Benefits of TEMPOL on ram semen motility and in vitro fertility: a preliminary study

Extending the preservation time of fresh semen is an important goal in artificial insemination programs particularly for ewes in natural oestrus, where insemination periods are longer than for ewes synchronized with hormonal treatments. The aim of this study was to evaluate the effect of the antioxidant TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) on the maintenance in long term storage of ram semen motility and fertility. Semen from Sarda breed rams was diluted in two extenders: sodium citrate buffer with TEMPOL and skimmed milk, used as control. Samples diluted with TEMPOL were cooled at either 15 °C or 22 °C, while those diluted with skimmed milk were cooled at 15 °C. Each sample was divided into four stocks, and stored for different times (5 min, 24, 48 and 72 h). Three aliquots were taken from each stock for every storage period. One was immediately evaluated under microscope; one was used for in vitro fertilization; one was incubated for 2 h in controlled humidified atmosphere (5% CO 2, 7% O 2 and 88% N 2) at 39 °C, then evaluated for motility and utilized for in vitro fertilization. Ram semen diluted with media containing TEMPOL demonstrated increased motility, fertility and an improved protective effect when it was stored at 15 °C.