Seasonal variations in the morphometric analysis of the ovary and uterus and in progesterone and 17 p-oestradiol production in the brown hare ( Lepus europaeus )

Seasonal variations in the morphometry o f the ovary and uterus, and in progesterone and 17(3-oestradiol production in 37 brown hares (Lepus europaeus), 10 in spring, 7 in summer, 14 in autumn, and 6 in winter, are reported. Evaluation o f the relative volume o f primary and growing follicles, and stroma in the hare ovary revealed a stable level o f primary follicles during the year. The highest relative volume o f growing follicles was found in the autumn, w i th a similar value in winter. As the relative volume o f growing follicles increased, the amount o f stroma decreased. The lowest relative volume o f stroma was found in the autumn (90.1 % ) and the highest in the spring (97.3%). N o significant differences were found in the relative volumes o f follicles and stroma in the ovary. The diameter o f primary follicles ranged from 30.8 to 35.9 u,m during the year, w i t h no significant seasonal difference. The diameter o f grow i n g follicles ranged from 139 to 222 | i m . The highest relative volume o f endometrium in the uterus occurred in the autumn and summer. This difference was significant in comparison wi th spring (P<0.05). The height o f the endometrium was highest in the summer and lowest in the winter. Detailed analysis o f the endometrium showed that the highest relative volume o f the surface epithelium was in the spring (6.5%), the highest relative volume o f glandular epithelium in the winter (22.4%), and the highest relative volume o f stroma in the autumn (85.8%). The highest surface epithelium was found in the spring. The average diameter o f uterine glands was highest in the spring (63.5%), but the differences between seasons were not significant. 698 VARIATIONS IN THE OVARY AND UTERUS IN HARE Analysis o f progesterone and 17P~oestradol in hare blood showed that the highest levels o f progesterone occurred in the spring (22.91 ng/ml) . Significant differences were found in the progesterone concentration between spring and autumn (P<0.001). For 17p-oestradiol, the highest concentrations occurred in the winter and the lowest in the summer. The results o f this study clearly demonstrate that the highest follicular growth in the brown hare occurs in the autumn, which corresponds w i th the highest relative volume o f uterine glandular epithelium in winter as we l l as w i th the highest diameter o f uterine glands in the spring. We suggest that in our conditions, the reproductive activity in the female brown hare starts in the autumn. K E Y W O R D S : hare (Lepus europaeus), ovary, uterus, progesterone, 17p-oestradiol


INTRODUCTION
Much attention is being paid to applied research on the brown hare, which had been the most plentiful game in Slovakia (Hell and Slamecka, 1999) in connection with the critical decline in its population during the last ten years (in the spring of 1989, the standard stock was 65.5 heads per 100 ha 1 , in 1992, only 58.5 heads per 100 ha 1 ; in the autumn of 1989, the standard stock was 114.8 heads per 100 ha 1 , in 1992 only 81.0 heads per 100 ha" 1 ).Attention should also be paid to fundamental research, supllemented with descriptions of microscopic structure, to solve this problem in practical terms.
Although there are many reports describing the quantitative structure of reproductive organs in farm animals (Jantosovicova et al., 1996;Kolodzieyski and Danko, 1995;Danko, 1997) there are few observations on the morphometry and secretory activity of sexual organs in the hare.Enlargement of the uterus (Calliol et al., 1989a) and increases in the levels of progesterone (Calliol and Martinet, 1976;Calliol etal., 1991) and oestradiol (Caillol et al., 1989a(Caillol et al., ,b, 1991;;Semizorova et al., 1990) have been documented.These authors studied mostly cage-bred brown hares, not animals from the wild.Knowledge of the natural reproductive changes that take place in the hare in the wild is interesting from the point of view of the venatical management of the species.
The purpose of this study was to identify seasonal variations in the microscopic structure of the ovary and uterus and in the blood concentrations of progesterone and 17(3-oestradiol in the brown hare {Lepus europaeus).

MATERIAL AND METHODS
The samples were taken from hares {Lepus europaeus), which were caught in the surroundings of the town of Trnava, West Slovakia.This is a hunting area with optimum soil and climatic conditions for this game, crop plant production, howe-ver, the ecology of the landscape is seriously affected by intensive, large-scale industrial production and its biodiversity is therefore markedly reduced.The hunting ground is situated on the table, which is in fact the northern prominence of the West Slovakian (Danube) Lowland.
Over a period of one year, 37 female hares were analysed.In the spring we collected the samples in March, April and May (10 animals), in the summer, in June, July and August (7 animals), in autumn, in September, October and November (14 animals), and in winter, in December, January and February (6 animals).The weight of animals was the highest in the spring (4.4±0.50 kg), lower in winter (4.10±0.33 kg) and autumn (3.84±0.75),and lowest in summer (3.23±1.70 kg), but the differences were not significant.In the spring the ovaries weighed 3.61 ±0.5 g, in winter 3.42±0.75g, in summer 3.03±0.66g and the least in autumn 1.99±1.02g.The weight of uterus was the highest in the summer (113.5±20.3g), lower in spring (29.3±5.0 g), winter (25.7± 6.3 g) and autumn (9.1±3.5 g).During the year, 5 females were pregnant (3 in summer, 1 in spring and 1 in winter).
Determination of the age (adult, juvenile) of the shot hares was based on the study of dry eye lens weights.This method is much more reliable that palpation examination of the Stroh's character, which does not enable identification of young from the first litters in December.The critical value of the eye lens weight, differentiating the yearlings and adult individuals was approximately 280 mg, and it ranged from 270 to 290 mg (Slamecka et al., 1997).
Immediately after killing the animals, samples of ovaries and uterus were fixed in 10% formol.After fixation the samples were dehydrated in a graded series of ethanol (70, 80, 90 and 100%), saturated in benzene, benzene-paraffin and embedded into paraffin.Blocks of samples were then sectioned on a microtome into 10 jum thick sections, which were stained with haematoxylin and eosin (Vacek et al., 1974).From microphotographs (Docuval, Carl Zeiss Jena) based on micromorphological criteria (Weibel et al., 1966;Uhrin, 1992;Massanyi and Uhrin, 1996a) the quantitative values of ovarian and uterine structures were evaluated with respect to each sample.
Ovarian tissue was investigated in terms of its qualitative microscopical structure, the relative volume (%) of primary and growing follicles and stroma, and the diameter (jum) of primary and growing follicles.The qualitative microscopical structure of the uterus was determined, so too was the relative volume (%) of the endometrium, myometrium, as well as of the surface epithelium, glandular epithelium and stroma in the endometrium.The height (|im) of the surface epithelium and the diameter (jum) of the uterine glands were determined and the data were analysed for seasonal effects.
The levels of progesterone and 17(3-oestradiol in the blood of hares were assessed by RIA (radioimmunoassay) using sets (Institute for Radioecology and Use of Nuclear Technique, Kosice).The sensitivity of RIA to progesterone was 2 pM/ml, the antibody against progesterone had 58.6% cross-reaction with 11 a-hydroprogesterone and lower than 0.01% with Cortisol, testosterone, estradiol and estrone.The sensitivity of RIA to estradiol was 2.5 pM/ml, the antibody had a cross-reaction with estrone 25%, with estradiol 1.84% and lower than 0.001 % with progesterone, testosterone and Cortisol.
To compare these treatment means the analysis of variance as well as Student's t-test and Scheffe's test were applied (SAS, 1989) and EXCEL (1998).

The ovary
The ovaries are small, flattened ovoid organs, laying in the right and left lateral pelvic cavities.The surface of the hare ovary is covered by a single layer of epithelium.A substantial basement membrane {tunica albuginea) separates the surface cells from the underlying ovarian tissue divided to the inner medulla and outer cortex, which consist of follicles and stroma (Figures 1, 2 and 3).An evaluation of the relative volume of primary follicles, growing follicles and stroma in the hare ovary (Table 1), indicated that the relative volume of primary follicles is very stable throughout the year, ranging from 0.53 to 0.86%.The highest relative volume of growing follicles occurred in the autumn (9.2%) with a similar value in the winter (7.9%).The difference between the autumn when the hare reproduction season starts and spring was 7.0%.On the other hand, with the increase in the relative volume of growing follicles the relative volume of stroma decreased.The lowest relative volume of stroma was found in the autumn and the highest in the spring (97.3%).In the evaluation of the relative volume of follicles and stroma, no significant differences were found, but a tendency towards increased folliculogenesis in the autumn and winter was evident (Figure 6).
The diameter of primary follicles ranged from 30.8 to 35.9 jj,m, with no significant seasonal differences.The diameter of growing follicles ranged from 139 to 222 jum.Since we did not study growing follicles further (follicles with less than 2 layers of granulosa cells; follicles with more than 2 layers of granulosa cells; follicles with antral formation; antral follicles), the standard deviation is high and no significant differences were found.

The uterus
The uterus is a muscular organ and receives the right and left fallopian tubes.It is lined by columnar surface epithelium, which forms glandular epithelium (Figures 4 and 5).The hare has a uterus duplex, i.e. the uterus has two bodies and two cervices.
Data reporting the relative volume of endometrium and myometrium are given in Table 2.The highest relative volume of the endometrium occurred in the autumn and summer (38.3%) and the lowest in the spring 23.9% (P<0.05).Although the highest values for the relative volume of the endometrium oc-   curred in autumn and the lowest in spring, the seasonal difference in this parameter was not significant.Detailed analysis of the endometrium showed that the relative volume of surface epithelium ranged from 3.5 to 6.5%, of glandular epithelium 10.0-22.4%, and of stroma 74.1-85.8%(Table 3).The highest relative volume of surface epithelium Hare uterine endometrium structures in relation to season was in the spring, the highest relative volume of glandular epithelium was in the winter and the highest relative volume of stroma was in the autumn.These data suggest that the highest activity of the endometrium is in the winter and spring.
The maximum height of surface epithelium was also found in the spring, but the seasonal differences were not significant.In the evaluation of the diameter of uterine glands responsible for secretory activity of endometrium, the highest average value was observed in the spring (63.5%), but again the differences were not significant.

Stew idogen es is
Seasonal variations in the morphometric analysis of the ovary and uterus were also confirmed by analysis of steroidogenesis.The highest level of progesterone in blood (Table 4) was found in the spring (22.91 ng/ml).Significant differences were found in the progesterone concentration between spring and autumn (P<0.001).The highest concentration of 17P~oestradiol occurred in the winter and the lowest in the summer, which corresponds with the morphometric values for the growing follicles found in the ovary.

DISCUSSION
Generally, there is very little data describing reproduction in the brown hare, especially data reporting microscopic structure.
In this study we report seasonal variations in the morphometry of the ovary and uterus.In the ovary we found the highest activity in autumn and winter, as shown by the highest relative volume of growing follicles.Changes were also observed in the uterus where the highest relative volume of glandular epithelium was found.The highest diameter of uterine glands occurred in the spring.Our results are supported by the analysis of steroids in the blood, where the highest concentration of progesterone was in the spring and the highest level of 17P-oestradiol in the winter.
It has been reported that in the brown hare, fertile matings take place from the beginning of December to September (Caillol et al., 1990).The different climatic condition in our region in comparison with those where these results were obtained (France) must be taken into account.The basal concentrations of LH remain undetectable until the end of January, rise from February to a maximum in July.On the other hand, Semizorova et al. (1990) reported that the progesterone level in the brown hare is low before pregnancy (0.41 ng/ml).During pregnancy it gradually increases, culminates on day 31-40 of pregnancy (41.9 ng/ml) and decreases immediately before parturition (2.8 ng/ml).Our results also correspond with the results reported by Slamecka et al. (1997) where they found the highest ovarian weight in the spring (March, April, May).
Microscopic analysis of female reproductive organs is mainly reported in farm animals (Kliment and Zitny, 1989;Pivko, 1995;Krajnicakova et al., 1999).In comparison with rabbits, our morphometric analysis of the hare ovary shows a lower relative volume of growing follicles.In our earlier study of the rabbit ovary (Massanyi and Uhrin, 1996a), 3.0% of its volume comprised primary follicles, 30.7% growing follicles and 66.3% formed stroma.The diameter of primary follicles in hares was 30.8-35.9 jum and in rabbits 29.6±4.9jum (Massanyi and Uhrin, 1996b).In the uterus, luminal epithelium formed 5.9%, glandular epithelium 5.8% and stroma 88.3% of the tptal volume of endometrium.