Post-thaw semen motility in different cryoprotectants after freezing in a CryogenⓇ automatic freezer compared to an isothermal box

Abstract

The objective of this study was to evaluate the effects of using the CryogenⓇ freezing machine (NEOVET, Uberaba, Brazil) compared to an isothermal equine semen box, in conjunction with new cryoprotectants and their combinations, on sperm motility parameters. Four stallions, aged 4 to 20 years, underwent four semen collections each. The ejaculates were divided into four groups with different cryoprotectants: GROUP 1: 2% glycerol + 1% methylformamide; GROUP 2: 2.5% ethylene glycol + 2.5% methylformamide; GROUP 3: 2.5% glycerol + 2.5% methylformamide, and BC Botu-CrioⓇ (Botupharma, Botucatu-SP, Brazil). The different cryoprotectants and their combinations were added to the standard Botucrio extender without the cryoprotectant. Each group was subjected to two cryopreservation methodologies: BOX [samples were cooled to 5°C for 20 minutes in a MinitubⓇ refrigerator (Minitub do Brasil Ltda) at a cooling rate of -3 to -5°C per minute. Freezing was performed in a styrofoam box, with the straws 6 cm above the liquid nitrogen level, at a rate of 10°C per minute between 5°C and -60°C, and 8°C per minute between -60°C and -110°C] and CryogenⓇ MACHINE: (standardized with the EQUINE 4 curve, with a cooling rate of -1°C/min, equilibrium temperature at +5°C for 1 minute; then, a freezing rate of -30°C/min down to -20°C; afterward, the second freezing phase starts at a rate of -30°C/min until reaching -100°C). All experimental groups used Botu-CrioⓇ and the BOX as the freezing standard. After freezing, straws were thawed at 37°C for 60 seconds and evaluated for sperm motility (CASA): total motility (TM), progressive motility (PM), number of rapid sperm cells (RAP), average path velocity (VAP), straight-line velocity (VSL), and curvilinear velocity (VCL). Statistical analysis was performed using the Kolmogorov-Smirnov test, ANOVA, and Tukey, with P<0.05. There were no statistical differences in TM, PM, RAP, and VAP. However, VSL in GROUP 2 BOX was lower (72.5±3.31b) compared to BC BOX (77.8±3.42a) and GROUP1 MACHINE (79.1±3.73a). As for VCL in the BC BOX protocol (178.3±4.39a), there was a difference compared to the groups and methodology GROUP 1 MACHINE (163±3.89b), GROUP 2 BOX (162.2±4.72b), GROUP 2 MACHINE (165.7±5.56b), GROUP 3 BOX (160.8±3.58b), and GROUP 3 MACHINE (161.1±4.62b). It can be concluded that both the box and the automated method (CryogenⓇ machine) were effective in cryopreservation, as well as the use of different cryoprotectants and their combinations. Nevertheless, the automated system provides greater control over all cryopreservation stages, while the isothermal box is subject to external influences such as temperature, nitrogen level, and the number of straws to be frozen.