Research in Veterinary Science 95 (2013) 362–367

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Research in Veterinary Science

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Risk factors for bacterial contamination during boar semen collection

Ana Maria G. Goldberg a, Laura E. Argenti a, Jamil E. Faccin a, Lídia Linck a, Mônica Santi a,
Mari Lourdes Bernardi b, Marisa R.I. Cardoso c, Ivo Wentz a, Fernando P. Bortolozzo a,⇑
a
Setor de Suínos da Faculdade de Veterinária da, Universidade Federal do Rio Grande do Sul (UFRGS), Av. Bento Gonçalves, 9090 CEP: 91540-000 Porto Alegre, RS, Brazil
b
Departamento de Zootecnia, Faculdade de Agronomia, UFRGS, Brazil
c
Departamento de Medicina Veterinária Preventiva, Faculdade de Veterinária, UFRGS, Porto Alegre, RS, Brazil

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

Article history: The aim of this study was to evaluate the influence of multiple factors on bacterial contamination in 213
Received 10 September 2012 ejaculates from four boar studs. Semen contamination by aerobic mesophiles increased in ejaculates
Accepted 25 June 2013 where the preputial fluid flowed into the collection container, collection glove was dirty, preputial hair
was long (>1.0 cm), the collection lasted >7 min and boars were older than 18 months. An increase in col-
iforms occurred when preputial fluid dripped into the collection container, collections lasted >7 min or
Keywords: when penis escaped during collection. Semen contamination increased when two or more factors related
Swine
to hygiene (poor hygiene of the boar, dirty preputial ostium, large preputial diverticulum, long preputial
Semen collection
Bacterial contamination
hair, dirty gloves, preputial liquid trickling from the hand of the technician into the semen container and
Boar stud penis escaping) were present. A vigilant protocol of collection must be followed to minimize bacterial
Artificial insemination contamination, especially avoiding dripping of preputial liquid into the semen container.
Extended porcine semen Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction sperm longevity) of the extended semen product (Althouse et al.,

Currently, artificial insemination (AI) is the technology of repro-
duction most used on swine breeding farms (Althouse et al., 2008).
However, to have success in AI programs it is essential that boar
studs produce semen doses that can keep both in vitro quality
and longevity of post-ejaculated semen (Althouse and Lu, 2005).
High quality semen doses, able to guarantee the reproductive per-
formance of sows, can only be produced when using ejaculates ap-
proved in morphology test, with sperm motility over 70% and
sperm agglutination below 40% (Bortolozzo and Wentz, 2005).
A critical point in extended semen production is the level of
bacterial contamination in raw semen (Waberski et al., 2008).
The boar semen collection process is far from being a sterile proce-
dure (Althouse and Lu, 2005); therefore, semen evaluation in a
boar stud should be the basis for detecting weak points in AI sta-
tions (Schulze et al., 2012). After ejaculate evaluation and process-
ing, boar semen doses are stored at 15–17 °C (Johnson et al., 2000),
a temperature that allows bacterial growth even when antimicro-
bials are added in the extender (Althouse et al., 2000; Althouse,
2008).
High levels of bacterial contamination are associated with a
high incidence of sperm-to-sperm agglutination, damaged acro-
somes, poor sperm motility, and reduced shelf life (i.e., decreased
⇑ Corresponding author. Tel.: +55 51 33166123; fax: +55 51 33167305.
E-mail address: fpbortol@ufrgs.br (F.P. Bortolozzo).
2000; Althouse and Lu, 2005). If bacterial contamination is left
uncontrolled, the end result is a decreased reproductive perfor-
mance of swine females (Althouse et al., 2000). For this reason, a
standard minimum contamination protocol must be followed to
reduce bacterial contamination during boar semen collection.
Among the factors considered important for proper semen collec-
tion are hygienic measures related to the male, proper preputial
pre-collection hygiene including an over-glove, proper penis fixa-
tion during collection, discarding the first jets of the ejaculate,
use of disposable gloves for each collection and ability to perform
the procedure (Althouse et al., 2000; Bortolozzo and Wentz, 2005).
Information about the specific contribution of semen collection
steps (interval to perform the mount, duration of semen collec-
tion), boar related issues (dirtiness, age, size of preputial diverticu-
lum, length of preputial hair), and cleanness of semen collection on
the degree of bacterial semen contamination is scarce. Dias et al.
(2000) demonstrated that a hygienic protocol during the collection
leads to ejaculates with a lower degree of contamination, although
the major factors determining the degree of bacterial contamina-
tion of the ejaculate were not identified. For instance, the knowl-
edge of specific procedures of the staff, which can be crucial to
prevent semen contamination, would contribute to a more focused
training of personnel. The aim of this study was to evaluate the
influence of multiple factors (at individual level) on bacterial con-
tamination of semen during routine semen collection in modern
boar studs.

0034-5288/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.rvsc.2013.06.022
 A.M.G. Goldberg et al. / Research in Veterinary Science 95 (2013) 362–367
2. Materials and methods
2.1. Boar stud description and semen collection
The study was conducted during 8 months and performed on
four commercial boar studs located in the south of Brazil, which
is the region with the major concentration of swine production.
At the moment of the study, 455 boars were kept in those studs,
representing about 4% of the boar population from Brazil.
The evaluated boar studs follow the rules for swine breeding
farms and are certified by the Brazilian ministry of agriculture.
To get certified, boar studs must be free of classical swine fever,
Aujeszky’s disease, brucellosis, tuberculosis and mange in addition
to be free or controlled for leptospirosis. Boars are monitored for
these diseases every 6 months and negative results must be ob-
tained in order to keep the mentioned certification. The replace-
ment animals are tested for these diseases before entering the
stud. They are submitted to a 30-days isolation period during
which they are trained to have semen collected off with the use
of an artificial dummy. Some characteristics of the four boar studs
evaluated are shown in Table 1. Two visits were performed, with
an interval of three to four months in each boar stud. A total of
53, 55, 53 and 52 semen collections were observed on boar studs
A, B, C and D, respectively, totalizing 213 ejaculates.
Ejaculates were collected by the gloved hand method, in a spe-
cific collection pen, after manually evacuating preputial fluids
using an over-glove. With boar studs A, B and D, penis fixation
was performed using the pressure of all the fingers on the tip of
the penis with the little finger extended, while with boar stud C,
the tip of the penis was placed between the thumb and extended
forefinger, and the other fingers remained on the side of the collec-
tion container. Disposable plastic bags inside insulated containers
were used for semen collection in all boar studs. A specific filter
to retain the gelatinous fraction of the bulbourethral glands was
also used.
Each semen collection was observed by the same examiner,
without any interference. In addition to aspects concerning the col-
lection procedure per se, some aspects concerning the boars were
also recorded (Table 2) and taken into account for the analysis.
2.2. Microbiological analysis of raw semen
After arrival at the laboratory of each boar stud facilities, a
10 mL sample of each ejaculate was collected in sterile tubes for
later analysis. Semen samples were transported at 5 °C and
processed within 12–18 h after collection. Microbiological evalua-
tion consisted of quantifying the aerobic mesophiles and coliforms
by the pour plate technique, in PCA (Plate Count Agar, Oxoid) and
VRB (Violet Red Bile Lactose Agar, Oxoid), respectively. Samples
were diluted up to 100 fold in 0.85% sterile saline and plated in
duplicate. After incubation (37 °C for 48 h) in PCA medium, all col-
onies were counted; in the case of VRB medium, only the colonies
considered typical of coliforms were counted (1–2 mm in diame-
ter, red and with a pink precipitation halo). The number of colony
forming units per milliliter (CFU mL 1) of semen was calculated by
multiplying the average number of colonies counted in duplicate
by the reciprocal of the dilution at which the count was performed.
2.3. Statistical analysis
All the statistical analyses were performed with Statistical Anal-
ysis System software (SAS, 2000), release 9.3. The effect of each
potential risk factor on the number of CFU mL 1 for aerobic meso-
philes and total coliforms was analyzed with the GLM procedure
after values were submitted to logarithmic transformation. Means
363
were compared by Student’s t test (two means) or Tukey–Kramer
(more than two means). Differences were considered as significant
at P < 0.05 or as trends for P-values between 0.05 and 0.10.
Evaluation of the effects of possible risk factors (Table 2) on the
degree of ejaculate contamination was carried out after separating
the semen ejaculates into two groups according to the number of
CFU observed in the microbiological evaluation: 6220 CFU mL 1
and >220 CFU mL 1 of aerobic mesophiles. The separation into
these two groups was based on the median value of CFU mL 1, so
that approximately 50% of samples would belong to each group.
Frequency distributions of ejaculates with >220 CFU mL 1 of
aerobic mesophiles according to each potential risk factor were ob-
tained through the FREQ procedure. Logistic regression models,
using the GLIMMIX procedure, were run to determine the relative
contribution of each factor to the probability of an ejaculate having
>220 CFU mL 1. Before running the model, possible collinearity
among factors was determined by the Chi-square test (FREQ proce-
dure; SAS, 2000). Due to collinearity among the explanatory vari-
ables, results of univariable logistic regression analysis for each
factor are also presented. The presence of collinearity was con-
firmed for six of the 11 factors studied and they were excluded
from the multivariable model: hygiene of the preputial ostium, size
of the preputial diverticulum, inclination of the semen container
during collection, interval between entry in the collection pen
and mount, duration of the semen collection, and boar age. The five
factors that did not show collinearity were the independent vari-
ables included in a multivariable hierarchical model as fixed ef-
fects: boar hygiene (clean or dirty); hygiene of glove collection
(clean or dirty); length of preputial hair (short or long); preputial
liquid trickling from the hand of the technician into the semen con-
tainer (yes or no), and penis escaping during semen collection (yes
or no). In addition to the described fixed effects the model con-
tained a random intercept term to account for the random effect
of the boar stud. The binary dependent variable was defined as
the presence or not of >220 CFU mL 1 in the ejaculate.
The isolated or combined effects of some factors directly related
to the hygiene of the semen collection procedure (poor hygiene of
boars, dirty preputial ostium, large preputial diverticulum, long
preputial hair, dirty collection glove, preputial liquid trickling from
the hand of the technician into the semen container and penis
escaping during the collection) on the percentage of ejaculates
with >220 CFU mL 1 were evaluated using the Chi-square test.
3. Results
The percentages of ejaculates with more than 220 CFU mL 1 of
aerobic mesophiles, according to the factors evaluated during se-
men collection, are shown in Table 3. With univariable models, sig-
nificant higher odds of ejaculates with >220 CFU mL 1 (P < 0.05)
were observed when preputial liquid trickled from the hand of
the technician into the semen container or in semen collections
longer than 7 min whereas a tendency (P < 0.10) was observed in
boars with long preputial hair (P1.0 cm), dirty collection glove,
or boars older than 18 m. Of the factors included in the multivari-
able logistic regression model, preputial liquid trickling from the
hand of the technician into the semen container was a significant
(P < 0.05) risk factor and long preputial hair (>1.0 cm) tended
(P < 0.07) to be a risk factor for the occurrence of ejaculates with
>220 CFU mL 1 of aerobic mesophiles (Table 4).
The number of aerobic mesophiles increased significantly
(P < 0.05) in ejaculates where the preputial fluid flowed into the
collection container, the collection glove was dirty, the collection
lasted >7 min and boars were older than 18 months (Table 5).
There was a tendency (P < 0.10) of an increase in aerobic meso-
philes when boars were dirty or when preputial hair was longer
364 A.M.G. Goldberg et al. / Research in Veterinary Science 95 (2013) 362–367

Table 1
Characteristics of boar studs evaluated.

Variables Boar stud

A B C D
Number of boars 144 87 105 119
Age of boarsa, mo 21.6 21.0 15.9 30.1
Breed of boarsb 1, 2, 3 1 2, 3 1, 4, 5
Annual replacement (%) 60 45 50 40
Collections/boar/week 1–1.5 1–1.5 1–1.5 1–1.5
Collections per montha 841 458 671 496
Semen doses per ejaculatec 28.9 28.5 27.0 34.1
Individual cages/pens Cages/pens Cages/pens Cages/pens Cages/pens
Pen size, m 2.5  2.5 2.0  2.7 2.0  2.0 2.5  2.5
Cage size, m 0.7  2.2 0.7  2.1 0.7  2.2 0.8  2.0
Floor of cages or pens Solid concrete and plastic slat Concrete slat Concrete slat Solid concrete and concrete slat
Separation between Partial Partial Partial Partial
animals
Type of dummyd 1, 2, 3 1 1 1
Floor under the dummy Rubber matting Rubber matting Rubber matting Rubber matting
Vaccination (every Leptospirosis, Parvovirus, Leptospirosis, Parvovirus, Leptospirosis, Parvovirus, Leptospirosis, Parvovirus,
6 months) Erysipela Erysipela Erysipela Erysipela
a
Based on the annual average which included the period of study.
b
1, Agroceres PIC; 2, Topigs; 3, DanBred; 4, Penarlan; 5, Genetiporc.
c
Annual average taking into account doses with approximately 3  109 sperm cells.
d
1, metal; 2, leather covered; 3, wood.

than 1.0 cm. In ejaculates where the preputial fluid flowed into the over-glove for hygiene of the preputial diverticulum and vinyl
collection container and the collection lasted >7 min, there was a gloves for the collection, discarding the first jets of the ejaculate,
significant (P < 0.05) increase in the number of coliforms whereas and removing the filter before entering the laboratory, were rou-
they tended (P < 0.10) to increase when penis escaped during se- tine actions performed for all collection samples evaluated.
men collection (Table 5). There was an increase in aerobic meso- The number of CFU mL 1 capable of causing damage to sperm
philes when two or more factors directly related to the hygiene possibly varies with bacterial species. According to Martín et al.
of semen collection were simultaneously present and an increase (2010), there is a positive correlation between the presence of
in coliforms if three or more factors were present (Table 6). E. coli and sperm agglutination, and a negative correlation between
sperm agglutination and litter size. The maximum of 3.5  103
CFU mL 1 of E. coli, considered an acceptable threshold to the use
4. Discussion of semen for AI (Martín et al., 2010), cannot be adopted to establish
direct comparisons because we measured the total number of
Contamination of raw swine semen is a common event during coliforms. Nevertheless, the importance of performing semen
routine collection from boar studs (Althouse and Lu, 2005; Waberski
et al., 2010), which was confirmed in the present study. In other Table 2
studies, up to 75% of semen samples were contaminated by bacteria, Description of the factors evaluated during boar semen collection.
especially Escherichia coli (Martín et al., 2010), and total aerobic bac- Factors Description
terial counts in raw boar ejaculates averaged 103–105 bacteria/mL
Boar hygiene Clean: no perceptible dirt on the
(Schulze et al., 2012). However, despite recommendations for mini- body
mal contamination (Althouse et al., 2000), the acceptable level in Dirty: presence of dirt on the body
terms of CFU mL 1 has not yet been determined. In the present Hygiene of preputial ostium Clean: no perceptible dirt
study, the influence of multiple factors on the predisposition to Dirty: presence of dirt
Length of preputial hair Short: <1 cm
semen contamination used an acceptability threshold of
Long: P1 cm
220 CFU mL 1, based on a previous study in which the importance Hygiene of glove collection Clean: did not touch anything
of semen collection under hygienic conditions was demonstrated. except the boar penis
In that study (Dias et al., 2000), a lower contamination level Dirty: touched something other
(217 CFU mL 1) was observed when boars were routinely cleaned than the boar penis
Preputial liquid trickling from the hand Into the semen container
two days before collection, emptying of the preputial diverticulum of the technician Out of the semen container
was complete and done using an over-glove, the preputial ostium Penis escaping during semen collection Yes
was dried with a paper towel, the first jets of ejaculate were No
discarded and the semen went straight into the collection container, Size of preputial diverticulum Small: <6.0 cm
Large: P6.0 cm
compared to ejaculates (18,862 CFU mL 1) obtained under less
Inclination of semen container Leaning: inclination of 45° during
strict hygienic measures. the collection
The higher number of aerobic mesophiles observed when the Not leaning: inclination <45° during
collection glove or boars were dirty, the preputial fluid flowed into the collection
the collection container, and long preputial hair was present is Interval between entry into the pen 0–2
and mount (min) 3–6
consistent with the standard minimum contamination protocol >6
(Althouse et al., 2000). In this protocol, it is recommended that Duration of semen collection (min) 2–5
the preputial hair be kept trimmed, clean gloves be used during 6–7
the collection, and the penis be held correctly so the preputial fluid >7
Boar age (months) 8–18
does not drain into the collection container and contaminate
>18
the ejaculate. Other recommendations, such as the use of an
 A.M.G. Goldberg et al. / Research in Veterinary Science 95 (2013) 362–367 365

Table 3
1
Univariable odds ratios of boar studs and potential risk factors for ejaculates with >220 CFU mL of aerobic mesophiles.
1
Factors Category n >220 CFU mL % (n) Odds ratio 95% CI P-value
Boar stud A 53 47.2 (25) 3.0 1.3–7.1 0.0099
B 55 54.5 (30) 4.1 1.8–9.5 0.0011
C 53 22.6 (12) 1.0 NA NA
D 52 71.1 (37) 8.4 3.5–20.4 <0.0001
Boar hygiene Clean 145 45.5 (66) 1.0 NA NA
Dirty 68 55.9 (38) 1.6 0.8–3.1 0.1469

Hygiene of preputial ostium Clean 197 48.2 (95) 1.0 NA NA

Dirty 16 56.3 (09) 1.4 0.4–4.6 0.5711
Length of preputial hair Short 160 45.0 (72) 1.0 NA NA
Long 53 60.4 (32) 1.9 0.9–3.9 0.0791
Hygiene of glove collection Clean 198 46.5 (92) 1.0 NA NA
Dirty 15 80.0 (12) 4.0 0.9–18.9 0.0691

Inclination of semen container Leaning 108 53.7 (58) 1.4 0.8–2.5 0.2811
Not leaning 105 43.8 (46) 1.0 NA NA
Preputial liquid trickling from the hand Out of the container 163 41.1 (67) 1.0 NA NA
Into the container 50 74.0 (37) 4.0 1.8–8.7 0.0013
Penis escaping during semen collection No 153 45.8 (70) 1.0 NA NA
Yes 60 56.7 (34) 1.6 0.8–3.2 0.1646
Size of preputial diverticulum Small 111 46.8 (52) 1.0 NA NA
Large 102 51.0 (52) 1.1 0.6–2.0 0.7120
Interval between entry into the pen and mount (min) 0–2 54 55.6 (30) 1.7 0.7–4.3 0.2161
3–6 112 49.1 (55) 1.4 0.6–3.0 0.3987
>6 47 40.4 (19) 1.0 NA NA
Duration of semen collection (min) 2–5 78 42.3 (33) 1.0 NA NA
6–7 70 42.9 (30) 1.0 0.5–2.0 0.9384
>7 65 63.1 (41) 2.2 1.1–4.7 0.0350
Boar age (months) 8–18 100 41.0 (41) 1.0 NA NA
>18 113 55.7 (63) 1.9 0.9–4.0 0.0953

collection without preputial liquid dripping into the semen con- Table 4
1
Odds ratios of potential risk factors for ejaculates with >220 CFU mL of aerobic
tainer in order to reduce contamination by coliforms was shown
mesophiles obtained in multivariable logistic regression model.
in the present study.
Bacterial contamination in boar ejaculate can be reduced by the Factors Category Odds 95% CI P-value
Ratio
use of a collection technique that avoids contact of semen with the
preputial fluid (Waberski et al., 2010). It is worth emphasizing that Boar hygiene Clean 1.0 NA NA
in boar stud C, where technicians held the penis between the Dirty 1.6 0.8–3.1 0.1986

thumb and extended forefinger, preventing the trickling of prepu- Length of preputial hair Short 1.0 NA NA
tial liquid into the collection container, 37.7% of the ejaculates Long 2.0 0.9–4.2 0.0685
were without aerobic mesophiles growth, contrasting with 7.5%,
3.6% and 1.9% of ejaculates without bacterial growth in boar studs Hygiene of glove collection Clean 1.0 NA NA
Dirty 3.4 0.7– 0.1261
A, B and D, respectively.
16.9
The higher risk of contamination observed when semen collec-
tion lasted >7 min is probably explained by the fact that preputial Preputial liquid trickling Out of the 1.0 NA NA
liquid dripped into the container in a higher percentage of ejacu- from the hand container
lates with longer (34%; 22/65) rather than shorter collection dura- Into the 4.3 1.9–9.8 0.001
container
tions (19%; 28/148). One way to reduce the time spent in the
collection procedure would be to collect only the rich fraction of Penis escaping during No 1.0 NA NA
the ejaculate, which would result in a decrease in the chance of semen collection Yes 1.5 0.7–3.1 0.2395
preputial fluid flowing into the collection container. However, this
In addition to the fixed effects, the model contained a random intercept term to
procedure is questionable because the sperm-poor fraction con- account for the random effect of the boar studs.
tains 10–30% of the total sperm cells (Bortolozzo and Wentz, Factors not included in the model due to collinearity: hygiene of preputial ostium,
2005) and much of the seminal plasma, which plays an important inclination of semen container, size of preputial diverticulum, interval between
entry into the pen and mount, duration of semen collection, and boar age.
role in the genital tract and the dilution ratio of the ejaculate
(Waberski, 1997).
The higher number of aerobic mesophiles in the semen of older The importance of boars being cleaned at the time of collection
boars could be related to the expectation that older animals have a as an aid in reducing semen contamination was previously reported
large preputial diverticulum, with the possibility of greater con- (Dias et al., 2000). However, the fact that neither the hygiene of the
tamination by dirt. Nevertheless, similar percentages (P > 0.05) of boars nor the hygiene of the preputial ostium increased the risk of
large diverticuli were observed between young and old boars semen contamination is probably explained by the presence of a
(44% vs. 51%). On the other hand, in the present study, the higher few small areas of dirtiness observed on the boars classified as
level of aerobic mesophiles contamination in ejaculates of older dirty. The dirty areas were usually smaller than 20 cm diameter
boars is probably associated with the greater incidence of preputial and located preferentially on the rear region of the body. The low
liquid dripping into the collection container as compared to young occurrence of dirtiness is due to the fact that cleaning the boars
boars (30% vs. 16%; P < 0.05). and facilities was a routine procedure in all boar studs.
366 A.M.G. Goldberg et al. / Research in Veterinary Science 95 (2013) 362–367

Table 5
1
Number of CFU ml of aerobic mesophiles and coliforms in the ejaculate according to the factors evaluated during boar semen collection (means ± SEM).
1 1
Factor Category n Mesophiles Log10 CFU mL P-value Coliforms Log10 CFU mL P-value
Boar hygiene Clean 145 2.1 ± 0.10 0.09 1.1 ± 0.10 0.26
Dirty 68 2.4 ± 0.13 1.3 ± 0.15
Hygiene of preputial ostium Clean 197 2.2 ± 0.08 0.36 1.1 ± 0.08 0.64
Dirty 16 2.5 ± 0.28 1.3 ± 0.29
Length of preputial hair Short 160 2.1 ± 0.09 0.08 1.1 ± 0.09 0.41
Long 53 2.4 ± 0.18 1.3 ± 0.17
Hygiene of glove collection Clean 198 2.2 ± 0.08a 0.02 1.1 ± 0.08 0.15
Dirty 15 2.9 ± 0.27b 1.6 ± 0.34
Inclination of semen container Leaning 108 2.3 ± 0.11 0.27 1.2 ± 0.12 0.89
Not leaning 105 2.1 ± 0.12 1.1 ± 0.11
Preputial liquid trickling from the hand of the technician Into the container 50 3.0 ± 0.14a <0.0001 1.7 ± 0.17a 0.0002
Out of the container 163 2.0 ± 0.09b 1.0 ± 0.09b
Penis escaping during semen collection Yes 60 2.4 ± 0.15 0.12 1.4 ± 0.16 0.06
No 153 2.1 ± 0.10 1.0 ± 0.09
Size of preputial diverticulum Small 111 2.1 ± 0.12 0.21 1.1 ± 0.12 0.34
Large 102 2.3 ± 0.11 1.2 ± 0.11
Interval between entry into the pen and mount (min) 0–2 54 2.3 ± 0.15 0.55 1.2 ± 0.17 0.76
3–6 112 2.2 ± 0.11 1.1 ± 0.11
>6 47 2.1 ± 0.19 1.1 ± 0.19
Duration of semen collection (min) 2–5 78 2.0 ± 0.14a 0.005 1.0 ± 0.13a <0.05
6–7 70 2.1 ± 0.12ab 0.9 ± 0.12a
>7 65 2.6 ± 0.15b 1.5 ± 0.17b
Boar age (months) 8–18 100 1.9 ± 0.12a 0.0003 1.0 ± 0.11 0.27
>18 113 2.5 ± 0.10b 1.2 ± 0.12

a and b in the column, for each variable, indicate significant differences.

Table 6 (P < 0.05) in collections where dirty gloves and preputial fluid drip-
Effects of isolated or associated factors assessed during boar semen collection on the ping into the collection container occurred together (100%; 6/6)
level of semen contamination (means ± SEM). than in samples with clean gloves and without preputial fluid drip-
No. of Ejaculates with Log10 CFU mL 1 ping into the collection container (39.6%; 61/154).
factors >220 CFU mL 1 Successful hygiene and biosecurity of boar studs are dependent
Aerobic Coliforms
mesophiles on the development of habitual actions by employees that will
only be possible by appropriate technical training of the
0 10/39 (25.6%) a 1.6 ± 0.18a 0.7 ± 0.16a
1 27/62 (43.6%) ab 2.1 ± 0.14ab 1.1 ± 0.14ab staff (Waberski et al., 2010). In the present study, it was shown that
2 31/56 (55.4%) bc 2.2 ± 0.17b 1.1 ± 0.16ab training of the staff should be focused on the correct grasping of
3 21/37 (56.8%) bc 2.5 ± 0.17bc 1.5 ± 0.21b the penis since the main contributor to semen contamination
4–5 15/19 (79.0%) c 3.2 ± 0.19c 1.7 ± 0.25b
was the occurrence of preputial liquid trickling into the semen
a,b,c in the column indicate significant differences (P < 0.05). container. Therefore, selecting, training and periodic retraining of
The following seven factors were considered in the analysis: poor hygiene of boar, a competent and dedicated staff should be a priority. Another pos-
dirty preputial ostium, large preputial diverticulum, long preputial hair, dirty sible way of reducing these failures is the use of automated collec-
collecting glove, preputial liquid trickling from the hand of the technician into the
semen container and penis escaping during semen collection. No more than five
tion systems, which do not differ from manual collection, with
factors occurred simultaneously. regard to ejaculate characteristics and number of CFU (Terlouw
et al., 2007). However, the use of these systems should also be
done under proper hygienic conditions to ensure a low semen
contamination.
The form of penis fixation, collection duration, preputial and The fact that the association of two or more factors related to
glove hygiene and all the other factors involving semen collection hygienic conditions significantly increased the numbers of bacteria
are dependent on the team that performs it. It has been reported shows that the greater the number of failures in the semen collec-
that personnel, besides being a source of contamination, must be tion procedure, the greater the likelihood of increasing contamina-
careful with their actions because they are important in transfer- tion of the raw semen. Therefore the steps described for boar
ring contamination from one site to another (Althouse and Lu, semen collection (Dias et al., 2000; Bortolozzo and Wentz, 2005;
2005; Bortolozzo and Wentz, 2005; Althouse, 2008). In the present Althouse, 2008) must be strictly followed to obtain ejaculates with
study many such failures were observed, and certainly their effects a low degree of contamination.
could be minimized if employees were better instructed, had in-
creased training and awareness about the consequences of their
actions. The contribution of careless actions by technicians to se- 5. Conclusions
men contamination was evidenced by the fact that more than
70% of the ejaculates with >220 CFU mL 1 occurred when the col- Trying to avoid contact of semen with preputial liquid flowing
lection glove was dirty because technicians touched something through the glove was shown as the most important procedure
other than the penis or when they failed to correctly grasp the pe- to obtain ejaculates with low bacterial contamination. Further-
nis, causing preputial fluid to drip from their hand into the collec- more, long preputial hair, dirty collection glove, penis escaping
tion container. Furthermore, the contribution of individual during semen collection, older boars, and longer semen collections
handling and carrying out the recommended procedures on the de- are also factors contributing to the increase of contamination level
gree of semen contamination was quite evident in ejaculates of boar semen. Semen contamination increases when two or more
where the two aforementioned factors occurred simultaneously. factors directly related to the hygiene of the semen collection pro-
The percentage of ejaculates with >220 CFU mL 1 was higher cedure are simultaneously present.
 A.M.G. Goldberg et al. / Research in Veterinary Science 95 (2013) 362–367
Conflict of interest
None of the authors has any financial or personal relationships
that could inappropriately influence or bias the content of this
manuscript.
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