Unraveling the effect of glycerol and amides and different cooling rates on post-thawed sperm parameters of equine frozen semen

This study assessed the effect of different cooling curves, stabilization periods, and permeating cryoprotectants on equine cryopreserved semen. Forty-eight ejaculates of six stallions were used for the study. After semen collection, the ejaculates were split into four aliquots, extended 1:1 (v:v, semen:extender) with a milk-based extender, and centrifuged at 600 × g for 10 min. After centrifugation, the sperm pellet was resuspended at 200 × 106 sperm/mL in a BotuCrioⓇ-based medium containing 5% of one of the following permeating cryoprotectant: glycerol (GLY), methylformamide (MF), dimethylformamide (DMF) and dimethylacetamide (DMA). Thereafter, semen was packed into 0.5 mL French straws and five straws from each group were subjected to four cooling rates using the Mini-Digitcool ZH 400 system: -1°C/min(C1), -0.25°C/min (C2), -4°C/min with stabilization at 5°C for 15min (C3), and -4°C/min with stabilization at 5°C for 75min (C4). Afterwards, the straws were frozen in nitrogen vapor (-140°C) at curve of ∼ -25°C/min with stabilization for 20min, and then immersed in liquid nitrogen and stored at -196°C. For sperm analysis, frozen semen samples were thawed at 46°C for 20s, incubated at 37°C/10min, and assessed for sperm kinetics by the CASA system (Hamilton Thorne Research – IVOS 12) and for plasma membrane stability (PMS) and high mitochondrial membrane potential (HMMP) by flow cytometry. Sperm parameters between cryoprotectants and cooling curves were assessed by ANOVA and Tukey test, and P was set as 〈 0.05. The C1 cooling curve produced superior sperm parameters independent of the cryoprotectant (TM, 60±12; PM, 21±12; RAP, 37±17; PMS, 50±13; HMMP, 6858±1230) compared to C2 (TM, 56±16; PM, 19±13; RAP, 33±18; PMS, 48±9; HMMP, 4827±1053), C3(TM, 54±15; PM, 17±11; RAP, 33±16; PMS, 41±9; HMMP, 2131±1043) e C4 (TM, 55±15; PM, 17±13; RAP, 33±16; PMS, 39±10; HMMP, 2112±1010). GLY samples had superior sperm kinetics (TM, 58±18; PM, 28±13; RAP, 40±19) and for HMMP (4504±2472), but lower for PMS (39±9) than DMF (TM, 60±10; PM, 17±7; RAP, 38±9; HMMP, 3709±1961; PMS 51±11) and DMA (TM, 51±15; PM, 9±6; RAP, 23±15; HMMP, 3719±2186; PMS, 46±11), but similar to MF (TM, 58±13; PM, 21±13; RAP, 36±17; HMMP, 4015±2377; PMS,41±9). DMA samples had the lowest values of sperm kinetics, whereas DMF presented the highest plasma membrane stability. In conclusion, GLY presented superior sperm kinetics and HMMP to the amides (DMF and DMA), and mainly DMF presented greater protection to the plasma sperm membrane. Regardless of the cryoprotectant added, the quickest freezing curve, -1°C/min, was more effective in preserving sperm quality.