Comparison of sperm parameters and fertility between sperm cryopreserved from ejaculated semen and from the epididymis of subfertile stallions

Abstract

Despite the benefits provided by seminal plasma during natural cover, its function is questionable when a biotechnology such artificial insemination is used. Studies employing spermatozoa from the cauda epididymis with or without the addition of seminal plasma showed no difference in conception rates. Furthermore, studies have reported deleterious effects of secretions from accessory glands on sperm viability and conception rate (Aurich et al., Theriogenology, 1996; 46, 791-797), especially in stallions with poor semen quality. Thus the aims of this study were: 1) compare the sperm kinetic parameters, viability and fertility of sperm recovered from the ejaculate (G1) and from the cauda epididymis (G2) of subfertile stallions (pregnancy rate<35%). For G1, one ejaculate from each of eight subfertile stallions was collected and then subjected to cryopreservation using the extender BotuCrioTM. One week after the last semen collection, the stallions underwent bilateral orchiectomy. Sperm from the cauda epididymis were harvested immediately after castration by retrograde flushing the caudal portion (G2) of the epididymis using a skim milk-based extender (BotuSemenTM). The recovered sperm were then cryopreserved using BotuCrioTM extender. The sperm samples were analyzed for kinetic parameters by computer-assisted analyses (CASAHTM-IVOS) and plasma membrane integrity (Propidium iodide, Sigma; Hoechst 33345, Sigma), acrosome integrity (FITC-PSA, Sigma; Hoechst 33345, Sigma) and degree of translocation of phosphatidylserine (Annexin-V–APC, Pharmingen; Hoechst 33345, Sigma) by flow cytometry (BD-LSRFortessa ). For analysis, straws were thawed in a 46 C water bath for 20 sec. Artificial insemination was performed in the tip of the uterine horn ipsilateral to