Results of the “test-freeze” approach in a commercial program of stallion sperm cryopreservation and the relationship between pre-freeze sperm quality and “freezability.”

Abstract

In the current study, we report the effect of different commercially available semen freezing extenders utilized for the “test-freeze” procedure for 13 years (2010–2023) as part of a commercial program of stallion sperm cryopreservation. Ejaculates obtained from sexually active and healthy stallions (n = 124) were cryopreserved using up to five commercially available semen freezing extenders (Lactose-EDTA [LE], MFR5, CryoMax LE [CMLE], CryoMax MFR5 [CMMFR5] or BotuCrio [BC]). Post-thaw sperm motility (total motility – TM [%]; progressive motility – PM [%]; and curvilinear velocity – VCL [μm/s]), sperm viability (VIAB [%]), and sperm DNA damage (COMP_ɑt [%]) were compared among semen freezing extenders. Overall, TM, PM, and VIAB were similar for extenders LE, CMLE, and BC (P > 0.05) while higher than for extenders MFR5 or CMMFR5 (P < 0.05). Mean VCL was lower for extender LE than for extender BC (P < 0.05). An effect of semen extenders on mean COMP_αt was not observed (P > 0.05). The proportion of ejaculates that yielded four different post-thaw sperm “freezability” indices, based on the average value between post-thaw TM and VIAB (MV; MV ≤ 41 % [LOW]; MV 42–54 % [AVERAGE]; or MV ≥ 55 % [HIGH]), or post-thaw PM: ≥30 %, was calculated. Extenders MFR5 and CMMFR5 yielded a higher proportion of stallions in the LOW category (36 and 37 %, respectively) compared to extenders LE (22 %), CMLE (20 %), or BC (21 %; P < 0.05). For the HIGH and PM ≥ 30 % categories, extenders LE (16 and 65 %), CMLE (15 and 66 %), and BC (15 and 61 %) yielded a higher proportion of stallions than extenders MFR5 (4 and 40 %) or CMMFR5 (2 and 45 %), respectively (P < 0.05). For the AVERAGE category, an effect of semen extenders was not observed (60–63 %; P > 0.05). The relationship between pre-freeze sperm quality parameters (i.e., sperm motility, morphology [Normal], viability, and COMP_ɑt) and the indices of post-thaw “freezability” (LOW, AVERAGE, HIGH, PM ≥ 30 %) was also determined. The percentages of TMOT and Normal in fresh semen yielded the highest ROC values related to “freezability,” irrespective of the index used: 1) LOW = % Normal – AUC 0.8328 (P < 0.0001); 2) AVERAGE = TMOT – AUC 0.5875 (P = 0.055); 3) HIGH = % TMOT – AUC: 0.8015 (P < 0.0001); 4) PM ≥ 30 % = % Normal – AUC 0.8392 (P < 0.0001). This study provides clinically relevant data regarding the effect of different semen freezing extenders on post-thaw stallion sperm quality and the potential relationship between some sperm quality parameters commonly assessed in practice and post-thaw sperm “freezability” in stallions.