Small Ruminant Research 144 (2016) 220–224

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Small Ruminant Research

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Short communication

Sperm quality in naturally infected rams with Brucella ovis

José Maria Carrera-Chávez (Professor-Researcher) a,∗,1 , Andrés Quezada-Casasola a ,
Eduardo Pérez-Eguia a , Mateo Fabián Itzá-Ortíz a , José Luis Gutiérrez-Hernández b ,
Juan Alberto Quintero-Elisea a , Jorge Luis Tórtora-Pérez c
a
Departamento de Ciencias Veterinarias, Instituto de Ciencias Biomédicas, Universidad Autónoma de Ciudad Juárez, Anillo Envolvente del Pronaf y
Estocolmo s/n, Zona Pronaf 35315 Ciudad Juárez, Chihuahua, Mexico
b
Centro Nacional de Investigación Disciplinaria Microbiología (CENID-Microbiología), Instituto Nacional de Investigaciones Forestales, Agrícolas y
Forestales, Km 15.5 Carretera Federal México-Toluca, Col. Palo Alto 05110, Ciudad de México, Mexico
c
Facultad de Estudios Superiores Cuautitlán, Universidad Nacional Autónoma de México, Km 2.5 Carretera Cuautitlán-Teoloyucan, 54714 Cuautitlán
Izcalli, Estado de México, Mexico

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

Article history: Ovine epididymitis caused by Brucella ovis is a chronic disease, characterized by testicular and epididymal
Received 10 March 2016 alterations, which cause loss of fertility in rams. However, infected rams without clinical signs, may
Received in revised form 9 August 2016 have decreased fertility and be excreting the bacteria. The aim of this study was to evaluate sperm
Accepted 24 September 2016
quality in serologically positive to B. ovis naturally infected rams. Seminal samples were obtained of
Available online 1 October 2016
207 rams to evaluate seminal volume, sperm concentration, mass motility, vitality, presence of somatic
cells and percentage of abnormalities. Clinical examination was performed by palpation. The serological
Keywords:
response against B. ovis was evaluated by double immunodiffusion in gel. The percentage variables were
Brucella ovis
Sperm quality
analyzed by GENMOD procedure and numerical variables were analyzed by ANOVA and differences were
Epididymal alterations assessed using Tukey’s test. 18.84% of the rams were positive against B. ovis. There was no difference in
scrotal circumference and volume (P > 0.05) between positive and negative animals for B. ovis. Sperm
concentration, mass motility, vitality, and the percentage of sperm with abnormalities, were modified
in positive animals (P < 0.05), regardless of the presence of palpable lesions (P < 0.05). The percentage of
rams with lesions in the epididymis and severe presence of somatic cells was higher in positive animals
(P < 0.05). B. ovis infection adversely affects semen characteristics, due to an increase in epididymal lesions
and by the severe presence of inflammatory cells in the ejaculate. Serological analysis to determine
possible infection by B. ovis, should be considered as a component of the evaluation of fertility in rams,
considering that sperm quality was affected in rams without lesions, but seropositive for B. ovis.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction
Ovine epididymitis caused by Brucella ovis is a clinical or sub-
clinical disease of chronic evolution, characterized by testicular
changes derived from the inflammatory process in the epididymis
(Machado et al., 2015), with seminal vesiculitis and ampulitis, fol-
lowed by orchitis (Paolicchi et al., 2000) with subsequent loss of
fertility in rams (Ridler and West, 2011). Although this species of
Brucella is not zoonotic, it is one of the main causes of reproduc-
tive failure in ovine flocks (Wiemer and Ruttle, 1987). It reduce
the reproductive life of rams, increases the sacrifice of rams due to
∗ Corresponding author.
E-mail address: jose.carrera@uacj.mx (J.M. Carrera-Chávez).
1 this evaluation is carried out before the start of the breeding season,
Present address.
chronic injuries (Ridler and West, 2011) and reduce herd produc-
tivity due to decreased fertility (Mejid et al., 2010).
For large scale diagnosis, the use of serological tests is recom-
mended, being gel diffusion test, enzyme linked immunosorbent
assay and complement fixation tests used mostly (Ridler and West,
2011). Infected rams may have intermittent excretion or no excre-
tion of bacteria, which makes bacteriological analysis not practical
or reliable. The clinical diagnosis by scrotal palpation is valid only
if the animal has developed lesions and should be considered that
epididymitis may be caused by other bacteria; moreover, infected
rams without apparent clinical signs, may excrete the bacteria
(Ficapal et al., 1998; Mejid et al., 2010).
The evaluation of fertility in rams usually includes examination
of the general health status, body condition, a careful examination
of the genitals and evaluation of semen quality (Ridler et al., 2012);

http://dx.doi.org/10.1016/j.smallrumres.2016.09.021
0921-4488/© 2016 Elsevier B.V. All rights reserved.
 J.M. Carrera-Chávez et al. / Small Ruminant Research 144 (2016) 220–224
Table 1
Seminal and spermatic characteristics in naturally infected rams serologically positive to Brucella ovis with or without the presence of palpable epididymal lesions and somatic cells.

Scrotal Volume (ml) Sperm Mass motility Vitality Abnormalities Abnormalities in Tailless
circumference (cm) concentration+ (percentage) (percentage) (percentage) the tail* (percentage)
(percentage)

Global 32.87 ± 0.28A 0.84 ± 0.04A 1897.4 ± 136.7A 0.54 ± 0.02A 71.12 ± 2.42A 6.98 ± 0.91A 3.54 ± 0.28A 3.49 ± 0.78A
Negatives 81.16% (168/207) Without lesions 32.70 ± 0.32a 0.72 ± 0.4a 1962.6 ± 139.8a 0.59 ± 0.01a 75.44 ± 2.18a 5.34 ± 0.53a 3.35 ± 0.28a 1.98 ± 0.33a
With lesions 33.80 ± 0.55a 0.86 ± 0.12a 1597.0 ± 418.0ab 0.30 ± 0.05b 48.76 ± 8.06b 11.90 ± 2.96ab 4.05 ± 0.98ab 10.97 ± 4.17b
Global 32.69 ± 0.56A 0.77 ± 0.10A 1188.0 ± 236.1B 0.27 ± 0.05B 49.64 ± 6.69 B 16.70 ± 3.15B 6.41 ± 1.84B 10.29 ± 2.50B
Positives 18.84% (39/207) Without lesions 32.84 ± 0.72a 0.87 ± 0.13a 1345.3 ± 327.4b 0.31 ± 0.06b 53.72 ± 8.49b 16.44 ± 4.03b 7.83 ± 2.81b 8.61 ± 2.58b
With lesions 32.43 ± 0.92a 0.78 ± 0.19a 916.4 ± 309.6b 0.21 ± 0.08b 42.30 ± 11.03b 17.15 ± 5.29b 3.85 ± 0.79a 13.30 ± 5.32b

Somatic cells
None 32.99 ± 0.31c 0.77 ± 0.04c 1935.4 ± 133.5c 0.54 ± 0.02c 69.81 ± 2.64c 8.51 ± 1.11cd 3.52 ± 0.28c 4.99 ± 0.97c
Moderate 33.55 ± 0.48c 0.73 ± 0.05c 1133.3 ± 356.3cd 0.28 ± 0.07d 67.99 ± 4.90c 5.94 ± 4.74c 4.12 ± 1.24c 1.81 ± 0.62c
Severe 32.07 ± 0.50c 0.72 ± 0.08c 519.2 ± 143.29d 0.18 ± 0.05d 47.65 ± 8.21d 17.75 ± 1.11d 11.60 ± 4.60d 6.15 ± 1.29c
A,B
Different letters within the same column indicate significant differences among positive and negative rams for B. ovis (Global) (P < 0.05).
a,b
Different letters within the same column indicate significant differences among rams with or without lesions in positive and negative rams for B. ovis (P < 0.05).
c,d
Different letters within the same column indicate significant differences among scores of presence of somatic cells; results are shown as pooled values of positive and negative rams (P < 0.05).
+Millions of sperm.
*Looped tail, bent tail and coiled tail.

221
222 J.M. Carrera-Chávez et al. / Small Ruminant Research 144 (2016) 220–224
during the selection of replacements, or when there are reproduc-
tive issues exist in the flock (Gouletsou and Fthenakis, 2010; Ridler
et al., 2012).
Although authors have evaluated the relationship between pal-
pable lesions of epididymitis and semen quality in rams (Carvalho
et al., 2012), according to the literature review, sperm quality in
rams serologically positive to B. ovis naturally infected has not been
evaluated before. Therefore, the aim of this study was to evaluate
sperm quality in serologically positive to B. ovis naturally infected
rams.
2. Material and methods
Sample sites are located in the state of Zacatecas, México (21◦ 04 43 – 25◦ 00 51
Latitude N and 101◦ 29 58 – 104◦ 20 03 Longitude W). All experimental proce-
dures were conducted according to protocols approved by the Unidad Académica
de Medicina Veterinaria y Zootecnia de la Universidad Autónoma de Zacatecas Ani-
mal Care and Use Committee. Serological and seminal samples were collected from
207 adult rams (1–6 years old). The breed frequency of the animals sampled was:
77 Dorper, 66 Katahdin, 27 Rambouillet, 8 Charolais, 7 Polipay, 7 Ile de France, 7
Blackbelly, 7 Suffolk and 1 Pelibuey.
In order to assess the relationship between prevalence of B. ovis and the age of
rams, the following categories were established: 1 to 2 years, 2 to 3 years, 3 to 4
years, 4 to 5 years, and over 5 years.
Clinical examination was performed by manual palpation of both testicles and
epididymis simultaneously, compared to each other; symmetry, size, shape and con-
sistency of the both testicles was assessed and recorded. The epididymides were
palpated, starting from the head, continuing to the body and the tail, and compared
to each other; their size and resilience was checked and recorded. Scrotal circumfer-
ence was measured using a metric tape at the widest point (Gouletsou and Fthenakis,
2010).
Collection of seminal samples was carried out using a 6 v manual ejaculator (Bai-
ley Ejaculator, Western Instrument Company, Denver, Colorado) and were evaluated
for volume, presence of somatic cells, sperm concentration by spectrophotome-
try (Acucell, IMV Technologies, France), mass motility, vitality and percentage of
abnormalities (tailless, looped, bent and coiled tails). Presence of somatic cells in
the ejaculate was determined by direct observation with light microscope (100x)
and subjectively scoring slides (None: 0 somatic cells; Moderate: 1 to 10 somatic
cells; and Severe: more than 10 somatic cells per microscopic field). To assess mass
motility, a semen sample without dilution was placed on a slide at 30 ◦ C, and was
subjectively evaluated on a scale of 0 to 100 under light microscopy (40x). Vitality
and abnormal sperm percentages were calculated in a count of 100 eosine-nigrosine
stained cells per sample (diluted 1:1 semen/stain). Stained cells in the smear were
counted as dead, while unstained cells were considered as live cells (Chemineau
et al., 1991).
Immediately after the semen collection, the blood samples were collected in
tubes with a clot activator (BD Vacutainer, Becton, Dickinson and Company, Franklin
Lakes, NJ, USA). The blood samples were centrifuged at 1500 × g for 10 min, and the
serum was separated and kept frozen at −20 ◦ C until laboratory analysis. Serum
was evaluated with the double immunodiffusion (DID) test in gel, using as antigen
a protein warm saline extract from the outer membrane of REO-198 B. ovis strain
(Alton et al., 1988). Tests were performed in the Laboratory of Diagnosis of the CENID
Animal Microbiology of INIFAP (México-Toluca Federal Highway 15.5 km Col. Palo
Alto, Cuajimalpa, México, D.F.).
Analysis of the all data was performed using SAS statistical package (SAS,
2009/STAT version 9.3, SAS Institute Inc., Cary, NC). Variables in the present study
were analyzed for normality using the UNIVARIATE procedure. To determine nor-
mality of data, descriptive statistics, histograms and Shapiro-Wilk tests (P > 0.05)
were considered. Categorical variables such as presence of palpable lesions and pres-
ence of somatic cells in the ejaculate were analyzed with a Chi square test using the
GENMOD procedure. This model included serological status (positive or negative as
statistical treatments) and breed of rams (Dorper, Katahdin, Rambouillet, Charol-
lais, Polipay, Ile de France, Blackbelly, Suffolk and Pelibuey), however, breed was
excluded from the analysis, since no effect was observed and some of the breeds
had very few animals. Additionally, to determine the influence of ram age on the
presence of the B. ovis seroprevalence, odds ratios (OR; probability that the disease
occurs in an age category against the risk of it occurring in another) were calculated,
using the Chi square Mantel-Haenszel test at a confidence interval (CI) of 95%.
Numerical data of scrotal circumference (cm), ejaculate volume (ml), sperm
concentration (millions of sperm), mass motility (percentage), vitality (percentage)
and percentage of abnormalities were considered as dependent variables and were
analyzed with an ANOVA using the GLM procedure. All percentage data were trans-
formed using arcsine transformation before analysis. Initially, a randomized block
design was used, with ram breed used as a blocking factor and serologic status of
the rams (positive or negative), detectable lesions (presence or absence) or somatic
cells (presence or absence) as independent variables (statistical treatments). Since
Table 2
Lesions and presence of somatic cells in semen from naturally infected rams sero-
logically positive to Brucella ovis (Percentage* ).
Lesion Negatives Positives
No lesion 84.52 (142/168)a 69.44 (25/39)b
Hypoplasia 2.97 (5/168)a 2.56 (1/39)a
Unilateral Epididymitis 8.93 (15/168)a 28.21 (11/39)b
Bilateral Epididymitis 5.95 (9/168)a 12.82 (5/39)b
Fibrosis 3.57 (4/168)a 17.95 (7/39)b
Somatic Cells
None 86.31 (145/168)a 71.80 (28/39) a
Moderate 6.55 (11/168)a 7.69 (3/39)a
Severe 7.14 (12/168)a 20.51 (8/39)b
*
The percentage is greater than 100% because some rams (26.92% of negative rams
and 71.43% of positive rams) had two lesions simultaneously.
a,b
Different letters within the same line indicate significant differences among pos-
itive and negative animals for B. ovis (P < 0.05).
no effect of the ram breed was observed, it was also excluded from this analysis and
a completely randomized design with the following model was used:
Y i,j =  + T i + D i,j
Yi , j = Dependent variable result,
= Overall mean,
Ti = Effect of serological status of rams, lesions or somatic cells,
D i , j = Experimental error.
Resulting data were compared with a Tukey test when differences between
treatments were observed (P ≤ 0.05).
3. Results
From the sampled rams, 18.84% were seropositive to B. ovis, and
30.56% of seropositive rams had testicular or epididymal lesions;
however, only 15.48% of seronegative rams showed lesions. There
was no significant difference for scrotal circumference and ejac-
ulate volume between serologically positive and negative B. ovis
rams (P > 0.05), regardless of presence of testicular and epididymal
lesions (P > 0.05). However, sperm concentration, mass motility,
vitality, percentage of abnormalities, percentage of sperm tail-
less and percentage of sperm with tail abnormalities (P < 0.05)
were negatively affected in serologically positive animals to B. ovis,
regardless of presence of epididymal lesions (P < 0.05) (Table 1).
Regarding lesions, a greater percentage of serologically positive
animals showed lesions compared to negative animals (P < 0.05).
These lesions occurred only in the epididymis; positive rams to B.
ovis presented unilateral or bilateral epididymitis, and fibrosis in
greater proportion than negative rams (P < 0.05). There was no sta-
tistical difference for hypoplasia testicular between serologically
positive and negative B. ovis rams (P > 0.05) (Table 2).
The percentage of rams that showed severe presence of somatic
cells was higher in serologically positive rams (P < 0.05), but there
was no difference in the percentage of rams that showed scant
somatic cells (P > 0.05) (Table 2).
A trend of greater prevalence of seropositive animals was
observed in rams over two years of age, especially in the range of
3–5 years of age (P = 0.08). Although the odds ratio increased to 3.37
from the second year, is even greater in rams that were between
4 and 5 years old (6.61) than in rams included in the category of
more than 5 years (5.18) (Table 3).
4. Discussion
Results of the present study indicate a significant affectation of
semen quality, decreasing sperm concentration, mass motility and
vitality, and increased sperm abnormalities in serologically posi-
tive rams to B. ovis, regardless of the presence or absence of clinical
epididymitis in positive rams, in comparison with negative rams
without lesions. Similarly, mass motility and vitality variables also
 J.M. Carrera-Chávez et al. / Small Ruminant Research 144 (2016) 220–224
Table 3
Percentage of serologically positive animals, odds ratio (OR) and confidence intervals
(CI) for prevalence of Brucella ovis regarding to ram age.
Ram age Percentage OR
1 to 2 years 5.00 (1/20) 1.00
2 to 3 years 15.07 (11/73) 3.37
3 to 4 years 22.22 (12/54) 5.43
4 to 5 years 29.03 (9/31) 6.61
Over 5 years 21.43 (6/28) 5.18
exists difference between negative without lesions compared to
negative with clinical lesion in the epididymis. In experimentally
infected rams, changes in sperm morphology was reported from
day 30 post-infection (PI), with a significant increase in tailless
sperm and sperm with abnormalities in the tail (Carvalho et al.,
2012). These abnormalities are related with decreased fertility,
since in the epididymis sperm cells acquire greater motility and
ability to fertilize (Samplaski et al., 2010). It has been suggested in
experimental infections the involvement of mast cells in the devel-
opment and maintenance of testicular disorders such as idiopathic
infertility associated to peritubular fibrosis, hypogonadism, dys-
function of the blood-testis barrier or testicular atrophy (Paolicchi,
2001).
In the present study, the infection with B. ovis occurred natu-
rally; considering this, the percentage of rams serologically positive
that presented any testicular and epididymal lesion was lower
(30.56%) than the one observed in other studies with experimen-
tally infected rams. Paolicchi et al. (2000) reported 71.4% of genital
abnormalities, while Carvalho et al. (2012) reported that 66.7% of
infected animals showed palpable changes from day 30 PI. Blasco
et al. (1982) reported that in naturally infected animals, 48% of
the rams showed observable physical changes; also, 35% of rams
evaluated and 73% of clinically affected were positive for B. ovis.
The most characteristic sign of the disease, epididymitis, usu-
ally occurs in the tail, although there may be lesions throughout
the epididymis. However, some infected rams may show palpa-
ble lesions in an exam and be clinically normal within weeks or
never show lesions (Mejid et al., 2010). In a retrospective study
done by Van Metre et al. (2012) indicated that 53.6% of seropositive
rams to B. ovis had a normal fertility evaluation, without abnor-
malities detected on physical examination or semen. Therefore, it
is essential that the diagnosis of B. ovis is carried out by serologi-
cal diagnosis, and be part of the evaluation of fertility in rams, since
other bacteria can cause epididymitis or there may be infected sires
without apparent clinical signs (Ficapal et al., 1998; Mejid et al.,
2010), which may have subfertility or maintain normal fertility,
increasing the risk of infection in the flock (Xavier et al., 2009).
Carvalho et al. (2012) reported that 44.4% of experimentally
infected rams showed the presence of somatic cells (neutrophils,
macrophages and lymphocytes) in the ejaculate from day 30 PI;
however, in that study, only one of nine animals showed abundant
somatic cells. In the present study, in seropositive animals, 28.20%
showed the presence of somatic cells in the semen, and 20.51%
showed severe presence of somatic cells. Infection with B. ovis in
addition to causing macroscopic and histological lesions, induces
autoimmune responses and increased presence of inflammatory
cells that produce cytokines and free radicals, alterations that may
be involved in the poor quality sperm present in infected animals
(Paolicchi et al., 2000). In this study, the rams which have severe
presence somatic cells showed a lower quality sperm (decreas-
ing sperm concentration, mass motility and vitality, and increased
sperm abnormalities) than those who did not present somatic cells
in semen; even those who had moderate somatic cells had lower
sperm mass motility. Reports indicate that the presence of leucocy-
tospermia in animals infected with B. ovis is related to a reduction in
the motility and fertilization ability of the sperm, since leukocytes
223
have a negative impact on semen quality as a result of the pres-
ence of reactive oxygen species, mainly produced by leukocytes,
which damages the sperm (Lackner et al., 2010). This oxidant effect
CI (95%) would be one of the causes of reduced semen quality in serologically
positive rams.
0.41–27.82 Regarding the age of the ram and the percentage of serologi-
0.65–44.81 cally positive animals to B. ovis, the odds ratio was increased from
0.76–57.64
the second year of age; however, rams included in the category of
0.57–46.96
between 3 and 5 years were the most affected, which had a higher
odds ratio even in comparison with rams included in the category
of over 5 years. Other authors have reported a similar trend; Ficapal
et al. (1998) indicate that animals between 2 and 5 years were most
affected by B. ovis, with more serological prevalence and increased
incidence of testicular macroscopic lesions that younger or older
animals. Also, Van Metre et al. (2012) report that there is a higher
odds ratio for failures in the evaluation of fertility by inflamma-
tory causes in rams of 2 to 5 years of age compared to younger or
older rams. This may be due to the fact that rams of this age group
are more sexually active, which increases the risk of infectious dis-
eases involving sexual transmission, while younger animals have
little sexual experience and are subordinated to older rams, which
may favor that they become infected by dominant animals positive
to B. ovis (the main route of infection is sexual contact between
males while they are separated from the females); in the case of
rams with more than 5 years of age, the lower odds ratio may be
due to an age-related decreased sexual activity, which reduces the
risk of infection.
5. Conclusions
Natural infection with B. ovis adversely affects semen quality,
in part due to an increase in epididymal lesions and the severe
presence of inflammatory cells in the ejaculate, especially in rams of
3 to 5 years of age. Since these clinical signs are often inconspicuous
or the lesions are not present, serological analysis is required, which
should be considered as a component of the evaluation of fertility
in rams, considering that sperm quality was affected in seropositive
for B. ovis rams without lesions, which were not different to rams
that were seronegative for B. ovis with lesions.
Conflict of interest
The authors confirm that there is no conflict of interest associ-
ated with the publication of this manuscript.
Acknowledgements
This study was partly supported by the Fundación Produce
Zacatecas A.C. (Zacatecas, México) (Project number: 32-2010-
0013). The authors thank participating farmers for their partici-
pation in the project.
References
Alton, G.G., Jones, L.M., Angus, R.D., Verger, J.M., 1988. Brucella ovis. Techniques for
the Brucellosis Laboratory. Institut National de la Recherche Agronomique
(INRA), Paris.
Blasco, J.M., Folch, J., Barberan, M., 1982. Características reproductivas de
moruecos Romanov afectados de epididimitis infecciosa. I. Observaciones
clínicas y alteraciones de la fertilidad. Anales del Instituto Nacional de
Investigaciones Agrarias. Serie Ganadería. 15, 67–76.
Carvalho, J.C.A., Moustacas, V.S., Xavier, M.N., Costa, E.A., Costa, L.F., Silva, T.M.A.,
Paixão, T.A., Borges, A.M., Gouveia, A.M.G., Santos, R.L., 2012. Andrological,
pathological, morphometric, and ultrasonographic findings in rams
experimentally infected with Brucella ovis. Small Rumin. Res. 102, 213–222.
Chemineau, P., Cagnié, Y., Guerin, V., Orgeur, P., Vallet, J.C., 1991. Collection and
Preservation of Spermatozoa. Training Manual on Artificial Insemination in
Sheep and Goats, first ed. Monnaie: FAO, Rome.
224 J.M. Carrera-Chávez et al. / Small Ruminant Research 144 (2016) 220–224
Ficapal, A., Jordana, J., Blasco, J.M., Moriyón, I., 1998. Diagnosis and epidemiology of
Brucella ovis infections in rams. Small Rumin. Res. 29, 13–19.
Gouletsou, P.G., Fthenakis, G.C., 2010. Clinical evaluation of reproductive ability of
rams. Small Rumin. Res. 92, 45–51.
Lackner, J.E., Agarwal, A., Mahfouz, R., du Plessis, S.S., Schatzl, G., 2010. The
association between leukocytes and sperm quality is concentration dependent.
Reprod. Biol. Endocrinol. 8, 12.
Machado, G., Santos, D.V., Kohek, I., Stein, M.C., Hein, H.E., Poeta, A.S., Vidor, A.C.M.,
Corbellini, L.G., 2015. Seroprevalence of Brucella ovis in rams and associated
flock level risk factors in the state of Rio Grande do Sul, Brazil. Prev. Vet. Med.
121, 183–187.
Mejid, J., Mathias, L.A., Robles, C.A., 2010. Clinical manifestations of brucelosis en
domestic animals and humans. Open Vet. Sci. J. 4, 119–126.
Paolicchi, F.A., Casaro, P.A., Gimeno, E.J., Kortebani, L.G., Mazzolli, A.B., 2000.
Antisperm response in rams experimentally infected with Brucella ovis. Small
Rumin. Res. 36, 7–15.
Paolicchi, F.A., 2001. Epididimitis ovina por Brucella ovis: lesiones genitales y
respuesta inmune antiespermática. Rev. Med. Vet. 82, 86–88.
Ridler, A.L., West, D.M., 2011. Control of Brucella ovis infection in sheep. Vet. Clin.
Food Anim. 27, 61–66.
Ridler, A.L., Smith, S.L., West, D.M., 2012. Ram and buck management. Anim.
Reprod. Sci. 130, 180–183.
Samplaski, M.K., Agarwal, A., Sharma, R., Sabanegh, E., 2010. New generation of
diagnostic tests for infertility: review of specialized semen tests. Int. J. Urol. 17,
839–847.
Van Metre, D.C., Rao, S., Kimberling, C.V., Morley, P.S., 2012. Factors associated with
failure in breeding soundness examination of Western USA rams. Prev. Vet.
Med. 105, 118–126.
Wiemer, K.E., Ruttle, J.L., 1987. Semen characteristics, scrotal circumference and
bacterial isolates of fine wool range rams. Theriogenology 28, 625–637.
Xavier, M.N., Azebedo, C.E., Alves, P.T., Lima, S.R., 2009. The genus Brucella and
clinical manifestations of brucelosis. Ciência Rural 39, 2252–2260.