Effect of the herbicide atrazine and its major metabolite, DACT, on bovine sperm cryotolerance

Abstract

During freezing and thawing procedures, sperm are exposed to chemical and/or physical stressors that may cause adverse and harmful changes to sperm membranes. Accurate evaluation of the structural and functional integrity of fresh as well as cryopreserved sperm is highly important in predicting sperm fertilization capacity and success of artificial insemination (AI). The herbicide atrazine (ATZ) and its major metabolite, diaminochlorotriazine (DACT) are considered a ubiquitous environmental contaminants and endocrine disruptors, which deleteriously effect sperm function. Taking into consideration possible damage caused by environmental contaminants to sperm membranes, additive effects during cryopreservation cannot be ruled out. The aim of the current study was to evaluate the effect of ATZ (0.1 or 1 μM) and DACT (1 or 10 μM) exposure during or after cryopreservation on bovine sperm cryotolerance. Sperm membrane integrity and functionality were evaluated using fluorimetric probes: (1) double-stranded DNA was examined by 4′,6-diamidino-2-phenylindole; (2) plasma membrane integrity was examined by propidium iodide; (3) acrosome reaction (AR) was examined by fluorescein isothiocyanate-conjugated Pisum sativum agglutinin; mitochondrial membrane potential (ΔΨm) was examined by 5,5′,6,6′-tetra-chloro-1,1′,3,3′-tetraethylbenzimidazolyl carbocyanine iodide fluorescent probe. The findings demonstrate, that exposure of sperm to ATZ (0.1 or 1 μM) or DACT (1 or 10 μM) during cryopreservation increased the proportion of dead sperm relative to the control (P < 0.09); exposure to DACT (1 or 10 μM) increased ΔΨm (P < 0.03). Neither ATZ nor DACT affected spontaneous AR. In contrast, the proportion of sperm with Ca++ ionophore-induced AR was lower after exposure to 1 μM DACT (P < 0.05). Following freezing and thawing procedures, exposing sperm to 1 μM ATZ increased the proportion of dead sperm relative to the control (P < 0.05), but had no significant effect on sperm ΔΨm or AR. In conclusion, exposing sperm to endocrine-disrupting chemicals such as ATZ or DACT during cryopreservation reduces sperm cryotolerance and resistance post-thawing.