The Veterinary Journal xxx (2013) xxx–xxx

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The Veterinary Journal

journal homepage: www.elsevier.com/locate/tvjl

Intratesticular injection of a zinc-based solution for contraception of domestic

cats: A randomized clinical trial of efficacy and safety
E.C.S. Oliveira a,⇑, A.K.F. Fagundes b, C.C.S. Melo c, L.T.B. Nery b, R.G. Rêvoredo a, T.F.G. Andrade a,
K. Oliveira-Esquerre d, J.P. Kastelic e, V.A. Silva Jr. b
a
Department of Veterinary Medicine, Federal Rural University of Pernambuco – UFRPE, Rua Dom Manoel de Medeiros s/n, Dois Irmãos, CEP 52171-900 Recife, PE, Brazil
b
Department of Animal Morphology and Physiology, UFRPE, Recife, PE, Brazil
c
Northeastern Biotechnology Network – RENORBIO, Recife, PE, Brazil
d
Department of Chemical Engineering, Polytechnic School, UFBA, Salvador, BA, Brazil
e
Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada

a r t i c l e i n f o a b s t r a c t

Article history: It has been reported that a commercial zinc gluconate preparation disrupts spermatogenesis and appar-
Accepted 17 January 2013 ently causes permanent sterilization in male dogs, but there is little information regarding similar
Available online xxxx approaches in the male cat. The objective of this study was to evaluate zinc gluconate as a permanent
contraceptive for domestic male cats. Sixteen sexually mature mixed breed cats were allocated at ran-
Keywords: dom, by replicate, into two groups and given a single injection into each testis of either isotonic saline
Cat or zinc gluconate, respectively. Clinical and reproductive parameters were assessed immediately before
Contraception
injection and after 60 and 120 days.
Semen
Sterilization
On day 120 the testis size of treated cats was decreased (P < 0.05). Azoospermia occurred in 8/11 (73%)
Testis cats, and penile spines were decreased in 6/11 (55%) and absent in 4/11 (36%) cats, and there were sub-
Zinc gluconate stantial reductions in male behavior. However, plasma testosterone concentrations (single samples col-
lected at each assessment) were not significantly different between treated and control cats at any
time point. Although additional studies are warranted, intratesticular injection of zinc gluconate might
have potential as a permanent contraceptive for cats.
Ó 2013 Elsevier Ltd. All rights reserved.

Introduction Cats

There are very few reports regarding chemical sterilization of
male cats although intra-epididymal injection of 4.5% chlorhexidine
digluconate caused azoospermia or severe oligospermia (Pineda
and Dooley, 1984); intratesticular injection of calcium chloride
has been shown to have potential for permanent contraception
(Jana and Samanta, 2011), and preliminary studies of an intratestic-
ular injection of zinc gluconate have been very promising (E.C.S.
Oliveira et al., unpublished data). The objective of the present study
was to investigate the effects of a single injection of intratesticular
zinc gluconate as a permanent contraceptive method for cats.
Materials and methods
Test compound
The test compound was a proprietary zinc gluconate solution for intratesticular
injection (Testoblock, BioRelease Technologies LCC). The product contained 0.2 M zinc
gluconate (13.1 mg zinc/mL), which was pH-neutralized in a physiological vehicle.
Sixteen intact shorthair male cats from two private colonies, approximately 9–
12 months old and 2.0–4.5 kg bodyweight, were used. The number of animals was
based on differences between groups in a previous study in dogs (Wang, 2002), as
well as logistics and available funding. The study was approved by the Animal
Experimentation Ethics Committee of Federal Rural University of Pernambuco (Pro-
tocol 008/2010). All cat owners were given detailed information and signed a re-
search consent form.
The work was undertaken between January and November in Recife, PE, Brazil
(8°040 South; 33°550 West). At this location, there is approximately 12 h of light per
day and a mean temperature of 26.4 °C. There were no clinical abnormalities in any
of the cats based on a physical examination, hematology and clinical chemistry. All
cats produced ejaculates with P80% progressively motile sperm; sperm counts
were within normal limits, and all males displayed sexual interest in an estrous
queen.
Cats were randomly allocated (in replicates of three; all assignments were made
by the primary investigator) into two groups, controls (n = 5) and treated (n = 11),
and each cat was given a single injection into each testis of either isotonic saline
or zinc gluconate, respectively. Group assignments were known to investigators
conducting animal-based procedures, but were not known to cat owners or to per-
sons assessing hematology, serum chemistry, or plasma testosterone concentra-
tions. All cats remained at their owners’ residence throughout the study and all
animal-based procedures were done there.

⇑ Corresponding author. Tel.: +55 81 3320 6432.

E-mail address: ecso21@uol.com.br (E.C.S. Oliveira).

1090-0233/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tvjl.2013.01.011

Please cite this article in press as: Oliveira, E.C.S., et al. Intratesticular injection of a zinc-based solution for contraception of domestic cats: A randomized
clinical trial of efficacy and safety. The Veterinary Journal (2013), http://dx.doi.org/10.1016/j.tvjl.2013.01.011
2 E.C.S. Oliveira et al. / The Veterinary Journal xxx (2013) xxx–xxx
Anesthesia
Cats were anesthetized for intratesticular injections and semen collection. Ac-
cess to food and water was withheld for 12 h and 7 h, respectively, before anesthe-
sia, which was induced with an injection of xylazine (0.5 mg/kg, IM; Rompun 2%,
Bayer) followed by ketamine (5.0 mg/kg, IM; Ketalar, Pfizer). If needed, additional
anesthetic drugs (approximately 25% of the original dose of each product) were
subsequently given.
Intratesticular injection
Anesthetized cats were restrained in a supine position and the scrotum was
cleaned (10% povidone iodine, SLF). The width of each testis was measured with cal-
ipers, and a single injection of isotonic saline or zinc gluconate was given into each
testis (1 mL of solution for every 27 mm of testis width; adapted from Wang, 2002).
The volume injected per testis ranged from 0.44 to 0.51 mL. Injections were per-
formed using a 0.5 mL U100 insulin syringe with a 28 G, 12 mm needle (a separate
needle was used for each testis). The injection was given in the cranial area of the
testis, lateral to the caput epididymis (near the ductuli efferentes), with the needle
parallel to the long axis of the testis.
Clinical and laboratory assessments
General attitude, bodyweight, ability to walk, scrotal changes, and rectal tem-
perature were evaluated on days 0 (immediately before treatment), 60, and 120.
Concurrently, testis width was measured (with calipers), the penis was examined
for the presence of testosterone-dependent spines, and blood samples were col-
lected (saphenous venipuncture) for complete hematology, hepatic and renal func-
tion (alanine and aspartine aminotransferase, urea and creatinine), and plasma
testosterone concentrations. The latter were measured using radioimmunoassay
kits (Coat-A-Count, Diagnostic Products Corporation), previously validated for
domestic cats (Levy et al., 2004).
Semen collection and evaluation
On days 0 (pre-treatment), 60, and 120, all cats were anesthetized (with xyla-
zine/ketamine, as previously described) and semen was collected with an electro-
ejaculator (Eletrojet, Eletrovet) connected to a 12 V source. A rectal probe
(10.0  0.8 cm) with two stainless steel electrodes was used and the procedure
was performed as described by Wildt et al. (1983). Semen was collected into plastic
tubes and immediately evaluated (by the same person). Total sperm count was as-
sessed on a scale of 0–3 (0, azoospermia; 3, apparently normal sperm count) and
progressively motile sperm (increments of 10%) were evaluated subjectively by
light microscopy (400). Following ejaculation, urine was collected (by cystocente-
sis) and evaluated for sperm (retroejaculation).
Behavior
Libido was assessed on days 0 (pre-treatment), 30, 60 and 120. Each male cat
was individually allowed access (for 30 min) to a female cat in spontaneous estrus
(confirmed by behavior) from the same private colony. It was subjectively assessed
(by a trained observer from the research team, who was responsible for all of these)
on a scale of 0–3, where 0 represented no interest, 3 represented rapid interest and
mounting, and 1 and 2 were gradations between these extremes. In addition, own-
ers completed a standardized questionnaire (regarding aggressive behavior, mount-
ing, roaming, urine marking, etc.) and were interviewed by the research team
throughout the experiment. Note that for assessment of sexual interest, mounting
behavior was considered the cardinal sign.
Statistical analyses
A mixed models ANOVA (for repeated measures) was used to determine the ef-
fects of group, time, and their interaction, on bodyweight, testis width and plasma
testosterone concentrations. For testis width, the average of the left and right mea-
surements was used in the statistical analysis. Testosterone concentrations were
highly variable and not normally distributed; therefore, a log 10 transformation
was done before analysis. Furthermore, for testosterone concentrations, due to
some missing values (lost samples), statistical analysis was restricted to days 60
and 120. For main effects and interactions, P < 0.05 was considered significant. A
Bonferroni test was used to locate significant differences. All analyses were done
with Statistical Analysis System (SAS) software (SAS Institute).
Results
Following treatment, rectal temperature remained normal.
Owners reported no biting or licking of the scrotum or testes, but
they did report transient testicular swelling (1 day after injection)
Please cite this article in press as: Oliveira, E.C.S., et al. Intratesticular injection of a zinc-based solution for contraception of domestic cats: A randomized
clinical trial of efficacy and safety. The Veterinary Journal (2013), http://dx.doi.org/10.1016/j.tvjl.2013.01.011
in all cats receiving zinc gluconate but in none of the cats receiving
saline. However, there was no apparent scrotal or testicular pain or
tenderness, except for one treated cat, with apparent discomfort
(reduced activity and feed intake) 2–3 h after injection. This cat
was given sodium dipyrone (25 mg/kg orally three times per day,
for 2 days) and the animal recovered uneventfully.
In both groups, hematology and clinical chemistry were consis-
tently within normal reference ranges for domestic cats (ÓBrien
et al., 1998). For bodyweight, neither main effect nor their interac-
tion was significant (data not shown).
Mean ± standard error (SEM) testis width on days 0, 60, and 120
was 10.5 ± 0.26, 10.5 ± 0.92, and 10.5 ± 1.03 mm in control cats,
and 12.4 ± 0.18, 8.7 ± 0.62, and 7.9 ± 0.65 mm in treated cats
(group x time interaction, P < 0.005). Testis width of treated cats
decreased over days 0–120 (P < 0.05). Furthermore, testis width
was smaller (P < 0.05) in treated cats compared to control cats on
days 60 and 120. However, testis width did not change over time
in control cats (P > 0.05; Fig. 1).
On day 120, testosterone-dependent penile spines were promi-
nent in control cats, but were absent in 4/11 (36%) and decreased
in 6/11 (55%) treated cats (Fig. 2A). One cat still had well-devel-
oped penile spines (Fig. 2B).
On day 60, 10/11 treated cats (91%) were azoospermic and the
remaining cat had reductions in both sperm count and sperm
motility. On day 120, eight cats (73%) were azoospermic, one with
well developed penile-spines had necrospermia, and two had
sperm in their ejaculates (scale 1/3 or 2/3), with reduced motility.
Urine collected by cystocentesis after eletroejaculation did not
contain sperm on day 60, although a few non-motile sperm were
present in 2/10 cats at day 120. In contrast, cats in the control
group consistently had excellent semen quality (normal sperm
counts and progressive motility) throughout the study. Further-
more, 3/5 cats in the control group (60%) had evidence of retro-
grade sperm flow. Regarding plasma testosterone concentration,
there were no significant differences for group or time, nor was
there a significant group by time interaction. On days 60 and
120, owners reported that treated cats had reduced aggression,
roaming, mounting and urine marking (spraying), whereas these
activities persisted in control cats.
18
15
Average testis width (mm)
12
9
6
Control
3
Treatment
0
0 30 60 90 120
Time (days)
Fig. 1. Testis width (mean ± SEM) of cats following intratesticular injection of
either isotonic saline (control, n = 5) or a zinc gluconate (treated n = 11) according
to the period of evaluation. Treated cats had a reduction (P < 0.05) in testis width
from day 60 to the end of the study.
 E.C.S. Oliveira et al. / The Veterinary Journal xxx (2013) xxx–xxx
Fig. 2. Penile spines were absent in a cat after the intratesticular injection of zinc gluconate (A) but were well-developed in a control cat injected with intratesticular isotonic
saline (B) at 60 days.
Discussion
To our knowledge, this is the first report of azoospermia in cats
treated with a single injection of zinc gluconate into each testis. At
120 days post-injection, ejaculates of eight cats (73%) were azoo-
spermic, one had necrospermia, and the remaining two ejaculates
had reductions in both sperm count and sperm motility. Further-
more, there were no indications of any adverse effects in treated
cats, consistent with a lack of adverse effects in previous studies
(Tepsumethanon et al., 2005; Soto et al., 2009; Oliveira et al.,
2012). Therefore, although a long-term study with a large number
of cats is necessary to positively establish safety and the long-term
irreversibility of these effects and to document changes in behav-
ior, this is evidence that intratesticular zinc gluconate has potential
as a permanent contraceptive for cats. Clearly, however, care will
be needed to identify cats that do not become azoospermic.
Transient testicular swelling was observed in zinc gluconate-
treated cats 1 day after treatment, presumably due to mild inflam-
mation. However, no injection site reactions (e.g. scrotal dermatitis
or ulceration) were noted during the 120 day observation period,
consistent with zinc gluconate being pH neutral and containing
zinc, which is present in male reproductive fluids and tissues (Fa-
him et al., 1993). Similarly, in previous studies of chemical castra-
tion of male dogs with intratesticular zinc gluconate, no injection-
site reactions were reported (Soto et al., 2009; Oliveira et al., 2012).
Intratesticular injection of zinc gluconate clearly suppressed
spermatogenesis in 91% (10/11) of cats at day 60 (the remaining
cat had reductions in sperm count and sperm motility). At day
120, azoospermia was present in 73% (8/11) of the treated cats.
Furthermore, at day 120, the two cats that were azoospermic at
day 60 had necrospermia or poor semen quality and semen quality
remained poor in the remaining cat (it also had poor semen quality
at day 60). Since the duration of spermatogenesis in cats is
46.8 days (França and Godinho, 2003), the effects of zinc gluconate
persisted for more than two spermatogenic cycles (120 days) in
most cats. Therefore, zinc-gluconate had a specific and prolonged
action on the seminiferous epithelium. Similarly, in sexually ma-
ture male dogs, intratesticular injections of zinc gluconate im-
paired spermatogenesis (changes in the seminiferous tubules
seemed irreversible; Oliveira et al., 2007).
On day 60, 73% (8/11) of cats injected with zinc gluconate had
substantial testicular atrophy (approximately 30% smaller than at
day 0). Furthermore, testis width was significantly lower in treated
cats on days 60 and 120 compared to controls. In contrast, intrates-
Please cite this article in press as: Oliveira, E.C.S., et al. Intratesticular injection of a zinc-based solution for contraception of domestic cats: A randomized
clinical trial of efficacy and safety. The Veterinary Journal (2013), http://dx.doi.org/10.1016/j.tvjl.2013.01.011
3
ticular zinc gluconate did not significantly affect testis width in
dogs compared to those given intratesticular isotonic saline (Oli-
veira et al., 2012). It is possible that zinc gluconate causes more se-
vere damage to the seminiferous tubules in cats than in dogs. The
three cats that were injected with zinc gluconate and had only a
modest decrease in testis width still had some sperm in their ejac-
ulate, consistent with incomplete suppression of spermatogenesis.
Intratesticular injection of zinc gluconate did not significantly
affect libido in dogs (Oliveira et al., 2012). Although many dog
owners require contraception but want to preserve behavior (e.g.
guarding behavior), for other species including cats, an ideal chem-
ical contraceptive would one that would render the animal infertile
and suppress androgenesis and libido, to mitigate secondary sex
characteristics (Jana and Samanta, 2011). In that regard, since zinc
gluconate did not alter sexual behavior in dogs, the dose used in
the present study was approximately twice that recommended
for intratesticular injection in dogs (Levy et al., 2008; Oliveira
et al., 2007). Wang (2002) recommended 0.3 mL of zinc gluconate
solution for a testis 12–14 mm wide, whereas in the present study,
0.44–0.51 mL was injected into each testis. Testicular atrophy and
apparent reductions in libido, mounting, aggression and urine
marking (spraying) in the present study were attributed to the
higher dose used. Furthermore, it was noteworthy that only one
treated cat had apparent mild discomfort following the procedure,
but responded well to symptomatic treatment.
Plasma testosterone concentrations were not significantly dif-
ferent between groups and remained within normal reference
ranges for domestic cats (Kirkpatrick, 1985). However, in both
groups, there was a general trend for testosterone concentrations
to decrease between days 60 and 120, perhaps due to an effect of
breeding season (Tsutsui et al., 2009). Only part of the experiment
reported here was performed during the breeding season. Regard-
less, it was noteworthy that at day 120, testosterone-dependent
penile spines were prominent in control cats, but absent in four
treated cats (36%) and decreased in a further six treated cats (55%).
Time savings for chemical castration versus orchidectomy in
male cats could be different to those in dogs, because surgical cas-
tration is a simpler and faster procedure in cats than in dogs.
Regardless, intratesticular injection of zinc gluconate is an inher-
ently less invasive contraceptive method for cats. Although no
behavioral changes were reported in dogs, in the present study
there were apparent effects on cat behavior (e.g. reduced urine
marking, vocalizing, fighting with other cats), which are important
benefits of this procedure.
4 E.C.S. Oliveira et al. / The Veterinary Journal xxx (2013) xxx–xxx
Conclusions
A single injection of zinc gluconate into each testis substantially
suppressed spermatogenesis in cats from 60 to 120 days after
treatment. It was effective in the majority of cats; was economical
and easy to perform, and did not require orchidectomy. Although
sedation is recommended and the current administration protocol
requires attention to detail, this method appeared to offer savings
in cost, time and facility requirements, relative to conventional
surgical castration.
Conflict of interest statement
None of the authors has any financial or personal relationships
that could inappropriately influence or bias the content of the
paper.
Acknowledgements
This study was supported by the Scientific and Technological
Support Foundation of State of Pernambuco (FACEPE). We thank
BioRelease Technologies LLC for providing zinc gluconate; B.E.T
Laboratories (USA and Brazil) for testosterone assays, and Dr. Telga
Almeida and Dr. Simone Vaz from the Clinical Analysis Laboratory
of UFRPE, and the staff from the Laboratory of Andrology (Andro-
lab) of UFRPE for their assistance.
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clinical trial of efficacy and safety. The Veterinary Journal (2013), http://dx.doi.org/10.1016/j.tvjl.2013.01.011
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