I De Pauw

In vitro survival of bovine spermatozoa stored at room temperature under epididymal conditions

In this study, environmental conditions mimicking those prevailing in the epididymis were used for storing ejaculated bull spermatozoa in vitro during 4 days at ambient temperature. These conditions were low pH, high osmolarity, high sperm concentration and low oxygen tension. Hepes-TALP was used as basic storage medium. Fresh spermatozoa were stored at a concentration of 10 x 10(6)spermatozoa/ml in Hepes-TALP of different pH (pH 4, 5, 6, 7 or 8), and osmolarity (100, 300, 400, 500, 600 or 800 mOsm/kg), and under different atmospheric conditions (nitrogen gassed or aerobic). Spermatozoa were also stored undiluted or at different concentrations: 10x 10(6), 100 x 10(6), 500 x 10(6) or 1 x 10(9)spermatozoa/ml. Sperm parameters such as membrane integrity, motility, mitochondrial membrane potential or DNA fragmentation were used to assess semen quality after storage. Adjustment of the pH of Hepes-TALP to pH 6 yielded significantly better results than storage at all other pH values. Isotonic Hepes-TALP (300 mOsm/kg) had a less detrimental effect on spermatozoa than hypo- and hyperosmotic versions. No differences in sperm parameters were observed when spermatozoa were incubated under aerobic or under nitrogen gassed storage conditions. Optimal sperm concentration in vitro is 10 x 10(6)spermatozoa/ml. This is in contrast with the in vivo situation, where spermatozoa are stored at high concentration. However, better results at high sperm concentrations were obtained when spermatozoa were diluted for less than 5 min in Triladyl-egg yolk-glycerol diluent immediately after ejaculation.

Migration of bovine spermatozoa in a synthetic medium and its relation to in vivo bull fertility

Although sperm migration has been extensively refined and validated in human infertility studies, its application to predict bovine fertility has been very limited, and a clear relation between the sperm migration distance and in vivo bull fertility has never been demonstrated. A synthetic medium based upon methyl cellulose (MC) was tested for its suitability to serve as a migration medium for frozen-thawed bovine spermatozoa. The effects of the concentration of MC, the incubation time, and sperm concentration on sperm migration capacity was determined. The relation between sperm migration capacity at different incubation times of the frozen-thawed spermatozoa of five bulls, and their 56 days nonreturn rates (NRRs) was assessed in order to evaluate its suitability as a tool to predict in vivo bull fertility. The highest repeatability of the sperm migration test (CV = 10.7%) was obtained when the sperm migration distance of the five vanguard motile spermatozoa was determined at 30 min incubation at 37 degrees C in a migration medium with 1.35% MC. No significant difference in migration distance was demonstrated when sperm concentrations of 100 x 10(6) and 150 x 10(6) spermatozoa/ml, respectively, were used. Despite the relatively high repeatability of the migration test, no relation was found between the sperm migration distance and the 56 days NRRs of five sire bulls. Therefore, the sperm migration test in 1.35% MC cannot be used to predict in vivo bull fertility accurately.

Ethylene Signaling: More Players in the Game

The Arabidopsis ethylene response pathway was established by characterization of ethylene mutants that were isolated exploiting the triple response of dark-grown seedlings. The various triple response screens were not yet exhaustive; however, a large number of ethylene-related loci have been identified, and for several of these an allelic series was isolated. To increase the chance of identifying new loci, screening for mutants at developmental stages other than the etiolated seedling stage might be a useful approach. We have isolated mutants from light-grown populations, by using the ethylene response of nutrient-deficient seedlings at two stages in development. Characterization of these mutants has resulted in the identification of new loci involved in ethylene signaling.