Analysis of Histopathological and Ultrasonographic Testicular Changes Following Intra Testicular Administration of Zinc Gluconate and Chlorhexidine Gluconate in Adult Dogs

Canine overpopulation especially regarding stray dogs and feral dogs represent a global issue, compromising public health and animal welfare. The control of canine population is a needed preventive public health action that has to be persistently performed at the municipality level as an approving measure for canine zoonoses control, specifically with respect to rabies (W.H.O., 1992).
       
Contraception set out to reduce the proportions of animal population. Moreover, it is also carried out because of medical indications as well as behavioral problems. The regularly cited infelicitous male behaviors are aggression towards people or other animals, fighting with other males, indecorous sexual behavior (mounting of other animals or people) (Maarschalkerweerd et al., 1997 and Munson et al., 2004). A classic non-surgical sterilizing agent would be one which seize androgenesis and spermatogenesis (Wiebe and Barr, 1984), besides maintaining the well-being, reducing the potency and the everlasting and predictable effects on behaviour and health. The chemical contraception is developed on bilateral intra-testicular injections of chemical contraceptive, eventually leading to pathological changes in the testes and infertility or sterility. This method of sterilization is uncomplicated and cheap in comparison to surgical approach and can be used in areas with colossal number of stray animals (Kutzler and Wood, 2006).
       
Intratesticular injection of neutralized zinc gluconate with arginine has been used for chemical contraception. This sort of sterilization is based on bilateral intratesticular injections of chemical compound, resulting into pathological changes in the testes and infertility. The injection of zinc solution into the testes of 3 to 10 months old dogs’ consequence into nearly complete fibrosis of the Leydig cells and seminiferous tubules within 2-3 months after injection (Tepsumethanon et al., 2005 and Kutzler and Wood, 2006). Different concentration of chlorhexidine digluconate i.e. 3.0% and 4.5% injected intratesticularly resulted in azoospermia on day 35 ad 28, respectively (Auidi et al., 2007). Percutaneous injection of 2-5% of chlorhexidine solution at dorso-cranial portion of testes resulted in reduced testosterone level and libido of the dog on day 60 (Auidi et al., 2010).
       
Keeping the aforesaid facts in view, the present study was designed to explore the contraceptive effect of intra-testicular injection of zinc gluconate neutralized with arginine and chlorhexidine gluconate in male dogs by analysis of ultrasonographic and histopathological changes in the testes.

Twenty-four apparently healthy mongrel stray male dogs with intact and well-developed testicles were used in the present study. There were no anatomical or reproductive disorders in each dog taken for study. Separate kennels were provided to each dog at TVCC, College of Veterinary Science and AH, Nanaji Deshmukh Veterinary Science University (NDVSU), Jabalpur, M.P., India and were fed on a balanced diet accompanying the ad-lib clean drinking water during the whole duration of the research i.e. November, 2018 to April, 2019. Animal experimentation was in alignment with Ethical Committee of aforesaid college.
       
The dogs were randomly divided into four groups each of six dogs each as G-I, G-II, G-III and G-IV (control) and the study was designed as mentioned in Table 1.
 

 
Intratesticular injection
 
Inj. Atropine sulphate (0.025 mg/kg b. wt. Kaptrop, Iskon Remedies) along with xylazine (1 mg/kg b.wt. Xylaxin, Indian Immunologicals Limited) was administered in all the dogs for sedation before the administration of intratesticular injection. The dogs were kept off-feed 6-12 hrs before the injection for sedation. Once sedated the dogs were physically controlled in a dorsal recumbent position followed by the cleaning of the testicular region using antiseptic solution (10% Povidone iodine solution Unidine, Unichem laboratories Limited).
       
The G-I received single bi-testicular injection of 2 ml of zinc gluconate neutralized with arginine while G-II and G-III animals received the bi-testicular injection of 2 ml of 5% and 4% chlorhexidine gluconate, respectively. The intratesticular injection was administered using a 26-gauge needle with sterile syringe with a 0.45×13 mm of needle, into the center of the testicle via dorso-cranial portion of testicle, parallel to the longitudinal axis and the animals in G-IV were kept as control and no treatment was given to them.
 
Testicular histopathology
 
Tissue samples were collected from each testis of the all animals using Brad Max Core biopsy instrument (16G) on day 0 and 30 post treatment (Fig 1, 2) and stored in 10% buffered formalin. These tissue samples were processed in acetone-benzene sequence, embedded and blocked in paraffin wax. Tissue sections of 5 µ thickness were obtained with the help of the microtome, mounted on clean glass slides with egg albumin and stained with the Haematoxylin and Eosin (Girdley, 1960).
 

 

 
Testicular ultrasonography
 
Ultrasound evaluation of the testes was conducted using 7.5-MHz dynamic B-mode real-time transducer (Philips HD 7XE). Sagittal sections of testes were used for morphometric analysis. The ultrasonic examination was performed on day 0, 5, 15, 24 and 30 and the ultrasonographic testicular length measurement was recorded on day 0, 15 and 30 of the study.
 
Statistical analysis
 
The quantitative data were analyzed by one-way ANOVA for ultrasonographic testicular length measurement to compare the mean value between the days and within the different groups using SPSS 24 software. Results were described as the mean±S.E. The significant level used was 5% (P<0.05) (Snedecor and Cochran, 1994).

Testicular histopathology
 
The biopsy tissue collected on day 0 (before treatment) and 30 from both right and left testicles of all the animals were processed and observed for microscopic changes.
       
In group I animals treated with intra-testicular injection of zinc gluconate neutralized with arginine had no microscopic lesions before start of treatment on day 0. Testicular tissues from all the dogs showed normal arrangement of germ cells in seminiferous tubules with regular interstitial spaces. The seminiferous tubules comprised multiple layers of spermatogenic cells with sertoli cells interspersed in between (Fig a and b).
       
On day 30 the microscopic section of testicular tissues showed prominent degenerative changes. Cellular necrosis in the tissue sections from the six animals ranged from pronounced to moderate. No marked difference in the severity of testicular tissue degeneration and necrosis was noted between the right or left testicular tissue of the same animal. Prominent tubular coagulative necrosis of germinal epithelium was observed with hyalinised eosinophilic mass collected in the centre of the tubules. There was loss of concentric arrangement of spermatogonia, swollen cells and irregular basement membranes. Discrete inflammatory infiltration comprising mainly neutrophils and few mononuclear cells was also observed in the interstitial space (Fig d). Thickening of inter-tubular stroma and discrete areas of inter-tubular fibrosis were present (Fig e).
       
Animals in group-II were treated with intra-testicular injection of 5% chlorhexidine gluconate. On day zero the cross section of testis showed no microscopic abnormality. Microscopically, multiple layers of healthy cells were observed with a few tubules showing spermatogenesis along with immature tubules lined by sertoli cells and immature germ cells (Fig c). The cross sections of testicular tissue obtained on 30^th day showed considerable degenerative changes. Haemorrhages were observed in the interstitium and a decrease in spermatogenesis which reached two or three layers of spermatogenic cells within the seminiferous tubules. Coagulative necrosis of seminiferous tubules, presence of eosinophilic mass, absence of nucleus in the germinative cells were also prominently observed (Fig f and g).
       
The animals of group-III treated with intra-testicular injection of 4% chlorhexidine gluconate had similar microscopic appearance of testicular tissue as observed in Group II. Normal testicular architecture was observed at day zero followed by marked degeneration and necrosis of seminiferous tubules on day 30. The germinal cells had a vacuolated appearance with loss of nuclei (Fig h). Empty seminiferous tubules, irregular basement membranes with severe depletion of both germinal and Sertoli cells was observed (Fig i).
       
The approx. 700 million global dog population (Hughes and Macdonald, 2013) raises public health concerns and affects environmental health that may foster the permanence of diseases in other species or the degradation of ecosystems (Brown, 2003).
       
The prominent degenerative changes in both right and left testicular tissue of zinc gluconate and chlorhexidine gluconate treated groups were evident via histo-pathological examination and right and left testicular showed nearly similar pathological lesions. The degenerative changes were exemplified by coagulative necrosis of seminiferous tubules, calcification, depletion of both germinal and sertoli cells and presence of eosinophilic mass in the lumen. Prominent tubular fibrosis was also seen and the germinal cell has a vacuolated appearance with loss of nuclei. All these findings are indicative of the non-reversible degeneration of the testes ultimately leading to sterility due to intratesticular administration of zinc gluconate and chlorhexidine gluconate. Similar pathological findings were also recorded in previous studies and were in agreement with the present study (Fahim et al., 1993; Tepsumethanon et al., 2005; Oliveira et al., 2007; Soto et al., 2009; Soto et al., 2018; Aiudi et al., 2010; Vannucchi et al., 2015).
       
The morphological changes in somatic and germ cells of dog testicular tissue after chemical treatment leading to compromise the function of testicular cells and atrophic seminiferous tubules with multinucleated gaint cells might have resulted from the inability of sertoli cells to support germ cells (Russel et al., 1990; Blanc et al., 1991; Munson et al. 2004). Various degree of pathological consequences such as decrease in germ cell number up to total testicular atrophy in zinc gluconate treated group based on histopathological evidence was also reported by Soto et al., (2009). Intra-testicular chemical administration might lead to permanent destruction of seminiferous tubules, that resulted in sterility (Hughes and Macdonald 2013; Vanderstichel et al., 2015).
 
Testicular ultrasonography
 
Ultrasonographic analysis of testes of all treatment groups on day 0 revealed normal testicular parenchyma with normal echotexture and size of the testicle (Fig i). On day 5 the hyperechoic line of the intra-testicular chemical was visible in the both the testicles (Fig ii, iii and iv). On day 15 the ultrasonographic examination revealed the formation of multiple hypoechoic nodules within parenchyma and non-homogenous parenchyma (Fig v, vi and vii) followed by elimination of the normal parenchyma by day 24 (Fig viii). At the end of the study both the testes appeared hypoechoic hard nodule like (Fig ix) upon ultrasound examination which was supported by the histopathological studies.
       
The results of mean ultrasonographic testicular length were presented in Table 2. The analysis of testicular length by ultrasonography on day 0, 15 and 30 revealed that in all the treatment groups the parameter was decreased non-significantly from day 0 to day 15 post treatment. Thereafter, they decreased significantly (p<0.05) from day 15 to 30 in all the treatment groups. However, in animals of Group IV no significant change was observed in mean ultrasonographic testicular length at all the time intervals.
 

       
In present study, the decrease in the testicular length in both the testis of all the treatment groups from day 0 to 30 may be attributed to fibrosis, calcification and atrophy of the testes leading to decrease in testicular mass. Similar findings were also reported in the previous studies and are in consensus with the findings of Leoci et al., (2009), Brito et al., (2011), Canpolat et al., (2016), Leoci et al., (2019) and Rafatmah et al., (2019).
       
The intra-testicular injection of zinc gluconate in adult dogs had led to atrophy and loss of the testicular parenchyma by day 37 after the treatment (Rafatmah et al., 2019). Similar findings were reported, in case of American black bears after the administration of intra-testicular injection of zinc gluconate, which led to severe changes in the echotexture of the testicle with a significant reduction in the testicular size (Brito et al., 2011).

The intratesticular injection of zinc gluconate neutralized with arginine, 5% chlorhexidine gluconate and 4% chlorhexidine gluconate reduced the male reproductive potential which was corroborated with histopathological and ultrasonographic findings and resulted in sub-fertile or infertile dogs. The procedure is least invasive and effective method of chemical castration. Moreover, it is cheap and easy to perform. Thus, this treatment can be used as an efficient, practical and economic method for spaying male dogs.

The authors are very thankful to NDVSU, Jabalpur, Madhya Pradesh for providing the basic facilities for the research.