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Morphological and Quantitative Aspects of the Development of Swine Embryos in Vitro^{1 ,2,}

Washington State University³, Pullman 99164

Abstract

Sixteen Yorkshire (Y) x Hampshire (H) gilts and two Y x H sows were used in this study. The gilts were hand mated at their first or second estrus, and the sows at their first estrus following weaning. Reproductive tracts were collected by slaughter 48 to 144 hr after the onset of estrus. One-hundred and seventy-two ova were recovered from 228 ovulations by flushing oviducts and uteri for a mean of (228/18) 12.6 ovulations per animal and a recovery rate of (172/228) 75.4%. Fertilization rate of recovered ova was (150/172) 87.2%. Embryos were placed singly in 50 µl (A drops of Whitten's culture medium supplemented with 1.5% (w/v) bovine serum albumin and incubated at 37 C under liquid paraffin oil in a humidified atmosphere of 90% N₂, 5% C0₂, 5% 0₂. Ova were classified as to stage of development, namely, unfertilized ova, one to two cells, three to four cells, five to eight cells, nine to 16 cells, morulae, blastocysts, and expanded blastocysts. Overall embryo diameter (OD) and zona pellucida thickness (ZPT) were measured at 100 x magnification with an ocular micrometer at 0 hr (in vivo measurement) and at subsequent 12-hr intervals (in vitro measurements) until the cessation of culture. Overall embryo diameters and ZPT varied from 150.3 to 157.1 µm and 13.5 to 18.0 µm, respectively, for unfertilized ova and embryos ranging from the one-cell to blastocyst stage of development. Little difference was observed in OD and ZPT among or between in vivo and in vitro cultured embryos prior to blastocyst expansion. Measurements of overall diameter and ZPT on a limited number of both in vivo and in vitro cultured expanded blastocysts suggests that their physical dimensions may be different. Thirty-five of the 150 embryos cultured in vitro developed into expanded blastocysts. Fifteen of these embryos initiated a hatching process in vitro with three embryos completely escaping from the zona pellucida. Two of the three hatched embryos were cultured from the four-cell stage. Observation of the in vitro hatching blastocyst suggests that the fracture of the zona pellucida is small with the embryo greatly changing shape in its movement out of the surrounding zona pellucida. This process is unlike that observed for blastocyst escape in the cow or sheep and is similar to that reported for the rabbit blastocyst.

¹ Scientific Paper No. 4851. College of Agriculture Research Center, Washington State University, Pullman Project 0313.

² The authors wish to thank the Washington State Swine Breeders Association for providing gifts and feed used in this study, Dr. E. L. Martin and Mr. Duncan Dunn for contributions to this experiment.

³ Department of Animal Sciences.