A significant decrease in the serum concentration of antioxidant enzymes (SOD, GPX and CAT) along with a significant increase in the serum concentration of MDA were found in dogs after OHE. According to the present results, melatonin treatment could increase the expression of antioxidant enzymes (SOD, GPX, and CAT) and significantly decrease the MDA level after OHE. Melatonin partially reduced the oxidative stress in dogs with OHE, as suggested by a decrease in the concentration of SOD, GPX, and CAT in all the groups that received melatonin. The activities of antioxidant enzymes significantly increased in the anesthesia group that received melatonin. In addition, in the melatonin-treated groups, melatonin led to a decrease in the level of MDA, which had reached its peak (MDA concentration) 3 days after surgery.
Other studies showed increased lipid peroxidation 24 h after surgery [2, 19]. In another study, the MDA level reached its peak 3 days after OHE in dogs but the CAT level did not differ significantly between experimental days. Also, the researchers indicated that the oxidative stress caused by OHE and plasma MDA was higher on day 3 post-OHE than before OHE and 3-h and 3–14 days post-OHE. Total antioxidant capacity on day 14 post-OHE was higher than pre-OHE, 3 h and 3–14 days post OHE. They also suggested the ovariohysterectomized dogs needed antioxidant for 7 days post-OHE to protect their bodies from free radicals [3].
Furthermore, researchers were observed that OHE and anesthesia, induced by a combination of Ketamine-Xylazine, could lead to an increased in the oxidative stress markers (Glutathione and MDA). The toxicity of drugs commonly used in anesthesia can induce oxidative stress. A significant decrease in antioxidative capacity was reported in dogs after enflurane anesthesia. It was observed that the serum level of vitamin E and beta-carotene significantly decreased whereas the serum level of vitamin A and MDA significantly increased during enflurane anesthesia in dogs [20].
Several studies have reported an oxidative stress induced by OHE in dogs and efforts should be made to identify potential antioxidant therapies [3, 18]. Melatonin has been shown in numerous studies to have antioxidant properties in dogs and human [14, 21]. In dogs, oral melatonin (0.3 mg/kg) was administrated daily for 1 month after castration [14]. Their findings indicated that antioxidant enzymes (GPX, CAT and SOD) increased and MDA concentration decreased in dogs treated with melatonin in compared with control and castrated dogs [14]. Melatonin increased antioxidant enzymes activity in different tissues (brain, liver, and kidney) during physiological (circadian rhythm) and pathological conditions. Furthermore, melatonin has been shown to regulate the activation or inhibition of several transcription factors related to antioxidant response, which can justify the antioxidative effects of this substance [21]. According to a study, male dogs were not under oxidative stress after castration due to the non-significant changes in the MDA and CAT concentrations. They were observed a decrease in total antioxidant capacity (TAC) on days 3, 7, and 10 compared to day 14, but their antioxidant system still functioned with high effectiveness [22]. OHE under ketamine-xylazine anesthesia was found to increase lipid peroxidation and decreased antioxidant enzyme activity in rats [23].
Other strategies have been previously studied in an attempt to identify potential treatments to reduce the oxidative stress induced by surgery or anesthesia. The researchers investigated the physiologic effects of hyperbaric oxygen (HBO) therapy on the antioxidant status in healthy dogs after OHE. They found that while this therapy, at the dose used in their study, had no adverse effects on the treated group, it was ineffective in reducing postoperative inflammation and improving the oxidative status in them [17]. In the study on the effects of flunixin meglumine (FM) and meloxicam on postoperative and oxidative stress after OHE, it is reported that while FM reduced postoperative oxidative stress, it had no influence on the oxidative stress status [24]. Elevated fraction of inspired oxygen during and after general anesthesia significantly increased MDA concentration 24 h after surgery and decreased the body antioxidant defense marker and glutathionyl hemoglobin in human [25].
In our previous study, we examined the effects of melatonin treatment on the levels of sexual hormones, serotonin, and cortisol in intact and castrated male dogs. The results showed that melatonin increased the level of serotonin but decreased the level of cortisol in intact and castrated dogs [13]. This study indicated that postoperative stress (increased cortisol concentration) continued for 1 week after castration and daily melatonin administration decreased cortisol concentration during 7 days after gonadectomy in castrated dogs. Thus, the authors suggested that melatonin administration considered pre- and post-operation period for about 7 days after OHE in the bitches. We also studied the effects of melatonin administration on the levels of thyroid hormones, leptin, and ghrelin in intact and castrated male dogs. Melatonin could regulate metabolic hormones and mitigate the metabolic side-effects of castration [15]. Long-term side effects of castration and beneficial effect of melatonin were reported in these studies [13, 15]. The melatonin treatment after gonadectomy may be suggested for prevention of metabolic disorders in gonadectomized dogs.
Malondialdehyde (MDA) represents one of the most investigated end products of lipid oxidation. Despite thiobarbituric acid test for MDA determination being the most frequently used method to evaluate lipid peroxidation, it has several pitfalls including thiobarbituric acid reaction with several compounds, including sugars, amino acids, bilirubin, and albumin, and the interference of hemolysis that falsely increases the measured MDA levels [26]. The most important antioxidant enzymes are SOD, CAT, and glutathione-dependent enzymes, such as GPX, and glutathione transferases. Changes of antioxidant enzymes activity are dependent on age, obesity, disease and stage of disease, different samples, processing and cryopreservation procedures and different cell types of blood [26]. Various methods were used for measuring nonenzymatic antioxidants (nonenzymatic antioxidant capacity, also named total antioxidant capacity) and included endogenous (e.g., urine analysis, bilirubin, and thiols) and nutritional (e.g., tocopherols, ascorbic acid, carotenoids, and phenolics) compounds in plasma. The measuring of the antioxidant enzymes activity can be replaced with total antioxidant status assay [26].
In this study, melatonin administration decreased oxidative stress in the ovariohysterectomized bitches. It may reduce the side effects of the surgery. It is suggested to measure melatonin concentration in dogs. Also, melatonin treatment included in more aggressive surgeries than castration or OHE, such as orthopedics and evaluated its effects in much more oxidative stress and stressful conditions. Bioavailability of oral melatonin following a 10 mg/kg dose is moderate in rats and high in dogs and monkeys. However, the bioavailability appears to be dose dependent, at least in dogs and monkeys [27]. Therefore, further studies are needed to determine the most appropriate therapeutic dose of oral melatonin in dogs to achieve an antioxidant effect.
The small sample size (n = 5) in this study was considered because of minimal number of repeats in each group to provide one of the essential condition for performing parametric statistical tests and regarding the standards in the protection of animals [28]. Increasing sample size will be helpful to decrease type II error. Injection of NSAIDs following surgery may influence oxidative stress by pain relief effect (pain can induce oxidative stress) [29]. Therefore, in the next studies, it needs to be considered and compared the effect of NSAIDs on oxidative stress with or without melatonin treatment in experimental design.