Cryoprotective effect of different glycerol concentrations on domestic cat spermatozoa

Abstract

Cryopreservation of spermatozoa is a pivotal tool in assisted reproduction, and studies aiming to establish optimal freezing/thawing protocols are essential to enhance sperm survival. The objectives of the present study were to (1) compare the cryoprotective efficiency of three different glycerol concentrations (3%, 5%, and 7%) on the basis of post-thaw sperm quality and (2) investigate whether the incidence of morphologically abnormal sperm in fresh samples is related to cryodamage sensitivity. Semen was collected from six tomcats using an artificial vagina (total 18 ejaculates). Each ejaculate was diluted using Tris-egg yolk-based extender (TEY), evaluated, equally divided into three aliquots, and rediluted using TEY with and without glycerol to achieve final concentrations of 3%, 5%, and 7%. Samples were loaded into 0.25 mL straws, equilibrated for 60 minutes at 5 °C, frozen, and then thawed at 46 °C for 12 seconds. Fresh and frozen-thawed samples were evaluated for sperm motion parameters (computer-assisted sperm analysis), plasma membrane integrity (PMI; propidium iodide and carboxyfluorescein diacetate), and DNA integrity (acridine orange). Plasma and acrosomal membrane integrity were assessed by flow cytometry (propidium iodide and fluorescein isothiocyanate-conjugated pea (Pisum sativum) agglutinin) immediately after thawing. Sperm motion parameters were also evaluated at 30 and 60 minutes of postincubation. For all treatment groups, cryopreservation significantly impaired the PMI and sperm motion parameters, except for straightness and amplitude of lateral head displacement. DNA integrity showed a slight reduction (P < 0.05) when 3% glycerol was used. The percentage of total motility, progressive motility, and rapid spermatozoa were significantly lower immediately after thawing and up to 60 minutes of incubation for the 3% glycerol group when compared with 5% and 7%. No difference (P > 0.05) was found for PMI, acrosome integrity, and DNA integrity among post-thaw groups. However, higher (P < 0.05) incidence of viable cells with reacted acrosome and dead cells with intact acrosome were observed with 7% and 3% glycerol, respectively. Percentage of morphologically abnormal spermatozoa in fresh sample was positively correlated with PMI only in the 3% glycerol group and negatively correlated with sperm motility in the 5% and 7% groups. In conclusion, the final concentration of 5% glycerol offered better cryoprotective effect for ejaculated cat sperm, and the relationship found between prefreezing sperm morphology and post-thaw sperm quality showed to be dependent on final glycerol concentration.

Successful Cryopreservation of Domestic Cat (Felis catus) Epididymal Sperm after Slow Equilibration to 15 or 10°C

“…The subsequent fast cooling step from 10 or even 15 to 4°C, which was in our case already part of the freezing process over liquid nitrogen, was well tolerated by cat epididymal sperm. In the samples only cooled to 15°C, the velocity of progressive sperm after cryopreservation was the only parameter which was immedi- AE 12 (Villaverde et al 2013) ately after thawing significantly reduced in relation to samples which had been further cooled to 10°C (and in relation to initial values). On the contrary, the linearity of progressive sperm after thawing was higher after cooling to only 15 than to 10°C.…”

“…Cooling rates between À0.5 and À0.1 K/min were probably used in most studies even if exact rates and final temperatures achieved during cooling are not always reported in detail. However, (Villaverde et al 2013) used a faster cooling rate of À3.4 K/min and (Zambelli et al 2002) was successful with a cooling rate of À12 K/min in the presence of glycerol followed by a subsequent holding step at 4°C (Table 3). This holding step can be attributed to sperm equilibration with glycerol, to sperm adaptation to 4°C or both.…”

Effects of supplementation of iodixanol to semen extender on quality and fertilization ability of frozen–thawed Thai native bull sperm

“…Iodixanol can be postulated to be a cryoprotectant similar to sugar, ethylene glycol and glycerol. Glycerol is widely accepted as having a lower freezing point in the intracellular process of spermatozoa in livestock (Villaverde et al 2013). In the present study, it was found that a low concentration of 2.50% iodixanol in the extender (together with glycerol in the TEY dilution) had a less toxic effect on the motility of frozen sperm; similar results have been previously reported (Saragusty et al 2009;Cirit et al 2013).…”

Recuperação e conservação de espermatozoides epididimários: Perspectivas e aplicações na espécie felina doméstica

“…Assim, recomenda-se para o gato, a adição do glicerol a 4ºC, a fim de minimizar os efeitos tóxicos do crioprotetor, em concentrações inferiores a 8%, sendo que o emprego deste a 5% confere melhor efeito crioprotetor à célula espermática (Deco- Souza et al, 2013;VillaVerde et al, 2013).…”

“…Quanto à descongelação, várias são as temperaturas (37ºC, 38ºC, 42ºC, 65ºC, 70ºC) e tempos (6 a 30 segundos) propostos (Axner et al, 2004;Chatdarong et al, 2007;Cocchia et al, 2010;Vick et al, 2012;VillaVerde et al, 2013;Vizuete et al, 2014;Buranaamnuay, 2015;Thuwanut et al, 2015;Klaus et al, 2016;Kunkitti et al, 2016). A escolha do protocolo de descongelação dependerá da curva de congelação estabelecida, sendo assim, espermatozoides submetidos à refrigeração lenta são submetidos a tempo maior de descongelação (Buranaamnuay, 2017).…”