Sperm concentration at freezing affects post-thaw quality and fertility of ram semen

We have investigated the effect of sperm concentration in the freezing doses 200, 400, 800, and 1600 × 106 mL−1 on the post-thaw quality and fertility of ram semen. Semen was collected from seven adult Churra rams by artificial vagina during the breeding season. The semen was diluted in an extender (TES-Tris-fructose, 20% egg yolk, and 4% glycerol), to a final concentration of 200, 400, 800, or 1600 × 106 mL−1 and frozen. Doses were analyzed post-thawing for motility (computer-assisted sperm analysis system [CASA]), viability, and acrosomal status (fluorescence probes propidium iodide [PI]/peanut agglutinin conjugated with fluorescein thiocyanate (PNA-FITC), SYBR-14/PI [Invitrogen; Barcelona, Spain] and YO-PRO-1/PI [Invitrogen; Barcelona, Spain]). Total motility and velocity were lower for 1600 × 106 mL−1 doses, while progressive motility and viability were lower both for 800 and 1600 × 106 mL−1. The proportion of viable spermatozoa showing increased membrane permeability (YO-PRO-1+) rose in 800 and 1200 × 106 mL−1. Intrauterine inseminations were performed with the 200, 400, and 800 × 106 mL−1 doses at a fixed sperm number (25 × 106 per uterine horn) in synchronized ewes. Fertility (lambing rate) was similar for semen frozen at 200 (57.5%) or 400 × 106 mL−1 (54.4%), whereas it was significantly lower for 800 × 106 mL−1 (45.5%). In conclusion, increasing sperm concentration in cryopreserved semen, at least at 800 × 106 mL−1 and more, adversely affects the postthawing quality and fertility of ram semen.