Morphology, morphometry and chromatin distribution in llama sperm nuclei.

The objectives of this study were as follows: (i) to describe and evaluate the frequencies of different morphologies of llama sperm nuclei, (ii) to determine morphometric values of nuclear parameters, (iii) to describe and estimate the frequencies of different classes of chromatin distribution and (iv) to measure haploid DNA content and analyse its nuclear distribution. The study was performed using ejaculates collected from seven males, and sperm nuclei were stained with the Feulgen reaction. Normal morphology ranged from 78.36% to 93.92%, and abnormalities included short, small, large, pyriform, narrow, micro and round nuclei. Important differences in nuclei considered normal were found between some males. The following average values were obtained for each sperm nuclear morphometric parameter analysed: area 11.64 µm2 , perimeter 13.16 µm, length 5.12 µm, width 2.81 µm, ellipticity 1.85 and form 0.83. Differences between males were significant for all the parameters (p < .01). Light microscope observations and cytophotometric determinations allowed discriminating between three classes of chromatin distribution: homogeneous, diffuse and showing a clear band. Significant differences between males were found for the frequencies of the three classes (p < .01). Cluster analysis methods were used to estimate the resemblance between males according to the characteristics of their sperm nuclei. A great intermale variability was found for morphological, morphometric and chromatin distribution data. These parameters would have low dependence between them.

Relationship between morphological abnormalities in commercial bull frozen semen doses and conception rate.

Commercial doses of frozen bull semen for artificial insemination may have a certain percentage of morphological defects, despite being subject to prior selection. The aims of this study were to determine the prevalence of morphological abnormalities in commercial doses (n = 55, r = 2) of dairy and beef bulls, from AI Centers and to determine the possible existence of differences between them, regarding the percentage of abnormal spermatozoa. At least 200 spermatozoa per sample were evaluated using Bengal Rose stain (3% m/v) and light microscopy (×1000 magnification). The mean percentage of abnormal sperm samples from dairy breeds was 7.19% ± 4.91% and from beef breeds was 15.83% ± 9.28%. Significant differences between biotypes were found in the proportion of abnormal spermatozoa, abnormal heads and abnormal midpieces; it could be due to different selection pressure. It was observed that the percentage of abnormal spermatozoa was not a good fertility level predictor for the commercial samples of frozen bovine semen used in this study. In both biotypes, the midpiece abnormalities were the most frequent, mainly its distal flexion (compensable defect). This could be as a result of the effects of freezing and thawing on spermatozoa.

DNA content of Ovis musimon spermatozoa.

To our knowledge, the value of the haploid DNA content (C-value) of Ovis musimon (mouflon) has not been previously published. Therefore, the aim of the present work was to determine the C-value and the nuclear area of O. musimon sperm cells and compare both parameters with those of Ovis aries. Feulgen reaction, which is specific and stoichiometric for DNA, was carried out on semen smears. The C-value and sperm nuclear area were determined using microspectrophotometry and Gallus domesticus erythrocytes as standard species. The C-value of O. musimon was 3.02 ± 0.04 pg, and the sperm nuclear area was 23.92 ± 0.89 µm(2). The C-value and the sperm nuclear area of O. aries were 3.07 ± 0.03 pg and 22.98 ± 0.86 µm(2) respectively. The O. musimon C-value was not significantly different (P > 0.05) from that of O. aries, indicating that both species may have a very close phylogenetic relation.

Major morphological sperm abnormalities in the bull are related to sperm DNA damage.

The influence of sperm morphology and chromatin integrity on bull fertility suggests a strong but undefined biological relationship between these two parameters. In this study we explore this relationship, making use of the Sperm Chromatin Dispersion test, which allows simultaneous observation of sperm abnormalities and DNA fragmentation. Based on spermatozoa from 17 Holstein-Friesian bulls, we determined a relationship between DNA fragmentation and the presence of the "so called" major-type sperm defects. Values for DNA fragmentation index (mean ± SEM) calculated from cells with major-type abnormalities were significantly (P < 0.05) higher (85.05 ± 5.00%) than those from abnormal forms classified as minor-type (17.89 ± 5.55%). Some of the sperm abnormalities, such as double forms, narrow base heads, small heads, shortened tails and proximal cytoplasmic droplets, were only associated with sperm showing fragmented DNA. The simultaneous assessment of sperm morphology and DNA fragmentation has the potential to improve the efficacy of sperm quality assessment in this species.

Ultrastructure of sperm 'tail stump' defect in wild boar.

Sperm 'tail stump' defect was found in ejaculates of a wild boar maintained in captivity. It was in good physical condition, the testes and genital tract were found to be of normal size and consistency. There was no evidence of macroscopic abnormalities at the clinical analysis and at necropsy. The volume and concentration of the semen samples obtained by electroejaculation were lower than normal. The slides examined contained a high level of abnormal spermatozoa (52.7%). The most frequent morphological finding was a droplet-like form attached to the base of the head or a very short stump. The non-stumped spermatozoa had no normal tail but a shortened one. Analysing the histological structure with light microscopy, no ring of spermatozoa was observed lining the lumen of the seminiferous tubules and the characteristically cellular structure was not conserved. The ultrastructural examination evidenced a disorganisation of the normal tubular structure of the flagellum, with lost of regular pattern of the axial bundle of fibrils and the mitochondrial helix. The origin of this abnormality is unknown.

Relationship between the nuclear morphology of the sperm of 10 bulls and their fertility.

The relationships between the fertility and nuclear morphology, chromatin maturity and chromatin condensation of the sperm of three bulls with a calving rate over a year of more than 65 per cent, four bulls with a calving rate between 65 per cent and 35 per cent, and three bulls with a calving rate of less than 35 per cent were studied. The sperm nuclei were stained with the Feulgen reaction, and chromatin condensation and maturation were evaluated in situ by staining with toluidine blue and acid aniline blue. Nuclear chromatin decondensation was induced with dithiothreitol; this showed that in the bulls with low fertility, more than 35 per cent of nuclei were decondensed, and that one of them had the lowest percentage of normal nuclei (64.9 per cent) and stronger positive reactions to the acid aniline blue and toluidine blue stains than the other bulls.

Enrichment of high-quality spermatozoa in bovine semen: relative effectiveness of three filtration matrixes.

To be practical, any method for improving bull semen must yield a large quantity of motile spermatozoa. Some separation methods based on physical properties, e.g. filtration, chromatography, centrifugation, washing and pooling, have been reported as satisfactory, but generally are not repeatable. Nevertheless, filtration methods appear to allow the attainment of an acceptable number of spermatozoa, thus allowing such a technique to be introduced in the production of standard bovine semen doses for artificial insemination. The aim of this work was to evaluate systematically the relative effects of three filtration matrixes (silica oxide, glass beads or Sephadexätrade mark) on the improvement of whole ejaculate quality. Analysis of the type of matrix and the volume and height of the filtration column was performed. The only characteristic of the columns that appears to influence ejaculate quality after filtering is the matrix volume. While all matrixes produced improvement of semen quality, SephadexTM was better than the other matrixes tested. An explanation for the mechanism of column filtration is proposed.