Prolonged post cooling but not pre-cooling equilibrium length improves the viability of ram sperm cryopreserved in an extender containing low-density lipoproteins

Silva, Maíra Corona da; Moura, Luís Cláudio de Oliveira; Melo, Maria Isabel Vaz de; Mambrini, Juliana Vaz de Melo; Neves, Mariana Machado; Henry, M.; Snoeck, Paola Pereira das Neves

doi:10.1016/j.smallrumres.2014.02.006

Cited by 12 publications

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“…This effect might be similar to that found by Strzeżek, Koziorowska-Gilun and Stawiszyńska (2012) who demonstrated that canine sperm cryopreservation impaired the functionality of the antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidase (GPx). Furthermore, the reduction in the activity of these two enzymes was higher using an egg yolk-based diluting extender than in extenders with LDL.…”

Section: Discussionsupporting

confidence: 85%

“…This concentration is smaller than those reported in previous studies (ALVAREZ et al, 2008;TONIETO, 2008;HU et al, 2010;MOUSTACAS et al, 2011). Furthermore, Silva et al (2014) reported that LDL concentrations below 4% are not ideal for preserving ovine sperm during freezing. However, the study on swine sperm cells by Bianchi et al (2011) reported no difference in post-thaw motility or sperm membrane integrity when LDL at 2% concentration were used during cooling and freezing.…”

Section: Discussionmentioning

confidence: 53%

“…The purification was performed according to the protocol described by Moussa et al (2002), and with the modifications proposed by Neves, Heneine and Henry (2014) and Silva et al (2014). Fresh egg yolk or purified fresh lipoproteins were used for extender preparation.…”

Section: Methodsmentioning

confidence: 99%

“…Therefore, many researchers have aimed to improve sperm cryopreservation. Recently, several studies have shown that replacing the egg yolk in extenders with low-density lipoproteins (LDL) enhanced motility, membrane integrity, and the fertilization ability of frozen sperm from various species compared to the use of commercial extenders containing total egg yolk (MOUSSA et al, 2002;AMIRAT et al, 2004;JIANG et al, 2007;BENCHARIF et al, 2008;VARELA JÚNIOR et al, 2009;MOUSTACAS et al, 2011;VERA-MUNOZ et al, 2009;HU et al, 2010;SILVA et al, 2014;HENEINE;.…”

Section: Introductionmentioning

confidence: 99%

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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

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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...

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Section: Discussionsupporting

confidence: 85%

Section: Discussionmentioning

confidence: 53%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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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

et al. 2015

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“…However, the addition of enzymatic antioxidants to diluents containing lowdensity lipoproteins (LDL) has not yet been tested. It has been shown that extenders containing LDL as a replacement for egg yolk have a cryoprotective effect that is similar or superior to extenders based on egg yolk (ALI AL AHMAD et al, 2008;VARELA JUNIOR et al, 2009;MOUSTACAS et al, 2011;SILVA et al, 2014). Therefore, the objective was to study the effects of the supplementation of extenders containing low-density lipoprotein with the antioxidant enzymes catalase, superoxide dismutase and reduced glutathione on ram sperm quality after cryopreservation.…”

Section: Introductionmentioning

confidence: 99%

Effect of catalase, superoxide dismutase and reduced glutathione in LDL extender on ovine cryopreserved sperm viability

et al. 2015

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The aim of this study was to evaluate the motility, kinetics and membrane integrity of ovine sperm cryopreserved in extenders containing 8% LDL with enzymatic antioxidants at different concentrations. Four Santa Inês rams were used to form four pools of semen (each pool containing ejaculates from four ram, totaling four ejaculates per animal). Each seminal pool was divided into eight aliquots for the following treatments: 1) Tris-glucose-glycerol (TGG) + (16%) egg yolk (control 1); 2) TGG + 8% (w/v) LDL (control 2); 3) TGG + 8% LDL + catalase 100 U/mL; 4) TGG + 8% LDL + catalase 200 U/mL; 5) TGG + 8% LDL + superoxide dismutase 100 U/mL; 6) TGG + 8% LDL + superoxide dismutase 200 U/mL; 7) TGG + 8% LDL + reduced glutathione 5 mM; and 8) TGG + 8% LDL + reduced glutathione 10 mM. The samples were packed into 0.25 mL straws, cooled (-0.25 °C/ min), maintained at 5 °C for 2 h and then frozen (-25 °C/ min) using a TK4000 ® . Immediately after thawing (38 °C/ 30 s), sperm motility and movement characteristics were assessed by computer sperm analysis (CASA). The structural integrity of the plasma and acrosomal membranes was analyzed using fluorescent dyes. The functional integrity of membranes was assessed using a hypoosmotic swelling test. As assessed by ANOVA, significant differences (P<0.05) among treatments were only observed for VCL, VSL and VAP. For the VCL variable, the 2, 3, 4, 5, 6 and 7 extenders were similar and higher than 1 and 8 extenders, the latter being similar to each other. For the VSL variable, the 3, 4, 5, 6 and 7 extenders were similar and higher than 1, 2 and 8 extenders, the latter being similar to each other. For the VAP variable, the 3, 4 and 6 extenders were similar and higher than 1, 2, 5, 7 and 8 extenders, the latter being similar to each other. In conclusion, enzymatic antioxidants as catalase and superoxide dismutase improve the protective activity of extenders containing LDL on frozen ovine sperm. concentrações. Quatro carneiros da raça Santa Inês foram utilizados para formar quatro pools de sêmen (cada pool contendo ejaculados provenientes dos quatro carneiros, totalizando quatro ejaculados por animal). Cada pool de sêmen foi dividido em oito alíquotas para os seguintes tratamentos: 1) Trisglicose-glicerol (TGG) + (16%) gema de ovo (controle 1); 2) TGG + 8% (g/L) LDL (controle 2); 3) TGG + 8% LDL + catalase 100 U/mL; 4) TGG + 8% LDL + catalase 200 U/mL; 5) TGG + 8% LDL + superóxido dismutase 100 U/mL; 6) TGG + 8% LDL + superóxido dismutase 200 U/mL; 7) TGG + 8% LDL + glutationa reduzida 5 mM; and 8) TGG + 8% LDL + glutationa reduzida 10 mM. As amostras foram envasadas em palhetas de 0,25 mL, resfriadas (-0,25 °C/min), mantidas a 5 °C por duas horas e em seguida congeladas (-25 °C/ min) usando uma máquina de congelar TK4000 ® . Imediatamente depois da descongelação (38 °C/30 s), as amostras foram submetidas à análise computadorizada (CASA) para avaliação da motilidade e cinética. A integridade estrutural das membranas plasmática e acrossomal foi analisada utilizando corantes fluor...

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Egg yolk plasma: Separation, characteristics and future prospects

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Prolonged post cooling but not pre-cooling equilibrium length improves the viability of ram sperm cryopreserved in an extender containing low-density lipoproteins

Cited by 12 publications

References 34 publications

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

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

Effect of catalase, superoxide dismutase and reduced glutathione in LDL extender on ovine cryopreserved sperm viability

Egg yolk plasma: Separation, characteristics and future prospects

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