Morphology and mucosal immunity of the oviduct and uterus during follicular and luteal phases in ewes

Reproductive diseases result in major production losses in the sheep industry. Although extensive studies on mucosal surfaces of the female reproductive tract had been conducted, very little information particularly the mechanism is known. Furthermore, defining the mucosal immunity is complicated by the critical interface between the endocrine and immune systems. Thus, an approach to gain sight into the pathogenesis of the reproductive diseases by investigating the local cellular immune response under the precise hormonal influences throughout the estrous cycle was undertaken. Fourteen ewes were synchronized into estrus and the plasma samples were collected every alternate day for hormonal profiles using radioimmunoassay (RIA) techniques. The ewes were divided into two groups: follicular and luteal phase groups (n=7). All the ewes were observed for three consecutive cycle before they were slaughtered at the peak of the follicular and luteal phases. The ewes started to exhibit signs of estrus between 24 to 36 hours following the intravaginal sponges’ removal. The average estradiol level during the peak of the follicular phase was 5.44 pg/ml for the first cycle, 4.85 pg/ml for the second and 4.25pg/ml for the third cycle. The ewes started the luteal phase which peaked at day 9 with the average progesterone value of 4.21ng/ml and 4.65 ng/ml respectively. The estrus occurred for 30.6 µ 0.65 hours, luteal phase period ranged between 12 to 14 days, making the complete cycle of 15 days. Average vaginal mucous resistances in ewes were recorded for three consecutive cycles using Draminski® estrous detector. The daily plasma concentrations of estradiol and progesterone were significantly correlated (p<0.01) with the estrus detector reading at (r = -0.924) and (r = 0.705), respectively. At slaughter, samples from the anterior, middle and posterior horns and the oviducts were taken and processed accordingly for light and electron microscopy. Under light microscopy, the number of lymphocytes in different parts of the uterus was significantly (p<0.05) higher during the follicular phase compared to the luteal phase. However, in the follicular phase group, the number of lymphocytes was not significantly different between the middle and anterior horn, while in the luteal phase group, the number of lymphocytes was not significantly different between the posterior and middle horn. Similarly, the quantity of plasma cell revealed that the number was significantly (p<0.05) higher in the follicular phase compared to the luteal phase for the different parts of the reproductive tract. However, in the luteal phase group, the number of plasma cells was not significantly different between the posterior and middle horn and between the anterior horn and oviduct. Ampullae were taken and processed accordingly for scanning, (SEM) and transmission electron microscopy (TEM). During the follicular phase, the population of secretory cells was less that the luteal phase, while the number of ciliated cells was higher than the luteal phase. The secretory cells were rounded, turgid with intact microvilli in the follicular phase, but in the luteal phase the surfaces were broken and some secretions were oozing out. The TEM examination revealed that during the follicular phase, the secretory cells had blunt processes at the apex intact microvilli, but during the luteal phase, the cytoplasmic protrusion on the secretory cells exhibited an increase involume. Numerous secretory granules with different sizes and electron density were found in both phases. During the follicular phase, the secretory cells were at the preparatory stage while they were actively secreting during luteal phase. The present study revealed marked cyclic changes and differences of the secretory cells during these two phases of the estrous cycle. Results of current study also suggested that the secretory granules were released by exocytosis and the apocrine would be the mode of secretion at estrus while the merocrine is the mode of secretion at luteal phase. In vitro experiments were conducted on the adhesion and colonization of E.coli to the uterus explants of both the follicular and luteal phases ewes. Scanning electron microscopy showed there were significant (p<0.05) differences between the groups at all different post-inoculaton times except for the negative control group and between 180 and 2 \3 60 minutes post-inoculation for both groups. Hence, the adhesion of E. coli to uterus during follicular phase was significantly (p<0.05) lower than the luteal phase. It seems that the intensity of adhesions increases with time. This present study contributes to the new knowledge on the endocrinology of Malin crossbres ewes where complete estradiol and progesterone hormonal profiles were obtained for the first time. In addition, the vaginal mucous resistance data recorded using the Draminski® estrous detector might be useful as the guideline in determining the exact timing of the estrus phase during the estrous cycle. Present results demonstrated that them mucosal immunity is lower during peak luteal phase than follicular phase in the ewes since the number of lymphocytes and plasma cells were higher during peak follicular phase. The morphological evaluations confirmed that during follicular phase, the uterus and the oviduct at the preparatory stage for the fertilization process while during luteal phase, the cellular immune response is modified to minimize rejection in the case if the ewe conceived. The in vitro challenged of the uterine explants confirmed the severity of the bacterial colonization during luteal phase than the follicular phase.

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