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.

This work was aimed to identify androgen receptors (AR) in the spermatozoa of wild and domestic ruminants and to assess the effect of testosterone on sperm localization of aquaporin-3 (AQP3) and cryopreservation process. Sperm samples from wild species were incubated with testosterone (T group), 1,3-propanediol (PDO group), phloretin (PHL group), PDO+T group, PHL+T group. Western blot identified the presence of AR as a single band of about 48 KDa. Immunolabelling of AR was located in the equatorial segment of the sperm head. In mouflons, the cryoresistance ratio for acrosome integrity was lower (p < 0.05) in the PHL+T than in Control and T groups. In ibexes, the cryoresistance ratio for acrosome integrity was lower (p < 0.05) in the PHL+T, PHL, and T group than in the Control group; the cryoresistance ratios for sperm kinematic variables were lower (p < 0.05) in PDO+T than in Control. No changes were found among treatments in the proportion of spermatozoa showing AQP3 in the different membrane domains after incubation and thawing in both mouflon and ibex. In conclusion, testosterone negatively affected sperm cryoresistance expressed as acrosome integrity, enhancing the effects of the AQP blocker PHL. Our findings provide a sound knowledge of the molecular mechanisms that explain the seasonal variation in sperm freezability from ruminants.

This study aimed to evaluate daily variations in sperm quality, testosterone levels, cryoresistance, and the relocation of AQP3 in plasma membrane domains in wild and domestic goats. Samples were collected from five Murciano-Granadina bucks and seven Iberian ibex at 12:00 h (noon) and 00:00 h (midnight), during the rutting season. Sperm motility, viability, acrosome integrity, DNA fragmentation, and immunolocalization of AQP3 were assessed in fresh and frozen-thawed semen samples. Serum testosterone concentrations were also determined at both times. In domestic goats, kinematic parameters were greater (p < 0.05) in semen collected at midnight, whereas the cryoresistance ratio was greater (p < 0.05) for these variables in semen recovered at noon. In ibexes, fresh semen collected at midnight was greater (p = 0.05) for VCL, VSL, VAP, and ALH values. After freezing-thawing, a significant increase in AQP3 labelling at the acrosomal region was observed in samples collected at midnight for both species, as well as in samples collected at noon for bucks (p < 0.05). An increase in the proportion of spermatozoa with AQP3 labelling in the mid-piece was detected in bucks in frozen-thawed samples collected at noon (p < 0.05). Testosterone concentrations were higher (p < 0.05) at midnight in bucks but no differences were found in ibexes. The findings reveal daily variations in sperm quality in both domestic and wild goats, which do not exactly coincide with the daily changes in cryoresistance, thus calling for attention when processing semen.

Background: Immunocontraception is a population management strategy based on vaccines that induce an immune response against reproductive targets such as GnRH. Objectives: This study aimed to investigate spermatogenic activity in adult baboons and adult dwarf bucks subject to repeated immunisation against GnRH for extended periods. Methods: In Experiment 1, histological findings in untreated dwarf bucks (CONTROL‐1) ( n =  10) were compared with those found in males repeatedly immunised against GnRH (Improvac) during 5 years (RI‐1; n =  15). In Experiment 2, one group of baboons received immunocontraceptive treatment against GnRH during 5–10 years until death (RI‑2; n =  6). The second group received the same treatment for 10 years before discontinuation, with the interval between treatment withdrawal and death ranging from 10 to 27 months (RI‑2+S; n =  19). As in Experiment 1, testicular tissue was collected post‐mortem and processed histologically. Results: In both species, the testicular tissue of animals treated with repeated immunisation against GnRH showed an absence of active spermatogenesis. The mean seminiferous tubule diameter in bucks was smaller in the RI‐1 group than in the CONTROL‐1 group (108.69 ± 13.17 µm vs. 187.35 ± 14.38 µm; p  < 0.05). In baboons, morphometric analysis showed similar seminiferous tubule diameters in the RI‐2 (82.63 ± 4.47 µm) and RI‐2+S (79.23 ± 5.89 µm) groups. Conclusions: In bucks and in more than 70% of baboons, the severity of degenerative changes in the germinal epithelium suggests that long‐term repeated immunisation against GnRH may lead to irreversible infertility.

The present study compares two protocols for the cryopreservation of chicken semen. Both protocols had an initial low cooling rate in the first step, followed by higher cooling rates around ice nucleation (Protocol 1) or following the dissipation of the latent heat of fusion (Protocol 2) in the second step. Semen ejaculates obtained from 12 roosters were diluted with Rootex with 6% dimethylformamide and frozen following either Protocol 1 (from +5°C to −10°C at 5°C/min and from −10°C to −130°C at 60°C/min) or Protocol 2 (from +5°C to −35°C at 7°C/min and from −35°C to −140°C at 60°C/min). Compared with fresh semen, following both protocols, cryopreservation resulted in reduced post‐thaw sperm quality (p < .001). Post‐thaw percentage of sperm with an intact plasma membrane was greater using Protocol 2 than Protocol 1 (p < .05). The results suggest that high cooling rates around the time of ice nucleation are not recommendable.