The relationship between semen evaluation methods

The

relationship

between

semen

evaluation methods

and

fertility

in the bull

Eileen

Linford,

F. A.

Glover,

C.

Bishop

and D. L. Stewart

Cattle

Breeding

Centre,

Ministry

of

Agriculture,

Fisheries and

Food, Shinfield,

Reading,

RG2

9BZ,

Berkshire and*_{National Institute}

for

_Researchin

Dairying,

Shinfield,

Reading,

RG2

9AT, Berkshire,

U.K.

Summary.

Theresultsare

presented

oftwo

experiments

inwhich the

quality

of

samples

of bullsemen wasassessed

by

anumberof

laboratory

testsandthencorrelatedwith the

112-day

non-return_percentageobtained

by

theuseofthose

samples

in A.I. The results

reveal the

inadequacy

of the

laboratory

testsusedasmethodsof

predicting

the

fertility

ofsemen

samples

but show that limits_maybesetoutsidewhichpoorsemen

samples

couldbediscarded.

Introduction

Laboratory

methodsof evaluation of bovinesemenareused

currently

toselect bulls forartificialin¬

semination andtoselectsemen

ejaculates

ofa

satisfactory

standard frombullsatA.I.centres.Itis

essential,

therefore,

to

quantify

the

relationship

between thevarious

laboratory

methods ofsemen

evaluationand the

fertility

obtained

by

theuseofthatsemen.

Bishop, Campbell,

Hancock & Walton

(1954)

investigated

this

relationship

at4inseminationcentres

using

168

ejaculates

from 76 bulls of7

breeds.Since that

time,

there have been

changes

incommercialA.I.

technology,

themost

important

of which is the

freezing

of bullsemenin

polyvinyl

chloridestrawsandthereductioninthe number of

spermatozoa per insemination

whereby

a

larger

number of inseminations _per

ejaculate

has been

achieved.

By

contrast,there have been few

changes

in themethods ofsemenevaluation. It seemed

appropriate

atthis timetore-examinethecorrelationofbullsemencharacteristicswith

fertility.

Theresults

presented

herearefromtwo

experiments.

Thefirst

experiment

was

designed

tocom¬

pare the correlation ofsemen evaluation methodswith

fertility

when eitherfreshorfrozen semen

wasused

(Stewart,

O'Hagan

&

Glover, 1972).

Semen used in this

experiment

wascollectedfrom bulls

used

routinely

at

Reading

in thecommercialA.I.service. Thelimitedrangeofnon-returndataob¬

tained in this

experiment

wastheresult of

using

semenfromasmallgroupof

regular

bulls. Therefore

asecond

experiment

was

designed

inwhicha

large

numberof frozen semen

samples

wasusedfrom

bullswitha_{wider range}of fertilities.Someofthese

bulls,

by

theirsemencharacteristics

alone,

would

nothavebeen

acceptable according

to current

practice

forcommercialA.I.

Materials and Methods

Semen

Experiment

1. Semenwascollectedonfiveoccasions from each of5Friesian bulls. The

samples

usedwereofa

single

ejaculate

or

occasionally

of

pooled ejaculates

and

provided

semenforaminimum

of160inseminations.Semen

samples

werethendividedintotwoforusein the

liquid

orfrozen

form,

both of whichwere

dispensed

in0-5-ml

capacity polyvinyl

chloridestrawseach

containing

atotal of

about 20 106_spermatozoa,

including

deadand livecells.

Samples

werediluted foruse as

liquid

semen in

92-5% (v/v)

ultra-heated

(UHT)

skim

milk, 6-0% (v/v)

_egg

yolk, 1-5% (v/v)

glycerol,

500i.u.

penicillin/ml

and 500pg

streptomycin sulphate/ml

and stored for

approximately

24 hrat5°C

beforeuse.Semen_tobe frozenin

liquid

nitrogen

wasdiluted in

83-0% (v/v)

UHT skim

milk,

10-0%

(v/v)

egg

yolk,

7-0

%

(v/v) glycerol,

towhichwasadded12-5_mg

fructose/ml,

500 i.u.

penicillin/ml

and 500µg

streptomycin

sulphate/ml.

Experiment

2. Fivesemen

samples

fromeach of 24 bulls

(13 Friesian,

7

Hereford,

2

Jersey

and 2

Charoláis)

werediluted with 83-0

% (v/v)

UHT skim

milk,

10-0

% (v/v)

_egg

yolk,

7-0

% (v/v) glycerol,

towhichwasadded12-5_mg

fructose/ml,

500 i.u.

penicillin/ml

and 500pg

streptomycin sulphate/ml.

Thedilutedsemenwas

dispensed

in0-5-mlstrawsandfrozen4cmabove

liquid nitrogen.

Eachstraw

contained about 20 106 spermatozoaandwasstored forat least 28

days

at—196°C. A minimum of200inseminations_persemen

sample

wasachieved.

Inseminations

Thesemenwasused

only

forfirst inseminationsin

Exp.

1 and forfirstand_repeatinseminationsin

Exp.

2. Theseinseminationswerecarriedout

by

the

Reading

Centre and the

Guildford,

Banbury

and

Faringdon

Sub-Centres. Thepercentageofcowsnot

returning

fora_repeatinsemination

(non-return)

at112

days

after the insemination datewasrecorded and usedasthemeasureof the

fertility

ofa semen

sample.

Semenevaluationmethods

Test A.

Sperm motility (vigour

of thewave

motion)

was

subjectively

scored

by

twoobservers

using

ascaleof0to5. Theundilutedand codedsemen

samples

wereexamined ina

microscope

withaheated

stagemaintainedat40°C.

Test B. As in Test

A,

butascale of 0to10wasused.

TestC. The

proportion

of_spermatozoa

(expressed

as a

percentage)

showing progressive motility

at40°Caftera20-fold dilutionofasemen

sub-sample

in

0-9% (w/v)

NaClwas

subjectively

assessed.

Test D. The

motility

ofthe spermatozoainundilutedsemen was

objectively

measured

by

meansof

the

Motility

Meter

(Glover, 1968)

whichis basedon an

optical-rheological principle;

shearing

forces are

imposed

onthesementoerasethe_patternofmovement,thewave_patternof the_{sperm movement}

gradually

returnswhentheseforcesareremoved and the

reciprocal

of thetimetaken for thereturnof

movementisrecordedas

motility.

TestE. The _percentageof 'dead' _spermatozoain undiluted semen, i.e. thosecells stained

by

a

nigrosin-eosin

stain

(Hancock, 1956),

was

subjectively

assessed.

Test F.Thepercentage of

morphologically

abnormalspermatozoawas

subjectively

assessed

using

the slides

prepared

for TestE.The abnormalspermatozoawerefurther classifiedasfollows:

(i)

ab¬

normal heads;

(ii)

detachedheads;

(iii)

cytoplasmic

droplets

attachedtothe anteriorpart ofthe

mid-piece; (iv) cytoplasmic

droplets

attachedtothe middle_partofthe

mid-piece;

(v) cytoplasmic

droplets

attached

distally; (vi)

coiled

tails;

(vii)

bent

tails;

and

(viii)

other abnormalities.

TestG. Thepercentage of motile spermatozoainsemen thawedforatleast 24 hr after

freezing

wasassessed

by

twoobservers.

TestH. As for Test

G,

butassessmentafter 1 hr ofincubation ofthethawedsemenshakenina

waterbathat40°C.

Test I.As for Test after 2 hrof incubation.

TestJ. Theamount of

glutamic-oxaloacetic

transaminase

(GOT)

in the seminal

plasma

ofun¬

dilutedsemenwasmeasured

spectrophotometrically.

This_assay indicated theextentof membrane

damage

of the_spermatozoaandwascarriedouton53ofthesemen

samples

only.

Test K. As for Test J butonthe

thawed,

dilutedsemen

samples.

TestL. For 114ofthesemen

samples

the heatof metabolism of spermatozoain

thawed,

diluted

samples

wasmeasuredon anLKB model 10700-1 microcalorimeter

by

Dr . J.

Berridge (Department

ofChemical

Microbiology,

NationalInstitute for Research in

Dairying, Shinfield, Reading).

Test M. Six of thesemen

samples

were

sub-sampled by

Dr R. C. Jones

(now

at

Department

of

Biological Sciences, University

of

Newcastle,

New South

Wales, Australia)

todetermine aberrations ofthe ultrastructureofthespermatozoaafter

cooling

and

freezing.

Nine

morphological

classes of

spermatozoawere

distinguished

according

tothe_presenceorabsenceofultrastructural features of

the

plasma

membraneandacrosome

(see

Jones &

Martin,

1973).

Results

Experiment

1

The distribution of the non-return data obtained

by

use of freshsemen

(mean

63-7,

standard deviation

70)

wassimilartothat of frozensemen

(mean 62-8,

standarddeviation

7-7)

even

though

the

number of

samples

ineach categorywassmall

(Text-fig.

1

).

The

degree

ofthe

relationship

between the

non-returnrate at112

days

afterinsemination andeachof the8 evaluationtestshasbeen

expressed

as acorrelation

coefficient,

thepercentageof variationofthenon-returnrateaccounted for

by

each

testandthe95

%

confidence limits ofthe

predicted

non-returnvalue ofa

single sample (Table

laand

b).

None ofthecorrelation coefficientswas

high.

Test

H,

the

post-thaw

% motility

afterincubationat

40°C for 1

hr,

and Test

D,

the

motility

meter

reading

at

collection,

_gavecorrelationcoefficients

sig¬

nificantly

different fromzero(P<

0-05)

for freshandfrozensemen

respectively. However,

the rank orderof thetestsin_respectof correlationwasnotthesameforfresh andfrozensemen.None of the

testscould be used forafreshorfrozensemen

sample

to

predict

anon-returnvaluewith95

%

con¬

fidence limitslessthan

±12-7%

(Table

la and

b).

60r 50 40 30 -20 -II) Exp. 1 _Exp.2 30 40 50 60 70 80 <30 30 40 50 60 70 80

112-daynon-return_{% groups}

Text-fig.1. Distribution ofthe 112-day non-return _{percentages obtained by} the use of bovinesemen

samples(-, fresh;-,frozen)in_Experiments1 and 2.

Experiment

2

In

comparison

with the results of

Exp. 1,

thedistribution ofnon-returndatainthis

experiment

(mean 57-9,standard deviation

10-7)

showeda

larger

number of

samples

ineachcategory,

particularly

those oflessthan 50

%

non-return

(Text-fig.

1).A

diagram showing

the

relationship

between thenon¬

return rateand Test G (the

post-thaw

motility

of

spermatozoa)

is

presented

inwhich each semen

sample

is

distinguished by

the breed of the bull fromwhichitwascollected

(Text-fig. 2).

TestGis that

most

commonly

usedtoevaluate bull semenafter

processing.

An

analysis

ofvariance ofthenon¬

Linford

Table 1. Relationshipbetweennon-return_{percentages and evaluation}testsforbovinesemen

Test Correlation coefficient(r) % Variationaccounted for innon-return(100

r2)

95%Confidence limits ofpredictednon-returnvalue

ofasingle_sample

(¡ï) Exp. 1,freshsemen

I G C E F A D +0-412* +0-377 -t-O-351 +0-312 +0-295 +0-179 +0-158 +0-094

(b)Exp. 1,frozensemen

D E G H C F I A (c) Exp. C B E A D F G L H J I K +0-408* +0-335 +0-253 +0-226 +0-222 -0-215 +0-158 +0-080 +0-698*** +0-672*** -0-666*** +0-641*** +0-608*** -0-604*** +0-596*** +0-548*** +0-472*** -0-443*** +0-350*** +0-002 17-0 14-2 12-3 9-7 8-7 3-2 2-5 0-9 16-6 11-2 6-4 5-1 4-9 4-6 2-5 0-6 48-7 45-2 44-4 41-1 37-0 36-5 35-5 300 22-3 19-6 12-3 0 12-7 130 131 13-1 13-1 13-5 13-6 13-7 13-6 14-3 14-9 150 14-8 14-8 15-2 15-1 15-1 15-5 15-7 161 16-9 16-9 16-9 17-1 18-6 18-6 19-8 21-0

Degreesof freedomfor correlationcoefficientswere23for(a)and(b)and 118 for(c).

Significancelevels:*_P<

0-05;***

<0001.

same breed

(P< 0001).

The variation between bulls was such that

significant

differences in non¬

returnratebetweenbreedscouldnotbeestablished.

Nevertheless,

ofthe 20semen

samples

that_gave anon-return rateof<

50%,

13

samples

werefrom 3 Hereford bulls.An

analysis

of variancewasalso

performed

forthesemenevaluationtests

when,

in all_exceptTest

(the

amountofGOT inthawed

samples),

therewere

highly

significant

differencesinsemencharacteristicsbetweenbulls ofthesame

breed

{P< 0-001).

Significant

breed differenceswere established at a

significance

level ofat least <

0-05,

mainly

between the Friesiansand

Herefords,

fortests

measuring motility

ortheextentof

abnormality

ofthe_spermhead but also betweentheHereford and

Jersey breeds,

particularly

forthe

subjective

assessmentof

motility

inthawedsemen

(Tests G,

and

I).

It_appearsfrom

Text-fig.

2 that thenon-return ratewas

positively

correlated with the

post-thaw

motility

ofsemen

samples.

Therewas someindication ofacurvilinear

relationship

butthenumber of

samples

in thelower_rangeofnon-returnwastoosmalltoestablishsucha

relationship

withconfidence.

Thecorrelation coefficients for all the testsusedare

presented

in Table

1(c).

The coefficientswere

higher

than in

Exp.

1 and allbutone were

highly significant

(P< 0001).

The

improvement

in the

correlationwas

mainly

dueto 10

samples

with low

motility

andlownon-return rates.

Alinear

regression analysis

ofthe datashowedthat the best

single

testwasthe

subjective

evalua¬

tion ofsperm

motility

after thesemenhad beendiluted in 0-9

%

NaCl

(Test

C).

The

relationship

be¬

of

Text-fig.2. Adiagramoftheassociationbetween the112-daynon-returnpercentageofa_sampleof bovine

semenand thepercentageof motilespermatozoainit after_{thawing(TestG).}Eachsampleisdistinguished bythe breed of bull fromwhichitwascollected.

meansof

measuring

_sperm

motility,

Test B.

Apart

from

motility

thenextmostreliable characteristic ofsemen fornon-return evaluationwas the

%

ofdeadcells

(Test E).

The othertwo methods of

motility

measurement

(Tests

A and

D)

followedTest E inrank-order.Allthesetestswerecorrelated with eachother. The correlation coefficient ofTest Cwas+0-94with Test

B,

-0-81with Test

E,

+0-91 withTest Aand +0-78with Test D.TestDwasthe

only objective

method of

motility

measurement

used in the

experiment.

%motilespermatozoaafter dilutionin saline(Test C)

Text-fig. 3. Therelationship between the 112-daynon-return_percentage and the_{percentage of motile}

spermatozoainbovinesemen_{diluted with saline(Test C).Theregressionline(-) and95%confidence}

limits(-)for_predictednon-returnrateofasingle sampleareshown.Numbersrepresent thelocationsat

Linfordet

A

multiple regression analysis provided

additional information. The _percentageofnon-return

varianceaccounted for could be increased

by

the combination of theresults oftwotests; the best

pair

being

TestE,the

%

of deadspermatozoa,and Test

F(iii),

the

%

of_spermatozoa

carrying

cyto¬

plasmic droplets

inananterior

position

(Table

2).

The lattertestwasnot

high

in the rank-orderof

single

testsbutsomefeature of the resultsit

provided

made it valuable in combination with either

Test E orTest C

(see

Table

2).

The correlationcoefficients ofTest

F(iii)

with Tests C and Ewere

—0-28and+0-17

respectively.

The

improvement

achieved

by combining

theresultsof Tests

C,

Eand

F(iii)

was

marginal

intermsof

discriminating

against

semen

samples

of lownon-return_rate,as

might

be

expected

fromthe

high

correlation betweenTests C and

E,

andnofurther

advantage

was

gained

by

thecombination ofmorethan threetests.

Table 2. The_{percentage of variance}accountedforbythe best threetests,alone and incombinations,for bovinesemen

Test

C E F(iii) E +F(iii) C +F(iii) E +F(iii)+C

Percentageofnon-returnvariance 48-3 43-8 22-3 57-4 56-5 60-5 accounted forbytest

95%Confidencelimits ofpredictednon- 15 16 19 14 14 13

returnvalueofa_singlesample

Theresultsof this

study

showed that

although

thetestswerecorrelated withnon-returnrate

they

wereoflimited valueas a meansof

predicting

thatrate.For thebest combinationsoftwoteststhe

following equations

havebeen

computed

fromwhicha_{non-return rate may}be estimated:

non-return rate=-0-42

{E}

-1-32

{F(iii)}

+78-2 or 0-44

{C}

-1-06

{F(iii)}

+40-9

Thus,

ifafter

testing

a semen

sample

we

predict

anon-return rateof 57-5

%

(the

meanvalueofthese

data),

weestimate

that,

with

probability

0-95,

theactualnon-return ratewillfallbetween 71 -5

%

and 43-5

%

(the

95

%

confidenceintervalofthe

predicted

non-returnvalue ofa

single sample).

The value

of these

regression equations

in

predicting

non-return rateis

clearly

limited because the confidence intervalsare_verywide in relationtothe overall_rangeof the data

(150%

to

77-2%).

Tests Jand

K,

theassayofGOT released into the seminal

plasma

by damaged

spermatozoa,were

carriedouton53 of thesemencollections andthesedatawere

analysed separately.

Thenon-return ratecorrelatedwellwith theamountofGOT in thesemenat

ejaculation

(r

=

—0-443)

butafterthe

semen wasfrozen

(Test

K)

therewas nocorrelation. Thenon-returnratewas

positively

correlated with

the heatof_sperm metabolism measuredon 114of thesemen

samples by calorimetry,

Test L

(/

=

+0-548).

The results of the ultrastructural studies on six ofthe semen

samples

are shown in Table 3.

Cooling

the dilutedsemento5°C causedanincrease in the

proportion

ofcells withswollenacrosomes

andafter

freezing

therewasevidence ofmoreextensive ultrastructural

damage

tothe spermhead.

Thespermatozoaindicated incolumns1 and 2 of Table3were

structurally

intact and contained all the

organdíes

required

for_{spermtransport}andfertilization.Asameansof

assessing fertility,

therefore,

thesumof thesetwocolumns showed that the

samples

from Bulls 3 and 6werenotas

good

asthe

other

samples.

Thenon-returnvalues obtained forthese

samples

confirmed this

prediction.

Discussion

Oneofthe aimsofthis

study

wastodetermineifbullsemenof

satisfactory quality

intermsofnon¬

return ratewas

being

discarded

by

routine

laboratory

tests.The results have shownthat thiswasthe caseand

provide

information

by

which limits of the

laboratory

tests_maybe

adjusted

toavoid dis¬

of

Table 3. The_{percentage distribution of}9 morphologicalclassesof spermatozoa, distinguished accordingtothe

presence(+)orabsence(—)of ultrastructuralfeatures of theplasmamembraneandacrosomeinfresh,cooledand frozensemenfrom 6 bulls

Class 5 Plasma membrane Intact Brokenorlost Acrosome Swollen Vesiculated Vacuolated

Loss ofoutermembrane Lossofcontents Freshsemen Bulli 2 3 4 5 6 Semen cooledto5°C Bulli 2 3 4 5 6 Frozensemen Bull 1 2 3 4 5 6 112-day non-return% 60-6 63-3 58-5 63-6 63-4 15-0 + + + + + + + -+ + + 96 70 67 79 86 18 23 21 6 26 7 2 6 9 15 20 0 0 24 3 0 0 12 62 63 30 40 74 34 31 19 15 14 30 0 0 4 11 3 0 2 + 1 13 9 4 4 0 + + 3 7 6 7 11 12 6 12 4 + + 17 12 11 9 6 6 + + + + -+ 2 3 10 9 3 50 6 3 30 17 11 43 25 40 43 46 25 88

what isan

unacceptable

non-returnrate;and

(b)

whetheritaimstodiscard aslittleas

possible

of

semenof

high

genetic

meritorto achievea

compromise

between

attaining

the

highest

non-return

rateand

discarding

a

larger

numberofsemen

samples.

Theresults of

Exp.

1onfreshandfrozensemen

might

havebeen

expected

to

yield

results similar

tothoseof the

previous study by Bishop

etal.

(1954).

However,the smallnumber ofsemen

samples

usedand thenarrowdistributionof thenon-returndata

provided

results oflittle value. For

example,

the

motility

meter

(Test D)

wasthe besttestforsemenused after

freezing

buttheworstforsemenused

fresh,

even

though

thetestwascarriedoutonrawundilutedsemen beforeitwas divided intotwo

batchesforthe

preparation

of fresh and frozeninseminates.

Furthermore,

positive

rather than_nega¬ tivecorrelationswereobtainedforthe

%

ofdead_spermatozoaandthenon-returnrateobtained

by

theuseof either freshorfrozensemen.Theseresults ledtonoconfidencein the

laboratory

testsand

indicated thatsemenshouldnotbe discardedonthis basis.

Nevertheless,

the

samples

usedwerefrom asmall number of bulls selected forA.I.from which_poorsemen

production

wouldnotbe

anticipated.

The

question

wasthen

posed

that

if, by

some

change

intheanimal's

health,

verypoorsemen wereob¬

tained fromabullatanA.I. Centrewouldthe

laboratory

testsreveal its

inadequacy?

Thereforea

larger

numberof

bulls,

someof whichwereknowntohavealownon-returnrate,wereselected for

Exp.

2. Theresults showed thatinmostcases_{very poor}

quality

semenwhich gaveanon-return rateof

Linford

This

study

shows

that,

atthis

time,

thebest evaluation of frozensemen_maybeachieved

by

mea¬

suring

the

motility

ofthe

sample

before

processing

andthe

%

of deadand

morphologically

abnormal

cells.

Bishop

etal.

(1954)

cametothesameconclusion forevaluationoffreshsemen.

By

thecombina¬ tion ofthree suchtests, 60

%

of thenon-returnvariancewasaccountedfor. Thiswasnot

high

anda new sementestwithabetter

regression

withnon-returnrateis

evidently

required. However,

thetrue

contribution of thesemen and thevariation in thenon-return rate is

unknown;

60%

may be the

maximum

proportion

attributable

solely

tothe maleandtherest maybe duetovariations in the

female. Until this information is availabletheassessmentofnewmethodsofsemenevaluationwill be

difficult.

Non-returnvalueswerecorrelated with allmeasuresof

motility.

Nevertheless,

itmustbestressed that the

subjective motility

measurementswerecarriedout

by

trained

personnel

with

experience

in

viewing

semenundera

microscope.

Ifsuch

personnel

werenotavailable itwouldbeadvisabletouse

the

motility

meter

although

this instrumentcannotbeused for dilutedsemen.Forexport,or

import,

for which

only

frozen

samples

canbe

used,

_sperm

motility

would havetobe assessed

subjectively

(Test G)

and these resultsshowthat

only

those

samples

inwhich

motility

after

thawing

is lessthan

20%

contributetoareductionin the averagenon-return rate.A

satisfactory

alternativeto

subjective

motility

assessment after

freezing

would be

by

calorimetry (Test L)

but the instrument

required

is

expensive.

Many

of the

motility

testsanddeadorabnormal celltestswere

inter-correlated,

i.e.adead

spermatozoon or onewith certain tailabnormalitieswasalsoanimmotile_{spermatozoon.} The_pre¬ senceofthe

cytoplasmic

droplet

onthe bullspermatozoonisanindication of

immaturity

of thecell

(Hancock,

1959)

andhasnocausal

relationship

withdeathor

immotility.

This

explains

why

the

%

of

spermatozoawithanterior

droplets (Test

F(iii))

was sovaluableincombinationwith othertestsbe¬

causeitcontributed additional information aboutthesemen.In thesearch fornewtests,

therefore,

emphasis

should belaid on

testing

otheraspectsof the_{spermatozoon,} suchas acrosome

integrity

(Saacke,

1972;Foulkes&

Watson,

1975),

ratherthanon

measuring

with_{greateraccuracy}characteris¬

tics

already

wellmonitored.

Although

thecorrelationsbetween thenon-return rate at 112

days

and thetestsusedherewere

relatively

high,

the

regressions

werenot

good,

showing

thatthetestswereof littlevalueas ameansof

predicting

non-return value. As in the

study

of Van

Duijn (1971),

formulae_may be

computed by

which estimatesof thenon-returnvaluesfromanumber ofsementests_maybe determined.

However,

the95

%

confidence limitsofourestimatesincluded almost thewholerangeof values obtained.What

justification

isthere thenfor either

rejecting

asemen

sample

or

rejecting

abullfor A.I.onthebasis

of thesetests?

Theresults of

Exp.

2areof valuetoillustrate thelimitforeachtestbelow whichsemenshould be

discardedto

improve

overall A.I. results.For

example,

lines_maybe drawnon

Text-fig.

3to_represent

acceptable

limits ofnon-returnvaluesand

%

motility

in saline

(Test

C). Thus,

if allsemen

samples

hav¬

ing

a

motility

of<20

%

werediscarded andanon-return rateof50

%

were

acceptable,

_any

samples

in

the hatchedareabounded

by

lines andy

(Text-fig. 3)

would be discardedeven

though

thenon¬

return ratewould havebeen

acceptable.

Thisamounts toone

sample

outsidetheconfidence interval

that would have been wasted

unnecessarily. By changing

the limits to

60%

non-returnand

40%

motility

(lines

x2andy2,

Text-fig. 3),

we can seethatthenumber of

good samples

wasted has increased

tofour.However itis

generally

more

important

from thefarmer's

point

ofviewto_preventtheuseof

inferiorsemenandso_acceptancelimits could bedefined.In this

experiment

alower limit of

40%

wouldexclude allsemenwithanon-return ratelessthan 50

%

andyetwouldwasteless than 10

%

of

good samples.

That

statistically highly significant

differenceswererevealed between bulls fornon-return

value,

%

motility

in

saline,

%

deadspermatozoa,

%

abnormal_spermatozoa,

%

_spermatozoawith anterior

droplets

and

% motility

after

freezing

raises doubtsastothe

validity

of

applying

onestandardtoall

bulls.Itwouldseemthat themost

satisfactory

method of

testing

abull'ssemen

quality

forA.I.would

betoconstructa

regression

lineforthat bull

by using

anumberof hissemen

samples

as

long

as

they

satisfied theminimum limit of

laboratory

tests. This

regression

may then be used tomonitor that

bull's

performance

during

his stayat theA.I.centre.Differences of statistical

significance

between

breeds,

particularly

between Friesians and

Herefords,

weredemonstrated for

motility

measurements

of

and forspermhead abnormalities. Sincenobreed differenceswere_apparentinnon-return ratethis suggeststhatdifferent standards_mayhavetobe

applied

todifferent breeds.

Theultrastructural studieswerecarriedouton

only

afew

samples.

The valueofelectronmicro¬

scopyas amethod ofsemenevaluation is limited

by

the expense and the

time-consuming procedures

involved.

However,

recent work has shown that transmission electron

microscopy (Morstin

&

Courot, 1974)

and

scanning

electron

microscopy (Lacy, Pettitt,

Pettitt &

Martin, 1974)

of_spermato¬

zoa_maybe usedtodetect

sub-fertility

in the bulland

infertility

inman.

We

acknowledge

theassistance of thestaffofthe

Reading

Cattle

Breeding

Centre andofMrA.J.

Cliffe,

andMrS. V. Morant of the National Institutefor Researchin

Dairying.

References Bishop, M.W.H., Campbell, R.C., Hancock, J.L. &

Walton, A. (1954) Semen characteristicsand fer¬

tilityin the bull. /.agrie. Sci.,Camb.44,227-248.

Foulkes, J.A. &Watson, P.A. (1975) Hyaluronidase

activityinseminal plasmaas amethod ofassessing

bull_spermintegrity./.Reprod.Fert.43,349-353.

Glover,F.A.(1968)Physicalmethod ofmeasuringthe

motility of bull spermatozoa. Nature, Lond. 219

1263-1264.

Hancock,J.L.₍₁₉₅₆₎The_morphologyof boarsperma¬ tozoa.JlR.microsc. Soc.76,84-97.

Hancock,J.L.(1959) Morphologicalcharacteristicsof spermatozoaandfertility.Int.J.Fert.4,347-359.

Jones, R.C. & Martin, I.C.A. (1973) The effects of

dilution,eggyolk andcoolingto 5°Con the

ultra-structure ofram _spermatozoa./. Reprod. Fert. 35,

311-320.

Lacy, D., Pettitt, A.J., Pettitt,J.M. &Martin,B.S.

(1974)Applicationofscanningelectronmicroscopy

tosemenanalysisof thesub-fertilemanutilisingdata obtainedbytransmission electronmicroscopyas an

aidto_{interpretation.Micron5,} 135-173.

Morstin, J. & Courot, M. (1974) Ultrastructure des

spermatozoïdesdetaureauxde différente fécondance

morphologie ultrastructurale, glycoprotéines

acro-somiqucs et _{membranaires, charges} négatives de surface. Annls Biol. anim.Biochim.Biophys.14, 581-590.

Saacke,R.G.(1972)Semenqualitytestsandtheirrela¬

tionship to fertility. Proc. 4th Tech. Confi A.I. &

Reprod.,N.A.A.B., Chicagopp.22-27.

Stewart, D.L., O'Hagan, C. &Glover, F.A.(1972)

Theprediction of thefertility of bull semen from

laboratorytests. Proc.7th Int.Congr.Anim.Reprod.

&A.I.,Munich_2, 1280-1283.

Van _{Duijn, C,} Jr ₍₁₉₇₁₎ Beziehungen zwischen den

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