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.
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