Characterization of sperm traits of donkey semen cryopreserved with different permeable cryoprotectants

Recent research has highlighted the potential advantages of dimethylformamide (DMF) over methylformamide (MF) as cryoprotectants for donkey semen (Bruno.et.al. JEVS 2024;136:105069). Higher concentrations of cryoprotectants could negatively affect mare fertility, underscoring the need for optimized protocols to balance cryoprotection with reproductive outcomes. The objective of this study was to assess (Experiment 1) the effects of different permeable cryoprotectants (glycerol [GLY], etilenoglycol [ETI], MF, and DMF) and (Experiment 2) to investigate the impact of reduced concentrations of cryoprotectants (5%, 3%, and 2%) on sperm traits of cryopreserved donkey semen. Semen was extended to 100 million sperm/mL with a sodium caseinate medium (BotuGoldⓇ), centrifuged (600xg/10min) and resuspended (100 million sperm/mL) in one of the tested extenders for cryopreservation (Oliveira.et.al., Theriogenology,2016;85:12-67-73). In experiment 1, four ejaculates of five Pêga donkeys (n=20) were cryopreserved using the BotuCrioⓇ-based medium (10% of egg yolk) added with 5% of one of the following cryoprotectants: GLY, ETI, MF, or DMF; and BotuCrioⓇ (BC, 1% GLY and 4% MF) was used as a Control. In experiment 2, five ejaculates of six Pêga donkeys (n=30) were cryopreserved using BotuCrioⓇ-based medium containing 5%, 3%, or 2% of the BC cryoprotectant combination (1:4; GLY:MF). Frozen-thawed semen (37°C/30s) was assessed by CASA for total motility (TM), progressive motility (PM), rapid sperm percentage (RAP), and by fluorescence microscopy for plasma membrane integrity (PMI). Data were assessed using mixed model/Bonferroni for parametric data, and Kruskal-Wallis/Dunns for non-parametric data with P<0.05. In experiment 1, BC and DMF had the highest TM (P<0.05) whereas ETI presented the lowest TM but similar to GLY. MF had intermediate (P>0.05) values of TM only similar to GLY (BC: 59.75±12.49; DMF:60.55±12.75, MF: 50.10±14.47; GLY:45.75±16.11; EG:39.40±17.24). PM was higher in BC compared to GLY and ETI (P<0.05), whereas MF and DMF had intermediate PM results similar to all groups (BC: 39.70±13.12; MF: 33.85±11.88; DMF: 33.30±8.47; GLY: 27.80±12.78; ETI: 29.65±20.25). DMF yielded the best results for RAP, which was similar to BC, whereas ETI and GLY had the lowest RAP values (P<0.05). MF had intermediate RAP parameters compared to all groups (DMF: 48.25±14.40; BC:45.50±16.14; MF: 38.50±15.23; GLY: 31.35±15.67; ETI: 29.55±16.51). DMF and BC better (P<0.05) preserved sperm PMI. MF and GLY had intermediate PMI compared to all groups (DMF: 44.35±9.68; BC: 41.25±10.44; MF: 35.05±10.81; GLY: 40.10±10.38; ETI: 33.55±12.67). In experiment 2, TM was similar across groups (5%: 57.70±4.82; 3%: 53.60±4.82; 2%: 49.50±4.82). However, the 5% yielded higher PM and RAP compared to 2% (P<0.05; PM: 5%: 37.00±6.11; 2%: 29.90±6.11; RAP: 5%: 42.40±7.15; 2%: 34.30±7.15), but similar to 3% (PM, 34.00±6.11; RAP, 39.90±7.15). The sperm PMI was unchanged between groups. In conclusion, DMF appears superior to other cryoprotectants for donkey semen cryopreservation, while the association of GLY:MF (1:4) yielded comparable outcomes. R reducing cryoprotectant concentration to 3% shows promise as a viable strategy for semen preservation.