Effect of factors affecting the embryo production of superovulated Holstein cows were evaluated. Data were obtained from 455 superovulation-collections using 84 donors. For all of the hormonal treatment, FSH and PGF_2α were used. Season (every 12 month), year (1988, 1989, 1990), age at calving (3, 4, 5, 6, ≥7 years old), time after calving (1st, 2nd, ≥3rd year), interval of treatment (1st treatment, 21-90, 91-150, ≥151 days) and interaction (season×year, age×time) were evaluated by analysis of variance for number of total ova/embryos (NT), number of fertilized ova (NF), number of normal embryos (NN) fertilization rate (%F) and normal rate (%N), and time after calving within each flush were evaluated by another model. The effects of season on NT, NF and NN, age at calving on NT, NF, %F and %N, and time after calving on NF, NN, %F and %N were significant (P<0.05), while effects of year, interval of treatment, season×year and age×time were not. Seasonal effects suggested the influence of heat. Effects of age at calving indicated a lag of peaks between NT (3 years) and %N (5 years), and no difference was observed among 3-6 years in NN. Influence of time after calving without the effect of repeated treatment was observed, and embryo production during the 3rd year or over was worse as compared with the 1st and 2nd year.

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In this study, some characteristics of cryoprotectant, polyalcohol used for vitrification such as toxicity, glass-forming tendency and stability of the amorphous state were estimated and the relationship of those characteristics with cryoprotection for vitrified rat morula was discussed. Glycerol was not harmful for rat morulae exposed for 5 minutes at room temperature to the examined concentration up to 50%. Both of 50% 1, 2-propandiol and 50% 1, 3-butandiol showed detrimental effect on the embryonic survival after exposure for 1 minute. However, combination of 25% propandiol or butandiol with 25% glycerol sustained the survival of embryos at 40-60% even after exposure for 5 minutes. Out of the three cryoprotective solutions, a vitrification solution (VS) of propandiol was easiest to transform into amorphous condition during cooling process. Devitrification (initial) temperature of VS was -92, -77 and -77C for 50% of propandiol, glycerol and butandiol, respectively. Seventy five percent of rat morulae in the solution containing more than 40% glycerol survived when directly plunged into liquid nitrogen, but none of the embryos survived in 50% propandiol or 50% butandiol. Both cryoprotectant, 25% propandiol or 25% butandiol combined with 25% glycerol had a comparable effect with 40-50% glycerol. In 5 percent percoll or 5% PVP supplemented to VS, the survival rate of embryos was improved.

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Two cryopreservation methods adapted to field use were investigated with bovine blastocysts produced by in vitro technique. In Method I, embryos were equilibrated with 1.36M glycerol and 0.25M trehalose for 10-15 min in modified phosphate buffered saline (mPBS) supplemented with 20% cow serum. Embryos were loaded into 0.25-ml plastic straws (2 embryos/straw) in a small volume of freezing medium separated by 2 air bubbles from the medium (0.25M trehalose with 20% cow serum in mPBS) filling the rest of the straw. The straws were placed directly into a cooling chamber kept at -7C and then seeded followed by holding for 10 min at this temperature. The straws were then cooled to -25C at 0.45C/min before being plunged into liquid nitrogen.
In Method II, embryos were equilibrated in a three-step procedure with 0.5M 1, 2-propanediol (PD) for 5 min, 1.0M PD for 5 min and 1.6M PD for 10-15 min in mPBS supplemented with 20% cow serum and loaded into straws separated by 2 air bubbles from the medium (0.2M sucrose with 20% cow serum in mPBS) filling the rest of the straw. The straws were placed directly into a cooling chamber kept at 0C and then cooled to -6C at 1C/min. After leaving for 10 min at this temperature, including seeding, the straws were then cooled to -30C at 0.3C/min. The straws were then left for 10 min at this temperature before being plunged into liquid nitrogen.
The straws containing embryos were thawed at 30C and the embryos were directly transferred into cow uterus. Pregnancy rates of transferred embryos cryopreserved by Methods I and II were 12.5% (3/24) and 37.8% (17/45), respectively. In Methods I, one out of 3 heifers has delivered a calf. The remaining 2 heifers have been maintaining pregnancy. In Method II, 4 (including one set of twins) out of 16 heifers lost their pregnancy between 2 and 4 months of gestation. One heifer (carrying twins) died due to disease and the another one died due to bad weather within 1 month before the expected days of delivery. Thirteen calves (including 2 sets of twins) were born from the remaining 11 heifers.

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The embryo production in cattle by superovulation treatment with the highly purified porcine FSH preparation containing reduced LH activity (FSH-R, Denka Pharm.) and with the commercially available crude porcine FSH preparation (FSH-P, Denka Pharm.) were compared. Twenty-four Holstein cows were treated with 36AU of FSH-P or FSH-R and PGF_2α in decreasing method. At 6, 7 or 8 days after estrus and insemination, all cows were slaughtered and their ovaries were observed visually (12, FSH-P group; 12, FSH-R group), and 21 cows were flushed and ova/embryos recovered (11, FSH-P group; 10, FSH-R group).
On the results, the mean number (+SD) of corpora lutea and total ova/embryos were not different between FSH-P group and FSH-R group (14.2±10.6 vs 14.7±7.4, 9.1±8.2 vs 10.7±6.8, respectively), however, the fertilized ratio and normal ratio were different significantly (86.0% vs 94.4%, P<0.05; 72.0% vs 82.2%, P<0.05, respectively). And the percentages of cows with 3 or more corpora lutea and normal embryos were both 100% in FSH-R group and 66.7% (P<0.05) and 45.5% (P<0.01) in FSH-P group. The difference was found in the number of follicles and the percentage of cows with unusual follicles between FSH-R group and FSH-P group (10.3±6.5 vs 5.5±3.6, P<0.05; 33.3% vs 0%, P<0.05, respectively).
These results suggested that FSH-R treatment was effective as compared with FSH-P treatment for embryo production in daily cows.

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Non-129 embryonic stem cells (F1/1) were transformed with the neomycin phosphot-ranscerase (neo) gene by electroporation and used as a vehicle for transgenesis. There was no difference between square wave and exponential decay in transformation efficiency of F1/1 cells by selection for G418 resistance. Southern blot hybridization analysis has confirmed that 4 out of 6 neomycin resistant ES cell lines have neo gene sequences in their genomic DNA. The karyotype of these cells appeared stable, as they showed euploidy after transformation. Overt coat color chimaeras were obtained from all 4 neo resistant ES cell lines tested.

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[in Japanese]

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