Effects of a lecithin and catalase containing semen extender and a second dilution with different enhancing buffers on the quality of cold-stored canine spermatozoa.

In the present study, a diluent containing 0.8% lecithin (Minitube®, Tiefenbach, G) for the cold storage of canine semen was compared to a Tris-egg yolk extender (TRIS-EY) containing 20% egg yolk. For this purpose, aliquots of 10 mixed ejaculates (main fractions) were either diluted with TRIS-EY or with three lecithin extenders containing 0.8% lecithin with or without catalase and tyrosine. All samples were examined by computer assisted sperm analysis (CASA), chlortetracycline assay (CTC) and flow cytometry, sperm chromatin structure assay (SCSA) and zona pellucida binding assay (ZBA). Samples were then cold stored for 8 d and examinations repeated at days 3 and 8. Measurement in the CASA were repeated daily and prior to measurement, each sample was diluted with each of 4 enhancers with or without acetylcarnitine. The use of an enhancer proved to be essential for all extenders and after 8 d of cooling, progressive motility (P) and viability (V) still averaged > 70% and > 80% with the lecithin extenders containing additives, whereas with TRIS-EY and without additives it was significantly lower (P < 0.05). The percentage of capacitated spermatozoa did not differ between extender groups and there was no significant increase in acrosome reactions (AR) within 3 d. The chromatin status of cells was not changed by cooling within 8 d. The ZBA showed that with additives, significantly more spermatozoa bound to oocytes when a lecithin extender with additives was used (P < 0.05). In conclusion, the 0.8% lecithin extender containing catalase, conserved P and V during 8 d of cold storage better than the TRIS-EY extender, however, only when an enhancer was used; addition of acetylcarnitine to the enhancer did not further improve semen quality. The here introduced lecithin extender / enhancer combination is a useful tool for prolonged storage of cooled semen with excellent longevity and binding ability; addition of tyrosine to the extender did not improve semen quality.

Comparison of different extenders with defined protein composition for storage of stallion spermatozoa at 5 degrees C.

To maintain the fertility of stallion spermatozoa during cooled storage, extender media are added to semen. In this study, three semen extenders were compared: EquiPro which contains defined caseinates and whey proteins instead of dried skim milk. The extender is provided in dry form and dissolved in distilled water prior to use. EquiPro TM has the same composition as EquiPro but is provided in a sterilized ready-to-use liquid form. AndroMed-E contains soybean lecithin as protein source. Semen was collected from seven stallions. Ejaculates were divided into three aliquots, diluted with the different extenders and stored at 5 degrees C for 4 days. Total motility, membrane integrity, average path velocity (VAP), curvilinear-velocity (VCL), straight-line velocity (VSL), distance average path (DAP), distance curved line (DCL) and distance straight line (DSL) were determined by computer-assisted analysis. Total motility decreased in all extenders during storage. The parameters VAP, VCL, VSL, DAP, DCL and DSL in semen diluted in EquiPro TM at most times and in semen diluted in AndroMed-E at some times were lower than in semen diluted in EquiPro (p < 0.05). Viability on days 0 and 4 was lowest in semen diluted in AndroMed-E (p < 0.05). Velocity decreased faster when semen had been diluted in the sterilized liquid extender EquiPro TM or in AndroMed-E compared with the dry formula of EquiPro. Therefore the liquid sterilized EquiPro despite no difference in its chemical composition differs from the dry, non-sterilized EquiPro extender. Heat sterilization apparently changes effects of the extender on spermatozoa.

[A thermodynamic study on bovine spermatozoa by microcalorimetry after Percoll density-gradient centrifugation - experimental probe of its utility in andrology]. / Mikrokalorimetrische Untersuchungen an bovinen Spermatozoen nach Percoll-Dichtegradienten-Zentrifugation - ein experimenteller Beitrag zum Einsatz in der Andrologie.

PURPOSE: Microcalorimetric measurements can be used for recording exothermic or endothermic summation effects of a great variety of biological processes. The aim of the present study was to examine the usefullness of the microcalorimetry method to characterise the biological activity of spermatozoa. MATERIALS AND METHODS: The heat flow of bovine fresh sperm as well as cryosperm samples were measured after Percoll density-gradient centrifugation in a 4-channel microcalorimeter. Various calibration times, volumes of samples and sperm concentrations were tested and analysed. Sperm concentration was recorded by a computer-assisted, computer-aided software system method (CASA). RESULTS: Using a calibration time of 15 minutes, the heat signal of the fresh and cryosperm samples showed a characteristic peak after 39.5 min and 38.1 min (mean), respectively, with a significant correlation to sample volume and sperm concentration (p < 0.05). For obtaining the best results, a sample volume of 1 ml and a sperm concentration of more than 50 x 10 (6)/mL was used. CONCLUSIONS: With microcalorimetric measurements the biological activity of spermatozoa could be recorded for reproducible results, thus opening the way to an automatised ejaculate analysis in the future. More investigations are necessary to correlate microcalorimetric parameters with semen function.

A new combined in-vitro test model for the identification of substances affecting essential sperm functions.

Binding of mammalian spermatozoa to the zona pellucida and the induction of the acrosome reaction are prerequisites for successful oocyte fertilization. It has been postulated that xenobiotics that are released in the environment as well as exposure to pharmaceutical medications may be associated with reproductive problems in men and wildlife. Examining physiological and non-physiological effects of particular compounds on sperm functions requires high quality in-vitro test systems. We established a reliable combined in-vitro test system with bovine gametes and evaluated if aliquots of pooled post-thaw spermatozoa are suitable for examining essential sperm functions. Using cryopreserved semen, the PSA-FITC/Hoechst 33258 staining procedure was applicable to evaluate the acrosomal status and cell viability. In the bovine hemizona assay, hemizona indices revealed no differences between cryopreserved and fresh semen. Treatment of post-thaw bovine spermatozoa with progesterone (1 microM or bovine follicular fluid (20%) induced the acrosome reaction from 12% (untreated spermatozoa) to 25% (P < 0.001) and to 22% [corrected] (P < 0.01), respectively. Incubation of both compounds (1 microM progesterone and 20% follicular fluid) raised the percentage of acrosome-reacted spermatozoa to 30% (P < 0001). Our results demonstrate that cryopreserved semen can be integrated into an in-vitro screening model for reproductive toxicology testing. Pooled, cryopreserved bovine spermatozoa will thus permit reproducible experiments for clinical and basic science purposes and may also be applicable for the human system.

[Investigations on egg yolk-free diluting medium for the cryopreservation of bull spermatozoa]. / Untersuchungen an einem eidotterfreien Verdünnungsmedium für die Tiefgefrier-Konservierung von Bullensperma.

On the basis of various laboratory researches and field experiments with 959 alternating first inseminations the traditional tris-extender and a yolk-free medium for semen freezing on soy lecithin base were compared in studies using a split-sample technique. Concerning important criteria such as non-return-rate, conservation quality, and standard of hygiene and practicability, the alternative product was found at least of equal quality. The renunciation of chicken yolk is to be seen as a general advantage. Here, the development of cryomedia has been enriched by a useful and sensitive new component. Apart from being competitive this product is also the first to conform to future demands in standardization through ISO-norm.

Identification of heterotrimeric G proteins in human sperm tail membranes.

Heterotrimeric G proteins play important roles as signal transducing components in various mammalian sperm functions. We were interested in the distribution of G proteins in human sperm tails. Prior to membrane preparation, spermatozoa were separated from contaminating cells which are frequently present in human ejaculates. Enriched human sperm tail membranes were generated by using hypoosmotic swelling and homogenization procedures. Antisera against synthetic peptides were used to identify G proteins in immunoblots. AS 8, an antiserum directed against an amino acid sequence that is found in most G protein alpha-subunits, and A 86, which detects all known pertussis toxin-sensitive alpha-subunits, reacted specifically with a 40-kDa protein. Antisera against individual G protein alpha-subunits failed to detect any specific antigens in enriched tail membranes. AS 36, recognizing the beta 2-subunit of G proteins, identified a 35-kDa protein in sperm tail membranes. Antisera against the 36-kDa beta 1-subunit did not detect any relevant proteins in the membrane fraction. Neither G protein alpha-subunits nor G protein beta-subunits were found in the cytosol. ADP ribosylation of spermatozoal membrane or cytosolic proteins revealed no pertussis toxin-sensitive alpha-subunits. However, membrane preparations of nonpurified human spermatozoa contained alpha i2 subunits, as shown immunologically and by ADP ribosylation; they most probably derived from somatic cells which are frequently present in human ejaculates. Our results stress the fact that spermatozoa need to be purified before sperm membrane preparation to avoid misinterpretations caused by contaminating cells. Furthermore, we suggest that G proteins in membranes of human sperm tails belong to a novel subtype of G protein alpha-subunits; the putative beta-subunit was identified as a beta 2-subunit.