Cryopreservation Effects on Ram Sperm Ultrastructure

Abstract

Cryoprotectants are known to have protective effects against cryodamage to spermatozoa. In this study, the cryoprotective effects of two cryoprotectants (glycerol, ethylene glycol) and cryoprotectants/trehalose combinations on frozen-thawed ram spermatozoa were investigated at the ultrastructural level. For this purpose, ejaculates collected from Konya Merino rams were pooled and diluted with a tris-based extender containing additives, including 5% glycerol, 3% glycerol +60 mM trehalose, 1.5% glycerol +100 mM trehalose, 5% ethylene glycol, 3% ethylene glycol +60 mM trehalose, and 1.5% ethylene glycol +100 mM trehalose. They were all cooled to 5°C and then frozen in 0.25 mL French straws in liquid nitrogen. The samples were thawed at 37°C and centrifuged to remove the diluents. Then, they were processed using a scanning transmission electron microscope. In the statistical analysis, the number of ultrastructurally cryodamaged and intact spermatozoa were counted in longitudinal and transverse ultrathin sections in all groups by electron microscopic examination. The amount of intact spermatozoa in the groups containing 5% ethylene glycol and 1.5% ethylene glycol +100 mM trehalose was found to be higher than other groups (p < 0.05). As a result, it was suggested that the groups of 5% ethylene glycol and 1.5% ethylene glycol +100 mM trehalose provided the highest protection for the ultrastructural morphology of frozen-thawed Konya Merino ram spermatozoa among the groups.

Enhancement impact of Moringa oleifera leaves extract–base extender on cryopreservation and fertilization of Barki ram sperms: comparative study with vitamin E and selenium combination

“…In addition, incubation of human spermatozoa with exogenous ROS for long time increases the sperm DNA fragmentation, while addition of antioxidants to semen decreases it (Lopes et al 1998). However, addition of antioxidants to Barki rams semen extender such as trehalose and raffinose (Elshamy et al 2019;Keskin et al 2020), GPx and SOD to dog semen extender (Chatterjee and Gagnon 2001), glutathione to Boer buck semen (Rawash et al 2018) and selenium to water buffalo semen (Dorostkar et al 2012) improves DNA integrity of spermatozoa and reduces cryopreservation induces DNA damage. Taken together it is became clear that addition of MOLE to rams semen extender in our study protected spermatozoa DNA against cryopreservation induced spermatozoa DNA damage via its antioxidant activity.…”

Combination of trehalose and low boron in presence of decreased glycerol improves post‐thawed ram sperm parameters: A model study in boron research

“…It is applicable in certain economically, biologically, or scientifically valuable males, consisting of livestock breeds or endangered animal species. [1][2][3] In spite of the extensive progress in this field, various physical and chemical stress factors during the freezing and thawing process, including ice crystal formation, oxidative stress, and chemical toxicity, could adversely affect sperm structure and physiological function, and thereby damaging the fertility of the spermatozoa. 4 The sperm plasma membrane of ruminants includes unsaturated phospholipids in high levels and cholesterol in low levels.…”

“…11,12 That's why the effectiveness of various agents has been investigated in the numerous cryopreservation of gamete cells of different species. 3,9 Rams have been reported to generate more cryosensitive spermatozoa than humans, rabbits, cats, and dogs during freezing-thawing methods because of their high content of polyunsaturated fatty acids and insufficient cytoplasmic antioxidant capacity. 7 When the inadequacy of the studies on the development of ram semen extenders is added, the importance of conducting studies in the field of ram sperm protectors becomes evident.…”

“…The cryopreservation of spermatozoa has been commonly used in branch of reproductive biotechnology as an effective procedure for male fertility management. It is applicable in certain economically, biologically, or scientifically valuable males, consisting of livestock breeds or endangered animal species 1–3 . In spite of the extensive progress in this field, various physical and chemical stress factors during the freezing and thawing process, including ice crystal formation, oxidative stress, and chemical toxicity, could adversely affect sperm structure and physiological function, and thereby damaging the fertility of the spermatozoa 4 .…”

The effect of L-glutamine and trehalose on dog sperm cryopreservation

“…Also, its cryoprotective effect is attributed to the increased sperm membrane fluidity before the freezing process occurs (Aboagla and Terada 2003;Zhang et al 2016). The post-thaw semen analysis determined that trehalose preserved motility, vitality, and membrane integrity in different species while also reducing oxidative stress (Iqbal et al 2016;Park et al 2018;Keskin et al 2020).…”