The role of different extenders in pre-centrifugation for optimizing equine semen cryopreservation

Cryopreservation can cause injuries to sperm, thus the inclusion of extenders is crucial in maintaining viability outside the reproductive tract. This study evaluated the effectiveness of 4 extenders: Skim Milk (G1), Skim Milk + Pentoxifylline (G2), Skim Milk + Cholesterol (G3), and Cholesterol + Casein (G4) in maintaining the kinetics and integrity of the cytoplasmic membrane of equine spermatozoa. Semen was collected from 4 stallions with a total of 28 ejaculates. Each ejaculate was divided into 4 aliquots, the samples were cryopreserved and thawed (Papa et al. 2008; Animal Reprod Sci 107:293-301). The evaluation of sperm kinetics, including total motility (TM), progressive motility (PM), percentage of sperm with rapid movement (RAP), and average path velocity (VAP), was performed using CASA (HTM-IVOS 12 Hamilton Thorne Research, Beverly, MA, USA). The integrity of the cytoplasmic membrane (IM) was evaluated using the technique of Harrison and Vickers (Reproduction. 1990; 88:342-352). Statistical analysis was performed using the Kolmogorov-Smirnov test, ANOVA, and Tukey, with P<0.05. After cryopreservation and thawing, the groups G3 (78.1 ± 1.5) and G4 (78.9 ± 1.3) showed higher total motility compared to G2 (73.9 ± 1.7). Progressive motility, percentage of rapid sperm, and plasma membrane integrity were higher in groups G3 (44.7 ± 2.3 and 64 ± 2.8) and G4 (45.1 ± 2.3 and 65 ± 2.5) compared to groups G1 (40.8 ± 2.6 and 60.1 ± 3.2) and G2 (41.6 ± 2.6 and 61.2 ± 2.8). The presence of cholesterol in the extenders G3 and G4 was essential for the preservation of membrane fluidity and stability. Cholesterol helps prevent membrane damage during cryopreservation, while the sodium caseinate present in G4 also contributes to the protection of sperm cells. On the other hand, the extender containing pentoxifylline (G2), which functions by inhibiting phosphodiesterase enzymatic activity, leading to an increase in intracellular cAMP, showed worse performance post-thawing, possibly due to rapid depletion of the energy substrate, generating an increase in ATP production, which can lead to cell death. The findings of this study indicate that extenders with the inclusion of cholesterol (G3 and G4) are more effective in cryopreservation of equine semen than extenders based solely on skim milk. It is known that kinetic parameters of total motility and membrane integrity have proven to be crucial components in fertility prognosis, as these characteristics are interrelated (Foster et al. Theriogenology. 2011; 75:1499-1505). Therefore, the incorporation of cholesterol into the sperm membrane is an effective strategy to increase the viability of cryopreserved semen, particularly in stallions with low resistance to thermal shock, ensuring better results in equine reproduction programs.