Vimentin expression in testes of Arabian stallions.

REASONS FOR PERFORMING STUDY: Specific patterns of cytoskeletal filaments reflect a functional state of the cell. In testicular cells intermediate filaments (IFs) are of the vimentin type. Since it is known that Sertoli cells regulate the spermatogenic function in the male gonad, it became important to propose a system that could quantify the state of seminiferous tubular quality. To date, a Johnsen score system has never been used to equine testes. OBJECTIVES: To demonstrate the expression pattern of vimentin in testes of mature Arabian stallions and correlate its distribution with grade of seminiferous tubule impairment as indicated by a Johnsen score. METHODS: For histological examination by the Johnsen method, routine haematoxylin-eosin staining was used. Vimentin expression and its presence in testicular sections and testicular homogenates were detected by immunohistochemistry and western blot, respectively. Both analyses were performed qualitatively and quantitatively and further validated by ANOVA tests. RESULTS: Distinct morphology of seminiferous tubules was found in testes harvested from 3 stallions. Vimentin in IFs was immunolocalised to the cytoplasm of Sertoli, Leydig and peritubular-myoid cells. The intensity and pattern of the IFs staining was different in individual seminiferous tubules suggesting a correlation between vimentin expression and the severity of tubule degeneration. Qualitative results by immunohistochemistry and western blot were confirmed by further quantitative analyses. CONCLUSIONS: In equine testes, differential expression of vimentin was found to be correlated with the impairment of seminiferous tubules indicated by a decrease in Johnsen score. POTENTIAL RELEVANCE: The Johnsen score system may be a useful method to facilitate the identification of tubular alterations in the stallion testes. Combined histological and immunohistochemical approach may provide a detailed phenotypic classification of stallions with decreased fertility.

Determination of the correlation between stallion's age and number of sex chromosome aberrations in spermatozoa.

The aim of this study was a cytogenetic analysis of stallions semen to find sex chromosome aberrations and to determine if there was an association between stallion's age and aberration frequency for the sex chromosomes. Sperm samples were collected from 22 stallions of various age from 3 to 23 years. Multicolour FISH was performed on each sample, using probes for the sex chromosomes and EGFR gene, localized on 4p12 in domestic horse. A total of 26199 sperm cells were analysed (from 1 070 to 1 532 per animal). Among the analysed cells, there were 50.318% with X chromosome, 48.543% with Y chromosome and 1.139% with aberrant chromosomes. The frequency of aberrations was: sex chromosomes nullisomy (0.466%), XY aneuploidy (0.454%), XX disomy (0.146%), YY disomy (0.041%), diploidy (0.024%) and trisomy XXY (0.008%). Additionally there was a correlation between the age of an animal and the frequency of sex chromosome aberration and a significant positive correlation between age and disomy of XY, XX, YY, trisomy of XXY, autosomal disomy was seen. A Correlation between the age of a stallion and the level of nullisomy was negative. The present study demonstrated that FISH technique is a powerful method to identify sex chromosome aberrations in equine spermatozoa and might be very helpful for a breeder during a selection for the best stallion.

Detection of sex chromosome aneuploidy in equine spermatozoa using fluorescence in situ hybridization.

The aim of our study was to diagnose aneuploidy in equine spermatozoa by multicolour fluorescence in situ hybridization (FISH) technique using specific molecular probes for equine sex chromosomes and autosome pair four (EGFR probe) labeled by different fluorochromes. These were applied on decondensed spermatozoa of four stallions. In total, more than 8800 sperm cells were examined. The total frequency of aberrant cells was 0.496%: aneuploidy of XX (0.135%), YY (0.023%), XY (0.102%), diploidy (0.057%), lack of sex chromosome (0.18%). In one stallion the ratio of normal X- and Y-bearing cells was different from the expected 1:1 ratio (p = 0.0002), in all three other stallions this ratio was close to 1:1. The present study demonstrated that the FISH technique is a powerful method to identify sex chromosome aberrations in equine spermatozoa and allows for the determination of the ratio between X-Y-spermatozoa.

Parthenogenetic activation of domestic cat oocytes using ethanol, calcium ionophore, cycloheximide and a magnetic field.

The objective of this study was to evaluate parthenogenetic activation of domestic cat oocytes after being exposed to either ethanol, magnetic field, calcium ionophore A23187, or cycloheximide and a combination of these agents. We also wished to evaluate the usefulness of the magnetic field for oocyte activation. In vitro matured oocytes subjected to artificial activation were randomly assigned into eight groups according to activating agents: (1) 10% ethanol; (2) the magnetic field (slow-changing, homogenous magnetic field with low values of induction); (3) 10% ethanol plus magnetic field; (4) 10 microM calcium ionophore A23187; (5) 10 microM calcium ionophore A23187 plus magnetic field; (6) 10% ethanol and 10 microg/mL of cycloheximide; (7) 10% ethanol and 10 microg/mL of cycloheximide plus magnetic field; (8) oocytes were not exposed to any of the activating agents. After activation oocytes were stained with Hoechst 33258 and parthenogenetic activation was defined as oocytes containing pronuclei and second polar bodies or two to four or six nuclei (embryonic cleavage). The total activation rate by using different activation treatments was 40%. The addition of the magnetic field to ethanol or calcium ionophore treatments resulted in increased parthenogenetic activation rates from 47% to 75%, and from 19% to 48%, respectively (P<0.001). Instead, when the magnetic field was added to ethanol and cycloheximide treatment, activation rate decreased from 48% to 30%. Oocytes activated with magnetic field only gave the lowest activation rate (12%). We concluded that a magnetic field can be used as an activating agent, and the combination of ethanol and magnetic field is an effective method for domestic cat oocyte activation.

Effect of increased daylight during late pregnancy on the reproductive performance of mares after parturition.

The aim of the present study was to determine the effect of prolonged photoperiod during late pregnancy on subsequent ovarian activity and fertility in mares. Pregnant mares (n=13) due to give birth in January and February were stimulated by a fixed photoperiod (16 h light: 8 h dark) from 15 November (during the last 2-3 months of gestation) until up to 1 month after parturition. A control group of mares (n=9) due to give birth at the same time were kept in the same stable and management regimen, but under natural light conditions. Light-treated mares ovulated during foal oestrus approximately 3 days earlier than did the control mares (P < 0.05). The incidence of winter ovarian inactivity in foaling mares was 7.6% in the treatment group and 33% in the control mares. The processes of involution and uterine fluid expulsion were similar in both groups. The final pregnancy rate was high in both the experimental and control groups, being 92 and 89%, respectively. However, the duration of the open period (period from parturition to next pregnancy) was significantly shorter in the mares that received the light treatment compared with the untreated control mares.

Maternal influence on pre- and postnatal growth of foals born after embryo transfer.

In the present study the growth and development of three pairs of matching gender foals from birth to maturity were compared. One Polish Pony embryo of each pair was transferred from a donor mare (mass 387-400 kg) to a much larger heavy type recipient mare (mass 561-780 kg). The other embryo of each pair underwent normal gestation (control). The transferred and control horses were examined at 9 and 13 years of age, and X-ray pictures were taken of their front legs to measure the length of the bones. On the basis of the results of this study, it is concluded that the processes of growth and development in transferred foals born to heavy type recipient mares can be divided into three phases. During the first fetal stage, body weight gain and growth are faster in transferred foals than in control foals. In the second phase, which comprises the suckling period, the transferred foals put on weight faster than control foals, but the growth rate is slower than during the first phase. In the third phase, after weaning, both body weight gain and growth rate are comparable to those of control foals.

Effect of transport on the intensity of stress reactions in mares and foals.

The aim of this study was to determine the effect of transportation on the behaviour and stress responses of the sympatho-adrenal and hypothalamo-pituitary-adrenal systems in mares and their foals by measuring the peripheral plasma concentrations of adrenaline, noradrenaline and cortisol. Control blood samples were taken from mares and their foals (n=5 pairs) on day 8 after parturition to establish whether blood sampling caused any changes in adrenaline, noradrenaline or cortisol concentrations in blood plasma. The experiment was conducted during foal oestrus (on day 9 after parturition). Adrenaline and noradrenaline concentrations increased in the blood plasma of mares after only 10 min transportation and the increased concentrations were maintained until 30 min after transportation. The adrenaline and noradrenaline concentrations in foal blood plasma increased after 10 min transportation and decreased rapidly to basal concentrations. Cortisol concentrations in the blood plasma of mares reached a peak after 60 min transportation. Peak cortisol concentrations in foal blood plasma were attained after transport was completed. The results of the present study indicate that transportation during foal oestrus is a stronger stress-causing factor for mares than for their foals.

Offspring from one-month-old lambs: studies on the developmental capability of prepubertal oocytes.

A wave of follicular growth in lamb ovaries occurs at about 4 weeks of age, generating a life-time peak in follicle numbers. In order to take advantage of the large number of oocytes available, and to substantially decrease the generation interval, embryos were derived from oocytes collected from 1-mo-old lambs. Animals were subjected to one of 3 regimes of hormonal stimulation: groups 1 and 2 were treated to obtain germinal vesicle-stage oocytes, and group 3 to produce mature metaphase II oocytes. Adult sheep stimulated by an appropriate dose of FSH served as control. The developmental ability of collected oocytes was evaluated by either in vivo or in vitro culture to the blastocyst stage after in vitro maturation and/or fertilization. Blastocysts were transferred immediately or after cryopreservation to suitable recipient sheep. In order to investigate the full developmental potential of these embryos, pregnancies were allowed to go to term. The results show significant differences (P < 0.001) between all experimental groups in blastocyst numbers produced. Embryos derived from group 1 animals produced the greatest number of blastocysts, under both in vivo (36. 7%), and in vitro (22.9%) culture systems. Group 2 gave lowest blastocyst production (5.0%), while group 3 yielded 13.2% blastocysts. The number of pregnant recipients carrying to term lamb-derived embryos was severely reduced for both in vivo- (2 of 9; 22.2%) and in vitro-cultured, fresh (3 of 10; 30.0%) and cryopreserved (1 of 6; 16.7%) lamb embryos. This study is the first report of the birth of live lambs derived from oocytes obtained from donors as young as 4 wk. Defects in the competence of lamb-derived embryos may account for the increased fetal loss during pregnancy and the occurrence of mummified fetuses delivered alongside normal healthy lambs.

Ovum pick-up in sheep: efficiency of in vitro embryo production, vitrification and birth of offspring.

The production of offspring involving available technologies like ovum pick-up, in vitro embryo production and cryopreservation has not been fully described in the sheep. We tested the overall efficiency of these procedures on 20 Sarda dairy ewes that were twice stimulated for recovery of follicular oocytes. In total, 415 oocytes were aspirated from 522 follicles (11.5 oocytes/ewe), and 328 of them (9.1 oocytes/ewe) were selected for in vitro embryo production procedure. Development into blastocysts occurred in 98 embryos (2.7 blastocysts/ewe), of which 64 were vitrified and 34 were transferred, in pairs, directly to recipients. The pregnancy rate, diagnosed at 80 d for fresh and vitrified embryos, did not differ significantly (47.1 vs 42.8%, respectively), but there were significant differences in lambing rates between the 2 groups (41.2 vs 23.8%, respectively). Overall, 24 lambs were born; all weighed within the range for the breed, but head deformities were observed in 2 cases. The results of this study show that with application of the above techniques, it is possible to obtain repeatedly embryos and viable offspring.

Superovulation of a low fecundity sheep breed using a porcine gonadotrophin extract with a defined LH content (Pluset).

The aim of this study was to determine the efficiency of a porcine pituitary gonadotrophin extract with a defined pLH content in the superovulation of sheep. Estrus was synchronized in 61 Polish Mountain ewes with intravaginal fluorogestone acetate sponges. Twenty-four hours before the sponges were removed, the ewes underwent different superovulatory treatments: Group I 250 IU of pFSH with 250 IU of pLH (n=19); Group II 500 IU of pFSH with 500 IU of pLH (n=19); and Group III 750 IU of pFSH and 750 IU of pLH (n=18). Gonadotrophine was administered intramuscularly twice a day over a 3-day period in decreasing dosages. A control group of ewes (n=5) was treated with saline. In most of the ewes estrus began about 20 hours after sponges were removed. All the ewes were bred naturally every 12 hours. Superovulation was confirmed in 75% of the treated animals. The ewes receiving 250 IU each of pFSH and pLH produced an average of 7.6 +/- 3.1 corpora lutea (CL), 6.3 +/- 2.4 ova and 4.3 +/- 4.1 transferable embryos. Group II (500 IU of pFSH and pLH) produced 8.5 +/- 4.0 CL, 7.6 +/- 4.1 ova, and 4.1 +/- 2.9 transferable embryos. Group III (750 IU each of pFSH and pLH) produced 8.3 +/- 5.2 CL, 7.5 +/- 5.5 ova and 5.2 +/- 5.1 transferable embryos. The mean embryo recovery rate was 87% for all three groups. Differences in superovulatory response and embryo recovery rate among the groups were not statistically significant (P>0.05).

Embryo recovery from mares exposed to a year-to-year artificially prolonged daylength.

The aim of the experiment was to determine the effect of a year-to-year prolonged daylength on the patterns of equine reproductive activity and results of embryo recovery. Experiments using Konik Polski mares were conducted over four reproduction seasons. Five mares were exposed to a regimen of artificially prolonged daylength (APD) and another five mares in a control group were kept under conditions of natural daylight. Both the control and experimental groups were examined for appearance of estrus, ovulation and also for the state of their coats. A single stallion was used for breeding all of the mares. The embryos were recovered nonsurgically 6 to 9 days after ovulation. All of the mares exposed to APD showed increased ovarian activity, which commenced earlier than in the control group. About 19% more ovulations were detected in the experimental group. The average number of ovulations per lighted mare per year was 15.3, while in the control group it was 12.4 ovulations (P<0.05). However, the embryo recovery rate and total number of embryos obtained from the mares exposed to APD did not exceed the number of embryos collected from the control mares (P<0.05). Modification of daylength had a visible effect on the mares by producing a change in their coats.

Evaluation of mare oocyte collection methods and stallion sperm penetration of zona-free hamster ova.

Comparisons were made between 2 methods of oocyte recovery from the ovarian follicles of slaughtered mares: 500 oocytes (3 per mare) were obtained by aspiration of follicular fluid from ovaries of 162 mares, and 120 oocytes (8 per mare) by isolation and rupture of follicles from ovaries of 14 mares. In the oocytes recovered after rupture of follicles, 89.2% were morphologically unchanged, in comparison to 29.3% obtained by aspiration of follicular fluid. Stallion spermatozoa capacitated in vitro were tested on zona-free hamster oocytes. The stallion spermatozoa were washed in TCM-199 and preincubated for 4-5 h at a concentration of 0.5-1.0 x 10(9)/ml. Of the 305 hamster oocytes fertilized by spermatozoa obtained from 8 stallions, 77.8% penetration was confirmed on the basis of the presence of enlarged heads of spermatozoa and male pronuclei with their corresponding tails. Spermatozoa from one of the stallions were tested 10 times, 8 months later. The percentage of penetrated oocytes averaged 52.8% (39.1-76.9%). In the control group of oocytes preincubated in the absence of spermatozoa, spontaneous activation and female pronuclei were found in 12 out of a total of 57.

Activity of delta(5)3beta-hydroxysteroid dehydrogenase and steroid hormones content in early preimplantation horse embryos.

The activity of delta (5)3 beta-hydroxysteroid dehydrogenase was examined histochemically in 6 to 10 days aged horse blastocysts. A positive reaction was noted in the blastomeres of all embryos incubated in medium with substrate. Measurable amounts of progesterone, androgens and estrogens were found in blastocysts on day 8th. The presence of enzyme and hormones suggests that steroid hormone production takes place in very early preimplantation horse embryos.

Patterns of stallion sexual behaviour in the absence of mares.

In the first experiment the response of 93 adult stallions to stimulated and natural sexual stimuli was observed just before and 2 weeks after the breeding season (December and July respectively). About 72% of the stallions reacted the same before and after the breeding season (P less than 0.01) while 9% behaved similarly and 19% differently. Also, 72% of the stallions reacted with sexual arousal (mounting) to one of 3 successive simulated sexual stimuli; 25% to a dummy, an additional 44% to a gentle stallion and another 3% to a dummy smeared with mucus and urine from a mare in oestrus. In another experiment, the sexual behaviour of 4 young stallions and 3 older stallions was observed while they were in the stable. Erection was observed an average of 7.4 times in 24 h, a full erection 3.8 times and attempts to masturbate 4.1 times. Complete masturbation with ejaculation was only sporadically noted. The total time of erection averaged 38 min/24 h. The peak of erection time occurred in the morning and the low point during the night and at 05:00-08:00 h. No significant differences were found between the young and older stallions; differences occurred only between individual stallions.

Non-surgical embryo collection in the mare and subsequent fertility of donor animals.

Observations were made on 14 mares which were non-surgically flushed on one or more occasions for recovery of embryos on Days 7-9 after ovulation. Flushing alone shortened the oestrous cycle by about 2.7 days but increased the number of mares failing to ovulate. Flushing followed by an injection of a prostaglandin analogue shortened the cycle by about 4.5 days and hence increased the frequency of ovulations. From a total of 70 flushings performed, 27 embryos were recovered and of 12 mares inseminated during the first oestrus after embryo collection, 6 conceived and foaled. The other 7 mares were used again as donors during the next seasons. They underwent a total of 21 non-surgical embryo collections and yielded 15 embryos. It is concluded that non-surgical embryo recovery and transfer provides a practical means of increasing the fecundity of valuable donor mares without depressing their fertility.