111 ESTIMATION OF SPERM QUALITY IN FRESH AND FROZEN–THAWED SEMEN FROM ASTURIANA DE LOS VALLES BULLS

Abstract

Artificial insemination and semen cryopreservation have significantly improved the breeding potential of male animals. However, current freezing techniques commonly result in reduced semen quality (Januskauskas et al. 1999 Theriogenology 52, 641-658), and surviving cells are affected post-thaw either structurally or functionally (Nagy et al. 2004 Anim. Reprod. Sci. 80, 225-235). In this work we analyze the impact of cryopreservation on Asturiana de los Valles bull sperm. Ejaculates (n = 373) from seven adult bulls were weekly collected by means of artificial vagina. Immediately after collection, routine parameters including volume (V), mass motility (MM), and concentration (C) of sperm cells were evaluated. Then the semen was extended with a commercial extender, loaded into 0.25-mL plastic straws at a concentration of 23 × 10⁶ per straw, frozen and stored for further analysis. Four straws per ejaculate were thawed, pooled and analyzed for motion characteristics by means of a CASA system (Sperm Class Analyzer, SCA 2002^® Microptic S. L., Barcelona, Spain) added to an optical phase-contrast microscope with heatable (37°C) stage. Immediately after thawing, we analyzed the % of motile spermatozoa (MS) and the % of progressively motile spermatozoa (PMS); then samples were incubated for 3 h at 37°C and MS and PMS were measured again (MS3 and PMS3, respectively). Functional integrity of the plasmallema was evaluated by the hypoosmotic swelling test (HOST) together with the % of typical tail coiling/swelling (percentage of HOST-positive spermatozoa, HOST-PS). The % of viable spermatozoa (VS) [membrane integrity was evaluated by fluorescence microscopy with a dual staining system (propidium iodide (PI) and 6-carboxyfluorescein diacetate (CFDA)]. Sperm showing partial or complete red fluorescence (PI staining) were considered nonviable, whereas sperm showing complete green fluorescence were considered viable. Altered acrosomes (AA) and morphological abnormalities were also determined. The % of morphological abnormalities was classified according to their location in head (HA), midpiece (MA), and tail (TA). Proximal and distal cytoplasmic droplets were counted as separate abnormalities (CD). Data were analyzed by the MEANS procedure of SAS (SAS Institute, Inc., Cary, NC, USA). A significant (P < 0.05) decrease in the sperm motility was observed after freezing/thawing (MS: 80.20 ± 0.75 vs. 47.36 ± 1.04, and PMS: 68.73 ± 0.73 vs. 42.14 ± 0.96 for fresh and frozen-thawed semen, respectively). Also, the frozen-thawed sperm showed increased % of HA, MA, AA, HOST-PS, and VS (P < 0.05). These morphological abnormalities could contribute to decreasing sperm motility. The new computer and video technologies provide useful information about sperm quality and can be used in the daily routine of processing semen.

This work was performed in collaboration with ASEAVA.