Fertility results using bovine semen cryopreserved with extenders based on egg yolk and soy bean extract

ELSEVIER

FERTILITY RESULTS USING BOVINE SEMEN CRYOPRESERVED WITH EXTENDERS BASED ON EGG YOLK AND SOY BEAN EXTRACT A.M. van Wagtendonk-de Leeuw,’ R.M. Haring, L.M.T.E. Kaal-Lansbergen and J.H.G. den Daas Holland Genetics, Research & Development P.O. Box 5073,6802 EB Arnhem, the Netherlands Received for publication: February Accepted: April

16, 26,

1999

1999

ABSTRACT Semenextenderscontaining componentssuch as egg yolk and skim milk are difficult to standardizeandthey introducethe risk of microbial contamination,A well-defined extendernot originating from animal tissueswould presenta valuable contribution to the AI industry. We evaluatedthe fertility of bovinesemencryopreservedwith 3 different extenders: 1)TRIS-Standard, preparedat 2 local AI laboratories,containing 20% (v/v) pasteurizedegg yolk, 2) TRISConcentrate,preparedby adding20% (v/v) pasteurizedeggyolk and 1:5(v/v) nonpyrogenicwater, and3) BiociphosPlus,a soybeanextract containingextender,preparedby adding1:5nonpyrogenic water. Ejaculatesof 4 Holstein bulls were split into 3 aliquots and cryopreservedwith the 3 extenders.Prior to this study,the semendose-response curve for eachof the 4 bullswasdeveloped in a field trial by freezing the semenand randomly distributing the straws throughout the Netherlandsfor insemination.Optimal semendoseswerethus establishedto detect the effect of extenderson fertility, evaluatedby 56&y non-returnrate(NR56), andby the estimatedconception rate and the calving rate, given a conception. We usedthe multiphasicmodel developedby Grossmanet al. (7). A total of 22,246first and secondinseminationswere recorded.The NR56 rangedamongbullsfrom 67.0to 70.1%for Tris-Standard,from 67.5to 69.9% for Tris-Concentrate and from 60.2 to 66.7 % for BiociphosPlus.No significant differencesin NR56 were detected between Tris-Standardand Tris-Concentrate(P=O.54), whereasBiociphos Plus resulted in a significantlylower NR56 thanTris-StandardandTris-Concentrate(P
by Elsevier

Science

Inc.

Key words: bovine semen,extender,cryopreservation,non-returnrate, conceptionrate

Acknowledgments We thank the staff at the AI laboratoriesof HollandGeneticsfor their excellent cooperation andperformance,andIMV, France,for supplyingthe BiociphosPlusextender. ’ Correspondence and reprint requests. Theriogenology 02000 Elsevier

.54:57-67,2000 Science Inc.

0093-691X/00/$-seefrontmatter PII: SOOQ3-691X(00)00324-1

58

Theriogenology INTRODUCTION

Two main challenges in the bovine AI industry today are the control of nonpathogenic, environmental contamination, and total quality management. Preparation and composition of the semen cryopreservation extender affects both factors. Standard extenders for cryopresetvation of bull spermatozoa usually consist of whole or skim milk or a TRIS solution to which egg yolk is added (2,10). Non-pasteurized fresh egg yolk introduces a risk of microbial contamination (1). To lower this sanitary risk, the use of industrially processed, pasteurized and microbiologically monitored egg yolk, processed according to established standards for food intended for human consumption, may serve as a good alternative. A welldefined and pathogen-free substitute of nonanimal origin for egg yolk would obviously be even more preferrable. Complete preparation of the extenders is usually carried out at the AI laboratory. The preparation is labor intensive and allows introduction of human errors and results in extenders of variable quality. Quality testing procedures are often required. Ready-to-use, manufacturer- certified extenders available on the market would minimize handling and accelerate preparation of the extender in the laboratory. For some years now, an extender concentrate containing soybean extract as a substitute for egg yolk of non-animal origin (Biociphos Plus, lMV, France) was commercially available. Studies with Biociphos Plus showed no significant differences in fertility compared with existing egg-yolk containing extenders (1,8). However, most field studies in which new extenders or procedures are tested apply high semen dose rates (e.g. 20 x 1O6total spermatozoa per dose ( 1,8)) for measuring effects on fertility in the field. The optimal dose rate, i.e. the semen dose containing a minimal number of spermatozoa and resulting in maximal fertility differs between bulls. This event occurs because the semen dose response curve can be described by an exponential function and is characterized by its asymptote, i.e. the maximum NR at high semen dose rates, and the rate of increase to this level of NR (5). Differences in the quality of the inseminated semen population will be reflected in differences in field fertility, only if optimal or suboptimal semen dose rates are used in a field trial (9). Recently, a multiphasic logistic model was developed (7) that estimates conception rate and calving rate from daily return data. The NR56 consists of both conception rate at or near time of insemination and the developmental capability of the zygote. Those two parameters are affected by different semen characteristics. Extrinsic semen factors related to the quality of the inseminated semen population such as motility, plasma membrane integrity, ability to capacitate, acrosome react and bind to the zona pellucida, are considered to affect conception rate; whereas intrinsic semen factors related to the quality of the penetrated spermatozoon such as degree of DNA condensation and DNA damage are, among others, related to the developmental capabiliy of the zygote. Conception rate and developmental capability of the zygote (=calving rate, given a conception) provide more relevant information for evaluation of the performance of AI technicians and bulls than the NR56 (7). In this study, the multiphasic model was applied for the first time to evaluate the effect of processing conditions (in the present study extender composition) on conception rate and developmental capability of the zygote, thus distinguishing effects on extrinsic and intrinsic semen characteristics.

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59

The aim of the presentstudy was to comparefield fertility, i.e. NR56, conceptionrate and calving rate,given a conception,at optimalsemendoserates,using3 different extendersfor bovine semencryopreservation:1) a standardTRIS extenderwith 20%(v/v) pasteurizedeggyolk, prepared at local AI laboratories,2) a five-fold concentratedTRIS extenderto which water andpasteurized eggyolk wereadded,and3) Biociphos-plusfive-fold concentrate,an extendernot of animalorigin, to which water wasadded.

MATERIALS AND METHODS SemenCollection and Processing Semenwascollectedfrom 4 provenHolsteinbullsat 2 AI stations(3 bullsat stationA and 1 bull at stationB). Within 5 to 10min after collectionandincubationat 31°C semenconcentrationwas determined by meansof particle counting (Coulter Zl, Beckman Coulter, Mijdrecht, the Netherlands).The raw ejaculatewasweighed,split-sampledinto 3 equalvolumesanddiluted with the appropriatepre-warmed(3 1“C) extenderto the relevantsemenconcentration.The percentage motile spermwas assessed subjectively by an experiencedtechnician, using a phasecontrast microscopeat 200 x magnificationat 37°C. Extendedsemenwith a motility of 65% or morewas usedfor further processing.At AI stationA, volumesof 100 mL extendedsemenbatcheswere placedfor 1.5h at 5°C beforeloadinginto 0.25 ml plasticstraws.During further equilibrationfor at least 1.5 h, strawswere spreadon precooled(5°C) freezing trays. Freezing was done by transferringfreezingtrays to a -95°C precooledtemperaturecontrolledchamber(Zuidberg,Raalte, Holland)for at least8 min. At AI stationB, extendedsemenwasloadedinto 0.25mL plasticstraws at ambienttemperature(18 to 20°C), storedin an insulatedbox andsubsequently placed at 5’C for at least3 h, at the endof whichtime strawswereplacedon precooled(5°C) freezingtrays. Freezing wasstartedby transferringthesetrays into a programmablefreezing device (IMV-Digitcool, L’ Aigle, France)at 5°C. Strawswere cooledto -1O’C at -4”C/min, and from -10°C to -145’C at -4O”Uminandweresubsequently plungedinto liquid nitrogen.Motility of spermatozoa of 2 pooled strawsper batch was assessed subjectively by an experiencedtechnician,usinga phasecontrast microscopeat 200x magnificationat 37°C.Semenbatcheswith a post-thawmotility of at least35% were usedfor insemination.The 3 split samples of eachbull’s ejaculatewerecodedanddistributed randomlyto AI technicians,which resultedin a blind trial. Extender Preparationand Storage Formulationsof Tris-Standardand Tris-Concentrateare shownin Table 1. Tris-Standardwas preparedin 1 batch accordingto the formulation of each Al station (Table 1). The necessary componentswere dissolvedin, or addedto, nonpyrogenicwater. Tris-Concentrate was custom made (Gibco-BRL, Life Technologies,Breda, the Netherlands)and delivered as a five-fold concentrateat 5°C. To 1 part of concentrate3 partsof nonpyrogenicwaterand 1part of pasteurized eggyolk wereadded.After preparationof 1 batch, portionsof Tris-StandardandTris-Concentrate werestoredfrozen at -20°C until the day of usage.The samebatchesof nonpyrogenicwater and pasteurizedeggyolk wereusedfor preparationof Tris-Standardand Tris-Concentrate.

60

Theriogenology

Biociphos Plus was custom made (IMV, L’ Aigle, France) and delivered as a five-fold concentrateat 5°C. This extendercontainssoy-beanextract. Preparationwasdoneon the day of usageaccordingto the manufacturer’srecommendationby adding 100mL of prewarmed(31“C) extenderconcentrateto 400 mL of prewarmednonpyrogenicwater. One batchwasusedthroughout the experiment. All 3 extenderscontainedantibiotic formulationsaccordingto the EC directive 88/407.

Table 1. Compositionof Tris-Standardpreparedand usedat Al StationA and Station B andof Tris-Concentrateusedat both StationsA and B. Component Tris-Standard Tris-Standard Tris-Concentrate StationA Station B StationsA & B Unit mM TRIS” 200 200 200 Citric Acid 66.7 64 64 mM 55.5 55.5 55.5 mM D-Fructose Glycerol 594 760 760 mM Benzylpenicillin-G 1.106 l.106 l.I06 W/Lb Streptomycinsulphate l.106 1.106 1.10” IU/Lh Lincomycin 0.51 0.51 0.51 mM mM Spectinomycin 0.228 0.280 0.280 Eggyolk” 200 200 200 mL/L -__ mL/L Green-Shaded 0.48 “TRIS(hydroxymethyl)aminomethane yntemational Units per liter “PasteurizedEgg-Yolk (Bouwhuis,Raalte,the Netherlands) dGreen-Shade (5% v/v), greendye

ExperimentalDesignand StatisticalAnalysis In orderto detectat leasta 2% differencein NR56(68 versus70%)with a reliability of 95%and a power of the statisticaltest of 80%, 6600recordedinseminations were requiredfor eachtested extendergroup( 11,13). Prior to the presentextenderfield trial, the semendose-response curve for eachbull wasdeterminedfor semenfrozen in Tris-Standardextenderusingsplit-sampleprocessed ejaculatesextendedto semenconcentrationsrangingbetween2.5 and20 x 10”total spermatozoa per dose (0.25 mL straws). Actual semendose rate of each ejaculate’ssplit sampleswere determinedpost-thawby meansof particlecounting(Coulter ZI) andNR56were relatedto “true” semendoserates. Table2 andFigure 1 showresultsof the dose-response curvesfor the 4 bullsused in the extenderfield trial. The NR56 in Tables2 and4 are(pre)correctedfor herd, season,day of the weekof insemination,Al technician,parity of the inseminated cow, liquid or frozen semen(11). Subsequently,theseprecorrectedNR56 wereanalyzedusingan exponentialcurve (NR56=a*exp(b/semendoserate) in caseof semendose-response curves,or usingthe statisticalmodeldescribed below in caseof the presentextender trial. Routine or optimal spermconcentrations(sperm concentrationsat the bendingpoint of the exponentialcurve, asestimatedby eye) for Bull 1,2,3 and 4 were 10, 15, 10and 15 x lo6 total spermper dose,respectively. In the extenderfield trial, spermconcentrationwassetbelow the optimal concentrationof thesebulls in order to be ableto

61

Theriogenology 74

74

73

73

72

72

71

71 NR70

NRi’o 56 % 69 66

;

69 66 67 66 65 64 0.00

A 5.00 .

.--.m~‘--+.

64 0.00

Bull 3

dose

15.00 rate

20.00

25.00

Bull 2

Bull 1

65

10.00 semen

i /

5.00

1

1 10.00 15.00 semen dose rate

20.00

25.00 0.00

5.00

io.on

15.00

20.00

Bull 4

Figure 1. Semen dose response curves for the 4 bulls used in the present extender field trial. n : NR56 at different semen dose rates; A: exponential curve fitted through NR56 points: Bull 1: NM6=72.8*exp (-0.24/dose); Bull 2: NR56=70.0*exp (-0.29/dose); Bull 3: no exponential fit; Bull 4: NR56=73,3*exp (-0.54/dose).

25.00

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Theriogenology

detect the effect of the extendersourceon fertility in the field. The spermconcentrationsusedin this studyfor Bull 1, 2, 3 and 4 were 5, 10,7.5 and 10 x IO6total spermper dose,respectively. Percentagepost-thawmotility andNR56 wereanalyzedby regressionusingGenstatstatistical software (6) with the following model: NR56 = u + bull + ejaculate(bull) + extender + bull*extender + error. Percentagemotility was consideredto be normally distributed,whereas NR56 wasconsideredto be binomially distributed. Conceptionrate and calving rate were estimatedfor eachbull extendercombinationby the multiphasicmodelby Grossmanet al. (7) from daily return datauntil Day 180post insemination suppliedby the Dutch Cattle Syndicate. Table 2. Dose-response for fertility in the field. Non Return(NR) ratesat 56 d postinseminationand numberof inseminations(n) of the 4 bullsusedin the presentextender-field study. Semendosea Bull 1 Bull 2 Bull 3 Bull4 -NR n NR n NR n NR n 2.5 66.3 1147 5 69.I 1171 67.0 1044 65.7 958 7.5 70.1 1167 65.7 1108 66.7 1265 10 72.5 1180 66.9 1142 67.8 1374 69.7 1068 12.5 70.8 1139 69.4 1014 70.2 1239 15 71.6 1182 69.3 1086 68.4 1363 70.5 981 20 64.9 1278 71.3 985 ax IO6total spermatozoaper dose(0.25 ml straw)

RESULTS For Bulls 1,2 and4 a total of 7, 12and8 ejaculatesweresplit-sampleprocessed at Al stationA, anda total of 9 ejaculateswere processedfor Bull 3 at AI stationB, respectively. None of the ejaculateswasrejectedbecauseof an inadequatepercentagemotilecellspost-thaw(~35%). A total of 22,246first andsecondinseminationswere recorded. Post-thawMotility In the statistical analysis, percentagepost thaw motility was significantly affected by the interactionbetweenbull andextender(PcO.05). Leastsquaremeansandstandarderrorsfor eachbull extender combination producedby the statistical model are shown in Table 3. No significant differencesin percentagepost-thaw motility were determinedbetweenTris-Standardand TrisConcentrate(overall P=O.7). The percentagepost-thawmotility wassignificantlydifferent (PcO.05) between Tris-Standardand BiociphosPlusand betweenTris-Concentrateand BiociphosPlus for Bulls 1 and 2. Bulls 3 and 4 showedno significant differencesin percentagepost-thawmotility betweenthe 3 extenders.

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Table 3. Least square means (LSM) and standard errors (SE) of percentage post-thaw motility for each bull extender combination produced by the statistical model. Overall LSM and SE for each extender and each bull are-given (number of ejaculates n). Bull Tris-Standard Tris-Concentrate Biociphos Plus Overall n LSM * SE LSM f SE LSM * SE LSM f SE 1 40.7i 1.1 a 42.1 f l.la 35.7i 1.1 b 39.5 f 0.7a 7 2 43.1 * 0.9” 42.7 * 0.9” 37.7 f 0.9 b 41.1 * 0.5”b 12 3 41.9* 1.0” 41.4i 1.0” 43.6 + 1.Oa 42.3 * 0.6 b 9 4 41.4 f 1.0” 42.5 i 1.0” 39.7 f 1.0” 41.2 l 0.6”’ 8 Overall 41.9 f 0.5” 42.2 i 0.5 a 39.2 f 0.5 b 36 a,b Different superscripts in the same row indicate significant differences (PcO.05)

Non-Return Rate In the logistic regression, NR56 was significantly affected by the interaction between bull and extender (P
Table 4. Least square means (LSM) and standard errors (SE) of Non Return rates 56 d post insemination for each bull extender combination produced by the statistical model (number of inseminations n). Biociphos Plus Overall Bull Tris-Standard Tris-Concentrate LSMiSE LSM*SE n LSM*SE n LSMiSE n 1 7O.k 1.2 a 1455 69.1* 1.2” 1502 60.21 l.3b 1408 66.6* 1.2” 2 69.51 1.1 ab 1821 69.9+ 1.1 a 1960 66.7* l.2b 1785 68.8* l.2b 3 67.0+ 1.1 ’ 2117 67.5i 1.1 a 2045 66.1+ 1.1 a 2035 66.9+ 1.2” 4 68.5* 1.1 a 2092 66.61 1.1 a 2148 63.1~ l.2b 1888 66.1* l.2a Overall 68.7* 0.6 a 7475 68.2* 0.6 a 7655 64.31 0.6b 7116 a,b Different superscripts in the same row indicate significant differences (PcO.05)

n 4355 5566 6197 6128 22246

No significant differences in NR56 were determined between Tris-Standard and TrisConcentrate (overall P=O.54). The NR56 was significantly higher (PcO.05) for Tris-Standard compared with Biociphos Plus for Bulls 1 and 4, whereas the NR56 of Tris-Concentrate was significantly higher compared with Biociphos Plus for Bulls 1, 2 and 4. Bull 3 showed no significant differences between the 3 extenders. Estimated Conception and Calving Rate Table 5 shows conception rate, calving rate and calving rate, given a conception, estimated by the multiphasic model from daily return data. The model does not produce standard errors of the parameters. Results in Table 5 show an overall lower conception and calving rate for Biociphos Pius compared with Tris-Standard and Tris-Concentrate, which is a reflection of results of Bull 1,

64

Theriogenology

2 and4. Calving rate, given a conception,waslowestfor BiociphosPluswithin Bull 1 and2, but highestfor BiociphosPlusin Bull 4 and intermediatein Bull 3. Bull 3 showedhighestconception ratefor Tris-Concentrateandhighestcalvingrate,given a conception,for Tris-Standard.Sincethere is only 1 estimateper extender, significancelevels can not be calculated. Figure 2 showsthe multiphasicmodelcurve of non-daily return datafor the 3 extenders. Table 5. The conceptionrate (per) andcalving rate (pcvr) were estimatedfor eachbull extender combinationfrom daily returndataprovidedby the Dutch Cattle Syndicate. Calvingrate, given a conception,wascalculated(pcvr/pcr). Tris-Standard Tris-Concentrate BiociphosPlus per pcvr pcvrlpcr per pcvr/pcr per pcvr pcvripcr Bull Pcvr 66. I 50.6 77.3 1 70.1 60.9 87.1 73.9 61.2 82.4 2 3 4

72.9 68.0 74.8

56.8 57.8 58.4 58.4

Overall 72.1

5

15

25

78.1 85.3 77.3 80.6

35

45

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57.4 58.0 57.9 58.5

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78. I 78.4 78.4 78.3

71.8 70.2 62.9 69.6

53.5 56.8 53.1 53.8

75.0 81.4 84. I 77.1

~5105115125135145155165175

since

service

Figure2. The multiphasicmodelcurve of daily nonreturndata(asa probability of nonreturn)for the 3 extenders(curves of Tris-StandardandTris-Concentrateoverlap).

Theriogenology

65 DISCUSSION

We have shown in this field trial that use of a commercially preparedextender (TRISConcentratewith pasteurizedegg yolk) resultsin fertility equivalentto that of locally prepared standardTRIS extenders.Sincewe testedonly onebatchof eachextender,we canonly assume that, due to reducedhandling,there will be lessvariation betweenbatchesof commercialextender. BiociphosPlusresultedin significantly reducedfertility in 3 out of 4 bulls. The BiociphosPlus extenderincludessoybeanlecithin asa substitutefor eggyolk to prevent or repair damageto the spermatozoa1 plasmamembraneduringcryopreservation.An earlierstudy(3) hasshownthat, based on percentageof spermatozoa with intact membranes after thawing,the beneficialeffect of eggyolk during cryopreservationis hardto copy or improve upon.The observeddecreasein fertility using BiociphosPlusconfirmsthe resultsof thisstudywith field data.Earlier field studieswith Biociphos Plus,however,haveshownno significanteffect of extendercompositionon NR56. As mentioned in the introduction, thesefield studiesusedhigh semendoserates(20 x IO6total spermatozoaper dose; 1,8). It is likely that this semendoserate is supraoptimalfor most bulls, meaningthat differencesin the quality of the spermatozoa populationwill not be reflectedin fertility results(9). Bull 3 of this trial showedno sigmiticanteffect of extendercompositionon NR56anda deviant responsein conceptionrate and calving rate given conceptioncomparedwith the other 3 bulls. Taking into accountthe doseresponse curve of this bull (Table2 andFigure I), which could not be fitted with an exponentialrelationbetweensemendoserateandNR56, we may well havefailed to usean optimal semendoserate for this bull. Bull 3 wasstationedat a different location than the other 3 bulls. Therefore, an interaction betweenbull managementand/or semenprocessingand extendercompositioncan not be excluded. The NR56 waslower than usuallyreported,which wasexpectedbecauseof the relatively low suboptimaldoserate usedin this trial. Percentagepostthaw motility wassignificantly lower for BiociphosPluscomparedwith Tris-Standardor Tris-Concentrate.This may be explainedby the higherviscosity andthe presenceof particulatedebrisin the BiociphosPlusextenderresultingin a relatively lower, subjectiveevaluationof motility (personalcommunicationT. LuimstraandK. Zuidberg).Whetherthis is the causeof reducedfertility isnot clear.In general,the predictive value of postthaw motility for field fertility is consideredto be limited (4). Washingthe spermatozoa out of the cryopreservationextenderand resuspension in a morephysiologicalmediumwill probably resultin a morereliableestimationof post-thawmotility. Extender compositionseemsto affect both conceptionrate andcalving rate given conception, i.e. both extrinsic and intrinsic factors (4,5). However, the trend amongthe 3 extendersfor conceptionrateandcalving rategiven conceptionwasnot asconsistentasfor NR56. If we compare proceduresandresultsfor liquid semenwith thosefor frozen-thawedsemen,we find that to obtain a NR56 of about70%, we would haveto useinseminationdosesof 1 to 5 x lo6 spermatozoaof which 90%are live andinseminationdosesof 10to 20 x lo6 spermatozoa,of which 40 to 50%are live, for liquid andfrozen-thawedsemen,respectively, This impliesthat not only half of the cells die during cryopreservation,but that also the quality of the remainingpopulationis negatively affected. This negative effect can act on both extrinsic as intrinsic sperm factors. To our knowledge, this is the first time that a change in the settings of a parameterwithin the cryopreservationprocess,in the presentstudy extendercomposition,affects both conceptionrate,

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66

relatedto extrinsic semenfactors,andcalving rate given conceptionor developmentalcapability of the zygote, relatedto intrinsic semenfactors. The minimum number of inseminationsrequired to obtain an accurate estimationof the parametersby the multiphasicmodelis 1000(personalcommunicationJ. Zonderland).Although difftcult to interpretbecauseof the lack of standarderrorsof the estimatesproducedby the model, the dataon conceptionrate andcalving rate given conceptionadd to the informationprovided by NR56. We will continueto usethe multiphasicmode1for interpretationof semenfield trials in order to better understandthe additive value. We concludethat usingTris-Concentrateextenderin combinationwith pasteurizedeggyolk for the cryopreservationof bovine semenprovides the advantagesof comparablefertility and less handlingin the laboratory whencomparedwith the locally madeTris-Standard. In addition, the Tris-Concentrateisdefined andtestedby the manufacturer,which from a quality control point of view is preferableover the Tris-Standard. Although BiociphosPlus containsno animal based productsand thus involves lowest sanitary risks,NR56, conceptionrate and, to a lesserextent, calving rategiven conceptionarelower comparedto Tris-ConcentrateandTris-Standard.Basedon theseconsiderations, the AI laboratoriesof HollandGeneticshaveimplementedTris-Concentrate extenderwith pasteurizedeggyolk in their routine semenprocessing. REFERENCES 1. BousseauS, Brillard JP. In vitro and in vivo results of fertility in cattle, following inseminationsperformedwith semendiluted and frozen in a diluent free of animal origin products(BiociphosPlus),in: Proceedings6th EuropeanAI V 1994. 2. ChenY, FooteRH, TobbackC, ZhangL, HoughS. Survival of bull spermatozoaseeded and frozen at different ratesin eggyolk-tris and whole milk extenders. J Dairy Sci 1993; 76: 1028-34.

De LeeuwFE, de LeeuwAM, denDaasJHG, ColenbranderB, Verkleij AJ. Effects of various ctyoprotectiveagentsandmembrane-stabilizing compounds on bull spermmembraneintegrity after cooling and freezing.Cryobiology 1993;30:32-44. 4. Den DaasJHG. Predictionof bovine malefertility. PhD Thesis,WageningenUniversity 1997, 168pages. 5. Den Daas JHG, de Jong G, LansbergenLMTE, van Wagtendonk-deLeeuw AM. The relationshipbetweenthe numberof spermatozoainseminatedandthe reproductiveefficiency of individual dairy bulls. J Dairy Sci 1998;81:1714-1723. 6. Genstat5, Release3 ReferenceManual. Genstat5 Committee,Oxford, ClarendonPress, 3.

1993.

GrossmanM, KoopsWJ, den DaasJHG. Multiphasic analysisof reproductiveefficiency of dairy bulls. J Dairy Sci 1995;78:2871-2876. HaardM. Semen.Processing, generalaspects,new diluents,cooling rates.In: Proceedings 9th EuropeanAI V 1997. Hammerstedt RH. Evaluationof spermqualtiy: identificationof the subfertilemaleandcourses of action. Anim ReprodSci 1996;42:77-87.

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IO. HinschE, HinschKD, BoehmJG, Schil WB, Mueller-Schloesser F. Functionalparameters and fertilization success of bovine semencryopreservedin egg-yolkfree andegg-yolk containing extenders.ReprodDom Anim 1997;32:143-149. Il. SnedecorGW, CochranWG. StatisticalMethods.Ames,IA. The Iowa StateUniversity Press, 1980;66-70,102-105. 12. Van der SchansGD, TimmermanAJ, den DaasJHG. An immunochemicalassayfor the assessment of bovine spermquality with respectto fertility efficiency. Proc 13thInt Congron Animal Reprod,1996;3:24-15. 13. Van Wagtendonk-deLeeuwAM, den DaasJHG, Rall WF. Field trial to comparepregnancy ratesof bovine embryocryopreservationmethods:vitrification and one-stepdilution versus slowfreezing andthree stepdilution. Theriogenology1997;48:1071-1084.