Rectal temperature, as well as the observation of possible local or general adverse reactions, is important for the evaluation of the innocuousness of vaccine or melatonin administration. We have not found any local or general clinical signs, including granulomas, after administration of the vaccine or subcutaneous implants of melatonin in our experimental groups. In addition, the rectal temperature was in the physiological range.
Serum concentration of melatonin in the different experimental groups is shown in Table
. In control animals (not vaccinated, not treated with melatonin), the plasma level of melatonin was quite stable with no significant changes during the performance of the experiment. Vaccination per se
had no significant effect on the serum level of melatonin. As expected administration of melatonin by implant induced a significant increase in the serum concentration of melatonin, which was maintained elevated for 60
days and then returned to basal levels in animals with melatonin implant.
Plasma melatonin concentration (pg/mL) at the time of the vaccination procedure at 10:00 A.M
Melatonin plays a role in synchronizing the reproductive responses of animals to environmental light conditions; it has been reported that serum melatonin levels during human pregnancy are higher than in a non-pregnant state [18
]. Here, we showed for the first time in sheep, the differences in plasma melatonin concentration between non-pregnant and pregnant sheep. As we showed previously, sheep in non-gestational state have an average plasma melatonin concentration of 15
], considerably smaller than pregnant sheep (125
). Differences in plasma melatonin concentration may be due to strong melatonin antioxidant properties, as well as, as previously postulated, high levels of melatonin during pregnancy may be one of the factors that reduce oxidative damage from ROS in the placenta and systemic endothelial cells [18
Plasma antibody concentration in the different experimental groups is shown in Table
. As expected, in control subjects and those treated by melatonin implants the plasma antibody concentration was unaltered during the experiment, while vaccine administration increased significantly plasma antibody concentration reaching the highest level after 60
days of vaccination (Table
). As shown in Table
, in animals treated with melatonin implants the concentration of plasma antibodies was significantly greater after vaccination, which further support a positive role of melatonin in antibody production upon vaccination, as described in non-pregnant sheep [10
]. Interestingly, while the effect of melatonin on the serum antibody level after vaccination of non-pregnant sheep was found to be slightly greater but not statistically different from the levels in vaccinated animals in the absence of melatonin [10
], when the vaccination takes place prepartum, the serum antibody concentration was significantly greater in vaccinated controls in the presence of melatonin; thus suggesting that the time of vaccination plays a important role in the effect of melatonin on the immune response, being more effective when administered prepartum.
Serum antibody levels at the different treatments
Melatonin has also been reported to induce a number of immune responses besides antibody production, including enhance antigen presentation to the immunocompetent cells, as described in mice, where melatonin improved antigen presentation by macrophages, by increasing the expression of MHC class II and stimulated activation of T helper cells [22
]. Furthermore, melatonin may modulate the production of cytokines such as IL-2, INF-γ and IL-6 and increasing production IL-12 by monocytes as demonstrated in cultured human mononuclear cells [24
], thus promoting Th1 cell response. In addition, melatonin may also bind to high affinity receptors present on Th2 lymphocytes from human bone marrow thus increasing the levels of IL-4 [25
], increasing the production of IL-10 and decreasing of TFN-α in mice sensitized with ovalbumin injected with complete Freund’s adjuvant, activating the Th2 response [26
We have further investigated whether vaccination against C. Perfringens
(D serotype) in the absence or presence of melatonin might have any effect on platelet function, and thus, hemostasis. Hence, we tested ex vivo
platelet aggregation in response to the physiological agonist thrombin in the four experimental groups. The percentage of aggregation upon stimulation with 0.5 U/mL thrombin decreased significantly after partum in all the experimental conditions (Figure ; P
10–12). Similar results were obtained when we calculated the rate of aggregation (Figure ; P
10–12). Interestingly, the lag-time in response to thrombin was not altered in any of the groups investigated (Figure ). Vaccination or treatment with melatonin did not modify agonist-induced aggregation in sheep during the last stages of pregnancy and postpartum period. We have found that in pregnant sheep serum melatonin concentration is almost 10 times greater than in non-pregnant subjects [8
]. Therefore, melatonin administration results in a 2- over 20-fold increase in melatonin concentration in pregnant and non-pregnant animals, respectively, where the serum melatonin concentration reached similar values after exogenous administration.
Figure 1 Effect of melatonin administration on the percentage of thrombin-induced platelet aggregation in vaccinated and non-vaccinated sheep. Platelets from sheep vaccinated or not against Clostridium perfringens type D and treated with melatonin or the vehicle, (more ...)
Figure 2 Effect of melatonin administration in the rate of thrombin-induced platelet aggregation in vaccinated and non-vaccinated sheep. Platelets from sheep vaccinated or not against Clostridium perfringens type D and treated with melatonin or the vehicle, as (more ...)
Figure 3 Effect of melatonin administration in the lag-time of thrombin-induced platelet aggregation in vaccinated and non-vaccinated sheep. Platelets from sheep vaccinated or not against Clostridium perfringens type D and treated with melatonin or the vehicle, (more ...)