Replicate and technician variation associated with computer aided bull sperm head morphometry analysis (ASMA)

Associations of abnormal spermatozoa with bull fertility have yielded varying results. Manual methods of analysis are subjective and highly variable within and between technicians, which may account for these differences. Computer-aided sperm head morphometry appears to be a precise method of assessing sperm head dimensions; however, the effects of replication and technician on sperm head morphometry have not been assessed. The objective of this study was to determine the inter- and intra-analysis and technician variation associated with computer-aided bull sperm head morphometry analysis. Semen from 10 bulls was diluted to 200 x 10(6) sperm/mL, and slide smears were prepared and stained using haematoxylin and rose bengal. Each of two technicians analysed 250 images from each slide, 3 times, using computer-aided sperm head morphometry analysis. The morphometric dimensions of area, perimeter, length, width and width/length for individual sperm heads of each analysis were assessed by GLM-ANOVA for effects of bulls, replications and technicians. The coefficient of variation was recorded for each analysis and across replications. The mean coefficients of variation within and between analyses were compared between technicians by GLM-ANOVA. No differences (p > 0.1) between technicians were found between or among bulls for area (29.63 vs. 29.26 micron 2), perimeter (23.73 vs. 23.86 microns), length (8.73 vs. 8.71 microns), width (4.47 vs. 4.46 microns), or width/length (0.51 vs. 0.51). No differences (p > 0.1) between replicates for sperm head dimension were detected within or among bulls for either technician. No intra- or inter-analysis differences (p > 0.1) between technicians on CVs were observed. The mean intra-analysis CVs for all bulls for both technicians were area = 6.9%, perimeter = 4.9%, length = 4.5%, width = 5.6% and width/length = 6.5%. The mean interanalysis CVs for both technicians were area = 3.0%, perimeter = 2.4%, length = 2.0%, width = 2.0%, and width/length = 1.7%. The results indicate that ASMA is a repeatable and objective method of assessing bull sperm head morphometry within and between technicians. No differences between replications were detected, and hence replicate analyses are not necessary to acquire accurate morphometric data.     

Effects of male accessory sex glands on sperm transport, fertilization and embryonic loss in golden hamsters

This study was performed to determine the cause of reduced fertility after selective or complete ablation of the male accessory sex glands (ASG) in the golden hamster. The ASG (ampullary gland, coagulating gland, dorsal prostate, ventral prostate and seminal vesicle) were removed bilaterally. The following results were observed in matings involving such surgically-treated males. (1) Fertilization rate was not changed. (2) Total removal of all ASG resulted in significantly fewer sperm reaching the oviduct 1.5 h after mating and a higher rate of embryonic death at day 9 of pregnancy in the mated females. (3) Absence of the ampullary gland and ventral prostate led to higher rates of embryonic death by day 9 of pregnancy. (4) Compared with the controls, fewer oviductal sperm were found in post-ovulatory matings involving males in which the seminal vesicles had been removed. (5) No relationship could be established between the size (weight) of sperm plugs and the number of sperm found in the uterus.     

Effects of male accessory sex glands on fertilization, polyspermy and morphometry of early embryos in the golden hamster

Preimplantation embryos sired by hamsters without accessory sex glands (ASG) were found to have a higher mortality rate and a slower cleavage rate than those sired by sham-operated males at 72 h post coitum (P.c.). A time-course study of fertilization in vivo was conducted to determine whether this effect was due to delayed fertilization. Ultrastructural morphometry of 48 h embryos was also undertaken to establish the earliest manifestation of developmental anomalies. Compared to sham-operated controls (SH), ablation of all the ASG (TX), or just the ventral prostate (VPX) or ampullary gland (AGX) had no effect on the timing of sperm penetration, extrusion of the second polar body and pronuclear formation. Females mated with AGX males tended to have more polyspermic embryos (9.7%; p < 0.05). The volumes, volume fractions ( V v) of the blastomere nuclei, mitochondria and yolk material of the four-cell embryos sired by these same groups of males were assessed using point counting techniques. No difference in the V_v of yolk and mitochondria could be observed between groups. However, the SH group did have a significantly larger proportion of the cell occupied by the nucleus ( p < 0.05), and the TX group had a higher proportion of the nucleus occupied by nucleoli when compared with the SH group (p <0.01). Smaller nuclei and larger nucleoli in the TX group was interpreted as an early manifestation of a slower division rate of the blastomeres.     

Fertility, fecundity, sex ratio and the accessory sex glands in male golden hamsters

The ventral prostates, dorsolateral prostates, coagulating glands (anterior prostates), ampullary glands and/or seminal vesicles of male golden hamsters were excised bilaterally. The effects of these treatments on fertility, fecundity and sex ratio of offspring were studied. Total removal of the glands or ablation of the ventral prostates alone reduced fertility. The lack of secretions from the coagulating glands and seminal vesicles in the seminal fluid all favoured a higher proportion of male pups born in a litter. Absence of all or any of these glands did not appear to affect litter size.     

Innervation of accessory sex glands in the adult male golden hamster and quantitative changes of nerve densities with age

Summary. Enzyme histochemistry and immunohistochemistry were used to determine the types of nerves supplying the ampullary gland, coagulating gland, dorsolateral prostate, ventral prostate and seminal vesicle of the male golden hamster. Quantitative change of dopamine β-hydroxylase (DβH), and neuropeptide Y (NPY) immunoreactive and acetylcholinesterase-stained (AChE-stained) nerves with age was also determined. Using an antibody against protein gene product 9.5, nerves were seen to distribute in subepithelial connective tissues, smooth muscles and adventitial connective tissues. Presumptive catecholaminergic nerves immunoreactive for DβH and tyrosine hydroxylase were found mainly in periacinar smooth muscles, while AChE-stained nerves predominantly ramified subepithelial connective tissues. In addition, nerves immunoreactive to NPY, calcitonin gene-related peptide, leu-enkephalin, galanin, substance P, and vasoactive intestinal peptide were also detected. Quantitative estimation at 10, 52 and 78 weeks of age showed that densities of DβH and NPY nerves were halved by 52 weeks of age. This level was maintained in older animals. The density of AChE-stained nerves in all glands did not change with age. The ampullary gland appeared to have more AChE-stained nerves. These results were discussed from a comparative viewpoint and with regard to possible implications of aging of peripheral nerves on functioning of the male accessory sex glands.     

Visco-elasticity of seminal fluid in relation to the epididymal and accessory sex gland function and its impact on sperm motility

Seminal viscopathy was shown to be associated with male infertility. However, our knowledge about the regulatory mechanism of this process is still limited. In semen samples from 411 men attending for fertility assessment, traditional semen parameters including visco-elasticity were assessed according to the World Health Organization guidelines. Sperm motility was evaluated by use of computer aided sperm analysis (CASA). Seminal activity of neutral alpha-glucosidase (NAG) and concentrations of prostate-specific antigen (PSA), zinc, and fructose were measured. The activity of NAG, and the concentrations of PSA and zinc were significantly lower in hyper-visco-elastic semen samples (medians: 5 vs. 8 mU/mL; 741 vs. 924 mg/L; 1 vs. 2 mM/L), than in those with normal visco-elasticity (p = 0.004, 0.005 and 0.011, respectively). When comparing the total amounts, only for seminal fructose there was a difference between samples with high visco-elasticity as compared with those of normal visco-elasticity (median: 74 vs. 53 microM/ejaculate, p = 0.007) This seminal marker was the only significant independent parameter in predicting seminal visco-elasticity in a multiple logistic regression analysis (odds ratio for the highest quartile = 4.67). Hyper-visco-elasticity was associated with a lower percentage of motile spermatozoa (43 vs. 50%, p = 0.045). Similar trend was found for the CASA motility characteristics curvilinear velocity (VCL), average path length (VAP), amplitude of lateral head displacement (ALH) (p = 0.008, 0.038 and 0.020, respectively). Our study demonstrated the interplay between the regulatory effect of post-testicular organs on semen visco-elasticity. Hyper-visco-elasticity was associated with asthenozoospermia and lower levels of VCL, VAP and ALH.     

Contents of fructose, citric acid, acid phosphatase, proteins and electrolytes in secretions of the accessory sex glands of the male golden hamster

Secretions were collected from the ampullary gland, dorsolateral prostate, ventral prostate, coagulating glands and seminal vesicles of male golden hamsters aged 15–20 weeks. The concentrations of total protein, citric acid, fructose, acid phosphatase, chloride, sodium, potassium, calcium, magnesium and zinc were determined. The ampullary gland secreted predominantly citric acid, sodium and acid phosphatase. Zinc was secreted only by the prostatic complex, with the largest quantity coming from the coagulating gland. The highest concentrations of potassium, calcium and magnesium were found in secretions of the ventral prostate. The coagulating gland and dorsolateral prostate were the principal contributors to total protein. The hamster appears to be more related to the mouse than to the rat in terms of the secretory functions of its accessory sex glands.