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.

The presence of a microbiome/microbiota in the placenta is hotly debated. In previous studies, the presence of bacteria in equine amniotic fluid and umbilical blood was independent of foal health. The objective of the present study was to determine if the same bacteria are present in the equine placenta as in amniotic fluid and umbilical blood. Samples were obtained from 24 parturient mares and foals. Placental bacterial DNA was extracted, and the microbiome was identified using 16S rRNA sequencing. All amniotic fluid samples contained some polymorphonucleocytes; bacteria were isolated from four samples. Aerobic or anaerobic growth was found in 18 and 3 umbilical blood samples, respectively. Serum amyloid A was <5 mg/L in all 24 samples, total WBC varied between 2900 and 10,700/µL, and fibrinogen varied between 0 and 5.16 g/L. In jugular blood, serum amyloid A was <5 mg/L in all 24 foals, total white blood count was 3200 to 8100/µL, and fibrinogen was 0.44 to 4.42 g/L. The diversity of bacterial microbiota was similar in all placental regions at the phylum level but differed at the genus level; the most abundant phyla were Proteobacteria (42–46.26%) and Actinobacteria (26.91–29.96%). In conclusion, bacteria were found in the fetal compartments and placenta of healthy equine pregnancies; however, we can neither prove nor disprove the hypothesis that the placenta has its own microbiome.

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 case of an 8-year-old, sexually active but infertile Przewalski’s stallion (Equus ferus przewalskii) was studied. Besides the infertility, the stallion also showed permanent problems with its body condition, being obviously weaker than all the other group members. The horse was kept in a separate place for two years with 12 mares in its harem group (six mares had foals earlier); however, none of the mares covered got pregnant. Andrological and cytogenetic investigations revealed underdeveloped testes, arrested spermatogenesis, azoospermia, and XY/XXY/X0 mosaicism. We classify the case as a mosaic Klinefelter syndrome, the first reported case in Przewalski’s horse. The case of an 8-year-old, sexually active but infertile Przewalski’s stallion (Equus ferus przewalskii) was studied. Besides the infertility, the stallion also showed permanent problems with its body condition, being obviously weaker than all the other group members. The horse was kept in a separate place for two years with 12 mares in its harem group (six mares had foals earlier); however, none of the mares covered got pregnant. Andrological and cytogenetic investigations revealed underdeveloped testes, arrested spermatogenesis, azoospermia, and XY/XXY/X0 mosaicism. We classify the case as a mosaic Klinefelter syndrome, the first reported case in Przewalski’s horse.