Influence of porcine spermadhesins on the susceptibility of boar spermatozoa to high dilution

The effect of heparin-binding and non-heparin-binding spermadhesins on the viability, motility, and mitochondrial activity of boar spermatozoa at the high dilution (300,000 sperm/ml) to which sperm are exposed during the process of sex sorting by flow cytometry was investigated. Incubation of spermatozoa with heparin-binding spermadhesins caused a time- and dose-dependent decrease in the percentage of functional spermatozoa. The percentage of viable spermatozoa incubated at 38 degrees C with heparin-binding spermadhesins diluted in PBS (1 mg/ml) dropped from 75% (0.5 h) to 4% (5 h), whereas the percentage of viable spermatozoa incubated in PBS without proteins (control) decreased from 85% (0.5 h) to 19% (5 h). Addition of non-heparin-binding PSP-I/PSP-II spermadhesin to the PBS resulted in a concentration-dependent increment of the percentage of viable cells (65% after 5-h incubation), with maximum effect at 1.5 mg/ml. The heparin-binding spermadhesins totally suppressed sperm motility and mitochondrial activity after 5 h of incubation. The same parameters of sperm incubated in the presence of 1.5 mg/ml of PSP-I/PSP-II were 50% and 58%, respectively, and the percentages of control sperm displaying motility and mitochondrial activity were 21% and 26%, respectively. Moreover, the viability, motility, and mitochondrial activity all decreased on incubation of spermatozoa with mixtures of PSP-I/PSP-II and heparin-binding spermadhesins as the concentration of the latter increased. We conclude that PSP-I/PSP-II and the heparin-binding spermadhesins exert antagonistic effects on the functionality of highly diluted boar spermatozoa. The finding that PSP-I/PSP-II contributes to maintaining sperm with high viability, motility, and mitochondrial activity for at least 5 h at physiological temperature points to its potential use as an additive for sperm preservation, specifically of highly diluted, flow-sorted spermatozoa for sex preselection.     

A note on the effect of dilution and temperature on the bactericidal activity of potassium sorbate

O ragui , J.I. M ara , D.D.1984. A note on a modified membrane-Bovis agar for the enumeration of Streptococcus bovis by membrane filtration. Journal of Applied Bacteriology 56 , 179–181.
A modified membrane-Bovis agar, containing a reduced quantity of sodium azide, for the isolation and enumeration of Streptococcus bovis is described and evaluated. Higher counts, with larger colonies, were obtained from water and sewage samples with this medium than with the original formulation.     

The effect of dilution rate variation on the performance of continuous fermentation

The effect of dilution rate variation on the performance of bioreactors used for continuous biomass producton is discussed. Calculations based on Powell's model of microbial dynamics show that such a mode of operation does not improve the performance of the process. Remarkable improvement of the process performance reported in literature is caused by incorrectness of the dynamic model of microbial growth which has been used in the calculations.     

Ultrastructure of trout spermatozoa: Changes after dilution and deep-freezing

Summary Trout spermatozoa taken from the testis, vas deferens and ejaculate are described at the ultrastructural level. The morphology of the spermatozoa head, changes in the middle-piece structure, and the relationship between the centrioles and the flagellum were studied under consideration of their role in the reproduction of this species. Morphological changes observed after dilution of the spermatozoa in freshwater or saline and subsequent freezing deserve attention in connection with certain manipulations used in fish farms and laboratories.     

Pink-eyed dilution alleles affect negative surface charges of mouse spermatozoa

Abnormal spermiogenesis in sterile pink-eyed dilution mutants results in spermatozoa with bizarre sperm heads. The spermatozoa of normal mice bind colloidal iron hydroxide (CIH) along the length of the tail, yet the spermatozoa of pink-eyed sterile mice show a great reduction in ability to bind CIH. This implies a loss of negative surface charges. The group(s) responsible for the charges are sensitive to methylation but resistant to neuraminidase treatment, even after deacetylation with alkaline treatment. The membrane components containing the negatively charged groups may be neuraminidase-resistant forms of gangliosides.     

Acrosin activity in turkey spermatozoa: assay by clinical method and effect of zinc and benzamidine on the activity

We optimized a clinical assay developed for measuring total acrosin activity for mammalian and fish semen for use in turkey spermatozoa. The main modifications included dilution of semen to a final concentration of 25 to 1000 x 10(3) spermatozoa, an increase of Triton X-100 concentration to 0.05% and 1 hr preincubation without substrate, Acrosin activity in turkey spermatozoa was much higher than in human spermatozoa (about 100-times) but similar to that of boar sperm. To optimize this assay for turkey spermatozoa, it was necessary to use higher Triton X-100 concentrations in the reaction mixture. There was a better catalytic efficiency at higher temperatures and a special requirement for a preincubation period for proacrosin activation. We observed high inhibition of acrosin activity by zinc added during preincubation (90% at 0.01 mM of zinc chloride). Benzamidine also inhibited turkey acrosin, and the extent of inhibition was similar for the incubation or preincubation period. When zinc ions were added during incubation, this inhibition was lower (24%). The results suggest that zinc influences proacrosin activation of turkey spermatozoa. This influence may be important for successful long-term storage of spermatozoa in the hen's oviduct.     

Evaluation of the effect of butyl p-hydroxybenzoate on the proteolytic activity and membrane function of human spermatozoa

The inhibition of the proteolytic activity of acrosin in human spermatozoa by butyl p-hydroxybenzoate was assessed by the gelatin substrate film method. Compared with a typical acrosin inhibitor, TLCK, the inhibitory activity of butyl p-hydroxybenzoate to acrosin was much more effective (20 times) than that of TLCK, proving that butyl p-hydroxybenzoate was a potent acrosin inhibitor. The effect of butyl p-hydroxybenzoate on membrane function of human spermatozoa was evaluated using a sperm-tail hypoosmotic swelling test and supravital stain method. A good correlation (r = 0.92) was observed between the % spermatozoa with normal membrane function and the % live spermatozoa after treatment of the spermatozoa with butyl p-hydroxybenzoate for 1 min, indicating that the death of spermatozoa caused by butyl p-hydroxybenzoate is probably due to impairment of sperm membrane function. Both the inhibitory effect on acrosin and the adverse effect on membrane function suggest that butyl p-hydroxybenzoate could be developed as a new vaginal contraceptive.     

Physiology of spermatozoa at high dilution rates: the influence of seminal plasma

Extensive dilution of spermatozoa, as occurs during flow-cytometric sperm sorting, can reduce their motility and viability. These effects may be minimized by the use of appropriate dilution and collection media, containing balanced salts, energy sources, egg yolk and some protein. Dilution and flow-cytometric sorting of spermatozoa, which involves the removal of seminal plasma, also destabilizes sperm membranes leading to functional capacitation. This membrane destabilization renders the spermatozoa immediately capable of fertilization in vitro, or in vivo after deposition close to the site of fertilization, but shortens their lifespan, resulting in premature death if the cells are deposited in the female tract distant from the site of fertilization or are held in vitro at standard storage temperatures. This functional capacitation can be reversed in boar spermatozoa by inclusion of seminal plasma in the medium used to collect the cells from the cell sorter and, consequently, reduces their in vitro fertility. It has yet to be determined whether seminal plasma would have similar effects on flow cytometrically sorted spermatozoa of other species, and what its effects might be on the in vivo fertility of flow sorted boar.     

The cryoprotective effect of trehalose supplementation on boar spermatozoa quality

The objective was to compare the reproductive performances associated with the first (Cycle-1), second (Cycle-2), and mid-season (MS-Cycle) ovulations of the breeding season in donor mares that were treated with equine-FSH (eFSH) in the early vernal transition. Mares (n = 15) kept under ambient light were examined ultrasonographically per-rectum starting January 30. When an ovarian follicle ≥25 mm in diameter was detected, twice daily eFSH treatments were initiated. The eFSH treatments ceased when a follicle ≥35 mm was detected, and 36 h later hCG was administered. Thereafter, mares were artificially inseminated every 48 h until ovulation (Day 0). Trans-cervical embryo recovery attempts were performed on Day 8, and subsequently PGF2α was administered. Equine FSH was not administered in the subsequent estrous cycles. In Cycle-2 and in the MS-Cycle, hCG was administered when a follicle ≥35 mm was detected; breeding, embryo recovery, and PGF2α administration, were similar to Cycle-1. Mares had an untreated estrous cycle (no treatment or breeding) between Cycle-2 and the MS-Cycle. All mares developed follicle(s) ≥35 mm after 4.9 ± 0.6 days of eFSH treatment, and subsequently ovulations occurred; mean (95% CI) interval from treatment initiation to ovulation was 7.9 (6.5–9.3) days. The number of preovulatory follicles (≥30 mm) at the time of hCG administration (Cycle-1: 2.2 ± 0.3 compared with Cycle-2: 1.0 ± 0 compared with MS-Cycle: 1.1 ± 0.1 follicles), and the number of ovulations (2.5 ± 0.4 compared with 1.0 ± 0 compared with 1.1 ± 0.1 ovulations) were greater (p < 0.05) in Cycle-1. Nevertheless, mean embryo numbers did not differ among cycles (0.8 ± 0.2 compared with 0.5 ± 0.1 compared with 0.5 ± 0.1 embryo/mare). On average, embryo morphology grade was less (p < 0.05) in Cycle-1 as compared to non-eFSH cycles (combined Cycle-2 and MS-Cycle). This impaired embryo quality could be due to a seasonal effect, or negative effect of the eFSH treatment, which was possibly related to alterations in the hormonal environment (estradiol-17β and progesterone). A prolonged IOI (>21 days) was recorded in 7 of 15 mares following the Cycle-1 ovulation, but not subsequently. In conclusion, eFSH treatment of vernal transitional donor mares stimulated ovulation within only few days of treatment, and the following embryo recovery rate was at least as good as in the subsequent estrous cycles; however, on average, embryos were morphologically impaired. In subsequent estrous cycles in the breeding season, ovulations, embryo recovery rates, and embryo variables did not appear to be negatively affected; however, the first inter-ovulatory interval of the breeding season was prolonged in approximately half of the mares.