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Avoidance of fracture damage to the zona pellucida during freezing and thawing is essential for the successful freezing of rabbit embryos. The aim of this study was to examine the effects of encapsulation of rabbit embryos in calcium alginate gel, prior to freezing, on damage to the non-cellular components (zona and mucin coat) caused by freezing and thawing, and on the survival of embryos after thawing in vivo as well as in vitro. Morulae immersed in 2% sodium alginate in Ringer solution were aspirated into a Pasteur pipette. The contents of the pipette were then ejected into a 110 mM solution of calcium chloride. After 30 min, 1-to 2-mm long semi-solid cylinders, each including one embryo, were cut off with a surgical blade. In the presence of 11% dimethyl sulfoxide, the encapsulated embryos were cooled by the standard method and stored in liquid nitrogen. After rapid thawing, the alginate gel was mechanically removed from around the embryos, and the embryos were morphologically evaluated and allocated to different groups for examination of their development in vitro or in vivo. The percentage of embryos with an intact zona was higher in the encapsulated group than in the non-encapsulated group (95.3% vs. 76.5%, P < 0.001). The encapsulation markedly reduced the occurrence of damage to the mucin coat (from 43.1% to 8.5%, P < 0.001). The frozen and thawed embryos that were released from alginate gel developed normally in vitro (86.3%) and in vivo (54.2%). The results suggest that encapsulation of rabbit embryos in alginate gel prior to freezing improves the percentage of transferable embryos after thawing and increases the number of offspring that originate from frozen and thawed embryos.

Abstract. The aims of the present study were twofold. The first aim was to examine whether or not early pregnancy-associated thrombocytopenia (EPAT) occurred in cows following nonsurgical embryo transfer by observing the daily patterns of the concentration of peripheral platelets (PLT) during the preimplantation period of pregnancy. The other goal was to assess the use of PLT count as a diagnosis of early pregnancy in cows. In Experiment 1, out of 50 Holstein parous cows, 40 had one embryo nonsurgically transferred to each uterine horn on Day 6 or 7 (Day 0=onset of standing estrus). The remaining 10 cows served as controls having no embryos transferred. Blood was taken daily at the same time, from Day 6 or 7 following estrus (the day when the embryos were transferred in the non-controls) to the day of return to estrus for control cows and non-pregnant cows, or to Day 36 for pregnant cows. Bilateral embryo transfers resulted in 9 twin, 14 single, and 17 nonpregnancies. Some recipient cows showed an increase in PLT following embryo transfer, while others displayed a decrease in PLT. If significant PLT change was characterized either by a decrease or an increase of >20% compared to the value of pre-transfer, the proportions of cows indicating significant change were 78% (7/9) in twin, 64% (9/14) in single, 76% (13/17) in nonpregnancies and 30% (3/10) in controls. The proportions in twin (P<0.05), single (P<0.05) and nonpregnancies (P<0.01) were higher than that in controls. The effect of the particular group on PLT was significant (P<0.0025). In Experiment 2, each of several in vitro-produced blastocysts were nosurgically transferred to 9 Japanese Black cows on Day 7 or 8 following onset of estrus. Seven days after embryo transfer, an attempt to recover the previously transferred embryos by nonsurgical flushing was performed. Embryos could be recovered on Day 14 or 15 from 5 animals, whose blood was taken from the day of embryo transfer to the next day of embryo recovery and during the corresponding days of the following estrous cycle. For PLT, differences were detected between the both phases of 3 out of the 5 cows. The individual PLT variation in response means that EPAT does not necessarily occur in cows following embryo transfer, and that the use of PLT counts is unlikely to be clinically useful in assessing early pregnancy in cows. These results may suggest, however, that maternal platelet activation would occur during early pregnancy in cattle.