsurgically transferred into separate uterine horns of the same recipient (55% pregnancy rate) and at SEI when one demi-embryo was surgically transferred into the uterine horn ipsilateral to the corpus luteum (65% pregnancy rate). Culturing demi-embryos more than 4 hours reduced fertility at both locations. Splitting embryos was a worthwhile addition to the commercial ET programs and further trials are in progress to improve survival $\text{in}$$\text{vitro}$ and pregnancy rates.     

Effects of slitting the zona pellucida and its subsequent sealing on freeze-thaw survival of Day 7 bovine embryos

The effects of slitting the zona pellucida and its subsequent sealing by either embedding in agar or surrounding with an additional zona pellucida on the development of frozen/thawed Day 7 bovine embryos were investigated in vitro and in vivo. A total of 225 embryos was frozen and thawed rapidly as controls (Group 1), after slitting the zona pellucida (Group 2), after slitting and subsequent sealing of the zona pellucida with agar (Group 3), or after slitting the zona pellucida (Grothen transferring the embryo into an additional zona pellucida (Group 4). The survival rate (embryos classified morphologically as excellent, good, or poor) was 95.1, 95.4, 92.2, and 94.3% for Groupsl, 2, 3, and 4, respectively. Culture of 145 embryos in vitro for 60 h revealed that 57.1, 59.5, 47.4, and 57.1% developed to hatching and hatched blastocysts in Groups 1, 2, 3, and 4, respectively. Within Group 3, however, a significantly (P < 0.05) lower percentage of the embryos continued to develop when the agar was not removed after thawing (31.8%) compared with embryos from which the agar had been removed (68.8%). After nonsurgical transfer of 78 embryos, the pregnancy rate was significantly (P < 0.05) lower (8.3%) with embryos of Group 3 compared with controls (61.5%) or embryos of Group 2 (42.9%). No significant difference existed between controls and embryos of Group 2. We conclude that an intact zona pellucida prior to rapid freezing is not essential for the survival of Day 7 bovine embryos.     

Pregnancy rates with bisected bovine embryos

A method for producing identical twin calves is described in which 5.5 to 7.5 day bovine embryos were bisected using a fine microsurgical blade. The resulting demi-embryos were transferred surgically or nonsurgically to the uterine horn ipsilateral to the corpus luteum of synchronous recipients (+/- 2 days), one demi-embryo per recipient. The mean pregnancy rate per demi-embryo was 48% (157/330), and per whole embryo split, it was 95% (157/165). Pregnancy rate was dependent on the stage of embryonic development. The pregnancy rate from bisected early morulae, 16% (7/44), was lower than that from bisected early blastocysts, 60% (58/96) P<0.01. The rate of identical twin production per embryo split reflected these pregnancy rates. Bisected blastocysts resulted in more identical twin sets, 39% (24/62), than early morulae, 9% (2/22) P<0.01. When used with normal embryo transfer procedures, this technique provides a method for routine production of identical twin calves. Furthermore, more calves are produced per two demi-embryos than per whole embryo.