t

{ Thenogenology
I
!*-.-,.,",-
ELSEVIER Theriogenology59 (2003) ll57-ll70

Fertility of ram semenfrozenin Bioexceil@

and usedfor cervicalartificialinsemination
J. Gilu'b, M. Rodriguez-bazoqui", N. Lundeheimd,
L. Sóderquistu,H. Rodríguez-Martínezu'*
"Faculty of VeterinaryMedicíne, Department of Obstetricsand Gynecology,Swedish University of
Agricultural Sciences(SLU), UllsvtigenI4C, Clinical CentenPO Box 7039,
Ultuna, SE-75007, Uppsala,Sweden
bveterina,r!Laboratories, "M.C.
Rubino" (DII^AVE-MGAP),Paysanclú,(Jntgucry
'Ftrculty
of VeterinaryMedicine, UDELAR, Paysandú,Uruguay
'tFaculty
of Agrícutttrre,Departmentof Animal BreeclínganclGenetics,Swe¿lishIlniversíty
of Agricultural Sciences(SLU), Ullsviigenl4C, Clinical Center PO Box 7A39,
Ultunu, SE-75007, Uppsala,Sweden
ReceivedI November2001;accepted22 Apnl2002

Abstract

The cunent useof ingredientsof animalorigin, suchaseggyolk, in semenextenderspresentsa risk

of rnicrobial contamination,and has led to the search for alternatives.Such an extender is
commerciallyavailabletbr bull semen(Bioexceli", IMV L'Aigle, France),and it has previously
beentestedin vitro for freezingram semen,with satisfactoryresults.The airnof ttrepresentstudywas
to comparethe fertility resultsof ewesin Ur-uguay,after cervicalinselninationwith ram sementhat
was tiozen in Bioexcell" versussemenfrozen in a conventionalrnilk-egg yolk extender(control).
Semenfrom five Corriedaleramswas frozen,usinga split sampledesign,in eitherrnilk-egg yolk or
Bioexcell" extender,using a two-stepextensionmethod.The spelm pararnetersassessedafler
thawingwere subjectivemotility, membraneintegrity(SYBR-l4lPl), andcapacitationstarus(CTC).
Thawed selnenwas inseminatedintracervicallyonce during spontaneous estrustn 910 Coniedale
ewesthat grazedin naturalpastures,underextensivemanagement conditions.Fertility wasrecorded
as nonreturnratesatZI days (NRR-21) and 36 days(NRR-36)after artificial insemination(AI), as
well as pregnancyrate (PR-US,diagnosedultrasonographically 50 days after AI of the last ewe).
Subjectivemotility wasslightlyhigherin Bioexcell" thanin thernilk extender(47 vs.46.57o:NS), as
was membraneintegrity(38 vs. 37.77o;NS) and the percentage of uncapacitated spennatozoa (28.5
vs.26.3Vo;NS). There were no statisticallysignificantdifferencesin f-ertilityratesfound between
Bioexcell" and the control extender:NRR-21 (35.9 vs.33.27o),NRR-36 (34.8 vs.32.6Vo),and
PR-US(28.4 vs.27.ZVo). In conclusion,Bioexcell"' appearsto be an alternativeto the conventional

--7_-
'
Corresponding author.Tel.: +46-18-6'12172; fax: +46-18-673545.
E-mail address: heriberto.rodriguez
@og.slu.se (H. Rodríguez-Martínez).

0093-691)002l$- seefront matter e 2002 ElsevierScienceInc. All riehts reserved.

P I I :S 0 0 9 3 - 6 9 l X ( 0 2 ) 0 1 17 8 - 0
I 158 J. Gil et al./Theriogenology59 (2003) 1157-1170

rnilk-egg yolk extenderfor freezingram semen,andprovidessimilar fertility resultsafter cervical AI

t'',
underextensivemanagement conditions.Thus,Bioexcell containingno additivesof animalorigin,
can offer a safer alternativewhen frozen semenis used for introducing new genetic material into a
flock or a countrY.
e) 2002 Elsevier ScienceInc. All rights reserved.

Milk-egg yolk; Soy-bean

Keywords:Sheep;CervicalAI; Extenders; lecithin

l. Introduction

Extenders that ate used for semen preservationat low temperaturescontain basic
ingredients and additives that provide a suitable environment for spermatozoa.The
extendershouldcontainenergysubstrates, buffers to maintaina suitableosmoticpressure
and pH, and compoundsthat protect spermatozoaagainstcold-shock,thus, allowing their
survivalduring extension,cooling,,freezingand thawing t1-31. Usually,the ingredientsin
an extender vary from pure chemical compoundsto undefinedproducts of animal or
vegetalorigin. Egg yolk and skim milk are two common additivesof animal origin that
have been used for years to freeze fam semen t4-8].
In attemptingto improve quality standards,the artificial insemination(AI) industryhas
raisedconcemsregardingthe use of such additives,which are diverseand often vary in
their compositionso that quality certificationbecomesvery difficult or evenimpossibleto
perform. In addition,they also representa potentialrisk for contaminatingthe extenderif
such contaminants(microbial or otherwise) are present in the raw product. In fact,
Bousseauet al. [9] demonstrated that egg yolk can presenta major risk of contamination.
Commercial extenderswith an egg yolk substitutehave recently become available for
freezingbull semen(Biociphos" andBioexcell" , IMV L'Aigle, France),anddocumenta-
tion of the fertility achievedby bull semenfrozenin suchextendersalreadyexistsI l0- 12].
Ram spermatozoaseemto be well protectedin a milk-basedextenderwith the addition
of SVoegg yolk, and suchan extenderhastraditionallybeenusedfor freezingram semenin
Norway and Swedenl4-7,12).The reportedfertility resultsusing this homemadeextender
under the prevailing conditionsin thesecountriesis good, but reportedto be somewhat
lower underthe moreextensivemanagement conditionsusedin Uruguay[13]. In Uruguay,
aswell asin otherwool producingcountries,sheepfarming is undereconomicpressureand
measuresare being taken to attain a solution.Implementationof AI for geneticimprove-
ment will becomean important tool for enhancementof the productivity of the national
flock, which representsa major componentof the nationalexport income.However,there
is a needto be certainthat useof this technologydoesnot involve any sanitaryrisks related
to the extendersused,particularly when trading frozen semen.
Bioexcell'i (IMV L'Aigle, France)has not yet been testedas an extenderfor freezing
ram semen.Recently,we studiedthe influenceof Bioexcell'" on spermquality parameters
post-thaw,in an in vitro study [14].We found that, when comparedto the conventional
milk-egg yolk extender [13-15], Bioexcell "' preservedsperm viability post-thaw at
similar levels to the control. Our results also indicate that the two-step extension
methodology, adding the glycerol to the second fraction at 5 oC, appearsto be the
 J. Gil et al./Theriogenology59 (2003) I157-l 170 I 159

procedureof choicefor processingram semenin theseextenders.However,no information

is yet availableregardingthe influenceof this extenderon fertility after AI.
Therefore,the aim of the presentstudy was to comparethe fertility resultsof ram semen
frozen in Bioexcell'li'with the conventionallyusedmilk-basedextender(containing57o
egg yolk) after cervical AI, under extensivemanagementconditions in a commercial
Corriedaleflock.

2. Materials and methods

2.1. Ramsand semencollectíon

Semenfrom five Con'iedalerams aged34 yearswas usedin this study.The rams were
locatedat the ExperimentalStationof the Faculty of Agriculture, University of Uruguay,
Paysandú,Uruguay (32"S, 58'W), grazingnaturalpastures(March-April), and subjected
to semencollection once a day using an artificial vagina.In total, six to eight ejaculates
were collectedfrom eachram on consecutivedays.All spermparametersin the ejaculates
were within what is consideredto be normal rangefor ram semen(volume:0.75-2ml;
spermconcentration:>3.2 x l0e spermatozoalml; spermmotility: >7\Vo; and frequen-
cies of total morphologicalabnormalities:Sl07o).

2.2. Extenders

The extenderswerepreparedasdescribedbelow,andaliquotsstoredat - 18 "C until use.

ExtenderI (M): waspreparedfrom non fatty milk powder(ll%o w/v) anddistilledwater.
The milk was heatedto 95'C for l0 min, stirredconstantlyon a heatedplate, and then
cooledto room temperature for furtherprocessing. FractionI (f/ l) waspreparedby adding
57oeggyolk to the milk base,andafterhomogenization of theextender,f/l wascentrifuged
for 20 min at 3600 x g at 5 oC, to removelarge particles.The clarifiedsupernatant was
supplemented with ¿rntibiotics(penicillin 0.03 g per100rnl and streptornycin0.04 g per
100ml). Fraction2 (flz) waspreparedasdescribedfor f7l but with the additionof 224 mM
of fructoseand glycerol(147av/v). Clarificationwas donebeforeadditionof fructoseand
glycerol.
Extender2 (B) was a specialpreparationof the commercialBioexcell" (IMV L'Aigle,
France) available for bull semen.Two fractions were manufacturedfor the two-step
extensionprocedure,f/l without glycerol and fl2 with 12.8Vo(v/v) glycerol.

2.3. Sentenprocessing

Immediatelyaftercollection,the ejaculateswereplacedin a portablewaterbathat 33 'C,

and spermconcentrationwas assessed in a photometer(Spermacue " , Minitüb, Landshut,
Germany).Eachejaculatewas then split into two equalfractionsand slowly extendedto a
final concentration of 1600 x 106spermatozoalmlwith fll of extendersM and B,
respectively.For further processing,the extendedsemenwas transferredto an isothermal
box with wrapped freeze-packsand transportedfrom the collection site to the freezing
1160 59 (2003)1157-1170
J. Gil et al./Theriogenology

'C within t h, a second

laboratory (DILAVE-MGAq paysandú). After cooling to 5
extensionstep to 800 x 106spermatozoa/mlwas performed with fl2 of each extender.
Semenwas then manually packedinto 0.25 ml mini-strawsand sealedwith PVC powder.
oC, the strawswere dried with papertissue,
After 2 h of equilibrationin a waterbathat 5
placed horizontally on a rack and transferredto an isothermalbox to be frozen in vapor
4 cm above liquid nitrogen (LNz). After 10 min, the strawswere plunged into LN2 and
storedin a LN2 containeruntil use for AI.

2.4. Semenevaluation
'C for 9 sec and
Five straws (from different freezing operations) were thawed at 50
poclled in a test tube. The parametersstudied were subjective sperm motility, sperm
membraneintegrity and capacitationstatus.Subjectivespermmotility was assessed from
an aliquot (10 pl) of the pooled semendiluted with 100 ¡rl sucrosebuffer [16], prepared
from l0mM NaCl,2.5mM KCl,20mM HEPES,222mM sucrose, and l0mM glucose,
supplementedwith cold water-solublepolyvinyl alcohol,pH 7.5 (SigmaAldrich, Tyresó,
Sweden).Five microliter of the diluted samplewere then placed in a Makler Counting
Chamber(Sefi-MedicalInstruments,Haifa, Israel),and spermmotility was assessed to the
nearest5Vousing a phasecontrastmicroscopeequippedwith a warm stage(38 "C, Nikon
Corporation,Tokyo, Japan).The averageof the percentages judged in four different fields
in the microscopewas used as the final sperm rnotility score.
Sperm membraneintegrity was assessed with the fluorochromecombinationSYBR-
14 andpropidiumiodide(PI, SpermViability Kit l-7011,MolecularProbesInc.,Eugene,
OR, USA), asdescribedby GarnerandJohnsont171.In brief, 50 pl of thawedsemenwas
mixed with 5 prlSYBR-14 and2.5 pl PI (10 pM SYBR-14and 120¡"rMPI), then gently
mixed and incubatedat37'C for l5 min. The smearswere preparedby placing 4 ¡,rlof
stainedsemenand I ¡"rlof 37o glutaraldehydesolution (EM-gradein cacodylatebuffer,
pfl 7.4) onto a warm slide just before the assessment in order to stop the motion and
facilitatecounting.Two hundredspermatozoawere countedusing a microscope(Nikon,
Tokyo, Japan) equipped with a set of two filters (420-490 nm excitation, 510 nm
ernission,and 530-560nm excitation,5S0nm emission).The spermatozoa were classi-
fied as intact when they stainedgreen,or membranedamagedwhen they stainedgreen-
red or just red.
The chlortetracycline(CTC) assaywas used to determinethe capacitationstatusof
viable spermatozoa as describedby Gil et al. [3-15]. Briefly, l8 prlof thawedsemenwas
incubatedfor5 min at37 "C with 2 ¡-rlethidiumhomodimer(EthD-L,23.3prM,Molecular
ProbesInc., Eugene,OR, USA). After incubation,the mixturewas fixed with 20 ptlof 37o
giutaraldehydesolution (EM-grade in cacodylatebuffer, pH 7.4), and then stainedwith
40 ¡tl of CTC working solution.The samplewas washedby centrifugationat 700 x g for
8 rnin with I ml of sucrosebuffer (as above).One milliliter of the supernatantwas then
removedand the pellet resuspended. The smearswere preparedusing an aliquot of 5 ¡rl of
the stainedsampleon a cleanmicroscopeslide,coveredwith a coverglass,sealedwith nail
varnish,and kept in the dark at 5'C. Microscopicevaluationswere done within 12h of
staining.Two hundred viable spermatozoa(unstainedwith EthD-1) were classifiedinto
threecategories:uniform fluorescenthead(uncapacitated: CTC-F), fluorescence-free band
 59 (2003)1157-1170
J. Gil et al./Tlterk;genologv 1t6l

on the postacrosomalregion (capacitated:CTC-B), and nonfluorescentheaclor a thin

fluorescentband on the equatorialsegment(acrosome-reacted:
CTC-AR).

2.5. AI procedures

The fertility trial involved 970 Coniedale ewes (2-6 years old), grazingon improved
pastures(Estancia "La Palma", Fraile Muerto, CerroLargo,Uruguay;33'S, 55"W) during
the breedingseason(18-27April 2001).Toconcentratethe AI programinto a few days,the
flock was treatedtwice (11 days apart)with 100¡-rgof a PGF2,,. analogue(Glandinex'":
Universal-Lab,Montevideo,Uruguay).Visual control of standingestrusbegan 10 days
after the second administrationof PGF2' in order to skip the estrus that followed the
pharmacologicalinduction of luteolysis. To detect ewes in estrus, 120 androgenized
wethers (TestosteronaUltra Lenta Fuerte"': testosteronecyclopropionate,200 rng per
week per wether,LaboratoriosDispert,Montevideo,Uruguay) were used.Marker paints
were usedon the wethers'chests,the flock gatheredtwice daily from the grazingfields to
the inseminationpaddockswhere thoseewesmarkedon the sacralregion were manually
separatedfrom the flock and inseminated'oover-the-rail"in a shelteredfacility. Ewes
detectedin estrusin the morning were inseminatedin the afternoon,and vice versa,
followingan a.m./p.m ., p.m.la.m.schedule
(7.00a.m.-6.00p.m.).All inseminations in the
trial were performedduring a l0 day period by one highly skilled operator.Strawswere
thawedone by one at 50 "C for 9 s, loadedinto an inseminationgun, and coveredwith a
plasticsheath(Minittib, Landshut.Germany)beforeAI. This preparatorywork w¿rsdone
by an assistantto the inseminator.With the aid of a tubular speculumequippedwith a
fi'ontallight sollrce(Walmur-Veterinary Instruments,Montevideo,Uruguay),the external
openingof the cervix was visualizedand the tip of the AI gun was caref-ullyinsertedas
deeply as possible into the cervical canal, v¿herethe semen w¿ls slowly deposited.
Recordingswere made of the ear tag numberof the ewe, the color (transparent, white,
or cheese-like)and the amounf(scarce,moderate,or abundant)of cervicalmucuspresent
in thevagina,thedepositiondepthinto thecervicalcanalachieved(shallow:<10 mm; mid:
l0-20 tnffr;or cleep:>20 rnm, usingthe colorecltip of the plasticsheathas ref'erence),
the
numberof the ram fiom which the semenwasfrozen,andthetypeof extenderthatwasused
at eachinsemination.

2.6. Fertilih, nleasurements

Fertility was recordedas the percentageof ewesthat had not returnedto estrusat 2l
(NRR-21) or 36 (NRR-36) days after AI. Additionally,transabdorninal ultrasonography
(US, 3.5 MHz sectorprobe, Pie Medical 1000,Maastricht,The Netherlands)was per-
formed to confirm pregnancy50 daysafter AI in thoseewesthat did not returnto estrus36
days after insemination(PR-US).

2.7. Experintentaldesign

Each ejaculatewas divided into equal numbersof AI dosesfrozen in each of the two
extenders.The ewes were taken at random and artificiallv inseminatedwith dosesfrom
ll62 .1.Gil et al./Theriogenology59 (2003) II57-l 170

eachextender.As far as possible,an equal number of dosesfrom each extenderand ram

were used per insemination day. In total, 970 ewes were inseminated(485 ewes per
ffeattnent,200 ewes per ram for rams I4,, and 170 ewes for ram 5).

2.8. Statisticalanalysis

The statisticalanalysesof the recordedparameterswere performedusing the GENMOD

procedureof the StatisticalAnalysis System package(SAS Institute Inc., 1989).The
variationin "NRR-21 NRR-36 and PR-US" was analyzedaccordingto a statisticalmodel
that included: the effects of the ram [5]; extender[2]; body condition scoreof the ewes
(groupedin 3 classes:low, below 2.9; mid, 3-3.9; and top, above4 score;insemination
depth(3 classes);mucusassessment (amountandcolor, 3 classeseach);and the interaction
betweenram x extender.The differencesbetweenextenders(within ram) for the assessed
spermparameterswere analyzedby paired r-test(SAS Version8). The differencesfor the
fertility results (within treatment)between the different depths of AI, mucus amount,
mucus color, and body condition score (classified),were analyzedby Xt test. Spearman
rank was used to analyzefhe relationshipbetweenthe spermparametersand the fertility
results.

3. Results

3.1. Spermparameters

The overall averageconcentrationin the AI dosewas 196.8* 6.3 x 106spermatozoal

straw.In general,all spermparameters appearedslightlyhigherin sementhatwasfrozenin
Bioexcell" (B), excepttbr the proportionof acrosome-reacted (AR) spermatozoa.There
were, however, flo significant differencesbetween extendersfor the post-thaw sperm
parametersevaluated(Table l).

3.2. Fertility results

Of the 970 ewesartificiallyinseminated,335(34.47o)did not return to estrus2l days

afterAI (NRR-21),and327 ewes(33.6Vo)did not returnto estrus36 daysafter AI (NRR-
36). Of the latter,262 ewes (2l .7Vo)were diagnosedas pregnant(PR) using ultrasono-
graphy (US) 50 days after the AI of the last ewe.

Table I
Overallmeans(%)+ S.D. for the spermparameters
evaluatedpost-thawfbr milk-basedor Bioexcellextenders

Spermparameters Milk (control) Bioexcell ( treatrnent)

Subjectivemotility 46.5+ 4.4 47.0t 6.0

Membraneintegrity 3 7 . 7+ 6.6 3 8 . 0+ 8.2
Uncapacitated(F) 2 6 . 3+ 4.r 28j + 6.4
Capacitated(B) 6 1 . 1+ 1.6 6t.3 + 2.7
Acrosome-reacted (AR) 12.6+ 4.7 10.2+ 3.9
 J. Gil et al./Theriogenologv59 (2003) lI57-1170 I 163

3.3. Extendereffect

Fertility was slightly higher for semenfrozenin extenderB than that frozen in extender
M f o r N R R - 2 1( 3 5 . 7+ 9 v s . 3 3 . 1t 5 . 5 7 o ) , N N R - 3 6 ( 3 4+. 78 . 7 v s . 3 2 . 5I 5 . 5 7 o ) , a n d P R -
US (28.3 + 7.2 vs. 27.2 L 6.57o),althoughthesedifferenceswere not statisticallysig-
nificant.

3.4. Ram effect

Regressionanalysisshowedthat the ram effect was the only factor that significantly
affected the fertility results (NRR-21, NRR-36 and PR-US (P < 0.05)). Within ram,
the fertility result (NRR-21, NRR-.36and PR-US, respectively),including both exten-
ders,was highestfor ram 2 (41,,40.5,33.3Vo), followed by ram I (39.5, 38.5, 32Vo),,
ram 4 (34, 32, 28.37o),ram 3 (30, 30, 24.3Vo),and was lowest for ram 5 (27.5, 27,
20.17a).

Table 2
Fertility results after cervical insemination for the different rarns and extenders,reported as raw percentages(%o)

Ram Extender NRR-2I NRR-36 PR-US

Milk 40.0 40.0 33.7

Bioexcell 39.0 37.0 30.3
Mean 39.5 38.s 32.0

Milk 32.0" 32.0" 26.9"

Bioexcell 50.0" 49.0r' 40.0h
Mean 4l.0 40.5 33.3

Milk 30.6 30.6 25.9

Bioexcell 29.4 29.4 22.6
Mean 30.0 30.0 24.3

Milk 37.0 35.0 32.3

Bioexcell 31.0 29.0 24.2
Mean 34.0 32.0 28.3

Mitk 26.0 25.0 17.3

Bioexcell 29.0 29.0 24.2
Mean 27.5 27.0 20.7

Overall Milk 33.2 32.6 27.2

Bioexcell 35.9 34.8 28.4
Mean 34.5 )J. I 27.8
NRR-21: nometurnrate 2l days after AI, NRR-36: nonreturnrate 36 days after AI, PR-US:pregnancyrate
assessed (US) 50 daysafter the last insernination;
by ultrasonography Differentsuperscripts
denotestatistically
significantdifferencesbetweenextenders(P < 0.05; within rarn 2).
tr64 J. Gil et al./Theriogenology59 (2003)1157-1170

3.5. Effict of the interaction extender x ram

Differences between rams are shown in Table 2. The semen frozen in extender M
(control) resultedin betterfertility for rams 1, 3 and 4, while semenfrozenin Bioexcell'R'
yielded better results for rams 2 and 5. The end points used to measurefertility did not
differ significantly betweenextenderswithin ram, exceptfbr ram 2, where Bioexcell'ft'gave
significantlybetter results (P < 0.05).

3.6. Effects of the depth of cervical semendeposition, and the amount and color
of the cervical mucusat AI

The inseminationcathetercould be easilyintroducedmore than 20 mm into the cervical

canal(deepinsemination)in 1401970ewes,while in 617 1970ewesthe cervicalpenetration
was between10 and20 mm (mid-insemination).In2l3l970 ewesthe cervicalpenetration
was less than 10 mm (shallow insemination).Fertility increasedoverall (including both
extenders)with the depthof the insemirration(NS). This trendwas consistentfor extender
B (significantonly at NRR-21 andNRR-36, P < 0.05),but not for extenderM, where the
highestfertility was recordedafter mid-inseminations,fbllowed by shallow,and the lowest
for deep,respectively(Table3). Fertility was not affectedby the amountof cervicalmucus
(NS, Table 4),,or by its color (NS, Table 5).

3.7. Relationshipbetweenpost-thaw spermparametersand fertility

The Spearmanrank analysisshowed a high correlation(r:0.87-0.90, P < 0.001)

betweenthe percentageof viable, uncapacitatedspermatozoapost-thaw(i.e., percentages
of motile spermatozoa,of spermafozoawith intact plasmalerruna,and of uncapacitated
spermatozoawith CTC) and fertility.

Table 3
Fenility resultsfor two semenextendersand the mean result for both extendersafter cervical AI, either deep
(>20 rnm), middle (10-20 mm) or shallow(<10 rnm) depositionof semen

Extender AI Depth NRR-21Votu\ NRR-36Vo(n) PR-US Vo(n\

Mitk Deep 24(r4t59\" 24 (r4t59)" 22(r3ts8)"

Mid 36(1r6t32s\^ 35 (l14t325)^ 28(90t3r7\"
Shallow 3 1( ¡ 1 l l 0 l ) " 30 (30/101)" 26 (26t99)"
Bioexcell Deep 43 (35/81)" 43 (35/81)" 34 (26t77)"
Mid 37 (108t292)"' 36 (r0s/292)" 30 (84/284\"
Shallow 28 (3vtr2)b', 26 (29nr2)t', 2t (231t07)"

Mean Deep 3s (4etr40) 3s (49tr40) 29(39tr3s)

Mid 36 (224t6r7) 3s (2r9t6r7) 29(174/60r)
Shallow 2e (62t2r3) 28 (s9t2r3) 24 (49t206)
NRR-21: nonreturn rate2l days after AI, NRR-36: nonreturnrate 36 days after AI, PR-US: pregnancyrate
assessedby transabdominalultrasonography(US) 50 days after the last AI; Different superscriptsdenote
statisticalsignificanceP < 0.05 within the extender.
 59 (2003)1157-1170
J. Gil et al./Theriogenology I 165

Table 4
Fertility resultsfor two extendersand the mean result for both extendersas relatedto the amount of cervical
mucuspresentat time of cervicalAI

Extender Mucus NRR-21 Va(n) NRR-36 Vo(n) PR-US 7o (n)

Milk Abundant 30 (24t79) 30 (24t79) 2s (20t1e)

Moderate 3s (r04t296) 34 (10r/296) 29 (84t288)
Scarce 30 (33/lr0) 30 (33/1r0) 23 (2str07)
Bioexcell Abundant 33 (23t69) 32 (22t6e) 24 (r6t66)
Moderate 3 7( r r 7 / 3 1 6 ) 36 (rr3t3t6) 24 (9r/377)
Scarce 34 (34tr00) 34 (34/100) 27 (26t96)

Mean Abundant 32 (47tr48\ 3r (46tr48) 25 (36tr4s)

Moderate 36 (22U6r2) 3s (2r4t6r2) 29 (r7sts94)
Scarce 32 (67t2r0) 32 (67t2r0) 2s (5U203)
NRR-21: nonreturnrate 21 days after AI, NRR-36: noffeturn rate 36 days after AI, PR-US: pregnancyrate
assessed
by transabdominal ultrasonography (US) 50 daysafter the last AI.

Table 5
Fertilityresultsfor two extendersandthe meanresultfbr bothextenders,
asrelatedto thelnucuscolor at tirneof
cervicalAI

Extender Mucus NRR-2I NRR-36 PR-US

Milk Transparent 34(84t249) 33 (82t249) 26 (63t244\

Wh i ti s h 30(s4lr82) 30 (s4l 182) 27 (41n76)
Cheese-like 43 (23t54\ 4t (221s4\ 35 ( r9154)
Bioex c ell Transparent 37 (98t263) 31 (96t263) 29 (73t254)
Wh i ti s h 34 (59n76) 32 (561n6) 27 (4s/168)
Cheese-like 37 (11146) 37 (t7t46) 33 ( l s/46)

Mean Transparent 36( 182/5 l2) 35 (178/5 l2) 21 (t36/498)

Wh i ti s h 32 (l l3/358) 3l t t l0/358) 27 (92/344)
Cheese-like 4 0 ( 40/100) 39 (39/r00) 34 (34/100)
NRR-21:nonretunrrate 2l claysafier AI. NRR-36:tlonreturnrate 36 daysafier AI, PR-US:pregnancyrate
assessed
by transabdoruinal
ultrasonography (US) 50 claysafier the last AI.

4. Discussion

Any effort to improvethe efficiencyof cervicalAI in sheepmust considerthe quality

standardsof the seffrendoses,anclthis includesthe useof substitutesfor productsof animal
origin addedto the extenders[9,18].The aim of this study was to comparethe fertility
resultsof ram semenfrozenin Bioexcellrr with a controlmilk-egg yolk extenderthat has
been proven adequatefor freezingram semen.Such an extenderwould reducethe risks
involved in conventionalextenderswhen semenis to be introducedto flocks or countries
free from diseasesthat might be transferredvia the semen dose. Furthermore,some
ingredientsof semenextenders,suchas egg yolk or milk powder,could be diversein their
composition,if they aredifferentbrandsor from differentregions.Bioexcell " representsa
l166 J. Gil et al./Theriogenology59 (2003) 1157-1170

suitablehomogeneousextenderthat can be used as a standardizedreference for further

comparativestudies.
From the results achieved,Bioexcell'lt' seemsto be useful as an alternativeto the
conventional milk-egg yolk extender for processingram semen for cervical AI in
extensivelymanagedlarge flocks.
The sperm parametersstudied after thawing did not differ betweenextenders,confirm-
ing previousresultsfound in a more detailedin vitro studyusing the sameextenderst141.
The concentrationmeasuredby hemocytometryin the AI dosesconfirmedthe photometric
assessment,which was more practical and as accurateas the manual counting in the
hemocytometer.Spermquality post-thawvariedbetweenrams but not betweenextenders,
indicating that both extenderscould be used for long term storageof ram semen.We
observed a positive correlation between subjective sperm motility, sperm membrane
integrity, the proportion of uncapacitated spermatozoa,and the fertility parameters
assessed (NRR-21, NRR-36, and PR-US). Confirmationof high spermquality post-thaw,
as a resultof a suitable freezingprocedure,is a prerequisitefor an acceptablefertility result
after AI was indeedfound. Conversely,we found that the proportionof acrosome-reacted
spermatozoapost-thawshoweda negativecorrelationto fertility.
Freezingram semenin milk-egg yolk extenderhas producedacceptableresultswhen
depositedintracervically,thoughthe techniquehasonly beenappliedin rathersmall flocks
under semi-intensivemanagementconditionsin Scandinavia.The fertility resultsafter AI
in natural estruswith this method rangedbetween29Voin Denmark I l9], 40-51.5Voin
Sweden[6,8], and 33-63.17oin Norway 15,7,20).Pregnancyratesof 28Voin rhe currenr
study, using the same extenderand a modified protocol [5], are similar to the lowest
resultsreportedfrom Scandinavia.However,our resultsare higher than previousreports
from trials performedin Uruguaywherethe useof a differentextender(Tris-glucose-citric
acid-egg yolk) and a total number of motile spermatozoaper AI dose of 90 x 106
(intracervicalAI, in natural estrus)resulted in a lambing rate below l07o tzll. Gil
et al. [13] achievedlambing rates of 217o QA} AI's and estrusdetectiononce a day)
in Uruguay.A number of other factors affect fertility: the female (breed,flock manage-
ment,body condition,environment);the technique(skills,depthof AI, stressinflictedto
the ewe); the semen used for AI (sire fertility, sperm quality post-thaw, number of
spermatozoa);and the environment(pasturequality and quantity,weather,predators).
The conditionsin our study were typical for extensivesheepmanagement,basedon a
gtazing system with improved pastureson a commercialfarm. One of the factors that
negativelyinfluencedthe conditionswas the stressinvolvedin the detectionand separation
of the ewesin estrusfrom the restof the flock. This took placetwice daily, in a flock of 1000
ewesthat were held in a l7O-hectarepen.They werethenwalkedby the gauchosmore than
4 km to the inseminationfacilities.
The weatherwas anotherfactor that affectedworking conditions.On the day when the
number of ewes to be inseminatedpeaked (364 ewes),a rainfall of 40 mm occured.In
earlierstudies,heavyrainswerereportedU4,22lto negativelyaffectthe fertility results.In
this study,however,we did not find any effect due to the weather,probably becausethe
rainfall was calm and lastedthe whole day.
We recordedthe depthof semendepositionand its eventualeffect on fertility, but found
no significanteffectof this factor regardlessof the extenderused.This may be becausethe
 J. Gil et al./Tlteriogenology 59 (2003) 1157-1170 tt67

categoriesin the score did not differ enough (<10, 10-20, and >20 mm) to observe
differences referred to depth, and because most semen deposition was mid-cervix.
Nevertheless,one might speculateabout the differencesseen between extenders.We
found that semenprocessedin milk extenderyieldedthe best(NS) fertility resultswhen the
semen was depositedin the middle region (from 10 to 20 mm). Shallow deposition
(<10 mm) ranked second,and deepdeposition(>20 mm) into the cervix gave the lowest
results.This is in agreementwith a reportby Andersenet al. [4] wherethe semendeposited
between2.5 and4 cmyieldedlower conceptionratesthanwhendepositedin the cervicalos
(57 vs. 457o).The deepinseminations in our studyweremorethan20 mm, butneverabove
40 mm. Therefore,our deep inseminationscan only be comparedwith the mid-cervix
depositionof the previousreport.Furthermore,what wasrecordedasshallowandmiddle in
the presentstudy ought to be comparableto the depositioninto the cervical os in the study
of Andersenet al. [4]. This tendencyis contradictoryto someother reportswhere deeper
inseminationsresultedin higherfertility results[23,24].One explanationfor the tendencies
seenin the fertility resultsbetweenextendersin relation to the depth of AI could be the
reactivityof the cervix to the extenderplus the semen.Thus,the deeperthe depositionof the
semen using the milk-based extender,the greater the reaction to this foreign matter,
resultingin a faster clearanceof the inseminate.Conversely,Bioexcell"' yielded consis-
tently increasingfertility with deepersemendeposition.Possibleexplanationsfor the latter
might be the absenceof egg yolk, or a bettermicrobiologicalquality comparedto the milk
extender.Bousseauet al. [9] demonstrated that extenderscontainingsubstitutes for eggyolk
had a better microbiologicalquality when comparedto the extenderscontainingraw egg
yolk and otheradditivesof animalorigin. The lack of suchsubstances might elicit a milder
response by the femalegenitaltractto Bioexcell". We alsonoticedthat semenextendedin
Bioexcell" was less viscousthan the milk-extendedsemen.The higher viscosityof the
latterextendermay callsea higherresistance whendepositedinto the cervicalcanal.Thus,
the propulsionmight be slower and the risk of causinga larger retrogradeflow of semen
fiom thecervicalcanalinto thevaginaincreases. A lessviscousfluid (Bioexcell" ) mightbe
propelledmore easily along the cervicalcanal in a cranialdirection.
Both the amount and appearance of the cervical mucus were recordedat AI, but no
significantrelationshipwas fbund betweentheseparameters and fertility. It is known that
the sperm population that survives the freezing-thawingproceduresshows alterations,
particularlyin the plasmamembrane.The occurrenceof capacitation-like changeswhen
comparedto theirinitial statusafterejaculation,resultsin a spermpopulationwith a shorter
lif-e span 125-291.Our results confirm these findings, with low proportions of intact
membraneand uncapacitatedspermatozoa,and high proportionsof both capacitatedand
acrosome-reacted spermatozoa recordedpost-thaw.This might be the reasonfor the better
fertility resultsobtained,albeit not significant,when AI was donein eweswhosemucus
was cheese-likein appearance.Cervical mucus is initially transparentduring initial
standingestrusand becomesmore cloudy as estrusproceeds,changingfrom a whitish
to a cheese-likeappearance. Close to ovulation,which is reportedto occur at the end of
estrus,approximately25-30 h afterthe onsetof standingestrus[30], the appearance of the
cervical mucus becomescreamy and cheese-like.This might explain why the fertility
resultswere higher althoughnot significantly,when AI was performedat this stage,since
frozen-thawedsemenhasa shorterlife span,makingthe timing of inseminationin relation
1168 J. Git et al./Theriogenoiogy 59 (2003) ll57-1170

to ovulation important. Although this is a tempting explanation,the lack of statistical

significancemight be relatedto the low numbersof ewesallotted to the different classes
and to different rams.
The ram semenusedwas not selectedfor freezability.The subjectivemotility post-thaw
of the dosesusedvariedbetween40 and 507owith a cut off limit for approvingthe dosesof
40Vo,and most probablythosedosesthat were closeto the acceptancethresholdwould not
be adequatefor commercialAI. Nevertheless, we useda split sampledesignto comparethe
extendersand their effect on fertility, which variedbetweenramsfrom 2l to 337o,showing
that theseextendersare useful for preservingram semenby this freezrng method. For the
sperm parametersassessedpost-thaw,we observedsome beneficial effect of the milk
extenderwhen comparedto Bioexcell''i in threeof the rams,and the oppositeeffect in the
other two rams, confirming the individual variation seenbetweenrams by Windsor [31].
However, similar fertility results were achieved with both extendersunder the field
conditionsprevailing in this study,which might be practically useful, e.g. in the progeny
testing program of rams in Uruguay [32).
It has been demonstratedthat the concentrationof the AI dose is the major effect
accountingfor variationin fertility after intrauterineinseminationt331.Thus, it shouldbe
notedthat the spermconcentrationusedto inseminatethe ewesin our study was,probably
anotherfactor that maskedany differencebetweenextenders.
Bioexcell ú' has proven suitable for freezing ram semen with acceptableresults in
extensivemanagementconditionswhen comparedto the control rnilk-egg yolk extender.
In the light of the results achievedby other researchersusing similar [5-8,19,20] or
differentmethods[34], further studiesof extendersfbr semenextensionmethodology,such
as the one used here appearto be important.

Acknowledgements

The authorsthankLaboratoriosDispert-Uruguayfor providingTestosterona Ultra Lenta

Fuerte'", Universalab-Uruguay for providing Glandinex" and Albanell Hnos-"La
Palma" and its field staff for their help. Thanks also to A. Ferraris,M. Heinzen, the
EEMAC's SheepProductionstaff,and the technicalstaffof SUl-Uruguay (P.Scremini,A.
Casarettoand J. Bonino) for local supportand invaluablehelp. Supportedby the Swedish
Agency for ResearchCooperationwith DevelopingCountries(SAREC), and Kóttbón-
dernasF'orskningsprogram, Stockholm,Sweden.

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