TRIS-lecithin Extender Supplemented with Antioxidant Catalase for Chilling of Canine Semen

Introduction - Extenders for chilled semen usually contain egg yolk (EY) that has an important role in protecting sperm cells from cold-shock. EY is a complex biological compound which is not chemically defined. A chemically defined extender would allow a reliable evaluation of effects when different compounds are tested. Among these, antioxidants could play an important role during conservation because a spontaneous generation of reactive oxygen species (ROS) occurs over time. ROS have been reported to cause poor semen quality because they induce peroxidation of polyunsaturated fatty acids of sperm membrane (8). Antioxidant-supplemented semen extenders have shown variable efficacy for improvement of canine semen. Catalase (CAT) in TRIS-EY extender improved post-thaw quality of canine spermatozoa (4), but did not improve semen quality at 5°C (3). The effect of CAT should be tested in a chemically defined extender. A commercially available extender in which EY is substituted by soybean lecithin has been recently tested in different species (1,2). In the dog, the post-thaw quality of sperm was lower than that obtained in the presence of EY (5). No information is, however, available on the effect of TRIS-lecithin extender during chilling. Thus, the present study evaluated the effect of different concentrations of CAT in non commercial TRIS-lecithin extender on motility, capacitation and zona binding capacity of canine spermatozoa stored at +5°C for 4 days.

Materials and Methods - All chemicals were purchased from the Sigma Chemical Company (St. Louis, MO, USA) unless otherwise stated. Four privately owned stud dogs were used. The sperm-rich fraction of the ejaculates was collected by digital manipulation. Only ejaculates containing a minimum of 70% progressively motile and morphologically normal spermatozoa were included. The sperm concentration was measured with a Bürker chamber and semen samples were divided into 4 aliquots. After centrifugation and removal of the seminal plasma, sperm pellets were diluted (200x106 sperm/ml) in TRIS buffer, citric acid, glucose, antibiotics, supplemented with 20% egg yolk (TRIS-EY) or 0.04% soybean lecithin (TRIS-LC; Solae Company, St. Louis, MO, USA) with CAT (150 or 450 UI/ml) or without CAT. Sperm motility was evaluated by microscopic observation at the time of collection, after dilution (Time 0), at days 1 and 4 of cold storage at +5°C. Capacitation status by Chlortetracycline assay (6) and zona pellucida (ZP) binding assay using intact, homologous, denuded oocytes recovered from frozen-stored ovaries (7), were performed at the time of collection, and at days 1 and 4 of cold storage. Sperm-oocyte complexes were stained with bis-benzimide (Hoechst 33342). The number of sperm bound was counted using a fluorescent microscope (Axiovert 100, Zeiss, Italy), and the mean number of sperm bound per ZP was used as the endpoint. Oocytes with >200 sperm bound to ZP were recorded as 200. Significant differences (P<0.05) were determined by Student’s t-test. Mean values are presented as mean+SEM.

Results – Mean ejaculate volume was 1.3±0.4 ml, sperm concentration was 349.8±112.4×106/ml, sperm motility (%) was 87.5±2.5, morphology (%) was 87±3.7 normal sperm, and uncapacitated spermatozoa (%) was 89.4±4.1, respectively. The mean number of ZP-bound sperm was >200. Motility rates, uncapacitated spermatozoa and ZP-bound spermatozoa did not differ between TRIS-EY and TRIS-LC at any of the time points (Table 1 and 2). CAT addition neither improved motility nor membrane integrity. The ZP assay showed that 4 days of chilling significantly reduced ZP binding capacity in general, but there was a strong tendency for increased binding at days 1 and 4 when CAT was added. [...]