- Email: [email protected]
Ivermectin does not interfere with seminal and hormonal parameters in male rabbits
Accepted Manuscript Ivermectin does not interfere with seminal and hormonal parameters in male rabbits
N. Moreira, M.A. Torres, P.E. Navas-Suárez, V. Gonçalves, P.C.F. Raspantini, L.E. R. Raspantini, A.T. Gotardo, A.F.C. Andrade, H.S. Spinosa PII:
S0093-691X(18)30866-5
DOI:
10.1016/j.theriogenology.2018.09.029
Reference:
THE 14714
To appear in:
Theriogenology
Received Date:
21 March 2018
Accepted Date:
26 September 2018
Please cite this article as: N. Moreira, M.A. Torres, P.E. Navas-Suárez, V. Gonçalves, P.C.F. Raspantini, L.E.R. Raspantini, A.T. Gotardo, A.F.C. Andrade, H.S. Spinosa, Ivermectin does not interfere with seminal and hormonal parameters in male rabbits, Theriogenology (2018), doi: 10.1016/j.theriogenology.2018.09.029
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ACCEPTED MANUSCRIPT 1
Revised
1 2
Ivermectin does not interfere with seminal and hormonal parameters in male rabbits
3 4 5
Moreira, N.a; Torres, M. A.b; Navas-Suárez, P. E.a; Gonçalves Jr, V. a; Raspantini, P. C. F.c;
6
Raspantini, L. E. R.c; Gotardo, A. T.c; Andrade, A. F. C.d; Spinosa, H. S.e
7 8
a
9
School of Veterinary Medicine and Animal Science, University of São Paulo, Av. Prof. Dr.
10
Graduate Program of Experimental and Comparative Pathology, Department of Pathology,
Orlando Marques de Paiva, 87, São Paulo, SP, 05508-270, Brazil.
11 12
b
13
Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil. Av.
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Duque de Caxias Norte, 225, Pirassununga, SP, 13635-900, Brazil.
Graduate Program of Animal Reproduction, Department of Animal Reproduction, School of
15 16
c
17
Veterinary Medicine and Animal Science, University of São Paulo, Av. Duque de Caxias Norte,
18
225, Pirassununga, SP, 13635-900, Brazil.
Research Center of Veterinary Toxicology (CEPTOX), Department of Pathology, School of
19 20
d
21
University of São Paulo, Av. Duque de Caxias Norte, 225, Pirassununga, SP, 13635-900, Brazil.
Department of Animal Reproduction, School of Veterinary Medicine and Animal Science,
22 23
e
24
São Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87, São Paulo, SP, 05508-270, Brazil.
Department of Pathology, School of Veterinary Medicine and Animal Science, University of
25 26
Conflict of Interest Statement: All authors declare that there are no conflicts of interest.
27
Source of funding: FAPESP (Grant number 2015/03131-4).
28
Corresponding
29
[email protected]
30
Present address: Natalia Moreira, Faculdade de Medicina Veterinária e Zootecnia da
31
Universidade de São Paulo, Avenida Professor Dr. Orlando Marques de Paiva, 87, Cidade
author:
Natalia
Moreira.
Tel:
+55-11-3091-7657;
e-mail:
ACCEPTED MANUSCRIPT 2 32
Universitária, São Paulo - SP, 05508-270, Brazil.
33 34
Abstract
35
Ivermectin (IVM) is a macrocyclic lactone used as a broad spectrum antiparasitic agent against
36
nematodes and arthropods. It is mainly used in the control of parasitic infections of domestic
37
animals, and recently has been used in humans to treat onchocerciasis, scabies, and pediculosis.
38
In mammals, evidence has indicated that macrocyclic lactones interact with gamma-
39
aminobutyric acid (GABA)-mediated chloride channels. The GABAergic system is known to be
40
involved in the manifestation of sexual behavior, and previous studies have shown that IVM
41
impaired sexual behavior in both male and female rats. Thus, considering that IVM may interfere
42
with the sexual sphere, this study evaluated the temporal (1 up 60 days) effects of exposure to
43
IVM (0.2 and 1.0 mg/kg, administered subcutaneously) on seminal and hormonal parameters of
44
male rabbits. In male rabbits, the spermatozoa concentration, motility and morphology, the
45
integrity of the plasmatic, acrosomal and mitochondrial membranes of the spermatozoa, the
46
organ weights, gonadosomatic index, serum testosterone concentrations, histopathological
47
findings were evaluated and hematological and serum biochemical analysis was conducted. No
48
changes were observed in male seminal parameters evaluated by spermatozoa concentration,
49
motility, and morphology, nor the potential for fertilization evaluated by the integrity of the
50
plasmatic, acrosomal, and mitochondrial membranes of the spermatozoa; there was also no
51
interference in serum testosterone concentration, serum biochemistry and hematological
52
parameters. The findings of this study using the artificial vagina for collection of semen and
53
computer-assisted semen analysis showed that IVM at doses of 0.2 and 1.0 mg/kg of SC did not
54
alter any of the semen parameters of rabbits evaluated for up to 60 days after administration.
55 56
Keywords: Avermectin, spermatozoa characteristics, andrological evaluation, testosterone
57 58
HIGHLIGHTS
59
Ivermectin did not impair spermatozoa motility and morphology.
60
Ivermectin was not able to injure the plasma and acrosomal membranes and did not alter the mitochondrial potential of spermatozoa.
61 62
Ivermectin did not interfere with the levels of serum testosterone.
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Ivermectin did not alter serum hematological and biochemical parameters.
64 65
1. INTRODUCTION
66
Ivermectin (IVM) is a semi-synthetic macrocyclic lactone, used as a broad spectrum
67
antiparasitic agent against nematodes and arthropods [1-3], mainly on the control of parasitic
68
infections of domestic animals, and recently in humans to treat onchocerciasis, scabies and
69
pediculosis [1,6].
70
In mammals, IVM is a well-tolerated drug with no side effects at therapeutic doses and,
71
acts blocks post-synaptic transmission of gamma aminobutyric acid (GABA)-mediated nerve
72
impulses, which is the main inhibitory neurotransmitter of the central nervous system (GABAA,
73
GABAB, and GABAC) [7-13].
74
Studies have been evaluating the role of GABAergic receptors in the sexual behavior of
75
mammals. On male rats, the administration of macrocyclic lactones, such as IVM, doramectin,
76
and moxidectin, showed a high correlation between behavioral alterations and the GABAergic
77
system [14-18]. Previous studies in our laboratories have shown that IVM [19] and doramectin
78
[20] act as GABAergic agonists and interfere with behaviors related to this neurotransmitter. In
79
female rats, IVM impaired sexual behavior during natural and hormone-induced estrus [21]. In
80
addition, another study showed that IVM impaired the appetitive phase of sexual behavior in
81
male rats at a dose of 1.0 mg/kg; this dose increased the latency for first mount and the first
82
intromission 15 minutes after administration [16].
83
Sexual behavior in males is regulated by the hypothalamic-pituitary-gonadal axis. The
84
hypothalamus
liberates
the
gonadotropin-releasing
hormone
(GnRH)
and
stimulates
85
gonadotropic cells in the anterior pituitary gland [22,23] to release luteinizing hormone (LH) and
86
follicle-stimulating hormone (FSH) [24,23]. LH stimulates the release of testosterone from
87
Leydig cells located in the testis [25]. Testosterone is the main male sex hormone responsible for
88
sexual behavior, and it plays a key role in the development of reproductive organs, such as the
89
testes and prostate, as well as in the production of spermatozoa [26].
90
Sperm production may be impaired by drugs capable of affecting the reproductive system
91
through different routes and mechanisms. These drugs may impair spermatogenesis and,
92
consequently, cause poor semen quality and sperm concentration, as well as morphological
93
changes in spermatozoa, which may lead to infertility or \reduced fertility [27,28].
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A recent study by Moreira et al. [18] it was observed that a 1.0 mg/kg dose of IVM
95
administered intraperitoneally reduced serum testosterone levels in male rats, and this effect was
96
attributed to the performance of the drug in GABAergic receptors. In fact, it is known that there
97
are peripheral GABAergic receptors present in the testes, which, when activated, reduce
98
testosterone release and, consequently, damage sperm production [29-31].
99
Due to testosterone responsibility on male sexual behavior, and the relevant role in sperm
100
production [26], the present study aims to investigate whether IVM is capable of interfering on
101
seminal parameters, serum testosterone levels and serum hematological and biochemical
102
parameters of rabbits for 1, 2, 3, 5, 7, 15, 30, 45, and 60 days after administration. The choice on
103
the animal species is justified by reduced population requirements (application of the principle of
104
3 Rs) [32,33], facility in semen collection and analysis methods and monitoring of seminal and
105
hormonal parameters in the same animal for up to 60 days after administration [34].
106 107
2. MATERIALS AND METHODS
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2.1.
Animals
109
Healthy male New Zealand rabbits were used. They were kept in the Research Center of
110
Veterinary Toxicology (CEPTOX), Department of Pathology, School of Veterinary Medicine
111
and Animal Science, University of São Paulo (FMVZ/USP). The animals were housed
112
individually in cages, under controlled room temperature (22 ± 2°C), humidity (45–65%), and
113
artificial lighting (12 h/12 h light/dark cycle). The animals received 60 g per day for each kg of
114
the Coelhão® feed (Guabi, Sales Oliveira, Brazil) and free access to the filtered water. All of the
115
procedures were reviewed and approved by the Animal Care Committee of FMVZ-USP
116
(protocol no. 1892040315) and conformed to the guidelines of the Committee on Care and Use
117
of Laboratory Animal Resources, National Research Council, USA (1996). All efforts were
118
made to minimize animal suffering.
119 120
2.2.
Drugs
121
IVM (1% Ivomec® injectable, Merial Animal Health Ltda., Paulínia, SP, Brazil) was
122
administered subcutaneously (SC) at a dose of 0.2 or 1.0 mg/kg. Saline solution was
123
administered as a control solution (NaCl 0.9% - 1 ml/kg). The 0.2 mg/kg IVM dose was chosen
124
because it is the standard therapeutic dose used in several animal models [35,36]. The 1.0 mg/kg
ACCEPTED MANUSCRIPT 5 125
dose was chosen due to a previous study of our laboratory, in which it was observed that this
126
dose promoted impairment in the sexual behavior of male and female rats [16,21].
127 128
2.3.
Experimental design
129
Before the experimental period, the rabbits were evaluated according to their seminal
130
profile and classified according their potential fertility through complete andrological
131
examinations. All animals were within the recommended limits for the species [37].
132
Male rabbits (N = 18) were divided into three equal groups according to their seminal
133
profile: 0.2 IVM, 1.0 mg/kg IVM, and control solution (control group). All the sperm evaluation
134
procedures were performed on days 1, 2, 3, 5, 7, 15, 30, 45, and 60 after the administration of
135
IVM, considering that in the bioavailability and pharmacokinetic studies, in plasma analysis,
136
IVM has a half-life of 5 to 7 days [11,38]. We also considered the duration of spermatogenesis of
137
rabbits, which, according to MORTON et al. [34], is approximately 60 days. Blood samples from
138
the marginal ear vein were collected 1, 2, 7, 15, 30, and 60 days after the administration of IVM
139
for testosterone levels. Hematological and biochemical analyses were performed on days 1, 2,
140
and 7 after administration of IVM. Organ samples were collected on the 60th day after
141
administration of IVM for histopathological analysis.
142 143
2.4.
Semen collection and analysis
144
On days 1, 2, 3, 5, 7, 15, 30, 45, and 60 of the experiment, rabbit semen collections were
145
performed with the support of heated artificial vagina as proposed by ANDRADE et al. [39]. The
146
hygienization of the prepuce was performed with water and neutral soap before harvest to avoid
147
possible semen contamination [40]. Immediately after collection, the spermatozoa concentration,
148
spermatozoa motility, integrity of plasma and acrosomal and mitochondrial membrane potential,
149
and morphological characteristics of spermatozoa were evaluated.
150 151
2.4.1. SPERMATOZOA CONCENTRATION
152
To determine the sperm concentration, an aliquot of raw semen was diluted to 1:500 in
153
buffered formalin saline. The count was performed in a Neubauer chamber under light
154
microscopy, with a magnification of 400x.
155
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2.4.2. COMPUTER-ASSISTED SEMEN ANALYSIS
157
Spermatozoa motility was assessed using a computer-assisted sperm analysis (CASA-
158
Microptic®, Microptic S.L., Barcelona, Espanha). Samples were diluted in tyrode albumin lactate
159
pyruvate (TALP) to 20 x 106 spermatozoa/ml. An aliquot (5 µl) was placed on a pre-warmed
160
(36ºC) cover-slide and evaluated by phase-contrast microscope (Nikon, Model Eclipse 80i) with
161
100x magnification. The CASA set-up was pre-adjusted for rabbit sperm analysis (minimum
162
particle size 19 microns2, maximum particle size 79 microns2; number of frames: 25; frames per
163
second: 24; velocity limit for slow sperm, 10 microns s−1; velocity limit for medium sperm, 25
164
microns s−1; velocity limit for rapid sperm, 50 microns s−1; minimal straightness for progressive
165
spermatozoa, 70%; maximal linearity for circular spermatozoa, 50%. Five good fields were
166
examined to evaluate the following parameters: total motility (TM, %), progressive motility
167
(PROM, %), average path velocity (VAP, μm/s), curvilinear velocity (VCL, μm/s), straight-line
168
velocity (VSL, μm/s), amplitude of lateral head displacement (ALH, μm), beat cross frequency
169
(BCF, Hz), straightness (STR, %) and linearity (LIN, %) [41].
170 171 172
2.4.3. EVALUATION OF PLASMA AND ACROSOMAL MEMBRANE INTEGRITY AND MITOCHONDRIAL MEMBRANE POTENTIAL
173
After dilution of semen in TALP, an aliquot was stained with Hoechst 33342 (1.25
174
μg/mL), propidium iodide (1.5 μg/mL), fluorescein-conjugated Pisum sativum agglutinin (7.5
175
μg/mL), and 5,5',6,6'-tetrachloro-1,1,3,3'-tetraethylbenzimidazolylcarbocyanine iodide (612 μM),
176
then incubated at 37°C/15 min. After incubation, an aliquot (8 μl) was placed on a pre-warmed
177
(37ºC) cover-slide and evaluated, and 200 cells were counted by epifluorescence microscopy
178
(Nikon Epifluorescence Microscope, Model Eclipse 80i) in a triple filter (D/F/R, C58420) with
179
UV-2E/C assemblies (340-380 nm excitation and 435-485 emission), B-2E/C (excitation 465-
180
495 and emission 515-555) and G-2E/C (excitation 540 -525 and issue 605-655) at 1000x
181
magnification. Cells were classified into eight categories according to Celeghini et al. [42].
182 183
2.4.4. MORPHOLOGICAL CHARACTERISTICS OF SPERMATOZOA
184
The morphological characteristics of the spermatozoa were evaluated using the wet
185
chamber technique. For this, the semen was diluted and fixed in pre-heated, buffered saline
186
formol at 37°C. A diluted semen aliquot (8 μl) was placed between the slide and coverslip, and a
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sperm count of 200 sperm was magnified by 1000x under differential interference contrast
188
microscopy (Nikon®, model 80i). The sperm characteristics were classified as normal, minor,
189
and major defects [43].
190 191
2.5.
Evaluation of weight gain, relative organ weight, gonadosomatic index, and
192
histopathological study
193
On days 1, 30, and 60 of the experiment, rabbits were weighed on a scale (MARTE®,
194
model AD 10K) to measure body weight gain.
195
The liver, testes, epididymis, ventral prostate, and seminal vesicle were weighed on a
196
scale (MARTE®, model AL 500C), and the relative weight (RW) was calculated: RW = (organ
197
weight/body weight).
198
Representative fragments of the testes and epididymis were fixed in Bouin (75%
199
saturated picric acid, 25% formalin, 5% glacial acetic acid), while the representative fragments
200
of the liver and adrenals were fixed in 10% formaldehyde, dehydrated, diaphanized, and
201
embedded in paraffin (Sigma Chemical Co., St. Louis, MO). The material was then cut into 5-
202
µm-thick sections and stained with hematoxylin-eosin (HE) for histopathological analysis.
203 204
2.6. Evaluation of serum testosterone concentration
205
Serum testosterone levels were assessed using commercially available enzyme-linked
206
immunosorbent assays according to the manufacturer’s instructions (testosterone kit, Cayman
207
Chemical, Ann Arbor, MI, USA; catalog no. 582701).
208 209
2.7. Hematological and biochemical analyses
210
The collected blood sample was immediately expelled into separate heparinized plastic
211
vials for hematological analysis. The biomarkers determined were: red blood cells (RBCs),
212
hemoglobin (Hb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular
213
hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), eosinophils,
214
basophils, lymphocytes, and monocytes. An aliquot of blood was collected for biochemical
215
analysis. The serum was separated by allowing the blood (taken in a plain tube) to clot and then
216
centrifuged at 4,000 rpm for 10 min at 4ºC. The serum was analyzed for biochemical analysis of
217
aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), gamma-
ACCEPTED MANUSCRIPT 8 218
glutamyl transferase (GGT), globulins, albumin, total cholesterol, triglycerides, high-density
219
lipoprotein (HDL), urea, and creatinine.
220 221
2.8. Statistical analysis
222
Instat software (Prism 6.01, GraphPad, San Diego, CA, USA) was used for the statistical
223
analyses. The homoscedasticity was verified by the F or Bartlet test. Normality was checked by
224
the Brown-Forsythe test. One-way analysis of variance (ANOVA) followed by Dunnett's
225
multiple comparison post hoc test was used to analyze relative organ weight and gonadosomatic
226
index. Sperm concentration, sperm motility, plasma and acrosomal membrane integrity,
227
mitochondrial membrane potential, sperm morphological characteristics, biochemical analysis,
228
and serum testosterone levels were analyzed by two-way ANOVA, followed by the Dunnett’s
229
post hoc test. In all analyses, differences were considered significant when p <0.05. Data were
230
expressed as mean ± standard error of the mean (SEM).
231 232
3. RESULTS
233
No significant difference was observed among the groups on the spermatozoa
234
concentration, on the evaluation of plasma and acrosomal membrane integrity and mitochondrial
235
membrane potential, on the sperm morphology and on the serum testosterone levels in rabbits
236
(Fig. 1).
237 238 239 240
No significant difference was observed among the groups on the sperm motility characteristics obtained by computer-assisted semen analysis (Fig. 2). No significant differences in body weight gain were observed between the IVM-treated groups and control group (Fig. 3).
241
Relative organ weights and the gonadosomatic index in rabbits after IVM administration
242
were similar to the control group, with the exception of the relative weight of the liver (Fig. 3).
243
The one-way ANOVA revealed a significant effect of treatment on the relative liver weight
244
(F2,15 = 7.616, p = 0.0052). The Dunnett post hoc test revealed a decrease in the relative weight
245
of the liver in rabbits that were treated with 1.0 mg/kg of IVM on the day 60 after administration
246
compared with the control group.
247
The histopathological examination of rabbits that were euthanized on day 60 after
248
treatment did not reveal significant changes in morphology after IVM exposure (data not shown).
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No significant differences in hematological and biochemical analysis were observed between the IVM-treated groups and control group (data not shown).
251 252
4. DISCUSSION
253
The findings of this study showed that IVM at doses of 0.2 and 1.0 mg/kg of SC did not
254
alter any of the semen parameters of New Zealand rabbits evaluated for up to 60 days after
255
administration, differently of previous study that observed adverse effect on reproductive
256
parameters of rabbits at 30 and 60 days post-injection of 0.2 mg/kg IVM [44].
257
When administered at therapeutic doses (0.2 mg/kg), avermectins IVM and moxidectin
258
were found to have adverse effects on male fertility in Baladi rabbits, according to Eman and El-
259
Abdalla [44]. The authors observed that, 30 and 60 days after administration of these
260
avermectins, there was a decrease in sperm concentration, percentage of progressive motility,
261
and number of live spermatozoa. Comparing this study with the results of the present study,
262
differences in rabbit breed and sperm collection techniques can be observed. Eman and El-
263
Abdalla [44] collected spermatozoa from the tail of the epididymis. This technique, according to
264
Robaire and Viger [45] and Cornwall [46], allows the collection of spermatozoa with progressive
265
motility. However, the passage of the spermatozoa along the epididymal duct is what determines
266
the acquisition of the fertilizing capacity and the progressive motility, as this process dominates
267
sperm maturation [47]. In addition, collection of spermatozoa from the tail of the epididymis
268
does not allow them to be collected with seminal plasma. This fluid is rich in nutrients, such as
269
proteins and enzymes, and is essential for attesting to the viability of spermatozoa [34];
270
therefore, the absence of seminal plasma interferes with sperm viability and, consequently, with
271
progressive motility [34,37]. In contrast, in the present study the technique of semen collection
272
was used by means of an artificial vagina, which ensures the sperm viability, since in the semen,
273
the indispensable components for the progressive motile spermatozoa are present [48-50]. Thus,
274
it can be stated that sperm collection techniques are responsible for the differences between the
275
studies in relation to the results observed in the progressive motile spermatozoa of rabbits.
276
In another study that used the artificial vagina for collection of semen, but using another
277
animal species, partially corroborates the present findings. Thus, Naoman [38] observed that the
278
SC administration of 0.2 mg/kg IVM did not impair the sperm motility of sexually experienced
279
sheep. However, the author observed that in this dose there was a decrease in sperm
ACCEPTED MANUSCRIPT 10 280
concentration, testosterone levels and alteration in sperm morphology. These changes were no
281
longer observed 5 days after administration, which may be associated with the half-life of IVM,
282
which varies between 5 and 7 days [11,38].
283
Damage to sperm motility and fertilization potential may occur due to injury to the
284
plasma, acrosomal, and mitochondrial membranes of spermatozoa, since the integrity of these
285
membranes guarantees, respectively, sperm capacity and viability, fertilization, and sperm
286
motility [42]. In order to evaluate the damage to these membranes, fluorescence probes (PI,
287
FITC-PSA and JC-1) were used in the present work. The results obtained herein show that the
288
administration of 0.2 and 1.0 mg/kg of IVM did not cause damage to the plasma, acrosomal, and
289
mitochondrial membranes, corroborating the result of absence of alteration in sperm motility,
290
since damage to these membranes may harm the fertilization potential.
291
Testis weight has been used as a quantitative indicator of testis normality due to the
292
correlation between the weight of the testis and the number of germinal cells of the seminiferous
293
epithelium [51]. The germinal cells of the seminiferous epithelium have been found to be more
294
abundant in the testis. The results of the present study did not reveal alterations in the relative
295
weight of the testicles and accessory sexual glands, nor in the IGS, nor did they reveal
296
histopathological alterations in the sexual organs. Notably, the accessory sexual glands, such as
297
the prostate and seminal vesicle, respond differently to male and female sex hormones, and the
298
weights of these organs are bioindicators of the circulating levels of these hormones [52]. The
299
absence of changes in the testosterone levels of rabbits treated with IVM and evaluated for up to
300
60 days corroborate the evidence indicating that this drug did not disturb the testicular function,
301
that is, the production and transport of spermatozoa, as well as the production of testosterone.
302
On the other hand, El-Far [53] investigated the effects of IVM at doses of 0.5 and 1.0
303
mg/kg (SC) on testosterone levels and on serum biochemical parameters in male rabbits of the
304
New Zealand breed on days 1, 3, and 7 after administration. This author observed an increase in
305
serum testosterone levels and changes in hepatic and renal function over the 3 days of evaluation.
306
In this paper, the author did not explain the analytical method used for the evaluation of serum
307
testosterone and attributed the changes in hepatic and renal functions to the overload caused by
308
the drug in these organs. In this respect, the results of the present study showed no alterations in
309
any of the serum biochemical parameters, as well as hematological parameters in rabbits treated
310
with 0.2 and 1.0 mg/kg of IVM evaluated on days 1, 2, and 7 after the administration. There are
ACCEPTED MANUSCRIPT 11 311
few findings in the literature on the effects of IVM on serum and hematological biochemical
312
parameters in rabbits; there is only information on serum biochemical parameters, but in repeated
313
exposure to IVM in rabbits, causing alterations in these parameters [54].
314 315
5. CONCLUSIONS
316
In this temporal study of the administration of IVM in rabbits, at SC doses of 0.2 and 1.0
317
mg/kg, no changes were observed in male seminal parameters, as assessed by the concentration,
318
sperm motility and morphology, or fertilization potential, evaluated by the integrity of the
319
spermatozoa's plasma, acrosomal, and mitochondrial membranes. Furthermore, even at the
320
highest dose, IVM did not interfere with serum levels of testosterone, serum biochemistry, and
321
blood count parameters.
322 323
6. ACKNOWLEDGMENTS
324
This work is part of the doctoral thesis of Natalia Moreira and Vagner Gonçalves Jr. to
325
the School of Veterinary Medicine and Animal Science, University of São Paulo, and was
326
supported by grants from Fundação de Apoio à Pesquisa do Estado de São Paulo (FAPESP;
327
grant number 2015/03131-4) and from Conselho Nacional de Desenvolvimento Científico e
328
Tecnológico (CNPq; grant number 305500/2013-9).
329 330 331 332 333 334 335 336 337 338
7. REFERENCES [1] Poul JM. Effects of Perinatal Ivermectin Exposure on Behavioral Development of Rats. Neurotoxicol Teratol 1988; 10: 267-72. [2] Lankas GR, Minsker DH, Robertson RT. Effects of ivermectin on reproduction and neonatal toxicity in rats. Fd Chem Toxic 1989; 27: 523-29. [3] Uhlir J, Volf P. Ivermectin: its effect on the immune system of rabbits and rats infested with ectoparasitos. Vet Immunol Immunopathol 1992; 34: 325-36. [4] Omura, S. Ivermectin: 25 years and still going strong. Intl J Antimicrob Agents 2008; 31: 918.
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[5] Cully DF, Vassilatis DK, Liu KK, Paress PS, Van Der Ploeg LH, Schaeffer JM, Arena JP.
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Cloning of an avermectin-sensitive glutamate-gated chloride channel from Caenorhabditis
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elegans. Nature 1994; 371: 707-11.
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[6] Yates DM, Portillo V, Wolstenholme AJ. The avermectin receptors of Haemonchus contortus and Caenorhabditis elegans. Int J Parasitol 2003; 33: 1183-93. [7] Geary TG. Ivermectin 20 years on: maturation of a wonder drug. Trends Parasitol 2005; 21: 530-32. [8] Pong SS, Wang CC. Avermectin Bla modulation of yaminobutyric acid receptors in rat brain membranes. J Neurochem 1982; 38: 375-79. [9] Trailovic SM, Ivanovic SR, Varagić VM. Ivermectin effects on motor coordination and contractions of isolated rat diaphragm. Res Vet Sci 2011; 426-33. [10] El-sawy AEF, El-maddawy ZK, Seed SA. Adverse Effects of Ivermectin in comparison with Rafoxanide on Male Rats. Alexandria J Vet Sci 2015; 47: 119-28. [11] Gupta RC. Ivermectin and selamectin. In: Gupta RC, editors. Veterinary Toxicology: Basic and Clinical Principles, San Diego: Academic Press Inc; 2007, p. 508-13. [12] Sivilotti L, Nistri A. GABA Receptor mechanism in the central nervous system. Prog Neurobiol 1991; 36: 35-92.
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[14] Rodrigues-Alves PSB, Lebrun I, Florio JC, Bernardi MM, Spinosa HS. Moxidectin
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interference on sexual behavior, penile erection and hypothalamic GABA levels of male
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rats. Res Vet Sci 2008; 84: 100-6.
360 361 362 363
[15] Rodrigues-Alves PSB, Florio JC, Lebrun I, Bernardi MM, Spinosa HS. Moxidectin Interference on Motor Activity of Rats. Braz Arch Biol Technol 2009; 52: 883-91. [16] Bernandi MM, Kirsten TB, Spinosa HS, Manzano H. Ivermectin impairs sexual behavior in sexually naïve, but not sexually experienced male rats. Res Vet Sci 2011; 91: 77-81.
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[17] Ferri R, Todon e Silva AFS, Cabral D, Moreira N, Spinosa HS, Bernardi MM. Doramectin
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reduces sexual behavior and penile erection in male rats. Neurotoxicol Teratol 2013; 39; 63-
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8.
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[18] Moreira N, Sandini TM, Reis-Silva TM, Navas-Suárez P, Auada AVV, Lebrun I, Florio JC,
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Bernandi MM, Spinosa HS. Ivermectin reduces motor coordination, serum testosterone, and
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central neurotransmitter levels but does not affect sexual motivation in male rats. Reprod
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Toxicol 2017; 74: 195-203.
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[19] Spinosa HS, Stilck SR, Bernardi MM. Possible anxiolytic effects of ivermectin in rats, Vet Res Commun 2002; 26: 309-21.
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[20] Spinosa HS, Gerenutti M, Bernardi MM. Anxiolytic and anticonvulsant properties of
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doramectin in rats: behavioral and neurochemistric evaluations. Comp Biochem Physiol C
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[21] Moreira N, Bernardi MM, Spinosa HS. Ivermectin reduces sexual behavior in female rats. Neurotoxicol Teratol 2014; 43: 33–8. [22] Johnson L, Petty CS, Neaves WB. A comparative study of daily sperm producion and testicular composition in humans and rats. Biol Reprod 1980; 22: 1233-44. [23] O’donnel L, Robertson KM, Jones ME, Simpson ER. Estrogen and spermatogenesis. Endocr Rev 2001; 22: 289-318.
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regulation of spermatogenesis: independent roles for testosterone and FSH. J Endocrinol
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[25] Shulman LM, Spritzer MD. Changes in the sexual behavior and testosterone levels of male rats in response to daily interactions with estrus females. Physiol Behav 2014; 133: 08–13. [26] Matos AFG, Moreira RO, Guedes EP. Neuroendocrinology of the metabolic syndrome. Arq Bras Endocrinol Metab 2003; 47: 410-20. [27] Bonde JP. Male reproductive organs are at risk from environmental hazards. Asian J Androl 2010; 12: 152-56. [28] Wong EWP, Cheng CY. Impacts of environmental toxicants on male reproductive dysfunction. Trends Pharmacol Sci 2011; 32: 290-99, 2011. [29] He XB, Hu JH, Wu Q, Yan YC, Koide SS. Identification of GABA(B) receptor in rat testis and sperm. Biochem Biophys Res Commun 2001; 283: 243-47. [30] He X, Zhang Y, Yan Y, Li Y, Koide SS. Identification of GABABR2 in rat testis and sperm. J Reprod Dev 2003; 49: 397-402. [31] Li S, Zhang Y, Liu H, Yan Y, Li Y. Identification and expression of GABAC receptor in rat testis and spermatozoa. Acta Biochim Biophys Sin 2008; 40: 761-67. [32] Presgrave OAF. Alternativas para animais de laboratório: do animal ao computador. 1st ed. Rio de Janeiro: FIOCRUZ; 2002.
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[33] Cazarin KCC, Corrêa CL, Zambrone FAD. Redução, refinamento e substituição do uso de
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animais em estudos toxicológicos: uma abordagem atual. Rev Bras Ciênc Farm 2004; 40:
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[34] Morton D. The Use of Rabbits in Male Reproductive Toxicology. Environ Health Perspect 1988; 77: 5-9.
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[35] Dadarkar SS, Deore MD, Gatne MM. Comparative evaluation of acute toxicity of
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ivermectin by two methods after single subcutaneous administration in rats. Regul Toxicol
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Pharmacol 2007; 47: 257-60.
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[36] Sartor IF, Santarém VA. Agentes empregados no controle de ectoparasitos. In: Spinosa HS,
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Górniak SL, Bernardi MM, editors. Farmacologia aplicada à medicina veterinária, Rio de
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Janeiro: Guanabara Koogan; 2017, p. 599-608.
412 413 414 415 416 417
[37] Foote RH, Carney EW. The rabbit as a model for reproductive and developmental toxicity studies. Reprod Toxicol 2000; 14: 477-93. [38] Naoman UD. Effect of ivermectin on semen characteristics of Iraqi Awassi ram. Al-Anbar J Vet Sci 2012; 5: 129-33. [39] Andrade AFC, Yonezawa LA, Celeghini ECC, Spers A, Arruda RP. Um novo modelo de vagina artificial para coelhos. Rev Bra Reprod Anim 2002; 26: 201-04.
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[40] Celeghini ECC, Arruda RP, Andrade AFC, Nascimento J, Raphael CF, Rodrigues PHM.
419
Effects that bovine sperm cryopreservation using two different extenders has on sperm
420
membranes and chromatin. Anim Reprod Sci 2008; 104: 119-31.
421
[41] Mortimer ST. CASA - practical aspects. J Androl 2000; 21: 515–24.
422
[42] Celeghini ECC, Nascimento J, Raphael CF, Andrade AFC, Arruda RP. Simultaneous
423
assessment of plasmatic, acrosomal, and mitochondrial membranes in ram sperm by
424
fluorescent probes. Arq Bras Med Vet Zootec 2010; 62: 536-43.
425
[43] Janett F, Thun R, Ryhiner A, Burger D, Hassig M, Hertzberg H. Influence of Eqvalan
426
(ivermectin) on quality and freezability of stallion semen. Theriogenology 2001; 55: 785-92;
427
[44] Emam EE, El-Abdalla O. Effects of ivermectin and moxidectin on fertility and some
428 429 430 431 432 433 434
biochemical parameters in male rabbits. Egyptian J Agric Res 2000; 78: 293-301. [45] Robaire B, Viger RS. Regulation of epididymal epithelial cell functions. Biol Reprod 1995; 52: 226-36. [46] Cornwall GA. New insights into epididymal biology and function. Hum Reprod Update 2009; 15: 213-27. [47] Oliva SU, Rinaldo PA, Stumpp T. Biologia epididimária: maturação espermática e expressão gênica. O Mundo da Saúde 2009; 33: 419-25.
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[48] Constantini FFA. Nel coniglio, sistemi de conservazione dello sperma. Riv di coniglicoltura 1989; 4: 14-8. [49] Rodriguez RM. Una vagina artificial para la extraccion de semen de conejo. Rev Cub Reprod Anim 1993; 17-18: 200-02. [50] Sinkovics G, Cenci T. Scuota, S, Dal Bosco A. Un’idea per la F.A. Riv di coniglicoltura 1993; 9: 35-6. [51] Russel L, Ettlin R, Hikin A, Clegg E. Histological and histopathological evaluation of the testis. Int J Androl 1990; 16: 83.
443
[52] Foote RH, Draddy PJ, Breite M, Oltenacu EAB. Action of androgen and estrone implants
444
on sexual behavior and reproductive organs of castrated male rabbits. Horm Behav 1977; 9:
445
57-68.
446
[53] El-Far AH. Effect of therapeutic and double therapeutic doses of ivermectin on oxidative
447
status and reproductive hormones in male rabbits. Am J Anim Vet Sci 2013; 8: 128-33.
448
[54] Al-Jassim KB, Jawad AADH, Al-Masoudi EA, Majeed SK. Histopathological and
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biochemical effects of ivermectin on kidney functions, lung and the ameliorative effects of
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vitamin c in rabbits (Lupus cuniculus). Basrah J Vet Res 2016; 14: 110-24.
451
ACCEPTED MANUSCRIPT 16 452
8. FIGURE LEGENDS
453
Figure 1. Effects of administration of ivermectin (IVM 0.2 or 1.0 mg/kg) or control solution (1.0
454
ml/kg - control group) on the spermatozoa concentration, on the evaluation of plasma and
455
acrosomal membrane integrity and mitochondrial membrane potential, on the sperm morphology
456
and on the serum testosterone levels in rabbits 1, 2, 3, 5, 7, 15, 30, 45, and 60 days after
457
administration of 0.2 or 1.0 mg/kg IVM or control solution (1.0 ml/kg). The data are expressed
458
as mean ± SEM. N = 6 animals per group. p > 0.05, compared with the control group (two-way
459
ANOVA followed by Dunnett post hoc test). Plasma membrane integrity
Sperm concentration 100
400
200
0
1
2
3
5
7
15
30
45
60
60 40 20 0
days
Acrosomal membrane integrity
2
3
5
100
50
1
2
3
5
7
15
30
45
60
80 60 40 20 0
days
1
2
3
Percentage (%)
Percentage (%)
60 40 20
2
3
5
7
15
30
45
60
days
45
60
days
1
2
3
15 10 5
1
2
3
5
7
15
30
5
7
15
30
45
60
days
Testosterone
45
60
days
Concentration of testosterone (ng/ml)
Percentage (%)
30
20
Minor defects
461
15
40
0 1
20
460
7
60
80
0
5
Major defects
Normal sperm 100
0
15 30 45 60 days
7
100
Percentage (%)
Percentage (%)
1
Active mitochondrial membrane
150
0
Control 0.2 mg/kg 1.0 mg/kg
80
Percentage (%)
106 sperm/ml
600
10 8 6 4 2 0
1
2
7
15
30
60
days
ACCEPTED MANUSCRIPT 17 462
Figure 2. Effects of administration of ivermectin (IVM 0.2 or 1.0 mg/kg) or control solution (1.0
463
ml/kg - control group) on the spermatozoa motility (CASA) in male rabbits 1, 2, 3, 5, 7, 15, 30,
464
45, and 60 days after administration of 0.2 or 1.0 mg/kg IVM or control solution (1.0 ml/kg). The
465
data are expressed as mean ± SEM. N = 6 animals per group. p > 0.05 compared with the control
466
group (two-way ANOVA followed by Dunnett post hoc test).
467 468
ACCEPTED MANUSCRIPT 18 469
Figure 3. Effects of administration of ivermectin (IVM 0.2 or 1.0 mg/kg) or control solution (1.0
470
ml/kg - control group) on the body weight gain, the relative weight of organs (liver, prostate,
471
testes, epididymis, adrenals, and seminal vesicle) and rabbit gonadosomatic index. The data are
472
expressed as mean ± SEM. N = 6 animals per group. **p < 0.01 compared with the control group
473
(one-way ANOVA followed by Dunnett post hoc test). Liver
Prostate Organs Relative weight (g)
Body weight gain (g)
250 200 150 100 50 0
0-30
31-60
days
4
**
3 2 1 0
Control
0.05
0.2 mg/kg
1.0 mg/kg
0.10
0.05
0.00
Control
0.2 mg/kg
1.0 mg/kg
0.010
0.005
0.000
1.0 mg/kg
Control
0.2 mg/kg
474
Control
0.06 0.04 0.02
Control
0.2 mg/kg
1.0 mg/kg
0.2 mg/kg
1.0 mg/kg
0.06
0.04
0.02
0.00
Control
1.0 mg/kg
1.0 mg/kg
0.010
0.005
0.000
Control
0.006
0.004
0.002
0.000
0.2 mg/kg
0.015
Gonadossomatic index Organs Relative weight (g)
Organs Relative weight (g)
Seminal vesicle 0.08
0.00
0.00
Left adrenal
0.015
Organs Relative weight (g)
Organs Relative weight (g)
Organs Relative weight (g)
0.02
0.2 mg/kg
0.01
Right adrenal
0.04
Control
0.02
Right epididymis
0.15
Left epididymis 0.06
0.00
Control 0.2 mg/kg 1.0 mg/kg
0.03
1.0 mg/kg
Organs Relative weight (g)
0.10
Control
0.2 mg/kg
0.04
Left testis Organs Relative weight (g)
Organs Relative weight (g)
Right testis 0.15
0.00
Organs Relative weight (g)
Body weight gain
Control
0.2 mg/kg
1.0 mg/kg
0.2 mg/kg
1.0 mg/kg
N. Moreira, M.A. Torres, P.E. Navas-Suárez, V. Gonçalves, P.C.F. Raspantini, L.E. R. Raspantini, A.T. Gotardo, A.F.C. Andrade, H.S. Spinosa PII:
S0093-691X(18)30866-5
DOI:
10.1016/j.theriogenology.2018.09.029
Reference:
THE 14714
To appear in:
Theriogenology
Received Date:
21 March 2018
Accepted Date:
26 September 2018
Please cite this article as: N. Moreira, M.A. Torres, P.E. Navas-Suárez, V. Gonçalves, P.C.F. Raspantini, L.E.R. Raspantini, A.T. Gotardo, A.F.C. Andrade, H.S. Spinosa, Ivermectin does not interfere with seminal and hormonal parameters in male rabbits, Theriogenology (2018), doi: 10.1016/j.theriogenology.2018.09.029
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Revised
1 2
Ivermectin does not interfere with seminal and hormonal parameters in male rabbits
3 4 5
Moreira, N.a; Torres, M. A.b; Navas-Suárez, P. E.a; Gonçalves Jr, V. a; Raspantini, P. C. F.c;
6
Raspantini, L. E. R.c; Gotardo, A. T.c; Andrade, A. F. C.d; Spinosa, H. S.e
7 8
a
9
School of Veterinary Medicine and Animal Science, University of São Paulo, Av. Prof. Dr.
10
Graduate Program of Experimental and Comparative Pathology, Department of Pathology,
Orlando Marques de Paiva, 87, São Paulo, SP, 05508-270, Brazil.
11 12
b
13
Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil. Av.
14
Duque de Caxias Norte, 225, Pirassununga, SP, 13635-900, Brazil.
Graduate Program of Animal Reproduction, Department of Animal Reproduction, School of
15 16
c
17
Veterinary Medicine and Animal Science, University of São Paulo, Av. Duque de Caxias Norte,
18
225, Pirassununga, SP, 13635-900, Brazil.
Research Center of Veterinary Toxicology (CEPTOX), Department of Pathology, School of
19 20
d
21
University of São Paulo, Av. Duque de Caxias Norte, 225, Pirassununga, SP, 13635-900, Brazil.
Department of Animal Reproduction, School of Veterinary Medicine and Animal Science,
22 23
e
24
São Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87, São Paulo, SP, 05508-270, Brazil.
Department of Pathology, School of Veterinary Medicine and Animal Science, University of
25 26
Conflict of Interest Statement: All authors declare that there are no conflicts of interest.
27
Source of funding: FAPESP (Grant number 2015/03131-4).
28
Corresponding
29
[email protected]
30
Present address: Natalia Moreira, Faculdade de Medicina Veterinária e Zootecnia da
31
Universidade de São Paulo, Avenida Professor Dr. Orlando Marques de Paiva, 87, Cidade
author:
Natalia
Moreira.
Tel:
+55-11-3091-7657;
e-mail:
ACCEPTED MANUSCRIPT 2 32
Universitária, São Paulo - SP, 05508-270, Brazil.
33 34
Abstract
35
Ivermectin (IVM) is a macrocyclic lactone used as a broad spectrum antiparasitic agent against
36
nematodes and arthropods. It is mainly used in the control of parasitic infections of domestic
37
animals, and recently has been used in humans to treat onchocerciasis, scabies, and pediculosis.
38
In mammals, evidence has indicated that macrocyclic lactones interact with gamma-
39
aminobutyric acid (GABA)-mediated chloride channels. The GABAergic system is known to be
40
involved in the manifestation of sexual behavior, and previous studies have shown that IVM
41
impaired sexual behavior in both male and female rats. Thus, considering that IVM may interfere
42
with the sexual sphere, this study evaluated the temporal (1 up 60 days) effects of exposure to
43
IVM (0.2 and 1.0 mg/kg, administered subcutaneously) on seminal and hormonal parameters of
44
male rabbits. In male rabbits, the spermatozoa concentration, motility and morphology, the
45
integrity of the plasmatic, acrosomal and mitochondrial membranes of the spermatozoa, the
46
organ weights, gonadosomatic index, serum testosterone concentrations, histopathological
47
findings were evaluated and hematological and serum biochemical analysis was conducted. No
48
changes were observed in male seminal parameters evaluated by spermatozoa concentration,
49
motility, and morphology, nor the potential for fertilization evaluated by the integrity of the
50
plasmatic, acrosomal, and mitochondrial membranes of the spermatozoa; there was also no
51
interference in serum testosterone concentration, serum biochemistry and hematological
52
parameters. The findings of this study using the artificial vagina for collection of semen and
53
computer-assisted semen analysis showed that IVM at doses of 0.2 and 1.0 mg/kg of SC did not
54
alter any of the semen parameters of rabbits evaluated for up to 60 days after administration.
55 56
Keywords: Avermectin, spermatozoa characteristics, andrological evaluation, testosterone
57 58
HIGHLIGHTS
59
Ivermectin did not impair spermatozoa motility and morphology.
60
Ivermectin was not able to injure the plasma and acrosomal membranes and did not alter the mitochondrial potential of spermatozoa.
61 62
Ivermectin did not interfere with the levels of serum testosterone.
ACCEPTED MANUSCRIPT 3 63
Ivermectin did not alter serum hematological and biochemical parameters.
64 65
1. INTRODUCTION
66
Ivermectin (IVM) is a semi-synthetic macrocyclic lactone, used as a broad spectrum
67
antiparasitic agent against nematodes and arthropods [1-3], mainly on the control of parasitic
68
infections of domestic animals, and recently in humans to treat onchocerciasis, scabies and
69
pediculosis [1,6].
70
In mammals, IVM is a well-tolerated drug with no side effects at therapeutic doses and,
71
acts blocks post-synaptic transmission of gamma aminobutyric acid (GABA)-mediated nerve
72
impulses, which is the main inhibitory neurotransmitter of the central nervous system (GABAA,
73
GABAB, and GABAC) [7-13].
74
Studies have been evaluating the role of GABAergic receptors in the sexual behavior of
75
mammals. On male rats, the administration of macrocyclic lactones, such as IVM, doramectin,
76
and moxidectin, showed a high correlation between behavioral alterations and the GABAergic
77
system [14-18]. Previous studies in our laboratories have shown that IVM [19] and doramectin
78
[20] act as GABAergic agonists and interfere with behaviors related to this neurotransmitter. In
79
female rats, IVM impaired sexual behavior during natural and hormone-induced estrus [21]. In
80
addition, another study showed that IVM impaired the appetitive phase of sexual behavior in
81
male rats at a dose of 1.0 mg/kg; this dose increased the latency for first mount and the first
82
intromission 15 minutes after administration [16].
83
Sexual behavior in males is regulated by the hypothalamic-pituitary-gonadal axis. The
84
hypothalamus
liberates
the
gonadotropin-releasing
hormone
(GnRH)
and
stimulates
85
gonadotropic cells in the anterior pituitary gland [22,23] to release luteinizing hormone (LH) and
86
follicle-stimulating hormone (FSH) [24,23]. LH stimulates the release of testosterone from
87
Leydig cells located in the testis [25]. Testosterone is the main male sex hormone responsible for
88
sexual behavior, and it plays a key role in the development of reproductive organs, such as the
89
testes and prostate, as well as in the production of spermatozoa [26].
90
Sperm production may be impaired by drugs capable of affecting the reproductive system
91
through different routes and mechanisms. These drugs may impair spermatogenesis and,
92
consequently, cause poor semen quality and sperm concentration, as well as morphological
93
changes in spermatozoa, which may lead to infertility or \reduced fertility [27,28].
ACCEPTED MANUSCRIPT 4 94
A recent study by Moreira et al. [18] it was observed that a 1.0 mg/kg dose of IVM
95
administered intraperitoneally reduced serum testosterone levels in male rats, and this effect was
96
attributed to the performance of the drug in GABAergic receptors. In fact, it is known that there
97
are peripheral GABAergic receptors present in the testes, which, when activated, reduce
98
testosterone release and, consequently, damage sperm production [29-31].
99
Due to testosterone responsibility on male sexual behavior, and the relevant role in sperm
100
production [26], the present study aims to investigate whether IVM is capable of interfering on
101
seminal parameters, serum testosterone levels and serum hematological and biochemical
102
parameters of rabbits for 1, 2, 3, 5, 7, 15, 30, 45, and 60 days after administration. The choice on
103
the animal species is justified by reduced population requirements (application of the principle of
104
3 Rs) [32,33], facility in semen collection and analysis methods and monitoring of seminal and
105
hormonal parameters in the same animal for up to 60 days after administration [34].
106 107
2. MATERIALS AND METHODS
108
2.1.
Animals
109
Healthy male New Zealand rabbits were used. They were kept in the Research Center of
110
Veterinary Toxicology (CEPTOX), Department of Pathology, School of Veterinary Medicine
111
and Animal Science, University of São Paulo (FMVZ/USP). The animals were housed
112
individually in cages, under controlled room temperature (22 ± 2°C), humidity (45–65%), and
113
artificial lighting (12 h/12 h light/dark cycle). The animals received 60 g per day for each kg of
114
the Coelhão® feed (Guabi, Sales Oliveira, Brazil) and free access to the filtered water. All of the
115
procedures were reviewed and approved by the Animal Care Committee of FMVZ-USP
116
(protocol no. 1892040315) and conformed to the guidelines of the Committee on Care and Use
117
of Laboratory Animal Resources, National Research Council, USA (1996). All efforts were
118
made to minimize animal suffering.
119 120
2.2.
Drugs
121
IVM (1% Ivomec® injectable, Merial Animal Health Ltda., Paulínia, SP, Brazil) was
122
administered subcutaneously (SC) at a dose of 0.2 or 1.0 mg/kg. Saline solution was
123
administered as a control solution (NaCl 0.9% - 1 ml/kg). The 0.2 mg/kg IVM dose was chosen
124
because it is the standard therapeutic dose used in several animal models [35,36]. The 1.0 mg/kg
ACCEPTED MANUSCRIPT 5 125
dose was chosen due to a previous study of our laboratory, in which it was observed that this
126
dose promoted impairment in the sexual behavior of male and female rats [16,21].
127 128
2.3.
Experimental design
129
Before the experimental period, the rabbits were evaluated according to their seminal
130
profile and classified according their potential fertility through complete andrological
131
examinations. All animals were within the recommended limits for the species [37].
132
Male rabbits (N = 18) were divided into three equal groups according to their seminal
133
profile: 0.2 IVM, 1.0 mg/kg IVM, and control solution (control group). All the sperm evaluation
134
procedures were performed on days 1, 2, 3, 5, 7, 15, 30, 45, and 60 after the administration of
135
IVM, considering that in the bioavailability and pharmacokinetic studies, in plasma analysis,
136
IVM has a half-life of 5 to 7 days [11,38]. We also considered the duration of spermatogenesis of
137
rabbits, which, according to MORTON et al. [34], is approximately 60 days. Blood samples from
138
the marginal ear vein were collected 1, 2, 7, 15, 30, and 60 days after the administration of IVM
139
for testosterone levels. Hematological and biochemical analyses were performed on days 1, 2,
140
and 7 after administration of IVM. Organ samples were collected on the 60th day after
141
administration of IVM for histopathological analysis.
142 143
2.4.
Semen collection and analysis
144
On days 1, 2, 3, 5, 7, 15, 30, 45, and 60 of the experiment, rabbit semen collections were
145
performed with the support of heated artificial vagina as proposed by ANDRADE et al. [39]. The
146
hygienization of the prepuce was performed with water and neutral soap before harvest to avoid
147
possible semen contamination [40]. Immediately after collection, the spermatozoa concentration,
148
spermatozoa motility, integrity of plasma and acrosomal and mitochondrial membrane potential,
149
and morphological characteristics of spermatozoa were evaluated.
150 151
2.4.1. SPERMATOZOA CONCENTRATION
152
To determine the sperm concentration, an aliquot of raw semen was diluted to 1:500 in
153
buffered formalin saline. The count was performed in a Neubauer chamber under light
154
microscopy, with a magnification of 400x.
155
ACCEPTED MANUSCRIPT 6 156
2.4.2. COMPUTER-ASSISTED SEMEN ANALYSIS
157
Spermatozoa motility was assessed using a computer-assisted sperm analysis (CASA-
158
Microptic®, Microptic S.L., Barcelona, Espanha). Samples were diluted in tyrode albumin lactate
159
pyruvate (TALP) to 20 x 106 spermatozoa/ml. An aliquot (5 µl) was placed on a pre-warmed
160
(36ºC) cover-slide and evaluated by phase-contrast microscope (Nikon, Model Eclipse 80i) with
161
100x magnification. The CASA set-up was pre-adjusted for rabbit sperm analysis (minimum
162
particle size 19 microns2, maximum particle size 79 microns2; number of frames: 25; frames per
163
second: 24; velocity limit for slow sperm, 10 microns s−1; velocity limit for medium sperm, 25
164
microns s−1; velocity limit for rapid sperm, 50 microns s−1; minimal straightness for progressive
165
spermatozoa, 70%; maximal linearity for circular spermatozoa, 50%. Five good fields were
166
examined to evaluate the following parameters: total motility (TM, %), progressive motility
167
(PROM, %), average path velocity (VAP, μm/s), curvilinear velocity (VCL, μm/s), straight-line
168
velocity (VSL, μm/s), amplitude of lateral head displacement (ALH, μm), beat cross frequency
169
(BCF, Hz), straightness (STR, %) and linearity (LIN, %) [41].
170 171 172
2.4.3. EVALUATION OF PLASMA AND ACROSOMAL MEMBRANE INTEGRITY AND MITOCHONDRIAL MEMBRANE POTENTIAL
173
After dilution of semen in TALP, an aliquot was stained with Hoechst 33342 (1.25
174
μg/mL), propidium iodide (1.5 μg/mL), fluorescein-conjugated Pisum sativum agglutinin (7.5
175
μg/mL), and 5,5',6,6'-tetrachloro-1,1,3,3'-tetraethylbenzimidazolylcarbocyanine iodide (612 μM),
176
then incubated at 37°C/15 min. After incubation, an aliquot (8 μl) was placed on a pre-warmed
177
(37ºC) cover-slide and evaluated, and 200 cells were counted by epifluorescence microscopy
178
(Nikon Epifluorescence Microscope, Model Eclipse 80i) in a triple filter (D/F/R, C58420) with
179
UV-2E/C assemblies (340-380 nm excitation and 435-485 emission), B-2E/C (excitation 465-
180
495 and emission 515-555) and G-2E/C (excitation 540 -525 and issue 605-655) at 1000x
181
magnification. Cells were classified into eight categories according to Celeghini et al. [42].
182 183
2.4.4. MORPHOLOGICAL CHARACTERISTICS OF SPERMATOZOA
184
The morphological characteristics of the spermatozoa were evaluated using the wet
185
chamber technique. For this, the semen was diluted and fixed in pre-heated, buffered saline
186
formol at 37°C. A diluted semen aliquot (8 μl) was placed between the slide and coverslip, and a
ACCEPTED MANUSCRIPT 7 187
sperm count of 200 sperm was magnified by 1000x under differential interference contrast
188
microscopy (Nikon®, model 80i). The sperm characteristics were classified as normal, minor,
189
and major defects [43].
190 191
2.5.
Evaluation of weight gain, relative organ weight, gonadosomatic index, and
192
histopathological study
193
On days 1, 30, and 60 of the experiment, rabbits were weighed on a scale (MARTE®,
194
model AD 10K) to measure body weight gain.
195
The liver, testes, epididymis, ventral prostate, and seminal vesicle were weighed on a
196
scale (MARTE®, model AL 500C), and the relative weight (RW) was calculated: RW = (organ
197
weight/body weight).
198
Representative fragments of the testes and epididymis were fixed in Bouin (75%
199
saturated picric acid, 25% formalin, 5% glacial acetic acid), while the representative fragments
200
of the liver and adrenals were fixed in 10% formaldehyde, dehydrated, diaphanized, and
201
embedded in paraffin (Sigma Chemical Co., St. Louis, MO). The material was then cut into 5-
202
µm-thick sections and stained with hematoxylin-eosin (HE) for histopathological analysis.
203 204
2.6. Evaluation of serum testosterone concentration
205
Serum testosterone levels were assessed using commercially available enzyme-linked
206
immunosorbent assays according to the manufacturer’s instructions (testosterone kit, Cayman
207
Chemical, Ann Arbor, MI, USA; catalog no. 582701).
208 209
2.7. Hematological and biochemical analyses
210
The collected blood sample was immediately expelled into separate heparinized plastic
211
vials for hematological analysis. The biomarkers determined were: red blood cells (RBCs),
212
hemoglobin (Hb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular
213
hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), eosinophils,
214
basophils, lymphocytes, and monocytes. An aliquot of blood was collected for biochemical
215
analysis. The serum was separated by allowing the blood (taken in a plain tube) to clot and then
216
centrifuged at 4,000 rpm for 10 min at 4ºC. The serum was analyzed for biochemical analysis of
217
aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), gamma-
ACCEPTED MANUSCRIPT 8 218
glutamyl transferase (GGT), globulins, albumin, total cholesterol, triglycerides, high-density
219
lipoprotein (HDL), urea, and creatinine.
220 221
2.8. Statistical analysis
222
Instat software (Prism 6.01, GraphPad, San Diego, CA, USA) was used for the statistical
223
analyses. The homoscedasticity was verified by the F or Bartlet test. Normality was checked by
224
the Brown-Forsythe test. One-way analysis of variance (ANOVA) followed by Dunnett's
225
multiple comparison post hoc test was used to analyze relative organ weight and gonadosomatic
226
index. Sperm concentration, sperm motility, plasma and acrosomal membrane integrity,
227
mitochondrial membrane potential, sperm morphological characteristics, biochemical analysis,
228
and serum testosterone levels were analyzed by two-way ANOVA, followed by the Dunnett’s
229
post hoc test. In all analyses, differences were considered significant when p <0.05. Data were
230
expressed as mean ± standard error of the mean (SEM).
231 232
3. RESULTS
233
No significant difference was observed among the groups on the spermatozoa
234
concentration, on the evaluation of plasma and acrosomal membrane integrity and mitochondrial
235
membrane potential, on the sperm morphology and on the serum testosterone levels in rabbits
236
(Fig. 1).
237 238 239 240
No significant difference was observed among the groups on the sperm motility characteristics obtained by computer-assisted semen analysis (Fig. 2). No significant differences in body weight gain were observed between the IVM-treated groups and control group (Fig. 3).
241
Relative organ weights and the gonadosomatic index in rabbits after IVM administration
242
were similar to the control group, with the exception of the relative weight of the liver (Fig. 3).
243
The one-way ANOVA revealed a significant effect of treatment on the relative liver weight
244
(F2,15 = 7.616, p = 0.0052). The Dunnett post hoc test revealed a decrease in the relative weight
245
of the liver in rabbits that were treated with 1.0 mg/kg of IVM on the day 60 after administration
246
compared with the control group.
247
The histopathological examination of rabbits that were euthanized on day 60 after
248
treatment did not reveal significant changes in morphology after IVM exposure (data not shown).
ACCEPTED MANUSCRIPT 9 249 250
No significant differences in hematological and biochemical analysis were observed between the IVM-treated groups and control group (data not shown).
251 252
4. DISCUSSION
253
The findings of this study showed that IVM at doses of 0.2 and 1.0 mg/kg of SC did not
254
alter any of the semen parameters of New Zealand rabbits evaluated for up to 60 days after
255
administration, differently of previous study that observed adverse effect on reproductive
256
parameters of rabbits at 30 and 60 days post-injection of 0.2 mg/kg IVM [44].
257
When administered at therapeutic doses (0.2 mg/kg), avermectins IVM and moxidectin
258
were found to have adverse effects on male fertility in Baladi rabbits, according to Eman and El-
259
Abdalla [44]. The authors observed that, 30 and 60 days after administration of these
260
avermectins, there was a decrease in sperm concentration, percentage of progressive motility,
261
and number of live spermatozoa. Comparing this study with the results of the present study,
262
differences in rabbit breed and sperm collection techniques can be observed. Eman and El-
263
Abdalla [44] collected spermatozoa from the tail of the epididymis. This technique, according to
264
Robaire and Viger [45] and Cornwall [46], allows the collection of spermatozoa with progressive
265
motility. However, the passage of the spermatozoa along the epididymal duct is what determines
266
the acquisition of the fertilizing capacity and the progressive motility, as this process dominates
267
sperm maturation [47]. In addition, collection of spermatozoa from the tail of the epididymis
268
does not allow them to be collected with seminal plasma. This fluid is rich in nutrients, such as
269
proteins and enzymes, and is essential for attesting to the viability of spermatozoa [34];
270
therefore, the absence of seminal plasma interferes with sperm viability and, consequently, with
271
progressive motility [34,37]. In contrast, in the present study the technique of semen collection
272
was used by means of an artificial vagina, which ensures the sperm viability, since in the semen,
273
the indispensable components for the progressive motile spermatozoa are present [48-50]. Thus,
274
it can be stated that sperm collection techniques are responsible for the differences between the
275
studies in relation to the results observed in the progressive motile spermatozoa of rabbits.
276
In another study that used the artificial vagina for collection of semen, but using another
277
animal species, partially corroborates the present findings. Thus, Naoman [38] observed that the
278
SC administration of 0.2 mg/kg IVM did not impair the sperm motility of sexually experienced
279
sheep. However, the author observed that in this dose there was a decrease in sperm
ACCEPTED MANUSCRIPT 10 280
concentration, testosterone levels and alteration in sperm morphology. These changes were no
281
longer observed 5 days after administration, which may be associated with the half-life of IVM,
282
which varies between 5 and 7 days [11,38].
283
Damage to sperm motility and fertilization potential may occur due to injury to the
284
plasma, acrosomal, and mitochondrial membranes of spermatozoa, since the integrity of these
285
membranes guarantees, respectively, sperm capacity and viability, fertilization, and sperm
286
motility [42]. In order to evaluate the damage to these membranes, fluorescence probes (PI,
287
FITC-PSA and JC-1) were used in the present work. The results obtained herein show that the
288
administration of 0.2 and 1.0 mg/kg of IVM did not cause damage to the plasma, acrosomal, and
289
mitochondrial membranes, corroborating the result of absence of alteration in sperm motility,
290
since damage to these membranes may harm the fertilization potential.
291
Testis weight has been used as a quantitative indicator of testis normality due to the
292
correlation between the weight of the testis and the number of germinal cells of the seminiferous
293
epithelium [51]. The germinal cells of the seminiferous epithelium have been found to be more
294
abundant in the testis. The results of the present study did not reveal alterations in the relative
295
weight of the testicles and accessory sexual glands, nor in the IGS, nor did they reveal
296
histopathological alterations in the sexual organs. Notably, the accessory sexual glands, such as
297
the prostate and seminal vesicle, respond differently to male and female sex hormones, and the
298
weights of these organs are bioindicators of the circulating levels of these hormones [52]. The
299
absence of changes in the testosterone levels of rabbits treated with IVM and evaluated for up to
300
60 days corroborate the evidence indicating that this drug did not disturb the testicular function,
301
that is, the production and transport of spermatozoa, as well as the production of testosterone.
302
On the other hand, El-Far [53] investigated the effects of IVM at doses of 0.5 and 1.0
303
mg/kg (SC) on testosterone levels and on serum biochemical parameters in male rabbits of the
304
New Zealand breed on days 1, 3, and 7 after administration. This author observed an increase in
305
serum testosterone levels and changes in hepatic and renal function over the 3 days of evaluation.
306
In this paper, the author did not explain the analytical method used for the evaluation of serum
307
testosterone and attributed the changes in hepatic and renal functions to the overload caused by
308
the drug in these organs. In this respect, the results of the present study showed no alterations in
309
any of the serum biochemical parameters, as well as hematological parameters in rabbits treated
310
with 0.2 and 1.0 mg/kg of IVM evaluated on days 1, 2, and 7 after the administration. There are
ACCEPTED MANUSCRIPT 11 311
few findings in the literature on the effects of IVM on serum and hematological biochemical
312
parameters in rabbits; there is only information on serum biochemical parameters, but in repeated
313
exposure to IVM in rabbits, causing alterations in these parameters [54].
314 315
5. CONCLUSIONS
316
In this temporal study of the administration of IVM in rabbits, at SC doses of 0.2 and 1.0
317
mg/kg, no changes were observed in male seminal parameters, as assessed by the concentration,
318
sperm motility and morphology, or fertilization potential, evaluated by the integrity of the
319
spermatozoa's plasma, acrosomal, and mitochondrial membranes. Furthermore, even at the
320
highest dose, IVM did not interfere with serum levels of testosterone, serum biochemistry, and
321
blood count parameters.
322 323
6. ACKNOWLEDGMENTS
324
This work is part of the doctoral thesis of Natalia Moreira and Vagner Gonçalves Jr. to
325
the School of Veterinary Medicine and Animal Science, University of São Paulo, and was
326
supported by grants from Fundação de Apoio à Pesquisa do Estado de São Paulo (FAPESP;
327
grant number 2015/03131-4) and from Conselho Nacional de Desenvolvimento Científico e
328
Tecnológico (CNPq; grant number 305500/2013-9).
329 330 331 332 333 334 335 336 337 338
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[13] Bormann J. The “ABC” of the GABA receptors. Trends Pharmacol Sci 2000; 21: 16-9.
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[14] Rodrigues-Alves PSB, Lebrun I, Florio JC, Bernardi MM, Spinosa HS. Moxidectin
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interference on sexual behavior, penile erection and hypothalamic GABA levels of male
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Bernandi MM, Spinosa HS. Ivermectin reduces motor coordination, serum testosterone, and
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[21] Moreira N, Bernardi MM, Spinosa HS. Ivermectin reduces sexual behavior in female rats. Neurotoxicol Teratol 2014; 43: 33–8. [22] Johnson L, Petty CS, Neaves WB. A comparative study of daily sperm producion and testicular composition in humans and rats. Biol Reprod 1980; 22: 1233-44. [23] O’donnel L, Robertson KM, Jones ME, Simpson ER. Estrogen and spermatogenesis. Endocr Rev 2001; 22: 289-318.
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[24] McLachlan RI, Wreford NG, O’donnell L, De Kretser DM, Robertson DM. The endocrine
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regulation of spermatogenesis: independent roles for testosterone and FSH. J Endocrinol
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1996; 148: 1-9.
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[25] Shulman LM, Spritzer MD. Changes in the sexual behavior and testosterone levels of male rats in response to daily interactions with estrus females. Physiol Behav 2014; 133: 08–13. [26] Matos AFG, Moreira RO, Guedes EP. Neuroendocrinology of the metabolic syndrome. Arq Bras Endocrinol Metab 2003; 47: 410-20. [27] Bonde JP. Male reproductive organs are at risk from environmental hazards. Asian J Androl 2010; 12: 152-56. [28] Wong EWP, Cheng CY. Impacts of environmental toxicants on male reproductive dysfunction. Trends Pharmacol Sci 2011; 32: 290-99, 2011. [29] He XB, Hu JH, Wu Q, Yan YC, Koide SS. Identification of GABA(B) receptor in rat testis and sperm. Biochem Biophys Res Commun 2001; 283: 243-47. [30] He X, Zhang Y, Yan Y, Li Y, Koide SS. Identification of GABABR2 in rat testis and sperm. J Reprod Dev 2003; 49: 397-402. [31] Li S, Zhang Y, Liu H, Yan Y, Li Y. Identification and expression of GABAC receptor in rat testis and spermatozoa. Acta Biochim Biophys Sin 2008; 40: 761-67. [32] Presgrave OAF. Alternativas para animais de laboratório: do animal ao computador. 1st ed. Rio de Janeiro: FIOCRUZ; 2002.
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[33] Cazarin KCC, Corrêa CL, Zambrone FAD. Redução, refinamento e substituição do uso de
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animais em estudos toxicológicos: uma abordagem atual. Rev Bras Ciênc Farm 2004; 40:
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[34] Morton D. The Use of Rabbits in Male Reproductive Toxicology. Environ Health Perspect 1988; 77: 5-9.
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[35] Dadarkar SS, Deore MD, Gatne MM. Comparative evaluation of acute toxicity of
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ivermectin by two methods after single subcutaneous administration in rats. Regul Toxicol
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Pharmacol 2007; 47: 257-60.
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[36] Sartor IF, Santarém VA. Agentes empregados no controle de ectoparasitos. In: Spinosa HS,
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Górniak SL, Bernardi MM, editors. Farmacologia aplicada à medicina veterinária, Rio de
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Janeiro: Guanabara Koogan; 2017, p. 599-608.
412 413 414 415 416 417
[37] Foote RH, Carney EW. The rabbit as a model for reproductive and developmental toxicity studies. Reprod Toxicol 2000; 14: 477-93. [38] Naoman UD. Effect of ivermectin on semen characteristics of Iraqi Awassi ram. Al-Anbar J Vet Sci 2012; 5: 129-33. [39] Andrade AFC, Yonezawa LA, Celeghini ECC, Spers A, Arruda RP. Um novo modelo de vagina artificial para coelhos. Rev Bra Reprod Anim 2002; 26: 201-04.
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[40] Celeghini ECC, Arruda RP, Andrade AFC, Nascimento J, Raphael CF, Rodrigues PHM.
419
Effects that bovine sperm cryopreservation using two different extenders has on sperm
420
membranes and chromatin. Anim Reprod Sci 2008; 104: 119-31.
421
[41] Mortimer ST. CASA - practical aspects. J Androl 2000; 21: 515–24.
422
[42] Celeghini ECC, Nascimento J, Raphael CF, Andrade AFC, Arruda RP. Simultaneous
423
assessment of plasmatic, acrosomal, and mitochondrial membranes in ram sperm by
424
fluorescent probes. Arq Bras Med Vet Zootec 2010; 62: 536-43.
425
[43] Janett F, Thun R, Ryhiner A, Burger D, Hassig M, Hertzberg H. Influence of Eqvalan
426
(ivermectin) on quality and freezability of stallion semen. Theriogenology 2001; 55: 785-92;
427
[44] Emam EE, El-Abdalla O. Effects of ivermectin and moxidectin on fertility and some
428 429 430 431 432 433 434
biochemical parameters in male rabbits. Egyptian J Agric Res 2000; 78: 293-301. [45] Robaire B, Viger RS. Regulation of epididymal epithelial cell functions. Biol Reprod 1995; 52: 226-36. [46] Cornwall GA. New insights into epididymal biology and function. Hum Reprod Update 2009; 15: 213-27. [47] Oliva SU, Rinaldo PA, Stumpp T. Biologia epididimária: maturação espermática e expressão gênica. O Mundo da Saúde 2009; 33: 419-25.
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[48] Constantini FFA. Nel coniglio, sistemi de conservazione dello sperma. Riv di coniglicoltura 1989; 4: 14-8. [49] Rodriguez RM. Una vagina artificial para la extraccion de semen de conejo. Rev Cub Reprod Anim 1993; 17-18: 200-02. [50] Sinkovics G, Cenci T. Scuota, S, Dal Bosco A. Un’idea per la F.A. Riv di coniglicoltura 1993; 9: 35-6. [51] Russel L, Ettlin R, Hikin A, Clegg E. Histological and histopathological evaluation of the testis. Int J Androl 1990; 16: 83.
443
[52] Foote RH, Draddy PJ, Breite M, Oltenacu EAB. Action of androgen and estrone implants
444
on sexual behavior and reproductive organs of castrated male rabbits. Horm Behav 1977; 9:
445
57-68.
446
[53] El-Far AH. Effect of therapeutic and double therapeutic doses of ivermectin on oxidative
447
status and reproductive hormones in male rabbits. Am J Anim Vet Sci 2013; 8: 128-33.
448
[54] Al-Jassim KB, Jawad AADH, Al-Masoudi EA, Majeed SK. Histopathological and
449
biochemical effects of ivermectin on kidney functions, lung and the ameliorative effects of
450
vitamin c in rabbits (Lupus cuniculus). Basrah J Vet Res 2016; 14: 110-24.
451
ACCEPTED MANUSCRIPT 16 452
8. FIGURE LEGENDS
453
Figure 1. Effects of administration of ivermectin (IVM 0.2 or 1.0 mg/kg) or control solution (1.0
454
ml/kg - control group) on the spermatozoa concentration, on the evaluation of plasma and
455
acrosomal membrane integrity and mitochondrial membrane potential, on the sperm morphology
456
and on the serum testosterone levels in rabbits 1, 2, 3, 5, 7, 15, 30, 45, and 60 days after
457
administration of 0.2 or 1.0 mg/kg IVM or control solution (1.0 ml/kg). The data are expressed
458
as mean ± SEM. N = 6 animals per group. p > 0.05, compared with the control group (two-way
459
ANOVA followed by Dunnett post hoc test). Plasma membrane integrity
Sperm concentration 100
400
200
0
1
2
3
5
7
15
30
45
60
60 40 20 0
days
Acrosomal membrane integrity
2
3
5
100
50
1
2
3
5
7
15
30
45
60
80 60 40 20 0
days
1
2
3
Percentage (%)
Percentage (%)
60 40 20
2
3
5
7
15
30
45
60
days
45
60
days
1
2
3
15 10 5
1
2
3
5
7
15
30
5
7
15
30
45
60
days
Testosterone
45
60
days
Concentration of testosterone (ng/ml)
Percentage (%)
30
20
Minor defects
461
15
40
0 1
20
460
7
60
80
0
5
Major defects
Normal sperm 100
0
15 30 45 60 days
7
100
Percentage (%)
Percentage (%)
1
Active mitochondrial membrane
150
0
Control 0.2 mg/kg 1.0 mg/kg
80
Percentage (%)
106 sperm/ml
600
10 8 6 4 2 0
1
2
7
15
30
60
days
ACCEPTED MANUSCRIPT 17 462
Figure 2. Effects of administration of ivermectin (IVM 0.2 or 1.0 mg/kg) or control solution (1.0
463
ml/kg - control group) on the spermatozoa motility (CASA) in male rabbits 1, 2, 3, 5, 7, 15, 30,
464
45, and 60 days after administration of 0.2 or 1.0 mg/kg IVM or control solution (1.0 ml/kg). The
465
data are expressed as mean ± SEM. N = 6 animals per group. p > 0.05 compared with the control
466
group (two-way ANOVA followed by Dunnett post hoc test).
467 468
ACCEPTED MANUSCRIPT 18 469
Figure 3. Effects of administration of ivermectin (IVM 0.2 or 1.0 mg/kg) or control solution (1.0
470
ml/kg - control group) on the body weight gain, the relative weight of organs (liver, prostate,
471
testes, epididymis, adrenals, and seminal vesicle) and rabbit gonadosomatic index. The data are
472
expressed as mean ± SEM. N = 6 animals per group. **p < 0.01 compared with the control group
473
(one-way ANOVA followed by Dunnett post hoc test). Liver
Prostate Organs Relative weight (g)
Body weight gain (g)
250 200 150 100 50 0
0-30
31-60
days
4
**
3 2 1 0
Control
0.05
0.2 mg/kg
1.0 mg/kg
0.10
0.05
0.00
Control
0.2 mg/kg
1.0 mg/kg
0.010
0.005
0.000
1.0 mg/kg
Control
0.2 mg/kg
474
Control
0.06 0.04 0.02
Control
0.2 mg/kg
1.0 mg/kg
0.2 mg/kg
1.0 mg/kg
0.06
0.04
0.02
0.00
Control
1.0 mg/kg
1.0 mg/kg
0.010
0.005
0.000
Control
0.006
0.004
0.002
0.000
0.2 mg/kg
0.015
Gonadossomatic index Organs Relative weight (g)
Organs Relative weight (g)
Seminal vesicle 0.08
0.00
0.00
Left adrenal
0.015
Organs Relative weight (g)
Organs Relative weight (g)
Organs Relative weight (g)
0.02
0.2 mg/kg
0.01
Right adrenal
0.04
Control
0.02
Right epididymis
0.15
Left epididymis 0.06
0.00
Control 0.2 mg/kg 1.0 mg/kg
0.03
1.0 mg/kg
Organs Relative weight (g)
0.10
Control
0.2 mg/kg
0.04
Left testis Organs Relative weight (g)
Organs Relative weight (g)
Right testis 0.15
0.00
Organs Relative weight (g)
Body weight gain
Control
0.2 mg/kg
1.0 mg/kg
0.2 mg/kg
1.0 mg/kg