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a Institute of Reproductive Biology & Veterinary Obstetrics, and
b Institute of Genetics, University of Bari, Bari, Italy
c Department of Public Health & Cell Biology, University of Rome "Tor Vergata", Rome, Italy
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present work, we studied whether the phenomenon of spontaneous hardening of the ZP could be responsible for the low fertilization rate of in vitro-matured equine oocytes. The ZP of mouse and rat oocytes has been shown to undergo spontaneous zona pellucida hardening (ZPH), defined as an increased resistance to protease digestion, when cultured for IVM in serum-free medium [26, 27]. Zona hardening occurs naturally after fertilization in oocytes from many species as a consequence of cortical granule release, constituting the primary block to polyspermy. On the other hand, spontaneous ZPH that occurs in vitro, probably following precocious cortical granule exocytosis, renders mouse and rat oocytes refractory to sperm penetration, thus preventing fertilization [27–29]. Fetuin, a major glycoprotein component of fetal calf serum (FCS) and a protease inhibitor, has been shown to inhibit ZPH during IVM of mouse oocytes by preventing the proteolytic conversion of ZP2 to ZP2f caused by precociously released cortical granules, and to allow fertilization at frequencies equivalent to those observed in oocytes cultured in serum-supplemented media [30]. We have recently demonstrated that the phenomenon of ZPH occurs also in equine oocytes matured in vitro and that it is related to the season, to the estrous cycle stage of the donor mare, and to culture conditions. In fact, oocytes recovered from mares in early diestrus and cultured in serum-free medium showed increased resistance of the ZP to protease digestion [31]. Moreover, oocytes cultured after removal of cumulus cells showed increased ZPH, whereas the ZP solubility of equine oocytes matured in vitro was not affected by culture duration [32], as in the mouse.
The main aims of this study were 1) to determine whether fetuin could inhibit ZPH in slaughter-derived and in vitro-matured equine oocytes; 2) to investigate the presence of fetuin in equine serum and in the preovulatory follicular fluid of the mare, which are currently used in IVM protocols in the horse; and 3) to evaluate whether the addition of fetuin in IVM medium could improve in vitro sperm penetration and fertilization by IVF, using ICSI as a control procedure for assessing the viability and fertilizability of fetuin-treated oocytes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Oocyte Collection
Ovaries from mares of unknown reproductive history were obtained at slaughterhouses located at a maximum distance of 30 km (half an hour) from the IVF laboratory. The ovaries were placed in saline (9 g/L NaCl containing 40 mg/L gentamycin sulfate) within 20–30 min of slaughter and were transported to the laboratory in thermal containers at 25–30°C. Follicular fluid was aspirated as previously described [3]. Cumulus-oocyte complexes (COCs) were collected in Hepes-buffered tissue culture medium (TCM 199) supplemented with additives for the corresponding experimental group, and those oocytes with a complete cumulus (compact or expanded) were selected for culture and washed four times in the same medium. The total time between slaughter and culture ranged between 2 and 4 h. Oocytes for evaluations were from pools of oocytes collected from several mares.
Additives for IVM Medium
Estrous mare serum (EMS) and mare's follicular fluid from preovulatory follicles (FF) were prepared as previously described [15]. BSA (A-4503), FCS (F-4135), fetal equine serum (FES, F-7763), and Pedersen's fetuin (F-6131) [33] were purchased from Sigma (Milan, Italy). Fetuin stock solution was prepared by reconstituting 250 mg fetuin in 10 ml Hepes-buffered TCM 199, and aliquots were stored at -20°C until use.
IVM
The basic medium was TCM 199 (Sigma M-0148) with Earle's salts, buffered with 4.43 mM Hepes (Sigma H-9136) and 33.9 mM sodium bicarbonate (Sigma S-5761) and supplemented with 0.1 g/L L-glutamine (Sigma G-7513), 2 mM sodium pyruvate (Sigma P-2256), 2.92 mM calcium lactate (Serva Feinbiochem GmbH, Heidelberg, Germany; no. 29760), and 50 µg/ml gentamycin (Sigma G-1272). After preparation, pH was adjusted to 7.18, and the medium was filtered through 0.22-µm filters (no. 5003-6; Lida Manufacturing Corp., Kenosha, WI). Then gonadotropins (10 µg/ml ovine FSH [Sigma F-4520] and 20 µg/ml bovine LH [NIADDK, NIH, Bethesda, MD]) and estradiol-17ß (1 µg/ml, Sigma E-2257) were added. The medium was further supplemented according to the experimental design. The medium was filtered again and allowed to equilibrate for 1 h under 5% CO2 in air before being used. Up to 20 COCs were placed in 400 µl of medium per well of a four-well multidish (no. 176740; Nunc Intermed, Roskilde, Denmark), covered with preequilibrated lightweight paraffin oil (Sigma M-3516), and cultured for 28–30 h at 38.5°C under 5% CO2 in air.
ZPH Assay
The test was performed as described by De Felici and Siracusa [26]. In vitro-matured oocytes were freed of cumulus cells by hyaluronidase digestion (see below). Figure 1 shows a group of equine oocytes, observed after 28–30 h of maturation in vitro followed by cumulus cell removal and morphological selection before ZPH testing. The oocytes were then washed twice in 50-µl drops of a solution of 3 mg/ml 
-chymotrypsin (Sigma C-7762) dissolved in protein-free Hepes-buffered TCM 199 and finally incubated in 100-µl drops of the same solution under paraffin oil at room temperature. The oocytes were examined at 5-min intervals under a Nikon (Garden City, NY) SMZ-2T stereomicroscope, and the fraction of ZP lysed at each time was recorded. A zona was considered lysed when it had completely disappeared or a few barely visible fragments remained. Figure 2 shows two oocytes during (Fig. 2A) and at the end (Fig. 2B) of -chymotrypsin digestion. The progressive dissolution of the zona and few barely visible remnants can be observed. Data were expressed as means ± SD of the minutes required for 50% of the oocytes to become zona-free (t50).
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Oocyte Preparation for IVF and ICSI
After 28–30 h of IVM culture, oocyte morphology was assessed after partial removal of cumulus cells. Those oocytes showing the perivitelline space, an extruded first polar body, and an intact oolemma were selected [3]. The cumulus and corona cells of oocytes undergoing ICSI were totally removed by incubation in TCM 199 with 20% FCS containing 80 IU/ml hyaluronidase (Sigma H-3506) and by aspiration in and out of finely drawn glass pipettes.
Semen Preparation for IVF and ICSI
Semen samples (0.4 ml/straw) from a single ejaculate frozen at a concentration of 1 x 108 sperm cells/ml were rapidly thawed (30 sec) in a water bath at 37°C. Total motility after thawing was 70%, with 50–60% progressive motility. Sperm cells for IVF were prepared using the swim-up procedure in modified Tyrode-lactate medium containing heparin (10 µg/ml; Sigma H-3393) as described previously [24]. Semen samples for ICSI were prepared by the swim-up procedure in Earle's Balanced Salt Solution (Sigma E-2888) supplemented with 0.4% BSA, 40 µg/ml streptomycin sulfate (Sigma S-9139), and 25 UI/ml penicillin G (Sigma P-3032) as described by Dell'Aquila et al. [3].
IVF and ICSI Procedures
A final sperm concentration of 1 x 106 sperm cells/ml was added to 400 µl of culture medium containing oocytes for insemination in four-well multidishes. The sperm cells and oocytes (5–20 oocytes per well) were kept together for 24 h at 38.5°C under 5% CO2 in air. ICSI was carried out basically according to the procedures described by Palermo et al. [34] and Van Steirteghem et al. [35] as applied to equine germ cells [3, 11]. All procedures were performed at 38.5°C in Hepes-buffered human tubal fluid (HTF-Hepes 9962; Irvine Scientific, Santa Ana, CA). The injected oocytes were then transferred to 25-µl drops of fresh HTF medium covered by lightweight paraffin oil and incubated at 38.5°C for 18–20 h under 5% CO2 in air.
Assessment of Fertilization
On the day after IVF or microinjection, oocytes were fixed with 3:1 ethanol:acetic acid, stained with 1% Lacmoid (Sigma L-7512) in 45% acetic acid, and assessed by phase-contrast microscopy. Normal fertilization was defined by the presence of 2 polar bodies with 2 pronuclei. The frequency of sperm head decondensation, the presence of a single pronucleus with or without signs of the sperm cell in the cytoplasm, and polyspermy were also assessed.
Immunoblotting
One sample of each serum type was denaturated in single-strength sample buffer (10 mM Tris/HCl, pH 8.0, 1 mM EDTA, 10% [v:v] glycerol, 2% [w:v] SDS, 5% [v:v] ß-mercaptoethanol, and 0.001% [w:v] bromophenol blue), heated for 5 min at 100°C, and subjected to SDS-PAGE [36] in minislab gels (100 x 80 x 0.75 mm) containing 12% acrylamide and 0.12% bisacrylamide. Molecular weight markers (bovine albumin A-7517, egg albumin A-7642, and glyceraldehyde 3-phosphate dehydrogenase G-5262; Sigma) were run to estimate the molecular weight of the immunoreactive bands. After electrophoresis, the proteins were transferred electrophoretically (100 V, 1 h) to a sheet of nitrocellulose using the mini Bio-Rad (Richmond, CA) Trans Blot electrophoretic blotting apparatus and 20 mM Tris, 150 mM glycine, 20% (v:v) methanol at pH 8.2 as transfer buffer. After protein transfer, the nitrocellulose sheet was incubated successively with 3% (w:v) BSA in 10 mM Tris/HCl (pH 7.5), 150 mM NaCl, and rabbit anti-fetuin serum (Chemicon Int., Temecula, CA, by Società Italiana Chimici, Rome, Italy; no. AB871) diluted 1:4000 in TNT buffer (10 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.5% Triton X-100). Alkaline phosphate-conjugated goat anti-rabbit IgG (Chemicon AP132A) diluted 1:1000 in 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Chemicon) in TNT buffer was used as the substrate for the alkaline phosphatase reaction.
Experiment 1
Groups of COCs (n = 2 to 10) were randomly assigned to the experimental groups and matured in vitro as described above. After 28–30 h of IVM culture, the cumulus and corona cells were totally removed by hyaluronidase incubation. The oocytes were then transferred into fresh medium and checked for morphology and maturation stage. Those showing the presence of an extruded first polar body were evaluated for ZP hardness by -chymotrypsin digestion as described above. For each group, 4 to 16 replicates were performed.
In experiment 1A, fetuin at concentrations ranging from 2500 to 0.001 µg/ml was added to 0.3% BSA-supplemented IVM medium during oocyte maturation. Oocytes cultured in medium supplemented with 0.3% BSA without fetuin were used as controls.
In experiment 1B, oocytes were cultured in 20% (v:v) EMS, FES, FCS, or FF in IVM medium. Oocytes cultured in protein-free medium were used as controls.
In experiment 1C, fetuin, at the concentrations showing maximal ZPH-reducing activity in experiment 1A (1000–10 µg/ml), was added to medium supplemented with 10% EMS and 10% FF. Oocytes cultured in the same medium without fetuin were used as controls.
In experiment 1D, fetuin, at the concentrations used in experiment 1C, was added to the medium without BSA or any other protein source. Oocytes cultured in the same medium without fetuin were used as controls.
Experiment 2
The presence of fetuin in EMS, in FES, and in FF was investigated by Western blot. Estrous cow serum (ECS) prepared as described in Dell'Aquila et al. [24], fetuin, and FCS were used as positive controls.
Experiment 3
In experiment 3A, fetuin was added to IVM medium, at the concentrations showing maximal ZPH-inhibiting activity (1000–10 µg/ml), to assess the effects of fetuin on sperm penetration and oocyte fertilizability. Oocytes were cultured in medium supplemented with 0.3% (w:v) BSA or 10% EMS plus 10% FF. Oocytes matured in similar medium, but without fetuin, were used as controls. Twenty-eight to thirty hours after IVM culture, the oocytes were submitted to IVF as described above. Eight replicates, of 8–20 oocytes each, were performed for each treatment.
In experiment 3B, oocytes matured in the presence of fetuin (0.1 mg/ml) in BSA- or EMS/FF-supplemented medium were submitted to ICSI to assess their fertilizability. Oocytes cultured in similar medium, but without fetuin, were used as controls. Four replicates, of 8–20 oocytes each, were performed for each treatment.
Data Analysis
The statistical significance of the results of IVM, IVF, and ICSI procedures was evaluated by the chi-square test with the Yates correction for continuity and by Fisher's exact test. The frequencies of matured oocytes, penetrated oocytes, and 2-pronuclear-stage oocytes were compared to evaluate the effects of the addition of fetuin on nuclear maturation, sperm penetration, and fertilization. The differences in the lysis times (t50) observed in the different groups were analyzed by Student's t-test. In experiments 1A, 1B, 1C, and 1D, comparisons were made among all groups in the same experiment. In experiment 3, comparisons were made between fetuin-treated and control oocytes and between corresponding values of IVF- and ICSI-treated oocytes. Values with p < 0.05 were considered statistically different.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In experiment 1, 929 COCs were harvested from the ovaries of 269 mares (average number of oocytes per mare = 3.4). Of these, 854 (91.9%) were evaluated. Lost oocytes (those damaged during handling or that degenerated) were excluded from the analysis.
Table 1 shows the maturation rates and the results of the ZPH assay of oocytes cultured in the presence of various fetuin concentrations added to 0.3% BSA-supplemented medium. There was no significant effect of fetuin on rates of maturation to metaphase II (first polar body extruded). Fetuin significantly inhibited ZPH during IVM of equine oocytes in a dose-dependent manner.
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The results obtained in oocytes cultured in the presence of FES, EMS, FF, or FCS or in protein-free medium are shown in Table 2. The t50 were significantly lower in oocytes cultured in medium supplemented with FES, FF, and FCS than in oocytes cultured in EMS or in protein-free medium (p < 0.05).
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The ZPH-reducing effect of fetuin was also observed when fetuin, 100 µg/ml and 1000 µg/ml, was added to medium supplemented with EMS and FF (experiment 1C, Table 3).
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In experiment 1D, in which the effect of fetuin alone was tested, t50 values were significantly lower in oocytes cultured in the medium containing fetuin than in control oocytes (1000 µg/ml vs. control: p < 0.001; 100 µg/ml and 10 µg/ml vs. control: p < 0.05, Table 4). In experiments 1C and 1D, there was no significant effect of fetuin on rates of maturation to metaphase II.
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Immunoblotting
Equine serum, FES and EMS, and ECS and FF were examined at 1:10 dilution. The control lanes contained FCS at 1:1000 and 0.025 µg of fetuin. The gel was repeated three times. The immunoreactivity band corresponding to fetuin appeared in all lanes. It was more evident in FES than in EMS and in FF than in EMS (Fig. 3). At a higher dilution (1:50), the band corresponding to fetuin appeared only in the lanes containing FCS and fetuin (data not shown).
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Effects of Fetuin on Sperm Penetrability of In Vitro-Matured Equine Oocytes by IVF
In experiment 3, 979 COCs were harvested from the ovaries of 237 mares (average number of oocytes per mare = 4.1). Of these, 783 were used for IVF, and 196 were submitted to ICSI. Lost oocytes (those damaged during handling or that degenerated) were excluded from the analysis. The fertilization rates of oocytes cultured with different fetuin concentrations in the presence of either BSA or EMS/FF are shown in Table 5. The highest fertilization rates were observed in oocytes cultured in media containing 100 µg/ml fetuin, both the EMS/FF-supplemented medium (12 of 90, 13.3%) and the BSA-supplemented medium (7 of 72, 9.7%). However, in both groups, the difference between fetuin-treated and control oocytes was not statistically significant (12 of 90, 13.3% vs. 1 of 36, 2.8%: not significant [NS]; 7 of 72, 9.7% vs. 0 of 33: NS, for oocytes cultured in EMS/FF-supplemented and BSA-supplemented medium, respectively). Cleavage was observed in two oocytes, and no polyspermy was observed with the IVF procedure.
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Effects of Fetuin on the Fertilizability of In Vitro-Matured Equine Oocytes by ICSI
The fertilization rates after ICSI are given in Table 6. Fertilization rates were not significantly different between oocytes cultured with and without fetuin, either in the presence of BSA or in the presence of EMS/FF. In one oocyte, 3 pronuclei and 1 polar body were found. No cleavage was observed after ICSI. The overall fertilization rate was significantly higher after ICSI than after IVF (34 of 80, 42.5% vs. 21 of 439, 4.8%; p < 0.001).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-chymotrypsin digestion, resulting in a longer time required to dissolve the ZP. ZPH was compared among groups of oocytes cultured in medium with various additives. The results indicate that there was no significant effect of fetuin on the rates of maturation to metaphase II stage (experiments 1A, 1C, 1D; see Tables 1, 3, 4). These findings indicate that, through addition of fetuin to IVM medium, procedures for optimal IVM could be established under chemically defined conditions. The use of a chemically defined medium instead of a medium supplemented with serum or albumin, could be valuable for accurately determining requirements of substrates for equine oocyte maturation.
Incubation of equine oocytes with fetuin reduced hardening of the ZP. This, together with our previous reports showing that equine oocytes undergo spontaneous ZPH in vitro [31, 32], suggests that fetuin in equine oocytes, as in mouse oocytes [30], prevents the modifications of the zona responsible for the phenomenon. Although the molecular events involved in ZPH are not fully understood, previous studies suggest that it is the result of a cleavage of the ZP2 zona protein, caused by proteolytic enzymes from cortical granules [37, 38]. Commercial preparations of fetuin, a major component of FCS and a protease inhibitor, were shown to inhibit such conversion in mouse oocytes matured in vitro [30]. In our study, Pedersen's bovine fetuin appeared to have a similar protectant effect on ZPH in equine oocytes. Little is known about the modifications occurring during IVM in equine oocytes. Recently, Willis et al. [39] and Goudet et al. [5] described cortical granule migration during IVM in the horse, and Grondahl et al. [16] described the releasing of cortical granules after ICSI; but no information is available to date on the zona reaction in either in vivo- or in vitro-matured oocytes or on the release of cortical granules and related ZP modifications. Our data add novel information on the modifications occurring in the ZP of equine oocytes during IVM.
The results of experiment 1B demonstrated that the time required for 50% of the oocytes to become ZP-free was significantly lower in the presence of FCS, FES, or FF than in the protein-free medium or in the presence of EMS or BSA. These observations are in agreement with the documented efficacy of FCS [27, 28, 38] and of pig and human FF [40] in preventing ZPH on mouse and rat oocytes.
In experiment 1C, the effect of fetuin on ZPH was tested in a medium supplemented with EMS and FF and thus containing factors useful for achievement of oocyte nuclear and cytoplasmic maturation (reviewed in Dell'Aquila et al. [15]). In these trials, maintenance of the protective effect of fetuin, 100 µg/ml and 1000 µg/ml, was observed in the presence of EMS and FF.
In experiment 1D, the effects of fetuin alone, i.e., in the absence of other biological protein sources, were tested to exclude any possible interference with other factors in the medium. In these trials, the effect of fetuin in reducing the time required for 50% of the oocytes to become zona-free was maintained in the absence of other proteins in the medium. However, since the main aim of this study was to evaluate the effects of fetuin on improving in vitro fertilizability of horse oocytes, the effect of fetuin on the fertilization rates was examined on oocytes cultured under conditions suitable for achievement of fertilization. The medium supplemented with EMS/FF was used to test the effects of fetuin in the presence of factors useful for reaching cytoplasmic maturation [15], and medium supplemented with BSA was used as a control.
The immunoblotting analysis demonstrated that fetuin was present in equine serum from both fetal and adult subjects. Fetuin was also evidenced in mare preovulatory FF. There was a higher intensity of the immunoreactive band in fetal serum than in serum from adult mares. The band also appeared more intense in FF than in serum from adult mares. Correspondingly, reduced ZPH was observed after IVM in medium supplemented with FES and FF, whereas longer t50 values were found in oocytes cultured in medium with EMS. Previously, little information on equine fetuin was available and was limited to the fetal period [41, 42]. These data provide further information on the chemical composition of blood serum and FF in the horse [43].
Although a wide range of fetuin concentrations was examined, its addition to the culture medium did not increase the fertilization rate with conventional IVF. There seems to be a trend related to the presence of 100 µg/ml fetuin, but the data were not significantly different from the data observed in the control groups, both in BSA-supplemented (7 of 72, 9.7% vs. 0 of 33: NS) and in EMS/FF-supplemented medium (12 of 90, 13.3% vs. 1 of 36 2.8%: NS). The fertilization rates observed in the column of oocytes cultured in the presence of 100 µg/ml, both in BSA- and in EMS/FF-supplemented medium, were low if compared with previously reported data [8, 24, 25]. Moreover, only 5 of the 12 fertilized oocytes (5.5%) were normally fertilized (2 polar bodies, 2 pronuclei), whereas the others showed signs of delayed completion of fertilization. These results are in contrast to those found in the mouse by Schroeder et al. [30], who reported the beneficial effect of fetuin added to IVM medium on fertilization rates. However, our data are in agreement with those obtained by Kito and Bavister [44] in hamster oocytes. These authors reported no effect of fetuin on fertilization rates using commercially available preparations of Pedersen's fetuin. When they used Spiro's purified fetuin preparation, an increased penetration rate was observed, but it related to very high levels of polyspermy [44]. Moreover, our data are in line with reports describing an inefficient use of both fetal [1, 22, 23, 45–47] and adult [8, 17, 24, 25] serum in protocols for equine IVM and IVF. Further studies are needed to investigate the sperm-binding ability of equine ZP following IVM in various culture conditions as reported in the mouse [48].
The oocytes submitted to ICSI yielded higher fertilization rates than oocytes inseminated with conventional IVF, confirming that problems related to sperm penetration through oocyte membranes persist in fetuin-treated oocytes. High rates of fertilization after ICSI in this study indicate that the cytoplasmic maturation and fertilizability of oocytes cultured in the medium supplemented with fetuin was acceptable. No polyspermic fertilization was observed after IVF. After ICSI, only one oocyte showed 3 pronuclei, probably a digynic fertilization and a consequence of failed second polar body extrusion.
The failure of fetuin to increase sperm penetration through the zona suggests that zona hardening is not the major barrier to fertilization in this species. Fetuin could have a role in inhibition of the protease activities that induce spontaneous ZPH in equine oocytes during IVM, but it is not directly involved in control of the sperm-oocyte receptor interaction. This interaction could be related to peculiarities in the ZP glycoprotein composition of the equine oocyte and/or in the carbohydrate-binding properties of the plasma membrane of stallion spermatozoa. Dolci et al. [49] and Miller et al. [50] investigated the involvement of carbohydrates in the hardening of the ZP of mouse oocytes. The characterization of ZP glycoproteins in the horse by PAGE and immunoblotting has been reported by Miller et al. [51, 52]. Recently, Dobrinsky et al. [53] found galactose-binding proteins in the periacrosomal plasma membrane of equine spermatozoa, which could mediate the attachment to oviductal cells useful for maintaining the fertilizing ability of the sperm cell. Other changes in ZP occurring during IVM are probably more deleterious for sperm penetration of the equine oocyte. Moreover, a modification of the ZP3 protein, not related to ZPH and occurring during oocyte maturation, has been reported in the mouse [54]. Our observations further characterize the ZP in equine species, in which biochemical investigations are limited by the scarcity of available oocytes.
In conclusion, the addition of fetuin to IVM media reduces ZPH, but it does not influence the subsequent sperm penetration of equine oocytes matured in vitro. This implies that, in the horse, spontaneous ZPH due to a premature reaction of cortical granules is unlikely to be the only factor responsible for inhibiting sperm penetration in vitro. This study also shows that fetuin treatment did not affect the cytoplasmic maturation of oocytes, as ICSI was successful. Further studies are required to investigate the biochemical modifications occurring after in vivo and in vitro oocyte maturation in order to improve IVF and to extend our knowledge of the molecular mechanisms of fertilization and of reproductive pathologies in the horse.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Maria Elena Dell'Aquila, Institute of Reproductive Biology & Veterinary Obstetrics, Faculty of Veterinary Medicine, University of Bari, Str. Prov. Casamassima Km 3° - 70010, Valenzano, Bari, Italy. FAX: 39 80 4679083; e.dellaquila{at}veterinaria.uniba.it
Accepted: April 12, 1999.
Received: October 22, 1998.
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