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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.

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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.

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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.

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This study was aimed at testing the hypothesis that different forms of fibronectin (FN), produced as a consequence of the alternative splicing of the precursor messenger RNA, play specific roles during development of the ovarian follicle. In particular, we were interested in determining the effect of the ED-I (also termed ED-A) type III repeat, which is absent in the plasma form. Analysis of FN levels in follicular fluids corresponding to different stages of development of bovine follicles revealed marked changes in the concentrations of ED-I+ FN, whereas total FN levels remained relatively constant. ED-I+ FN levels were higher in small follicles, corresponding to the phase of granulosa cell proliferation. The hypothesis of a physiological role for ED-I+ FN was further supported by the finding of a regulation of the alternative splicing of FN in primary cultures of bovine granulosa cells by factors known to control ovarian follicular development. cAMP produced a 10-fold decrease in the relative proportion of the ED-I region. In contrast, transforming growth factor-beta elicited a 2-fold stimulation of overall FN synthesis and a 4-fold increase in the synthesis of ED-I containing FN. This effect was evident at the protein (Western blots) and messenger RNA (Northern blots) levels. Although a negative correlation (P < 0.001) was detected between ED-I+ FN and estradiol levels in follicular fluid, this steroid was unable to modulate in vitro the alternative splicing of FN. A possible mitogenic effect of ED-I+ FN was suggested by the observation that a recombinant peptide corresponding to the ED-I domain stimulated DNA synthesis in a bovine granulosa cell line (BGC-1), whereas a peptide corresponding to the flanking type III sequences had no effect. The hypothesis of ED-I+ FN as a growth regulatory factor was further strengthened by the fact that depletion of FN from BGC-1-conditioned medium, which contained ED-I+ FN, abrogated its mitogenic activity, whereas plasma FN was without effect. We propose that changes in the primary structure of FN may mediate some of the effects of gonadotropin and intraovarian factors during follicular development.

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An increasing body of evidence indicates that the oocyte plays an active role in the control of ovarian follicle development in mammals. In the present study, we have examined the role of oocytes in regulating granulosa cell proliferation. Rat and bovine oocytes cocultured with rat granulosa cells stimulated granulosa cell DNA synthesis and DNA content in the cultures. FSH or cAMP further amplified this effect. Poor-quality oocytes showed a marked decrease in their stimulatory effect. Stimulation of DNA synthesis by bovine oocytes seems to be cell-type specific, since Swiss 3T3 fibroblasts and CCL-64 mink lung epithelial cells were not responsive, while primary cultures of rat and bovine granulosa cells and the bovine granulosa cell line BGC-1 showed significant responses. Oocyte-conditioned medium produced only a slight stimulation of rat granulosa cell DNA synthesis. However, the effect of oocyte coculture was dependent on the total incubation volume, suggesting that the growth promoting activity was mediated by a soluble factor. The stimulation elicited by bovine oocytes was evident even in the presence of maximally effective doses of transforming growth factor-beta or tumor necrosis factor-alpha, indicating that neither of these growth factors was responsible for this effect. In vitro maturation of bovine oocytes was associated with a marked decrease in the stimulatory activity. This decrease was partially prevented when maturation was blocked by addition of cycloheximide. Comparison of the developmental pattern of the secretion of the growth promoting activity with that of the cumulus expansion-enabling factor indicated that both activities can be dissociated. Our data suggest the existence of a very labile factor produced by the oocyte before completion of the first meiotic division that promotes granulosa cell proliferation.

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This study was aimed at testing the hypothesis that different forms of fibronectin (FN), produced as a consequence of the alternative splicing of the precursor mRNA, play specific roles during follicular development. In particular, we analyzed the presence of the ED-I region, which is absent in the plasma form. Analysis of FN levels in follicular fluids corresponding to different stages of development of bovine follicles revealed marked changes in the concentrations of ED-I + FN whereas total FN levels remained relatively constant. A negative correlation (P < 0.001) was detected between ED-I + FN and estradiol levels. This steroid was without effect on the alternative splicing of FN in primary cultures of bovine granulosa cells. However, cAMP produced a marked decrease in the incorporation of the ED-I region. In contrast, transforming growth factor beta (TGF-beta) elicited both a stimulation on overall FN synthesis and an increase in the inclusion of ED-I. This effect was evident at the protein level (Western blots) and also in the mRNAs (Northern blots). A peptide corresponding to the ED-I region stimulated DNA synthesis in a bovine granulosa cell line (BGC-1) whereas the peptide corresponding to the flanking sequences was without effect. Data presented herein suggest a novel form of regulation by which changes in the primary structure of FN may mediate some of the effects of gonadotropin and intraovarian factors during follicular development.

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