Vitrification of Canine Embryos at Various Developmental Stages.

Abstract

The cryopreservation of the genetic resource materials such as oocytes and embryos can be useful for improved breeding of companion and working dog, including guide dog for the blind. Vitrification has been widely developed to apply to cryopreservation of mammalian embryos. In the mouse, vitrification of the embryos was succeeded by DAP213 method, moreover, it was available to vitrify the canine ovarian tissues. While, porcine oocytes were vitrified successfully using the E30S method. However, the suitability of both vitrification methods for canine embryos has not been investigated. To improve the breeding management programs in guide dogs for the blind, the objective of the present study was to compare DAP213 with E30S method for vitlification of the embryos at 1-cell to blastocyst stages. Female Labrador Retrievers were inseminated on day 4-6 after LH surge. On day 10-15 after LH surge (day 4-10 after insemination), embryos were collected from excised oviducts and uteri of bitches by flushing, and were vitrified DAP213 or E30S method. In the DAP213 method, embryos were transferred into a cryotube containing 1 M DMSO. Subsequently, DAP213 solution, composed of 2 M DMSO, 1 M acetamide and 3 M propylene glycol, was added to each cryotube. After placing the cryotube in ice water for 5min, they were plunged directly into liquid nitrogen. In the E30S method, the embryos were exposed to PB1 containing 5, 10 and 20% ethylene glycol (EG), and 30% EG supplemented with 0.5 M sucrose for 5, 2, 2 and 1 min, respectively, at room temperature (23 ± 2 °C). They were then placed on a cryotop sheet (Kitazato Supplies, Japan), and the cryotop was immediately plunged into liquid nitrogen. After thawing, embryos were stained with propidium iodide for assessment of plasma membrane integrity of blastomeres. The blastomeres with disrupted plasma membrane were dyed red with PI. The E30S method showed fewer morphological abnormalities of vitrified embryos than the DAP213 method, and a few abnormalities of vitrified embryos in which integral blastomeres accounted for more than 75% at 1-cell to morula stages. However, blastomeres were mostly damaged at blastocyst stage embryos in the E30S group. While, almost blastomeres of vitrified embryos in the DAP213 group had disrupted plasma membrane regardless of developmental stage. These results suggest that the E30S method of vitrification is suitable for cryopreservation of canine embryos, and embryos at earlier developmental stage have a high tolerance to cryopreservation comparing with them at blastocyst stage. Although further study seems to be required to develop an optimal cryopreservation method for canine blastocysts and it is necessary to produce the pups derived from vitrified embryos, our findings may contribute to the efficient production of guide dogs for the blind.