Chemical castration in cattle with intratesticular injection of sodium
chloride: Effects on stress and inﬂammatory markers
Fernando C. Oliveira
, Carlos E.R. Ferreira
, Cristina S. Haas
Leonardo G. Oliveira
, Rafael G. Mondadori
, Augusto Schneider
Monique T. Rovani
, Paulo B.D. Gonçalves
, Arnaldo D. Vieira
Bernardo G. Gasperin
, Thomaz Lucia Jr.
ReproPel, Universidade Federal de Pelotas, 96010-900, Pelotas, RS, Brazil
Faculdade de Veterin
aria, Universidade Federal de Pelotas, 96010-900, Pelotas, RS, Brazil
Instituto de Biologia, Universidade Federal de Pelotas, 96010-900, Pelotas, RS, Brazil
Faculdade de Nutriç~
ao, Universidade Federal de Pelotas, 96010-900, Pelotas, RS, Brazil
Biorep, Centro de Ci^
encias Rurais, Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brazil
Received 8 September 2016
Received in revised form
30 November 2016
Accepted 1 December 2016
Available online 2 December 2016
Intratesticular injection (ITI) of sodium chloride (NaCl) is efﬁcient for chemical castration of young calves,
but its effects on calves welfare are unknown. Two experiments were conducted to evaluate the effects of
ITI of 20% NaCl on stress and inﬂammatory markers in calves less than 20 days old and to assess the
efﬁciency of ITI of 30% NaCl in 5 months old calves. In Experiment 1, control calves were only restrained
and compared to calves submitted to castration through surgery (SC) and ITI with 20% NaCl (n ¼9/
group). No differences were observed for the eye corner temperature measured by thermography from
60 s before to 60 s after the procedures (P >0.05). In the SC group, acute serum cortisol levels increased
at 30 and 60 min after the procedure, but increased levels in the ITI group occurred only at 30 min
(P <0.05). Chronic discomfort markers were measured at 0, 24, 48, 72 and 96 h after the procedures (D0,
D1, D2, D3 and D4, respectively). The serum levels of the paraoxonase 1 (PON1) enzyme and cortisol did
not differ among groups (P >0.05). Scrotal temperature was higher at D1 in the SC group than for the
other groups, but lowest at D4 compared to the control (both P <0.05). In Experiment 2, histological
sections of testes were compared after ITI with either 30% NaCl or 30% calcium chloride (CaCl
), to intact
calves (control). After 60 days, intact seminiferous tubules and mediastinum were observed after ITI with
30% NaCl, whereas coagulative necrosis, inﬂammatory inﬁltration and calciﬁcation occurred after ITI
with 30% CaCl
.Efﬁcient chemical castration through ITI of 20% NaCl in young calves was followed by
slight stress and inﬂammatory responses compared to surgical castration. However, ITI of 30% NaCl was
ineffective for chemical castration of 5 months old calves.
©2016 Elsevier Inc. All rights reserved.
Castration of calves is a widespread practice among beef
breeders to prevent aggressive behavior, sexual activity and bull
breeding , with additional beneﬁts for carcass ﬁnishing, since
decreased circulating testosterone levels prevent the pH increase in
the meat [2e4]. Castration can be done using physical,
immunological or chemical methods. Physical castration can be
performed through either surgery (orchiectomy) or emasculation,
using Burdizzo clamps, stenotic elastic rings or strips to interrupt
the blood supply to the testicles . Immunological castration is
based on reducing testosterone levels through immuno-
contraception, inducing the production of antibodies against
GnRH . Chemical castration can be accomplished by intra-
testicular injection (ITI) of compounds that induce destruction of
testicular cells through caustic or osmotic processes, such as lactic
acid , CaCl
 and NaCl .
Surgical castration is widely used in cattle, even though it is
often performed under inadequate conditions, resulting in a
*Corresponding author. ReproPel, Faculdade de Veterin
aria, Universidade Federal
de Pelotas, 96010-900, Pelotas, RS, Brazil.
E-mail address: email@example.com (T. Lucia).
Contents lists available at ScienceDirect
journal homepage: www.theriojournal.com
0093-691X/©2016 Elsevier Inc. All rights reserved.
Theriogenology 90 (2017) 114e119
signiﬁcant degree of pain and bacterial contamination [10,11]. Be-
sides the pain, calves show various behavioral changes during and
after the surgical procedure such as: agitation, leg movements, tail
swing, disoriented walk, prostration, reduced interest in their
mothers, and decreased feed intake . Such signs of discomfort
can be assessed by markers capable of identifying processes of
stress, pain and inﬂammation, such as the serum levels of cortisol
, the eye corner temperature  and acute phase proteins such
as the paraoxonase 1 (PON1) enzyme .
Currently, consumers are increasingly questioning management
practices that may be associated with pain and discomfort on the
animals that provide the meat they consume [16,17]. Thus, the use
of alternative castration methods that can improve animal welfare
is of interest for the cattle industry. The ITI is a minimally invasive
chemical castration technique that is efﬁcient when using NaCl on
calves that are 30 days old or younger  and CaCl
in 7e8 months
old buffalos . However, the effects of ITI with NaCl on the welfare
of castrated young calves are still unknown and its effectiveness for
chemical castration of older calves has not yet been evaluated. The
objectives of this study were to evaluate the effects of ITI with 20%
NaCl on markers of stress and inﬂammatory responses in calves up
to two weeks old, and to test the efﬁciency of 30% NaCl ITI for
chemical castration of 5-months old calves.
2. Material and methods
All procedures were approved by the Ethics in Animal Experi-
mentation Committee (CEEA-UFPel; process # 2258).
2.1. Experiment 1
2.1.1. Experimental design
This experiment was conducted with 27 beef calves from 4 to 20
days of age, with average 36 kg of body weight and kept with their
mothers in ryegrass pasture with ad libitum access to water. All
calves were submitted to the same manual restraining procedures
(calves were put down on the ground by gently pulling their legs)
and randomly assigned to three groups (n ¼9 each). Calves in the
control group were only restrained, calves in the second group
were castrated through orchiectomy and those in the third group
were submitted to ITI of 20% NaCl.
Surgical castration was performed as described elsewhere .
Antisepsis of the scrotum was performed with 2% iodine-ethanol
solution and 2% chlorhexidine digluconate solution (both from
e do Rio Preto-SP, Brazil). Local anesthesia was
conducted with 5 ml 2% lidocaine. The orchiectomy was conducted
after incision of the scrotum, removal of both testicles and section
of the spermatic cords.
The ITI was conducted by dissolving NaCl (Synth
, Diadema, SP,
Brazil)  and lidocaine  in ultrapure water. The ﬁnal solution
with 20% NaCl and 2% lidocaine was sterilized by 0.22
and kept in sterile vials at 5
C. After scrotum antisepsis, each
testicle was immobilized and the solution was injected with a 21
G1½needle, at its distal end. The injected volume (1.5e4.0 ml)
varied according to the size of each testicle, as long as the gonad
presented ﬁrm consistency. For all calves, the ITI was performed by
the same technician, using the same criteria to deﬁne the injected
2.1.2. Acute discomfort markers
Blood samples were collected from calves of the three groups, at
the following time points: during the procedure; 30 min after; and
60 min after. Samples were collected through puncture of the ju-
gular vein with a 21G needle connected to a vacuum collection
system (BD Vacutainer
), into 10 ml tubes without anticoagulant.
Immediately after collection, samples were centrifuged (1500 g
for 10 min). Serum samples were subsequently stored in liquid
Cortisol levels were quantiﬁed by the electro-
chemiluminescence assay Cortisol II Cobas (Roche Diagnostics,
Mannheim, Germany; REF 06687733), in a commercial laboratory,
with intra and inter-assay coefﬁcient of variability lower than 10%.
The eye corner temperature was determined by thermography
to identify changes in temperature caused by reduced blood ﬂow in
the eye corner due to vasoconstriction of the sympathetic nervous
in response to pain . Thermography was conducted at the
following time points: before (60 s and 30 s); during (0); and
after the procedures (30 s and 60 s). Thermographic images were
obtained with the thermograph FLIR
E25 and analyzed by the
software (FLIR QuickReport™PC software).
2.1.3. Chronic discomfort markers
Blood samples were collected from the time of the procedures
up to four days after, as described above. Chronic serum cortisol
levels were determined as described above for acute cortisol.
Scrotal thermography was conducted as described above for eye
The quantiﬁcation of serum PON1 was performed as described
elsewhere [15,19]. Brieﬂy, samples were previously diluted in a 1:3
ratio and mixed with a working solution (3.3
L of the diluted
sample in 500
L of working solution). The working solution con-
sisted of 20 mM Tris/HCI buffer; 1.0 mM CaCl
; and 4.0 mM phe-
nylacetate. The reading was performed in a Cirrus 80ST
spectrophotometer, at 270 nm wavelength for 60 s. Enzyme activity
was determined by the following formula:
Absorbance x 115 3.
The activity of PON1 was expressed in U/L.
Scrotal temperature and serum levels of PON1 were determined
at the time of the procedures (D0); after 24 h (D1); 48 h (D2); 72 h
(D3); and 96 h (D4). Chronic serum cortisol levels were determined
at D0, D2 and D4.
2.2. Experiment 2
Nine calves aging between 120 and 150 days and average live
weight of 125 kg (from 119 to 137 kg) were randomly assigned to
three groups (n ¼3 each): in two groups, calves were submitted to
ITI with either 30% NaCl or 30% CaCl
; whereas calves in the control
group were only restrained. The restraining procedures for calves in
all three groups were the same described for Experiment 1. Both
hypertonic solutions were prepared using 2% lidocaine (20 mg/ml)
in saline, ﬁltered in 0.22
mﬁlter and stored in sterile vials at 5
until use. After scrotum antisepsis, each testicle was immobilized
and the solution was injected with a 21 G1½needle, at its distal
end. The injected volume varied from a minimum of 4.0 ml to a
maximum of 8.0 ml, which was deﬁned considering the size of each
testicle, as long as the gonad presented ﬁrm consistency. As
occurred in Experiment 1, that procedure was always conducted by
the same technician. The group submitted to ITI with CaCl
considered as a positive control, since such chemical is known to be
effective on promoting sterility in older animals .
Sixty days after the procedures, calves from all three groups
were surgically castrated, as described for Experiment 1. Immedi-
ately after castration, samples of testicular parenchyma were
placed in 10% formalin buffered solution. Thereafter, fragments
were removed from that solution, dehydrated in alcohol, cleared in
xylene and embedded in parafﬁn. Sections of 5
m were obtained
using an automatic microtome (RM2245, Leica Biosystems, San
Diego, CA, USA), stained with hematoxylin and eosin (HE) and
blindly evaluated by an experienced veterinary pathologist. Testicle
samples from calves of the control group were considered as
F.C. Oliveira et al. / Theriogenology 90 (2017) 114e119 115
2.3. Statistical analysis
The effects of the castration methods on serum levels of cortisol
(both acute and chronic) and PON1, and on eye corner and scrotal
temperatures were evaluated by analysis of variance with repeated
measures, using the SAS
Mixed Procedure. Data without normal
distribution were transformed, but the results were presented in
the original scales.
In Experiment 1, the average eye corner temperature varied
between 38 and 39
C. There were no differences in eye corner
temperature among castration methods (P >0.05), at any period
Scrotal temperature declined in all treatments (P <0.001) after
the procedures (Fig. 1B). A treatment per moment interaction
indicated that scrotal temperature was higher at D1 for surgically
castrated calves than for calves at other groups, but lowest at D4
only for calves in the control group (P ¼0.05).
An interaction between treatment and moment inﬂuenced the
acute cortisol concentration, indicating that surgically castrated
calves had higher serum cortisol levels 30 min and 60 min after
castration (P <0.05), compared to the levels observed during sur-
gery (Fig. 2A). However, serum cortisol levels of calves submitted to
20% NaCl ITI were higher after 30 min (P <0.05), but declined after
60 min to levels similar to those observed at 0 min, during the ITI
(P >0.05). There was no effect (P >0.05), of the castration method
on the serum levels of chronic cortisol (Fig. 2B) and PON1 (Fig. 2C).
The histological sections of testes samples from calves of
Experiment 2 are shown in Fig. 3. Out of the six testicles submitted
to ITI of 30% NaCl, all presented intact seminiferous tubules and ﬁve
(83.3%) presented intact mediastinum, whereas only one out of six
(16.7%) samples showed both such features after ITI of 30% CaCl
(Fig. 4). Coagulative necrosis and inﬁltration of inﬂammatory cells
were observed in testicles submitted to both chemical castration
methods, but no testicle in the control group presented such le-
sions. Calciﬁcation was observed only in testicles of calves sub-
mitted to CaCl
ITI (Fig. 4).
Although it is known that 20% NaCl ITI impairs testosterone
secretion and testicular development in beef calves younger than
20 days of age , the present study is the ﬁrst one to report that
such chemical castration method is also beneﬁcial for animal
Fig. 1. Means ±SEM for eye corner (A) and scrotal (B) temperature (C) in 4e20 days old male calves submitted to surgical castration, chemical castration through intratesticular
injection of 20% NaCl or only restrained (control), at distinct time points relative to the procedures - Experiment 1: (n ¼9 per group). *At D1, surgical castration differs from control
and chemical castration; at D4 surgical and chemical castration differ from control (P <0.0001).
F.C. Oliveira et al. / Theriogenology 90 (2017) 114e119116
welfare. Pain and stress manifestations are expected to occur after
castration through physical methods [10,20], after either surgery
 or emasculation using Burdizzo clamps or rubber rings .
Increased cortisol is an indicator of the occurrence of pain and
stress in such situations , although cortisol levels may eventually
be similar after castration through different methods .In
Experiment 1, no relevant pain manifestation was observed in
chemically castrated calves, which showed only a mild reaction at
the time of needle insertion in the scrotal skin, similarly to that
described elsewhere for water buffalos . Calves submitted to 20%
Fig. 2. Means ±SEM for serum levels of acute (A) and chronic (B) cortisol and paraoxonase activity (C) in 4e20 days old male calves submitted to surgical castration, chemical
castration through intratesticular injection of 20% NaCl or only restrained (control), at distinct time points relative to the procedures - Experiment 1: paraoxonase and acute cortisol
(n ¼9 per group); chronic cortisol (n ¼6 per group)
Differences among time points for chemical castration (P <0.05).
Differences among time points for surgical castration
F.C. Oliveira et al. / Theriogenology 90 (2017) 114e119 117
NaCl ITI had an acute response after the procedure, restoring their
serum cortisol levels up to physiological levels earlier than surgi-
cally castrated calves. Chemically castrated calves also presented a
more favorable response to chronic stress compared to surgically
castrated calves, since their scrotal temperature was lower 24 h
after 20% NaCl ITI and similar throughout the remaining evaluated
periods. However, calves castrated through both methods pre-
sented higher scrotal temperatures 96 h after the procedure than
calves in the control group. Such temperature increase could be
attributed to an increase in the local blood supply due to the release
of inﬂammatory mediators. Furthermore, surgically castrated
calves do not have normal scrotal thermoregulation due to
anatomical changes after orchiectomy. Similarly, after intra-
testicular injections, testicles move closer to abdominal wall (per-
sonal observation), potentially affecting thermoregulation. Agents
other than NaCl have also been used for chemical castration in
cattle, such as CaCl
, ethanol  and lactic acid .Both
 and lactic acid  were associated with lower cortisol
release than castration by emasculation, but were also followed by
pain manifestation. Nonetheless, to our knowledge, there are few
studies investigating the impact of chemicals used for ITI on stress
and inﬂammation markers in calves.
The lack of effect of the tested castration methods in the eye
corner temperature in our study also suggests that the acute pain
manifestation after 20% NaCl ITI was mild. Decreased eye corner
temperature related to painful and stressful procedures was
detected by thermography in dogs by Travain et al. , supposedly
initiated by vasoconstriction induced by the sympathetic system
activated by acute pain . Rapid reduction in the eye corner
temperature was also reported in bulls 2 min after surgical
castration, but such temperature was raised after 15 min concom-
itantly with an increase on circulating cortisol levels, indicating an
activation of the parasympathetic nervous system, leading to an
increase on peripheral blood ﬂow .
None of the tested castration methods induced inﬂammatory
reaction in young calves capable to alter systemic markers, as
indicated by the similar serum level of PON1 across groups.
Although both PON1  and cortisol  are related to situations
of inﬂammation and discomfort, there are currently no markers
considered speciﬁc and reliable for stress and pain . Also, the
secretion of both markers may be affected by the management
practices conducted prior to castration, which may by themselves
constitute sources of anxiety for the animals . For this reason,
the experimental design applied in our study tried to control such
factor, by submitting the non-castrated control calves to the same
restraining procedures applied to the calves in the other groups.
When applied to calves up to 20 d-old, ITI of 20% NaCl resulted in
extensive testicular ﬁbrosis, replacement of parenchymal
Fig. 3. Histological sections of the testicular parenchyma (hematoxylin-eosin staining) for 5-months old male calves submitted to chemical castration through intratesticular
injection of 30% NaCl (B) or 30% CaCl
(C) and only restrained (control) (A) - Experiment 2. *Typical prepubertal seminiferous tubes; #Typical rete testis; I: inﬂammatory cells; CN:
extensive coagulative necrosis; Arrows: calciﬁcation areas.
Fig. 4. Histological features in testicular tissues of 5-months old male calves submitted to chemical castration through intratesticular injection of 30% NaCl or 30% CaCl
restrained (control) - Experiment 2 (n ¼6 testicles per group).
F.C. Oliveira et al. / Theriogenology 90 (2017) 114e119118
components by dense connective tissue and coagulative necrosis of
Leydig cells and seminiferous tubules. Nevertheless, in Experiment
2, most testicles submitted to 30% NaCl ITI presented intact areas of
seminiferous tubules and mediastinum 60 days after the procedure,
indicating that such method is not effective for chemical castration
of older calves, even with a higher concentration of NaCl (increased
by 10% points compared to Experiment 1). Testicles from calves in
the control group did not present the histological characteristics
observed in testicles submitted to ITI of CaCl
, such as coagulative
necrosis, inﬂammatory cell inﬁltration and calciﬁcation, as also
observed in testicles of chemically castrated buffaloes . Chemical
castration with CaCl
ITI was also effective in domestic cats,
although with lower concentration of CaCl
and with distinct his-
tological features observed in the testicles . Our results agree
with the ﬁndings of Andrade Neto et al. , which reported that
testosterone secretion at 12 months of age was fully impaired after
GnRH injection when ITI with 20% NaCl was conducted in calves at
most 20 days-old, but were similar to basal levels from intact males
when ITI was performed in calves older than 20 days of age. Thus,
compared to ITI with 30% CaCl
, ITI with NaCl is less effective for
chemical castration of 5-months old calves, even when using con-
centrations greater than 20%, since it appears unable to induce
damages in the testicular parenchyma extensive enough to warrant
permanent sterilization after 20 days of age.
In conclusion, efﬁcient chemical castration through ITI of 20%
NaCl in young calves was followed by slight stress and inﬂamma-
tory responses compared to surgical castration. Chemical castration
using ITI of NaCl with concentration increased to 30% was inefﬁ-
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