Animals
Clinically healthy 30 male cats (Felis catus) weighing 2-3 kg, aged from 09 to 12 months with normal libido in the breeding season (December-January) were acclimatized initially for one month in the animal house. Free roaming partially socialized cats were collected from the housing of animal lovers. The privately owned cats were temporarily donated for the study with known consent as per the guidelines of "Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA)", Government of India. Breeding soundness in all selected cats were judged and every cats showed sexual interest for the oestrus queen cat. The animals were routinely de-wormed with giving PRAZICON (Concept Pharmaceuticals, Mumbai, India; Paziquantel- 5 mg/kg body weight, Pyrantel pamoate- 15 mg/kg body weight, Febantel- 15 mg/kg body weight) and vaccinated with NOBIVAC® Rabies (intervet, Hyderabad, India; Inactivated adjuvant vaccine against Rabies vaccination in cats) and NOBIVAC® Tricat (intervet, Hyderabad, India; Combined live vaccine for cats against feline viral rhinotracheitis calcivirus infection and panleucopenia) prior to arrival in the animal housing area. The animal house has artificial lighting and controlled temperatures (22°C, ranging from 19 to 25°C and 12 h:12 h light dark cycles). The experimental protocol was approved by the ethical committee of the Faculty of Veterinary Surgery and Radiology, West Bengal University of Animal and Fishery Sciences. The cats were housed in pairs in indoor and outdoor runs, fed a standard commercial cat food and given water ad libitum. Investigations were conducted in accordance with the "Principles for the Care and Use of Research Animals" recommended by the Society for the Study of Reproduction. Guidelines for Ethical Conduct in the Care and Use of animals (American Psychological Association) were followed throughout the experimental duration. The animal house was registered with "Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA)", Government of India.
Experimental protocol
The maximum effective dose of calcium chloride for induction of sterilization was estimated by dividing the 30 animals by random selection into 5 groups (Group I-V). Every animal in each of the 3 groups (Group I, II and III) received a single bilateral intratesticular injection of 0.25 ml of 5%, 10% or 20% sterile analytical grade of calcium chloride dihydrate in saline solution per testis (CaCl2, 2H2O, Merck, Mumbai, India) containing 1% lignocaine hydrochloride (a local anaesthetic agent, Astra IDL, Bangalore, India) respectively (Figure 1) under standard humane manual restraint condition guided by experienced Veterinary Surgeons. The injection volume of 0.25 ml was selected by standardization with a dose dependent study and in that volume of high dose calcium chloride solution caused necrosis of entire testicular gland parenchyma without any leakage from the gland. So that 0.25 ml of different concentration of calcium chloride were considered to evaluate the effectiveness of calcium chloride for the sterilization in cats. The animals in the control group (Group IV) each received a single bilateral intra-testicular injection of 0.25 ml sterile normal saline per testis containing 1% lignocaine hydrochloride. Parallel surgical castration was done in another group (Group-V) of animals according to the standard procedure.
Intratesticular injection of calcium chloride solutions
Each intratesticular injection was performed using a sterile 27-guage 1/2 in. needle 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. Necessary care should be taken to prevent the seepage of the solution from the injection site. In the previous study we have seen that if the solution was spilled in the scrotal skin then there was a skin lesion due to tissue necrosis. But if the solution immediately wiped away then the complications were avoided.
Routine clinical observation
All the animals were kept under routine clinical observations for 60 days. We performed a routine clinical observation before and subsequent follow-up exams (Continuous observations for first 72 hours and after that with 1 hr. intervals up to 7 days, 6 hr intervals up to 30 days and of 12 hr intervals up to 60 days) after chemical and surgical sterilization procedure and must paid attention to the palpation of the testis. Following few parameters were sincerely considered during clinical observation.
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1.
Body weight 2. General attitude 3. Appetite 4. Rectal temperature 5. Scrotal and inguinal integument 6. palpation of testis and 7. Heart and Respiratory rate.
Measurement of testicular volume
Length and width of the right and left testis were measured using laboratory callipers, and the volume of each testis was quantified by multiplying the length (cm) by width (cm2) by 0.524. Total testicular volume (cm3) was calculated by adding together the volume of the right and left testis [18].
Collection of blood and testes
60 days following intra-testicular injection, both testes were obtained from all cats by castration under anaesthesia. Each animal was anesthetized with an intra-muscular injection of 20 mg/kg ketamine hydrochloride combined with 0.06 mg/kg diazepam. The both testicular weights were recorded and the gonado-somatic index was calculated with the ratio of testes weight and body weight. The right testis from each animal was used for histo-morphological studies, while the left one was used for biochemical assays. Blood was collected in 0, 30, 45 and 60 days after intratesticular injection before castration in fasted animals between 8.00 a.m. and 8.30 a.m. from the saphenous vein of each cat into a clean tube. A portion of blood was allowed to stand for 10-15 min at 4°C. The serum was aspirated into clean tubes and centrifuged 1500 × g for 10 min at 4°C. The serum samples were stored at -20°C until assayed for hormone concentrations.
Epididymal sperm count
Sperms were collected from an equal length of the caudae of the excised epididymis of each cat by flushing through vas deferens with same volume (10 ml) of suspension medium containing 140 mmol NaCl, 0.3 mmol KCl, 0.8 mmol Na2HPO4, 0.2 mmol KH2PO4 and 1.5 mmol D-glucose (pH adjusted to 7.3 by adding 0.1 (N) NaOH) (E Merck). Collected sample was centrifuged at 100 × g for 2 min, and the precipitate part was re-suspended in 10 ml of fresh suspension medium. A fraction of suspension (100 μl) was mixed with an equal volume of 1% Trypan blue in the same medium, and numbers of sperms were counted in four chambers (used for counting of WBC) of the hemocytometer slide. At this concentration of Trypan blue (0.5%), the dye was completely excluded by intact sperms, which appeared bright and colourless, but taken up by dead and damaged sperms, which showed blue heads. The sperms number was expressed per ml of suspension [19].
Histopathological studies on the testes
The right testis from each animal was fixed in Bouin's fixative and embedded in paraffin wax. A section 5 μm thick was cut from the middle portion of each testis, stained with hematoxylin-eosin and examined under light microscopy at 100X and 400X magnifications. The structures of the seminiferous tubules and interstitial spaces in the testis were examined.
Assays for serum and intratesticular testosterone and cortisol concentrations
The testicular tissue was homogenized in 0.5 ml of water using Teflon homogenizer that was fitted into a microfuge tube chilled in ice. Each sample was centrifuged at 10000 rpm for 10 min. The supernatant was removed, frozen, and stored until the hormone assay [20]. The serum and intratesticular concentrations of testosterone and cortisol were measured using an ELISA reader (Merck, Japan) according to the standard protocol given by National Institute of Health and Family Welfare (NIHFW, New Delhi, India) [21]. The ELISA kit for testosterone was supplied by IBL (Hamburg, Germany) and the cortisol kit was supplied by NIHFW. Horseradish peroxidase was used as an enzyme-labelled antigen in both assays that made a competition with unlabelled antigen for binding with a limited number of antibody sites on the micro-plates (solid phase). Assays were performed following standardized instructions. Each testosterone concentration was calculated from a standard curve with 5 standards supplied by IBL whereas each cortisol concentration was calculated from 6 standards supplied by NIHFW with the absorbance of the standards and samples monitored against a blank at 450 nm. The stated cross-reaction of the testosterone antibody with dehydrotestosterone was 10% and the intra-assay CV was 6.2%. The stated cross-reaction of the cortisol antibody with corticosterone was 10% and intra-assay CV was 5.5%. All the samples were included in a single assay.
Estimation of total serum protein concentrations, blood urea nitrogen and fasting blood sugar levels
Total serum protein concentration was measured according to the standard method of Lowry et al. [22] and the level was expressed as gm/dl. Blood urea nitrogen was measured using the kit supplied by Dr. Reddy's Laboratories (diagnostic division), Hyderabad, India, according to the manufacturer's instructions. Fasting blood sugar level was measured using a single touch glucometer (Blood Life Scan, Johnson and Johnson, Milpitas, California, USA) and the concentration was expressed as mg/100 ml.
Steroidogenic enzymes activities
3β-Hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase (17β-HSD) are the two crucial enzymes in the steroid biosynthesis pathway and they are the key androgenic enzymes in the testis. These two enzymes were assayed according to the methods described earlier [19]. One unit of the enzyme activity was equivalent to a change in the absorbance of 0.001 units/min at 340 nm.
Western Blot Analysis of testicular steroidogenic enzymes
The testicular tissue was homogenized in approximately 20 volumes of buffer using a tissue protein extraction kit (Pierce Chemical Co. Rockford, IL, USA) and cleared by centrifugation at 10000 × g for 10 min at 4°C to produce a protein extract. The protein content of the preparation was determined with a BCA kit (Pierce) using BSA as a standard. An equal concentration of each sample (100 μg protein) was resolved on 10% sodium dodecyl sulfate- poly-acrylamide gels (SDS- PAGE) and electrophoretically transferred to nitrocellulose membranes. The membrane was blocked for 1 hr. in TBST (0.05% Tween-20 in Tris-buffered saline, pH 7.6) with 5% non-fat milk and then incubated with 1: 100 specific primary antibodies (rabbit polyclonal 3β-HSD and 17β-HSD antiserums, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 4°C overnight and subsequently exposed to horseradish peroxidase- conjugated anti rabbit IgG goat poly-clonal secondary antibody. In between each step, the membrane was washed with TBST about 10 minutes for three times. The bands were visualized with the Amersham Pharmacia Biotech enhanced chemiluminescence system (Uppsala, Sweden) according to the manufacturer's instructions. Finally the developed blots were subjected to densitometry using the β-actin (Specific β- actin antibody, Cell Signalling Technology, Beverly, MA, USA) as an internal control [23].
Assay of testicular glutathione peroxidase (GPx) activity
The activity of glutathione peroxidase was determined by the slightly modified method of Rotruck et al. [24]. Briefly, the assay mixture containing 0.5 ml of sodium phosphate buffer, 0.1 ml of 10 mM sodium azide, 0.2 ml of 4 mM reduced glutathione, 0.1 ml of 2.5 mM H2O2, and 0.5 ml 1:10 tissue extract was taken and the total volume was made up to 2 ml with distilled water. The tubes were incubated at 37°C for 3 min and the reaction was terminated by the addition of 0.5 ml 10% TCA. To determine the residual glutathione content, the supernatant was removed after centrifugation and to this, 4.0 ml of disodium hydrogen phosphate (0.3 M) solution and 1 ml of DTNB reagent were added. The colour developed was measured at 412 nm in a spectrophotometer against a blank containing only phosphate solution and DTNB reagents. The enzyme activity was expressed as units/mg of protein (1 unit is the amount of enzyme that converts 1 μmol GSH to GSSG in the presence of hydrogen peroxide/min).
Assay of testicular glutathione reductase (GR) activity
The activity of glutathione reductase was determined using the method of Staal et al. [25]. Briefly, the assay mixture containing 0.2 ml of tissue extract, 1.5 ml of sodium phosphate buffer, 0.5 ml of 25 mM EDTA, 0.2 ml of 12.5 mM oxidized glutathione, and 0.1 ml of 3 mM NADPH were prepared, and immediately read at 340 nm in a spectrophotometer against a blank containing all the components except the enzyme at 3 min at 30 s interval. The activity of GR was expressed as μmol of NADPH oxidized/min/mg of protein.
Assay of testicular glutathione-S-transferase (GST) activity
The enzymatic activity was determined with the method of Habig et al. [26]. Briefly, the assay mixture containing 0.4 ml of potassium phosphate buffer, 0.1 ml of tissue extract, 1.2 ml of distilled water and 0.1 ml of 1-chloro-2, 4-dinitrobenzene (CDNB) was added and incubated in a water bath at 37°C for 10 min. After incubation, 0.1 ml of 30 mM reduced glutathione was added. Immediately, the optical density was measured against a blank at 340 nm on a spectrophotometer at 30 s interval for 3 min. The activity of GST was expressed as units/mg of protein (1 unit is the amount of enzyme that conjugate 1 nmol of CDNB with GSH/min).
Assay of testicular glucose-6-phosphate dehydrogenase (G-6-PDH) activity
The enzyme was assayed by the method of Beutler [27]. Briefly, the assay mixture containing 0.1 ml each of Tris-HCl buffer, NADP and MgCl2, 0.5 ml of water and 0.1 ml of 1:5 diluted tissue extract was taken in a cuvette. The reaction was started by the addition 0.1 ml of glucose-6-phosphate and the increase in optical density was measured at 340 nm against a blank. The activity of G-6-PDH was expressed as μmol of glucose-6-phosphate to 6-phosphogluconate/min/mg protein.
Assay of testicular γ- glutamyl transpeptidase (γ- GT) activity
The testicular tissue was homogenized in ice-cold 0.1 mol Tris Hcl buffer, pH 7.4 at a tissue concentration of 10%/ml and then the homogenized mixture was centrifuged at 10,000 × g for 30 min at 4°C. The supernatant was used for the enzyme estimation. The enzyme activity determined by the modified method of Orlowski and Meister [28] and was expressed as μmol of p-nitroaniline formed/min/mg protein.
Determination of lipid peroxidation
The level of lipid peroxidation was measured by the slightly modified method of Devasagayam and Tarachand [29]. In brief, the reaction mixture consisted of 1.0 ml of 0.15 M Tris-HCl buffer (pH 7.4), 0.3 ml of 10 mM KH2PO4, and 0.2 ml cell extract in a total volume of 2 ml. The tubes were incubated at 37°C for 20 min with constant shaking. The reaction was stopped by the addition of 1 ml 10% trichloroacetic acid. The tubes were shaken well, followed by addition of 1.5 ml thiobarbituric acid (TBA) and were heated in a boiling water bath for 20 min. The standard tubes containing 10, 20, 30, 40, and 50 nmol/ml were also run simultaneously. The tubes were centrifuged and the colour developed was measured at 532 nm. The malondialdehyde (MDA) content of the sample was expressed as nmoles of MDA formed per milligram protein.
Quantification of testicular contents of reduced glutathione (GSH) and glutathione disulphide (GSSG)
GSH (reduced glutathione) and GSSG (oxidized glutathione) levels of testes were estimated as described by Hissin and Hilf [30]. To 0.5 ml of the tissue extract (10000 × g supernatant), 4.5 ml of the phosphate-EDTA buffer, pH 8.0, was added. The final assay mixture (2.0 ml) contained 100 μl of the diluted tissue supernatant, 1.8 ml of phosphate-EDTA buffer, and 100 μl of the 0.1% orthopthalaldehyde (OPT) solution. For GSSG estimation, 0.5 ml portion of the tissue extract was incubated at room temperature with 200 μl of 0.04 M N-ethyl maleimide (NEM) for 30 min to interact with GSH present in the tissue. To this mixture, 4.3 ml of 0.1 N NaOH was added. 100 μl of this mixture was taken and added to 1.8 ml of 0.1 N NaOH and 100 μl of the 0.1% OPT solution. After thorough mixing and incubation at room temperature for 15 min, the solution was transferred to a quartz cuvette. The fluorescence at 420 nm was determined with the activation at 350 nm. The tissue GSH and GSSG levels were obtained from a standard curve prepared using GSH and GSSG standards. The results were expressed as GSH/GSSG ratio.
Determination of mitochondrial membrane potential (Δψm)
Mitochondrial membrane potential (Δψm) was measured using JC-1 probe according to the method described by Mishra and Shaha [31]. JC-1 is a vital cationic mitochondrial dye that is lipophilic and becomes concentrated in the mitochondria in proportion to the membrane potential; more dye accumulates in mitochondria with greater Δψm and ATP generating capacity. Therefore, the fluorescence of JC-1 can be considered as an indicator of relative mitochondrial energy state. The dye exists as a monomer at low concentrations (emission, 530 nm, and green fluorescence) but at higher concentrations forms J-aggregates (emission, 590 nm, red fluorescence). The 530:590 ratio in fluorescence spectrophotometer (Perkin-Elmer) was considered as the relative Δψm value.
Statistical Analysis
The results were expressed as mean ± standard error (S.E.). One-way analysis of variance (ANOVA) with Bonferroni modification was first carried out to test for any differences between the mean values of all groups. If any differences between groups were established, the values of the treated groups were compared with those of the control group by a multiple two tail t-test. A value of p < 0.05 was interpreted as statistically significant [32].
