New insights on pig seminal cytokines

Resum

Pig seminal plasma contains measurable concentrations of several cytokines, whose quantification could be of interest to predict and/or diagnose reproductive disorders, sperm functionality and fertility outcomes. Little else is known about pig seminal cytokines. Therefore, more studies are timely. The objective of this PhD Thesis was to improve the current knowledge of porcine seminal cytokines, which was addressed through four independent studies. The first focused on evaluating how semen handling and seminal plasma storage influenced cytokine measurements. The second study evaluated whether the period of the year of ejaculate collection influenced the seminal measurements of cytokines. The third study evaluated whether the measurement of seminal cytokines had predictive value of the capability of pig sperm to withstand preservation. The fourth study focused on evaluating the distribution of a specific cytokine, the granulocyte macrophage colony stimulating factor (GM-CSF), in the male pig reproductive tract. The cytokines measured in the first three studies were interferon gamma (IFN?), GM-CSF, interleukin (IL)-1?, IL-1?, IL-1ra, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18 and tumor necrosis factor alpha (TNF?). Additionally, the transforming growth factor beta (TGF) -?1, -?2 and -?3 were also measured in the third study. The first study contemplated three separate trials exploring the delay (2 h and 24 h) in the harvest of seminal plasma after ejaculation collection (trial 1) and the storage of seminal plasma before cytokine measurements, either for short (5 ºC, -20 ºC and -80 ºC for 72 h, Trial 2) or prolonged period (-20 ºC and -80 ºC for two months, Trial 3). The results showed that the 24-hours delay in seminal plasma harvesting did not influence the seminal cytokine measurements. However, the storage of seminal plasma, both short- and long-term, generated instability in many cytokines. Therefore, if storage of seminal plasma is required, the storage conditions must be adjusted to each cytokine. In the second study, the seminal plasma samples were grouped according to the period of the year of ejaculate collection. Two periods were contemplated, with decreasing (summer and autumn) and increasing (winter and spring) daylight. Both periods also showed clear differences in air temperature, being highest in the decreasing period. The seminal cytokines showed differences in concentration between both periods. The highest were measured during the increasing period. In summary, the period of ejaculate collection influences seminal cytokine measurements. The third study included two separate experiments, one for semen stored in liquid state and the other for frozen-thawed semen. In addition to the measurement of seminal cytokines, semen samples were evaluated for sperm quality and functionality. The multiple linear regression models with a Bayesian approach revealed that the cytokines TGF- ?2, TGF- ?3, IL-1ra, IL-4, IL-8 and IL-18 have the ability to predict the motility changes experienced by sperm during liquid storage. The same cytokines plus IFN? also have the ability to predict the functional changes experienced by sperm during freezing and thawing. In conclusion, seminal cytokines would contribute to modulate the functional changes undergoing pig sperm during preservation. The last study, focused in GM-CSF, demonstrated positive labelling of this cytokine in all the male pig reproductive tissues (testes, epididymis and accessory sex glands), fluids (epididymal and seminal plasma) and in mature sperm from both cauda epididymis and ejaculate. Western blot revealed three forms of GM-CSF with different glycosylation degrees (15, 31 and 40 kDa) in the tissues. Fluids expressed only the GM-CSF of 15 kDa while the GM-CSF forms expressed in sperm varied among sperm source. In sum, GM-CSF is widely present in the reproductive tract of male pigs, attached to the spermatozoa already in the epididymis as well as verted to seminal plasma.