Sterilization of Male Dogs by Intra-testicular Administration of Calcium Chloride Solution

Canine overpopulation has become a global problem with significant public health impacts (Ahmed, 2015). India has approximately 25 million dogs, with an estimated dog to man ratio of 1:36 and approximately 80% dogs are community dogs which are unrestricted and partially dependent on others (Menezes, 2008). Dogs also play an evident role as a vector in transmission of rabies which is a major zoonotic disease in humans and livestock.

For controlling stray dog population, the most widely accepted and followed protocol is castration or bilateral orchidectomy. The main methods of castration are surgical, hormonal, immunological and chemical castration. The present common method of dog’s population control is surgical sterilization which requires anaesthesia, medical equipment, a suitable surgical facility, post-operative care and the advanced training of a veterinarian and bears different risks. However, chemical castration has been suggested as a fast and low cost alternative that can be used in a wide range of canine population, especially in regions where the problem is more intense (Soto et al., 2007). Hence, present study was conducted to evaluate the efficacy of intra-testicular administration of 20% calcium chloride solution on testicular degeneration and serum testosterone concentration in dogs.

The study was performed for a period of nine months from August, 2018 to April, 2019 in Department of Veterinary Gynaecology and Obstetrics, College of Veterinary Science and A.H., Nanaji Deshmukh Veterinary Science University Jabalpur and approved by Animal Ethics Committee of College. A total of 12 fertile, healthy adult stray male dogs were selected for the present study and randomly divided into two groups consisting six animals in each. The animals of group-I were administered bilateral intra-testicular 1.0 ml of 20% w/v calcium chloride solution in normal saline  while in group-II, 1.0 ml of normal saline was injected intra-testicular as placebo. Clinical signs following treatment, if any, were recorded.

For the assessment of effects of calcium chloride, testicular morphology and scrotal circumference were measured in both the groups on day 0, 7, 15 and 30. Measurement of testicular length, width, thickness and scrotal circumference were taken by vernier callipers and testicular volume was calculated by the following formula.
 

Where
TL= Testicular length.
SC= Scrotal circumference.

Histo-morphological studies were also conducted on the testicular biopsy samples obtained from each dog on day 0 and 30 using 18 G biopsy gun. Serum testosterone was measured on day 0, 15 and 30 using commercial sandwich ELISA kit.

The quantitative data were analyzed by two way ANOVA for testicular morphometry and scrotal circumference and one way ANOVA for testosterone concentration to compare the mean values between the days and within the group using SAS 9.4 and SPSS 24 software.

Testicular morphometry
 
In this study, the mean values of all the parameters of testicular morphometry were found to be increased significantly on day 7 as compared to day 0, i.e. start of treatment and then decreased gradually and significantly in group I on day 15 and 30 post-treatment, but no such changes were noted in group II (Table 1). Literature is scant regarding effect of intra-testicular injection of 20% calcium chloride in normal saline on testicular morphometry. However, few reports are published where calcium chloride was used for chemical sterilization in animals using different concentration for different study periods (Ahmed, 2015; Chatterjee et al., 2009; Jana and Samanta, 2007).



The results of decreased testicular volume after intra-testicular injection of calcium chloride in dogs indicated degenerative effect of this chemical in testicles. Silva et al., (2017) also reported reduction in testicular volume using 7.5% calcium chloride in 0.5% DMSO during 60 days of study in dogs. However, Pereira et al., (2018) reported no change in testicular volume after intra testicular injection of 20% and 40% calcium chloride solution. Leoci et al., (2014) reported decrease in testicular width upto 50% for a period of 12 months in dogs. However, it was only 23-25% in this study which was due to comparatively short period of observation, i.e., upto 30 days only.

In present study, no significant side effects were observed in any of the treatment groups, except temporary local testicular inflammation due to injection and biopsy which disappeared within a week. Discomfort to dogs was found to disappear within 3 days. General symptomatic treatment along with intra-testicular injection can overcome this problem. It was also reported that single bilateral injection of calcium chloride solution resulted in necrosis along with concomitant production of reactive oxygen species at testicular level without imposing testicular pain, general stress response, metabolic toxicity or any toxic and untoward side effects (Jana et al., 2002; 2005; Jana and Samanta, 2006).

Testicular atrophy found in the present study was also responsible for change in testicular volume and it supported the findings of Canpolat et al., (2006) in bull, Jana and Samanta (2006) in rats, Chatterjee et al., (2009) in stray dogs and Ahmed (2015) in dogs. The probable mechanism of action of intra-testicular injection of calcium chloride solution is stated to be higher extracellular calcium that leads to higher level of intracellular calcium which activates calcium dependent protease chaplain and initiates necrosis and thus leads to atrophy of testicles (McGinnins et al., 1999; Jana and Samanta 2007).
 
Histo-morphological changes
 
The histo-morphological changes in testicles of all dogs of both the groups were studied at day 0 (pre injection) and day 30 (post injection). At day 0 there was normal histo-morphological pattern with intact germinal epithelium and distinct interstitial spaces. The Leydig cells were demarcated with no cellular changes (Plate 1 and 2 shows day 0 image of group I and Plate 3 and 4 shows day 0 image of group II).









However, at day 30 of treatment in group I, the microscopic sections showed significant morphological changes including complete derangement of seminiferous tubules. Out of six animals the testes of four animals represented complete destruction of outline of seminiferous tubules. The germinal epithelium and Leydig cells were degenerated and parenchyma was replaced by fibro-collagenous tissue which was evident by the presence of numerous fibrocytes and fibroblast cells. There was no evidence of mature germ cells in section of both the testes.  In some areas small blood capillaries were observed which may indicate neo-vascularization. Discrete inflammatory infiltration of leucocytes was also observed in the parenchyma. In one case, the testicular section showed comparatively less fibrosis with vacuolation in some areas and complete derangement of seminiferous tubule (Plate 5 and 6).





In the present study germinal epithelium were completely degenerated, the chances of regeneration of epithelium were negligible. Secondly, there was extensive fibrosis in the parenchyma which was evident by presence of numerous fibroblasts and fibrocytes. This is in accordance with the findings of Jana and Samanta (2007), Leoci et al., (2014), Puri et al., (2018) and Silva et al., (2018) in dogs. Similar finding was also reported by Jana et al., (2005) in male goat, Jana et al., (2006) in albino rat, Canpolat et al., (2006) in bull, Jana and Samanta (2011) in tom cat and Pereira et al., (2018) in bull after chemical castration by calcium chloride. It can be concluded that chemical sterilization with calcium chloride in normal saline showed complete derangement of seminiferous tubules in animals of group I. Similar findings were also reported by Jana et al., (2002) in rats and Chatterjee et al., (2009) in dog by using intra-testicular injection of calcium chloride with the greatest effect at dose rate of 20 mg.

The occurrence of lesions may be due to oxidative damage caused by calcium chloride in the testicular tissue. This damage increased calcium influx into the cells, due to ion channels opening, destabilizing plasma membrane by lipid peroxidation. Calcium chloride is also responsible for promoting the disruption of cellular integrity leading to apoptosis. This functional correlation supports the presence of areas of vacuolation in the testicular sections of group I.

In animals of group II on day 30, the microscopic sections did not show any remarkable change in histo-morphology of testicular tissue parenchyma (Plate 7 and 8). This may be due to the fact that normal saline do not produce any inflammatory response in the testes.




 
Serum testosterone assay
 
In this study decrease in serum testosterone level at the end of study (day 30) in treatment group I indicated degeneration of Leydig cells of testicles along with other cells (Table 2). Similar results were also reported by Chatterjee et al., (2009) where significantly decreased serum concentration of testosterone was in graded manner at the doses of 100 mg, 150 mg and 200 mg in stray dogs. Leoci et al., (2014) reported same trend in dogs after using graded concentration of calcium chloride (10, 20, 30 and 60 %) in saline solution. Ahmed (2015) also reported similar results in dogs treated with 20% calcium chloride or clove oil. However, Silva et al., (2018) reported no change in serum testosterone level after chemical sterilization of dogs using intra testicular 7.5% calcium chloride (15 mg/kg) combined with dimethyl sulphoxide.

Jana et al. (2002), Jana and Samanta (2006) and Das and Karmakar (2016) studied chemo-sterilization using intra-testicular calcium chloride in different doses in adult albino rats and found significant decrease in the plasma testosterone concentration. Jana et al., (2005) reported same results in male goats by intra-testicular injection of calcium chloride solution at the doses of 10, 20 or 40 mg/kg body weight in 2.0 ml normal saline. Canpolat et al., (2006) also reported fall in testosterone concentration in bulls after injection of 10.0 ml absolute ethanol or 10.0 ml of a 30% calcium chloride solution. However, Jana and Samanta (2011) in male cats reported 70% reduction in testosterone concentration after 60 days of 20% calcium chloride administration. The decrease in the testosterone concentration can be attributed to the mass destruction of the testicular tissue especially Leydig cells which are responsible of secretion of the testosterone as reported by Das and Karmakar (2016).

It can be concluded that bilateral 1.0 ml intra-testicular injection of 20% calcium chloride in saline causes testicular degeneration along with decrease in a serum testosterone on day 30 post-treatment indicating its efficacy for chemical castration in male dogs.

The authors are extremely grateful to the authorities of College of Veterinary Sciences and A.H. NDVSU, Jabalpur for their support and MPCST Bhopal for providing financial assistant to conduct the research work.