Evaluation of alterations in physical, hematobiochemical and ultrasonography images following chemosterilization using calcium chloride intratesticular injection in Wistar rats

Introduction

Street animal overpopulation is a significant issue in many places all over the world because they can serve as reservoirs or vectors for diseases that are deadly to humans as well as economically valuable domestic species. This causes the public health issues, even the vaccination against these diseases to stray animals is practically not possible. So to combat it there should be control on the increasing population which can be achieved through sterilization of animals. Sterilization can be done by Surgical, Chemical and hormonal methods. Surgical, hormonal or by Vaccination methods are costly and time-consuming. Chemical sterilization can be the alternative to both Surgical and hormonal sterilization being less expensive and less labor intensive, which allows mass-scale sterilization quickly and safely (Taylor et al., 2017). Various chemical agents such as ferric chloride (Kar et al.,1965), danazol (Dixit et al., 1975), BCG (Das et al., 1982), zinc tannate (Ansari et al., 2017), zinc gluconate (Oliveira et al., 2007), zinc arginine (Chaudhary et al., 2018), glycerol (Weinbauer et al., 1985;Immegart and Threlfall, 2000;Gunay et al., 2004), glucose, NaCl (Heath and Arowolo, 1987;Russell et al., 1987) dibromochloropropane (Shemi et al., 1988), lactic acid (Fordyce et al.,1989), zinc arginine (Fahim et al., 1993), sodium fluoride (Sprando et al., 1996), formalin (Bakir et al.,2002), calcium chloride (Jana et al., 2002;Jana and Samanta, 2011;Hami et al., 2020), ethanol (Canpolat et al., 2006a), potassium permanganate plus glacial acetic (Giri et al., 2002), mannitol (Maadi et al., 2021) and cetrimide (Fesseha and Negash, 2020) [2, 3, 4, 8, 11, 15, 16, 18, 19, 20, 21, 22, 24, 28, 35, 43, 44, 50, 52, 55, 57, 59] .

And in ruminant animals, chemicals like lactic acid (Hill et al., 1985), tannic acid and zinc sulphate (Feher et al., 1985), alpha-hydroxy propionic acid (Cohen et al.,1990), formalin (Ijaz et al., 2000), Ethanol (Canpolat et al., 2006b) and castrate-Quin (Soerensen et al., 2001) are been used till date for the chemical sterilization [5, 10, 17, 25, 27, 56] . Calcium chloride (CaCl2) is one of the most promising chemical agents, calcium chloride, has been used to chemically castrate a variety of species (Koger, 1978) [37] . Following intratesticular injection, calcium chloride induces necrosis, fibrosis, and degeneration of seminiferous tubules and Leydig cells, which, in a dose-dependent manner, reduces the production of spermatozoa, testosterone, and sperm counts. It results in both local and systemic reactions, including scrotal ulcers, dermatitis, abscesses, and fibrosis in the injected area, as well as peripheral edema, nausea, vomiting, diarrhoea, lethargy, and leukocytosis, which is followed by intratesticular injection (Canpolat et al., 2006b, Leoci, 2012, Kutzler and Wood, 2006) [5, 38, 40] . After intratesticular injection of CaCl2, signs of pain, pyrexia and even severe inflammation were observed (Jana and Samantha, 2006). Many a time anti-inflammatory drugs are used to reduce pain and inflammation. So, it is possible that anti-inflammatory treatment disturbs the desired action as a sterilizer [29] .

Materials and Methods

Ethical approval:

This experimental protocol (VETCOLL/IAEC/2022/19/ PROTOCOL-04) was approved by the Institutional Animal Ethics Committee (IAEC) of the College of Veterinary Science and Animal Husbandry, Kamdhenu University (KU), Sardarkrushinagar, Gujarat, India.

Procurement of Rats: Total 18 Wistar rats were procured from the Laboratory Animal Facility of Torrent Pharmaceuticals, Gandhinagar, and Gujarat, India. All the rats were acclimatized for 7 days before enrolling in the studies. Animal management and treatment procedures compiled with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals, New Delhi, India. Rats were kept in polypropylene cages at the Laboratory Animal Facility in an environmentally controlled room with 22±3 ºC temperature and 30-70% humidity. Light/dark cycles of 12/12 hours were given. Standard pellet food and Water were provided ad libidum.

Experimental design: Rats were divided in three groups, each comprising of 6 rats. The group were named as control group (Normal saline), treatment group T1 (CaCl2) and treatment group T2 (CaCl2 + Meloxicam). Meloxicam at the dose rate of 2 mg/kg body weight, was given by subcutaneous route.

Intratesticular injections:

After applying local anesthetic intratesticular injection of either normal saline or 20% CaCl2 was given with the use of insulin syringes at the dose rate of 0.1ml/100gram body weight as described by Jana et al., 2002. Clinical observations: All animals were observed for morbidity and mortality twice daily throughout the study. During the acclimatization period, the clinical observation was recorded once daily. Clinical observations like rectal temperature and behavioral observations like activeness were recorded twice for 5 days. Following intratesticular injection presence of swelling was checked.

Body Weight:

The body weight of all rats was recorded on day one of initiation of dosing and thereafter on days 5, 10, 20 and 30.

Blood Collection: Blood was collected on 0, 10th and 30th day of the study after anesthetizing with isoflurane. Blood was collected from retro-orbital plexus as described by Parasuraman et al. (2010) [47] with the help of a capillary tube in the sterilized vial containing 4.0 per cent Potassium Ethylene Diamine Tetra Acetic Acid (K3 EDTA) for the estimation of hematological parameters and in serum clot activator vial for biochemical estimates

Aspartate aminotransferase (AST) (U/L)

Its estimation was carried out by the colorimetric method devised by DeVries et al., 1976 using standard procedure and a diagnostic kit (Agappe, India). The working reagent was made by mixing 4 volumes of Reagent 1 (R1) with 1 volume of Reagent 2 (R2). For absorbance of blank only 500 µL working solution's OD was taken and for sample absorbance 500 µLand 50 µL was mixed and incubated for 1 minute, then 490 µL mixture aspired and change in absorbance per minute during 3 minutes was noted at 340 nm wavelength on Microlab RX-50V semiautomatic biochemical analyzer (Fig. 3.8).

Lipid peroxidation (LPO)

Oxidative stress in serum was determined in terms of malondialdehyde (MDA) by using the method of Buege and Aust (1978).

Ultrasonographic Observation

Testis of rats was scanned with a frequency of 7.5 MHz Micro convex probe and Sonosite M-Turbo Fujifilm Ultrasonographic device. Ultrasonographic picture of testis were observed on 0, 5, 10, 20 and 30 days. Echogenicity was graded based on low brightness 1, medium brightness 2, high brightness 3 and very high brightness 4 (Hami et al., 2020) [22] .

Statistical Analysis

The data pertaining to Haematological, Biochemical, Rectal temperature and Body weights were compiled as mean±S.E. and analyzed by analysis of variance method, ANOVA in SPSS 20.0 software and significant differences among the treatments were determined using DMRT.

Results: Animals of the group T1 and T2 showed a slight decrease in their activeness for the first two days. But the rats of the control group had not expressed any change in their active behavior. Further, no mortality was observed during the whole study of 30 days in any of the groups.

Swelling of Testis:

The presence of swelling in the testis after the intratesticular inject groups group T1 and T2 group was noticed for 48 hours, (Fig. 4.1 and 4.2) which diminished gradually and disappeared totally after 5 days. While no such pronounced swelling was evident in any of the testis of control group rats (Fig. 4.3). There was no difference in the swelling of the testis between the T1 and T2 groups. There was no pain on palpation, but there was a slight increase in the firmness in the testis following intratesticular injection for up to 24 hours.

change

Rectal Temperature: There was no significant difference in the rectal temperature on day 0 among the different groups. However, on day 1 highly significant elevation of rectal temperature was observed in the T1 and T2 groups as compared to the control group. But there was no difference between the T1 and T2 groups. Further, on days 2 and 3 the highly significant rise in rectal temperature was noticed only in the group T1. And there was no difference in rectal temperature between the control and T2 groups. During the period of observation which is from day 0 to day 5, no significant change was observed in the control group. In the T1 group, the temperature remained elevated up to day 3 which comes equal to day 0 on day 4. But in the T2 group, the elevated temperature was seen only on day 1, which reduced on day 2 and come to the temperature recorded on day 0 from day 4 onwards.

Body Weight

Body weight is a frequent but important indicator. A drug or treatment's impact might be seen in the body weight change.

The mean body weight in grams of rats of control, T1 and T2 groups taken on days 0, 5, 10, 20 and 30 is mentioned in Table 4.2 and Fig. 4.5. The mean body weight of rats of group T1 and T2 showed no significant difference with the control group on day 0, 5, 10, 20 and 30, even no significant difference was seen between group T1 and T2. While looking at the period no significant difference between body weight Day 0, 5, 10, 20, and 30 in control and groups T1 and T2 was recorded. On day 0, the echogenicity of testis noticed was hyperechogenic (grade 3, Fig. 4.11) with few anechoic areas in all rats of three groups. On day 5, the echogenicity of control group rats was showing anechoic area in the centre surrounded by hyperechoic spots (nearly grade 3, Fig. 4.12). However, in T1 and T2 group's rats the noticeable area with hyperechogenicity (grade 4, Fig.

Haematology

No significant difference was observed in TEC, HB, PCV, Platelet count within the group and between the group. There was a significant there was no significant difference in the values of total leucocyte count on day 0, day 10 and day 30 in control group. But in group T1 and T2 highly significant (P<0.01) difference in the value of total leucocyte count on day 0, day 10 and day 30 was observed.

Total leucocyte count on day 0 was within normal limit but on day 10 highly significant (P<0.01) increase in the total leukocyte count was observed which then reached to normal value on day 30.

Biochemical Estimation 4.4.2.1 Serum Aspartate aminotransferase (AST) level

The mean of the serum AST level of three groups on day 0, day 10 and day 30 is presented in Table 4.9. The mean serum AST level in control group on day 0, day 10 and day 30 showed no significant difference, similarly in group T1 and T2 on day 0, day 10 and day 30 no significant difference was recorded. The mean serum AST level on day 0, day 10 and day 30 did not differ significantly between the groups.

Serum Malondialdehyde level

The mean of the serum MDA level of three groups on day 0, day 10 and day 30 is presented in Table 4.10 and Fig. 4.30. The mean serum MDA level in control, T1 and T2 group showed significant difference, value on day 0 differed highly significantly (P<0.01) from day 10 but not with value on day 30 and MDA level on day 10 also differed significantly from day 30 level. The mean serum MDA level on day 0, day 10 and day 30 did not differ significantly between the group. But, serum MDA level on day 10 shows the non-significant increase in T1 and T2 group as compared to control group. This elevation in serum MDA level on day 10 may be due to the damage caused by intratesticular injection leading to lipid peroxidation of the cell membrane. [1] in dogs there were no similar findings as of this study were recorded. De Macedo et al. (2018) used the dipyrone + meloxicam following the intratesticular injection of Zinc gluconate and opined that there was no suppression of local inflammatory response in rats. This supports the present study. They have used various agents like dipyrone, meloxicam, celecoxib and dexamethasone to control the local reactions following intratesticular injection. But none was found effective to control the inflammatory for analgesic effect following intratesticular injection of zinc gluconate. Similarly, Oliveira et al. (2012b) also did not find beneficial effects of dipyrone (anti-inflammatory, analgesic and antipyretic) following intra-testicular injection of Zinc gluconate in dogs [6, 13, 14, 23, 26, 29-33, 40-42, 43, 45, 46, 48, 53, 58] . Jana et al. (2002) found an elevation in the rectal temperature in rats by 3rd day after intratesticular injection of calcium chloride, which supports the findings of the present study, Jana and Samantha (2006) In rats of the T2 group, rectal temperature was elevated only on the next day after intratesticular injection and not on the following days. This may be attributed to the effect of the meloxicam injection given subcutaneously as meloxicam is having an antipyretic effect [6, 23, 26, 29-33, 43, 45, 48, 51, 58] . Similar results to those of the current study were found in rats by Jana and Samanta (2006), Smita (2011), Das and Karmakar (2016), Karmakar and Das (2017), and De brito et al. (2020): no significant variation in body weight was seen after intratesticular injection of a different sterilizing agent. Fesseha and Negash (2020) in rats and Sen et al. (2017) in guinea pigs both found a significant increase in body weight in the treatment group animals following intratesticular injection of different chemo-sterilant which contradict the present work [12, 13, 18, 35, 36, 51, 54] . Alterations in echogenicity over the time were recorded where it first increased and then the gradual decrease in echogenity. Similar trends of variation in echogenicity were observed by Canpolat et al. (2006b) Cicirelli et al. (2022) in dogs, found reduction in the echogenicity but no acute increase in echogenicity was observed by them [4-6, 9, 22, 39, 49] . Total leucocyte count increase was also observed by Smita (2011) following intratesticular injection of sterilizing agent in rats. While no similar findings was observed by Kadam (2014) and De brito et al. (2020) following intratesticular injection of sterilizing agent in rats [13] . Serum AST level observed in the present study was found to be in agreement with the observations of Smita found elevation in the MDA content in testicular tissue following intratesticular injection [7, 12, 29-33, 34, 35, 36, 44, 54] .

Discussion

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