268 IN VITRO EVALUATION OF FRESH SPERM QUALITY IN TOMCATS: A COMPARISON OF COLLECTION TECHNIQUES

Abstract

Semen can be collected from tomcats by urethral catheterization (CT) after medetomidine administration, offering a novel approach to obtain sperm for in vitro fertilization. This study was designed to determine motion characteristics of CT sperm samples by means of computer-assisted sperm analysis (CASA) and to compare sperm quality parameters and in vitro fertilizing capacity of CT spermatozoa with those of spermatozoa retrieved after epididymal slicing (EP). Semen was collected in 17 adult cats by urethral catheterization as reported by Zambelli et al. (2008 Theriogenology 69, 485-490), after which the cat was orchidectomized. Motility (subjective and objective by means of CASA), morphology [eosin/nigrosin (E/N)], plasma membrane integrity (E/N and SYBR14-PI staining), and acrosomal status (FITC-PSA staining) of fresh CT and EP samples were evaluated and compared between both methods with a paired t-test or the Wilcoxon Rank test, dependent on normality of the data. In vitro maturation (24 h), fertilization (18 h), and culture (6h) of grade I to III oocytes were carried out as described by Pope et al. (2009 Theriogenology 71, 864-871). Twenty-four hours after in vitro insemination, fertilization rates were assessed for group 1 (CT; n = 148) and group 2 (EP; n = 159) presumptive zygotes. The distribution of presumptive zygotes between CT and EP over the different developmental stages was compared using Pearson chi-square test. Results showed that total and progressive motility as well as the percentage of normal spermatozoa were higher for EP sperm compared with CT sperm (P < 0.01). Epididymal sperm had a lower percentage of spermatozoa with an intact acrosome (P < 0.01), whereas CT sperm contained more spermatozoa with tail abnormalities (P < 0.01). Other sperm parameters did not differ (P > 0.05) between collection techniques. In group 1, 84% of in vitro-matured oocytes (metaphase II) were penetrated (40.2% cleaved, 24.4% with 2 pronuclei, 12.2% with >2 pronuclei, 7.3% with expanded sperm head), whereas in group 2, penetration rate was 88.5% (42.7% cleaved, 21.8% with 2 pronuclei, 16.7% with >2 pronuclei, 7.3% with expanded sperm head). No difference (P > 0.05) in in vitro fertilizing capacity between spermatozoa collected by means of the 2 methods was found. In conclusion, semen collection by means of CT yields fertilization results similar to epididymal slicing, despite the fact that several sperm variables were different. Because CT is repeatable and easy to perform and does not require a trained male/queen in heat, it may be preferable for routine IVF experiments with fresh spermatozoa.

The first author is a research fellow of the Fund for Scientific Research-Flanders (Belgium).