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An evaluation of the outcome of bull castration
by intra-testicular injection of ethanol and calcium chloride
I. CANPOLAT1*, S. GUR2, C. GUNAY1, S. BULUT1, H. EROKSUZ3
1
2
3
Department of Surgery, Veterinary Faculty, Firat University, Elazig- Turkey
Department of Reproduction and Artificial Insemination, Veterinary Faculty, Firat University, Elazig- Turkey
Department of Pathology, Veterinary Faculty, Firat University, Elazig- Turkey
*Corresponding author : Firat University, Veterinary Faculty, Department of Surgery, 23119-Elazig-TURKEY
Fax : +90 4222388173 - Tel : +90 42223700-4079 - E-mail : [email protected]
SUMMARY
RESUME
In this study, the effectiveness of intra-testicular ethanol and calcium
chloride administrations was compared for chemical-castration in 12 mixbred young bulls (12-15 months old and 250-400 kg). Ten ml of absolute
ethanol or 10 ml of a 30 % calcium chloride solution were administered intra-testicularly in each testicle which were removed with the open surgical
technique after 60 days for histopathologic evaluation. Testicular swelling
was evident in both group bulls following injection and reached peak within
48 hours. While testicular volume decreased significantly in ethanol group
after 3 weeks, no significant change occurred in calcium chloride group. The
testicles underwent atrophy at the 60th day in ethanol group with no marked
alteration in calcium chloride group. Semen characteristics of only 3 bulls in
ethanol group were very poor. Though only 3 bulls were infertile in ethanol
injected group, other bulls were fertile.
Évaluation de méthodes de castration de taurillons par injection intratesticulaire d’éthanol et de chlorure de calcium.
Dans cette étude, deux méthodes de stérilisation chimique basées respectivement sur l’administration intratesticulaire d’éthanol ou de chlorure de
calcium ont été évaluées séparément sur 2 groupes de 6 taurillons âgés de 12
à 15 mois et pesant de 250 à 400 kg. Dix ml d’éthanol absolu ou 10 ml d’une
solution à 30 % de chlorure de calcium ont été administrés dans chaque testicule. Les testicules ont été prélevés chirurgicalement 60 jours après l’administration pour une évaluation histopathologique. Un gonflement des
testicules a été mis en évidence suite à l’administration des deux produits
avec un effet maximal observé dans les 48 h suivant l’administration. Alors
que le volume testiculaire a été réduit dans le groupe traité à l’éthanol, 3 semaines après l’administration, aucun changement n’a été observé dans le
groupe traité au chlorure de calcium. Les testicules sont devenus atrophiés
60 jours après l’administration d’éthanol alors qu’ils ne présentaient pas
d’altération marquée dans le groupe traité au chlorure de calcium. Les caractéristiques du sperme de seulement 3 taurillons traités à l’éthanol ont été très
mauvaises et associées à une infertilité. Les 3 autres taurillons traités à
l’éthanol étaient fertiles.
The method is readily applicable with no major harms and adverse effects.
If 50 % rate is considered satisfactory it can be used then in large flocks, in
particularly where no chance of operation exists.
Key words : Chemo sterilization, ethanol, calcium chloride, bull.
En conclusion, la stérilisation chimique basée sur l’administration d’éthanol pourrait être applicable dans de grands élevages sans effets néfastes si un
taux de 50 % de réussite est considéré satisfaisant en l’absence d’intervention possible.
Mots-clés : Stérilisation chimique, éthanol, chlorure de
calcium, taurillon
Introduction
Chemo sterilization was experimented in males
monkeys, hamsters, rabbits, rats and dogs by intratesticular injection of some agents such as ferric chloride [19],
danazol [6], BCG [5), zinc tannate [8], glycerol
[14,17,30], glucose, NaCl [15,25] dibromochloropropane
[27], lactic acid [11], zinc arginine [9], sodium fluoride
[29], formaline [1], calcium chloride [18,26], ethanol [3],
and potassium permanganate plus glacial acetic [13]. In
male ruminants, intratesticularly lactic acid [16] tannic
acid and zinc sulphate [10] alpha-hydroxypropionic acid
[4], formalin [21] Castrate-Quin 14 [28] have been used,
but because of many complications following the use of
these chemicals, an effective chemo sterilization method
has yet to be established.
The purpose of the study was to determine the efficacy of
intratesticular ethanol and calcium chloride injections for
chemo sterilization in bulls.
Materials and methods
In this study, 12 mix-breed young bulls (250-400 kg, 1215 months old) were used. They were randomly divided into
two groups, each group containing 6 bulls, and called EG
(ethanol group) and CCG (calcium chloride group). Each
intratesticular injection was performed using a sterile 21
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BULL CASTRATION BY INTRA-TESTICULAR INJECTION OF ETHANOL AND CALCIUM CHLORIDE
gauge needle directed from the caudoventral aspect of each
testis approximately 1 cm from the epididymal tail towards
the dorsocranial aspect of the testis after restrained per
animal. After 2 ml local anaesthetic (Citanest % 2) injections, 10 ml of absolute ethanol or 10 ml of a 30 % calcium
chloride solution per testis were injected intra-testicularly.
ULTRASONOGRAPHIC EXAMINATIONS :
Trans-scrotal testicular ultrasonography was performed
with 7.5-5 MHz linear probe (Pie medical-scanvet 200) prior
to the intratesticular injection and once a day for week
following intratesticular injection and then one week intervals up to the end of the study. During this examination, the
ultrasonographic appearance of a testis was evaluated and its
height (h), width (W) and length (l) was measured. The
volume of the testis was estimated using :
Volume = (Ï€/6) X (w X h X l) equation.
SEMEN COLLECTION AND EVALUATION :
Semen samples were collected using an artificial vagina
prior to, and 15, 30 and 60 days after intratesticular drug
injection. Semen volume was determined by direct reading
the graduations of collection tubes [from 0.5 to 15 ml]. For
determination of mass activity, non-cover slipped drop of
fresh non-diluted semen was placed on a warm slide (37 ºC)
under light microscope with heater stage at 100 x magnification. The condenser diaphragm of microscope was
lowered in order to increase the contrast. The following
descriptors were used for mass activity : [5] rapid dark
swirls ; [4] slower dark swirls and eddies ; [3] little slower
swirls ; [2] no swirls, but prominent individual cell motion ;
[1] little individual cell motion ; and [0] no individual cell
motion.
Sperm concentration was determined with a haemocytometer. Semen samples were decimally diluted with
isotonic sodium citrate solution at 37 ˚C [3 %, w/v
dissolved in distilled water] at the rate of 1:10 for the determination of sperm motility. A slide was placed on light
microscope with heater stage and allowed to warm up to 37
ºC, and then a small droplet of diluted semen was placed on
the slide and the percentage of motile sperm was evaluated
visually at a magnification of 400 x. Motility estimations
were performed from 5 different fields in each sample. The
mean of the five estimations was used as the final motility
score. The abnormal sperm rate was determined from slides
prepared with an Indian ink. A total of 300 sperm cells were
counted on each slide under light microscope at 400 x
magnification.
BLOOD COLLECTION
MEASUREMENT :
AND
TESTOSTERONE
Blood samples were collected prior to, and 15, 30 and 60
days after intratesticular drug injection. Then all sera were
removed by centrifuging the blood samples at 5000 rpm for
5 min, and stored at -20 oC until assayed.
Revue Méd. Vét., 2006, 157, 8-9, 420-425
421
The serum testosterone level was determined by CoatedTube Radioimmunoassay method using Active® Testosterone RIA DSL – 4 000 kit (Diagnostic System Laboratories Inc. Texas, USA) in gamma counter (LKB-Wallac
Multigamma) according to the report of the kit manufacturer. Briefly ; one vial of standard, labelled A, containing
0 ng/ml of testosterone was reconstituted with 1 ml deionised water. Other five vials of standards, labelled B-F,
containing approximately 0.1, 0.5, 2.5, 10.0 and 25.0 ng/
ml of testosterone were also reconstituted with 0,5 ml
deionised water. Similarly two vials of controls, Levels I
and II, containing low (0.3 ng/ml) and high (5.4 ng/ml)
concentrations of testosterone were reconstituted with
0,5 ml deionised water. Then the sera were resolved in
room temperature (~250 C), and liquid reagents and sera
were mixed thoroughly by gentle inversion before use.
Two plain uncoated tubes were labelled for Total Counts.
All of the anti-testosterone-coated tubes were labelled and
arranged in duplicate. Fifty μl of the standards, controls
and sera was added to the bottom of appropriate tubes.
Immediately 500 μl of testosterone [I125] reagent was
added to the all tubes which were placed the test tube rack.
The contents of tubes were mixed by shaking the test tube
rack gently by hand. All tubes were incubated in a water
bath at 37±2 oC for 60 to 70 minutes. After the incubation,
all tubes were decanted except Total Count Tubes by
simultaneous inversion with a sponge rack into a radioactive waste receptacle. The tubes were stroked sharply on
absorbent material to facilitate complete drainage and then
allow them to drain on absorbent material for a minimum
of 2 minutes. The tubes were then blotted to remove any
droplets adhering to the rim before returning them to the
upright position. As soon as the blotting, all of the tubes
were counted in a gamma counter for one minute. The
amount of testosterone was read in computer RIA data
analysis program and expressed as ng/ml. The calibration
range and sensitivity of kit were 0.1 to 25 and 0.08 ng/ml,
respectively. The intra-assay and inter-assay coefficient
variations of kit were lower than 9.6 % and 9.1 %, respectively.
HISTOPATHOLOGIC EXAMINATIONS :
The testicles were removed with open surgical technique
60 days after drug injection for histopathologic evaluation.
The testis from each animal were fixed in formalin and
embedded in paraffin wax. A section 5 μm thick was cut
from the middle portion of each testis, stained with haematoxylin-eosin and examined under light microscopy at 200x
magnification. The structures of the seminiferous tubules
and interstitial spaces in the testis were examined.
STATISTICAL ANALYSES :
All data are presented as mean ± SEM (Standard Error of
the Mean). The level of significance was set at p<0.05.
Paired sample t-test was used to determine the differences
between mean control and treatment values of mass
activity, semen volume and sperm concentration. The
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422
differences between control and treatment values for the
percentages of motile and abnormal sperm were compared
using a Chi-square (2) test. All the data were analyzed by
using the SPSS (Version 12.0) and MNTAB® software
package program (Version 14.0).
Results
Every animal tolerated the intratesticular injections of
ethanol and calcium chloride. They did not suffer from any
agitation and fever except for a slight increase of firmness of
a testis on palpation. Food consumption remained unaffected
among the two groups of animals.
Ultrasonographic examination following drug injection
revealed that the testicular tissue of both group animals had a
diffuse echotexture and increased echogenicity. The necrotized regions were differentiated ultrasonographically from
the normal testicular tissue with a hypoechogenic area
(Figure 1). Testicular swelling was evident in both group
bulls following injection and reached a peak within 48 hours.
While testicular volume decreased significantly (P<0.05) in
EG group after 3 weeks, no significant change occurred in
CCG group (P>0.05). The testicles underwent atrophy at the
60th day in EG group while no marked alteration could be
observed in CCG group (Figure 2). In two cases of the CCG
group, testicular swelling was associated with orchitis and
scrotal sloughing. At necropsy, their testicular tissues were
found to have been necrotized. These problems were not
observed in any animal of the EG group.
In this study, mean baseline value of serum testosterone
concentration was 13.2 ± 2.6 ng/ml in EG group and 13.0 ±
2.4 in CCG group. After drug administration, these concentration values were significantly decreased in EG group
(P<0.05) and remained almost constant in CCG group.
Intratesticular administration of ethanol induced a gradual
fall of serum testosterone concentrations from baseline 13.2
± 2.6 ng/ml to 5.7 ± 3.9, 3.5 ± 0.7 and 2.6 ± 0.7 ng/ml on
day 15, 30 and 60 after ethanol administration respectively.
The respective serum testosterone concentrations observed
in the CCG group were 12.9± 1.8, 13.0 ± 2.2 and 12.9± 2.4
ng/ml on day 15, 30 and 60 after ethanol administration,
respectively. Semen characteristics of only 3 bulls in
ethanol group were very poor (p<0.01, p<0.001). Though
only 3 bulls were infertile in ethanol injected group, other
bulls were fertile. No statistically significant differences of
semen characteristics were observed after calcium chloride
treatment (Tables I and II).
Microscopic examination of testicles from ethanol treated
bulls showed severe diffuse tubular necrosis along with
varying degree of inflammatory response (Figure 3). Inflammatory cells consisted of mainly mononuclear cells. Intertubular oedema, fibrosis, haemorrhage were also detected.
Some of the necrotic cells showed desquamation, or even
calcification. Intertubular vessels were severely congested.
Although similar lesions were noted in CCG treated bulls, the
severity and distribution of the lesions were not so
pronounced as in EG group.
CANPOLAT (I) AND COLLABORATORS
Discussion
Chemical, surgical, and mechanical castration were previously used in bulls [16,28]. For chemical castration in male
ruminants intratesticular lactic acid [16] tannic acid and zinc
sulphate [10] alpha-hydroxypropionic acid [4], formaline
[21] Castrate-Quin 14 [28) have been experimented. A few
studies on the clinical use of absolute ethanol have been
reported. These studies involved in the treatment of simple
renal cysts, tumours and renal angioinfarction [7,20] and
benign prostate hyperplasia [31]. The actual physiological
effect of this treatment has previously been speculated.
Necrosis and infarction leading to fibrosis, shrinkage, or
sloughing of tissue are presumed to be due to its mechanism
of action [2,31]. In our study ; severe diffuse tubular necrosis
along with varying degree of inflammatory response were
observed as a main finding (Figure 4).
B-mod ultrasonography was shown to produce adequate
image quality to evaluate size and tissue changes. Measurements of testicular size have been used in the bull [23].
Similar measurements have been suggested as being of value
in the dog [17]. Our study evaluated ultrasonographically the
size and structural alterations in the testicle tissue following
drug injection. Ultrasound imaging provides a rapid and
accurate method of measuring testicular volume. Using this
approach, we have detected a diffuse and increased echogenicity in the testicle tissue at acute period of drug administration, which was replaced by a decreased echogenicity in
association with inflammatory reactions and necrotic formation.
For chemical sterilization ethanol and formaldehyde
mixture were also used [12,24]. Gardner [1980] used intratesticularly a solution consisting of 3.6 % formaldehyde in
90 % ethanol for sterilization purpose in 10 bulls ; 3 among
them showed active sperm after 85 days. We previously used
a dose of 7 ml ethanol in male dogs [3] and judged the result
as satisfactory. Therefore, we thought that the use of 10 ml
drug in bulls because of their larger testicle sizes compared
with dogs could be enough. In the present study, absolute
ethanol was used in 6 bulls ; 3 bulls became infertile while
the others maintained fertile despite marked testicular
atrophy. This situation was attributed to the differences
between the life weights and testicle weights of the bulls and
their specific susceptibilities.
Optimal effective doses of calcium chloride in rat were
considered to range between 10-20 mg/kg, however, the best
results were obtained using 20 mg/kg [18]. The former
authors and others [18,26] found marked necrosis in the
seminiferous tubules following the use of this agent. In a
study, calcium chloride intratesticular administration was
shown to reduce serum testosterone level in bulls [22]. The
results of the present study, however disagree with the former
one, since we could not evidenced marked alterations of
serum testosterone levels and histopathological damages in
testicles from CCG treated bulls. Thus, this agent appeared to
be ineffective for chemo sterilization of bulls. On the other
hand, in EG group, the serum testosterone levels declined
markedly and meanwhile important findings were deterRevue Méd. Vét., 2006, 157, 8-9, 420-425
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BULL CASTRATION BY INTRA-TESTICULAR INJECTION OF ETHANOL AND CALCIUM CHLORIDE
Mass activity
Semen volume
(ml)
Sperm Concentration
(…..x 10 9 / ml)
Sperm Motility
(%)
423
Abnormal Sperm Rate
(%)
Bull
Number
Control
Treatment
Control
Treatment
Control
Treatment
Control
Treatment
Control
Treatment
1008
4.0 ± 0.2
3.1 ± 0.3
4.8 ± 0.3
3.6 ± 0.9
0.90 ± 0.11
0.82 ± 0.23
78.3 ± 2.1
70.8 ± 3.7
5.6 ± 0.2
18.3 ± 7.4
1010
3.6 ± 0.3
1.8 ± 0.8*
4.5 ± 0.7
2.1 ± 0.3*
1.05 ± 0.26 0.33 ± 0.50**
72.5 ± 2.5
27.5 ±6.5**
5.9 ± 0.3
39.8 ± 2.7**
1012
4.1 ± 0.3
1.9 ± 0.7*
4.6 ± 0.7
2.0 ± 0.4*
0.91 ± 0.25 0.34 ± 0.49**
75.3 ± 3.5
37.5 ± .5**
5.7 ± 0.2
46.2 ± 1.8**
1014
4.2 ± 0.5
3.4 ± 0.2
4.3 ± 0.8
3.5 ± 0.8
0.98 ± 0.24
0.88 ± 0.5
70.5 ± 2.5
61.7 ± 3.33
6.0 ± 0.2
12.7 ± 0.8
1016
3.9 ± 0.2
1.3 ± 0.6*
4.5 ± 0.7
2.0 ± 0.2*
0.90 ± 0.2
0.24 ± 0.4**
76.6 ± 2.1
25.0 ± 7.1**
4.9 ± 0.2
45.5 ± 2.8**
1018
4.3 ± 0.3
3.8 ± 0.3
4.5 ± 0.2
3.9 ± 0.2
0.86 ± 0.3
0.79 ± 0.2
81.6 ± 2.1
70.0 ± 3.4
9.6 ± 0.2
19.5 ± 4.5
Mean
4.02 ± 0.1
2.55 ± 0.41
4.53 ± 0.06
2.85 ± 0.36
0.93 ± 0.02
0.56 ± 0.11
75.8 ± 1.6
48.8 ± 8.7
6.3 ±0.7
30.3 ± 6.2
* : p<0.01 versus control, ** : p<0.001 versus control.
TABLE I. Control and treatment values of mass activity, semen volume, sperm concentration, motility and abnormal sperm rate in bulls 60 days after intratesticular
administration of ethanol. Data are presented as mean ± SEM.
Mass activity
Semen volume
(ml)
Sperm Concentration
(…..x 10 9 / ml)
Sperm Motility
(%)
Abnormal Sperm Rate
(%)
Bull
Number
Control
Treatment
Control
Treatment
Control
Treatment
Control
Treatment
Control
Treatment
1020
4.5 ± 0.2
4.3 ± 0.2
4.5 ± 0.2
4.2 ± 0.3
0.93 ± 0.02
0.92 ± 0.02
81.7 ± 2.1
80.0 ± 0.0
5.5 ± 0.2
4.8 ± 0.3
1021
4.2 ± 0.3
4.3 ± 0.4
4.6 ± 0.5
5.0 ± 0.5
0.91 ± 0.25
0.90 ± 0.15
77.5 ± 1.9
80.8 ± 0.8
6.4 ± 0.9
5.9 ± 0.5
1022
3.8 ± 0.5
4.0 ± 0.3
5.0 ± 0.9
5.2 ± 0.8
1.08 ± 0.35
0.98 ± 0.17
76.2 ± 1.7
78.3 ± 2.1
8.3 ± 0.2
9.1 ± 0.9
1024
4.0 ± 0.7
3.9 ± 0.2
5.3 ± 0.4
4.8 ± 0.5
0.96 ± 0.22
0.90 ± 0.02
75.8 ± 3.7
77.5 ± 2.6
5.6 ± 0.5
7.2 ± 0.3
1025
4.6 ± 0.1
3.8 ± 0.9
6.5 ± 0.6
6.0 ± 0.4
1.12 ± 0.21
1. 01 ± 0.25
71.8 ± 2.0
68.3 ± 1.6
7.9 ± 0.7
6.4 ± 0.2
1026
3.6 ± 0.4
3.5 ± 0.5
5.8 ± 0.2
5.2 ± 0.3
0.88 ± 0.28
0.80 ± 0.12
80.8 ± 2.8
75.0 ± 0.9
4.1 ± 0.4
5.1 ± 0.3
Mean
4.12 ± 0.16
3.97 ± 0.12
5.28 ± 0.31
5.07 ± 0.24
0.98 ± 0.04
0.92 ± 0.03
77.3 ± 1.48
76.7 ± 1.9
6.3 ± 0.7
6.4 ± 0.6
No significant differences were observed between control and treatment (p>0.05).
TABLE II. Control and treatment values of mass activity, semen volume, sperm concentration, motility and abnormal sperm rate in bulls 60 days after intratesticular administration of calcium chloride. Data are presented as mean ± SEM.
mined histopathologically. Infertility rate was 50 % ; we
postulate that this rate could have been improved if this
process had been performed on much younger bulls.
The method of chemo sterilization based on ethanol intratesticular administration is readily applicable with no major
harms and adverse effects. If 50 % rate is considered satisfactory it can be used then in large flocks, in particularly where
no chance of operation exists.
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CANPOLAT (I) AND COLLABORATORS
a
FIGURE 2 : Left : representative testicle of a bull from the CCG group.
Right : representative atrophied testicle of a ethanol-treated bull, 60 days
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b
FIGURE 1 : Ultrasonic appearance of testicles after intratesticular ethanol
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