69 REACTIVE OXYGEN SPECIES EVALUATION OF DONKEY FROZEN SEMEN ADDED TO HOMOLOGOUS SEMINAL PLASMA ON POST-THAW

Abstract

For many years the pregnancy rate of donkey frozen semen presented lower results in donkey jennies; however, a recent study showed an increase in pregnancy rates using frozen semen added to seminal plasma on post-thaw. A hypothesis for this result is the higher uterine inflammation response after breeding when using seminal plasma. The same studies demonstrated higher uterine inflammation in the presence of higher reactive oxygen species concentration. The aim of the present study was to evaluate the content of reactive oxygen species in donkey frozen semen added to homologous seminal plasma on post-thaw. Five ejaculates from each 3 donkeys were used. Semen was diluted (1 : 1) with a skim milk-based extender (Botu-Semen^TM, Botupharma, Brazil). The semen was frozen with Botu-Cryo^TM extender (Botupharma, Brazil) in an isothermal box in straws containing 100 × 10⁶ of total sperm. The samples were thawed at 46°C for 20 s. After this, the straws of each donkey were divided in 2 group: control group (CG), in which the semen was incubated at 37°C for 5 min, and plasma seminal group (PG), in which the semen was incubated at 37°C for 5 min with 70% of homologous seminal plasma. Sperm kinetic parameters were evaluated by computer-assisted semen analysis, and the plasma membrane integrity (propidium iodide and fluorescein isothiocyanate -PSA) and reactive oxygen species (5–6-carboxi-2,7-diclorodihidrofluoresceindiacetate) were evaluated by flow cytometer. Comparison of sperm parameters was performed by t-test. Total motility (%, CG = 75.4 ± 8.2^a v. PG = 57.5 ± 16.4^b), progressive motility (%, CG = 42.0 ± 8.7^a v. PG = 33.3 ± 13.2^b), progressive angular velocity (μm/s, CG = 95.8 ± 10.8^a v. PG = 88.9 ± 10.9^b), and percentage of rapid sperm (%, CG = 58.4 ± 12.5^a v. PG = 41.0 ± 17.3^b) were higher in CG compare with PG. No difference (P < 0.05) was observed in membrane integrity (%, CG = 20.7 ± 7.4 v. PG = 20.6 ± 7.8); however, reactive oxygen species (%, CG = 12.3 ± 10.6^a v. PG = 81.8 ± 32.5^b) were higher in PG. The results of this study showed that the addition of homologous seminal plasma on post-thaw decreases the sperm kinetic parameters and viability of donkey frozen semen but increases reactive oxygen species, and this may cause higher uterine inflammation response in donkey jennies and increase their fertility.