Comparative Evaluation of Single, Bilateral IntraTesticular Injection of Hypertonic Saline Solution and Calcium Chloride as Chemical Sterilizing Agents in Male Cats

et al., Comparative Evaluation of Single, Bilateral Intra-Testicular Injection of Hypertonic Saline Solution and Calcium Chloride as Chemical Sterilizing Agents in Male Cats. Biomed J Sci Tech Res 21(3)-2019.


Introduction
Homeless cats and dogs pose a major threat for the spreading of zoonotic diseases around the globe. 27 million cats and dogs are bounded annually in United States alone. In 1990, it was predicted that in United States 5.7 to 9.5 million cats and 5.4 to 9.1 million dogs were euthanatized in animal houses. To maintain the pet population, sterilization is expressed as the most adequate method.
Sterilization is an approach that impairs the testicular tissues by various chemicals, causing damage and atrophy of the testicular cells by deducing or stopping the blood delivery or their surgically excision [1]. Neutering of male animals is more significant than female counterparts since males have greater capability of producing offspring as compared to females [2]. In addition to the fore-mentioned primary objective, sterilization is also used for the eradication of the secondary sex characteristics of animal like urine marking, mounting and aggressive behavior towards other males [3]. With evolving research, scientists have deduced that myths like lethargy of neutered toms or urethral obstruction has no relation with neutering instead these complications are mainly related to dietary plan of the animal and urinary tract disorders [3]. It is generally perceived that un-owned and free roaming cats suffer more parasitism and diseases and have short span of life as compared to owned cats. Therefore, to deduce the population of such un-owned cats is a worthwhile goal.
Various techniques of sterilization are being practiced which include hormonal, surgical, mechanical and chemical sterilization [4]. Castration by chemical agents is a nonsurgical approach for the male sterilization [5]. It has been practiced by injecting some necrotic toxic agent into the testicles in animals belonging to different families like Felidae, Canidae, Rodentia etc. The chemicals which are being utilized for sterilization are, calcium chloride, formalin, zinc gluconate + arginine, hypertonic saline solution, potassium permanganate, chlorhexidinegluconate + cetrimide, glycerol, ethanol, sodium fluoride [6,7] while concentrated sodium chloride was used by [8]. Administration of these chemical agents into the epididymidis, testes and ductus deference ultimately lead perpetual azoospermia, reduce testosterone production and alteration in physical structure of testes. It also causes a reduction in the androgen production and improves the androgen-dependent disorders such as prostatic disease, gonadal disease and behavioral problem (mounting, aggression, urine marking). The injection of these chemicals sterilants into the epididymis, testicles, or ductus deferens induces sterility by damaging sperm cells transportation and producing fibrous occlusions that cause azoospermia. The intratesticular injection leads to testicular necrosis and decreases the spermatogenesis as well as reduction in the serum concentration of testosterone [3]. Therefore, the chemical approach of castration is considered as a suitable alternate of surgical method. It is less time consuming and demands less technical staff also. Moreover, it is convenient for practice and no extra post-operative care is required [9]. Hypertonic saline solution (HSS) has not yet been evaluated for chemical castration in male cats. Therefore, the current study was planned with the objective to evaluate and compare the efficacy of hypertonic saline solution (HSS) with calcium chloride as chemosterilizing agent in male cats.

Experimental Animals and Treatment Protocol
Experimental animals (n=20 toms) were equally and randomly allocated to four groups containing five animals in each. The toms of group A and B were injected a single, bilateral, intra-testicular injection of hypertonic saline solution respectively at concentration of 20% and 30%. Calcium chloride was administered in testes of animals of group C. While, in the D group, intra-testicular injection of sterilized normal saline solution was given, and this group was considered as a control group. The amount of testing solutions administered was according to the width of testes in each animal [10].

Intra-Testicular Injection
Before injection, the scrotum was shaved and cleaned with an antiseptic of povidone-iodine solution. Intra-testicular injection was given with a sterile 21-gauge needle. Needle was inserted from the caudoventral aspect of testis approximately 1cm from the epididymal tail and directed towards the dorso-cranial aspect of that testis so that when needle is withdrawn from the proximal to distal end, the solution was uniformly administered throughout the entire route. Table 1 gives an estimate of the amount of solution to be injected with respect to the weight of the testes

Evaluation Criteria
Clinical Assessment: Alterations in scrotum (e.g. pain, swelling, dermatitis) were noted once daily for 3 days and afterwards once a week until the completion of the experiment. Vernier's calipers were used to measure width of the testes post injection with the same time interval as described above [11].
Serum Testosterone Concentration Analysis: Blood was obtained in Gel vaccutainers from the cephalic vein and was centrifuged @2000 rpm for 15 minutes to separate serum. Serum was preserved at -20°C until the completion of the experiment.
Histological Examination: After the removal of testes, samples were washed with normal saline and fixed in Bouin's fixative solution in 50ml plastic containers for each right and left testis, separately [16]. Conventional tissue processing of the samples was done. Tissue sections with thickness 5µm were cut with the help of rotary microtome and mounted afterwards on the glass slides. Slides were then stained with Hematoxylin and Eosin stain and its histological features were compared with the help of ImageJ® Software.

Statistical Analysis
Data thus generated were analyzed by Generalized Linear Model Complete randomized design (CRD-2 factor) and difference in means was compared by Least Significance Difference (LSD) at 5% level of significance [17,18].

Testis Width
Regarding testicular width a statistically non-significant

Conclusion
The findings of the study lead to a conclusion that HSS have potential to replace other (physical or surgical) methods in practice