Fertilization and Early Embryogenesis
(M349) ASSESSMENT OF PRONUCLEAR FORMATION FOLLOWING IVF WITH BOVINE SPERMATOZOA HAVING ABNORMAL MORPHOLOGY AFTER THERMAL INSULATION OF THE TESTIS.
Walters, Anneke 1, Saacke, Richard1, Pearson, Ronald1, Gwazdauskas, Francis1, 1 Virginia Polytechnic Institute and State University, Blacksburg, VA
ABSTRACT- Fertilization encompasses a series of stages which need to be performed in a well-orchestrated manner. This study was conducted to analyze the chronology of pronuclear formation in bovine zygotes after in vitro insemination with a population of spermatozoa having abnormal morphology. Semen samples were obtained and cryopreserved from four Holstein bulls before and after a scrotal insulation period of 48 h (d 0) and a pre– (d −5) and a d 20 post–insult semen sample were used. Pronuclear formation was scored using a DIC microscope evaluation on subpopulations of presumptive zygotes after they were fixed and stained with methanol:acetic acid mixture at 3 h time intervals from 6 to 18 h post insemination (hpi). Post–thaw morphological evaluation of semen samples revealed a decrease (P < 0.01) in the percentages of normal spermatozoa in the postinsult samples compared with the pre–insult samples for Bulls I and Bull III (74 to 22.3% and 67.7 to 0.5 %, respectively). Pronuclear formation was significantly (P < 0.01) affected by the three–way interaction between bull, semen sample collection day, and hpi. The pronuclear formation rates for post–insult zygotes for Bull II and Bull IV had comparable increases in development over time (1.1 ± 0.19 to 5.1 ± 0.23 and 1.2 ± 0.23 to 4.9 ± 0.18, respectively), while there was no increase (P < 0.01) in the pronuclear development for the zygotes from the post–insult samples for Bulls I and III and they remained predominantly at PN1 stage (1.5 ± 0.17; 1.8 ± 0.22, respectively). In conclusion, our results suggested that the failure in normal pronuclear formation was associated with an absence of normal decondensation of the penetrating spermatozoon and provides compelling evidence that the effect of morphologically abnormal spermatozoa occurred prior to cleavage, thus limiting early embryonic development.
KEY WORDS: pronuclear formation, abnormal spermatozoa, scrotal insulation, zygote