The aim of the present study was to examine the effects of SP, spermatozoa in extender (BTS), fresh semen in extender and extender only (control) on the presence of the cytokines IL-6, IL-10 and TGF-β1 in ovarian follicles and follicular fluid shortly after insemination in gilts. The present study showed that the concentration of TGF-β1 was much higher than the concentrations of IL-10 and IL-6 in the fluid from mature porcine follicles. The patterns of TGF-β1, IL-10 and IL-6 presence as shown by IHC-labeling of the follicles were similar; the scores of the labeling varied less for TGF-β1 than for IL-10 and IL-6.
The presence of TGF-β1 and IL-10 in porcine follicular fluid has, to our knowledge, not been reported previously. The high level of TGF-β1 (mean levels about 800–1650 pg/mL) is similar to levels reported in bovine follicular fluid  and the low concentration of IL-10 (range of 4–85 pg/ml) is comparable to the results found by Geva et al. in women (range of 10–350 pg/mL). The detected concentration of IL-6 (23–35 pg/mL) agrees with the low level (around100 pg/mL) found earlier from normally developed follicles in ovaries from pigs  and women .
Apparent IHC-labeling has previously been demonstrated for TGF-β1 in porcine granulosa cells of preovulatory follicles . Positive IHC- labeling of the cytokines investigated in the present study (TGF-β1, IL-10 and IL-6) was found primarily in the granulosa cells of all antral follicles, independent of developmental stage, as well as in the oocytes. In mature follicles, theca interna cells were labeled to a higher degree than in follicles less developed. May et al. found TGF-β1 mRNA expression by in situ hybridization in both granulosa and theca cells in the follicles of prepubertal gilts, although their results on cell cultures suggested that only the theca cells translated and secreted this cytokine. They also showed, from culture of intact hemi-follicle linings, that the secretion of TGF-β1 increased with increasing follicular size. In the present study, the labeling of the different cytokines in the cells of mature follicles seemed to be similar while the cytokines in follicular fluid varied with the TGF-β1 levels being high and the IL-10 and IL-6 levels low or undetectable. The results on IL-6 were notable, since only two gilts had detectable concentrations of IL-6 in follicular fluid whereas their follicular walls (granulosa and theca cells) were positively labeled for IL-6, indicating that that there is no correlation between IHC presence of the cytokine in granulosa cells and content of the same cytokine in follicular fluid.
As reviewed by Knight and Glister , studies in several species, especially in rodents, indicate that receptors, signaling intermediaries and binding proteins associated with the TGF-β superfamily, are expressed by oocytes and ovarian somatic cells in a developmental stage-related manner. The present study showed that oocytes, independent of follicular development, demonstrated positive labeling not only for TGF-β1 but also for IL-10 and IL-6, suggesting that these two cytokines may also play a role in the bi-directional communication between oocytes and granulosa cells.
Waberski et al.[7–9] found that seminal plasma (SP) stimulated induction of ovulation in inseminated gilts early during oestrus (immediately after the start of the standing reflex). In the present study, comparing inseminated fluids with BTS used as control, no differences were found between the groups inseminated with seminal plasma, spermatozoa in BTS, and fresh semen in BTS, either in IHC-labeling of the follicular cells, or in levels of IL-6, IL-10 and TGF-β1 in follicular fluid. Several possible reasons may explain the non-appearance of SP-effect in the present study. First, the cytokines studied may not be involved in the final maturation process of follicles leading to ovulation even if e.g. the TGF-superfamily, including TGF-β1, is suggested to be important for intra ovarian communication between cells see . Furthermore, the interval between treatment and collection may have been too short (5–6 h) or the time for collection of ovarian tissue may have been too close to ovulation to detect any differences based on treatment. The latter suggestion is strengthened by the earlier study of Waberski et al., who found that SP did not shorten the interval to ovulation if the treatment was done too close to ovulation.
The presence of TGF-β1, Il-10 and Il-6 in porcine follicles indicate that these cytokines have a role in the process of follicular and oocyte development and therefore need to be further investigated.