Influence of centrifugation or low extension rates prefreezing on the fertility of ram semen after cervical insemination.

We compared the fertility of thawed ram semen, frozen according to different prefreezing semen handling protocols and previously well-defined in vitro, after cervical artificial insemination (AI) during natural estrus in Corriedale sheep. Following primary extension 1 + 1, we adjusted the final sperm concentration before packaging (200 x 10(6)/straw) either by centrifugation, in order to reconcentrate the extended semen (Protocol 1: P1), or without centrifugation, by adjusting the final sperm number by stepwise extension (Protocol 2: P2). We evaluated sperm motility (assessed both subjectively and with a computer-assisted sperm analysis instrument [CASA]), membrane integrity (SYBR-14/PI), and capacitation status (chlortetracycline [CTC]) in vitro in three pooled straws of frozen-thawed semen. Three hundred Corriedale ewes, having shown spontaneous estrus during the breeding season (i.e., April, in the southern hemisphere) under extensive management conditions in Uruguay, were cervically inseminated with thawed semen from the same freezing operations as studied in vitro. The semen evaluation in vitro yielded higher percentages (P < 0.05) of damaged spermatozoa in the samples where sperm numbers were adjusted by extension before freezing (P2), compared with when adjustment was done by centrifugation (P1). However, due to the higher sperm concentration finally achieved by P2, the calculated total number of viable spermatozoa was almost equal in the two AI doses. We observed no differences in fertility between P1 and P2 for either nonreturn rates (NRRs) 21 (30.8 vs. 29.7%) and 36 (28.5 vs. 27.8%) days after AI or lambing rate (21.9 vs. 21.4%), respectively. Fertility did not differ significantly between the two different procedures of adjusting sperm numbers prior to freezing. This may indicate that the simplified protocol with adjusted extension of the semen, resulting in higher numbers of viable spermatozoa, should be the procedure of choice when freezing ram semen under field conditions. Further studies aimed at improving the modified protocol need to be performed.