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Storage of cryopreserved sperm in liquid nitrogen is believed to maintain semen fertility for decades, but recently, information on declining sperm characteristics after long term storage was reported. We investigated differences in frozen-thawed stallion semen stored in our laboratory between zero and 26 years. We hypothesized that although semen motility is not severely affected, differences regarding mRNA characteristics might exist among frozen horse semen batches stored for different time periods in liquid nitrogen. Ejaculates from 18 stallions collected and cryopreserved between 1998 and 2024 were included in the study. The semen was cryopreserved for commercial purposes from donor stallions (Warmblood, Thoroughbred and Lipizzaner), aged between 2 and 18 years at semen collection and meeting requirements for semen production according to European Union regulations. Directly after semen processing, frozen-thawed semen characteristics met minimal requirements defined for commercial use. Semen was stored in liquid nitrogen (-196°C) until assessment, where straws were thawed in a water bath (37°C) for 20 s. Frozen-thawed semen was assessed for sperm concentration (Nucleocounter), motility (CASA), membrane integrity (viadent staining), mitochondrial membrane potential (JC1) and chromatin condensation (aniline blue). Semen mRNA was extracted, and RNA quality analysed for contamination by solvents (260/230 ratio), by proteins (260/280 ratio), RNA integrity (DV200 Index), RNA yield (TapeStation), with three repetitions (straws) per sample. In 12 semen batches, the mRNA abundance of ten genes previously proven to be associated with fertility was determined by RT-qPCR. Correlation analysis of all endpoints with storage time of samples at analysis was done by Spearman-Rho test (IBM-SPSS statistics 29.0.1.0). Results were considered significant with P<0.05. There were no significant correlations between storage time and any of the sperm quality characteristics or any endpoints of mRNA quality. Significant correlations were determined for storage time of semen and mRNA abundance of the genes PRM1 (-0.338), PRM3 (-0.373), PRDX6 (-0.336) and CRISP3 (-0.507). Major modifications in sperm quality are induced by semen processing at cryopreservation, but the present results suggest no relevant further changes over a storage time of up to 26 years. Samples included into the study were continuously stored in our laboratory and storage conditions were closely monitored. Although the quality of semen RNA remained unaffected by long term storage, the present data suggest possible changes in mRNA abundance of some candidate genes relevant for fertility. The number of samples included into this analysis will be increased to further assure the quality of results. [ABSTRACT FROM AUTHOR]

The external genital tract of stallions is physiologically colonized by a variety of bacteria, most of which are considered non-pathogenic, with a small proportion classified as pathogenic or potentially pathogenic. When collecting stallion semen, proper hygiene is essential because bacterial contamination can affect semen quality and the reproductive health of mares. Currently, there is no consensus regarding the best practice for washing the stallion's penis before semen collection. The most common approaches are (1) no washing, (2) washing with warm water or a mild soap, and (3) washing with chlorhexidine soap before semen collection for export purposes. Our aim was to determine effects of washing the stallion's penis before semen collection on the bacterial load of the genital tract and semen and on semen characteristics. Five stallions were included and randomly assigned to one of three treatments: control (NW), washing with warm water (WW), and washing with chlorhexidine soap and warm water (WC). Treatments lasted for two consecutive weeks, with three semen collections per week. After each treatment period, stallions underwent a two-week rest interval before switching to the next treatment in a crossover design. On the first day of each treatment period, semen and swabs from the urethral fossa were collected for bacteriological analysis without prior cleaning procedure. Sampling was repeated after the two week-washing protocols. Samples were serially diluted up to 1 × 10-8 and plated on Columbia Agar with 5% sheep blood and Schaedler Agar with vitamin K 1% and 5% sheep blood. Colony forming units (CFU) were counted after 72 hours of aerobic and anaerobic incubation at 37°C. Native semen was analyzed for pH, sperm concentration, motility and membrane integrity (CASA), and morphology. Semen was subsequently diluted with Equiplus extender (Minitube) without antibiotics and an aliquot of 2 ml was stored at 5°C for 72 hours and analyzed every 24 hours for motility and membrane integrity. For statistical analysis, CFU and sperm characteristics were compared among treatments by a general linear model for repeated measures. Total bacterial load decreased over time (P<0.01) and an effect of treatment (P< 0.05) and a treatment x time interaction was observed (P<0.05). The number of CFU/ml after treatment was already decreased in WW and even more in WC compared to NW (CFU/ml for aerobic and anaerobic culture, respectively, WW: 5.2 × 105 and 5.8 × 105, WC: 3.0 × 104 and 6-8 × 104, NW: 4.5 × 106 and 6.2 × 106). Total motility and percentage of membrane-intact sperm decreased over time (P<0.001), but there was no treatment effect. In conclusion, washing the stallion's penis with warm water or chlorhexidine soap before semen collection reduces the total bacterial count in semen without compromising semen characteristics. [ABSTRACT FROM AUTHOR]

Insemination of mares with cryopreserved semen requires specialized equipment for semen storage and thawing as well as intensive mare management. This often limits its use under field conditions. Thawing frozen stallion semen on demand at a laboratory and sending it to the insemination site in a cooled state would reduce these efforts. In the present study, we investigated the suitability of different commercial semen extenders for storage of frozen-thawed stallion semen. For this purpose, frozen semen from five Warmblood stallions (three repetitions per stallion) was thawed at 38°C for 20 s and thereafter split in four aliquots that were assigned to one of the following treatments: i) dilution in EquiPlus (Minitube, Tiefenbach, Germany), storage at 5°C; ii) dilution in SperVital White Cap (Spervital, Toldijk, Netherlands), storage at 5°C; iii) dilution in Beyond (Minitube), storage at 5°C; iv) dilution in Beyond (Minitube), storage at 17°C. Semen was always diluted to a final concentration of 50 × 106 sperm/ml and stored for 72h. Total motility, progressive motility and membrane integrity of sperm were determined by CASA (IVOS II, IMV, L'Aigle, France), at 24 h intervals for 72h. Data were analysed (SPSS 28.0, IBM-SPSS, Armonk, NY, USA) by two-way repeated measures ANOVA with storage time (0h, 24h, 48h, 72h), and semen treatment as within subject factors and Duncan test for post hoc comparisons; significance was set a P<0.05. All values given are means ± standard error of the mean (SEM). The semen characteristics total motility, progressive motility, and membrane integrity decreased over time (P< 0.001) with clear effects of the storage conditions (time x group P<0.001). Mean total motility, progressive motility and membrane integrity was better maintained (P<0.05) in extenders EquiPlus and SperVital than in Beyond, irrespective of the storage temperature (5 vs. 17°C). Immediately after thawing, total motility was 28±2% (EquiPlus), 27±2% (SperVital), 26±2% (Beyond, 5°C) and 26±2% (Beyond, 17°C), corresponding values after 48h of storage were 22±2%, 23±2%, 13±1% and 18±1%. In conclusion, the present study confirms that frozen stallion semen maintains motility and membrane integrity if stored for prolonged time after thawing. The results provide further information that storage conditions (extender composition and temperature) considerably influence these semen characteristics. Further research into sperm metabolism may be a key to improve storage conditions for frozen-thawed semen to allow for its successful shipment and use in AI programs. [ABSTRACT FROM AUTHOR]