Search

International audience

Summary Fertilisation and early embryonic development were studied in the following sequence: (1) detection of the pre-ovulatory follicle by daily ultrasonography; (2) induction of ovulation by injection of pituitary extract: (3) artificial insemination 19 to 25 h after injection; (4) precise detection of ovulation by hourly ultrasonography from 34 or 35 h post injection (pi); (5) surgical collection of the ovary and oviduct at different intervals from ovulation (6, 12, 24, 48, 72, 96 h); (6) flushing of the embryo and immediate evaluation by microscopy; (7) either fixation for later morphological study or culture for 12 to 84 h. Induction of ovulation was attempted in 44 mares by intravenous injection of 25 mg crude horse pituitary extract when a growing follicle over 33 mm was detected. Twenty four mares ovulated between 34 and 40 h pi. Five ovulated before 35 h but had not been scanned at 34 h. Five ovulated before 34 h, three between 40 and 47 h and seven did not ovulate. Recovery of ova by flushing the oviduct with PBS containing antibiotics and 100 iu/ml heparin was successful in 24 of 32 attempts. In vivo development of the embryos was estimated at collection. At 6 h after ovulation (n=6), ova were in cumulus and fertilisation could not be determined. At 12 h after ovulation (n=4), ova had two polar bodies and had not yet cleaved. At 24 h (n=4), three had two cells but one had not cleaved. At 48 h (n=3), embryos had four to six cells. At 72 h (n=5), four embryos had seven to twelve cells and one had not divided. At 96 h (n=2), embryos had eight and twelve cells. From a total of 18 ova or embryos collected between 12 and 96 h after ovulation, 16 (89 per cent) were fertilised and close synchronisation of cleavage divisions was observed. In vitro culture of the embryos was performed at 38.5°C in tubes containing 0.5 ml of B2 medium with 15 per cent heat-inactivated foetal calf serum under air and 5 per cent CO2. Of nine ova collected at 6 or at 12 h and cultured, six had cleaved at 24 h after ovulation; the other three did not divide and were not considered further. From 24 to 48 h, all seven embryos divided from two cells to four to six cells. From 48 to 72 h, three of four embryos divided from four to six to seven to eight cells and one did not develop. The two embryos cultured from 72 to 96 h divided from eight to twelve cells. In vitro cell divisions in embryos less than 96 h seemed to occur at a similar schedule as those in vivo. Six ova which divided following an attempt at in vitro fertilisation of 20 oocytes were compared for their division rate and aspect to the in vivo controls. Division rate seemed advanced in at least three of them but two had normal aspects and stages of division (two cells at 48 h and seven to eight cells at 70 h). All ova had been penetrated by spermatozoa which were found in the zona pellucida, under it or between blastomeres. A mid-piece of a flagellum was found in the ooplasm providing proof of fertilisation rather than parthenogenesis. The reason for the abnormal development is suspected to be polyspermy.

Adult transgenic mice overexpressing human insulin-like growth factor-binding protein-1 in the liver present reproductive abnormalities in both sexes. In the present work, we have investigated the mechanisms responsible for limiting breeding capacity in these transgenic male mice. Homozygous adult transgenic male mice (3–6 months old) exhibited irregular copulatory behavior and a reduction of the number of pregnancies per female as well as of litter size per pregnancy. Genital tract weight, more specifically epididymal and seminal vesicle weights, were reduced by 45% in homozygous transgenic vs. nontransgenic mice. Homozygous transgenic mice exhibited a 30% reduction of the length of seminiferous tubules (P = 0.007), a 30% decrease in daily sperm production per testis (P = 0.019), and a 50% decrease in the number of spermatozoa in testis (P = 0.037), associated with morphological abnormalities of the sperm heads leading to an approximately 50% reduction of fertilized two-cell eggs (P = 0.002) and of implanted embryos on d 5.5 after mating (P = 0.004). The round spermatids also appeared altered in their morphology. In addition, Leydig cells in homozygous transgenic mice exhibited an altered appearance, with a 1.8-fold increase in lipid droplets in their cytoplasm (P < 0.001). Moreover, the concentration of 3β-hydroxysteroid dehydrogenase was 66% lower in testis from transgenics compared with those from normal mice (P = 0.01), leading to a tendency toward lower plasma testosterone levels (P = 0.1). Interestingly, LH concentrations were increased by 40% in transgenic pituitary extracts (P = 0.02), and basal LH secretion by pituitary explants in vitro was increased by 60% in homozygous transgenic vs. normal mice (P = 0.04), suggesting an alteration of LH pulsatile secretion in vivo. In conclusion, these data suggest that the breeding impairment of human insulin-like growth factor-binding protein-1 transgenic males is due at least in part to an alteration of the process of spermatogenesis, leading to a diminution of sperm production and of its quality. Minor impairment of steroidogenesis may also contribute to the reduced reproductive capacity of these animals. Our observations are consistent with the idea that normal spermatogenesis and perhaps also steroidogenesis are dependent on the actions of sufficient concentrations of unbound IGF-I.

The fertilization capacity of goat sperm stored in milk extenders is approximately 12-24h. Long-term storage of goat sperm (up to 3 days) is desirable as it would confer greater flexibility to breeding farms. The aim of this study was to evaluate in vitro motility parameters of buck spermatozoa for up to 7 days of storage using skim milk or chemically defined extender supplemented with native phosphocaseinate (NPPC). Four experiments were conducted to determine optimum temperature (4 or 15 degrees C) and storage conditions (aerobic versus anaerobic), the effect of seminal plasma on sperm survival, the optimal concentration of NPPC and the effect of beta lactoglobulin (BL). Both skim milk and NPPC were found to be more efficient for preserving goat sperm at 4 degrees C than at 15 degrees C (P

International audience; The aims of this study were to evaluate the effects of cooling rate to 4 °C and temperature at the time of centrifugation/glycerol-addition (freezing extender: INRA82 + 2% egg yolk + 2.5% glycerol) on postcentrifugation recovery rate, post-thaw motility and per-cycle fertility. When centrifugation/glycerol-addition was performed at 4 °C (14 ejaculates), a moderate cooling rate (37 °C to 4 °C in 1 h) resulted in higher post-thaw motility (45%) than when using a slow cooling rate (37 °C to 4 °C in 4 h) (39%; P

In order to extend the duration of boar sperm survival at 15 degrees C for artificial insemination, we tested the effect of indole-3-acetic acid (IAA), which appeared to be the main sperm protective substance present in the Coco nucifera endosperm (coconut water). Two IAA concentrations (10 and 100 ng/mL) in Beltsville extender (BTS) were studied for their in vitro effects. The motility, the percentage of motile spermatozoa and the acrosome morphology of sperm were recorded each day over 13 days of storage at 15 degrees C, after 5 min and 3 h of incubation at 39 degrees C. The IAA effect on sperm preservation was also studied in vivo at a concentration of 10 ng/mL in BTS by inseminating groups of females twice at 24 h intervals either at D0 (day of sperm collection) and D1 (D0/1) or at D5 and D6 (D5/6). At D0/1, the two groups of females (control and IAA) were inseminated with a total of 6.3 x 10(9) spermatozoa (3.15 x 10(9) at D0 and the same dose at D1) whereas at D5/6, on IAA group was inseminated with a total of 6.3 x 10(9) spermatozoa and another one with 12.6 x 10(9) spermatozoa. The animals in the D5/6 control group were inseminated each with a total of 12.6 x 10(9) spermatozoa. For each group of females (n = 106-140), fertility rate (% farrowing) and prolificacy rate (litter size) were recorded. No effect of IAA in vitro on the motility rate and on the percentage of motile spermatozoa was observed over a 13 day storage. However, IAA (10 ng/mL) had a significant positive effect on the percentage of living spermatozoa with intact acrosomes after 13 days (66 vs 54%, P < 0.05). The fertility and prolificacy rates after 5-6 days of sperm preservation in BTS extender alone did not differ significantly between D5/6 and D0/1 but the total number of inseminated spermatozoa was 12.6 x 10(9) at D5/6 instead of 6.3 x 10(9) at D0/1. When the spermatozoa were stored in the presence of 10 ng/mL IAA for 5-6 days at 15 degrees C, the fertility and prolificacy of the females inseminated with only 6.3 x 10(9) spermatozoa were identical to those of the females inseminated with an equal number of spermatozoa at D0/1 in the presence or absence of IAA.

Hen egg yolk is normally used as a cryoprotective agent in semen freezing extenders, but its use has sanitary and practical disadvantages. Moreover the protection afforded by egg yolk has not yet been completely elucidated. The objective of this study was to compare the egg yolk plasma fraction to whole egg yolk in stallion freezing extender. Plasma contains mainly Low Density Lipoproteins (LDL), which are widely presumed to be the cryoprotective agent in egg yolk. Plasma can be produced on an industrial scale, sterilised by gamma-irradiation and incorporated in a ready-to-use extender (our ultimate objective). Plasma samples were subjected to different doses of gamma-irradiation (3, 5, 10 kGy) without dramatic chemical changes that may affect their cryoprotective properties. Stallion semen was frozen with whole egg yolk as a control and with sterilised egg yolk plasma. A fertility trial was conducted on a total of 70 mares' cycles. Fertility per cycle was 60% after insemination of semen frozen in our control extender containing egg yolk (EY), compared to 69% for the extender containing sterilised egg yolk plasma (EYP) (P0.05). Post-thaw motility and membrane integrity of spermatozoa were also analysed. Motility parameters were not significantly different between extenders except for the variable VAP (for EY versus EYP, VAP: 63 μm.s(-1) versus 59 μm.s(-1), a, b: P0.001; PROG: 41% versus 39%, RAP30: 56% versus 54%; RAP40: 51% versus 48%, P0.05). Membrane integrity was better preserved in EY than in EYP but the difference between extenders was small (P0.05). Our results demonstrated that sterilised egg yolk plasma has the potential to replace egg yolk in stallion freezing extender. This experiment led to the development of a ready-to-use extender called INRA-Freeze(®) (IMV-Technologies, France).

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.

International audience

Hypoosmotic swelling test (HOS) has been proposed by many authors to evaluate the functional integrity of the sperm membrane. Our approach in this experiment has consisted in exposing spermatozoa to a wide range of osmotic pressures then evaluating the reacted sperm cells by flow cytometry and finally modelling the sperm cell responses. Semen samples were diluted in skim milk or NPPC (native phosphocaseinate) extenders, and stored at 4 °C for 3 days. At D0 and D3 aliquots from each ejaculate (n = 12) were submitted to seven hypoosmotic solutions varying from 230 to 10 mOsm/kg. Sperm samples were analyzed using flow cytometry to determine two populations of spermatozoa identified by propidium iodide (PI): PI+ (including PI, red fluorescence) and PI− (excluding PI, no fluorescence). Spermatozoa PI+ were considered as spermatozoa with membrane damages. PI+ exhibited a high variation from 230 to 10 mOsm/kg which was considered as a dose–response curve. Data were modelled using Mixed procedure and probit analysis to a sigmoid curve. Each model curve characterized the profile of response of the variable PI+ to the range of osmotic pressure from 230 to 10 mOsm/kg. The estimated parameters modelling the sigmoid curves are discussed in order to evaluate the effect of extender (skim milk versus NPPC) and duration of preservation (D0 versus D3). Such modelling could help to differentiate storage method ejaculates within males or between male, contributing therefore to improve semen technology.

Semen of Turkeys between 31 and 52 weeks of age was analyzed to investigate the cause of reduction in Turkey fertility at the end of the reproductive period. Sperm motility and viability, lipid concentration, fatty acid composition and lipid peroxides were evaluated on fresh spermatozoa or spermatozoa stored for 48h at 4 degrees C. Fertility of fresh semen was also evaluated. Fertility obtained with fresh semen decreased at 44-47 weeks of age. Ageing was also accompanied by a decrease in sperm viability (at 47 weeks) and later by a decrease in motility of spermatozoa (at 52 weeks). Polyunsaturated fatty acids (PUFAs) were the first lipids of fresh spermatozoa affected by age, especially n-3 and n-9 PUFAs. Changes in these PUFAs were followed by a 30% increase in lipid peroxidation at 47 and 52 weeks of age and a reduction in phospholipid content at 52 weeks. In vitro storage did not cause lipid peroxidation in sperm obtained during the first half of the reproductive period but malondialdehyde (MDA) levels significantly increased in sperm obtained during the second half of this period. In vitro storage also decreased phospholipid content of spermatozoa from 41 weeks of age, and viability and motility regardless of age. In conclusion, lipid alteration mainly originating from PUFAs peroxidation could partly explain the decrease in semen quality and fertility observed with ageing. In addition, lipid peroxidation was increased during in vitro storage of spermatozoa from older Turkeys.

The role of the proto-oncogene Kit expression during gonadal development, then in differentiated spermatogonia has been thoroughly established. The present study was designed to investigate the consequences of a partial defect in Kit gene expression on sperm fertilizing ability, using Kit haplodeficient mice (kitW-lacZ/+). Same inbred mice (kit+/+) were used as controls. Epididymal sperm characteristics and in vivo fertility were assessed, then in vitro-fertilization experiments were carried out for mice of both genotypes. Epididymal sperm count was drastically reduced, and sperm motility was also decreased in kitW-lacZ/+ compared with kit+/+ males. Both in vivo or in vitro fertility were greatly reduced in kitW-lacZ/+ compared with kit+/+ males. By contrast, the fertility of kitW-lacZ/+ females was apparently unaffected. Additionally, a higher number of spermatozoa with undetected acrosomal contents was revealed by fluorescein isothiocyanate-labelled Pisum sativum agglutinin acrosomal staining after epididymal sperm retrieval in kitW-lacZ/+ mice, whereas no difference was observed after induction of acrosomal reaction in mice of either genotype. Ultra-structural data confirmed the higher frequency of abnormal acrosome in spermatozoa of kitW-lacZ/+ mice. Thus, sperm production is impaired in Kit haplodeficient mice both on a quantitative and a qualitative basis. Finally, we show that one single copy of Kit gene is not sufficient to maintain genuine fertility in male mice.

Sperm cryopreservation is used for preservation and diffusion of genetic diversity and genetic progress. One approach to improve cryopreservation is to identify and control the cellular parameters responsible for intrinsic sperm fitness for cryopreservation. The aim of this study was to determine if a relationship exists between sperm cholesterol/phospholipid molar ratio (CHO/PL) and sperm fitness for cryopreservation. CHO/PL in goat and boar semen were respectively 0.281 ± 0.048 (n = 112) and 0.375 ± 0.043 (n = 47). CHO/PL of washed stallion spermatozoa was 0.541 ± 0.072 (n = 17). In stallion spermatozoa and goat semen, CHO/PL and sperm motility after thawing were negatively correlated, although the significance of this relation fluctuated between ejaculates (stallion) and between months of collection (goat). In boar, mobility parameters after thawing were not related to CHO/PL, but a positive correlation was noted between CHO/PL and the percentage of spermatozoa alive and normal 120 min after thawing. No relationship was found between blood plasma cholesterol (goat: 1160 nmol.mL(−1) ± 186; boar: 1410 nmol.mL(−1) ± 200) or seminal plasma cholesterol (boar sperm rich fraction: 104 nmol.mL(−1) ±52) and sperm CHO/PL. To conclude, sperm CHO/PL is not a direct indicator of sperm fitness for cryopreservation. However, as supported by the observed correlation, it could not be ruled out that CHO/PL is involved in the cryopreservation success.

The sexual behavior of 42 stallions from French national and private studs was examined in two contexts: semen collection for artificial insemination (AI) and in-hand natural service (NS). Each stallion was observed twice in the same context. Erection and ejaculation latencies, the number of mounts leading to ejaculation, dismount latency and total breeding time were measured and compared between AI and NS. Mount without erection was rare (6/83 observations). Erection latency was 89+/-11s, and was not different between NS (62+/-22s) and AI (100+/-13s, P=0.128). Stallions ejaculated after either one mount (62/83 observations), or two (11/83 observations) or three mounts (10/83 observations). Ejaculation latency was 85+/-15s (84+/-19 in AI and 86+/-28 in NS). If 1st mount did not lead to ejaculation, then ejaculation latency increased several fold following the 2nd mount during both AI and NS. The results provide reference measures for semen collection in French studs. Difference in erection latency between AI and NS, although not statistically significant, may reflect different contributions of excitatory inputs from the brain and the genital area to the activation of spinal networks controlling erection. In contrast, lack of difference in ejaculation latency between AI and NS suggests that the spinal network that controls ejaculation follows a more rigid motor pattern.

The aims of this study were to evaluate the effects of cooling rate to 4 degrees C and temperature at the time of centrifugation/glycerol-addition (freezing extender: INRA82 + 2% egg yolk + 2.5% glycerol) on postcentrifugation recovery rate, post-thaw motility and per-cycle fertility. When centrifugation/glycerol-addition was performed at 4 degrees C (14 ejaculates), a moderate cooling rate (37 degrees C to 4 degrees C in I h) resulted in higher post-thaw motility (45%) than when using a slow cooling rate (37 degrees C to 4 degrees C in 4 h) (39%; P0.05). When centrifugation/glycerol-addition was performed at 22 degrees C (37 degrees C to 22 degrees C in 10 min) (10 ejaculates), post-thaw motility was lower when spermatozoa were frozen directly from 22 degrees C (23%) than when spermatozoa were cooled to 4 degrees C (22 degrees C to 4 degrees C in 1 h) before freezing (47%; P0.0001). When centrifugation/glycerol-addition was performed at 22 degrees C (before cooling at a moderate rate), as opposed to 4 degrees C (after cooling at a moderate rate), a significant improvement of 1) recovery of spermatozoa after centrifugation (P0,0001), 2) post-thaw motility of spermatozoa at thawing (40% vs 36% (nor = 291 ejaculates/group), P0.0001) and 3) per-cycle fertility (56% vs 42% (nor = 190 cycles/group), P0.01) was observed. In conclusion, centrifugation/glycerol-addition at 22 degrees C followed by cooling to 4 degrees C at a moderate rate results in an improvement of post-thaw motility, spermatozoa recovery rate and per cycle fertility.

Several reports have suggested that human semen quality has declined throughout the world over the last few decades. Chemicals in the environment acting as endocrine disruptors have been implicated as a possible cause. If this is indeed the case, then similar effects may be observed in animals. We report data on secular trends in semen quality of stallions collected during the last two decades by French National Studs.We analyzed 1489 ejaculates collected from 390 Breton draught stallions between 1981 and 1996 and 341 ejaculates from 86 anglo-arab thoroughbred stallions from 1985 and 1995. We employed a standardized semen collection and analysis protocol for evaluating the semen quality.For both stallion breeds studied, we observed a decreased seminal volume (around 2% per year) whereas total sperm production remains unchanged.Seminal fluid volume is controlled by accessory sex glands, which are regulated by androgens. Chemicals with anti-androgenic properties have been detected in the environment. By affecting the development or function of accessory sex glands, these chemicals may be at least partly responsible for the observed decrease in semen volume.

Several reports have suggested that human semen quality has declined throughout the world over the last few decades. Chemicals in the environment acting as endocrine disrupters have been implicated as a possible cause. If this is indeed the case, then similar effects may be observed in animals. We analyzed 1489 ejaculates collected from 390 Breton draught stallions between 1981 and 1996. Semen was collected from all the stallions at a single center, according to standardized semen collection protocols and laboratory methods. Semen volume decreased slightly but significantly and there was an increase in sperm concentration over the study period. However, total sperm production was unchanged. Seminal fluid volume is controlled by accessory sex glands, which are regulated by androgens. Chemicals with antiandrogenic properties have been detected in the environment. By affecting the development or function of accessory sex glands, these chemicals may be at least partly responsible for the observed decrease in semen volume.

The high-molecular-weight proteins of equine follicular fluid were examined to determine whether some polypeptides are unique to certain physiological conditions. Fluids from ovarian follicles of various diameters and physiological stages during the follicular phase were recovered by ultrasound-guided follicular aspiration. Granulosa cells and cumulus-oocyte complexes (COC) were recovered by scraping the intrafollicular wall during puncture. Follicular fluids and corresponding serum, as well as granulosa cell lysates, were analyzed by one-dimensional SDS-PAGE and silver staining. COC morphology was assessed microscopically. A 200-kDa protein band was demonstrated in fluids from preovulatory follicles, in natural conditions or after induction of ovulation. This protein band was absent in fluids from follicles at earlier stages, subordinate follicles, and serum. The presence of this protein at the preovulatory (PO) stage was ascertained through recovery of the fluid from follicles twice during their growth. Its appearance was time dependent after induction of ovulation but was not induced by an intrafollicular injection of a physiological dose of progesterone. We also demonstrated the presence of this 200-kDa protein in granulosa cells lysates recovered from preovulatory follicles. The expression of this protein in the follicular fluid was related to the cumulus aspect and chromatin configuration of the enclosed COC. No relation was found between its presence in the follicular fluid at the PO stage and subsequent ovulation of the punctured follicle or embryo production. The identification of this molecule is approached and discussed. These results show a novel PO stage-related protein in equine follicular fluid, which may be involved in the differentiation and maturation mechanisms occurring in the follicle during the preovulatory period.

The motility of ejaculated spermatozoa (% of progressive spermatozoa) has been evaluated visually on 26 adult rams. Half the animals were immunized with a secretory epididymal protein (prealbumin epididymal-specific (PES) ovine) injected according to immunizing protocols (Freund or Hunter adjuvant) intraperitoneally or intradermally (Freund or Hunter adjuvant) or intramuscularly (Hunter adjuvant or PES only). The other animals received the same product without the protein (controls). The product resulted in a strong asthenospermia which parallels the transient presence of anti-PES antibodies in the seminal plasma. A study with 64 ewes showed that the fertility of 10 immunized rams that had recovered sufficient forward motility to ensure fertilization, did not differ from that of control rams.

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.

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.

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.

The meiotic spindle of ovulated mouse oocytes are cold sensitive similar to the mitotic spindles of somatic cells and unicellular organisms. On crushed ice, the spindle microtubules were disorganized between 15 and 60 min of treatment: continuous microtubules depolymerized at first, then kinetochore fibers followed. MTOCs also dispersed during the cold treatment while kinetochores remained intact. The effects of isopropyl-N-phenylcarbamate (IPC) on the reorganization of the meiotic spindle of mouse oocytes were studied after cold disorganization. This drug is an antimitotic agent which appears to act on cells lacking centrioles (higher plants, amoeba, alga) as oocytes do. In ovulated mouse oocytes, IPC disturbed microtubule orientation and the relative distance between MTOCs and chromosomes. However this herbicide did not seem to act on polymerized microtubules and its action was apparently reversible. The disturbances in microtubule orientation are probably due to the displacement of chromosomes and MTOCs, which are loci of microtubule polymerization.