Morphological changes of in-vitro-produced bovine blastocysts after vitrification,in-straw direct rehydration,and culture

Morphological signs of injury and regeneration following vitrification and warming of bovine embryos were studied by light and electron microscopy. In-vitro-produced Day 7 expanded blastocysts (Day 0 = day of insemination) were vitrified by a two-step equilibration method using ethylene glycol and dimethyl sulphoxide as cryoprotectants. Thawing was performed by in-straw direct rehydration, followed by in vitro culture on a granulosa cell monolayer. Embryos were processed for transmission electron microscopy immediately after warming (0 hr) as well as after 4 hr or 24 hr of culture following warming. A control group of unfrozen embryos was also processed. At 0 hr after warming, except for a rapid collapse of the blastocoele, only minor changes were detectable by stereomicroscope. However, at the ultrastructural level, signs of extensive injury were seen, including a general distension or shrinkage of mitochondria, disintegration of cell adhesions between adjacent trophoblastic cells, and complete rupture of some cells. At 4 hr, stereomicroscopic investigation revealed collapsed blastocoele and a darkened granular appearance of the cell mass. At the ultrastructural level, signs of regeneration were also observable: cells with minor injuries were re-assembled in a central area forming a small blastocoele, cell adhesion structures were re-established, and damage of mitochondria was less severe. The majority of irreversibly damaged cells or cell debris was accumulated in the perivitelline space. At 24 hr, stereomicroscopic investigation of surviving blastocysts showed no signs of the previous injury. At the ultrastructural level, cellular debris in the perivitelline space and some degenerated cells in the blastocoele were the only signs of previous injuries. In conclusion, ultrastructural investigation revealed unexpectedly extensive damage followed by a rapid regeneration and reorganization of the embryonic structure. Mol. Reprod. Dev. 48:9–17, 1997. © 1997 Wiley-Liss, Inc.     

In vitro evaluation and pregnancy rates after vitrification of in vitro produced bovine embryos

The efficacy of different vitrification solutions to cryopreserve in vitro-produced bovine blastocysts was evaluated based on in vitro development of embryos in culture and on in vivo development of embryos transferred into recipients. In the first experiment, 2 vitrification solutions were compared: propylene glycol + glycerol (Pg + Gly) and ethylene glycol + Ficoll + sucrose (EFS). Differences in the overall development and hatching rates in favor of EFS were found (56.4 vs 33.3% and 35.4 vs 13.3%; P < 0.05). In the second experiment, 3 vitrification solutions were compared: EFS, modified EFS (EFSm) and ethylene glycol + glycerol (Eg + Gly). The vitrification solutions EFSm and Eg + Gly yield higher hatching rates than did EFS (57.7 vs 59.6 vs 35.7%; P < 0.05). The last experiment was designed to compare in vivo 2 vitrification solutions: EFSm and Eg + Gly. There were no differences between them based on the results obtained after transfer (35.2 vs 43.7%). The vitrification solutions EFSm and Eg + Gly have resulted in good pregnancy rates. These results demonstrated that vitrification can be used successfully in the cryopreservation of in-vitro produced bovine embryos, and it might be considered for use in commercial programs.     

New device for the vitrification and in-straw warming of in vitro produced bovine embryos

Two experiments were designed to test the use of a new device designed to vitrify and in-straw warm in vitro produced (IVP) embryos, which can potentially be used for their direct transfer to recipient females in field conditions. In experiment 1, IVP embryos from both prepubertal and adult animals were vitrified on cryotops and warmed in steps (1, 0.5 and 0 M sucrose; protocol W3) or directly in 0.5 M (protocol W1/0.5) or 0 M sucrose (protocol W1/0). Similar survival rates were recorded 24 h after warming for calf embryos irrespective of the warming procedure (W3: 79.2%, W1/0.5: 62.5%, W1/0: 66.7%). For cow embryos, survival rates at 24 h post-warming were significantly higher when embryos were warmed using the W3 (85.7%) or W1/0.5 (89.1%) protocols compared to the W1/0 protocol (70.5%). In experiment 2, IVP embryos were vitrified on the new designed device followed by their in-straw cryoprotectant (0.5 M sucrose) dilution/warming and different warming temperatures (45, 50, 60 and 70 °C) were tested. When warming solution passed through the new vitrification/warming device at 45 °C, 61.5% of blastocysts were fully re-expanded or hatched at 24 h post-warming, being not significantly different to the control (65%). Other warming temperatures triggered significantly lower survival rates at 24 h post-warming. No significant differences were detected in total cell numbers and blastocyst apoptosis indices in response to vitrification followed by warming at 45 °C respect to the control. Our findings indicate that the new device allows vitrification and in-straw warming of IVP bovine embryos, being a useful option for their direct transfer in field conditions.     

Factors affecting the survival of mouse embryos cryopreserved by vitrification

Preimplantation stage mouse embryos have been used to examine the response of a simple multicellular system to cryopreservation by the complete vitrification of the suspension. Successful vitrification requires the use of a solution of cryoprotectants that is sufficiently concentrated to supercool and solidify into a glass at practicable cooling rates. Factors that influence the survival of embryos include the concentration and composition of the vitrification solution, the procedure used to equilibrate embryos in this solution, the cooling and warming conditions, and the procedure used to dilute embryos from the vitrification solution. High rates of survival are obtained when embryos are dehydrated prior to vitrification in solutions composed of saline plus multimolar concentrations of either mixtures of permeating cryoprotectants (e.g. dimethyl sulphoxide-acetamide-propylene glycol) or single permeating cryoprotectants (propylene glycol or glycerol). Full permeation of cryoprotectants into the cells is not necessary and may lead to chemical toxicity and osmotic injury. Partial permeation and osmotic shrinkage concentrates the endogenous cytoplasmic macromolecules and greatly increases the likelihood of intracellular vitrification. Vitrification is a practical approach for embryo cryopreservation and offers new opportunities to examine fundamental aspects of cryoprotection and cryoinjury in the absence of freezing.     

Factors affecting the survival of one- and two-cell rabbit embryos cryopreserved by vitrification

The effects of equilibration time, glycerol (GLY), and 1,2-propanediol (PROH) concentration, and of vitrification and sucrose solution on the viability of 1- and 2-cell rabbit embryos were investigated. After collection, the embryos were equilibrated for 5 or 10 minutes in phosphate buffered saline (PBS) containing 10% GLY-20% PROH and were exposed for 30 seconds at 4 degrees C or were exposed and vitrified in one of two vitrification solutions 35% GLY-35% PROH or 20% GLY-50% PROH. The in vitro survival rates of 1-cell embryos equilibrated for both 5 and 10 minutes were lower (34.0 and 48.0%, respectively) than those of 2-cell embryos (78.8 and 68.5%, respectively; P<0.01). No differences were noted in the viability of embryos exposed to the 2 vitrification solutions. Following vitrification in a mixture of 35% GLY-35% PROH, the survival rates of 1- and 2-cell embryos were 18.3 and 13.7% and 19.6 and 10.4% for 5 and 10 minutes of equilibration, respectively. The survival rates of 1- and 2-cell embryos vitrified in a solution of 20% GLY-50% PROH were 25.7 and 35.4% and 26.2 and 21.3% for 5 and 10 minutes of equilibration, respectively. The survival rates of 1-and 2-cell embryos stored in 1M sucrose solution were 63.8 and 84.0%, respectively. In conclusion, the viability of vitrified 1- and 2-cell rabbit embryos was reduced as a consequence of their equilibration before vitrification, the exposure to vitrification solution and the dilution in a sucrose solution rather than of the vitrification process itself.     

In vitro assessment of a direct transfer vitrification procedure for bovine embryos

We developed a simple vitrification technique for bovine embryos that could permit direct transfer. Embryos were produced in-vitro by standard procedures. The base medium for cryopreservation was a chemically defined medium similar to SOF + 25 mM Hepes and 0.25% fatty acid free bovine serum albumin (FAF-BSA) (HCDM2). In experiment 1, embryos were first exposed to 3.5M ethylene glycol (V1) for 1, 2 or 3 min at room temperature (20-24 degrees C), and then moved to 7 M ethylene glycol (V2) at 4 or 20-24 degrees C and loaded in 0.25-mL straws. After 45 s in 7 M ethylene glycol, straws were placed in liquid nitrogen. Embryos that were loaded at 20-24 degrees C had higher survival rates than those loaded at 4 degrees C (P<0.05). Exposure for 1 min was best for morulae, while 3 min was best for blastocysts. In experiment 2, blastocysts were handled at 24 degrees C and exposed to two concentrations of ethylene glycol in V1 (3.5 or 5 M) followed by V2 as in experiment 1, two warming temperatures (20 or 37 degrees C) and two post-warming holding times until culture (5 or 15 min). Exposure to 5 M ethylene glycol and warming at 37 degrees C was the optimal combination of procedures, and embryos survived well after 15 min in straws if warmed at 37 degrees C. In experiment 3, ethylene glycol concentration (3, 4 or 5 M) and exposure time (0.5 or 1 min) during two-step addition of cryoprotectant were studied for bovine morulae. In experiment 4, morulae were exposed to V2 for 30 or 45 s in HCDM2 or Vigro holding medium and then held in 22-24 degrees C air or 37 degrees C water post-warming. Experiment 5 was like experiment 4 except blastocysts were used. Overall survival rates of blastocysts in experiment 5 averaged 80% of non-vitrified controls after 48 h culture. The survival rates with in vitro-produced morulae in experiments 1, 3 and 4 were unacceptable. Vitrification solutions based on Vigro tended to result in higher survival than HCDM2 for blastocysts, but not morulae. In experiment 6, the survival rate in vitro of in vivo-produced morulae and blastocysts after two-step vitrification was nearly 100%. Our vitrification technique was very effective for in vitro produced blastocysts, but not for in vitro-produced morulae.     

Comparison of two co-culture systems to assess the survival of in vitro produced bovine blastocysts after vitrification

The ability of bovine blastocysts to recover after cryopreservation and thawing procedures is often assessed by evaluating their re-expansion during in vitro co-culture. However, the influence of factors such as feeder cell type and gas atmosphere on blastocyst survival and evolution have never been considered. This study therefore compared two cell co-culture systems and two different gas atmospheres to assess survival of in vitro produced bovine blastocysts after vitrification. Day-7 blastocysts (n=181) were vitrified in a mixture of 25% glycerol/25% ethylene glycol. After warming and dilution, they were co-cultured either on Buffalo rat liver cells (BRL CC cell line) or on granulosa cells (GR CC primary culture) in TCM 199 supplemented with 10% FCS and under an atmosphere of 5% or 20% O₂. Surviving and hatching rates were recorded at 24 h intervals for 3 days. After 72 h of culture, surviving blastocysts were treated for differential counting of inner cell mass (ICM) and trophectoderm cells. Blastocyst survival rates were higher when BRL and granulosa co-culture were performed under 20% oxygen as compared to 5% oxygen (20% O₂: 62% vs. 5% O₂: 25%, P<0.0001). However, the quality of blastocysts surviving in the granulosa co-culture condition was lower under 20% O₂ than under 5% O₂ as indicated by lower total and trophectoderm cell numbers (respectively 79±6 and 56±6 at 20% O₂ vs. 100±10 and 74±10 at 5% O₂, P<0.05), by an altered ICM/trophectoderm ratio (20% O₂: 28% vs. 5% O₂: 23%, P<0.05), by a higher total nuclear fragmentation (20% O₂: 3.7% vs. 5% O₂: 1.5%, P<0.05) and a trend to decreased hatching (20% O₂: 32% vs. 5% O₂: 81%, P=0.07). Whereas, for BRL co-culture, 20% O₂ yielded higher quality blastocysts than 5% O₂ as evaluated by higher ICM and trophectoderm cell numbers (19±1 and 71±5 at 20% O₂ vs. 15±2 and 48±9 at 5% O₂, respectively, P<0.05), by lower nuclear fragmentation in the ICM (20% O₂: 2.2% vs. 5% O₂: 6.7%, P<0.05). In conclusion, co-culture conditions may influence blastocysts survival and quality after cryopreservation. In our conditions, co-culture with BRL cells under 20% O₂ seems to be the best combination to evaluate blastocyst survival and quality after vitrification.     

Pregnancy rates and births after unilateral or bilateral transfer of bovine embryos produced in vitro.

Late morulae and blastocysts produced in vitro were nonsurgically transferred to heifers by unilateral (n = 184) or bilateral (n = 94) transfer. Of the recipients, 58% had serum progesterone values greater than 1.4 ng ml-1 on day 21 and rectal palpation on day 35 showed that 50% (138 of 278) were pregnant. The embryonic mortality rate between days 21 and 35 was estimated to be about 14% and between days 36 and 90 to be about 12%. Of the animals, 8% aborted between days 91 and 250 of pregnancy. No difference was observed in pregnancy rates between unilateral transfer of one (47%) or two embryos (49%) and bilateral transfer (53%), or in the twinning rate between bilateral transfer (42%) and unilateral transfer of two embryos (33%). The pregnancy rate was 54% with embryos evaluated as morphologically excellent or good, 51% with fair embryos and 26% with poor ones. A higher pregnancy rate (60%) was obtained after embryo transfer when the synchrony between recipient and embryo was -1 day.     

Cryopreservation of bovine in vitro produced embryos using ethylene glycol in controlled freezing or vitrification

In this study, the cryoprotectant ethylene glycol (EG) was tested for its ability to improve and facilitate the cryopreservation of in vitro produced (IVP) bovine embryos. Embryos were cryopreserved in EG solutions supplemented with either newborn calf serum (NBCS) or polyvinyl alcohol (PVA). To assess EG toxicity, the embryos were equilibrated in EG concentrations from 1.8 to 8.9 M at room temperature for 10 min and then cultured for 72 h on a cumulus cell monolayer. The hatching rate was highest for day 7 blastocysts frozen in 3.6 M EG (98%) and was not different from the control group (85%). The controlled freezing (0.3 degrees C/min to -35 degrees C) of expanded day 7 blastocysts resulted in a hatching rate of 81%, which was similar to that of the nonfrozen controls (76%). Differential staining revealed only very few degenerate blastomeres attributed to freezing and thawing. Upon direct nonsurgical transfer of day 7 expanded blastocysts frozen in 3.6 M EG, a pregnancy rate of 43% was achieved, while the pregnancy rate after transfer of other developmental stages was significantly lower (22% with expanded day 8 blastocysts). When bovine IVP embryos were incubated at room temperature in 7.2 M EG preceded by preequilibration in 3.6 M EG, the hatching rate of day 7 expanded blastocysts reached 93%. Upon vitrification of IVP day 7 and day 8 blastocysts and expanded blastocysts in 7.2 M EG, the latter showed a higher hatching rate (42%) than blastocysts (12%). Overall, PVA as supplement to the basic freezing solution instead of NBCS had deleterious effects on survival after controlled freezing or vitrification. The simple cryopreservation protocol employed in this study and the low toxicity of ethylene glycol highlight the usefulness of this approach for controlled freezing of IVP embryos. However, further experiments are needed to improve the pregnancy rate following embryo transfer and to enhance survival after vitrification.     

Sex-related differences in developmental rates of bovine embryos produced and cultured in vitro.

The classical concept of sex determination in mammals is that a Y chromosomal gene controls the development of the indifferent gonad into a testis. Subsequent divergence of sexual phenotypes is secondary to this gonadal determination. The most likely candidate gene is SRY (sex-determining region Y) in humans, and Sry in mouse. However, several lines of evidence indicate that sexual dimorphism occurs even before the indifferent gonad appears. Here we present evidence that bovine male embryos generally develop to more advanced stages than do females during the first 8 days after insemination in vitro. Corresponding relationships between both cell numbers and mitotic indices and sex were also seen. Although it is not clear whether this phenomenon involves factors originating before or after fertilization, these findings suggest that sex-related gene expression affects the development of embryos soon after activation of the embryonic genome and well before gonadal differentiation.     

Effects of vitrification medium composition on the survival of bovine in vitro produced embryos, following in straw-dilution, in vitro and in vivo following transfer

This study examined the effects of adding a macromolecule, polyvinylpyrrolidone (10% PVP) and a sugar (0.3 M trehalose) to vitrification solutions (VS) containing either one (40% ethylene glycol [EG], two (25% EG+25% DMSO) or three (20% EG+20% DMSO+10% 1, 3-butanediol [BD]) permeable cryoprotectants on the survival and hatching of IVP bovine embryos, following vitrification, warming and in-straw cryoprotectant dilution. Grade 1 and 2 compact morulae and blastocysts were selected on Day 7 (Day 0=IVF) of culture in SOFaaBSA and equilibrated for 10 min at room temperature in 10% EG. Following exposure, for up to 1 min at 4 degrees C, to one of the above VS (with or without PVP+trehalose), the embryos were loaded into straws and immersed in liquid nitrogen. Following warming and in-straw cryoprotectant dilution, the embryos were cultured for 48 h to assess hatching. There was no effect of VS on the survival of embryos after 24 h, however fewer compact morulae than blastocysts survived after 24 h (24% vs. 75%; P<0.001) or hatched after 48 h (15% vs. 59%; P<0.001). When blastocysts only were considered, an interaction between VS and additional PVP+trehalose was also observed (P<0.01). Hatching was reduced when they were added to 25% EG+25% DMSO (70% vs. 45%) but was not affected for either 40% EG (44 and 49%) or to 20% EG+20% DMSO+10% BD (72 and 72%). Pregnancy rates (Day 90 ultrasound) of recipients that were transferred either two non-vitrified or two vitrified (20% EG+20% DMSO+10% BD) blastocysts, did not differ (3/6 [50%] and 11/20 [55%]). However, significantly (P<0.02) fewer recipients that received compact morulae maintained pregnancy to Day 90 although this was not affected by vitrification (fresh vs. vitrified; 1/5 [20%] vs. 3/18 [17]). These data demonstrate that a VS comprising three cryoprotectants, rather than one, enables more embryos to hatch during post-thaw culture and that the survival, following direct transfer of these vitrified embryos, is not different to non-vitrified embryos.     

Factors affecting in vivo viability of DNA-injected bovine blastocysts produced in vitro

In vitro matured and fertilized bovine ova were microinjected with pBL1, which consisted of the bovine beta-casein gene promoter, human lactoferrin cDNA and SV40 polyadenylation signal. Of the 2931 zygotes injected, 2505 (85.5%) survived 1 h after DNA injection and were cultured in 50-microl drops of CR1aa medium containing 3 mg/ml BSA under mineral oil at 39 degrees C, 5% CO2 in air. Cleaved (2- to 8-cell) embryos were selected at approximately 48 h after DNA injection and then cultured further in 50-microl drops of CR1aa medium supplemented with 10% (v/v) FBS. Blastocysts were classified into 4 quality grades and 3 developmental stages by morphological criteria. Then all but poor quality blastocysts were nonsurgically transferred to the uterus of heifers 7 to 8 d after natural estrus. Following transfer, the recipients were observed for signs of estrus, and pregnancy was confirmed by palpation per rectum at approximately 60 d of gestation. Although 72.0% (1804/2505 ) of the DNA-injected zygotes reached 2- to 8-cell stages only 5.2% (131/2505) developed to blastocysts. A total of 75 DNA-injected, in vitro cultured blastocysts were transferred to 59 recipients. When 2 blastocysts were transferred to a single recipient, only the better quality embryo was counted. The overall pregnancy rate was 30.5% (18/59 ) and reflected 1) an apparent correlation between the quality of embryos and the pregnancy rate. However, the difference was not statistically significant. 2) expanded blastocysts had a higher pregnancy rate (50.0%, 11/22 ) than early (13.3%, 2 15 ) or mid (22.7%, 5/22 ) blastocysts with a significant difference between expanded and early blastocysts (P < 0.05). 3) the pregnancy rate of DNA-injected blastocysts was higher when they were transferred at Day 7 (34.5%, 10/29 ) or 8 (36.8%, 7/19 ) than at Day 6 (9.0%, 1/11 ). The results indicate that the developmental stage of DNA-injected bovine embryos may be one of contributing factors in improving the pregnancy rate after transfer, although the effects of the quality and culture period of the embryos may not be inconsequential.     

Effect of culture system on survival rate of vitrified bovine embryos produced in vitro

This study was designed to evaluate the effect of in vitro culture system on bovine blastocyst yield and quality after vitrification. In Experiment 1, IVM/IVF zygotes were allocated to three culture conditions: (I) Oviductal cells-SOF (OCM-SOF); (II) Oviductal cells-TCM (OCM-TCM); and (III) SOF for 8 days. There was no significant difference between blastocyst rates among groups.In Experiment 2, the IVP-blastocysts in three above culture conditions were vitrified within groups segregated according to age (Day 7 and 8) and blastocoelic cavity size (early and expanded blastocysts). A trend of higher survival rate was obtained in vitrified/warmed early blastocysts compared with expanded ones, so that the difference in OCM-TCM group was significant (P < 0.001). Higher survival and hatching rates (P < 0.001) were obtained in OCM-SOF and OCM-TCM groups (co-culture) compared with SOF group and the age of blastocyst had no effect on post-thaw survival and hatching rates. In Experiment 3, after staining of blastocysts, in fresh blastocysts the highest number of trophectoderm cells was observed in OCM-TCM group and the number of inner cell mass (ICM) was higher in co-culture groups than SOF group (P < 0.001). In vitrified/warmed blastocysts the number of ICM and trophectoderm cells in co-culture groups was higher than SOF group (P < 0.001) except for the ICM of expanded blastocysts. In conclusion, in our culture conditions, the blastocyst yield is not influenced by culture system, while the cryotolerance of IVP-blastocysts is positively influenced by the presence of somatic cells. Moreover, the expanded blastocysts are more susceptible to cryoinjury than early blastocysts.     

Effect of culture system on survival rate of vitrified bovine embryos produced in vitro

This study was designed to evaluate the effect of in vitro culture system on bovine blastocyst yield and quality after vitrification. In Experiment 1, IVM/IVF zygotes were allocated to three culture conditions: (I) Oviductal cells-SOF (OCM-SOF); (II) Oviductal cells-TCM (OCM-TCM); and (III) SOF for 8 days. There was no significant difference between blastocyst rates among groups.In Experiment 2, the IVP-blastocysts in three above culture conditions were vitrified within groups segregated according to age (Day 7 and 8) and blastocoelic cavity size (early and expanded blastocysts). A trend of higher survival rate was obtained in vitrified/warmed early blastocysts compared with expanded ones, so that the difference in OCM-TCM group was significant (P < 0.001). Higher survival and hatching rates (P < 0.001) were obtained in OCM-SOF and OCM-TCM groups (co-culture) compared with SOF group and the age of blastocyst had no effect on post-thaw survival and hatching rates. In Experiment 3, after staining of blastocysts, in fresh blastocysts the highest number of trophectoderm cells was observed in OCM-TCM group and the number of inner cell mass (ICM) was higher in co-culture groups than SOF group (P < 0.001). In vitrified/warmed blastocysts the number of ICM and trophectoderm cells in co-culture groups was higher than SOF group (P < 0.001) except for the ICM of expanded blastocysts. In conclusion, in our culture conditions, the blastocyst yield is not influenced by culture system, while the cryotolerance of IVP-blastocysts is positively influenced by the presence of somatic cells. Moreover, the expanded blastocysts are more susceptible to cryoinjury than early blastocysts.     

Factors affecting survival of deep frozen bovine embryos in vitro: The effect of freezing container and method of removing cryoprotectant

The effect of freezing container and method of glycerol removal on in vitro survival of frozen-thawed Day 7 bovine embryos was investigated. Two hundred and fifteen embryos were frozen in ampules or straws, in either vertical or horizontal position and at a cooling rate of 0.3 degrees C/minute from -7 degrees C to -35 degrees C, before being plunged into liquid nitrogen. Samples were thawed in a water bath at +35 degrees C and glycerol was removed by either step-wise dilution (increments 0.25 M) or by exposure to 1.0 M sucrose for 10 minutes. A total of 197 embryos was recovered post-thaw (91%) with an overall survival after 1, 3, 6 and 24 hours in culture of 87, 81, 71, and 23%, respectively. Embryonic quality and percent survival, as assessed morphologically, did not change significantly between 1 and 3 hours but decreased significantly between 6 and 24 hours in culture (p < 0.05). Survival at 24 hours was significantly higher after removal of the cryoprotectant with sucrose when compared to the step-wise glycerol dilution (p < 0.05). Overall, embryonic survival in straws equaled that in ampules; freezing orientation of straws did not affect results. Further, glycerol removal with sucrose tended to yield survival superior to that provided by a step-wise dilution technique.     

Factors affecting sperm recovery rates and survival after centrifugation of equine semen

Conventional centrifugation protocols result in important sperm losses during removal of the supernatant. In this study, the effect of centrifugation force (400 or 900 × g), duration (5 or 10 min), and column height (20 or 40 mL; Experiment 1); sperm concentration (25, 50, and 100 × 10⁶/mL; Experiment 2), and centrifugation medium (EZ-Mixin CST [Animal Reproduction Systems, Chino, CA, USA], INRA96 [IMV Technologies, Maple Grove, MN, USA], or VMDZ [Partnar Animal Health, Port Huron, MI, USA]; Experiment 3) on sperm recovery and survival after centrifugation and cooling and storage were evaluated. Overall, sperm survival was not affected by the combination of centrifugation protocol and cooling. Total sperm yield was highest after centrifugation for 10 min at 400 × g in 20-mL columns (95.6 ± 5%, mean ± SD) or 900 × g in 20-mL (99.2 ± 0.8%) or 40-mL (91.4 ± 4.5%) columns, and at 900 × g for 5 min in 20-mL columns (93.8 ± 8.9%; P < 0.0001). Total (TMY) and progressively motile sperm yield followed a similar pattern (P < 0.0001). Sperm yields were not significantly different among samples centrifuged at various sperm concentrations. However, centrifugation at 100 × 10⁶/mL resulted in significantly lower total sperm yield (83.8 ± 10.7%) and TMY (81.7 ± 6.8%) compared with noncentrifuged semen. Centrifugation in VMDZ resulted in significantly lower TMY (69.3 ± 22.6%), progressively motile sperm yield (63.5 ± 18.2%), viable yield (60.9 ± 36.5%), and survival of progressively motile sperm after cooling (21 ± 10.8%) compared with noncentrifuged semen. In conclusion, centrifuging volumes of ≤ 20 mL minimized sperm losses with conventional protocols. With 40-mL columns, it may be recommended to increase the centrifugal force to 900 × g for 10 min and dilute the semen to a sperm concentration of 25 to 50 × 10⁶/mL in a milk- or fractionated milk-based medium. The semen extender VMDZ did not seem well suited for centrifugation of equine semen.     

Pregnancies, calves and calf viability after transfer of in vitro produced bovine embryos

Pregnancy, parturition and calf survival following the transfer of embryos produced in vitro were monitored. A total of 44 blastocysts was transferred in pairs to 1 uterine horn ipsilateral to the corpus luteum (CL) of 22 synchronized heifers. At Day 42 of development 14 recipients (64%) were pregnant; the calving rate was also 64%. The twinning rate was 9/14 at Day 42 and 7/14 at birth, for an overall fetal mortality rate of 9%. The average gestation length was 281 and 275 d for single and twin pregnancies, respectively. Blood samples from recipients were collected for determination of bovine pregnancy associated glycoprotein (bPAG) from 2 wk after transfer and throughout the pregnancy. During the first trimester of pregnancy, the bPAG concentration was significantly higher in twin than in single bearing heifers, and the perinatal increase in bPAG was correlated positively with the total weight of the fetus(es). The percentage of male calves was 43%. The birth weight of twin individuals was 25 +/- 1 kg, which was 78% of the birthweight of the singletons (32 +/- 2 kg). One singleton calf was oversized, weighing 58 kg (80% more than the median weight of the other singletons). Stillbirths occurred in 21% of the twins, butin none of the singletons. Calf mortality during the first 14 d was higher for twins (4/11) than for singletons (1/7) due to infections and cerebellar hypoplasia. Karyotyping the calves detected no cytogenetically recognizable abnormalities. All calves were negative for BVD virus and IBR antibodies. The results of this study showed that although the incidence of fetal loss was low, there was an unacceptable high perinatal mortality of the calves. Thus it is likely that the blood supply through the placenta of animals pregnant with twins was impaired or it is possible that these fetuses and calves had increased stress susceptibility caused by the in vitro conditions. Furthermore, the birth of 1 oversized calf, 2 calves with cerebellar hypoplasia and 5 calves succumbing to infections seems to indicate that a proportion of in vitro produced calves may suffer from factors inherent in the in vitro production system.     

Embryo survival and birth rate after minimum volume vitrification or slow freezing of in vivo and in vitro produced ovine embryos

The objective was to evaluate pregnancy outcomes and birth rate of in vivo derived vs. in vitro produced ovine embryos submitted to different cryopreservation methods. A total of 197 in vivo and 240 in vitro produced embryos were cryopreserved either by conventional freezing, or by vitrification with Cryotop or Spatula MVD methods on Day 6 after insemination/fertilization. After thawing/warming and transfer, embryo survival rate on Day 30 of gestation was affected by the source of the embryos (in vivo 53.3%, in vitro 20.8%; P < 0.05) and by the method of cryopreservation (conventional freezing 26.5%, Cryotop 52.0%, Spatula MVD 22.2%; P < 0.05). For in vivo derived embryos, survival rate after embryo transfer was 45.6% for conventional freezing, 67.1% for Cryotop, and 40.4% for Spatula MVD. For in vitro produced embryos, survival rate was 7.3% for conventional freezing, 38.7% for Cryotop, and 11.4% for Spatula MVD. Fetal loss from Day 30 to birth showed a tendency to be greater for in vitro (15.0%) rather than for in vivo produced embryos (5.7%), and was not affected by the cryopreservation method. Gestation length, weight at birth and lamb survival rate after birth were not affected by the source of the embryo, the cryopreservation method or stage of development (average: 150.5 ± 1.8 days; 4232.8 ± 102.8 g; 85.4%; respectively). This study demonstrates that embryo survival and birth rate of both in vivo and in vitro produced ovine embryos are improved by vitrification with the minimum volume Cryotop method.