Sterilization has long been recognized as the most effective means of controlling pet populations. Yet with the huge numbers of owned and un-owned cats in the developing countries like India, the sterilization programs currently available are not enough. The mainstay of population control for male cats has been accomplished through surgical sterilization, namely orchidectomy (castration) . However, for many reasons, surgical sterilization may not be effective as the sole method for population control. It requires anesthesia, medical equipment, a sterile surgical suite, a trained veterinarian, recovery time, incision site observation, and more . It carries the risks that inherent in any surgical procedure. Furthermore, many people are unwilling to subject their pets to what they perceive to be a painful and invasive procedure. The cost of surgery is prohibitive for many owners, particularly in developing countries. In addition, when considering cat populations where permanent sterilization is desired, surgical methods can be expensive to be performed on a large scale . Presently, a viable alternative to surgical sterilization is being intensively investigated. A non-surgical form of contraception is a promising additional method of pet population control. An ideal non-surgical sterilizing agent would be one which is safe, effective, affordable, permanent, and delivered in a single injection, with predictable effects on behavior and health [2, 3].
In humans, an ideal chemo-sterilizing agent would be one which effectively arrests the production of sperm (spermatogenesis) or blocks their fertilizing capacity without inhibiting or altering the production of male steroid hormones (steroidogenesis), libido, accessory sex glands activities and pituitary function. But for other animals such as cats, an ideal chemical sterilizing agent would be one which arrest androgenesis and libido as well as spermatogenesis . Otherwise the development of secondary sex characteristics may cause management problems of animals in the community, especially at their breeding season.
Sterilization by chemicals (chemical castration) is a non-surgical approach to male contraception. Chemical agents injected into the testis, epididymis or vas deferens cause infertility by inducing azoospermia in male animals. The technique is not technically demanding, is inexpensive and is suitable for large scale sterilizing programs in both domestic and wild animals .
Intra-testicular injection has been investigated as a method of inducing aspermatogenic orchitis and male contraception for more than five decades [1, 5]. For example, injection of 10-25 units of freund's complete adjuvant (FCA) or Bacillus Calmette Guerin (BCG) directly into testis of male dogs resulted in severe oligospermia or azoospermia without the development of circulating anti-sperm antibodies. Infertility occurred within 6 weeks and lasted for several months [5, 6]. In addition, intra-testicular administration of a combined solution of methallibure, dexamethasone, metopiron, niridazole, α-chlorohydrin or danazole caused testicular and epididymal atrophy and azoospermia in dogs . Injection of 3.5% formalin in phosphate buffered saline or 1.5% chlorhexidine gluconate in 50% dimethyl sulfoxide into the tail of both epididymis in dogs resulted in irreversible azoospermia with chemical occlusion and secondary testicular atrophy . Although intratesticular injection of glycerol resulted in sustained azospermia in squirrel monkey or Saimiri sciureus , even 70% glycerol solution did not result in azoospermia and sterility in dogs . Intra-epididymal treatment with formalin alone induced only temporary azoospermia or oligospermia in treated dogs . A single injection directly into the vas deferens with sclerosing chemical agents like 10% silver nitrate, 3.6% formaldehyde in ethanol, 5% potassium permaganate, 100% ethanol, or 3.6% formaldehyde resulted in irreversible infertility in male dogs  but no elimination of unwanted reproductive behavior. Intra-epididymal administration of zinc arginine also resulted in azoospermia within 90 days following injection in male dogs . In 2003, the FDA approved a product Neutersol®, labeled for chemical castration via intra-testicular injection in male puppies. Neutersol®/Esterisol®, is a zinc-gluconate solution neutralized to a pH of 7 by arginine . The procedure involves injecting a predetermined amount of zinc solution based on scrotal width into each testis of puppies 3-10 months of age . Histological findings within 2.5 months of injection induced almost complete fibrosis of the seminiferous tubules and Leydig cells [1, 12]. Though Neutersol® is not currently available in the U.S., a similar compound, Esterisol®, is on the market in Mexico. However, neither compound produces a decline in testosterone long-lasting enough to significantly reduce nuisance behavior. Studies using these models of male contraception report no or minimal signs of discomfort following injection, but a transient increase in testicular diameter may follow the injection, resulting scrotum swelling. Additional local and systemic reactions reported after intra-testicular injections include scrotal ulceration and dermatitis, scrotal self-mutilation, preputial swelling, vomiting, diarrhea, anorexia, lethargy and leukocytosis . Also, unlike surgical castration, this kind of chemical sterilization does not eliminate gonadal sources of testosterone . In another study on Isabella Island of the Galapagos, 3.9% of 103 dogs given zinc gluconate developed necrotizing injection site reactions. By comparison, 3.4% of 58 dogs experienced wound dehiscence after surgical castration. The zinc gluconate reactions were more severe and required more extensive surgical repair than the traditional surgical complications. The dogs that experienced zinc gluconate reactions were large, mature dogs that received near maximum volume doses . However, Neutersol®, the product is appealing to owners who do not want their dogs to have surgery or who want their dogs to retain the "masculine" look and presence of testicles.
Moreover, there are very few reports available in the literature related to the chemical sterilization of cats. Intra-epididymal injection of a 4.5% solution of chlorhexidine digluconate resulted in azoospermia or severe oligospermia in tom cats . Unlike in the dog, transient scrotal swelling and pain persisted for up to 2 weeks following intra-epididymal injection in cats. Due to the above negative aspect following the use of the chemical, an effective chemosterilizing agent has yet to be established in cats. We thus began testing calcium chloride solution, reported in the U.S. veterinary literature as early as 1978 . Based on reports and on its chemistry, we expected calcium chloride to be less toxic and carry less risk of skin necrosis and injection site reaction than previously-tested compounds, including zinc gluconate. We have reported that single bilateral intra-testicular injection of calcium chloride solution, with or without added local anesthetic, indeed induces permanent (irreversible) sterilization in male albino rats, goats and dogs through testicular degeneration, along with significant and dose-dependent diminution in testosterone concentration, without imposition of any apparent pain, general stress response, metabolic toxicity, or toxic and untoward side effects [2, 3, 16, 17]. This simple technique thus fulfills the criteria of a method for nonsurgical sterilization in male animals. Previously, to study the mode of action of calcium chloride for induction of sterilization in other mammals we have seen that calcium chloride caused oxidative testicular tissue damage [2, 3, 16, 17]. Nowadays, it is clear that the oxidative tissue damage occurred due to the increased production of reactive oxygen species (ROS) and ROS damages lipids, proteins, carbohydrates and DNA and also caused lipid peroxidation and affects enzyme activity and cellular genetic machinery . Indeed, it has been shown that ROS inhibits steroidogenesis, steroidogenic enzyme activity and testosterone production in testis [16, 17]. However, the biological systems possess a number of mechanisms to remove ROS, such as the integrated antioxidant defense systems. Different enzymatic and non-enzymatic scavengers of ROS, may protect the cellular system from the deleterious effects of the oxidative stress [16, 17]. In continuation of our previous work, in this present study we investigate the effectiveness and also the mode of action of calcium chloride solution for chemical sterilization of male cats.