Low density lipoproteins added to an extender frozen or lyophilized are evenly efficient in cryoprotecting ovine sperm cells than when 16% whole egg yolk was added

Abstract

Low density lipoproteins added to an extender frozen or lyophilizedare evenly efficient in cryoprotecting ovine sperm cells than when 16% whole egg yolk was added AbstractThe aim of this study was to test different concentrations of low density lipoprotein (LDL) in replacement of whole egg yolk in extenders preserved in the aqueous or lyophilized form, for ram sperm cryopreservation using two freezing curves (-40°C/min from 5 to -140°C and nitrogen vapor). One ejaculate from six Santa Inês rams was collected. Each ejaculate was divided into nine different diluents as follows: Tris-16% yolk (control), and Tris with 2, 4, 6 and 8% fresh LDL, and criopreserved in the aqueous or lyophilized form. The samples were diluted to a final concentration of 100 x 10 6 sperm/mL and filled into 0.25 ml straws. After thaw, sperm cells were evaluated for motility and sperm kinetics (CASA), and submitted to the hypoosmotic swelling test and the evaluation of the structural integrity of sperm membranes using fluorescent dyes (CFDA: PI), as well as sperm morphology and longevity. The experimental design was randomized blocks, and results were submitted to ANOVA and the averages were compared using the Scott-Knott test. There were no differences in progressive motility and functional and structural integrity of the membrane evaluated when different concentrations of aqueous or lyophilized low density lipoproteins or egg yolk were added to the extender (P>0.05). As for the velocity of sperm movement, the control medium had some kinetic scores similar to the extender containing LDL, both aqueous and lyophilized (P> 0.05). Results were similar between cooling curves. Therefore, we conclude that the media containing all concentrations of LDL, aqueous or lyophilized, were able to protect the ram sperm cells during the cryopreservation process, as whole egg yolk did. ResumoO objetivo deste trabalho foi testar para criopreservação de sêmen ovino diferentes concentrações de lipoproteína de baixa densidade (LBD) em substituição à gema de ovo em meios diluidores, armazenados na forma aquosa e liofilizada, utilizando-se duas curvas de congelação (-40°C/min de 5 à -140°C ou vapor de nitrogênio). Os ejaculados de seis carneiros da raça Santa Inês foram fracionados e distribuídos em nove tratamentos, sendo o primeiro o meio controle (Tris-gema 16%) e os demais meios Tris contendo lipoproteínas de baixa densidade nas concentrações de 2, 4, 6 e 8% (v/v), criopreservados tanto na forma aquosa quanto liofilizada. As amostras foram diluídas para a concentração final de 100 x 10 6 espermatozoides/mL e envasadas em palhetas de 0,25 mL. Após a descongelação foram avaliadas a motilidade e cinética espermática (CASA), morfologia e longevidade espermáticas, além da integridade funcional (HOST) e estrutural (CFDA/IP) das membranas. O delineamento experimental foi blocos ao acaso e os resultados foram submetidos a ANOVA, sendo as médias comparadas pelo teste de Scott-Knott. Quanto aos parâmetros de motilidade progressiva e integridade funcional e estrutural...

Post-thaw quality of ram sperm frozen with different concentrations of low-density lipoproteins associated with non-enzymatic antioxidants

“…The efficacy of both 2 and 8% LDL instead of 16% egg yolk in Tris-Glucose base extender to cryopreserve ram sperm was demonstrated in the present study, corroborating previous reports ( Tonieto et al, 2010 ; Moustacas et al, 2011 ; Vera-Munoz et al, 2011 ; Silva et al, 2014 ; Loaiza-Echeverri et al, 2015 ). It was shown that even at a low LDL concentration (2%) it was possible to obtain a high percentage of sperm with functional plasma membrane and post-thaw kinematic parameters similar or superior to Control extender.…”

Preservation of boar semen: An update

“…However, egg yolk is one of the most commonly used components to prepare freezing extenders for semen of different species in a concentration of 20% (v/v) (Hu et al, ; Loaiza‐Echeverri et al, ; Strzezek, Koziorowska‐Gilun, & Stawiszyńska, ). Their beneficial effects are associated with the low‐density lipoprotein (LDL) content in the egg yolk (Bergeron & Manjunath, ).…”

“…In freezing extenders, disaccharides such as lactose are preferred (Frydrychová, Lustyková, Václavková, Lipenský, & Rozkot, 2015;Gómez-Fernández, Gómez-Izquierdo, Tomás, Mocé, & Mercado, 2012), but trehalose also improves the viability and in vitro fertilization of boar ejaculates (Malo et al, 2010). Its use in a concentration of 100 mM makes it possible to obtain better sperm parameters in terms of motility, mitochondrial activity and plasma and acrosomal membrane integrity compared to concentrations of 25, 50 and 200 mM (Hu et al, 2009) However, egg yolk is one of the most commonly used components to prepare freezing extenders for semen of different species in a concentration of 20% (v/v) (Hu et al, 2010;Loaiza-Echeverri et al, 2015;Strzezek, Koziorowska-Gilun, & Stawiszyńska, 2012). Their beneficial effects are associated with the low-density lipoprotein (LDL) content in the egg yolk (Bergeron & Manjunath, 2006).…”

Impact of in vitro fertilization by refrigerated versus frozen buffalo semen on developmental competence of buffalo embryos

“…Thereafter samples were fixed adding 200 μL of buffered formol saline. Two hundred cells were examined using a phase contrast microscope (1000x) ( Loaiza-Echeverri et al, 2015 ).…”