Effect of exogenous lactoferrin and phosphoglycerate mutase 2 on the cryopreservation of wild ruminant epididymal/ejaculated sperm and testicular tissue

Spermatozoa collected from the cauda epididymis of wild ruminants are more cryoresistant than are ejaculated spermatozoa. This work examines the effects of lactoferrin (LF) and phosphoglycerate mutase 2 (PGAM2), which are abundant in the epididymal sperm of wild ruminants, as additives in Iberian ibex and mouflon sperm extenders. In addition, LF was added to a vitrification medium to determine whether it also provided protection during the cryopreservation of testicular tissue. Epididymal sperm samples and testes were recovered post-mortem from ibexes (n=13) and mouflons (n=8) during the breeding season. Ejaculates were collected from both species (n=10 each) using the transrectal ultrasound-guided massage technique. Four aliquots were taken from each sample, diluted in the appropriate freezing extender for each species, and subjected to the following treatments: control, LF (100 μg/ml), PGAM2 (25 μg/ml), and LF+PGAM2. Testicular tissue was cut into small pieces and cryopreserved by needle-immersed vitrification in a medium with or without LF. In vivo fertilization capacity was assessed using frozen-thawed ejaculated sperm from the ibexes. Supplementation of extenders with LF or PGAM2, and their combination, had no beneficial or harmful effect on any sperm variable after freezing-thawing. Artificial insemination of ibexes showed that the fertility rate in controls was 62.5%, but this fell to 20% in females inseminated with sperm treated with LF (p = 0.06), suggesting a putative negative effect of LF on fertility. The viability of elongated spermatids and spermatozoa from mouflon testes was greater (p < 0.05) in samples that were vitrified-warmed with LF. Thus, the hypothesis that supplementing ejaculated sperm with LF and/or PGAM2 improves sperm quality after freeze-thawing was not upheld. However, LF would seem an appropriate additive when vitrifying mouflon testicular tissue.