Congenitally caused fused labia in the common marmoset (Callithrix jacchus).

In this paper, the occurrence of an external genital abnormality in female marmoset monkeys (fused labia) is discussed. This malformation was detected, for the first time, in a group of animals at the German Primate Center (GPC), Goettingen. The malformed vulva was completely sealed except for an opening of 1.5-2.5 mm around the urethra sufficient for urination. Because of this defect the animals were not able to copulate. As a consequence, the affected females were functionally infertile although they had a normal genital tract and a regular cycle. This vulvar abnormality was found in 12 females, offspring of 10 pairs in which either one or both came to the German Primate Center from two genetically related colonies in Munich, Germany, and one colony in Basel, Switzerland. The abnormality appeared to be recessive and inheritable from either parent. In pairs in which both animals were from one of the mentioned colonies, 45% of the female offspring were affected. In pairs where only one partner came from these colonies, 26% of female offspring had the malformation. These results indicate that avoidance of inbreeding, which is frequently performed in primate colonies, may reduce, but not eliminate the expression of abnormalities of genetic origin. Therefore selective breeding is required, and, in colonies where these recessive mutations are widespread, the development of genetic screening tests would be advantageous.

Ultra-rapid freezing and storage of rat embryos in an electric refrigerator at -130 degree C without liquid cryo-agents, with ultra-short exposure in the freezing medium and direct rehydration after thawing.

An ultra-rapid freezing technique for embryos has been developed. This procedure involves: ultra-short exposure to cryoprotectants; freezing of embryos in a metallic powder pre-cooled in an electric refrigerator at -130 degree C; storage of embryos ina refrigerator at -130 degree C; direct rehydration after thawing.

Vitrification of mouse germinal vesicle oocytes: effect of treatment temperature and egg yolk on chromatin and spindle normality and cumulus integrity.

The success rates for cryopreservation of immature oocytes from several species including human remain low, in contrast to major improvements with mature oocytes. In this study, a new approach has been developed using a short exposure ultra-rapid freezing protocol, examining the effect of temperature and egg yolk (two factors which may be expected to influence membrane flexibility) on the cryostability of immature mouse oocytes and cumulus complexes. These two factors were tested in various patterns for their cryoprotective effect using ethylene glycol as the principal cryoprotectant. The results showed that 37 degrees C pre- and post-freeze exposure significantly improved both survival and normal spindle configuration after in-vitro maturation. Egg yolk was found to produce further beneficial effects on both the oocyte and cumulus cell integrity, with the best effects being obtained at 37 degrees C with inclusion of egg yolk both before and after the freezing. This protocol produced > 80% normal survival post-thaw with intact and attached cumulus complex, 84% maturation rate and 45% normal metaphase configuration. In summary, a unique combination of high survival and meiotic normality together with good preservation of the attached cumulus cell mass has been achieved using a simple new vitrification procedure.

Ultrarapid freezing of rat embryos with rapid dilution of permeable cryoprotectants.

The purpose of this study was to determine the feasibility of ultrarapid freezing of rat morulae with rapid postthaw dilution of permeable cryoprotectants in isotonic culture medium. Four experiments were carried out. Experiment 1 examined the possibility of using vitrification with postthaw dilution of permeable cryoprotectants in an isotonic solution. Embryos were exposed first to 10% glycerol + 20% propylene glycol and then to the final vitrification solution which contained 25% glycerol + 25% propylene glycol. Embryo survival was very low when the subsequent dilution was in a solution that did not contain sucrose. In Experiment 2. three mixtures were tested: 15% glycerol + 15% ethylene glycol + 0.7 M sucrose, 15% glycerol + 15% propylene glycol + 0.7 M sucrose, and 30% glycerol + 0.7 M sucrose. The third mixture, which contained only glycerol and sucrose, produced the best results with 88% embryo survival. In Experiment 3, the embryos were frozen in 30% glycerol plus 0.7 M sucrose and in addition were exposed to 1 M sucrose for 7 min following thawing. The survival rate was 85% with the sucrose dilution step, 91% when dilution was in isotonic medium, and 95% in controls not exposed to the cryoprotective mixture. Experiment 4 examined the effect of the time and temperature of exposure of the embryos to 30% glycerol + 0.7 M sucrose. The highest rates of embryo development followed exposure at 4 degrees C for 2-3 min (95-84%) or at 24 degrees C for 0.5-3.0 min (90-88%). These results indicate that it is possible to develop a method for the ultrarapid freezing of mammalian embryos that does not require dilution of permeable cryoprotectants in a hypertonic sucrose solution.

Improvement of attachment rate of bovine embryos after transfer with trophoblastic fragments.

Fragments of trophoblastic vesicles have been shown to improve embryo development when used in co-culture. We wished to investigate the effect of trophoblastic fragments on establishment of pregnancy after embryo transfer. Embryos (n=129) frozen by direct immersion in liquid nitrogen (Group 1), 82 embryos frozen by the slow freezing method (Group 2) and 55 fresh embryos stored at 20-22 degrees C for 21-22 h (Group 3) were transferred into recipient heifers. The embryos of each group were divided in 2 batches. Batch 1 embryos (control) were transferred without trophoblastic fragments, Batch 2 embryos of each group (experimental) were transferred in combination with frozen-thawed trophoblastic fragments at a rate of 5 to 7 fragments per embryo. The fragments were produced from 16-17 day bovine embryos frozen by vitrification. Two months following the embryo transfers it was established that the attachament rate for Batch 1 embryos in Groups 1, 2, 3 was 33%, 39% and 48%, respectively; for Batch 2 embryos in the same Groups the rate was 55%, 56% and 74%, respectively. We conclude that trophoblastic fragments promote embryo attachment, but their effect does not continue beyond the time of attachment.

Survival of trophoblastic fragments and vesicles after vitrification, ultrarapid freezing, and storage at 4 degrees C.

Embryos recovered from superovulated cows on Day 13 or Day 17 of their sexual cycles were cut into fragments to make trophoblastic vesicles. The fragments were suspended in a cryoprotectant solution consisting of glycerol and sucrose and frozen by direct immersion into liquid nitrogen: alternatively, they were vitrified using the Massip method in a mixture of glycerol and 1,2-propanediol. Trophoblasts and fragments of a chilled embryo were placed into a temperature-controlled humidified chamber for culture. Vesicles which developed from trophoblastic fragments were subjected to vitrification according to the method of Massip. We observed good survival of trophoblastic fragments which had been subjected to chilling, freezing, or vitrification. Their survival did not differ from the survival of "fresh" trophoblastic fragments.