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Animal Reproduction Science 122 (2010) 142–149

Contents lists available atScienceDirect

Animal Reproduction Science

journal homepage:www.elsevier.com/locate/anireprosci

Bacterial contamination of ram semen, antibiotic sensitivities, andeffects on sperm quality during storage at 15

◦

Jesús Luis Yániz

∗

, María Angeles Marco-Aguado, José Angel Mateos, Pilar Santolaria

Institute of Environmental Sciences (IUCA) and Departamento de Producción Animal y Ciencia de los Alimentos, Universidad de Zaragoza, Huesca, Spain

a r t i c l e i n f o

Article history:

Received 4 January 2010Received in revised form 23 July 2010Accepted 6 August 2010Available online 13 August 2010

Keywords:

RamSemenBacteriospermiaSperm quality

a b s t r a c t

This study was designed to determine the degree and type of bacterial contamination of ejaculated semen samples in fertile rams and its consequences on sperm quality duringstorage. In experiment 1, 68 ejaculates from 36 rams were divided into two aliquots, oneof which was used for bacterial culture, while the other one was diluted, stored at 15

◦

Cand assessed for plasma membrane integrity and motility at 0, 24 and 48h after dilution.From the 68 ejaculates, 66 were positive for aerobic bacteria, including 20 species of bac-teria from 14 genera. The most frequently isolated bacteria were

Escherichia coli

Proteusmirabilis

Enterobacter cloacae

Staphylococcus epidermis

, and

Staphylococcus aureus

species.These 5 bacteria were present in 97% of all contaminated samples. All contaminant bac-teria were found to be sensitive to gentamicin and to ceftiofur, with variable percentagesof resistance to the other antibiotics evaluated. In samples with total enterobacteria countlower than 100 colony-forming units (CFU)/ml, higher proportions of motile and progres-sive sperm and higher velocities of spermatozoa were observed at different times duringstorage. In experiment 2, pure cultures of the most frequently isolated bacteria were indi-vidually added to fresh semen samples of low contamination and tested for their effectson sperm quality during storage at 15

◦

C. Semen with

E. coli

showed a drastic reduction inmotility, velocity and viability during storage. This reduction was also signiﬁcant, but lessdrastic,insemenwith

E.cloacae

and

P.mirabilis

,whereasitwaspartialandlesspronouncedin the other groups (

S. epidermidis

and

S. aureus

). In conclusion, the contamination of ramsemen with enterobacterial species reduced sperm quality during storage at 15

◦

1. Introduction

Semen collection in farm animal species is not a ster-ile procedure, and some degree of contamination withbacteria cannot be avoided (Clément et al., 1995; Varneret al., 1998; Althouse et al., 2000; Thibier and Guerin,2000; Althouse and Lu, 2005; Aurich and Spergser, 2007;

∗

Corresponding author at: Departamento de Producción Animal yCiencia de los Alimentos, Escuela Politécnica Superior de Huesca, Uni-versidad de Zaragoza, Ctra. Cuarte S/N, 22071 Huesca, Spain.Tel.: +34 74 239312; fax: +34 74 239302.

E-mail address:

jyaniz@unizar.es(J.L. Yániz).

Bielanski, 2007).In rams, semen is usually collected withan open-ended artiﬁcial vagina that may be contaminatedwith bacteria from the surface of the penis and prepuce,collection area, equipment and people. As a consequence,bacteria might compromise semen quality during storageand contaminate the female’s reproductive tract. To mini-mize these adverse effects, antibiotics are included in thecomposition of the ram semen extenders to prevent bac-terial growth (Maxwell and Salamon, 1993; Salamon andMaxwell, 2000).Some bacteria are deleterious to the spermatozoa ina concentration-dependent manner. The most extensivelystudied is the effect of

Escherichia coli

in human spermato-zoasurvival.ThisGram-negativebacteriumreducessperm

J.L. Yániz et al. / Animal Reproduction Science 122 (2010) 142–149

143

motility through sperm adhesion and agglutination (Wolff etal.,1993;MongaandRoberts,1994),causesmorphologi-calchangesatthelevelofthemidpiece,plasmamembrane,and acrosome (Diemer et al., 2000),alters sperm function, andincreasesphosphatidylserinetranslocation(Villegasetal., 2005).In animal species, a variety of bacteria from dif-ferent genera have been identiﬁed after aerobic culture of extended semen samples, and certain bacteria had detri-mental effects on semen quality during low temperaturestorage (Althouse et al., 2000; Aurich and Spergser, 2007;Akhter et al., 2008).Limited information is currently available describingthe bacterial contamination of ejaculated semen samplesin fertile rams. The aim of this study was to determinethe degree and type of bacterial contamination of ejacu-lated semen samples in fertile rams and its consequenceson sperm quality during storage at 15

◦

2. Materials and methods

2.1. Experiment 1 2.1.1. Animals, semen collection and preparation of samples

All animal procedures were performed in accor-dance with the Spanish Animal Protection RegulationRD223/1988, which conforms to European Union Regula-tion 86/609. A total of 68 ejaculates were collected from36 genitally healthy adult Rasa Aragonesa rams (2–6 yearsold)usingsterilizedartiﬁcialvaginasandglasstubes.RamswerefromtheonlyinseminationcentreforRasaAragonesa(25 rams) and from a public regional research centre (11rams),andwereselectedonthebasisofasuccessfulrepro-ductive history and healthy appearance during generalexploration and local examination of the genital organs.The undiluted semen was evaluated for motility undera phase contrast microscope at 400

(Olympus BX40,Olympus Optical Co., Ltd., Japan) and sperm concentra-tioncalculatedbyloading6



lintoaNeubauer(Marienfeld,Germany)chamberinduplicate,andcountingunder200

magniﬁcation.Thesemenfromeachejaculatewasdividedinto two aliquots, one of which was used for bacterial cul-ture(storedat5

◦

Cinsterileglasstubes),andtheotherwasdilutedinlong-life,ultra-heat-treated(UHT)milk(0.7%fat)without antibiotics to 800

sperm/ml, kept in sterileglasstubesandstoredinarefrigeratorat15

◦

C.At0,24and48h after dilution, semen samples were carefully mixedanddilutedto50

sperm/mlinacitrate–glucosesolu-tion to assess plasma membrane integrity and motility.

2.1.2. Bacterial culture

Cultures were taken from single-sire whole ejaculatealiquots (

=68; 36 rams) within 2h after recovery. Eachsample was plated on blood agar, chocolate agar and Mac-Conkey agar, and the plates were incubated in aerobicconditions at 37

◦

C. Aerobic cultures were inspected andbacterial growth recorded after 24 and 48h of incubation.Bacterial contamination (expressed as colony-formingunits—CFU/ml)wascategorizedintofourgroupsforstatis-tical analysis: <100, 100–999,

≥

1000. Nonenteric bacteriawere identiﬁed using the API 20NE system (BioMérieuxS.A., Madrid, Spain), and enteric bacteria were identiﬁedusing the API 20E (BioMérieux S.A., Madrid, Spain) or BBLEnterotube II (Becton Dickinson S.A., Madrid, Spain) sys-tems. Antibiotic sensitivities were performed using theKirby-Bauer Disk Diffusion Susceptibility Method (BectonDickinsonS.A.,Madrid,Spain)followingestablishedguide-lines (Watts, 1999).

2.1.3. Assessment of stored semen samples 2.1.3.1. Assessment of plasma membrane integrity.

Spermviability (membrane integrity) was assessed as describedbyYániz et al. (2008).Brieﬂy, semen samples diluted in a citrate-based extender (80.6mM sodium citrate titratedto pH 7.0 using a 1M citric acid solution, 55.6mM glu-cose) to 50

sperm/ml. Aliquots (0.5ml) of dilutedsamples were pipetted into 1ml Eppendorf centrifugetubes, and 5



l of PI solution (0.5mg/ml in PBS) wereadded to the samples. Each aliquot was incubated for8min in the dark at 30

◦

C, and spermatozoa were immobi-lized with formaldehyde at a ﬁnal concentration of 0.35%.Spermatozoa were examined and photographed underan Olympus BX40 ﬂuorescence microscope at 100

. Twopictures where taken of each field under negative-phasecontrast and fluorescence microscopy (Olympus BX40,Olympus Optical Co., Ltd., Japan; UMWIG3 filter block)using a digital camera adapted to the microscope (CanonEos 400D controlled using the computer through a remotecontrol program). The images were processed using UTH-SCSA Image-Tool open software (Version 3.0, availableon-line athttp://ddsdx.uthscsa.edu/dig/download.html).The percentage of membrane-damaged spermatozoa wascalculatedasthenumberofpropidium-iodidepositivecells(inthefluorescencemicroscopyimage)dividedbythetotalnumber of cells in the same field (negative-phase contrastmicroscopy image). At least 500 cells were examined persample.

2.1.3.2. Sperm motility determination by CASA (computer-assisted sperm analysis).

Computer-assisted sperm anal-yser (ISAS

, Version 1.0, PROISER, Valencia, Spain) wasused to assess sperm motility, as described byYániz etal. (2008).Brieﬂy, sample aliquots (5



l) were placed ina pre-warmed Makler chamber, and at least 500 spermcells were analysed by CASA for each sample. The semenvariables recorded were motility percentage (MS, %), pro-gressive motility percentage (PS, %), straight line velocity(VSL,



m/s),curvilinearvelocity(VCL,



m/s),averagepathvelocity(VAP,



m/s);linearity(LIN,asameasureofacurvi-linear path, VSL/VCL), straightness (STR, as the linearity of theaveragepath,VSL/VAP),wobble(WOB,oscillationmea-sure of the actual path about the average path, VAP/VCL),and amplitude of lateral head displacement (ALH,



m).

2.2. Experiment 2

A total of 16 ejaculates were collected from 6 genitallyhealthy rams from an experimental centre as describedpreviously,usingmeticuloushygienemeasurestodecreasebacterial contamination. The minimum visual motility cri-terion was 80%. Native semen was checked for bacterialcontamination immediately after collection as in experi-

144

J.L. Yániz et al. / Animal Reproduction Science 122 (2010) 142–149

ment 1. Seminal samples with bacterial growth in nativesemen higher than 100CFU/ml (

=6) were not processed.Pure cultures of the most frequently isolated bacteria (

E.coli

Enterobacter cloacae

Proteus mirabilis

Staphylococ-cus aureus

and

Staphylococcus epidermis

)wereindividuallytested for conﬁrmation of effects on sperm quality dur-ingstorageat15

◦

C.Testingwasperformedbytransferringeach of the above bacteria diluted in sterilized salinesolution or saline solution alone (control) into a samplealiquotoffreshlymilk-extendedsemen(8

sperm/ml)to a ﬁnal concentration of 10

bacteria/ml. Bacteria con-centration was determined by turbidity (Sutton, 2006).Immediately after addition of the bacteria or saline solu-tion, and after cooled storage (15

◦

C) for 24 and 48h,membrane integrity (viability), and motility of dilutedsemen were determined as previously described.

2.3. Statistical analysis

Thevaluesobtainedwereexpressedasmean

standarderrorofthemean(SEM).StatisticanalyseswereperformedusingtheSPSSpackage,version15.0(SPSSInc.,Chicago,IL,USA). Normality distributions and variance homogeneityof the median value score for each set were checked bythe Kolmogorov–Smirnov and Levene tests, respectively.Asalldatawerenormallydistributedparametrictestswereused throughout. Differences in membrane integrity andmotilitybetweengroupswereexaminedthroughone-wayanalysisofvariance(ANOVA)usinggeneralizedlinearmod-els. If the

value was signiﬁcant, a Tukey test was used foraposteriorimultiplecomparisons.Statisticalanalysiswereperformed considering the parameters of sperm quality ata given time during storage as the dependent variablesand bacterial contamination with aerobic bacteria (<100,100–999,

≥

1000), enterobacteria (<100, 100–999,

≥

1000)(experiment 1), or the effect of different bacteria added tothe semen aliquots (experiment 2) as independent factors.

3. Results

3.1. Experiment 1

Microbial results indicated that, from the 68 ejaculates,66werepositiveforaerobicbacteria.Asinglebacterialcon-taminantwasobtainedfrom43%(29/68)oftheejaculates;54% (37/68) contained 2 or more bacterial species, with10 specimens containing 3–6 contaminants. The degree of bacterial contamination is shown inFig. 1.Table 1lists the bacteria isolated from contaminated semen samples withtheir respective antibiotic sensitivities. After aerobic cul-ture, the growth of 20 species of bacteria from 14 generawasregistered(Table1).Themostfrequentlyisolatedbac- teria were from both enteric (

E. coli

P. mirabilis

E. cloacae

)and nonenteric (

S. epidermis

S. aureus

) groups. Some of these 5 bacteria were present in 97% of all contaminatedsamples (64/66). Different enterobacteria were isolated in69.1%(47/68)ofsamples,and

E.coli

in60.3%(41/68)ofsam-ples. All contaminant bacteria were found to be sensitivetotheaminoglycosidegentamicinandtoceftiofur.Thefol-lowing percentages of bacteria were found to be resistanttotheotherantibioticsevaluated:20%toampicillin,53%to

Fig.1.

Level of contaminationwithtotal aerobicbacteriain68 ejaculatesof Rasa Aragonesa rams.

penicillin, 15% to streptomycin, 4% to spectinomycin, 47%to erythromycin, 33% to oxytetracycline, 13% to polymixinB, and 15% to co-trimoxazole (SXT).No signiﬁcant relationships were observed betweenthe total aerobic bacterial counts, determined using stan-dard plate count agar (PCA) method, and the parametersof sperm motility and viability during storage. However,when the total enterobacteria counts in MacConkey plateswere lower than 100CFU/ml, higher proportions of motile(Fig. 2a) and progressive (Fig. 2b) sperm and higher velocities (Fig. 2c–e) of spermatozoa were observed at different timesduringstorage(

<0.001),althoughnosigniﬁcantdif-ferences were observed in the other kinematic variablesanalysed.

3.2. Experiment 2

The effects of specific bacteria on the most significantparametersofspermmotilitydeterminedbyCASAandvia-bility are shown inFig. 3.Immediately after addition of bacteria,onlythepercentageofspermatozoawithprogres-sive motility was significantly higher in the control groupcomparedtoallothergroups(Fig.3b).After24hofstorage, semen with

E. coli

showed a drastic reduction in motility(Fig.3a),velocity(Fig.3c–i)andviability(Fig.3 j)compared totheothergroups.Thisreductionwasalsoimportant,butlessapparent,insemenwith

E.cloacae

.Theothergroups(

P.mirabilis

S. epidermidis

and

S. aureus

) differed signiﬁcantlywith the control in some kinematic variables at this timeperiod(progressivemotility,VSL,VAP,WOB,STR,ALH),butdifferences were less pronounced (Fig. 3).At 48h, all spermatozoa in the

E. coli

group were immotile (Fig. 3a) and had damaged membranes (Fig. 3 j). Signiﬁcantly (

<0.001)lower motility (Fig. 3a) and viability (Fig. 3 j)parameters were found in semen with

E. cloacae

and

P. mirabilis

incomparison with the other groups. Differences in viabil-ity, motility and velocity parameters between semen withGram-positivebacteriaandthecontrolwerenotsigniﬁcantat this time (Fig. 3).

4. Discussion

Semen is normally colonized by a variety of microor-ganisms that may reduce semen preservation and fertility,