Effects of bovine sperm cryopreservation using different freezing techniques and cryoprotective agents on plasma, acrosomal and mitochondrial membranes

Abstract

The success of semen cryopreservation is influenced by several factors, such as freezing curves and cryoprotectants. These two factors are of special interest once they may lead to many important physical-chemical changes resulting in different degrees of damage in spermatozoa structure. This experiment was designed to compare the effect of bull semen cryopreservation using two freezing techniques: conventional (CT--cooling rate of -0.55 °C min(-1) and freezing rate of -19.1 °C min(-1) and automated (AT--cooling rate of -0.23 °C min(-1) and freezing rate of -15 °C min(-1)), performed with different curves, and with three cryoprotectants (glycerol, ethylene glycol and dimethyl formamide) on bovine sperm motility and integrity of plasma, acrosomal and mitochondrial membranes. These variables were simultaneously evaluated using the fluorescence probes propidium iodide, fluorescein-conjugated Pisum sativum agglutinin and MitoTracker Green FM. The effects of freezing techniques, as well as of different cryoprotectants were analysed by the analysis of variance. The means were compared by Fisher's test. There were no significant differences between freezing techniques (P > 0.05). Glycerol showed higher percentages of motility, vigour and integrity of plasma, acrosomal and mitochondrial membranes than other two cryoprotectants (P < 0.05). Ethylene glycol preserved higher motility and integrity of plasma and mitochondrial membranes than dimethyl formamide (P < 0.05). Sperm motility with glycerol was 30.67 ± 1.41% and 30.50 ± 1.06%, with ethylene glycol was 21.17 ± 1.66% and 21.67 ± 1.13% and with dimethyl formamide was 8.33 ± 0.65% and 9.17 ± 0.72% to CT and AT curves, respectively. The percentage of spermatozoa with simultaneously intact plasma membrane, intact acrosome and mitochondrial function (IPIAH) was 14.82 ± 1.49% (CT) and 15.83 ± 1.26% (AT) to glycerol, 9.20 ± 1.31% (CT) and 9.92 ± 1.29% (AT) to ethylene glycol 4.65 ± 0.93% (CT) and 5.17 ± 0.87% (AT) to dimethyl formamide. Glycerol provided the best results, although nearly 85% of spermatozoa showed some degree of injury in their membranes, suggesting that further studies are required to improve the results of cryopreservation of bovine semen.

Equilibration and freezing interactions affecting bull sperm characteristics after thawing

“…Summarily, the length of equilibration as well as the freezing curve type are responsible for many important physicochemical changes leading to different degrees of spermatozoa structure damage (Forero-Gonzalez et al 2012) deteriorating spermatozoa characteristics after thawing (Spalekova et al 2014). Despite this fact, interactions mentioned have not been sufficiently known yet.…”

Serum- and albumin-free cryopreservation of endothelial monolayers with a new solution

“…14,35,36 Loss of mitochondrial membrane potential was also described after cryopreservation, for example in sperm, 37–39 mouse embryos, 40 muscle, 41,42 hepatocytes 43 and in isolated mitochondria. 44,45 Also, ultrastructural changes in mitochondria, like mitochondrial swelling and rupture have been described to occur in endothelial cells after vascular cryopreservation, 46,47 however, little is known about mitochondrial membrane potential in this cell type.…”

Cryopreservation increases coating of bull sperm by seminal plasma binder of sperm proteins BSP1, BSP3, and BSP5

“…Cryopreserved bull sperm show ultrastructural damage of the plasma membrane (PonsRejraji et al 2009, Forero-Gonzalez et al 2010) and cytoskeleton (Felipe-Pérez et al 2011), increased levels of intracellular Ca 2C (Bailey & Buhr 1993, Pons-Rejraji et al 2009) and K C (Blässe et al 2012), redistribution of plasma membrane proteins, reduced tolerance to hypotonic stress, and a decrease in intracellular pH and cAMP (Pons-Rejraji et al 2009, Blässe et al 2012. The sub-lethal damage and modifications induced by cryopreservation translate into a shortened life span (Bailey et al 2000).…”