The present study evaluated the effect of storage conditions on the LDL efficacy for cryopreserving ovine sperm. In this way, we compared egg yolk extender with three different forms for LDL storing, LDL diluted in Tris-glucose extender and stored in frozen (i) and freeze-dried (ii) states and LDL stored pure and added into the extender prior to use (iii). We also tested the effect of two storage temperatures (-20 and -80 °C) and three storage times (30, 60, 120 d). Frozen and freeze-dried extenders containing LDL, as well as LDL stored pure, improved post-thaw sperm quality. Storage temperatures did not influence negatively the cryoprotectiveness of LDL extenders. Furthermore, lyophilised LDL extenders stored at -20 °C were more effective in preserving sperm longevity than the other extenders stored at -20 °C. Finally, LDL extenders stored for 30 and 120 d were more efficient than 60 d in preserving ram sperm freezability.

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...

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...

Egg yolk is the most widely used cryoprotectant in the composition of extenders for cryopreservation of mammalian spermatozoa; yet, efforts have been made to find ways to substitute it due to the possibility of transporting pathogenic microorganisms, the lack of standardization, and the presence of substances that inhibit metabolic exchanges or decrease the motility of sperm. The protective effect of egg yolk was attributed to the presence of low density lipoproteins (LDL) that prevent cholesterol efflux by increasing membrane stability and resistance to low temperatures. (Moussa et al. 2002 Theriogenology 57, 1695–1706) demonstrated that the purification of LDL is possible, thereby allowing its use as a replacement for integral egg yolk in extender. Then, this study aimed to evaluate the effect of substitution of egg yolk by different LDL concentrations during cryopreservation of ram semen. A total of 6 sheep, 3 ejaculates per animal, were collected. After collection, the ejaculates were evaluated and diluted in different extenders: E1) Tris, glucose, 15% egg yolk, and 5% glycerol; E2) Tris, glucose, 5% LDL (Moussa et al. 2002 Theriogenology 57, 1695–1706), and 5% glycerol; E3) Tris, glucose, 10% LDL, and 5% glycerol; and E4) Tris, glucose, 20% LDL, and 5% glycerol. The samples were adjusted to a final concentration of 600 × 106 sptz mL–1 and filled into 0.25-mL straws and frozen in a TK 4000® machine. After thawing, sperm motility and spermatic vigor were evaluated, and the test of thermo-resistance (TTR) was conducted. Functional and structural integrity of the spermatic membrane were evaluated through the hypoosmotic test and the use of fluorescent dyes. The kinetic parameters of sperm were assessed by the computerized system (CASA). The statistical analysis was performed using the statistical program SAS (Statistical Analysis System), and the averages were compared using the Duncan multiple test. No difference (P > 0.05) was found between extenders for progressive motility after thawing. After 3 h of TTR, E1 showed higher values (P < 0.05) than E2, not differing from E4. The percentage of cells reactive to the hypoosmotic test was lower with the use of E2 (P < 0.05) than with other groups. Regarding the fluorescence technique, the average percentage of cells with intact membrane after thawing was higher in samples preserved in the extenders E1, E3, and E4 (P < 0.05) than in E2. Velocity average pathway (VAP), velocity straight line (VSL), and linearity of cryopreserved ram semen were (P < 0.05) significantly higher in E1 than in E2 and E3. The other kinetic parameters were similar in all groups tested. The results indicate that the extenders containing 10 and 20% of LDL are capable of protecting the spermatic cells during cryopreservation. Research supported by the FAPESB.