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Milk-based diluents are generally considered efficient for survival of stallion spermatozoa in vitro. However, milk is a complex and variable medium and native phosphocaseinate is a milk component that is more efficient for preservation of sperm motility and fertility, although the mechanisms involved in this protection have not yet been elucidated. The aim of the present study was to characterize the interactions between native phosphocaseinate and equine spermatozoa. No binding between sperm membranes and native phosphocaseinate was observed using indirect immunofluorescent staining or electron microscopy and native phosphocaseinate showed no indirect protective effect on spermatozoa after incubation in two distinct storage chambers separated by a dialysis membrane. In addition, the intracellular Ca2+ concentrations in spermatozoa did not alter after incubation in native phosphocaseinate. The favourable influence of the native micelle structure of the casein was observed only for spermatozoa stored at 15 degrees C. In conclusion, the results of the present study indicate that native phosphocaseinate has a direct protective effect on equine spermatozoa, without any evidence of binding to sperm membranes.

The composition of seminal plasma must be determined to assess the possible roles of sex gland secretions in survival of stallion spermatozoa. In the present study, an automated semen collection device and 1H magnetic resonance spectroscopy were used to analyse and compare the composition of seminal plasma from fractionated and nonfractionated stallion ejaculates. The contribution of each semen component to the ejaculate (sequence of production of component and concentration) was evaluated and its relationship to biophysical parameters was determined. 1H magnetic resonance spectroscopy was used to quantify molecules defined as markers of sex gland secretions: carnitine, glycerophosphorylcholine and choline for the epididymides; N-acetyl function of glycoproteins and spermine for the ampullae; acetic acid for the bulbourethral glands; and citric acid for seminal vesicles. The results from 32 ejaculates (four ejaculates from each of four stallions by two collection methods) demonstrated the reliability of the 1H magnetic resonance spectroscopy quantitation, the sequence of sex gland secretion contributions to the ejaculate (bulbourethral glands, epididymides, ampullae and seminal vesicles) and the concomitant appearance of the sperm-rich fraction with secretions from the epididymides and ampullae.

Since the first successful collection of oocytes by non-surgical puncture, there have been numerous attempts to fertilize them but few segmented embryos have resulted. The latest attempts at follicular puncture (Palmer et al., 1987) provided 159 oocytes. Oocytes found broken (18%) were probably already broken, or at least fragile, before puncture. The 41 oocytes were fertilized only with semen treated with Ionophore A23187. Following ionophore treatment of semen, 16 ova segmented (of 113 inseminated oocytes) indicating fertilization, and another 7 showed signs of fertilization but not segmentation. Our basic protocol, when applied to 60 oocytes, yielded 11 (18%) embryos and 5 (8%) that were fertilized without segmentation. Modifications to the protocol produced no improvement in the results. Eight embryos fertilized in vitro were transferred into the ampulla of 8 recipient mares. One pregnancy resulted and foaling occurred June 14, 1990. This is the first equine pregnancy to continue to full term following in vitro fertilization.

Semen from three ejaculates from each of 4 stallions was frozen in liquid nitrogen. Morphology was evaluated by coating the spermatozoa with fluorescein-labelled Concanavalin A (FITC-ConA2) and motility was measured by computer-assisted image analysis. Coating was performed at each step of the freezing procedure (dilution, cooling, addition of glycerol and freeze-thawing) and observations were made after each step, to evaluate changes, or after subsequent steps, to determine protection provided by the coating method. All the parameters showed progressive changes during the freezing procedure. Mean track speed of the spermatozoa, the proportions of normal and motile spermatozoa and lateral head displacement all decreased, whereas the proportions of spermatozoa exhibiting non-fluorescence in the equatorial segment and wrinkled acrosome membranes increased. The results demonstrated that coating stallion spermatozoa with ConA2 provides some degree of protection for acrosome membranes and it helps to preserve motility after freezing and subsequent thawing.

The ovaries of 23 mares were collected at slaughter during April-June and follicles (4-40 mm in diameter) were dissected and punctured to obtain oocytes for culture. The follicles were grouped according to histology: (a) normal, (b) showing primary and (c) secondary atresia. Antral fluid was analyzed for steroid content; oestradiol and testosterone (but not progesterone or androstenediol) were closely correlated with follicle size and histological state. Oocytes were cultured early after slaughter in Medium 199 (Difco OSI, France) or Medium B2, the highest percentage of oocytes reaching Metaphase 2 (53%) occurred after 36-38 h in culture. Among the 52 oocytes cultured and inseminated 1 case was confirmed with a 4-blastomere-like structure, 10 cases had structures resembling pronuclei, 9 oocytes were in Metaphase 1, 14 were in Metaphase 2 and 18 were degenerated.

In an attempt to define optimal season and ejaculation frequency for frozen semen, semen was collected from 6 stallions (3 horses and 3 ponies) 3 times per week or every day, alternating every week, for 1 year. The semen was evaluated and frozen. All the samples were thawed at the end of the experiment. At collection, fresh semen evaluations showed that winter (as opposed to spring and summer) was associated with low sexual behaviour, small volumes of spermatozoa and gel, high sperm concentration and lower motility. The high ejaculation frequency yielded a decreased volume, concentration of spermatozoa in the ejaculate and slightly improved motility. The quality of thawed semen was analysed by video and microscope estimations for motility and by two staining methods for vitality. No variation was observed according to the ejaculation frequency; the best freezability was obtained in winter but the difference was small compared to between-stallion variability and optimization of frequency and season did not change a 'bad freezer' into a good one.

(1) Various estimations of motility (subjective appreciation, count on video record, Doppler laser apparatus and optic-microcomputer analyser) or percentage of live spermatozoa (eosin-nigrosin staining) showed a higher repeatability for measurements of a same sample than for straws of a same ejaculate: the values were high (respectively greater than 0.78 and greater than 0.69) except for the optical analyser and staining. (2) Semen samples were collected from 80 stallions 5 times at 24-h intervals. The repeatability varied from 0.37 to 0.69 for gel-free volume, concentration, total sperm number, % of motile spermatozoa in raw and extended semen at collection or after storage. Therefore, to assess a stallion, 2-7 ejaculates, according to the characteristic being measured, are needed to get a repeatability of 0.80 for the mean value. (3) A study on 79 stallions serving mares over 4-152 oestrous cycles per year showed that 17 seasons are necessary to measure fertility based on the pregnancy rate per oestrous cycle with the same accuracy. If they served more than 25 cycles every year, 6 seasons only were required. In both cases, it was less than for % of live foals per season.