The effect of cooling to different subzero temperatures on dog sperm cryosurvival

Abstract

The objective was to assess the effect of cooling to different subzero temperatures around ice formation (-5°C) on dog sperm cryosurvival and plasma membrane fluidity. Semen was centrifuged, and sperm were resuspended in a Tris-egg yolk medium (3% glycerol). Diluted sperm were cooled from 22 to 5°C, and then, a Tris-egg yolk medium containing 7% glycerol was added (final concentration of 5% glycerol and 200 × 10 cells/ml). Sperm were packaged in 0.5-ml plastic straws, and equilibration was done 16 hr at 5°C before freezing. I. Straws (n = 47) at 5°C were exposed to nitrogen vapours to determine the freezing point. II. Other straws (from different ejaculates) processed as mentioned, were further cooled to -3, -5 or -7°C and immediately rewarmed in a water bath at 37°C. Motility, plasma membrane functionality and acrosome integrity were assessed. III. Other straws (from different ejaculates) processed as mentioned were further cooled to -3 or -5°C, frozen over nitrogen vapours and stored in liquid nitrogen for one month. Straws were thawed in a water bath at 38°C for 30 s. Motility, plasma membrane functionality, plasma membrane integrity, acrosome integrity, capacitation status and plasma membrane fluidity were assessed. Ice nucleation temperature was -14.3 ± 2.05°C (mean ± SD); cooling to +5, -3, -5 and -7°C, without freezing, produces no differences on sperm quality between target temperatures; cooling to +5, -3, and -5°C produced no differences on sperm survival and plasma membrane fluidity after freeze-thawing. In conclusion, cooling of dog spermatozoa to different subzero temperatures did not improve sperm cryosurvival and had no effect on plasma membrane fluidity after thawing.

The effect of non-traditional cooling on dog sperm cryosurvival and ability to perform the acrosome reaction

“…In physiological conditions, plasma membrane fluidity increases during sperm capacitation to prepare sperm membranes to suffer the acrosome reaction and facilitates the sperm-oocyte interaction (Flesch and Gadella 2000); however, in cryopreserved sperm, hyper-fluidity shortens the window of sperm fertility (Watson 1995). Studying the cryopreservation of dog spermatozoa, Alcantar-Rodriguez and Medrano (2017) found no differences in sperm quality and plasma membrane fluidity between sperm cooled to either +5 °C or -5 °C. In that work, approximately 52% of spermatozoa were classified as Merocyanine high-binding cells (hyper-fluidity).…”

“…In contrast, in our work, values were 69.5 and 63.8% for +5 °C and -5 °C, respectively. In the study by Alcantar-Rodriguez and Medrano (2017), sperm were frozen on the next day after collection. In contrast, in our work sperm were collected and frozen on the same day.…”

“…In this way, the occurrence of severe changes in plasma membrane fluidity could be reduced. Some researchers have been exploring the effect of cooling to sub-zero temperatures on sperm cryosurvival (Garzon-Perez et al 2010, Contreras-Mendez and Medrano 2016, Alcantar-Rodriguez and Medrano 2017. However, they have obtained either positive or null effects on sperm cryosurvival.…”

Spermadonatie en de start van een open spermabank voor honden: een nieuw hulpmiddel om inteelt bij rashonden te voorkomen

“…It allows the different components of the extender to carry out several modifications on the spermatozoa in order to prepare them to survive at low temperatures (Barbas et al, 2009). The plasma membrane suffers, however, from different stressors and some harmful cellular modifications may lead to the death of the spermatozoa, especially because of the toxic effect of glycerol (Barbas et al, 2009;Belala et al, 2016;Alcantar-Rodriguez et al, 2017).…”