Effects of cryopreservation on glucose metabolism and survival of bovine morulae and blastocysts derived in vitro or in vivo

Bovine morulae and blastocysts were either produced in vitro through maturation, fertilization and culture of immature oocytes recovered from slaughterhouse-derived ovaries, collected in vivo or obtained after 24 h in vitro culture of in vivo collected embryos. The morulae and blastocysts were classified into four categories of embryo quality and two stages of embryonic development. Embryos were frozen by a controlled freezing method using 10% glycerol as a cryoprotectant. The ability of individual embryos to withstand freeze/thawing was measured immediately before and after cryopreservation by changes in CO2 production from (U-14C)glucose during a 2 h incubation period in a non-invasive closed system immediately before and after cryopreservation. Post-thaw survival was assessed by development in vitro during a 48 h culture period. Survival rates and oxidative metabolism after freeze/thawing were similar in embryos of the two developmental stages. However, after freeze/thawing, the rate of CO2 production of in vitro produced embryos was reduced to one half of their pre-freeze levels and was associated with poor survival rates. In vivo collected embryos had a significantly better tolerance to freezing and higher survival rates. However, when in vivo embryos were exposed to in vitro culture conditions, the rates of CO2 production and survival were significantly reduced. Pre-freeze embryo quality affected post-thaw in vitro development and metabolic activity markedly in embryos produced in vitro or pre-exposed to in vitro culture conditions. While there was no relationship between pre-freeze levels of CO2 production and post-thaw in vitro embryo development, all embryos which developed in vitro after freezing/thawing retained at least 58% of the pre-freeze levels of CO2 production regardless of their origin. Results of the present study indicate that embryos produced in vitro or pre-exposed to in vitro culture conditions are more sensitive to cryo-injury. This sensitivity is affected by embryo quality and is similarly reflected at the biochemical level. Determination of oxidative metabolism offers a feasibility for selection of viable morulae/blastocysts after freezing/thawing.     

Cryopreservation of bovine blastocysts obtained by intracytoplasmic sperm injection

The freezability and survivability of zona-intact and zona-free (hatched) bovine blastocysts obtained by intracytoplasmic sperm injection (ICSI) were assessed. Day 7 or 8 blastocysts were cryopreserved by slow freezing using 1.5 M glycerol and 0.2 M sucrose. Embryos were exposed to solutions in a 2-step procedure at room temperature and frozen in a programmed cell freezer. Blastocysts that re-expanded within 6 h of post-thaw culture were considered viable. The cleavage, morula and blastocyst development rates after ICSI were 52.4 (131/250), 39.7 (52/131), and 24.4% (32/131), respectively. Blastocyst stage embryos were randomly divided into 2 groups. The first group of embryos was frozen with their zonae intact, while the second group was allowed to hatch from their zonae during the additional 18 h culture, after which they were frozen. The data showed that more Group 2 blastocysts (14/16, 87.5%) than Group 1 (12/16; 75.0%; P<0.05) survived, and more zona-free bovine blastocysts frozen with glycerol as the cryoprotective agent (CPA) than zona-intact blastocysts after slow freezing retained their viability.     

Commercial freezing of bovine embryos in straws

Bovine embryos were frozen commercially in clear double length $\text{1}\text{2}$ cc French straws with the wick and powder plug in the center of the straw. One-half of the double length straw serves as a handle and contains a color coded $\text{1}\text{4}$ cc straw around which an adhesive backed label has been applied. After plunging into liquid nitrogen, straws are transferred into goblets on canes while under liquid nitrogen. The straws are stored in the liquid phase of a nitrogen tank and canes containing straws are not transferred from one container to another unless the goblet containing the straws is full of liquid nitrogen.Embryos held for longer than 4 hours after collection prior to freezing showed a steady decline in pregnancy rate related to the length of time held prior to freezing. The percentage of embryos thawed and then evaluated as being transferrable was related to the quality of the embryos prior to freeze (Grade 1–93.6%, Grade 2–87.0%, Grade 3–63.8%). There was no statistical difference in pregnancy rates obtained from prefreeze Grade 1 embryos when comparing advanced blastocysts (45.2%), blastocysts (38.7%), early blastoclyst (43.1%) and advanced morula (41.6%).     

Effect of lecithin on in vitro and in vivo survival of in vitro produced bovine blastocysts after cryopreservation

The use of soybean lecithin in an glycerol-based solution for slow freezing of in vitro matured, fertilized and cultured (IVMFC) bovine embryos was examined. Embryos were developed in vitro in INRA Menezo's B2 medium supplemented with 10% fetal calf serum (FCS) on Vero cells monolayers. Day 7 blastocysts were frozen in a two-step protocol consisting of exposure to 5% glycerol and 9% glycerol containing 0.2 M sucrose in F1 medium + 20% FCS. Soybean lecithin was either added or not to the freezing solutions at a final concentration of 0.1% (w/v). In Experiment 1, blastocysts were equilibrated in cryoprotectant solutions without cooling. Cryoprotectant was diluted from embryos with 0.5 M and 0.2 M sucrose. The percentages of fully expanded and hatched blastocysts treated with or without lecithin after 24 and 48 h in culture were not significantly different (100 versus 100% and 93.3 versus 100%, respectively). In Experiment 2, the in vitro survival of frozen-thawed IVMFC blastocysts was compared when cryoprotectant solutions were either supplemented or not with lecithin. No significant effect of lecithin was found on the ability of frozen-thawed blastocysts to re-expand after 48 h in culture (65.6 and 54.2%, respectively). However, the post-thaw hatching rate of embryos cryopreserved in the presence of 0.1% lecithin was significantly higher after 72 h in culture (52 and 31.8%, respectively). In Experiment 3, the ability of frozen-thawed IVMFC blastocysts to establish pregnancy following single embryo transfer was determined. Transfers of 58 and 66 frozen-thawed embryos cryopreserved with or without lecithin resulted in 6 and 10 (10.3 and 15.1%, respectively) confirmed pregnancies at Day 60. Addition of lecithin to cryoprotectants did not improve the in vivo development rate of cryopreserved IVMFC bovine blastocysts.     

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.     

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.     

A method for one-step freezing of mouse embryos

A simple one-step method of freezing mouse embryos directly in liquid nitrogen is described. The main objective of this study was to assess post-thaw survival following predehydration in various mixtures of glycerol and sucrose. Also investigated was pretreatment with glycerol prior to dehydration and effects of embryo stage. When sucrose was held constant (0.25 M) and glycerol concentration varied (1.0-4.0 M), post-thaw survival was best (67%) in 2.0 M glycerol. Pretreatment in glycerol provided no advantage over no pretreatment. When glycerol was held constant (2.0 M) and sucrose concentration varied (0-1.0 M), optimum post-thaw survival (81%) was found in 0.5 M sucrose. Morulae survived better than blastocysts (86% vs 72%, respectively). Transfer of thawed embryos frozen by the optimum treatment (2.0 M glycerol + 0.5 M sucrose) resulted in a birthrate of 41%, compared to 54% for fresh controls. This technique could find application in freezing and thawing of livestock embryos on the farm.     

A serum-free, cell-free culture system for development of bovine one-cell embryos up to blastocyst stage with improved viability

With the aim of developing a serum-free, cell-free culture system for embryo development, in vitro-matured (IVM) and -fertilized (IVF) bovine oocytes were cultured in TCM 199 with the following supplements: 1) BSA alone (10 mg/ml); 2) BSA with ITS (5 mug/ml insulin, 5 mug/ml transferrin and 5 ng/ml selenium; BSAITS medium); 3) estrous cow serum alone (ECS; 10%); or 4) ECS with BOEC (bovine oviduct epithelial cells) (Experiment 1). In Experiment 2, embryos were cultured in BSAITS medium with or without feeding with fresh medium on Day 4 (day of insemination = Day 0). Embryos were evaluated on Day 2 for first cleavage, on Day 7 for morulae and blastocysts, and on Day 8 for blastocysts. Blastocysts from Experiment 1 were frozen in 10% glycerol in PBS, thawed and further cultured in ECS medium with BOEC for 48 h, and evaluated for formation of a distinct blastocoel, or expansion and hatching of blastocysts. In vivo-developed, Grade-1 and Grade-2, 7-d-old embryos served as control for the freezing, thawing and subsequent culture procedures. The percentage of first cleavage did not differ between the treatments (74 to 79% in Experiment 1 and 80 to 83% in Experiment 2). The percentage of blastocysts developed in BSAITS medium did not differ from that in ECS medium whether BOEC were present or not. However, medium with BSA alone had fewer blastocysts than any other culture system (P<0.05). Feeding embryos with fresh BSAITS medium on Day 4 did not lead to any further increase in the proportion of blastocysts. The culture systems had a significant effect on the post-thaw viability of blastocysts developed in them (P<0.001). Blastocysts developed in BSAITS medium had better (P<0.05) viability (14/38) than those from medium with ECS alone (1/27) or with ECS and BOEC (3/37). The post-thaw survival of control embryos was 80% (n=30). One of the three transfers of BSAITS-treated, frozen-thawed blastocysts resulted in a pregnancy. The results indicate that a serum-free, cell-free culture system can support the development of IVM-IVF bovine oocytes up to the blastocyst stage with better viability than a complex co-culture system.     

Vitrification of bovine IVF blastocysts in an ethylene glycol/sucrose solution and heat-stable plant-extracted proteins

The use of heat-stable plant proteins in an ethylene glycol-based solution for the vitrification of in vitro-derived embryos was examined. Day 7, 8 and 9 bovine in vitro matured, fertilized and cultured (IVMFC), full and expanded blastocysts were vitrified in solutions composed of 40% ethylene glycol (EG) plus 0.3 M sucrose supplemented with 20% Ficoll and 0.3% BSA (VF-1), 25 mg/ml heat-stable plant proteins (HSPP; VF-2), or with no supplement (VF-3). In Experiment 1, embryos were expelled from the straw after thawing, and EG was diluted from embryos with 0.5 M sucrose. There were no differences in post-thaw embryo survival rates or in hatching/hatched rates after 24 h of culture between the VF-1, VF-2 and VF-3 solutions (40.1, 54.1 and 50.8% and 10.7, 16.4 and 17.5%, respectively). Transfer of 12 frozen/thawed embryos to 6 recipients (2 recipients per treatment) resulted in 2 pregnancies from the VF-2 group and 1 pregnancy from the VF-3 group. In Experiment 2, EG was diluted from embryos after thawing within the straw with 0.5 M sucrose. There were no differences in post-thaw survival or hatching/hatched rates after 24 h of culture (19.0, 13.6 and 23.8% and 9.5, 9.0 and 14.4% for VF-1, VF-2 and VF-3, respectively). Transfer of 6 frozen/thawed embryos to 3 recipients (1 recipient per treatment) resulted in no pregnancies. The post-thaw histology of Day 7, 8 and 9 IVMFC blastocysts showed typical ultrastructure with well preserved cell-to-cell contacts. There were no major differences in the fine structure of blastocysts regardless of treatment. The use of HSPP at a concentration of 25 mg/ml in the vitrification medium did not affect the post-thaw embryo survival over that of no protein supplementation. The presence of macro molecules in a 40% EG/sucrose vitrification solution also did not improve post-thaw viability of IVMFC-derived blastocysts.     

Cryopreservation of in vitro-produced bovine embryos: effects of protein type and concentration during freezing or of liposomes during culture on post-thaw survival

Two experiments were conducted to examine the effect of membrane stabilization through the modification of in vitro culture medium or freezing medium on post-thaw survival of in vitro-produced bovine embryos. In Experiment 1, Day 7 (Day 0 = day of IVF) late morulae and blastocysts that developed following culture in SOF/aa/BSA (IVC medium) were frozen slowly to -35 degrees C in the presence of 1.5 M ethylene glycol prepared in ovum culture medium (OCM) or in OCM supplemented with 10, 25 or 50% fetal calf serum (FCS) or 5, 10 or 25 mg/mL BSA. Post-thaw survival was assessed by re-expansion and/or hatching following 48 h of culture in IVC medium + 10% FCS. Overall, survival was significantly (P < 0.01) affected by embryo stage, with more hatched blastocysts surviving (71%) than blastocysts (59%) or late morulae (51%). Addition of FCS significantly (P < 0.01) reduced survival compared with control embryos or those frozen in BSA-supplemented medium (50.48 vs 68.01 vs 63.53%, respectively). There was also a significant interaction between embryo stage and protein type (P < 0.05). The survival of late morulae/early blastocysts following freezing was improved in the presence of additional BSA but not FCS. In Experiment 2, the IVC medium was supplemented with liposomes containing lecithin, sphingomyelin and cholesterol. Sphingomyelin and cholesterol at ratios of 1:1, 1:4 and 4:1 were added to 50, 100 or 150 micrograms/mL lecithin to yield a final lipid concentration of 200 micrograms/mL. A further group contained 200 micrograms/mL lecithin only. Blastocysts were frozen in 1.5 M ethylene glycol in OCM, then thawed and assessed as in Experiment 1. The presence of liposomes during IVC did not affect the proportion of cleaved embryos that developed to blastocysts or survival following freezing. However, the survival of blastocysts that developed in the presence of 200 micrograms/mL lecithin only was significantly lower than in any other treatment (6%; P < 0.03). These studies demonstrate that the protein composition of the freezing medium can significantly affect survival after thawing and that the survival of late morulae can be improved with additional BSA. The presence of lecithin only in the liposome preparation did not affect embryo development, but significantly reduced survival after freezing, suggesting it can affect post-thaw embryo survival, perhaps by altering embryonic membrane composition.     

Freezability of porcine blastocysts at different peri-hatching stages

The freezability of porcine peri-hatching stage blastocysts was investigated by the cryopreservation of embryos at -196 degrees C with 1.5 M glycerol and by thawing, followed by in vitro culture. Of 66 expanded blastocysts frozen, 34 (51.5%) developed in vitro after thawing, while only 2 (6.7%, P<0.05) of 30 earlier stage blastocysts survived freezing. After freezing of 85 hatched blastocysts with an embryonic diameter of 150 to 300 mum, 59 (69.4%) surviving embryos were obtained, whereas none of the 78 advanced staged hatched blastocysts (>300 mum) survived the cryopreservation. High post-thaw survival (32 39 , 82..1%) was obtained with in vitro-hatched blastocysts precultured in Whittingham's M-16 medium containing 12mg/ml bovine serum albumin (BSA). In contrast, none of the 14 in vitro-hatched blastocysts precultured in the M-16 medium supplemented with 15% fetal calf serum (FCS) survived freezing. Similarly 51 of 56 hatced blastocysts (diameter = 150 to 300 mum) precultured in the M-16 medium supplemented with BSA survived cryopreservation, compared with 3 of 26 embryos precultured in the medium supplemented with FCS (P<0.001). Because both groups of the embryos precultured with BSA or FCS possessed normal ability to develop after transfer (developmental rate = 61.1 and 93.3%), the improved freezability of the embryos precultured with BSA may relate to a favorable change of embryonic cell membranes during the culture period. It was concluded that in vitro-hatched blastocysts precultured in medium containing BSA and in vivo-hatched blastocysts at the appropriate stage of development could both tolerate deep freezing to -196 degrees C; however, a differece in the freezability of embryos between breeds of pig was suggested from a further experiment performed with German Landrace embryos.     

A new procedure for the cryopreservation of equine embryos

Early equine blastocysts and blastocysts were collected nonsurgically at six days post-ovulation. Thirty-two embryos were randomly assigned to a 2x2 factorial design. Factors were: 1) 0.5-ml straws or 1-ml glass ampules; and 2) plunging into liquid nitrogen (IN(2)) at -33 C or -38 C. Cryoprotectant, 10% glycerol in PBS plus 5% fetal calf serum (FCS) was added in two steps, 5% then 10%. Embryos were cooled at 4 C/min to -6 C and then seeded, 0.3 C/min to -30 or -35 C and 0.1 C/min to -33 or -38 C. Samples were thawed in 37 C water and glycerol removed in six steps, 10 min per step. Embryo quality and stage of development were evaluated prior to freezing, immediately post-thaw and after 24 h culture in Ham's F10 with 5% FCS. The mean post-thaw quality of embryos plunged at -33 C was superior (P<0.05) to that of embryos plunged at -38 C (2.0 vs 2.9). Embryos frozen in ampules and plunged at -38 C were of poorer quality (P<0.05) than those frozen in ampules and plunged at -33 C or frozen in straws and plunged at -33 C. After 24 h of culture, more embryos developed if frozen in straws compared to ampules, and plunging at -33 C resulted in higher quality embryos than plunging at -38 C. In Experiment 2, 23 embryos were packaged in straws and plunged at -33 C as described in Experiment 1. Six of the 23 surgically transferred frozen embryos were degenerate at thawing and the remaining 17 surgically transferred were via flank incision. Pregnancy rate at 50 days post-ovulation was 53% (nine of 17). Early blastocysts resulted in a higher (P<0.05) pregnancy rate (8 10 , 80%) than expanded blastocysts (1 7 , 14%).     

Comparison on in vitro fertilized bovine embryos cultured in KSOM or SOF and cryopreserved by slow freezing or vitrification

The objectives of this study were to identify an improved in vitro cell-free embryo culture system and to compare post-warming development of in vitro produced (IVP) bovine embryos following vitrification versus slow freezing. In Experiment 1, non-selected presumptive zygotes were randomly allocated to four medium treatments without co-culture: (1) SOF + 5% FCS for 9 days; (2) KSOM + 0.1% BSA for 4 days and then KSOM + 1% BSA to Day 9; (3) SOF + 5% FCS for 4 days and then KSOM + 1% BSA to Day 9; and (4) KSOM + 0.1% BSA for 4 days and then SOF + 5% FCS to Day 9. Treatment 4 (sequential KSOM-SOF culture system) improved (P > 0.05) morulae (47%), early blastocysts (26%), Day-7 blastocysts (36%), cell numbers, as well as total hatching rate (79%) compared to KSOM alone (Treatment 2). Embryos cultured in KSOM + BSA alone developed slowly and most of them hatched late on Day 9, compared to other treatments. In Experiment 2, the sequential KSOM-SOF culture system was used and Day-7 blastocysts were subjected to following cryopreservation comparison: (1) vitrification (VS3a, 6.5 M glycerol); or (2) slow freezing (1.36 M glycerol). Warmed embryos were cultured in SOF with 7.5% FCS. Higher embryo development and hatching rates (P < 0.05) were obtained by vitrification at 6h (71%), 24h (64%), and 48h (60%) post-warming compared to slow freezing (48, 40, and 31%, respectively). Following transfer of vitrified embryos to synchronized recipients, a 30% pregnancy rate was obtained. In conclusion, replacing KSOM with SOF after 4 days of culture produced better quality blastocysts. Vitrification using VS3a may be used more effectively to cryopreserve in vitro produced embryos than the conventional slow freezing method.     

Viability of frozen-thawed bovine IVM/IVF embryos in relation to aging using various cryoprotectants

Bovine IVF embryos developed on Days 7, 8 and 9 were equilibrated with 1.6 M propylene glycol (PG), 1.8 M ethylene glycol (EG), 1.1 M diethylene glycol (DEG) or 1.3 M ethylene glycol monomethyl ether (EME) for 10 to 20 min in modified phosphate buffered saline. (mPBS) supplemented with 10% superovulated cow serum. The embryos were loaded into 0.25-ml plastic straws and were placed directly into a 0 degrees C alcohol bath chamber and held for 2 min. They were cooled from 0 degrees C to -5.5 degrees C at 1 degrees C/min and then seeded, followed by a 10-min holding period at -5.5 degrees C. The straws were then cooled to -30 degrees C at 0.3 degrees C/min before plunging into liquid nitrogen. Embryos were thawed and placed directly into the culture medium and washed 3 times. The survival rates of the Day-9 embryos based on reappearance of blastocoele, expansion, and hatching after 48 h of post-thaw culture were significantly lower (P<0.01) than those of the Day-7 and 8 embryos, in all of the cryoprotectants tested. On the other hand, while the reappearance of blastocoele and expansion of blastocysts after 48 h of post-thaw culture were not significantly different among each cryoprotectant, the percentage of hatching blastocysts were significantly different between DEG and EME (P<0.05), between DEG and EG (P<0.01) and between PG and EG (P<0.05). These findings demonstrate that the age of the embryo (Day 7 and 8) is very important for the successful freezing of IVF bovine embryos. Also, as to the hatching rates, EME and EG are superior as cryoprotectants than the other 2 cryoprotectants tested.     

Human leukemia inhibitory factor improves the viability of cultured ovine embryos.

Embryos were collected from ewes on Day 6 after estrus (Day 0 = estrus), placed in M2 culture medium, and assigned to 1 of 4 treatment groups. Some embryos were transferred to recipient ewes on Day 6 of their estrous cycle either in pairs (group 1) or singularly (group 2) within 3 h of collection. The remaining embryos were individually cultured for 48 h in an atmosphere of 5% CO2 in humidified air in either synthetic oviduct fluid (SOF) medium (group 3) or SOF containing 1,000 U/ml of recombinant human leukemia inhibitory factor (hLIF) (SOF + hLIF: group 4). These embryos were then transferred to recipient ewes on Day 8 of their estrous cycle. The addition of hLIF to culture medium significantly improved the development of the embryos compared with control embryos prior to transfer (blastocysts hatching from the zona pellucida: group 3 = 16% vs. group 4 = 64%, p less than 0.05; those degenerative: group 3 = 27% vs. group 4 = 9%, p less than 0.05) and the subsequent pregnancy rates of the recipient ewes, receiving a single embryo, at Day 70 of pregnancy (group 3 = 16% vs. group 4 = 50%, p less than 0.05). The pregnancy rate of ewes given embryos cultured for 48 h in SOF + hLIF prior to transfer (50%; group 4) was similar to the group 2 ewes receiving a single embryo soon after collection (52%), but the pregnancy rate for both groups was significantly lower than that for the group 1 ewes receiving two embryos soon after collection (89%: 53% twins, 36% singles; p less than 0.05).     

Early development and location of embryos in the reproductive tract of Nili Ravi buffalo (Bubalus bubalis): a retrospective analysis

One year data on embryo recovery were analyzed to study the development and descent of preimplantation embryos in Nili Ravi water buffalo. Forty-five superovulatory attempts were performed on 23 buffalo. A total of 45 embryos were recovered either nonsurgically or after slaughtering the animals at various time intervals (85 to 176 h) post estrus. Embryos were located in the oviducts at 85 h after estrus. At 108 h post estrus, most of them (78%) were recovered from the uteri. The embryos had 8 to 16 cells at 85 h post estrus, grew to morulae at 108 h and to compact morulae at 125 h post estrus. Early blastocysts were observed at 141 h post estrus. Blastocysts were predominant (69%) at 156 to 176 h after estrus; no hatched blastocysts were recovered during this time interval. Based on our findings, embryo recovery at around 150 h post estrus (i.e., Day 6 of the cycle) is recommended for compact morulae or blastocysts in the water buffalo.     

Survival of rapidly frozen hatched mouse blastocysts

The objective of the present study was to examine the effect of rapid freezing on the in vitro and in vivo survival of zona-pellucida-free hatched mouse blastocysts. Hatched blastocysts were rapidly frozen in a freezing medium containing either ethylene glycol (EG) or glycerol (G) in 1.5 M or 3 M concentration. Prior to freezing, embryos were equilibrated in the freezing medium for 2 min, 10 min, 20 min or 30 min at room temperature. To freeze them, embryos were held in liquid nitrogen vapour [approximately 1 cm above the surface of the liquid nitrogen (LN2)] for 2 minutes and then immersed into LN2. After thawing, embryos were transferred either to rehydration medium (DPBS + 10% foetal calf serum +0.5 M sucrose) for 10 minutes or rehydrated directly in DPBS supplemented with foetal calf serum. In vitro survival of embryos frozen with EG was higher than those frozen with G. The highest survival was obtained with 3 M EG and 2 min or 10 min equilibration prior to freezing, combined with direct rehydration after thawing. Frozen blastocysts developed into normal foetuses as well as unfrozen control ones did, with averages of 30% (control), 26% (EG) and 15% (G). The results show that hatching and hatched mouse blastocysts can be cryopreserved by a simple rapid freezing protocol in EG without significant loss of viability. Our data indicate that the mechanical protection of the zona pellucida is not needed during freezing in these stages.     

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.     

Direct freezing of cattle embryos after partial dehydration at room temperature

A new and simple method for freezing of bovine morulae and blastocysts was developed. Embryos were predehydrated at room temperature, frozen at -30 degrees C (cooling rate = 12 degrees C/min), and plunged into liquid nitrogen. This method was compared in vitro and in vivo to the slow freezing method (0.3 degrees C/min to -30 degrees C). Predehydration of the embryos in 1.5M glycerol was achieved by sucrose solution that makes the cells osmotically shrink. After the predehydrated morulae and blastocysts were frozen and thawed, 6 .4% (33 52 ) were developed in vitro for 48h and 44.2% (23 52 ) were hatched. Development obtained with slowly frozen embryos were 70.8% (17 24 ) and 58.3% (14 24 ) respectively. After transfer to recipient heifers, 33.3% (7 21 ) of the embryos frozen according this new method developed normally into viable foetuses or calves. This was the case for 48.5% (16 33 ) of the slowly frozen embryos.     

Analysis of cryoprotectant, cooling rate and in situ dilution using conventional freezing or vitrification for cryopreserving sheep embryos

Two studies were conducted to evaluate the influence of cryoprotectant, cooling rate, container and cryopreservation procedure on the post-thaw viability of sheep embryos. In Study 1, late morula- to blastocyst-stage embryos were exposed to 1 of 10 cryoprotectant (1.5 M, glycerol vs propylene glycol)-plunge temperature treatments. Embryos were placed in glass ampules and cooled at 1 degrees C/min to -5 degrees C, seeded and further cooled at 0.3 degrees C/min to -15, -20, -25, -30 and -35 degrees C before rapid cooling by direct placement in liquid nitrogen (LN(2)). Post-thaw embryo viability was improved (P<0.01) when embryos were cooled to at least -30 degrees C before LN(2) plunging. Although there were no overt differences in embryo viability between cryoprotectant treatments (each resulted in live offspring after embryo transfer), there was a lower (P<0.01) incidence of zona pellucida damage using propylene glycol (4%) compared to glycerol (40%). In Study 2, embryos were equilibrated in 1.5 M propylene glycol or glycerol or a vitrification solution (VS3a). Embryos treated in propylene glycol or glycerol were divided into ampule or one-step((R)) straw treatments, cooled to -6 degrees C at 1 degrees C/min, seeded, cooled at 0.5 degrees C/min to -35 degrees C, held for 15 minutes and then transferred to LN(2). Embryos vitrified in the highly concentrated VS3a (6.5 M glycerol + 6% bovine serum albumin) were transferred from room air to LN(2) vapor, and then stored in LN(2). Propylene glycol- and glycerol-treated embryos in straws experienced lower (P<0.05) degeneration rates (27%) and yielded more (P<0.05) hatched blastocysts (73 and 60%, respectively) at 48 hours of culture and more (P<0.05) trophoblastic outgrowths (67 and 53%, respectively) after 1 week than vitrified embryos (47, 40 and 20%, respectively). In vitro development rate for VS3a-treated embryos was similar (P>0.10) to that of ampule controls, which had fewer (P<0.05) expanded blastocysts compared to similar straw treatments. Live offspring were produced from embryos cryopreserved by each straw treatment (propylene glycol, 3 of 7; glycerol, 1 of 7; VS3a, 2 of 7). In summary, freeze-preservation of sheep embryos was more effective in one-step straws than glass ampules and propylene glycol tended to be the optimum cryoprotectant. Furthermore, these findings demonstrate, for the first time, the biological competence of sheep embryos cryopreserved using the simple and rapid procedure of vitrification.