60 HISTOLOGICAL COMPARISONS BETWEEN NUCLEAR TRANSFER AND IN VIVO PORCINE EMBRYOS

Abstract

Due to the high rate of embryonic loss during a nuclear transfer pregnancy, cloning is considered a relatively inefficient process. However, as the only method of producing knockout domestic animals it is considered an invaluable tool for the biotechnical industry. By histologically comparing embryos at significant stages in the porcine pregnancy (Days 10, 12, and 14), factors contributing to embryonic loss may be revealed. Many consider the period between days 10 and 14 to be critical for determining survivability as the embryos must undergo rapid changes to signal for maternal recognition of pregnancy as well as adapt to a changing environment. This study included three gilts per stage of pregnancy and four different experimental groups for each stage studied: nonpregnant animals, in vivo-pregnant animals, nuclear transfer (NT) recipients, and in vitro-manipulated recipients (IVM). IVM embryos were in vitro-produced embryos upon which a mock nuclear transfer has been performed in an effort to account for the variability introduced by the actual technique. Animals either were bred or underwent a surgical embryo transfer on Day 1 of the estrous cycle according to their assigned experimental group. Fifty embryos were transferred per embryo transfer. Embryos were flushed from the uterine horns at time of collection (Day 10, 12, or 14) and preserved in 10% neutral buffered formalin. All embryonic disc diameters and gross morphology were evaluated as parameters for normal development. Embryos were then dehydrated in ethanol, paraffin-imbedded, sectioned, stained with hemotoxylin and eosin, and Day 14 embryos were evaluated for abnormalities such as higher-than-normal nucleoli numbers, increased cytoplasmic vacuoles, and higher than normal numbers of mitotic figures. All results were analyzed using ANOVA. There were significant differences (P < .0001) between diameters of the embryonic disks, with the diameters of the NT embryonic disks being smaller than those of the in vivo controls at all stages studied. Morphologically, the in vivo controls were more developmentally competent than their NT counterparts by the time they reached Day 14 (P = 0.0002) in that most had achieved the more advanced elongated form of growth as opposed to remaining spherical in shape. Significant histological differences in the number of nucleoli per nuclei were also found between in vivo and NT embryos (P = 0.05) as well as between MNT and NT embryos (P = 0.05). Therefore, nuclear transfer embryos develop at a much slower rate than their in vivo counterparts and often exhibit histological abnormalities that could contribute to this slow growth. Due to the apparent increase in nucleoli, it is possible that NT embryos are being arrested at a specific stage in the cell cycle.

The authors would like to acknowledge the Research Animal Diagnostic Laboratory for their help in imbedding, sectioning, and staining the embryos; Dr. Duane Keisler for running the hormone assays; and Kristin Whitworth, Melissa Samuel, Aaron Bonk, Jin-Geol Kim, Emily Fergason, David Wax, Tom Cantley, August Rieke, Randy Farrell, and Lacey Griesbaum for all their help.