Estudo de diferentes protocolos utilizando lipoproteínas de baixa densidade na criopreservação do sêmen eqüino

Abstract

Equine semen freeze protocols are not standardized and thus the success of this technique varies substantially. Experiment 1 aimed to determine if the substitution of the total egg yolk for the low density lipoprotein fraction of the egg in the diluent exerts a more effective cryoprotective effect on the equine sperm cell and what concentration is indicated. Experiment 2 sought to evaluate the addition of dimethylformamide in the diluents tested in Experiment 1, as well as the effect of base media osmolarity on sperm quality after thawing. For Experiment 1, the ejaculates of 7 stallions were frozen in two freezing curves, one rapid (reeds kept 3 cm from the liquid nitrogen level for 15 minutes) and one slow (falling from 0.5 ° C / min to 5, 0oC, and fall of 20oC / min up to - 120oC, obtained in computerized machine, Model TK 2000 - TETAKON - NUTRICELL); Using two cryoprotectants, glycerol or dimethylformamide at different concentrations of low density lipoproteins. The results of the study were: T1: INRA 82, with 5% glycerol (control), T2: INRA 82, 5% glycerol and 5% LBD, T3: INRA 82, 5% glycerol and 10% LBD, T4: INRA 82, 5% glycerol and 20% LBD, T5: INRA 82, 5% dimethylformamide and 5% LBD, T6: INRA 82, 5% dimethylformamide and 10% LBD, T7: INRA 82, 5% dimethylformamide and 20% LBD, T8: INRA 82, with 5% dimethylformamide (control). For Experiment 2, six ejaculates were frozen in the following diluents tested: D1: INRA 82, with 20% LBD at 300 mOsmol / L and 5% DMF; D2: INRA 82, with 20% LBD at 400mOsmol / L and 5% DMF; D3: INRA 82, with 2% egg yolk at 300 mOsmol / L and 5% DMF; D4: INRA 82, with 2% egg yolk at 400mOsmol / L and 5% DMF. In this case the 4 diluents tested were made in duplicate, with dimethylformamide being added in single or fractional fraction over time. In both experiments the thawing was done at 52oC for 10 seconds, followed by immersion in a water bath at 37oC for 30 seconds. After thawing, the parameters of total motility, progressive motility and sperm vigor in light microscopy were evaluated. Functional and structural integrity of the spermatozoa were evaluated through hyposmotic and fluorescence tests, respectively. The spermatozoa were submitted to the endurance test. Statistical analysis was performed using ANOVA, with a randomized block design, and comparison between means by Tukey Test. In Experiment 1, low-density lipoproteins used at concentrations of 10 and 20% were shown to be as efficient in protecting the spermatozoa as in the whole egg yolk used in INRA 82. The slow curve was superior to the rapid curve for the parameters total motility, progressive motility, spermatic vigor, hyposmotic test and thermo resistance test, independent of the cryoprotectant used. Dimethylformamide was superior to glycerol in the two freezing curves, in evaluations of total motility, progressive motility, sperm morphology, hyposmotic test, number of spermatozoa injured and endurance test. For Experiment 2, all viability parameters of cryopreserved equine spermatozoa except motility were increased by the fractionated addition of dimethylformamide together with the use of higher initial osmolarity of the base medium.