Effect of different methods of cryopreservation on the cytoskeletal integrity of dromedary camel (Camelus dromedarius) embryos

This study examined the effect of different methods of cryopreservation on the cytoskeletal integrity of camel embryos. A total of 32 embryos were recovered on Days 6 and 7 after ovulation and measured before being frozen using either a conventional slow-cooling technique (n=12: six Day 6 and six Day 7 embryos) or vitrification (n=12: four Day 6 and eight Day 7). The remaining 8 'control' embryos (four Day 6 and four Day 7) were not cryopreserved but instead incubated in holding medium for 30 min. After thawing, warming or incubation, the embryos were stained with 4,6-diamino-2-phenylindole dihydrochloride (DAPI) to identify dead cells. Subsequently, the embryos were fixed in 4% paraformaldehyde, permeabilized and labelled with Alexa Fluor 488-Phalloidin to enable assessment of cytoskeleton integrity. Vitrified-warmed embryos contained a significantly higher percentage of dead cells than either conventionally frozen embryos or controls (P<0.05). Although the proportion of dead cells in conventionally frozen embryos tended to be higher than in controls, the difference was not significant (P> or =0.07). Whereas embryo size did not affect the number of dead cells in conventionally frozen embryos, vitrified-warmed embryos >300 microm in diameter had a significantly higher percentage of dead cells than embryos < or =300 microm (P=0.01). Cytoskeleton integrity was also affected by both freezing method and embryo diameter. All 8 control embryos had a Grade I cytoskeleton, compared with only 2/24 (8.3%) frozen or vitrified embryos. Of the 8 slow-frozen or vitrified embryos with a Grade III cytoskeleton post-thaw, 7 had been vitrified and 6 were larger (Day 7) embryos. These results indicate that while both slow-freezing and vitrification of camel embryos lead to cytoskeleton disruption and cell death, embryo quality is better preserved by slow-freezing.     

Two step freezing of cow embryos in 1.4 M glycerol

Day 6 1 2 -7 1 2 cow embryos were frozen in 1.4 M glycerol in PBS, at 0.3 degrees C/min to -30 (group I), -35 (group II), and -40 degrees C (group III) before being plunged into liquid nitrogen. They were subsequently thawed by direct transfer to water at 37 degrees C. In Experiment I, embryos frozen and thawed were cultured in vitro, 12 out of 19 embryos (63%) survived and there were no significant (p > 0.05) differences in survival rates among the three freezing groups. In Experiment II, 29 embryos frozen to -30 or -35 degrees C were transferred non-surgically to heifers on day 7. Seventeen of 29 recipients (59%) were pregnant at day 60. Five embryos frozen to -35 degrees C resulted in 5 pregnancies (100%) after thawing and surgical transfer.     

Distribution of polymorphic forms at the porcine GH locus in a population of day-10 pig embryos

The present study describes an analysis of genotype and allele distribution at the porcine GH locus among day-10 pig embryos. Embryos were collected post mortem from 6 crossbred (Danish Landrace x Yorkshire) sows inseminated with mixed Duroc semen and individually frozen for later analysis. After extraction, DNA was subjected to PCR amplification and restriction analysis with Msp I and Hae II enzymes. The genotype frequencies were: Msp I CD 0.17, DD 0.83; and Hae II AA 0.33, AB 0.58; and BB 0.09. The Msp I CC genotype was not found among analysed embryos. To our knowledge, this is the first report on the genotype and allele distribution at the GH locus among early pig embryos.     

Evaluation of cryopreservation techniques for bovine embryos

A total of 228 embryos was nonsurgically collected from superovulated cows and dehydrated in dimethyl sulfoxide (DMSO) or glycerol by a three-step procedure or a (T.I.T.) timed interval titration procedure. Embryos were loaded in straws, frozen by cooling to -6.0 degrees C at 1.0 degrees C/min, and seeded, followed by cooling to -30 degrees C at 0.3 degrees C/min and to -38 degrees C at 0.1 degrees C/min. At this time the straws were plunged into liquid nitrogen at -195 degrees C. Embryos were thawed in a 27 degrees C or 37 degrees C water bath and rehydrated by a six-step, three-step (sucrose) or one-step (sucrose) procedure. This yielded a 2 x 2 x 2 x 3 factorial treatment structure. Survival was based on development after 12 h in in vitro culture. The only significant single factor affecting survival was the initial quality grade of the embryo. Grades 1 and 2 embryos survived more often than Grade 3 embryos (P < 0.05). Using DMSO as the cryoprotectant resulted in better scores for the post dehydration to post thawing interval (P = 0.02). For both intervals, post dehydration to post thawing and post thawing to post rehydration, the previous quality grade was significant in determining the subsequent quality grade (P < 0.01). At each step of the freeze-thaw process, the embryos became progressively less morphologically intact.     

Effects of varying the holding temperature and interval from collection to freezing on post-thaw development of bovine embryos in vitro

The objective of this study was to evaluate the in vitro development of frozen-thawed bovine embryos held at room temperature or refrigerated for 2, 6 or 12 h prior to freezing. After recovery, embryos were randomly assigned to be placed in holding media for 2 h (n=131), 6 h (n=136) or 12h (n=133) prior to freezing. Approximately one-half of the embryos were refrigerated (5 degrees C; n=203) while the remaining half were held at room temperature (22 degrees C; n = 197) until freezing. Embryos were frozen in 10% ethylene glycol and stored in liquid nitrogen. After thawing, embryos were cultured for 72 h in Ham's F-10 media supplemented with 4% fetal bovine serum. Embryos were evaluated for quality and stage of development prior to freezing and after culture. At the end of culture, it was determined if each embryo had developed beyond the stage recorded prior to freezing and if the embryo had hatched from the zona pellucida. The percentage of embryos that developed during culture was greater (P < 0.001) for Grade 1 (81%) than for either Grade 2 (65%) or Grade 3 (48%) embryos. Likewise, a greater proportion (P < 0.001) of Grade 1 embryos developed to hatched blastocysts (60%) than either Grade 2 (40%) or Grade 3 (24%) embryos. The holding temperature from collection to freezing did not influence embryo development, regardless of the interval from embryo collection to freezing. The proportion of embryos that developed to expanded blastocysts and hatched was greater (P < 0.005) for embryos held 2 h prior to freezing (64%) than for embryos held for 12 h (33%). Hatching rate of embryos held 6 h prior to freezing (54%) tended (P < 0.08) to be lower than the hatching percentage for embryos held for 2 h. Thus, post-thaw embryonic development was impaired the longer embryos were held prior to freezing and temperature during the interval from collection to freezing did not affect post-thaw development.     

The integration of the transfer of frozen embryos with artificial insemination in a commercial beef herd

This trial was designed to examine the integration of the transfer of frozen embryos and artificial insemination (A.I.) in a commercial herd of beef cows. Embryos were collected nonsurgically from 15 superovulated cows and all embryos judged viable (n = 69) were frozen in 0.25 ml plastic A.I. straws. Glycerol was used as a cryoprotectant and the embryos were frozen with omission of the traditional "seeding" step. On three consecutive days immediately preceding the onset of a 60-day breeding season, 54 embryos were transferred to multiparous cows with calves at foot. The remaining 82 cows in the same herd were used as controls. Cows receiving embryos were maintained under observation, but not inseminated for 21 days, after which cows exhibiting signs of estrus were inseminated up to Day 60. Cows in the control group were inseminated at observed estrus throughout the 60-day period. The overall pregnancy rate for the experimental and control groups did not differ (77.8 % and 73.2 % respectively). Of the experimental cows, 24.1 % conceived to the embryo transfer, and 53.7 % to A.I. The mean calving to conception interval for the experimental cows was 96.3 days (median 101 days) and for the control cows it was 92.3 days (median 88 days). The calving pattern in the experimental cows was biphasic, calvings resulting from embryo transfer of A.I. accounting for the two peaks. Reconception rates in the following breeding season were not different between the embryo transfer or A.I. groups or between experimental and control herds. Weaning masses of calves born of embryo transfer were significantly higher than the other groups, but calves resulting from A.I. in the experimental herd were not different from calves in the control herd. It was concluded that the transfer of frozen embryos could be successfully integrated into an A.I. program with a limited breeding season without detrimentally affecting the overall reproductive performance of the recipient herd.     

Use of ethylene glycol as a cryoprotectant for bovine embryos allowing direct transfer of frozen-thawed embryos to recipient females

Four experiments were conducted to define a system for the direct transfer of frozen-thawed bovine embryos to recipient females. In Experiment I, nonsurgically recovered embryos were frozen in 1.5 M ethylene glycol (EG), 1.5 M propylene glycol (PG), 1.5 M DMSO or 1.4 M glycerol (GLY), and then thawed and placed directly into holding medium. Viability at 72 hours of post-thaw culture was 70, 11, 25 and 30% for the four groups, respectively. In Experiments II and III, 1.0, 1.5 and 2.0 M concentrations of EG were compared; a concentration of 1.5 M appeared to provide optimal cryopreservation and survival after direct rehydration. In Experiment IV, embryos were packaged in straws containing only 1.5 M EG, in straws containing a column of 1.5 M EG and the embryo and two columns of PB1 in a 1:3 ratio of volumes (EG PB1 ), or were frozen in 1.4 M glycerol. After thawing, embryos in EG and EG PB1 treatments were transferred directly to recipient females, while embryos frozen in GLY were rehydrated using a three-step procedure. In the first trial, pregnancy rates at approximately 60 days of gestation for embryos frozen in EG and GLY groups were 39 and 62%, respectively (P<0.10). In the second trial, the pregnancy rate for embryos frozen in EG PB1 was equal to that of embryos frozen in GLY (50% in both groups). These experiments demonstrate the potential for using ethylene glycol as a cryoprotectant for bovine embryos, thus permitting direct transfer of frozen-thawed embryos to recipient females.     

Pregnancy rate and birth rate of calves from a large-scale IVF program using reverse-sorted semen in Bos indicus, Bos indicus-taurus, and Bos taurus cattle

Obtaining sexed sperm from previously frozen doses (reverse-sorted semen [RSS]) provides an important advantage because of the possibility of using the semen of bulls with desired genetic attributes that have died or have become infertile but from whom frozen semen is available. We report the efficiency of RSS on the pregnancy rate and birth rate of calves in a large-scale program using ovum pick-up and in vitro embryo production (IVEP) from Bos indicus, Bos indicus-taurus, and Bos taurus cattle. From 645 ovum pick-up procedures (Holstein, Gir, and Nelore), 9438 viable oocytes were recovered. A dose of frozen semen (Holstein, Nelore, Brahman, Gir, and Braford) was thawed, and the sperm were sex-sorted and cooled for use in IVF. Additionally, IVF with sperm from three Holstein bulls with freeze-thawed, sex-sorted (RSS) or sex-sorted, freeze-thawed (control) was tested. A total of 2729 embryos were produced, exhibiting a mean blastocyst rate of 29%. Heifers and cows selected for adequate body condition, estrus, and health received 2404 embryos, and 60 days later, a 41% average pregnancy rate was observed. A total of 966 calves were born, and 910 were of a predetermined sex, with an average of 94% accuracy in determining the sex. Despite the lower blastocyst rate with freeze-thawed, sex-sorted semen compared with sex-sorted semen, (P < 0.05), the pregnancy rate (bull I, 45% vs. 40%; II, 35% vs. 50%; and III, 47% vs. 48% for RSS and control, respectively; P > 0.05) and sex-sorted efficiency (bull I, 93% vs. 98%; II, 96% vs. 94%; and III, 96% vs. 97% for RSS and control, respectively; P > 0.05) were similar for each of the three bulls regardless of the sperm type used in the IVF. The sexing of previously frozen semen, associated with IVEP, produces viable embryos with a pregnancy rate of up to 40%, and calves of the desired sex are born even if the paternal bull has acquired some infertility, died, or is located a long distance from the sexing laboratory. Furthermore, these data show the feasibility of the process even when used in a large-scale IVEP program.     

Differential mRNA expression in in vivo produced pre-implantation embryos of dairy heifers and mature cows

Decreasing fertility with increasing parity is considered to be a major constraint in the reproductive management of dairy cows. Even though pregnancy rates (PR) in mature cows have declined drastically in the last 50 years, it has remained constant in heifers. Early embryonic loss is a major cause for the loss of pregnancy in cows. Expression of developmentally important genes is vital for the function and survival of embryos. Hence, in this study, we compared the mRNA abundance of GLUT5, INFτ, HSP70, Na/K-ATPase, BAX, and BCL2 genes in the pre-implantation embryos of dairy heifers and mature cows. Heifers (n = 25) and cows (n = 20) were superovulated and artificially inseminated on the day of estrus. On day 7, the embryos were flushed and morphologically graded and RT-PCR was performed. HSP70 was expressed more in the grade I embryos in heifers than in cows, and in the grade I embryos of heifers than in grade II embryos of heifers. In pooled embryos (both grades I and II) of heifers and cows, expression for INFτ was greater in heifers than in cows. Grade I embryos had a higher expression of GLUT5 and Na/K-ATPase than the grade II embryos of cows. From this study, we conclude that there is differential expression of some developmentally important genes between embryos of heifers versus cows and between grades I and II embryos regardless of the embryo source. Future research will be necessary to elucidate any potential cause and effect between these genes and reduced PR observed in dairy cows. Mol. Reprod. Dev. 76: 1165–1172, 2009. © 2009 Wiley-Liss, Inc.     

Batchwise assessment of porcine embryos for cryotolerance

The viability or developmental ability of porcine embryos after slow-freezing and thawing differs depending on the embryonic stage or the batch, which is defined as a group of embryos obtained from one donor at one time. We froze porcine blastocysts in batches and assessed their cryotolerance by using two expanded blastocysts (EBs) as samples to predict the developmental potential of other blastocysts from the same batch at different stages. Two EBs from the same batch that had been separately frozen were thawed and cultured in vitro for 48 h to examine their in vitro ability to develop to the hatched blastocyst stage. Thereafter, each batch was assigned to Grade A, B, or C according to the viability of the two EBs, i.e., 100% viability (2/2: number of hatched blastocysts/number of cultured EBs) was Grade A; 50% (1/2) was Grade B; and 0% (0/2) was Grade C. The viability of EBs after freeze-thawing and in vitro culture varied depending on the batch and was lower (31.0+/-10.2%, mean+/-S.E.M.; P<0.01) than that of unfrozen controls (96.8+/-2.3%). The viability of frozen-thawed hatched blastocysts (HBs) did not differ among the graded batches, but the blastocyst diameter decreased (from 409 to 326 microm) as the batch grade decreased (from A to C). When both EBs and HBs from batches of the same grade were transferred to recipients (average 11.7 EBs and 16.0 HBs per recipient), the rate of pregnancy and farrowing in recipients decreased (from 77.8% to 0%) and the number of piglets obtained decreased (from 15.3 to 0) as the batch grade decreased. However, when not only frozen-thawed EBs from Grade B or C batches, but also four helper embryos at the morula to early blastocyst stage (which were expected to support the pregnancy) were transferred, the number of piglets generated was higher from EBs from Grade B batches (16.0) than from EBs from Grade C batches (0.0). When frozen-thawed HBs and helper embryos were transferred, the number of piglets generated was higher from HBs from Grade B batches (12.7) than that from HBs from Grade C batches (1.9). After slow-freezing of porcine blastocysts, their rate of survival to the piglet stage differs batchwise, and in vitro viability assessment of sample EBs after freezing and thawing may help in assessing the post-freezing and post-thawing developmental potential of other blastocysts at different stages from the same batch.     

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.     

Occurrence of a non-cellular suprazonary layer in embryos collected from prepuberal calves

Twenty Angus calves between 4 and 7 months of age were randomly assigned to one of two superovulation treatment groups. Group I consisted of ten calves which were injected intramuscularly with 50 mg of progesterone 4 and 2 days before injection with 1200 I.U. of PMSG followed 72 hrs later by 50 mg of LH given intravenously. Group II consisted of ten calves which were not injected with progesterone before receiving PMSG and LH as in Group I. Both groups of calves were inseminated by the rectal fixation method with two straws of frozen semen 72 hrs after PMSG injection and at subsequent 12 hr intervals for a total of four inseminations. All semen was extended from a single ejaculate from one bull. Embryos were recovered by surgery or slaughter 48 to 72 hrs following the last insemination. A total of 80 and 70 ovulations were recorded from treatment Group I and II, respectively. Recovery and fertilization rates were 66 and 57% following progesterone treatment and 67 and 51% in calves without progesterone pretreatment. Seventy-seven percent ($\text{23}\text{30}$) of the fertilized embryos recovered in treatment Group I exhibited a suprazonary layer. This suprazonary layer appeared to be non-cellular on the basis of eosin-hematoxylin staining and ranged in thickness from 3 to 24 μm. All fertilized embryos in treatment Group II and unfertilized eggs in treatment Groups I and II, possessed completely smooth zonae pellucidae with no evidence of a suprazonary layer. These observations suggest that the conditions of the progesterone treated prepuberal tract, coupled with the process of sperm penetration of the oocyte, result in the formation of a non-cellular layer which surrounds the zona pellucida to varying degrees of thickness. The influence of this suprazonary layer on embryo viability in prepuberal calves remains to be determined.     

Effect of gas atmosphere on the development of one-cell bovine embryos in two culture systems

The effect of two concentrations of oxygen on the development of bovine embryos was compared using two separate co-culture systems. In Experiment I, bovine oocytes were matured and fertilized in vitro and were then co-cultured for 7 days in 20 mul drops of M199 with 10% fetal calf serum containing oviduct cells. When cultures were performed in an atmosphere of 5% CO(2) in air (20% O(2)) or in a mixture of 5% CO(2), 5% O(2) and 90% N(2) (5% O(2)), 22 of 179 (12%) and 56 of 179 (31%) zygotes developed to or beyond the late morula stage (P<0.0001), respectively. After freezing, thawing and 48 hours of additional culture, 2 of 21 (10%) and 18 of 53 (34%) embryos were judged viable (P<0.001) within the respective treatment groups. In Experiment II, zygotes produced by the same means were co-cultured in 0.5 ml of M199 containing 10% fetal calf serum with monolayers of buffalo rat liver (BRL) cells. In 20% O(2), 51 of 177 (29%) zygotes developed into viable embryos, while in 5% O(2) only 9 of 177 (5%) were judged viable after 7 days of culture (P<0.0001). Post-freezing survival rates were 53% and 67% for embryos from the two respective oxygen concentration treatment groups. The transfer of 20 Grade 1 frozen/thawed embryos produced by co-culture with BRL cells produced six pregnancies (30%). These experiments show that the critical effect of oxygen concentration on embryo development in vitro and the ability of embryos produced by in vitro procedures to survive freezing can be influenced by the type of culture system employed.     

Developmental capacity of bovine oocytes cryopreserved after maturation in vitro and of frozen-thawed bovine embryos derived from frozen mature oocytes

The present study was conducted 1) to investigate the post-thaw developmental capacity of in vitro mature bovine oocytes (Metaphase II) frozen by 1.6 M of 1,2-propanediol and 2) to confirm the viability of frozen bovine embryos derived from frozen mature oocytes. The cleavage and developmental rates to the blastocyst stage of frozen-thawed mature oocytes were significantly lower (P<0.01) than that of nonfrozen oocytes. When mature oocytes were treated with hyaluronidase, trypsin, or base solution (solution control) before processing to remove the cumulus cells, the developmental rates to the blastocyst stage of frozen-thawed oocytes were 2.8% (5 180 ), 3.1% (9 295 ) and 1.1% (1 89 ), respectively. The viability and developmental capacity of frozen-thawed bovine embryos derived from frozen mature oocytes were not different from those of frozen-thawed bovine embryos derived from nonfrozen mature oocytes (control). Furthermore, nonfrozen and frozen-thawed embryos derived from frozen-thawed mature oocytes were nonsurgically transferred to recipient cows. One of the four and one of the two recipient cows became pregnant, respectively. The results of this study demonstrated the viability of embryos obtained from frozen-thawed bovine oocytes at Metaphase II followed by in vitro fertilization and culture to the blastocyst stage in vitro.     

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 an asynchrony between ovulation and insemination on the results obtained after insemination with fresh or frozen sperm in rabbits

The effects of the introduction of an 8-h asynchrony between ovulation and insemination on litter size components from rabbits were assessed. A total of 202 females belonging to a maternal line were used. Fresh and frozen sperm were used to perform the inseminations. Sperm was frozen with an extender composed of 1.75 M DMSO and 0.05 M sucrose. Four experimental groups were obtained depending on the type of sperm used (fresh or frozen) and on the moment that ovulation had been induced relative to the insemination (at the same time as insemination (t(0)) or 8 h before insemination (t(8))). Laparoscopy was performed on 12th day of pregnancy in pregnant females, and the ovulation rate, normal and total implanted embryos were noted. At kindling, total and live-born rabbits were noted. Results showed that better results were obtained after insemination with fresh semen than with frozen sperm (for females in the group t(0): 79% versus 61% fertility rate, 10.2 versus 6.4 normal implanted embryos and 8.1 versus 5.2 total number born, for fresh and frozen sperm, respectively). On the other hand, after the introduction of an 8-h asynchrony between ovulation and insemination, results were lower for both fresh (50% fertility rate, 7.5 normal implanted embryos and 5.7 total number born for the group of the asynchrony) and frozen sperm (31% fertility rate, 4.6 normal implanted embryos and 3.4 total number born for the group of the asynchrony). Although an approach between the moment of insemination and ovulation is justified when sperm survival could be compromised, results observed after the induction of an 8-h asynchrony were not those expected, perhaps due to the ageing of the oocytes before being fertilised, leading to both lack of fertilisation or early embryonic mortality.     

Fast freezing of cow embryos in French straws with an automatic program

Cow embryos between day 6.5 and 9 were frozen in 1.5M DMSO in PBS at 2 degrees C/min from seeding to -25 degrees C before being plunged into liquid nitrogen directly or after 10 min at -25 degrees C. Cooling rate from 20 degrees C to -5 degrees C was 9 degrees C/min. Seeding was induced automatically at -5 degrees C by injection of liquid nitrogen vapour. Embryos were subsequently thawed by direct transfer to water at 20 degrees C (group I) or at 37 degrees C (group II). Survival was assessed by culture in vitro and by transfer. In group I, 35.7% were degenerated after thawing (compared to 35.4% in group II). Survival rate after culture in vitro for 24h was not significantly different (48.3% vs 42.8%) and hatching rate after 96h culture was quite similar (33.3% vs 34.4%). In group II, four pregnancies were obtained from 10 embryos transferred. Time at -25 degrees C did not improve the results. Automatic seeding did not impair survival. These results show that the quality of the embryo is the determinant factor for survival after freezing and that the plastic straw is the most suitable vessel for freezing, storage and transfer of embryos.     

Stage-dependent viability of vitrified rabbit embryos

The aim of the work was to determine the susceptibility of rabbit embryos to vitrification at different developmental stages. The experiment was carried out on 676 embryos at 1-, 2- and 8-to 16-cell stages as well as the morula and blastocyst stages. As a vitrification medium, a mixture of 30% 1,2-propanediol + 30% glycerol (Solution I), or 35% 1,2-propanediol + 35% glycerol (Solution II), was used. The embryos were frozen in glass ampules placed in nitrogen vapour for 5 min before being plunged into liquid nitrogen. Dilution after rapid thawing was done in one step in a 1-M sucrose solution. After vitrification in Solution I, none of the 1- or 2-cell embryos survived, whereas the survival rate of 8-to 16-cell embryos, morula and blastocysts, was 23.0, 82.7 and 78.5%, respectively. After vitrification in Solution II, the survival rate of 1-, 2- and 8-to 16-cell embryos was 20.0, 43.8 and 92.9%, respectively. The proportion of live offspring on the Day 28 after transfer of 68 vitrified morula was 26.5% compared with 24.0% in the control group. Thus, the proposed vitrification procedures can be useful in the cryopreservation of rabbit embryos.     

Pregnancy rates for grade 2 embryos following administration of synthetic GnRH at the time of transfer in embryo-recipient cattle

To succeed with pregnancy a bovine embryo must overcome the luteolytic mechanism and achieve recognition of pregnancy. It is understood that well developed embryos are more successful in achieving recognition of pregnancy than poorly developed ones. Attempts have been made to assist this recognition of pregnancy by utilising a number of hormonal supplements with varying levels of success. A study was undertaken to test the hypothesis that supplementation with synthetic GnRH at the time of transfer of Grade 2 embryos will enhance pregnancy rates in recipients receiving this category of embryo. Pairs of fresh and frozen Grade 2 embryos (n = 38) from 34 donor animals were allocated to the trial. Thirty eight pairs of recipients were used and one of each pair was randomly assigned to receive treatment on the day of embryo transfer (Day 7) with 5 ml of gonadorelin, containing a synthetic gonadotrophin releasing hormone, 0.1 mg/ml. Pregnancy diagnosis was carried out from 42 days post-transfer by either palpation per rectum or ultrasound scanning. Treatment, embryo processing, side of transfer, parity of recipient, breed of recipient and breed of donor dam showed no statistically significant effect on pregnancy rate. The overall pregnancy rate in this study was within commercially accepted limits for Grade 2 embryos at 38.2%. The pregnancy rates were 34.2 and 42.1% for the GnRH-treated and control groups, respectively and were not significantly different at P < 0.05. The failure of this treatment to improve pregnancy rates could be due to its effect being transitory therefore allowing subsequent pregnancy loss. The timing of the treatment post-transfer, treatment dose and potency of the GnRH analogue may also play a role in this. Further study is required to determine the hormonal or follicular status of prospective candidates for treatment before applying this as a whole herd regime.     

Increased NO bioavailability in aging male rats by genistein and exercise training: using 4, 5-diaminofluorescein diacetate

Background

Some studies have suggested that the suppression of endogenous LH secretion does not seem to affect the majority of patients who are undergoing assisted reproduction and stimulation with recombinant FSH (r-FSH). Other studies have indicated that a group of normogonadotrophic women down-regulated and stimulated with pure FSH preparations may experience low LH concentrations that compromise the IVF parameters. The present study aimed to compare the efficacy of recombinant LH (r-LH) supplementation for controlled ovarian stimulation in r-FSH and GnRH-agonist (GnRH-a) protocol in ICSI cycles.

Methods

A total of 244 patients without ovulatory dysfunction, aged <40 years and at the first ICSI cycle were divided into two groups matched by age according to an ovarian stimulation scheme: Group I (n = 122): Down-regulation with GnRH-a + r-FSH and Group II (n = 122): Down-regulation with GnRH-a + r-FSH and r-LH (beginning simultaneously).

Result(s)

The number of oocytes collected, the number of oocytes in metaphase II and fertilization rate were significantly lower in the Group I than in Group II (P = 0.036, P = 0.0014 and P = 0.017, respectively). In addition, the mean number of embryos produced per cycle and the mean number of frozen embryos per cycle were statistically lower (P = 0.0092 and P = 0.0008, respectively) in Group I than in Group II. Finally the cumulative implantation rate (fresh+thaw ed embryos) was significantly lower (P = 0.04) in Group I than in Group II. The other clinical and laboratory results analyzed did not show difference between groups.

Conclusion

These data support r-LH supplementation in ovarian stimulation protocols with r-FSH and GnRH-a for assisted reproduction treatment.