61 PLANT EXTRACTS REDUCE DNA FRAGMENTATION IN FROZEN–THAWED STALLION SPERM

Abstract

Mares inseminated with frozen–thawed sperm have reduced pregnancy rates compared with mares inseminated with fresh sperm. Processing mammalian sperm for cryopreservation increases the concentration of free radicals and induces oxidative stress, which can result in DNA damage and may lead to lower fertility. The objective of this experiment was to examine the effects of several plant antioxidant extracts on stallion sperm post-thaw motility and DNA quality. Single ejaculates were collected from 4 stallions and the concentration of sperm cells in each ejaculate was determined spectrophotometrically. Semen was centrifuged at 300g for 10 min at room temperature and seminal plasma was removed. Sperm pellets were resuspended to a final concentration of 200 × 10⁶ cells mL^–1 in E-Z Freezin LE (ARS, Chino, CA) extender (control) or extender containing 1 of 3 plant extracts (3% v/v) from 2 different commercial sources. Extended sperm cells were loaded into 0.5-mL straws and frozen over liquid nitrogen vapor for 10 min. Straws were then plunged into liquid nitrogen and stored until further evaluation. Motility and velocity parameters were determined at 0, 30, and 60 min post-thaw using a computer-assisted sperm analyzer. DNA fragmentation was determined immediately after thawing using a single-cell gel electrophoresis (Comet) assay. Motility (total and progressive) and velocity parameters of sperm cells did not differ between controls and plant extract treatments (P > 0.05). However, total Comet length and tail length were reduced in sperm cells stored in extender containing each plant extract (P < 0.05). Tail and olive moment tended to be reduced (P < 0.10) in sperm cells stored in plant extracts. In conclusion, sperm cells stored in plant extracts had reduced post-thaw DNA damage. The addition of plant extracts to commercial freezing extenders may be a practical method for improving sperm quality.