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The time of the first ovulation depends on the sheep breed, feeding conditions, year season, length of lamb nursing and following galactopoiesis. The objective of the present study was to analyse the follicle status after parturition in non-lactating Improved Valachian ewes. Laparotomy with following ovariectomy was performed after lamb weaning (spring) on days 17, 24 and 32 after parturition. The ovaries were USG analysed with 5.0 MHz linear and 7.5 MHz convex transducer. Follicular data were analysed quantitatively and qualitatively. The ovaries were collected at the end of laparotomy and fixed in 10% formalin. The sections of the ovarian tissue were stained with haematoxylin and eosin and Azan staining. Ovarian slides were microscopically studied and analysed by LUCIA-G ver. 4.71. The differences in the ovary size were not significant. The number of follicles < 3 mm monitored by USG on day 32 was higher than that on day 17 after parturition. The image analysis of the ovary sections showed significantly higher numbers in the total follicles (P < 0.05) and the follicles > 3 mm (P < 0.001). The rate of atresia was 82% and 89% on day 17 and on day 32 post partum, respectively. We observed single ovulation on day 17 and double ovulations on days 24 and 32 post partum. More than half of the total number of antral follicles visible on the ovary surface was prepared for recruitment and this number was higher on day 32 post partum. Follicle selection connected with a relatively low rate of atresia and responsible preovulatory follicle sizes opened the way for selecting more follicles for the dominance process and thus also for the occurrence of double ovulations.

Hredzak, R., A. Ostro, I. Maraaek, J. Kaamarik, V. Idilova, J. Vesela: Influence of Slow-rate Freezing and Vitrification on Mouse Embryos. Acta Vet. Brno 2005, 74: 23-27. The aim of the study was to compare the effect of two different methods of embryo freezing (slow-rate freezing employing programmable freezing equipment and ultra-rapid freezing by vitrification) on developmental capacity of two-cell mouse embryos on the basis of their development to blastocyst stage and implantation rate of blastocysts. Two-cell embryos were obtained from superovulated female mice and divided to three groups. The first group of embryos was frozen by the slow controlled-rate method using a programmable freezing equipment with propanediol as a cryoprotectant. Embryos from the second group were vitrified employing ethylene glycol as a cryoprotectant. The third group of embryos was cultivated in vitro without cryopreservation in a cultivation medium in an atmosphere of 95% air and 5% CO2. After thawing, the embryos from the first two groups were cultivated in vitro under conditions identical to those used for fresh embryos. The blastocysts that developed in vitro from embryos of all three groups were transferred to uteri of pseudo-gravid female mice to determine their implantation capacity. The percentage of vitrified embryos that developed into blastocysts was significantly lower than that of the fresh and slow-rate frozen embryos. The morphological appearance of embryos from all three groups was the same. The implantation rate of blastocysts that developed from vitrified embryos was significantly lower compared to the fresh and slow-rate frozen embryos. The results obtained indicate that freezing of embryos affects negatively their further development the negative effect of vitrification being more detrimental. As a “universal” vitrification protocol has not yet been defined, additional studies are needed to achieve its optimisation. Cryopreservation, cryoprotectant, implantation, slow-rate freezing, vitrification

The aim of the paper was to observe the activity of alkaline and acidic phosphatase and non-specific esterase in the epithelial and glandular uterine cells of postpartum goats. Fifteen Slovak short-haired does were used in the experiment. The animals were slaughtered on days 3, 7, 21, 36 and 40 postpartum. The density of alkaline phosphatase in the cylindrical cells of the glandular epithelium of the uterus tended to increase; with a significant (P

Maraaek I . , M. Krajniaakova, M. Kosteck , G. Greserova, I . Valock : Tertiary Follicular Growth Wave Dynamics after Oestrus Synchronization and Superovulation in Ewes and Goats. Acta Vet. Brno 2002, 71: 481-486. The quality and quantity of morphological and functional changes in ovarian tertiary follicles during their growth and development waves after synchronisation of oestrus and superovulation treatment is described. Within 48 and 120 h after injection of 125 μg cloprostenol simultaneous amplification of healthy non-atretic tertiary follicles as well as those recruited and selected as dominant on the ovarian surface was observed. The mean size of the largest growing dominant, i.e., ovulatory follicle significantly increased in ewes 24 and 48 h after cloprostenol administration compared not only to controls but also to experimental groups at 72 and 120 h after luteolytic treatment. Eleven (73.3%) goats and 45 (88.24%) ewes were found to respond to superovulatory treatment. Significant differences (P < 0.01) in ovulation response were noted between FSH (9.6 ± 3.8) and PMSG (4.6 ± 1.1 ) preparations in ewes. After superovulatory treatment the oestradiol17β levels in follicular fluid of the selected dominant follicles increased significantly as compared to the large atretic follicles (P < 0.001). Oestrus synchronisation in ewes was carried out by cloprostenol induction of the new follicular wave. These results suggest that luteolysis has a key role both in the process of recruitment and in the induction of selection during folliculogenesis in ewes. Evaluation of the superovulatory response in ewes and goats demonstrates that FSHand PMSG-based preparations affect follicles after recruitment in the process of selection. Ovarian follicles, recruitment, selection, cloprostenol, chlorsuperlutin, FSH, PMSG The ovary of an adult ewe contains 12 000-86 000 primordial follicles and 100-400 growing follicles of which 10-40 are visible on the ovarian surface (Cahi l l et al. 1979; Mc Natty et al. 1982; et al. 1985). The differentiation of one mature preovulatory follicle, the so-called dominant follicle, is the result of a complex interaction between the cohort of ovarian follicles on the growth trajectory and the hypothalamo-pituitary system with intraovarian endo-, paraand autocrine regulatory factors and mechanisms (Driancourt et al. 1985; Murdoch 1985, 1988; Erickson 1986, Presl and Bukovsk 1989ab; Greenwald and Terranova 1988; Driancourt 1994). Differentiation of the dominant tertiary follicle is a two-stage process (Di Zerega and Hodgen 1981) in which, as a result of gonadotrophic stimulation, anthral follicles are advantaged by more intensive growth. The development of normal healthy tertiary follicles with a diameter surpassing 2 mm is termed recruitment and the developing, growing anthral follicles with a diameter of 2 5 mm are termed recruited. Differentiation and maturation advance to the process of selection of those tertiary follicles that reach a diameter of more than 5 mm. This process of selection usually results in one, seldom in two and only exceptionally in multiple, 3 or 4 dominant ovulatory follicles. All other follicles undergo atresia (Driancourt et al. 1985; Murdoch 1985, 1988; DoleIel 1995ab). Literary data point at the fact that in sheep, the process of recruitment occurs at a variable time around luteolysis (Mc Nat ty et al. 1981, 1982; Murdoch 1985). ACTA VET. BRNO 2002, 71: 481–486 Address for correspondence: Prof. MVDr. I. Maraaek, DrSc. Department of Physiology University of Veterinary Medicine Komenskeho 73, 041 81 Ko‰ice, Slovak Republic Phone: +421 55 638 249 Fax: +421 55 63 318 53 E-mail: maracek@vuvm.sk http://www.vfu.cz/acta-vet/actavet.htm In this work quantitative analysis was carried out of the tertiary follicular populations in the ovaries of sheep and goats from the viewpoint of the process of follicle recruitment and selection after (1) luteolytic treatment of Czigaya sheep with a preparation on the basis of cloprostenol and (2) superovulation induction by means of FSH or PMSG preparations in Merino sheep and white goats. It was our aim to materialize and extend our knowledge (1) of the selection of the dominant ovulatory tertiary follicle and (2) of the influence of cloprostenol racemate-, follitropinand pregnant mare serum gonadotropin-based preparations upon tertiary follicles in sheep and goats. Materials and Methods Experiment 1 Observations and experiments with the cloprostenol racemate-based preparation were carried out during the breeding (mating) season in the autumn oestrous period. Forty-one Slovak Czigaya sheep aged 2 5 years and bred under standard commercial conditions were divided into 5 groups. Group I (n = 6) comprised the control animals on day 10 12 of their sexual cycles. In the luteal phase of their oestrous cycles the experimental animals were divided into 4 groups, viz, Groups II (n = 8), III (n = 9), IV (n = 8) and V (n = 10); they were i. m. treated with 125 g cloprostenol in 0.5 ml of Oestrophan inj. ad us. vet. (Leaiva Prague, Czech Rep.) per animal. The ewes in Groups II – V were killed 24, 48, 72 and 120 h after treatment, respectively. After excision, the ovaries were fixed in 10% neutral formalin for 48 h; then, surface follicles were counted and measured. Fixation of the ovarian tissues was followed by cutting the ovaries into 4 mm segments that were postfixed for 5 days, rinsed, dehydrated and subsequently paraffin-embedded. Five-to-seven m thick simultaneous transverse serial sections made at 150-210 m intervals were stained with haematoxylin-eosin or the PAS reaction with nuclear staining using Harris haematoxylin. Qualitative evaluation of the developing growing and healthy follicles and their differentiation from the atretic ones as well as quantitative evaluations were made in the way described earlier (Maraaek et al. 1983; 1993). According to this classification there are three stages of atretic follicles in addition to normal developing tertiary follicles, i. e., 1 early atresia, 2 definite atresia and 3 late atresia. A tertiary follicle in early atresia shows the following histological features: the membrana limitans interna disappears, atretic bodies are formed, the membrana basalis disappears or is absent. In definite atresia the follicle is subjected to many changes that prevent the production of the ovum. In this stage of atresia collapsing, contracting, cystic and luteinized cystic atresia can be differentiated. The stage of late atresia reveals changes typical of the terminal stage of the individual types of definite atresia with a typical decrease in size and lumen. Experiment 2 In the second experiment we evaluated the ovaries of 51 ewes of the Slovak Merino breed and 15 Slovak white goats. For superovulation treatment, folitropin (FSH) in the preparation Folicotropin inj. ad us. vet. (Leaiva, Prague, Czech Rep.) (n = 24) or pregnant mare serum gonadotropin (PMSG) in the preparations Folligon (Intervet) (n = 6) and Sergon (Bioveta, a. s., Czech Rep.) (n = 21) were used, respectively. The superovulation treatment was carried out after synchronization of the oestrus by means of chlorsuperlutin (Agelin ad us. vet., Spofa, Prague, Czech Rep.). The schemes for the preparation of donors and recipients were designed in the way described earlier (Maraaek et al. 1989; et al. 1997). The ovarian tertiary surface follicles and ovulation (the number of corpus luteum) were evaluated at washing up and collection of oocytes and zygotes during laparotomy or laparoscopy. Experiment 3 In the third experiment we determined the level of oestradiol-17β (E2) and progesterone in the follicular fluid of the largest follicles from the ovaries of 9 Slovak white goats. Follicular fluid was aspirated from the largest follicles on the 2nd day after PMSG (n = 4, goats) administration or after the last injection of FSH (n = 5, goats). After centrifugation at 3000 g the supernatant was stored at -18 oC until processing. In order to determine oestradiol-17s and progesterone levels in the follicular fluid the RIA-test-Estra (SI-125-9) and RIA-test-Prog (SI-125-6) commercial kits manufactured by Huma-Lab Ko‰ice (Slovak Republic) were used, respectively. The fluid was diluted with 1.5% bovine gammaglobulin. The intraand inter-assay coefficient of variation presented 10.1 and 13.3%, respectively, for the oestradiol kit and 11.4 and 13.7%, respectively, for the progesterone kit. After determining the significance of differences between the standard deviations by the F-test (Sokal and Rohl f 1969) the significance of differences between the two experimental groups was calculated by the unpaired Student’ s t-test.

Krajniaakova M., V. Cigankova, a. Lenhardt, M. Kosteck˘, I. Maraaek: Functional and Morphological Evaluation of the Mucosa of Endometrium and Oviduct in Postpartum Goats. Acta Vet. Brno 2002, 71: 517-522. The aim of this work was to characterize the structural changes in the surface epithelium and endometrial glands as well as oviduct mucosa in goats after parturition using the light and transmission electron microscopy. Fifteen Slovak short-haired goats were used. They were killed on days 3, 21, 36, and 40 after parturition. The evaluation of semithin sections from day 3 postpartum revealed impaired epithelial cells. Lymphocytes and neutrophil granulocytes were present in the lumen of uterine glands. Ciliated cells and sporadic secretory cells that were released into the oviduct lumen were found on semithin sections from the ampullary part of oviducts (day 3 postpartum). Cells in the cytoplasm contained mitochondria and cisternae of endoplasmic reticulum. Impaired cells with a vacuolised cytoplasm were present on ultrathin sections from the caruncular region of endometrium on day 21 postpartum. Uterine glands were in the stage of proliferation. Ultrathin sections from day 21 postpartum showed that ciliated cells were present and secretory cells with secretory granules increased in the epithelium of oviduct ampulla. The surface epithelium on day 36 was continuous, simple columnar; in some locations pseudostratified columnar. No signs of morphological damage of cells were found. On the free border of ciliated cells of the oviduct mucosa on day 36, the presence of numerous kinocilia was observed. Glandular cells contained a great amount of dense secretory granules in the cytoplasm (day 36 postpartum). The microscopic and submicroscopic appearance of the uterus and oviduct on day 40 postpartum was similar to that on day 36 of the period observed. The results obtained can be used in synchronization or induction of estrus in the intensification of reproduction and increasing the reproductive turnover in goat husbandry.

Krajniaakova M., E. Bekeova, a. Lenhardt, V. Cigankova, I. Valock˘, I. Maraaek: Microscopic Analysis of the Uterine Endometrium in Postparturient Ewes. Acta Vet. Brno 1999, 68: 9-12. Earlier we found that the puerperal period of ewes lambing in February was finished by day 34 post partum.The aim of this paper was to study the puerperal changes in ewes that lambed in September. Structural changes in the endometrium of the caruncular region were studied in 12 Slovak Merino ewes that lambed in September. The animals were killed on days 7, 17, 25 and 34 post partum. Samples were taken from the caruncular region of their uterine horns. The tissue samples were fixed, dehydrated and embedded in paraffin, and 7-10 mm sections were stained with hematoxylin-eosin. Microscopic analysis was performed using a projection microscope. Simultaneously the collected material was stained with toluidine-blue, and examined on the semi-thin sections. The epithelium above the caruncles was entirely destroyed on day 7. Damaged mitochondria and dilated cisterns of the endoplasmic reticulum were found in the electron micrographs. On day 17, the epithelium covered gradually a sizeable caruncle. The glandular epithelium was considerably degenerated. The caruncle was markedly reduced on day 25, and the endometrium was covered with epithelium. The endometrium was totally covered with epithelium, and cellular ultrastructure was no more damaged on day 34 post partum. The study of micromorphology of endometrial structure in caruncular area revealed that also in ewes that lambed in September, the puerperium is finished by day 34 post partum. Its course and timing did not differ from that seen in ewes lambing in February. The results extend the knowledge of the puerperal changes in different season of the year. Caruncular region, endometrium, ewe, histological structures, post partum, season of the year One of the factors that may unfavourably affect fertility is the course of the involutional and reparatory processes of the uterus in the postpartal period. At the onset of postpartal reproductive activity it is necessary to take into account the breed, the season of the year, nutrition, and the length of suckling (Kudlaa 1985; DoleIel 1989). The macroscopic changes occurring during involution of the sheep uterus were reported by F oote and C all (1969); Crowder et al. (1982); Botha (1976); Van Wyk et al. (1972); Krajniaakova (1990); Greyling and V an Niekerk (1991) in goat, and Massanyi (1996) in rabbits. A comprehensive histological study of the sheep uterus post partum was carried out by Uren (1935); and Van Wyk et al. (1972) who found that the involution process of the uterus was completed by day 28 post partum. B otha (1976) reported that both the season and lactation influence epithelisation of the caruncles. The author observed sheep that lambed during the out-of-mating season (August) to have epithelisation in the caruncular region finished on day 34 post partum whereas those that lambed in the lambing season (March) on day 30 post partum.

The aim of the study was to determine the relationship between the rate of cooling of eight-cell mouse embryos to the temperature of liquid nitrogen (-196°C) and their developmental capacity after thawing on the basis of their ability to leave the zona pellucida ('hatching') during in vitroculturing. Eight-cell embryos were obtained from superovulated female mice and divided into three experimental and one control group. Embryos from the experimental groups were cryopreserved by the vitrification method using ethylene glycol as cryoprotectant. The vitrification protocols used in the study differed in the rate of cooling of the cryoprotectant solution. Embryos from the first group were frozen in conventional 0.25-ml plastic straws, those from the second group in pipetting 'tips', and embryos from the third group, placed in vitrification solution, were introduced dropwise directly into liquid nitrogen. The control group of embryos was cultured in vitrowithout freezing in a culturing medium in an environment consisting of 95% air and 5% CO2. The developmental capacity of thawed embryos was assessed on the basis of their ability to leave the zona pellucida ('hatching') after three days of in vitroculturing. In the control group 95.1% of embryos 'hatched'. A significantly higher number of embryos that 'hatched' after thawing was observed in the group introduced dropwise directly into liquid nitrogen (60.0%) compared to the group frozen in pipetting 'tips' (37.9%). The group frozen in straws yielded significantly the lowest proportion of 'hatching' embryos (8.1%). These results showed that increasing cooling rates during vitrification of embryos improved their survival.