Submission Number: YAS-4-25-24

Abstract Number: 372

BLASTOCYST DEVELOPMENT OF IN VITRO PRODUCED MORULA-STAGE BOVINE EMBRYOS BRIEFLY EXPOSED TO ULTRAVIOLET LIGHT FOLLOWING HOECHST STAINING.

Yassin M Elhassan, X Zhang, Duane C Kramer* and Mark E Westhusin*

Department of Veterinary Physiology and Pharmacology, Texas A & M University, College Station, TX ¹

Abstract:
In vitro exposure of oocytes or embryos to Hoechst (HQ) stain and Ultraviolet (UV) light for visualizing of nuclear materials for different purposes in nuclear transfer and other embryo micromanipulation work is common. Ultraviolet (UV) light is known to inflict harmful effects on mammalian cells including DNA damage, apoptosis and cancer. In the present study we investigated the effect of exposing in vitro produced bovine morula-stage embryos to HQ, UV or both on subsequent development. Following routine in vitro maturation and fertilization of slaughterhouse oocytes, the presumptive zygotes were cultured to the morula stage in a defined culture medium (KSOM) supplemented with PVA (0.1%), MEM-E (0.5X) and NE (1x) amino acids, Na lactate (10 mM), Na pyruvate (0.2 mM) and glucose (0.2mM). Embryos were then randomly assigned to one of 4 treatments and cultured in drops under oil at 38.5 C and humidified atmosphere of 5% O₂, 5%CO₂ and 90% N₂. Treatments included the exposure of the morula-stage embryos to HQ staining (5 ug/ml; Hoechst 33342, Sigma) for 20 min, HQ staining followed by 15 sec UV light or 15 sec UV light. An additional group of untreated embryos served as a control. Following the exposure treatment, embryos in different treatment groups were transferred to fresh culture medium and allowed to develop to the blastocyst stage. The experiment was repeated 4 times using a total number of 72 morulae, and the data were analyzed by Chi Square (P<0.05). The results showed that the exposure of in vitro produced morula-stage bovine embryos to either HQ staining or UV light had no (P>0.05) detrimental effect on percent development into blastoctysts compared with the control (83, 78 and 88%, respectively). However, exposure of the morula-stage embryos to HQ staining followed by 15 sec UV light resulted in significantly (P<0.05) lower percentages of blastocyst development compared with the control (11 and 89%, respectively). We conclude that Hoechst staining coupled with brief UV light exposure, a common practice in micromanipulation of mammalian embryos, is harmful to in vitro developing bovine embryos at the morula stage, and possibly other earlier stages including metaphase II oocytes.

Keywords: UV light, blastocyst, bovine

This abstract is being presented at: 8:00 AM in session:
Preimplantation Embryo/Fetus