Efeito da adição in vitro de ácido docosahexaenóico (DHA) e fator de crescimento semelhante à insulina tipo I (IGF-I) na qualidade do sêmen de garanhões

Abstract

Semen quality after cooling, freezing and thawing processes decreases, which consequently impairs the pregnancy rate following artificial insemination. Compound as docosahexaenoic acid (DHA) and insulin-like growth factor-I (IGF-I) can eliminate this loss of quality caused by low temperature. The aim of this study was to assess the effect of the in vitro addition of DHA and IGF-I on the quality of cooled and cryopreserved stallion semen. In Experiment 1, semen from 10 Irish Sport Horse stallions was collected (3 ejaculates per stallion) during 2014 in Ireland. Semen was transported to Laboratory of Animal Reproduction (School of Natural Sciences, Faculty of Science and Engineering, University of Limerick), diluted to 100 x 106 spermatozoa/mL, supplemented with 0.02 mM of vitamin E (VE) and 0, 1, 10 or 20 ng of DHA/mL and frozen. Semen was thawed and total motility (TM), rapid progressive motility (PM), acrosome integrity, membrane fluidity and morphology were assessed. In Experiment 2, semen from 3 stallions was collected (3 ejaculates per stallion) during 2015 season breeding and frozen as in Experiment 1, but VE and DHA were added after thawing. TM and PM were assessed at 30, 60 and 120 min and viability, acrosome integrity and membrane fluidity at 30 min after addition of DHA. In Experiment 3, semen from 5 stallions was collected (1-3 ejaculates per stallion) during 2015 season breeding and diluted to 20 x 106 spermatozoa/mL. Posteriorly DHA and VE were added and semen was stored at 4ºC. After 1, 24, 48 and 72 h, TM, PM, viability, membrane fluidity and lipid peroxidation were assessed. In Experiment 4, semen from 3 stallions (3 ejaculate per stallion) was collected between February and March 2016. Ejaculates were processed as per Experiment 1 and supplemented with VE. After freezing and thawing processes, semen was diluted to 25 x 106 spermatozoa/mL and split in 4 treatments, namely: DHA0 (0 ng of DHA/mL; control), DHA0 + IGF-I (control + 100 ng of IGF-I/mL), DHA1 (1 ng of DHA/mL) and DHA1 + IGF-I (1 ng of DHA/mL + 100 ng of IGF-I/mL). Addition of 20 ng/mL of DHA to cooled semen (Experiment 3) increased the TM compared to the control without VE (52.9 ± 7.99% X 25.7 ± 5.23%; P<0.05); addition of any concentration of DHA improved the PM compared to the controls (20.8 ± 5.86% X 4.3 ± 1.48%; P <0.001) and addition of 10 and 20 ng/mL of DHA increased the membrane fluidity compared to other treatments (28.7 ± 4.35% and 29.4 ± 5.18%, respectively X 19.0 ± 3.85%; P<0.001). However, addition of DHA did not affect frozen semen (Experiment 1 and 2). The simultaneous addition of DHA and IGF-I to stallion semen after thawing (Experiment 4) improved PM (7.2 ± 1.56% X 4.1 ± 1.15%; P<0.05), but did not affect the TM, viability and acrosome integrity.