IN VITRO EFFECTS OF THE CHLAMYDOMONAS REINHARDTII EXTRACT ON BOVINE SPERMATOZOA

Abstract

In vitro storage and processing of animal semen is represents a risk factor to spermatozoa vitality, potentially leading to reduced fertility. A variety of substances isolated from natural sources may exhibit protective or antioxidant properties on the spermatozoon, thus extending the lifespan of stored ejaculates. Chlamydomonas has been shown to possess antimicrobial, anti-inflammatory and antioxidant properties, turning the extract into a potential candidate for preserving liquid animal semen during in vitro storage. This study compared the ability of different concentrations of the Chlamydomonas reinhardtii extract on the motility, viability and superoxide production of bovine spermatozoa during different time periods (0, 2, 6, 12 and 24h) of in vitro culture. Spermatozoa motility was assessed using the SpermVisionTM CASA (Computer aided sperm analysis) system. Cell viability was examined using the metabolic activity MTT assay and the nitroblue-tetrazolium (NBT) test was applied to quantify the intracellular superoxide formation. The CASA analysis revealed that the Chlamydomonas extract supplementation was able to prevent a rapid decline of spermatozoa motility, especially in the case of concentrations ranging between 1 and 5 µg/mL (P<0.001 with respect to Times 6h, 12h and 24h). At the same time, concentrations ranging between 5 and 10 µg/mL of the extract led to a significant preservation of the cell viability throughout short-term (P<0.05 in case of Time 6h) as well as long-term periods of the experiment (P<0.01 with respect to Time 12h, and P<0.001 in case of Time 24h). 5 and 10 µg/mL of the extract exhibited antioxidant characteristics, translated into a significant reduction of the intracellular superoxide production, particularly notable at Times 12h (P<0.01 with respect to10 µg/mL and P<0.05 in case of 5 µg/mL) and 24h (P<0.01). The results indicate that the Chlamydomonas extract is capable of delaying the damage inflicted to the spermatozoon by the in vitro environment.