Effect of long-term immunization against inhibin on sperm output in bulls.

To determine the effect of neutralization of inhibin on sperm output, 12 Holstein bulls were paired by birth date and weight on Day 1 of age. Each bull was actively immunized against bovine inhibin alpha1-26 gly-tyr (bINH) conjugated to human alpha globulin (HAG, n = 6 bulls) or HAG alone (controls, n = 6) at 60 days of age; booster immunizations were administered at 90, 104, 124, 270, and 395 days of age. Body weights and scrotal circumferences were measured at the time of primary immunization and at 10 days after each booster. In addition, jugular blood was obtained at 60, 70, 100, 114, 134, 280, and 405 days of age, during the 3-wk sperm collection period, and during a 6-h blood-sampling period after sperm collection to determine bINH antibody titer and concentrations of FSH, LH, testosterone, and estradiol. Beginning at 405 days of age, sperm output was measured 3 days/wk for 3 wk with two successive ejaculates collected each day for a total of 18 ejaculates per bull. During Days 60-405 of age, the increase in titer of bINH antibodies, scrotal circumference, and serum concentration of FSH was greater (p < 0.01) for the bINH-immunized compared with control bulls. There were significant (p < 0.01) pair x treatment interactions for sperm output and serum FSH and LH concentrations. Specifically, bINH-immunized bulls for four of the six pairs had nearly 50% greater serum FSH concentrations and sperm output. For the remaining two pairs, sperm output was lower and FSH was either lower or only marginally higher in the bINH-immunized bulls compared with controls. Also, the control bulls for the two remaining pairs produced more sperm than all but one bINH-immunized bull, and had markedly higher serum LH concentrations than all other bulls. To summarize, enhancement of sperm output after immunization against inhibin depends on the subsequent increment in FSH concentrations. We conclude that inhibin suppresses spermatogenesis. Thus, methods to immunoneutralize inhibin may have merit as a therapeutic route to enhance sperm production in reproductively maturing bulls.

A technique for the evaluation of sperm penetrating ability and quality of bovine semen processed in an extender made with Brackett-Oliphant medium and egg yolk.

Egg yolk-sodium citrate (EYC) semen extender was compared with an extender made of Brackett-Oliphant medium and egg yolk (BOEY). Ejaculates were divided into equal portions, processed and frozen. Semen was thawed and evaluated for quality. Additional semen was thawed, stained with Hoechst 33342 and the spermatozoa capacitated, after which they were co-incubated with zona-free hamster oocytes to determine their penetrating ability. Sperm penetration of non-compressed, unfixed oocytes was evaluated using an optical sectioning technique on a standard research microscope. Sperm penetration was considered successful if a fluorescing sperm head was observed within the living oocyte in a hanging drop of fertilization medium. There were small differences in percentage of secondary abnormalities and percentage of progressive motility immediately after thawing between spermatozoa extended in EYC or BOEY diluent. There were no differences due to by extender composition in percentage of spermatozoa with intact acrosomes or percent of progressively motile after a 3 h incubation at 37 degrees C, nor the percentage of spermatozoa with head abnormalities. While there were significant correlations between all seminal quality characteristics, no quality measurements were correlated to percentage of oocyte penetration. The new penetration evaluation method allowed for examination of the fertilized oocytes using fluorescent microscopy initially and again after re-incubation for further development.

Nanovid microscopy for assessing sperm membrane changes induced by in vitro capacitating and acrosomal reacting procedures.

This study was to verify the usefulness of Nanovid microscopy techniques for evaluating induced modifications in bovine spermatozoal membranes. Frozen thawed bovine sperm were labeled with 20-nm colloidal gold particles bound to concanavalin A (ConA) or heparin ligands. Sperm membrane changes were induced in vitro by capacitating and acrosome-reacting procedures. Capacitation was induced by incubation with 10 micrograms/ml of heparin for 4 hours at 37 degrees C, 5% CO2, and high humidity. Membrane changes associated with the acrosome reaction were induced by addition of lysophosphatidylcholine (100 micrograms/ml) and incubation for 15 minutes at 37 degrees C, 5% CO2, and high humidity. Gray intensity (black = 0; white = 255) of sperm (ONCELL) and background (OFFCELL) were evaluated with computer-enhanced videomicroscopy with either differential interference contrast (DIC) or Nanovid optics. A high gold concentration on a membrane region produced blacker video pictures with Nanovid microscopy. Gray intensity of video pictures of a region with little or no gold would have a gray intensity equal to or greater than that of the background, that is, toward white. Weighted least squares methods were used to analyze ONCELL data using OFFCELL as a covariate. In experiment 1, ONCELL intensities of cells labeled with ConA-gold complex were lower than those labeled with heparin-gold at the same treatment level. In experiment 2, ONCELL intensity decreased as the concentration of heparin-gold increased from 0 to 0.041 microgram/microliter heparin. ONCELL intensity significantly decreased after sperm were treated with the highest heparin-gold level and acrosome reacted.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of freezing semen and dosage of sperm on number of accessory sperm, fertility, and embryo quality in artificially inseminated cattle.

This experiment was conducted to determine whether use of fresh or frozen semen at either 20 x 10(6) (low) or 100 x 10(6) (high) sperm per dose affects the number of accessory sperm and fertilization status/embryo quality as determined from ova/embryos recovered nonsurgically 6 d after insemination. Ejaculates of four bulls were split and prepared for use as fresh or frozen semen at either the high or low dose. From 129 inseminations to normally cycling cows, 98 ova/embryos were recovered. To reduce male effects, ova/embryos used were randomly balanced across treatments, by ejaculate within bull for evaluation of frozen vs fresh semen (n = 80) and by bull for evaluation of high vs low dosage treatments (n = 76). Distribution of accessory sperm was highly skewed downward; thus, median values were more meaningful than means. Freezing semen had no significant effect on fertility status/embryo quality or number of accessory sperm at either dosage. Increasing dosage improved the number of accessory sperm per ovum or embryo (median value) and fertility status/embryo quality (P < .05). Mean +/- SD and median values for accessory sperm were 37.8 +/- 38.3 and 27.5; 28.9 +/- 62.8 and 3.0 for the high and low dose, respectively. Percentage of unfertilized ova, degenerate embryos, and embryos classified poor to fair and good to excellent were 3, 5, 24, 68, and 21, 16, 18, 45, for the high and low dose, respectively. We conclude that number of accessory sperm and fertility status/embryo quality respond favorably to increased dosage of semen and that freezing semen in this study was not detrimental to these parameters.