journal

In vitro sperm penetration through the zona
pellucida of immature and in vitro matured oocytes
using fresh, chilled and frozen canine semen

Monica De los Reyes a,∗, Jaime Palomino a, Johanna de Lange a, Carla Anguita a, Claudio Barros ba Laboratory of Animal Reproduction, Faculty of Veterinary Sciences, University of Chile, Casilla 2 Correo 15, Santiago, Chile b Laboratory of Embriology, Faculty of Biological Sciences, P. Catholic University of Chile, Santiago, Chile Received 28 June 2007; received in revised form 5 December 2007; accepted 12 December 2007
Available online 28 December 2007

Abstract

The aim of this study was to evaluate the effect of sperm cryopreservation and the maturation state of the oocyte on the time course of canine gamete interaction during co-culture for periods of 1–10 h. Semen samples were obtained by digital stimulation and ejaculates processed as fresh, chilled and frozen samples. Sperm were co-cultured with immature or in vitro mature bitch oocytes for up to 10 h. At hourly intervals, oocytes were evaluated for sperm penetration with epifluorescence microscopy. The results were analyzed statistically using generalized linear models. Spermatozoa treatments had a significant effect on the total percentage of oocyte penetration for both types of oocytes; fresh spermatozoa showed the highest average penetration rate, while frozen sperm showed the lowest value (p < 0.05). At the 1st hour of co-culture, chilled and frozen dog sperm had a higher penetration percentage (p < 0.05) of in vitro matured canine oocytes (43.6% and 45.7%, respectively) than the fresh sperm had (33.8%). Sperm penetration was directly proportional to the time of incubation, when fresh or chilled sperm were used (P < 0.05); in contrast, frozen dog sperm did not change penetration rates with either immature or in vitro matured oocytes over time. Therewas a significant difference in the average of penetration rate between immature (47.3%) and in vitro matured oocytes (56.6%) throughout the 10 h of culturing; irrespective of sperm treatment. The optimal incubation time in terms of maximizing penetration rates probably are dependent on how spermatozoa were processed prior to fertilization.

© 2007 Elsevier B.V. All rights reserved.
Keywords: Capacitation; Cryopreservation; Dog sperm; Gamete interaction

∗ Corresponding author. Tel.: +56 2 9785534; fax: +56 2 9785611.
E-mail address: mdlreyes@uchile.cl (M. De los Reyes).
0378-4320/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.anireprosci.2007.12.010