Effect of the addition of two superoxide dismutase analogues (Tempo and Tempol) to alpaca semen extender for cryopreservation

Abstract

The main objective was to study the effects, on sperm function, of the addition of two superoxide dismutase (SOD) analogues (Tempo and Tempol) to alpaca semen extender for cryopreservation. Twelve alpaca semen samples were collected using an artificial vagina and then diluted at a 1:3 ratio in an extender based on skim milk, egg yolk, and fructose. Each semen sample was divided into three equal parts to form the following groups: control, Tempo (1 mM), and Tempol (1 mM). Groups were cooled to 5 °C in 90 minutes (-1 °C in 3 minutes); when samples reached approximately 10 °C, SOD analogues were added to the respective groups. At 5 °C, ethylene glycol (final concentration, 0.1 M) was added to each group. After 30 minutes at 5 °C, samples were loaded in 0.25 mL plastic straws, placed in liquid nitrogen vapor for 15 minutes, and then plunged. Percentages of sperm motility, functional sperm membrane integrity, and viable sperm with intact acrosomes were evaluated before and after freeze-thaw using visual analysis, the hypoosmotic swelling test, and the double-stain trypan blue/giemsa technique, respectively. The Terminal deoxymucleotidyl transferase dUTP Nick End Labeling assay was performed for evaluation of sperm DNA fragmentation of frozen-thawed sperm. Sperm motility was higher (P < 0.05) in the Tempol and Tempo groups than in the control group (mean, 22.1%, 19.7%, and 11.2%, respectively), with similar results for functional sperm membrane integrity. Additionally, DNA fragmentation was lower (P < 0.05) in the Tempol group (16.7%) than in the control group (38.8%). Viable sperm with intact acrosomes were not affected by the use of SOD analogues. There was a negative correlation (r = -0.58) between DNA fragmentation of alpaca sperm and sperm motility after freeze-thawing, but DNA damage was neither related to functional membrane integrity nor viable sperm with intact acrosomes. We concluded that DNA fragmentation and loss of motility during cryopreservation of alpaca sperm could be partially prevented by supplementation of the semen extender with 1 mM Tempo or Tempol.