Effects of two freezing methods and two cryopreservation media on post‐thaw quality of stallion spermatozoa

Abstract

Glycerol-based extenders are widely utilized for freezing equine semen, but media combining methylformamide may better preserve sperm motility and mitochondrial function. Semen is cryopreserved utilizing either a Styrofoam box filled with liquid nitrogen or an automatic freezer. The objective of this experiment was to compare the post-thaw characteristics of the same ejaculates cryopreserved in a Styrofoam box or in an automatic freezer, utilizing a glycerol-based extender (Gent) and an extender that combines methylformamide and glycerol (BotuCrio ). For that, one ejaculate from 30 stallions collected in two different centres was used. For data analysis, a mixed linear model with laboratory, medium and freezing method and respective interactions as fixed effects was used. Stallion was taken into account as a random effect. There was no influence (p > .05) of laboratory, while stallion effect was marked. Semen frozen in BotuCrio in the automatic freezer had higher (p < .001) VCL than semen cryopreserved in Gent using the Styrofoam box. VCL was also higher (p = .068) for semen frozen in BotuCrio in the Styrofoam box than for semen cryopreserved in Gent using the same method. The difference between percentage of sperm with intact plasma membrane frozen in Gent using the Styrofoam box (44.43% ± 2.44%) compared to spermatozoa cryopreserved in BotuCrio using the same method (40.78% ± 2.42%) approached significance (p = .0507). The percentage of sperm with intact acrosome membrane was higher (p < .05) in semen frozen in BotuCrio (79.08% ± 1.79%) than semen frozen in Gent (75.15% ± 1.80%). A higher (p = .0125) percentage (32.24% ± 2.18%) of semen extended in Gent and cryopreserved in the Styrofoam box had high mitochondrial membrane potential than semen frozen in BotuCrio using the same method (26.02% ± 2.15%). Fertility studies are warranted to assess whether differences found have any effect on the fertility of inseminated mares.

Freezing Stallion Semen—What Do We Need to Focus on for the Future?

“…However, when the egg yolk was replaced by soya bean lecithin, pregnancy rates were reduced, although sperm characteristics were not affected [ 63 ]. There were considerable differences between the methods of freezing (controlled versus uncontrolled rate freezing) and the types of extenders that were used, i.e., the glycerol-containing extender, Gent, and BotuCrio, which contains amides [ 64 ]. The proportion of spermatozoa with high mitochondrial membrane potential was higher in the Gent extender than in the BotuCrio, and membrane integrity was almost statistically significant in favor of the Gent extender.…”

Stallion semen cryopreservation: Frozen semen preparation, handling and breeding

“…Amides (methyl formamide or dimethyl formamide) are another category of penetrating cryoprotectants. Amides are of lower molecular weight than glycerol, allowing faster diffusion and less osmotic damage (Alvarenga et al, 2005;Macedo et al, 2018;Squires et al, 2004).…”

“…Glycerol is the most common penetrating cryoprotectant for sperm and enters the cell via passive diffusion. Glycerol also causes injury directly to cells, including protein denaturation and alteration of the plasma membrane (Macedo et al., 2018; Moore et al., 2006; Vafaei et al., 2019). The search for a freezing medium which incites minimal osmotic damage on the sperm cell is ongoing.…”

“…Amides (methyl formamide or dimethyl formamide) are another category of penetrating cryoprotectants. Amides are of lower molecular weight than glycerol, allowing faster diffusion and less osmotic damage (Alvarenga et al., 2005; Macedo et al., 2018; Squires et al., 2004). Commercial freezing extenders generally contain a combination of glycerol and an amide, as well as non‐penetrating cryoprotectants (Moffett et al., 2016; Sieme, 2011b).…”

Slow Freezing of Preserved Boar Sperm: Comparison of Conventional and Automated Techniques on Post-Thaw Functional Quality by a New Combination of Sperm Function Tests

“…Slow freezing is widely used to cryopreserve boar sperm and can be performed by controlling or not controlling the temperature descent curve. The conventional slow freezing technique consists of placing semen straws (10–20 min) on racks suspended a few centimeters (3–5 cm) above LN 2 , which are kept in a Styrofoam box and frozen in liquid nitrogen vapors [ 7 , 8 ], the approximate temperature range of which is between −60 and −70 °C/min [ 9 ]. The automated slow freezing technique, using a biofreezer, controls the freezing rate, which is extremely important in maintaining a wide variety of cellular functions.…”