Clinicopathological and Reproductive Studies on The Use of Ivermectin in Ewes

This study aimed to investigate the effect of Ivermectin (IVM) on the reproductive hormones and hemato-biochemical parameters of twenty apparently healthy ewes weighing 30-40 kg and 2-3 years old. Ewes were randomly divided into two groups (ten for each). The first group was left without treatment (control group), and the second was treated with the recommended therapeutic dose of IVM (0.2 mg/kg, S/C) one day after parturition (treated group). The study continued for three months. Blood samples were collected from the two groups at the 1 st , 30 th , 60 th , and 90 th days after IVM treatment. The current study revealed that IVM injection delayed estrous for up to 3 months (absence of estrous signs and no ovarian structures were observed by sonar examination). There was a significant decrease in hemoglobin concentration (Hb), red blood cells (RBCs) count, and packed cell volume (PCV), with a significant increase in total leukocytic count (TLC) at 30 th and 60 th days post-treatment (p.t.). In addition to a significant decreased at (P<0.05) in the activity of glutathione peroxidase (GSH) and concentrations of total antioxidants (TAC), copper (Cu), phosphorus (P), estradiol, triiodothyronine(T3), and tetraiodothyronine (T4) for up to 3 months. In contrast, a significant increase in concentrations of calcium (Ca), progesterone and cortisol, and activity of malondialdehyde (MDA), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) enzymes were recorded at 30 th and 60 th days p.t. It has been concluded that IVM delayed estrous in ewes for three months via disturbing the female reproductive hormones and the hemato-biochemical parameters. Therefore, it is recommended that IVM not be injected at least three months before the reproductive season. ـــــــــــــــــــــــــــــــــــــــــ


INTRODUCTION
Ivermectin (IVM) is an anthelmintic drug widely used to control both internal and external parasites in humans and animals (Makhlouf et al., 2020). IVM is a form of insecticide Ivermectin, abamectin, eprinomectin, and doramectin are among them. IVM consists of two compounds; dihydroavermectin B1a (H2B1a) and dihydro-avermectin B1b (H2B1b), similar to macrolide antibiotics but it does not seem to have any antibacterial or antifungal properties. IVM is approved for use in 46 countries and is being used to treat cattle, sheep, horses, and pigs worldwide. On the other hand, IVM has a wide range of action against gastrointestinal, lung nematodes, and ectoparasites in domestic animals (Suarez et al., 2013). Sheep is a highly prized animal; its meat is much relished, and it is very easy to market; it allows the farmer to meet unexpected expenditure quickly (Sania et al., 2016). Its raising has a social role which takes many forms as performing some religious rites, in Muslim ceremonies, in marriage feasts, slaughtered to honor a respected visitor or parent.
The fertility study results revealed unfavorable effects of Ivermectin on fertility and blocked the pregnancy (Al-jassim et al., 2015). The endocrine disruptor resulted from the excess production of free radicals, and it is very clear that Ivermectin attends to accumulate in fatty tissues, particularly ovarian tissues. Shkolnik et al., (2011) found that Ivermectin has a negative impact on female reproductive efficacy, especially during ovulation.
In cows, Ivermectin affects fertility, healthy status, immunity, estrous and reproduction via delaying the estrous, disturbing the female reproductive hormones, and calcium/phosphorus homeostasis. Others believe that, IVM has a positive impact on animal reproductive production (Kadry and Hazem, 2015). This study aimed to investigate the effect of Ivermectin (IVM) on the reproductive hormones and hemato-biochemical parameters of twenty apparently healthy ewes

Experimental Study
In this experiment, ewes weighing 30-40 kg and aged 2-3 years were used. Two weeks prior to the experiment, all ewes were kept under observation and were subjected to fecal examinations to ensure they are free from any parasite. Twenty mature apparently healthy ewes and free from any diseases, especially parasitic ones, were randomly divided into two groups, comprising ten animals. The first group was left without treatment and considered as a control group. The second was treated with the recommended therapeutic dose of IVM one day after parturition (treated group) (1% W/V solution of IVM, VMD Ltd, Arendonk, Belgium, 0.2 mg/kg, S/C). The experiment continued for three months (Garg et al., 2007). All ewes were examined by ultrasound scanner (200 pie Medical Co -Netherlands -Holland).

Hematological Studies
Blood samples (10 ml) were collected via jugular venipuncture at the 1 st , 30 th , 60 th , and 90 th days p.t. The first blood sample was anticoagulated using dipotassium salt of ethylene diamine tetra-acetic acid (EDTA); It was used for the evaluation of RBCs count, Hb concentration, PCV and TLC according to Feldman et al., (2000).

Biochemical And Hormonal Assays
The second blood sample was collected in a clean centrifuge tube and was allowed to clot. Sera were extracted from blood samples by centrifuging them at 3000 rpm for 15 minutes, and clear nonhemolyzed supernatant serum was harvested and stored at -20 °C until carrying the biochemical analysis. All biochemical parameters were analyzed using commercially available kit methods. The biochemical analysis measures the following parameters: reduced glutathione peroxidase (GSH) (Sedlak and Lindsay, 1968), total antioxidant capacity (TAC) (Cortassa et al., 2004) and malondialdehyde (MDA) (Zhang, 1992). Estimation of serum calcium and copper (Ahmad et al., 2007). Serum transaminases, including (AST) and (ALT) were measured according to the method described by Kaneko et al., (1997). All the before-mentioned parameters were measured colorimetrically using commercial kits supplied by Biodiagnostic® company, Egypt. Concentrations of estradiol, progesterone, cortisol, T 3 and T 4 were measured by using enzyme-linked immunosorbent assay (ELISA) according to Maxey et al., (1992) using commercial diagnostic ELISA kits (Nova Tec. Immudiagnostica GmbH, WaldstraBe 23 A6, D-63128 Dietzenbach, Germany)

Statistical Analysis
All data were subjected to statistical analysis (ANOVA) according to Sendecor and Cochran, (1982) using a computer program "COSTAT."

General signs
Estrous signs including redness and swelling around the vulva, were absent. The expression of estrous in ewes is not as easily detected when she has been separated from the ram for a period of time. When mature ewes are in heat, they will seek out the ram and stand still for him to mount them. Ultrasound examinations revealed no ovarian structures (follicular and luteal structures) were observed for three months post IVM injection.

Effects of IVM's on hematological parameters
By comparing the treated group results with those of the control group, normocytic normochromic anemia was observed in treated ewes at the 30 th and 60 th days p.t. This anemia was manifested by the significant decrease (P<0.05) in RBCs count, Hb concentration, and PCV% associated with the insignificant changes in MCV and MCHC values. However, a significant increase (P<0.05) has been recorded in TLC in treated ewes at 30 th and 60 th days p.t. compared with the control group. At the 90 th day p.t., all these results were changed toward control values (Table 1).

Effects of IVM's on antioxidants and oxidant parameters
By comparing the mean values of the treated group with those of the control group, serum activity of GSH, and concentration TAC were significantly decreased (P<0.05) at days 30 th and 60 th p.t. Moreover, it was returned to control values at the 90 th -day p.t. Significant increase (P<0.05) has been indicated in MDA activity in treated ewes at the 30 th and 60 th days. These values were directed toward the control values at the 90 th -day p.t. (Table 2).

Effects of IVM's on serum biochemical parameters
By comparing the obtained results of the treated group with those of the control group, there was a significant increase (P<0.05) in the concentration of Ca and activity of AST and ALT enzymes in treated ewes at days 30 th and 60 th days p.t. Significant decrease (P<0.05) in concentrations of P and Cu in treated ewes at day 30 th and 60 th days p.t. were recorded while, these results were returned to normal control values at the 90 th -day p.t. (Table, 3

Effects of IVM's on serum reproductive hormones
By comparing the results of this treated group with those of the control one, a significant decrease (P<0.05) has been encountered in estradiol concentrations, T 3 and T 4 at the 30 th and 60 th days p.t and returned to its normal values at 90 th . On the other hand, serum progesterone and cortisol concentrations show a significant (P<0.05) increase in treated ewes at the 30 th and 60 th days p.t. which towered to normal control concentration at the day 90 th p.t. (Table, 4).

DISCUSSION
Since Ivermectin is a lipophilic substance, it tends to settle in fatty tissue, especially in the ovaries, which is an essential reservoir for the medication. This may affect how long it lasts in the body and how it acts pharmacologically. Ivermectin's lipophilic nature also speeds up its distribution from the bloodstream to various tissues (Al-jassim et al., 2015). On the other hand, the significant increase in TLC of the treated animals is consistent with previous findings and is most likely the result of underlying stress (Saqib et al., 2015). Treatment with IVM appeared to substantially increase the number of leukocytes as compared to the control group. This increase may have resulted from body response to any injury (Wanji et al., 2017).
As the demand for calcium grows, the parathyroid gland secretes parathormone into the blood, raising calcium levels and causing hypercalcemia (Schmitz DG 2007). At the 30 th and 60 th days after drug injection, there was a significant increase in calcium and a significant decrease in phosphorus levels, implying that IVM disrupted calcium/phosphorus ratio and its long duration of action contribute to this effect (Kadry and Hazem, 2015).
The investigated ewes' liver enzymes (AST, ALT) were elevated at the 30 th and 60 th -day posttreatment; this may be attributed to the liver having the most IVM residues. Ashraf and Ausama (2007) reported that the level of these enzymes is increased, an increase in AST and ALT levels may be attributed to hepatic cell damage caused by the drug's direct effect resulting in the escape of these enzymes into the plasma. ALT and AST are generally assessed clinically to assess liver health as part of a hepatocellular injury diagnostic assessment (Wang et al., 2012).
Asmaa and Mohamed, (2020) concluded that, the use of Ivermectin for the treatment of camel mange has some adverse effects on liver function tests due to oxidative stress that could last for a long time. Antioxidant administration with Ivermectin was highly suggested to reduce the drug's side effects.
The copper concentration of the treated group at 30 th and 60 th days p.t. was decreased than the control group. This deficiency has been related to bone disorders, diarrhea, infertility, and tachycardia, and susceptibility to anemia in ruminants (Mohamed et al.,  2014). These diseases are linked to Cu-containing enzymes' functions in cellular respiration, immune function, and erythropoiesis formation. As a result, there was a potential connection between low Cu levels and the anemia seen in sheep. The biological significance of copper deficiency and abundance in the mammalian system has piqued researchers' interest. Various diseases are caused by abnormally high copper levels in the liver. The reciprocal rivalry between Zn and Cu in sheep may be affected by a genetic factor (Abdou and Sharkawy, 2004).
Oxidative stress induced by the injection of the treated group with IVM resulted in a significant decrease in the concentrations of both TAC and GSH and increased MDA (Mahmoud et al., 2014). Ivermectin causes damage and lowers antioxidant enzyme activity, and produces free radicals. Behera et al., 2011 andTurkan et al., 2018 mentioned that, IVM affects the balance between oxidants and antioxidants and significantly suppresses the release of GSH, with an inhibitory effect on the antioxidant's enzymes.
The effects of IVM on several reproductive hormones in ewes were studied. The current findings indicate that, after IVM injection, concentrations of estradiol, triiodothyronine, and tetra-iodothyronine hormones were decreased at the 30 th and 60 th -day p.t., and then return to normal values at the 90 th -day p.t. Furthermore, because of the detrimental impact on the animals' fertility criteria, IVM should not be used in breeding bucks and rams. There was a substantial increase in serum progesterone levels when IVM was given to ewes during the breeding season (Seri et al.,  2000). The cause of the rise in progesterone level is unclear, but it may stem from the ovary or adrenal glands (Kadry and Hazem, 2015).
Reduced FSH and LH, and as a result, reduced estrogen associated with delayed estrous after IVM injection may be due to progesterone's hypothalamihypophyseal negative feedback as observed in the current study (Kadry and Hazem, 2015). In the meantime, the cortisol hormone was elevated in Rabbits. Cortisol is a steroid hormone released by the adrenal cortex's zona fasciculata. Cortisol levels elevated in IVM treated rabbits indicate that, those animals are stressed (Muehlenbein et al., 2010 and El-Sawy et al., 2016). Mejía et al., (1999) reported that, IVM treatment in dairy heifers might increase growth rate during development, advance the onset of ovarian function (earlier puberty), and positively affect the yearling pelvic region. When heifers were given IVM at weaning, the number of animals in estrous at the end of the feeding period increased. The same authors predicted that, any change in reproductive success would lead to an increase in fertility.
In heifers, antiparasitic therapy tends to be linked to early puberty and increased fertility (Purvis and Whittier, 1996). Cortisol is a steroid hormone generated by the zona fasciculata of the adrenal cortex in response to stress. Although it serves to restore homeostasis, prolonged cortisol secretion, whether due to excessive secretion or chronic stress, causes significant physiological changes, suppressing immune and reproductive functions (Muehlenbein and Watts, 2010).
Hormones are required for the normal growth, development, and metabolism of cells (Yen, 2001 andPuri, 2011). Hormones are produced in the blood via several glands for example, the thyroid gland; is the largest gland that produces two principal hormones: tetraiodothyronine hormone (T 4 ) and triiodothyronine hormone (T 3 ) (Puri, 2011). Both T 3 and T 4 are bioindicators of the hypothalamus and pituitary glands activities (Kirsten, 2000 andMebis et al., 2008). These hormones have an important function in the body, specifically the stimulation of metabolism (Puri, 2011 andQuraishi et al., 2015). T 3 and T 4 help acquire the element iodine and convert it into the biologically available form (Granner, 2003). Abamectin significantly decreases the content of both T 4 and T 3 hormones compared to control. Oxidative stress has an impact on thyroid physiologies ( Omidi et al., 2015). Ivermectin's free radicals, according to some researchers, can cause oxidative stress, which can lead to sperm or ovum damage, deformity, endometriosis, preeclampsia, miscarriage, intrauterine growth retardation, and infertility (Bansal and Bilaspuri, 2011 and Al-Jassim et al., 2015).
When Ivermectin was given during the hormone decline process, there was no effect. Sania and colleagues, (2016) showed that, Ivermectin is an effective insecticide that enhanced the reproductive efficiency of ewes in her trials, as ovulation occurred at the drug's highest dosage. This result may be due to a change in hormones triggered by an endocrine disruptor as a result of free radical synthesis, resulting in reproductive toxicity.
Reactive oxygen species (ROS) could be generated directly from oocytes and embryos or from their surroundings, which leads to mediating the processes of embryonic development. This ROS could cause multiple physiological processes from oocyte maturation to fertilization and pregnancy (Agarwal et  al., 2005). This study provides first-hand information on adverse reactions of Ivermectin on ewes' reproduction.

CONCLUSION
This study's results identify adverse reactions of Ivermectin on ewe's reproduction via fertility troubles, alteration in hemato-biochemical parameters, and reproductive hormones. IVM delayed estrous in ewes for three months. As a result, IVM should not be injected after delivery. Moreover, the drug induced some degree of harm in the liver. So, we should use Ivermectin carefully to avoid possible adverse effects, especially during the reproductive season. We believe that more research is required to understand the relationship between IVM and reproductive hormones fully.

Declaration of Competing interest
On behalf of all authors, I hereby declare that no conflict of interest may interfere with the publication of the manuscript.