Ar Mahmoudzadeh

Compaction rate of in vitro fertilized bovine embryos related to the interval from insemination to first cleavage

A study was conducted on early cleavage divisions and timing of compaction in bovine preimplantation-stage embryos. Zygotes were produced using conventional in vitro maturation and fertilization procedures. Twenty hours post insemination, the zygotes were denuded and cultured with oviduct epithelial cells in B2 medium + 10% estrous cow serum. Starting at 24 hours post insemination, the embryos (n=657) were evaluated every 6 hours and then were put into different co-culture drops according to their cell number. Starting from 78 hours post insemination, the cleavage rate was evaluated every 12 hours. Embryos were stained with Hoechst 33342 at the compacted morula stage or when they were degenerated, at 162 hours post insemination. Developmentally capable embryos were characterized by a rapid cleavage rate in the first 3 cell cycles and by an extended 8- to 16-cell stage. Peak concentrations of 2-, 4-, 8- and 16-cell stages emerged at 36, 42, 60 and 102 hours post insemination, respectively. Compaction did not occur until 126 hours post insemination. The rate of compaction was significantly higher in embryos that were at the 2-cell stage before or at 36 hours post insemination (P < 0.05). The mean cell numbers of compacted morulae that were identified at 126 and 138 hours post insemination were 30.9 +/- 6.8 and 31.6 +/- 7.7, respectively. These results indicate that developmentally capable bovine embryos reach the 2-cell stage at 36 hours post insemination, and that they become compacted at the 32-cell stage, which usually occurs between 126 and 138 hours post insemination.

Birth of double-muscled Belgian Blue calves after transfer of in vitro produced embryos into dairy cattle

The possible application of the bovine in vitro fertilization technique for economical beef production was evaluated by transferring in vitro produced Belgian Blue embryos to synchronized dairy cows and heifers. In total, 4167 oocytes, collected in the slaughterhouse from double-muscled Belgian Blue cows, were matured in vitro. Frozen-thawed semen from 3 Belgian Blue bulls was used for in vitro fertilization. Zygotes were cultured in B(2) + 10% estrous cow serum together with oviductal cells at 39 degrees C in 5% CO(2) in air. After 7 days, 576 (13.8%) transferable embryos were obtained. One hundred and eighteen of the most advanced embryos were selected for fresh transfer into 90 recipients. Some of the remaining embryos were frozen using conventional methods. After fresh transfer, 50 recipients (55.6%) had elevated progesterone at day 23. Thirty cows (33.3%) calved after a mean gestation length of 282.8+/-6.0 days and produced 25 single births and 5 twins. The sex ratio was 71.4%. The mean birth weight was 45.1+/-8.3 kg. Three calves were of the conventional type instead of double-muscled and 2 calves died of congenital malformations. After transfer of in vitro produced frozen-thawed Belgian Blue embryos into 27 recipients (1 embryo/recipient), 2 bull calves (7.4%) were born. Bovine embryo production by in vitro techniques could form a low-cost supply of beef calves. However, to render it commercially attractive, selection of sires and dams has to be performed with great care.

A comparative study of the effect of one-step addition of different vitrification solutions on in vitro survival of vitrified bovine embryos

Three experiments were conducted to study the effect of vitrifying in vivo-produced bovine embryos using one-step addition of different vitrification solutions. In Experiment 1, 23 compact morulae to early blastocyst stage embryos were vitrified using a solution consisting of DMSO (2.6 M), acetamide (2.6 M), propylene glycol (1.3 M), and polyethylene glycol (7.5 mM). Only 1 embryo expanded after a 30-second exposure period. In Experiment 2, 11 compact morulae to early blastocysts were exposed for 2 minutes to a vitrification solution containing glycerol (3.4 M) and propylene glycol (3.4 M). None of the embryos survived after vitrification and post-thaw in vitro culture. Dilution of the cryoprotectants in experiments 1 and 2 were carried out in 1 M sucrose for 10 minutes. In Experiment 3, 20 compact morulae-early blastocysts were vitrified after 3 minutes of exposure to a vitrification solution consisting of 7.15 M ethylene glycol, 2.5 mM ficoll and 0.3 M sucrose prepared in embryo transfer freezing medium. As recommended for mouse and rabbit embryos, the cryoprotectant in Experiment 3 was diluted in 0.5 M sucrose. Fifteen compact morulae-early blastocysts expanded or hatched after 48 hours post-thaw in the in vitro culture. It is concluded that ethylene glycol is less toxic following one-step addition of vitrification solution to in vivo-produced bovine compact morulae-early blastocysts than the other vitrification solutions tested. A low concentration of sucrose for dilution of ethylene glycol was also found to reduce the chance of possible osmotic injuries due to dehydration.

Comparison of two-step vitrification versus controlled freezing on survival of in vitro produced cattle embryos

Abstract Grade 1 in vitro produced bovine day 7 embryos at the compact morula-early blastocyst, blastocyst and expanded blastocyst stage were selected for cryopreservation. From 7 replicates, 572 embryos were randomly divided into two groups. One group was cryopreserved by controlled freezing after 20 minutes equilibration in 10% v/v glycerol and slow cooling (0.3 °C / minute) from −7 °C to −30 °C. The other group was vitrified after 3 minutes exposure to 20% v/v ethylene glycol and 30 to 45 seconds exposure to a vitrification solution consisting of 40% v/v ethylene glycol, 18% w/v ficoll and 10.26% w/v sucrose. Embryos from both groups were thawed in a water bath at 20±1 °C. Frozen-thawed embryos were diluted in three successive solutions consisting of 10.26% w/v sucrose and 6.6%, 3.3% and 0% glycerol, respectively, during 5 minutes for each step. Vitrified-warmed embryos were diluted in a 8.5% w/v sucrose solution during 5 minutes. All diluted embryos were cultured in Menezo-B2 medium supplemented with bovine oviduct epithelial cells. The survival rate of all developmental stages of embryos was significantly higher after vitrification than after controlled freezing (P

Salvage of oocytes from sterile genetically valuable cows, resulting in the birth of a calf

The objective of this study was to determine the possible application of in vitro fertilization techniques for calf production in incurable sterility patients or in involuntary cull cows. Ten sterile or fatally injured dams of high genetic value were submitted to in vitro fertilization procedures. Ovaries were recovered after slaughter in eight animals or after ovariectomy in two cows. A total of 261 oocytes were obtained and matured in vitro. Frozen-thawed sperm from eight different bulls of high breeding value (selected to match with one particular cow) was separated with Percoll and used for in vitro fertilization in the presence of heparin. Seventy four embryos started to cleave and 7 days post-insemination, 13 transferable embryos were obtained. Eleven embryos were transferred fresh into nine recipients and produced two pregnancies. One pregnant recipient died of hydro-allantois, the other delivered a live bull calf 288 days after in vitro fertilization.