20 SPERM CHARACTERIZATION OF ASTURCON PONIES AFTER COLLECTION AND EQUILIBRATION/REFRIGERATION BEFORE FREEZING

Abstract

The need to conserve farm animal biodiversity is accepted by many countries through the ratification of the convention of biological diversity, and sperm quality is known to be an important criterion in the evaluation of breeding soundness. The aim of this work was to characterize the semen of a local breed of ponies 'Asturcon' (maintained free over the mountains all year around) before its incorporation into a germplasm bank. Semen was obtained from six stallions (6–17 years of age) using an artificial vagina, 3 days/week, during 12 weeks. Immediately after collection, gel-free semen was evaluated for volume, sperm concentration, and motility. Semen motility was again evaluated after equilibration/refrigeration. For evaluation of individual (IM) and progressive motility (PM) rates, semen was diluted (20 × 10⁶ spermatozoa/mL) and analyzed with a CASA System (SCA; Microptic S.L., Barcelona, Spain). Five fields per sample were evaluated (minimum 500 spermatozoa/sample) under a phase contrast microscope (100×). Semen samples were subjected to a hypo-osmotic swelling test (HOS) test to detect the presence of swollen tails in a 100 mM citrate–fructose solution. Percentages of altered acrosomes and morphological abnormalities were determined by counting 100 spermatozoa (1000×). Then, semen was diluted and centrifuged for 10 min at 600g. After the supernatant was discarded, the pellet was re-suspended in freezing medium (skim milk extender containing 2% egg yolk and 2.5% glycerol) to a final concentration of 100 × 10⁶ spermatozoa/mL, and equilibrated/cooled (60 min) to 4°C. Statistical analysis was carried out by means of the GLM and CORR procedures and Duncan test for means (SAS Institute, Inc., Cary, NC, USA). A significant effect between males (P < 0.05) on semen quality, such as volume of the ejaculate, sperm concentration, and morphological abnormalities, were detected among stallions. On the other hand, positive and significant correlations were found between the sperm motility immediately after collection and after equilbration/refrigeration (r = 0.73; P < 0.05); moreover, sperm motilities (both fresh and refrigerated) correlated with the results of the HOS test (r = 0.56; P < 0.001, and r = 0.27, P < 0.05, respectively). These preliminary results confirm that the sperm of the Asturcon ponies breed can be collected and will survive the equilibration/refrigeration procedures. Conservation and development of local breeds is important because they represent a unique source of genes for improving health and performance traits of industrial breeds. However, complementary studies on the ability of the stallion sperm to survive freezing/thawing procedures in rates higher than 30% are needed to ensure that genetic banks are correctly created.

This work was performed in collaboration with ACPRA and Depósito de Sementales de Santander (Spain), and supported by RZ2004-00031-C02-01.