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.

Heat stress negatively affects bovine fertility and embryonic development, while cryopreservation imposes additional cellular stress that may impair embryo survival. The present study investigated whether controlled heat treatment applied during the early morula stage could influence subsequent blastocyst development, molecular stress responses, and post-thaw survival of bovine embryos. In vitro-produced bovine embryos were exposed to controlled thermal treatment involving gradual elevation in temperature from 38.8 °C to 40.0 °C followed by subsequent cooling. Blastocyst formation, post-thaw survival after slow freezing, and the expression of stress-associated genes and microRNAs were evaluated. Heat treatment did not significantly affect blastocyst formation rates or post-thaw embryo survival compared to the control groups. However, significant alterations in the expression of stress-associated markers were observed. The expression of HSP90AA1 and HSPB11 was significantly reduced following heat treatment, while cryopreservation induced increased expression of these markers compared to embryos exposed only to heat treatment. Significant alterations were also detected in the expression of several stress-associated microRNAs. These findings suggest that controlled heat exposure during the morula stage may induce molecular changes associated with the embryonic response to heat stress without compromising developmental competence or cryosurvival. The results contribute to a better understanding of the molecular mechanisms underlying embryonic responses to thermal and cryogenic stress and may support future optimization of embryo production and cryopreservation strategies.