Interrelationships among fluorometric analyses of spermatozoal function, classical semen quality parameters and the fertility of frozen-thawed bovine spermatozoa.

Cryopreserved spermatozoa from 8 bulls were used to examine the interrelationships among flow cytometric spermatozoal quality assessments and classical semen quality parameters and nonreturn rate estimates of fertility. The integrity of the sperm cell membrane and the functional capacity of the mitochondria were quantified by flow cytometry after concurrent staining with carboxydimethylfluorescein diacetate (CDMFDA), propidium iodide (PI), and rhodamine 123 (R123). For each sample a total of 10,000 stained spermatozoa were simultaneously quantified for the intensity of their green and red fluorescence. Three straws from each bull were each examined initially and following incubation at 37 degrees C for 3 hours to assess the rate of senescence. The proportion of spermatozoa retaining membrane integrity and having functional mitochondria, as determined by CDMFDA and R123 staining, were compared with classical semen quality assessments (sperm motility, acrosomal status, cellular and head morphology, presence of vacuoles/craters and cytoplasmic droplets) and with fertility (nonreturn to estrus rates). For individual ejaculates nonreturn rates, the range was from 61.8 to 78.8%, whereas the cumulative rates of several ejaculates for each bull ranged from 71.3 to 83.5%. The proportion of spermatozoa with functional membranes and mitochondria were positively correlated with the percentage of spermatozoa with normal morphology (r=0.82; P=0.01) and motility after 4 hours of incubation (r=0.78; P=0.02), but not with the estimates of fertility. The actual number of spermatozoa per straw staining with CDMFDA and R123 after 4 hours of incubation at 37 degrees C was correlated with the percentage of spermatozoa with normal morphology (r=0.73; P=0.04). Multiple regression equations indicated that combinations of semen quality measurements could be useful in estimating fertilizing potential.

Metabolic toxicity of fluorescent stains on thawed cryopreserved bovine sperm cells.

Several fluorescent probes, including derivatives of carboxyfluorescein, carbocyanine, ethidium, and rhodamine, have been used to assess sperm viability. However, the effects of these fluorescent dyes on the metabolic activity of sperm cells have not been systematically examined. This study was conducted to determine the effect of specific fluorescent stains on the metabolic processes of sperm. Cryopreserved bovine sperm cells were thawed, fluorescently stained, and examined using metabolic and flow cytometric techniques. Sperm were stained with either rhodamine 123 (Rhod-123), the aliphatic cell-tracking compound PKH2-GL, dihydro-ethidium (HED), the bisbenzimide stain Hoechst 33342 (Ho33342), or left unstained. The stained samples were compared for metabolic activity, cell staining pattern, and fluorescent intensity over a 180-min period. Samples stained with HED, Ho33342, and PKH2-GL had less oxygen uptake when compared with the unstained sperm samples (p greater than 0.05). Unstained samples and samples stained with Rhod-123 had similar oxygen consumption. The carbon dioxide produced during the 180 min was not different between controls and stained samples. Therefore, some fluorescent probes inhibit the oxygen metabolism of thawed, cryopreserved bovine sperm cells.

The effect of cottonseed components on testicular development in pubescent rams.

The effect of gossypol on testicular development in rams was examined using 2 experimental approaches. Trial I utilized direct ruminal deposition of gossypol acetic acid (GAA; 95% purity), whereas Trial II compared 2 types of dietary cottonseed, regular and glandless. For Trial I, 18 Polypay or Polypay x Dorset ram lambs, aged 4 to 6 mo, were surgically fitted with rumen cannulae. These 18 ram lambs were divided into groups of 6 which were given either 0, 2.2 or 6.6 mg of GAA/kg body weight daily via gelatin capsules directly into the rumen. All rams in Trial I were slaughtered after 4 or 8 w of treatment to allow recovery of their scrotal contents. The mean testicular weight for the group of rams given 6.6 mg GAA/kg/d was 8% (p less than .05) less than for the rams given 0 mg GAA/kg/d. The caudal epididymal sperm were examined microscopically for percentage of progressively motile sperm and flow cytometrically for mitochondrial function using rhodamine 123 (R123). Differences in sperm motility were not evident among the treatment groups, nor were differences detectable in the 2 major sperm populations, B and C, that were identified by flow cytometry. In Trial II, 21 Polypay and Polypay-Dorset ram lambs, aged 10 mo, were randomly assigned into 3 groups (n = 7) and fed either glandless fuzzy cottonseed (GFC), glandless flake cottonseed (GFL) or regular fuzzy cottonseed (RFC). All rams were fed for 5 w a total daily ration (TDR) equivalent to 3.5% of their body weight daily with 25% being cottonseed. After 8 w the scrotal contents were recovered by castration.(ABSTRACT TRUNCATED AT 250 WORDS)