Clinical evaluation of non-surgical sterilization of male cats with single intra-testicular injection of calcium chloride

Abstract

Background: Calcium chloride solution is an established injectable sterilant in dogs and other mammals. With cat populations a continuing problem, we sought to explore its first use in cats. Six cats per group were injected with 5%, 10% or 20% calcium chloride dihydrate in saline solution with lignocaine hydrochloride, a local anaesthetic.

Results: At the 60th day post-injection, cat testes were collected and showed complete testicular necrosis and replacement by fibrous tissue; very low sperm counts; and reduction of serum testosterone by at least 70% in 20% dose. Androgenic enzyme activities and their expressions were also reduced in all the treated groups along with intra-testicular testosterone concentration was also low. Increased testicular lipid peroxidation, with reduced antioxidants and mitochondrial membrane potential, were evident following calcium chloride treatments. However, there were no apparent changes in serum concentrations of cortisol, fasting blood sugar level, blood urea nitrogen, packed cell volume, or total serum protein following calcium chloride injection, suggesting that this method of sterilization is not associated with any general stress response.

Conclusion: Calcium chloride solution demonstrates potential for androgenesis-eliminating nonsurgical sterilization of male cats in addition to its proven efficacy in dogs and other mammals.

Figure 1

Photograph shows the procedure of single bilateral intra-testicular injection of 0.25 ml sterile analytical grade of different concentrations of calcium chloride solution containing 1% lignocaine hydrochloride for sterilization of mature male cats. Each intra-testicular injection was performed using a sterile 27-guage 1/2 in. needle that was directed from the ventral aspect of each testis approximately 0.5 cm from the epididymal tail towards the cranial aspect of that testis. The solution was carefully deposited along the entire route by linear infiltration while withdrawing the needle from proximal to distal end. Care was taken to prevent the seepage of the solution from the injection site.

Figure 2

Photograph showing comparative state of the testicular volume following 60 days of single bilateral intra-testicular injection of 0.25 ml normal saline, or 5%, 10%, or 20% sterile analytical grade of calcium chloride solution containing 1% lignocaine hydrochloride in mature male cats. All the animals were kept for 60 days under routine clinical observations. Both the testes were collected from all cats by castration under anaesthesia.

Figure 3

Photomicrographs showing testicular histology following 60 days after single bilateral intra-testicular injection of 0.25 ml normal saline containing 1% lignocaine hydrochloride in mature male cats showed normal arrangement of germ cells in seminiferous tubules with distinct interstitial spaces (A & B) or 5% sterile analytical grade of calcium chloride solution containing 1% lignocaine hydrochloride in mature male cats induced derangement of germ cell association in seminiferous tubules along with washing out of germ cells from the tubules (C & D).

Figure 4

Representative photomicrographs showing testicular histology following 60 days after single bilateral intra-testicular injection of 0.25 ml 10% sterile analytical grade of calcium chloride solution containing 1% lignocaine hydrochloride in mature male cats showed coagulative necrosis in the seminiferous epithelium and the interstitial spaces as well as presence of degenerated and coagulated germ cells in combination with fibrous tissue and infiltration of leucocytes in tubular and interstitial spaces (E & F) or 20% sterile analytical grade of calcium chloride solution containing 1% lignocaine hydrochloride in mature male cats resulted complete testicular necrosis along with only presence of fibrous tissue and hyaline tissue and neither presence of germ cells or Leydig cells (G & H).

Figure 5

Diagrammatic representations of the changes in serum testosterone (ng/ml) and intra-testicular testosterone concentrations (ng/mg of testicular tissue) following surgical castration as well as single bilateral intra-testicular injection of 0.25 ml 5% or 10% or 20% sterile analytical grade of calcium chloride solution containing 1% lignocaine hydrochloride in different time points (after 30 or 45 or 60 days) in mature male cats.

Figure 6

Western blot analysis of testicular androgenic enzymes (3β-HSD and 17β-HSD) expressions and diagrammatic representations of the changes in these enzymes activities (units/mg of protein/hr.) following 60 days after single bilateral intra-testicular injection of 0.25 ml normal saline or sterile analytical grade of different concentrations of calcium chloride solution containing 1% lignocaine hydrochloride in mature male cats for induction of sterilization.

Figure 7

Representative histograms showing the changes in testicular activities of glutathione peroxidase (GPx; units/mg of protein), glutathione reductase (GRd; units/mg of protein), glutathione S-transferase (GST; units/mg of protein), glucose-6-phosphate dehydrogenase (G-6-PDH; units/mg of protein) following 60 days after single bilateral intra-testicular injection of 0.25 ml normal saline or sterile analytical grade of different concentrations of calcium chloride solution containing 1% lignocaine hydrochloride in mature male cats for sterilization.

Figure 8

Representative histograms showing the changes in testicular γ- glutamyl transpeptidase (γ-GT; units/mg of protein) activity, MDA levels (nmoles of MDA fomed/mg of protein), GSH/GSSG ratio (Reduced glutathione/oxidised glutathione ratio) and mitochondrial membrane potential (Δψm; fluorescence ratio at 530:590 nm) following 60 days after single bilateral intra-testicular injection of 0.25 ml normal saline or sterile analytical grade of different concentrations of calcium chloride solution containing 1% lignocaine hydrochloride in mature male cats for sterilization.

Figure 9

Diagrammatic representation of the mechanism related to induction of sterilization of male cats following single bilateral intra-testicular injection of calcium chloride.