Intratesticular and intraepididymal injections to sterilize male cats

Abstract

Aim and rationale:

The aim of intratesticular and intraepididymal injections is to provide an inexpensive non-surgical method for sterilizing tom cats. Intratesticular and intraepididymal injections have been studied for decades and warrant continued investigation. While both methods result in azoospermia, intratesticular injection of sclerosing agents induces orchitis, resulting in decreased spermatogenesis, whereas intraepididymal injection blocks sperm transport but does not alter spermatogenesis.

Evidence base:

Sclerosing agents that have been used effectively for intratesticular injections in cats include calcium chloride dihydrate and zinc gluconate. For sclerosis by intraepididymal injections, chlorhexidine digluconate has been used successfully in cats. The volume, formulation and concentration of sclerosing agents for intratesticular and intraepididymal injections in cats have not been standardized.

Challenges:

Neither intratesticular nor intraepididymal injections entirely eliminate gonadal testosterone production, which may be undesirable for pet cats and therefore may restrict the application of this method of sterilization to feral cats with limited human contact. In addition, both methods may require sedation or general anesthesia, leading some to support routine castration over these non-surgical methods. Lastly, even if the technique is successful in inducing permanent sterility, normal fertility may persist in treated males for 1–2 months after treatment because of sperm present within the epididymis and vas deferens.

Intratesticular injections

Intratesticular injections have been investigated as a method of male contraception for more than six decades. ¹ Improvements in the injection technique and refinements in the solutions injected have greatly reduced the incidence and severity of adverse reactions. In search of an ideal non-surgical sterilant for intratesticular injection, a broad range of agents has been tested in a wide range of species. For the purposes of this review, only those substances used for intratesticular injection in cats will be discussed.

The goal of intratesticular injection is to induce aspermatogenic orchitis, resulting in permanent azoospermia. According to current research, intratesticular injections in cats induce prolonged (and theoretically permanent) azoospermia (or teratospermia or necrospermia) following a 4–6 week delay.^2,3 This treatment efficacy delay is most likely attributable to sperm reserves present in the epididymis. ⁴ Depending on the agent injected, there may also be a reduction in androgen concentrations. However, complete elimination of gonadal sources of testosterone is unlikely, irrespective of treatment.

Rationale

The technique of intratesticular injection is precise but not technically challenging, and it is inexpensive compared with surgical castration. However, the various agents used for feline intratesticular injection are still under research and development, and currently not yet suitable for large-scale sterilization programs in cats. With respect to restraint, recommendations vary from general anesthesia^3,5,6 to ‘standard humane manual restraint’. ⁷ The scrotal hair may need to be clipped to allow for proper injection technique.

The goal is to inject enough chemical to cause necrosis in the majority of the testicular tissue without injecting excess solution that can leak out and result in skin necrosis or adversely affect adjacent tissues.

Studies using this method for male sterilization in cats report no or minimal signs of discomfort following injection, which can be explained by the fact that afferent nerve endings associated with pain sensation are located only on the scrotal skin and in the testicular capsule rather than within the testicular parenchyma. ⁸ Within 24 h following the injection, a transient increase in testicular diameter may occur, resulting in pain secondary to swelling of the testicular capsule. None of the systemic reactions reported after intratesticular injections in dogs (neutrophilia, vomiting, anorexia and lethargy) ⁹ have been reported in cats.

Evidence base

Calcium chloride dihydrate (CaCl₂)

Intratesticular injection of a CaCl₂ solution induces sterilization via two mechanisms:

• Intratesticular edema following the injection leads to necrosis and fibrosis, which causes degeneration of seminiferous tubules (and germ cells) and the interstitial (Leydig) cells;

• Free radicals produced within testicular tissue following the injection lead to lipid peroxidation and destruction of other cellular structures, which also directly impairs spermatogenesis. ²

CaCl₂ intratesticular injections should be performed using a sterile 27 gauge needle directed from the ventral aspect of each testis approximately 0.5 cm from the epididymal tail towards the cranial aspect of that testis (Figure 1). The CaCl₂ should be carefully deposited along the entire route by linear infiltration while withdrawing the needle from the proximal to distal end.

Figure 1

Injection of calcium chloride dihydrate (CaCl2) solution into the right and left testis of a male cat. Reproduced from Baran et al, ¹⁰ with permission

Although there has been considerable research in other species (including dogs) using CaCl₂ intratesticular injections for sterilization, there have only been two studies in cats. First, Baran and colleagues injected a volume of 0.2 ml per testis of 0% (n = 1 cat), 5% (n = 1 cat), 10% (n = 1 cat) or 20% (n = 1 cat) CaCl₂ and found that the 5% and 10% treated cats were oligospermic (<20 million sperm/ml in ejaculate), whereas the 0% treated cat had a normal ejaculate (>20 million sperm/ml). ¹⁰ The 20% treated cat did not ejaculate any sperm, and histologic evaluation showed degeneration and calcification of the seminiferous tubules and interstitial cells along with significant fibrosis at 60 days post-treatment. ¹⁰

In a similar study, Jana and Samanta injected a volume of 0.25 ml per testis of 0% (n = 6 cats), 5% (n = 6 cats), 10% (n = 6 cats) or 20% (n = 6 cats) CaCl₂ in saline with 1% lidocaine. ⁷ The intratesticular injection of 5% CaCl₂ induced dissolution of germ cells associated with atrophy of the seminiferous tubules. ⁷ However, the effects were uneven and inconsistent throughout the testes. ⁷ The intratesticular injection of 10% CaCl₂ induced coagulative necrosis in the seminiferous epithelium and interstitial spaces. ⁷ Degenerated and coagulated germ cells were present in combination with fibrous tissue in tubular and interstitial spaces. ⁷ Intratesticular injection of 20% CaCl₂ solution resulted in complete testicular necrosis of the entire germinal epithelium, with only fibrous and hyaline tissue remaining. ⁷ At 60 days post-treatment, serum testosterone concentrations were an average of 2.15 ng/ml in the 20% CaCl₂ group compared with 7.82 ng/ml in the 0% CaCl₂ group. Anecdotal reports also note a reduction in sex-linked behaviors following treatment.

Intratesticular CaCl₂ injection is reported to be well tolerated. ¹⁰ Mild discomfort can be observed 1–5 mins after injection. ⁷ Testicular swelling is evident by 24 h, peaking 2–4 days following injection and then decreasing over a period of 3–4 weeks. ⁷ Care should be taken to prevent seepage of CaCl₂ solution from the injection site because, if the solution remains on or under the scrotal skin, tissue necrosis occurs. ⁷ If the solution is immediately wiped away, then complications may be avoided. ⁷ Scrotal skin necrosis can also develop if an excessive volume is injected or leakage occurs outside of the tunic. ¹¹ Although sample sizes are small, no serious adverse effects have been reported.

Ingredients to prepare the CaCl₂ solution for intratesticular injection (eg, sterile analytical grade CaCl₂) are readily available in many countries where commercially manufactured products for canine intratesticular injection are not available or are too expensive. ¹² However, the formulation, concentration and volume of the CaCl₂ solution for intratesticular injection in cats are not yet standardized. Studies to date suggest that alcohol or lidocaine are superior to saline or water as a vehicle. ¹²

Effectiveness of the CaCl₂ solution appears to vary from no change to complete destruction of sperm production depending on volume, vehicle and CaCl₂ concentration. ¹² The final volume (0.2–0.25 ml/testis) administered depends upon testicular mass. The injection volume of 0.25 ml was selected by standardization with a concentration-dependent study; ⁷ in that study, this volume of a high-concentration CaCl₂ solution caused necrosis of the entire testicular parenchyma without any leakage. ⁷ At the highest concentrations, sterility appears to be permanent based on the type of testicular destruction seen, but few studies have allowed these chemically sterilized toms to mate or have followed the histopathological results past 3 months.

Zinc gluconate

Ark Sciences (New York, USA) manufactures a proprietary zinc gluconate solution for intratesticular injection. This product contains 0.2 M zinc gluconate (13.1 mg zinc/ml), which is neutralized to pH 7.0 with 0.2 M L-arginine. Zeuterin™ (approved by the US Food and Drug Administration [FDA] for use in male dogs 3–10 months of age) and EsterilSol™ (the formulation name marketed in select Latin American countries) have been used off-label in cats and in older dogs. BioRelease Technologies (Birmingham, Alabama, USA) also manufactures a proprietary zinc gluconate solution for intratesticular injection (Testoblock^®). This product contains 0.2 M zinc gluconate (13.1 mg zinc/ml), which is pH-neutralized with arginine in a physiologic proprietary vehicle. ¹³

Irrespective of the product, the procedure for intratesticular injection of zinc gluconate involves inserting a 27 gauge needle connected to a 0.5 ml U100 insulin syringe into one pole of the testis and gently pushing it towards the other pole. The solution is deposited homogeneously throughout the testis as the needle is withdrawn. Similar to CaCl₂, the needle for zinc gluconate should be inserted parallel to the long axis of the testis. However, unlike CaCl₂, the needle for zinc gluconate should be inserted into the dorsal-cranial area of each testis, lateral to the caput epididymis (close to the rete testis and efferent ducts). No explanation is provided by investigators using either CaCl₂ or zinc gluconate for the difference in direction of needle insertion.

More extensive research with zinc gluconate has taken place in dogs than in cats; the incidence of side effects using the Ark Sciences product to sterilize male dogs is lower than reported for surgical castration (incidence of injection-site reactions reported in 270 dogs was 1.1%). ¹² Levy reported that 115 cats were treated with a dose of 0.3–0.4 ml/testis of EsterilSol at 6 months of age, with no injection-site reactions developing during 12 months of monitoring. Treatment resulted in reduced serum testosterone concentrations, testicular atrophy and absence of sperm. ¹⁴ Using Testoblock in cats, no biting or licking of the scrotum or testes following intratesticular injection have been reported, but a transient testicular swelling 1 day after injection does occur. ³ No apparent scrotal or testicular pain or tenderness was noted with Testoblock, except for one treated cat that displayed reduced activity and feed intake for 2–3 h post-injection. ³

Oliveira and coworkers injected 0.44–0.51 ml/testis of Testoblock on the basis of testis width (measured with calipers). The zinc gluconate solution was administered at a rate of 1 ml for every 27 mm of testis width. ³ The dose of zinc gluconate for intratesticular injection is non-linear. According to the authors, this dose was adapted from research in dogs using 0.3 ml of zinc gluconate solution for a testis 12–14 mm wide. ¹⁵ The testicular width of zinc gluconate-treated cats decreased over 120 days, and testosterone-dependent penile spines were absent in 36% (4/11) and decreased in 54% (6/11) of treated cats. One cat still had well-developed penile spines. ³ On day 60, 91% (10/11) of treated cats were azoospermic, and the remaining cat had reductions in both sperm count and sperm motility. ³ However, on day 120, only 73% (8/11) of treated cats were azoospermic. ³ One treated cat had necrospermia and two had asthenospermia. ³ Plasma testosterone concentrations were not significantly different between controls and cats treated with zinc gluconate ³ and remained within the normal reference interval for domestic cats. ¹⁶ Despite normal testosterone levels on days 60 and 120, owners reported that treated cats showed reduced aggression, roaming, mounting and urine marking (spraying), whereas these activities persisted in control cats. ³

The testes from the treated cats in the Oliveira and coworkers’ study were evaluated histopathologically by Fagundes and colleagues. ⁵ Cats treated with zinc gluconate had few or no germ cells present in the basal and adluminal compartments of the seminiferous epithelium and most of the seminiferous tubules were atrophied. ⁵ Conversely, some tubules had increased in size owing to an expanded lumen, but these tubules also had fewer germ cells and incompletely formed spermatids. ⁵ Sertoli cells displayed intracytoplasmic vacuolation and most of the seminiferous tubules had a thicker basement membrane. ⁵ Hyalinized formations were present within the intertubular tissue along with clusters of Leydig cells surrounded by collagen, fibroblasts and inflammatory cells. ⁵

Challenges

To date, less research has taken place on intratesticular injection in cats than in dogs. Large scale (eg, 100–1000 cats) field experiments with a methodology that will allow for determination of the appropriate dose (and formulation with respect to CaCl₂), as well as long-term (eg, 2–3 years) follow-up on fertility outcomes (eg, sperm counts, testicular histology), are needed.

None of the intratesticular injection products described herein entirely eliminates gonadal testosterone production. The minimum threshold for circulating testosterone concentration for behavior change on a par with castration is not known, and additional behavioral studies following intratesticular injections in cats are warranted. Elimination of gonadal testosterone production would be preferential for indoor cats, as even low levels of circulating testosterone may be associated with characteristic tom cat behaviors (eg, urine marking/spraying, inter-cat aggression). For pet cats (either owned or in shelters available for adoption) and unowned, free-roaming/feral cats close to human habitation, this behavior would be unacceptable. A reduction in male reproductive behaviors has, however, been anecdotally reported with intratesticular injections, which warrants additional research in this area.

Intraepididymal injections

The use of a broad range of sclerosing agents to chemically vasectomize and induce permanent sterility has also been investigated in a wide range of species. For the purposes of this review, only research on intraepididymal injection in cats will be discussed.

Rationale

The site of injection is important to the mechanism of action: intraepididymal injection blocks sperm transport, whereas intratesticular injection decreases spermatogenesis and results in azoospermia. ¹⁷ Unlike the dog, ¹⁸ the tail of the epididymis in cats is very small and may be more difficult to locate. For this procedure, a 30 gauge needle should be used. ⁶

Evidence base

Pineda and Dooley administered intraepididymal injections of 4.5% chlorhexidine digluconate to mature tom cats and collected weekly semen samples for 36 weeks. ⁶ There was considerable variation in the response to treatment among cats, both within and between treatment groups (0.05 ml vs 0.10 ml), including inconsistent and transient severe oligospermia or azoospermia. ⁶ At various time points following the intraepididymal injection, three of the four (75%) cats in the 0.05 ml treatment group became azoospermic compared with only one of the four (25%) given 0.10 ml of the sclerosing agent. ⁶ A transient scrotal swelling was reported within the first 2 weeks following injection. ⁶ During this same period, cats demonstrated a withdrawal reaction when pressure was applied to the epididymal area of the scrotum. ⁶ However, open wounds and other undesirable clinical signs were not observed and the intraepididymal injections did not impair ambulation or alter the general behavior of the cats. ⁶

At the conclusion of the study, toms were castrated and the testes and epididymides were histologically evaluated. Cats given intraepididymal injections of chlorhexidine digluconate had normal germinal epithelium and active spermatogenesis. ⁶ Microscopic sperm granulomas were found bilaterally in the epididymides of the treated cats irrespective of whether or not they became azoospermic. ⁶

Challenges

Intraepididymal injections for sterilizing male cats have demonstrated both successes and failures. Intraepididymal injections do not reduce gonadal testosterone production. This may be advantageous for feral cat trap– neuter–return (TNR) programs because dominant, high libido, sterilized males will mate with queens and induce pseudopregnancy, possibly limiting opportunities for queens to actually become pregnant by non-sterilized toms. By contrast, this behavior would be unacceptable for pet cats and unowned, free-roaming/feral cats close to human habitation.

Key Points

• Intratesticular and intraepididymal injections are inexpensive compared with surgical sterilization. Once optimized for success with additional research, intratesticular injection may be suitable for large-scale sterilization programs.

• Although both methods may require sedation or general anesthesia, the technique of intratesticular injection is not technically challenging; intraepididymal injection is more difficult owing to the relatively smaller size of the tail of the epididymis compared with the testis.

• Normal fertility may persist in treated males for 1–2 months after either intratesticular or intraepididymal treatment because of sperm present within the epididymis and vas deferens.

• Intraepididymal injections do not affect testosterone production, which is undesirable for pet cats and feral cats close to human habitation. Although intratesticular injections do not entirely eliminate testosterone production, a reduction in male reproductive behaviors has been anecdotally reported, which warrants additional research.