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A 9-month-old Yorkshire terrier was admitted to the clinic because of abnormal sexual behaviour and clitoral hypertrophy. External examination confirmed standard development of caudal genital organs: vagina, vulva and cervix uteri. Serum profile of gonadotropin hormones 17 β-estradiol (<10.0 pg.ml(-1)) and testosterone (9.1 ng.ml(-1)) revealed the presence of testicular tissue. A midline laparotomy was performed to detect the cranial parts of the genital system. Gonads resembling testicles, structures indicating epididymis and rudimentary deferent ducts were resected, along with adherent part of the uterus. Cytogenetic analysis showed a male chromosomal complement 78, XY in all metaphases of the studied Yorkshire terrier dog. The chromosomal constitution was confirmed by fluorescence in situ hybridisation (FISH) with whole-chromosome painting probes specific for chromosomes X and Y, as well as by polymerase chain reaction (PCR) amplification of the 271-bp Y-linked fragment of SRY (the sex-determining region on the Y chromosome) gene. Sequencing of the dog's SRY gene coding region did not reveal any mutation. To search for potential mutation in the SOX9 gene (Sry-box containing gene 9), which is considered to be one of the key genes involved in the sex determination process, the PCR fragments of exons 1, 2 and 3 originating from the canine patient were sequenced in order to compare with both male and female healthy control dogs. In the analysed regions of the SOX9 gene, no mutation was found.

The objective of the present study was to compare the concentrations of 17 beta-estradiol, progesterone, cyclic adenosine monophosphate and cyclic quinosine monophosphate in the largest follicles of cows that persist for seven days after insemination following the preceding synchronization of oestrus and superovulation and in follicles of the luteal phase of cycle (5th-10th days). Animals included in the experiment were selected on the basis of rectal examination. Synchronization of oestrus was achieved in 24 crossbreds of Slovak Pied x Lowland Black Pied breeds (SS x Nc) using two doses of cloprostenol of Czechoslovak provenience Oestrophan Spofa, 500 micrograms in each, within 11 days. Serum gonadotrophin at the amount of 2500 I. U. was administered forty-eight hours before administration of the second dose PGF2 alpha. Experimental animals were inseminated after 72 hours. On the 7th day after mating the cows were killed at a slaughterhouse. Evaluated were only the ovaries of the 14 cows in which the persistent large follicles occurred. Ovaries of the 13 control cows in the luteal phase between the 5th-10th days were obtained at the slaughterhouse by the method after Ireland et al. (1980). Correct determination of the phase of sexual cycle was substantiated by determination of progesterone concentrations in blood serum. Follicular fluid was obtained from the largest follicles by aspiration and centrifuged in a cooled centrifuge at 3000 G. The concentrations of 17 beta-estradiol and progesterone in follicular fluid were determined using kits from URVJT at Kosice, designated RIA-test-ESTRA (SI-125-9) or RIA-test-Prog (SI-125-6).2+ persistent follicles (9.15 +/- 5.47 nmol.l-1).(ABSTRACT TRUNCATED AT 250 WORDS)

The objective of this study was to compare the concentrations of 17 beta-estradiol, progesterone, cAMP and cGMP in the follicular fluid of the largest cow follicle from the follicular phase of physiological sexual cycle and of follicles after synchronization of fut by cloprostenol (PGF2 alpha) and superovulation treatment with serum gonadotrophin (PMSG), in dependence on steroidal dominance of follicles. 2 x 25 cows, Slovak Pied x Lowland Black-Pied crossbreds with active corpus luteum, were subjected to superovulation treatment on the basis of rectal examination. Rut synchronization was achieved by cloprostenol of Czechoslovak provenience (Oestrophan Spofa), administered at the amount of 500 micrograms per dose. Serum gonadotrophin (Bioveta Concern, Ivanovice na Hané) at the amount of 2500 I. U. was administered forty-eight hours before the second dose of closprostenol. The animals were killed in slaughterhouses 48 hours later, or 72 hours later, since administration of the second dose of cloprostenol. The phase of the sexual cycle of control animals was determined by the method after Ireland et al. (1980) on the basis of morphological appearance of corpus luteum, presence of large preovulation follicle and by means of average concentrations of progesterone in blood serum. Aspirated follicular fluid was centrifuged using a cooling centrifuge at 3000 G. After separation, the supernatant was stored in a freezer at -18 degrees C until further treatment. 17 beta-estradiol and progesterone in the follicular fluid were determined by means of kits under the brand-names RIA-test-ESTRA (SI-125-9), or RIA-test-Prog (SI-125-6). Concentrations of cyclic nucleotides were determined by the RIA kits from the Institute vor Radioisotope Research, Production and Use (Prague), cAMP by 125J RIA kit (RIO12) and cGMP by 125J RIA (RIO42).(ABSTRACT TRUNCATED AT 250 WORDS)

Repeated radio-immunological assays of progesterone in milk and ovary rectal palpation were used for indirect determination of embryonal mortality in cows. Milk sampling and rectal examination were made in the insemination period and then in two- to three-day intervals, mostly from the 21st day after insemination till the early pregnancy determination made after the 42nd day on the basis of the double wall phenomenon. Observation was made in 185 animals from two herds. Embryonal mortality was found in 9.06% out of 289 evaluated inseminations, which prolongs the oestral cycle over 25 days. The monitoring corpus luteum function in connection with rectal ovary palpation supplemented by oestrus, ovulation and insemination control enable a more objective indirect diagnosis of embryonal losses prolonging the oestral cycle.

Trials were conducted to study and describe the micromorphological parameters of endometrium in five cows of the Black-Pied breed after the expiration of the synchronizing effect of cloprostenol in the Oestrophan inj. Spofa preparation. Two animals four to five days after oestrus were used as control and three cows on the sixth day of sexual cycle with rectally palpated corpora lutea were treated intramuscularly with 0.5 mg cloprostenol in 2 ml of Oestrophan. On the eighth day from the administration of the product, samples of uterine horns were obtained by necropsy and were subjected to histological preparation. Paraffin slices, 7 micron thick, were stained with haematoxylin-eosine by the PAS reaction. Changes were observed in the ipsilateral and contralateral uterine horns in relation to active corpus luteum. A state resulting from previous oedematization, with ample thin connective tissue and fading cell infiltration after the administered preparation, was described in the subepithelial layer of endometrium. The occurrence of intraepithelial lymphoid cells was observed in the surface epithelium of endometrium. A significant increase in the thickness of endometrium (P less than 0.001) was observed to persist on the eighth day after the administration of cloprostenol. An increase in the thickness of surface epithelium, ipsilateral with the corresponding corpus luteum, was on the level of significance (P less than 0.05). The subsurface cells of endometrial glands shrunk significantly after the administration of the preparation, both ipsilaterally (P less than 0.001) and contralaterally (P less than 0.001) to the corpus luteum, like in the glands in the depth of the mucous membrane just above myometrium (P less than 0.01). The occurrence of intraepithelial lymphoid cells decreased significantly after the administration of cloprostenol.

Thyroxine and progesterone levels were studied in cows and heifers in the luteal stage of the cycle after the application of the synthetic analogue of prostaglandine of group F2 alpha: Oestrophan Spofa. The preparation was administered at a rate of 0.5 mg in 2 ml per animal. The thyroxine and progesterone levels were determined radioimmunologically. The administration of Oestrophan led to a reduction in the progesterone level from the initial value of 17.33 +/- 2.73 nmol/l of serum to 7.69 +/- 1.17 nmol/l of serum an hour after the application, followed by slow decrease below the value 1.59 +/- 0.22 nmol/l of serum at the 24th hour. The initial levels of thyroxine with the average values of 122.11 +/- 23.41 nmol/l of serum were comparatively high. An hour from application of the preparation, a significant decrease to the average levels of 88.14 +/- 16.26 nmol/l of serum (P less than 0.05) was recorded with a significant negative correlation. The correlation coefficient r = -83316, P less than 0.01. The thyroxine level decreases to the values of 24.20 +/- 9.26 nmol/l of serum and 24.12 +/- 6.68 nmol/l of serum, P less than 0.01, at the 52nd to 56th hour, i. e. at the time when the pre-ovulation peak of oestrogens can be expected. A statistically significant correlation--even despite the fact that a highly significant decline in thyroxine and progesterone had been observed--was recorded in none of the cases in the subsequent period of study, except the first hour. It can be assumed from the results that the thyroid hormones are among the links acting at more levels of the regulatory biological circuit responsible for the physiological course of ovulation and reproduction process.

We determined the thyroxine (T4) concentration in 63 heifers on 0th (day of insemination), 6th and 21st day after first and in repeat-breeder cows after second and third insemination in relation with the breed, age and weight of animals. The examination was carried out with a feed ration balanced according to the Czechoslovak State Standard CSN 46 7070, with all-the-year-round housing, with keeping the uniform time of insemination (8.00--9.00 a. m.) as well as the time of blood sampling (10.00--12.00 a. m.). The animals were inseminated from August to November. The average T4 concentrations, with a successful insemination, fluctuated on 0th day after first, second and third insemination at levels of 56.15 +/- 13.6; 84.6 +/- 10.66 and 89.75 +/- 13.62 nmol X l-1 of serum. As the results show, the lowest T4 concentrations (P less than 0.001; P less than 0.001) on 0th day were recorded in animals becoming pregnant in first insemination. The comparison with non-pregnant animals did not show any statistically significant difference in T4 concentrations. On 0th day of the second insemination, the T4 concentrations in pregnant animals were higher (P less than 0.01) as compared with non-pregnant. On 6th and 21st day after successful first insemination the T4 content increased significantly (P less than 0.001). After second and third successful insemination we recorded in T4 concentrations a moderate decrease on 6th day (P less than 0.01 for third insemination), with insignificant increase on 21st day.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of beta-carotene was studied, as exerted on the amount, ductility, pH value, arborization and sperm survival time in the medium of the cervical mucus of heifers included in the breeding stock. The measurements were taken during oestrus and mating. The animals were divided into four groups. The first group was given diet containing no carotene. In the other groups (II, III, IV) the animals were fed a supplement of synthetic beta-carotene, the doses being 100, 200 and 400 mg per head/day. The survival of spermatozoa in the medium of cervical mucus was found to be significantly longer in the fourth group (115.2%) as compared with the first group (61.8%). In the statistical comparison of these results the P level was less than 0.05. Statistically significant differences were observed in the amount, ductility and arborization between the pregnant heifers and oestrus return ones in the first group. These differences were reduced after the addition of beta-carotene to feed. Beta-carotene saturation in the feed ration was not found to exert any significant influence on the pH value of cervical mucus. As suggested by the evaluation of the results and their comparison with literary data, sperm survival time in cervical mucus was the only trait influenced significantly by beta-carotene: no significant effect was exerted on the other parameters under study. Beta-carotene seems to influence the role of polyfactorial factors in the control of steroidogenesis.

We evaluated the ovaries of 34 cows cross-bred of the Slovak Pied and Lowland Black-pied breeds which were culled and intended for slaughter during the winter type of feed rations. For superovulation treatment we used PMSG in the preparation Serum Gonadotropin (Bioveta, Nat. Ent., Ivanovice na Hané) and cloprostenol in the preparation Oestrophan inj. Spofa. We weighed the excised ovaries, fixed them in formalin 10% and made a quantitative evaluation of the surface follicles and differentiated them into recruited and selected or dominant follicles. We determined the concentration of 17 beta-estradiol and testosterone with the aid of the 3H RIA set from the firm Sorin after extraction by diethylether with separation of free and bound hormone with active charcoal. 72 hours after the giving of cloprostenol 43% reacted positively to superovulation treatment and after seven days a 50% positive response was recorded. After a dose of 2000 i. u. of PMSG (n = 6) embryo was obtained, whereas after 3000 i. u. of PMSG (n = 8) we flushed out eight embryos, of which four zygotes were suitable for transfer. After a higher dosage of PMSG there was an increase in the average weight of the ovaries, in right-hand ovaries significantly with P less than 0.05. After super-ovulation treatment the concentration of 17 beta-estradiol (E2) in the follicular fluid from follicles seven days after insemination was found to be so low as to be below the limit of detection, with the exception of four samples (mean = 8.60 nmol.l-1). The greatest concentration of E2 was from animals (n = 5) 72 hours after the giving of cloprostenol--1099.61 nmol.l-1) of follicular fluid. The concentration of testosterone was lower in the follicles of untreated cows in the follicular phase (mean = 5.92 nmol.l-1) compared with the follicles of super-ovulated animals the seventh day after insemination (mean = 14.12 nmol.l-1). The number of recruited and especially selected surface antral follicles 72 hours (n = 7) after the giving of cloprostenol and seven days (n = 8) after insemination was significantly higher in the group of brood cows reacting positively to superovulation in comparison with the animals which did not respond. It appears that the simultaneous monitoring of hormonal and morphological changes in the follicular system will help in objectivising the evaluation of the functional activity of stimulated follicles.

Serum concentrations of thyroxine (T4), beta-carotene (beta K), vitamin A and conception after the first insemination on the zero, sixth and 21st day were studied in 63 heifers fed synthetic beta K supplement in groups I to IV at stanchion housing and low-carotene diet. The following amount of beta K was supplemented per head/day: I--0 mg (control, n = 17), II--100 mg (n = 16), III--200 mg (n = 14) and IV--400 mg (n = 16). The parameters were evaluated separately according to groups and conception. By comparing the T4 concentration between experimental groups and control, separately in non-gravid and gravid animals, a significant increase (P less than 0.05) was determined only on the zero day in the fourth group. At the same time, in the fourth group, in comparison with the others, insignificantly lower T4 values were recorded in non-gravid animals. beta K values on the zero day in gravid animals increased in a direct proportion to the higher supplement (P less than 0.05; P less than 0.01; P less than 0.001); in non-gravid animals the increase was slower (P less than 0.05; P less than 0.05; P less than 0.01), but differences in relation to gravidity were not significant. An increase of beta K had no significant effect on serum concentrations of vitamin A. Differences between groups as far as conception was concerned were insignificant. It follows from the results that increasing rates of beta K, with the exception of serum concentrations, had no significant effect on other parameters. Higher concentrations of T4 and a slower increase of beta K in non-gravid animals, however, point at mutual co-operation relation and its positive effect on conception in animals.