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Studies revealed a global loss of genetic resources for local sheep breeds. Therefore, the current study aimed to introduce and highlight the progress made on Hungary’s existing gene conservation program (small Gene Bank). Furthermore, we evaluated breed (Tsigai, Cikta, and Racka), season, and individual variabilities (n = 24) of the pre-freeze and post-thaw semen stored in the Gene Bank to enhance the gene conservation of the breeds. The samples were cryopreserved manually, and post-thaw spermatozoa were analyzed for motility (CASA), viability, chromatin structure, and morphometry of the sperm nuclei. Ejaculate volume, spermatozoa concentration, subjective motility and standard motility, kinematic parameters, and spermatozoa’s head area standard deviation of the post-thaw samples differed significantly among breeds (p < 0.05). Season affected ejaculate volume, total spermatozoa number/ejaculate, STR, BCF, and ALH. We observed a significant (p < 0.001; 0.05) breed and season interaction on concentration, total spermatozoa number/ejaculate, VCL, LIN, WOB, spermatozoa’s head average perimeter and nucleus length (Tsigai and Cikta differed but were statistically the same as Racka). Similarly, season significantly (p < 0.05) affected the proportion of ejaculate suitable for freezing. There was a significant (p < 0.05) difference in kinematic parameters and viability among the rams across the breeds. The spermatozoa’s head morphometry of the Tsigai and Cikta breeds differed significantly (p < 0.05) among the rams. There were individual and breed differences in many spermatozoa quality parameters. The stored samples are of good quality, with more than 40% having intact membranes and low abnormal chromatin condensation.

There are limited studies on the factors affecting the success of ram epididymal spermatozoa (REPS) cryopreservation. On this note, the current study assessed the influence of three commercial soy lecithin-based semen extenders, AndroMed® (AND), BioXcell® (BIO), and OviXcell® (OVI), and two concentrations (400 × 106 vs. 200 × 106 spermatozoa/mL) on the pre-freeze and post-thaw quality of REPS. The REPS were retrieved from nine adult rams’ testes and diluted with each of the three extenders to both concentrations. Straws were frozen manually. Standard motility (SMP) and kinematic parameters (KPs) were assessed via a CASA, while spermatozoa viability, morphology, and acrosomal integrity were assessed via the Kovács–Foote staining technique. The concentration did not significantly affect the pre-freeze and post-thaw SMP and KPs of REPS. BIO and OVI had significantly higher pre-freeze and post-thaw BCFs, post-thaw VAP, and the percentage of all intact heads than AND. In contrast, AND had a significantly lower percentage of REPS with tail defects than BIO and OVI. The 400 × 106 spermatozoa/mL concentration resulted in a significantly higher percentage of all intact heads than the 200 × 106 spermatozoa/mL concentration. Freezing significantly increased tail defects and decreased the percentage of REPS with distal cytoplasmic droplets. The cryopreservation of REPS at the 400 × 106 spermatozoa/mL concentration is recommended. All three extenders must be optimized to preserve the viability, membrane integrity, and better normal morphology of REPS; the reason for increased tail abnormality after the freezing/thawing process needs to be studied.

The semen of domestic mammals is conventionally collected with an artificial vagina (AV) for artificial insemination (AI) or for short- or long-term storage. However, the procedure has certain drawbacks: animal training is not feasible in extensive animal care systems nor among wild species, as the trained animals sometimes fail to mount. Hence, there is a need for alternative semen collection methods. Electroejaculation (EEJ) and epididymal sperm recovery (ESR) are the two effective alternatives to AV. However, in recent years, animal welfare campaigners have called for the ban, in certain EU countries, of EEJ due to its inhumane nature. In this review, alternative methods of sperm collection (by EEJ and ESR, their qualities, and their freezing techniques) are highlighted, as well as the effects of EEJ on pre-freeze and post-thaw ram sperm quality parameters and the animal welfare progress made in EEJ between the 20th and 21st centuries. Additionally, the techniques for enhancing post-thaw sperm quality prior to freezing and for the freezing of EEJ and ESR spermatozoa are explored. ESR and EEJ are reliable alternatives to AV on certain occasions. EEJ is ideal for semen collection in wild or untrained animals, breeding soundness examinations, collection outside of the breeding season, and culling. At the same time, ESR is ideal in cases of castration, accidental death of elite sire, or postmortem for gene conservation purposes or assisted reproductive technologies (ARTs) studies.