Determination of effects of antioxidants addition in food on bull's sperm quality parameters in heat stress condition

Abstract

Oksidativni stres rezultat je neravnoteņe izmeĎu proizvodnje reaktivnih kiseoničnih vrsta (ROS) i zańtitnog efekta antioksidativnog mehanizma odgovornog za njihovu neutralizaciju i uklanjanje. Prekomerno stvaranje ROS u organizmu negativno utiče na pokretljivost spermatozoida i oplodnu sposobnost. Sem toga, mnogobrojni uticaji okoline, fiziolońki i genetski faktori mogu uticati na lońu funkciju spermatozoida i neplodnost. Značajnu ulogu u zańtiti spermatozoida od oksidativnog stresa imaju antioksidanti superoksid dismutaza (SOD) i glutation peroksidaza (GPx) te se stoga smatraju korisnim indikatorima antioksidativnog statusa. Cilj ove doktorske disertacije je bio ispitivanje efekata dodavanja prirodnih antioksidanasa u hrani za priplodne bikove (inaktivisanih ņivih ćelija kvasca Saccharomycess cerevisae [soj R397] sa visokim nivoom organski vezanog selena (izvor selen zavisne glutation-peroksidaze-Se-GPx) i liofilizovanog preparata pulpe dinje Cucumis melo (izvor superoksid dismutaze-SOD) na kvalitet ejakulata priplodnih bikova u uslovima toplotnog stresa. U ogled je bilo uključeno 15 bikova podeljenih u tri grupe (kontrolna grupa – C, ogledna grupa -M kojoj je dodavan izvor SOD – i ogledna grupa – A tretirana izvorom Se-GPx). Ispitivani su biohemijski parametri u krvnom serumu, parametri antioksidativne zańtite (SOD i Se-GPx) u seminalnoj plazmi kao i kvalitet semena u svim grupama. TakoĎe, ispitivana je povezanost izmeĎu sredinskih faktora (temperatura i vlaņnost), aktivnosti antioksidativnih enzima i kvaliteta sperme. Kod tretiranih bikova uočeno je povećanje aktivnosti SOD i GPx u odnosu na kontrolnu grupu, ńto ukazuje da su oba dodatka hrani povećala antioksidativni kapacitet semene plazme. Sem toga, antioksidansi u hrani su imali pozitivan uticaj na ukupnu pokretljivost spermatozoida jer je zabeleņeno značajno povećanje ukupne pokretljvosti kod tretiranih grupa. Analizom korelacije izmeĎu aktivnosti svakog enzima i procenta ukupno pokretnih i progresivno pokrenih spermatozoida kod svakog bika ponaosob nije ustanovljena značajna povezanost. Ispitivanjem uticaja THI (temperature-humidity index) na aktivnost antioksidativnih enzima uočeno je da je sa porastom THI dońlo do povećanja aktivnosti SOD i GPx, ali ne statistički značajnog. Pokazano je da ambijentalna temperatura i vlaņnost nisu značajno uticali na kvalitet semena u svim oglednim grupama. Moņe se zaključiti da je kod sve tri grupe bikova dońlo do povećanja aktivnosti oba enzima u semenoj plazmi, ali je povećanje značajno niņe u kontrolnoj grupi nego u tretiranim grupama. Ovo nam govori da je antioksidativni kapacitet semene plazme netretiranih bikova bio niņi nego kod tretiranih grupa bikova. Citomorfolońke analize semena su sprovoĎene u cilju utvrĎivanja odnosa ņivih/mrtvih ćelija, nalaza intaktnih i ońtećenih akrozoma, protoplazmatskih kapljica, kao i primarnih, sekundarnih i ukupno patolońkih formi spermatozoida specifičnim supravitalnim bojenjem po Blomu. Nije utvrĎeno postojanje značajna razlika izmeĎu ispitivanih grupa bikova prema prosečnom učeńću ņivih i mrtvih spermatozoida.Oxidative stress is a result of disbalance between reactive oxygen species (ROS) production and protection effect of antioxidative mechanism responsible for their neutralization and removal. Overproduction of ROS in body has negative influence on sperm motility and fertility. Besides, numerous effects of environment, physiological and genetic factors could impair function of sperm and cause infertility. Superoxide dismutase (SOD) and glutathione peroxidase (GPx) have key role in protection of oxidative stress and they are useful indicators of antioxidative stress. The aim of the current research was to assess the effects of the feed additive made of lyophilized melon juice (source of superoxide dismutase, SOD) and inactivated live Saccharomyces cerevisiae (strain R397) cells added to feed via the product containing high levels of organically bound selenium (source of selenium-dependent glutathione peroxidase, Se-GPx) on the semen quality of bulls in heat stress conditions. The 15 bulls chosen for the experiment were assigned to three equal groups (control –group C; treated group E-I, given the source of SOD; and treated group E-II, treated with the source of Se-GPx).The biochemical parameters in blood serum and the parameters of antoxidative protection (SOD and Se-GPx) in seminal plasma were determined spectrophotometrically. Whilst semen quality parameters were determined by CASA (computer assysted sperm analysis) and by cytopatomorphological analyzes. Relationship between environmental factors (temperature and humidity), activity of antioxidative enzymes and semen quality were determined also. The average SOD and GPx activity in the treated bulls was significantly higher than in control group and showed that both feed additives increased the antioxidative capacity of the seminal fluid. Besides, the antioxidative additives had positive influence on sperm motility because higher sperm motility was determined in treated groups. The analysis of relations between the activity of each enzyme and sperm motility and progressive motility in each of the bulls failed to detect significant correlation. The analysis of the relation between THI (temperature-humidity index) and the activity of the antioxidative enzymes revealed that the increase in THI coincided with the increase in the SOD and GPx activity but not significantly. Results showed that environmental temperature and humidity had no significant influence on semen quality in treated groups. It can be concluded that in all of the three groups of the bulls there was an increase in the activity of both enzymes in the seminal plasma, but the increase was significantly higher in the treated groups than in control group. Thus, the antioxidative capacity of the seminal plasma of treated bulls was proven to be higher (M and A groups). Cytomorphology analyzes of semen showed that there is no significant differences between groups according to the average of live and dead cells ratio