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AN INV EST IGATION INTO THE MECHAN ISMS INVOLVED IN THE DEPRES S ION OF

OVULATION RATES IN EWES GRAZING OESTROGENIC LUCERNE

A thes i s p r e s en t ed in pa rt ial ful f il ment of the requ iremen t s for

the degree o f Ma s t er of Agr icul ture Sc ienc e

in An ima l S c i ence at

Ma s sey Un iver s i ty

BERNARD JOSEPH McLEOD

1 9 7 8

Ot-\�1-0R

A B S T R A C T

The r eproduc t ive per f o rmance o f 123 Romney and Border Leic e s t er

x Romney ewe s wa s compar ed a f t e r they gra zed o e s t rogenic l uc erne or

non -oestrogen ic ryegra s s / c lover pa s t ur e .

i i

T o synchron ize oestrus pr ior to t h e exper imen tal t r ea tmen t ,

progestagen-impr egna t ed in t ravag inal sponges wer e inser t ed in a l l ewes

for 14 days . In Exper imen t I , wh ich invo lved 42 Romn ey and Border

Leic ester x Romney ewes , the an imal s gra z ed trea tmen t pa stures for one

comp l e t e oes trous cyc l e . B lood samples wer e taken a t interva l s

thro ughout t he cyc l e and l u t ein i z ing ho rmone concen t ra t ions wer e

d e t e rmined . Ovula t ion r a t e s and the numbers o f fol l ic l es presen t on

the surface of the ovaries wer e r ec orded a t laparotomy , three days po s t -

oes trus .

In Exper iment I I , which invo lved 81 Romney ewes , oes trogen ic lucerne

or non-oes trogenic ryegra s s / c lover pa s tu r e wa s grazed for a comp l e t e

o e s trous c yc l e , or t ea tmen t s wer e int erchanged i n mid-cyc l e . Al l ewes

wer e slaugh tered three days po s t -oes trus and their r eproduc t ive trac t s wer e

r ecovered and individual ly id en t i f i ed . Ovula tion r a t e s and the

numbers o f fo l l ic les on t he sur face of t he ovar i e s were r ecorded .

Af t er sec t ioning the ovar ies , a l l fo l l icles of a d iame ter gr eater t han

2.0mm wer e recorded . Sec t ions o f t i ssue from t he vag ina , c ervix , u t erus

and fa llopian tubes of each ewe were moun t ed , sta ined wi th ha ema toxyl in

and eosin , and the height of their ep i thel ial cel l s mea sur ed .

The inge s t ion o f oes trogenic lucerne for one comp l e t e oes trous

c yc l e depr e s s ed ovula t ion r a t e s by 29 per c en t (0.67 ovula t ions per ewe)

in Exper imen t I and by 22 percent (0.40 ovulat ion s per ewe) in Exper iment

II. The inge s t ion o f o es trog enic luc erne for pa r t o f the o e s t ro u s c yc l e ,

depr essed ovulat ion r a t e s only i f i t wa s consumed for the l a t t er

hal f o f t h e cyc l e .

i i i

O e s t ro genic luc erne d id no t sign i f ican tly in f luence t h e s ec r e t ion

of l u t e in i z ing hormone over the pre-ovula tory per iod , or a t any o ther

s tage of t h e oestrous cyc l e .

The r e wer e no s ignificant d i f f erenc es in t he to tal numb ers o f

ovar ian f o l l icles presen t , i n the numbers o f fol l ic l es o n the sur fac e

o f the ova r ie s , o r in the numbers o f large f o l l ic l e s ( wi t h a d iame ter

o f gr ea t er than 3 .5mm) p r e s en t , b e tween the trea tmen t group s .

The inges t ion of o e s t ro g enic lucerne did no t inc r ea s e sign i f ican tly ,

the h eight o f epi thel ia l c el l s in gen i ta l trac t t issue , when compared

with tha t in ewes graz ing non -oest rogenic ryegra s s / c l ov er pa s ture .

S imilar l y , there was no sign i f icant d i f f erence in u terine we ight s .

Th is i s in contrast to pub l i shed da ta where luc erne i s f ed to speyed

ewe s .

iv

A C K N 0 W L E D G E M E N T S

The author is spe c ia l ly indeb ted to his supervisor

Dr . M . F. McDona l d for his invaluab l e gui dance and a s s i s tance in

expe r ime n t a l work and adv ice dur ing the preparat ion o f this manu s c r ip t .

Special thanks are due t o D r . K . R. Lapwood for assis t ance with

radio immunoassays and for his he l p fu l d i s cuss ions and adv ice ; t o

Mr . M . J . Bir t les for a s s i s t ance with histo logica l preparat ions ; and

to Dr . R . W . Ke l ly , Inve rmay Agricu l t ural Research Centre , for the

coume s tan assays . Special apprec iat ion for t e chnical assis tance is

due to Mr. C . Mu i r , Mr . M. Wyc h e r l ey and Mr . B . Tha t cher and t o a l l

those who assisted with the co l l e c t ion o f b lood samp l e s , especi a l l y

my f e l l ow s t udents o f reprod u c t ive physio logy , Ma rk Fishe r , S t ua r t

Mc C u t cheon , Bi l l McMi l lan and Al a s t a i r Gibson .

Acknowl edgement i s mad e t o Mr . P.H. Whi t ehead and farm s t a f f

and to Miss J . McKegg for the typ ing o f t h i s manus crip t .

Financial assistance f rom the D . J . McGowan Tru s t and the

J . Hur ley Trus t , is gra t e f u l ly acknowledged .

Final ly , special t hanks a r e due t o my fami ly for s upport and

encouragement throughout this s t udy .

v.

P R E F A C E

This inv e s t iga t ion wa s c onduc t ed a t the Fer t il i ty Cen tr e , par t

o f the Depa r tmen t of S heep Hu sband ry , Ma s s ey Univer s i t y . The

experimen tal work wa s c a r r i ed out in April and May of 1 9 77 and represen t s

or ig ina l r es earch b y t h e au thor under the supervis ion o f Dr . M . F .

McDona ld , Reader , Sheep Husbandry Depa r tmen t , Ma s s ey Univer s i ty .

T A B L E 0 F C 0 N T E N T S

ABSTRACT • . . .

ACKNOWLEDGEMENTS

PREFACE

LIST OF TABLES

LIST OF FIGURES

INTRODUCTION . .

CHAPTER I

REVIEW OF LITERATURE .

(A) PHYTO-OES TROGENS

( i ) Phyto-oestrogen s in Pas ture Plants

( i i ) Fa c tors Inf luenc ing Phy to-oest rogen Levels

(iii) The Me tabo l ism o f Phy to-o e s t rogens in Sheep

( iv ) Phyto-oes trogens and Repro duc t ive Fa i lure in Sheep . . . . . . . . . . . . . . .

( B ) THE MECHANI SM O F ACTION OF OES TROGENS

( C ) EXOGENOUS OESTROGENS AND REPRODUCTION IN THE EWE

( i ) The Oes t rous Cyc l e

( i i ) Gonado trophin S ec r e t ion

( ii i ) The Reproduc t ive Trac t

( iv) Oes t rogen Recep t o r s

( D ) FOLLICLE MATURATION I N THE EWE

( i ) Pa t terns o f Fo l l ic l e G rowth and Atresia

( i i ) Hormonal Con trol o f Fo l l i c l e Ma tura t ion

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V

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xi

xii

1

1

1

4

6

8

12

13

13

1 5

1 8

19

2 0

20

21

( E )

( F )

OVULATI ON I N THE EWE . .

( i )

( i i )

( i i i )

Fac to r s Influenc ing Ovulat ion Ra tes

P o s tulated Mechanisms of Ovula t ion

Blockade of Ovulation

THE PURPOSE AND SCOPE OF THE INVESTIGATION

CHAPTER I I

MATERIALS AND METHODS .

(A) EXPERIMENTAL PLAN

( B )

( C )

( D )

( E )

( F )

( G )

( H )

( I )

LUCERNE AND CONTROL PASTURES

AN IMALS

OESTRUS SYNCHRONI ZATI ON

HORMONE ANALY S ES .

CERVICAL MUCUS ANALY S I S

UTERINE AND OVARIAN WEIGHTS

FOLLI CLE POPULATI ONS .

REPRODUCTIVE TRACT HISTOLOGY

( J ) ANALY S I S OF DATA

RESULTS . . . . . . .

CHAPTER Ill

(A) LUCERNE OESTROGENIC POTENCY

( i ) Luc erne Coume s tan Con t ent

( i i ) Daily Coumes tan Intake

( B ) EWE LIVEWEIGHTS AND OESTRUS

( i ) Ewe Livewe igh t s . . . .

( i i ) Oes trus Synchroni z a t ion

( i i i ) Oe s t rus-Ovulat ion Interval

Page

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25

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2 8

29

30

3 0

3 1

3 1

3 2

3 2

33

34

3 4

3 4

3 4

36

36

36

36

37

37

39

39

(C) LUTEINIZ ING HORMONE S ECRETION

( i ) Dur ing Oes t rous Cyc le

( i i) Pre-Ovul atory LH Peak

(D) OVULAT ION RATES

( i ) Experiment I

( i i ) Experimen t I I

( i i i ) Corpora Lu tea Diame t e r

(E) FOLLICLE POPULATIONS

( i ) Surface Fol l i c l e s

( i i ) To tal Fo llicle Popula t ions

(F) OVARIAN AND REPRODUCTIVE TRACT RESPONSES

( i ) Ovar ian and Ut erine Weigh t s

( i i ) Tra c t His tology

(G) SUMMARY . . . . . .

DI SCUSS ION

(A) OES TROGENIC LUCERNE

( B ) OES TRUS PHENOMENA .

CHAPTER IV

( i ) Synchron iza t ion of Oes trus

( ii ) Oes trus To Ovula t ion Int erval

( C ) LUTEINIZING HORMONE S ECRETION

( D ) DEPRESS ION O F OVULATION RATES

(E) FOLL ICLE POPULATIONS

(F) P ROGESTERONE S ECRETION

( G ) COUMESTANS AND THE REPRODUCTIVE TRACT

( H ) CONCLUS IONS

BIBLIOGRAPHY . .

Page

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5 5

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56

5 7

5 9

6 1

6 3

64

6 5

7 1

Tabl e 1 - 1

Tabl e 1-2

Tab l e 3- 1

Table 3-2

Table 3-3

Table 3-4

Table 3-5

Table 3-6

Table 3-7

Table 3-8

Tabl e 3-9

Tabl e 3- 1 0

Tabl e 3- 1 1

L I S T 0 F T A B L E S

The relat ive po t enc ies o f phyto-oes t rogens

admin i s tered orally or intramuscularl y .

Oestrogenic luc erne and repro duc t ive

per formanc e

Lucerne coume s tan conten t

The daily coume s t an intake (mg/day) o f ewes

inges t ing 1 kg ( DM) of lucerne .

Mean ewe l iveweights (kg) . .

Mean plasma LH concentrations (ng/ml ) during

the oes trous cyc l e , in ewes graz ing pas ture or

Page

3

1 1

3 6

3 7

3 8

oe s t rogenic luc e rne . 4 1

P re-ovulatory LH concentrations (ng/ml ) in ewes

graz ing pas ture or o e s t ro genic luc erne . 42

Mean ovula t ion ra t e s for ewe s grazing pas ture

o r oes trogenic luc erne - Experiment I . . 43

Mean ovulation ra t e s for ewe s grazing pas ture

o r oes t ro genic lucern e - Experimen t II

Compar i sons o f the d i f f er enc e s in ovulation rates

be tween t r ea tmen t group s - Experimen t II

Mean corpo ra lutea d iame ter (mm) three days

po s t-oe s trus

Surface f o l l i c l e s o f greater than 3 . 5mm d iamet e r,

corpora lutea and follicles p e r ovula t ion for

ewes grazing pas ture o r o e s t ro genic lucern e -

44

44

4 5

Exp erimen t I . . . . . . . . . . . . . . .. 46

Sur face f o l l i c l e s of grea t er t han 2 . 0mm

d iamet er , corpora lutea and fol licl e s p e r

ovula t ion f o r ewes graz ing pas ture o r

o e s t rogenic lucerne - Experiment I • • • • • • 4 7

Tabl e 3- 12

Tab l e 3- 1 3

Tab l e 3-14

Tab l e 3- 15

Tabl e 3- 1 6

Tabl e 3- 1 7

Page

Ovar ian fol l ic l e populat ions of ewes graz ing

pasture or oestrogenic l ucerne - Experimen t I I 4 9

To tal number o f f o l l i c l e s o f greater t han

3 . 5mm diameter , corpo ra l u t ea and f o l l i c l e s

p e r ovula t ion - Exper imen t I I

To tal numb er o f f o l l i c l e s o f grea ter than

2.0mm diameter , corpo ra l u t ea and f o l l i c l e s

p e r ovulat ion - Experiment I I

Fo l l i c l e s grea t er than 2.0mm d iame t er , p e r

gram o f ova rian t issue

Ovarian and uterine we ight s (g) of ewes

grazing pa s ture or o e s t ro gen ic lucerne

Ep ithel ial cell he igh t (�) o f reproduc t ive

t rac t t issue f rom ewe s graz ing pa s ture or

o e s t rogenic lucerne . . . . . . . . . . .

50

5 1

48

52

52

Fig . 1 - 1

Fig . 1 - 2

Fig . 1 - 3

Fig . 2 - 1

Fi g . 2-2

Fig . 3- 1

F i g . 3-2

Fig . 3-3

Fig . 3-4

Fig . 3-5

Fig . 3-6

Fig . 3-7

L I S T 0 F F I G U R E S

Phyto -o e s t rogens . . . . . . .

The metabol ism of fo rmononet in in sheep

Po stulated mechan ism o f o e s t rogen a c t ion

wi thin tar get cel l s

Exper imen tal plan - Exper imen t I .

Exper imen tal plan - Exper imen t I I

D i s t r ibut ion o f t he onset o f oes t rus-

Exp er imen t I . . . . . . . . .

Dis t r ibut ion o f t he onset o f oestrus-

Exper imen t II . . . . . .

Onset of Oest rus-LH peak int erval

LH secre tion over the oes trous cyc l e

Pr e-ovula tory L H s ecretion

Ovulat ion rates

Po s t-ovula t ion fo l l i c l e popula tions

f refers to following page

Page

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f 51

INTRODUCTION

Luc erne (medicago sa t iva) has become incr eas ingly impor tant as a

forage crop in tho s e par t s o f New Zeal and which exper i ence dry summer

cond i t ion s . I t s abil i ty to p roduc e high yields o f fo rage , o f high

nutr i t ive value , whether grazed in situ or f ed as hay or silage , has

led to i t s popul arity . In these areas luc erne produc t ion frequently

exceeds tha t o f convent ional pa s ture s .

xii

Because i t produces forage o f high nut r i t ive value in late summer

and autumn , lucerne would appear t o be ideal for ' f l ushin g ' ewe s prior

to ma t ing . However , lucerne has b een shown to con tain oes t ro genic

compound s which impa ir the reproduc tive performance of ewes . The phyto-

oe s t ro gens , the coumes tans , and to a le sser degree the isoflavones , may

reach high concen t r a t ions in lucerne l ea f ma terial , and the coumes tans

have been shown to lower lamb ing perc en tages . This d epres s ion in lamb ing

percentage is rela t ed to a redu c t ion in ovulat ion r a t e s .

An unders tand ing o f tho se mechanisms involved in the luc erne -

induced depression o f ovulat ion rates would be o f cons iderable advantage .

I t may enab l e management pra c t i c e s to be devised , tha t would al low lucerne

to be u t il i z ed in achieving opt imum ewe l iveweights prior to tupping ,

wit hout incurr ing any lo ss in r eproduc t ive performance .

C H A P T E R I

REVIEW OF LITERATURE

(A) PHYTO-OESTROGENS

C H A P T E R I

REVI EW OF LITERATURE

The t erm ' o estrogen ' originally re ferred to c ertain s tero id

hormones secre t ed by the ovary . The dev elopmen t o f the Al l en -Do isy

l.

vaginal smear t es t in 1923 provid ed a wider definition o f o e s t ro genic i ty .

' An o e s t ro gen i s any pure chemical subs tance which , when inj ec ted into

adul t ovar iec tomized mice or ra t s , produc es co rmf ica t ion of the vag ina ,

s imilar to tha t occurring during oes trus in the normal in t a c t an imal '

(Al l en and Da i sy , 1923) .

The o ccurrenc e o f o e s t ro genic subs tances in plan t s was f i r s t

repo r t ed i n 1 9 26 ( Dorhn e t al . , 19 26; Fellner , 1926; Lo ewe , e t a l . , 19 27) ani

the f ir s t oes trogen to be isol a t ed and iden t ified in the p lan t world wa s

o e s t rone , extrac ted from the oil palm ( Butenandt and Jacob i, 1 9 3 3 ) .

Cons equen tly several compoun ds clo s ely resemb l ing the na tural s t eroid

o e s t ro gens wer e isola ted f rom a number of plan t s. An o e s triol-l ike

compo und wa s ext rac ted f rom wil low ca tkins (Skarzynski , 19 3 3), and

f rom l iquorice roo t ( Costello and Lynn , 1 9 50) . A c r eeper c l a imed by

the Chine se to possess aphro d i s iac propert ies was shown to con tain mire­

oestral ( Jones and Pope , 1961) and plan t s used for con t racep t ive

purpo ses in Ind ia and China contain ed p so ral idin ( Da t tagup ta et al . , 19 60) .

( i ) Phyto-Oest rogens in Pasture Plan t s

When B enne t t s , Underwood and Shier ( 1 94 6) demon strated a n in f er t il i ty

syndrome a f f ec t ing ewes gra z ing sub terranean clover pas ture ( Tr i f o l ium

sub t e rraneum L var . Dwalganup) , they provided s trong presump t ive eviden c e

o f t h e presence o f a n oes t ro gen . This was conf irmed by Curnow e t a l . ,

( 19 4 8) who showed the o e s t ro genic a c t ivity o f e ther extrac t s o f

sub t erranean clover ; and suppo r t ed ind irec t ly b y Eas t e t a l . , ( 1949)

with the d emons t ra t ion o f a p ro te c t ive a c t ion o f androg en inj ec t ions t o

2 .

sheep grazing clover pastures . Oestrogenic activity was also attributed

to red clover ( Pope, 1954), lucerne ( Coop and Clark, 19 60) and red clover

and lucerne hay ( Cheng et al . , 1953).

In 19 51 Bradbury and White succeeded in isolating two isoflavones

from subterranean clover extract; 7-hydroxy-4 '-methoxy isoflavone

( formononetin) and 5:7: 4'-trihydroxy isoflavone ( genistein). Genistein

was also isolated from red clover ( Pope and Wright, 1 954; Curnow and

Rossiter, 1955; Wong, 19 62), and from white clover and lucerne

( Guggolz et al . , 19 61). Subsequently the isoflavones 7 : 4 '-dihydroxy

isoflavone ( diadzein), 5 : 7-dihydroxy-4'-methoxy isoflavone ( biochanin A),

and 5 : 7 : 3'-trihydroxy-4'-methoxy isoflavone were isolated from both

subterranean and red clovers (Livingstone et al . , 19 6 1). These isoflavones

were all shown to be oestrogenic ( Bradbury and White, 1954; Moule et al . ,

19 6 3 ), and their synthetic analogues were shown to be of equal oestrogenic

potency ( Cheng et al., 1 9 54 ) .

Bickoff et al . , ( 1 957; 1 958) isolated a crystalline substance of

oestrogenic nature from ladino clover, strawberry clover and alfalfa

( lucerne). This compound w�s not an isoflavone but a benzofurocoumarin

derivative - a class later named 'coumestans' . Bickoff proposed the

name 'coumestrol' for this oestrogenic compound which proved to be 30

times more potent than genistein ( Bickoff et al., 1958) . Coumestrol

and the non-oestrogenic 4'-methoxycoumestrol, have since been detected

in red clover, subterranean clover and burr clover ( Lyman et al. , 1959).

In addition, six other coumestans have been isolated from lucerne

( Bickoff et al . , 19 64) and from ladino clover ( Bickoff et al . , 19 6 5) .

The coumestans were later sho\vn to be biogenetically related to the

isoflavones ( Grisebach and Barz, 19 6 3 ) .

Until recently the most commonly used method o f measuring the

oestrogenic potency of a phyto-oestrogen has been the mouse uterine

3 .

response. However difficulties have often arisen in attempting to

correlate results obtained with mice and other laboratory animals, to

the results obtained under field conditions with farm animals (Morley

et al . , 1 9 6 8) . Bickoff and eo-workers ( 19 6 2 ) compared the oestrogenic

potencies of orally fed phyto-oestrogens and steroid oestrogens, with

injected admin::istration in mice. Braden et al . , (19 6 7 ) made similar

comparisons using ovariectomized ewes . The comparative potencies are

shown in Table 1- 1 .

TABLE 1-1: THE RELATIVE POTENCIES OF PHYTO-OESTROGENS ADMINISTERED

ORALLY OR INTRAMUSCULARLY

OESTROGEN MOUSE EWE

ORAL I/M ORAL

Diethylstilboestrol 1 00, 000 100, 000 1 00, 000

Oestrone 6, 900 6, 900 6,900

Coumestrol 3 5 3 5 9

Genistein 1 . 5 1 . 5 0 . 0 7

Diadzein 0.4 0 . 4 0 . 02

Biochanin A 1 . 0 1 . 0 0 . 05

Formononetin negligible negligible 0 . 06

from Bickoff et al., (1 9 6 2), Braden, et al . , (1 9 6 7) and

Wong and Flux, ( 1 9 6 2 )

I/M

1 00, 000

6 , 900

35

1 . 5

0 . 4

1 . 0

negligible

Despite the relatively low potencies of the phyto-oestrogens, they

are present in certain forages in sufficient quantities to influence

animal physiology . The structures of the phyto-oestrogens are shown

in FIG 1 - 1.

I· I

F ig. 1·1 PHYTO- OESTROGE NS

HO

HO

HO

COU M ESTANS

0 ISOF LAVON ES

OESTRADIOL -17/3

OH

Cournestrol : R = H 4'-Methoxy coumestrol: R:CH

3

OR 2

Genistein; R1=0H R2=H Biochanin A: RfOH R2:CH3 Diadzein : R1 = H R2=H Formononef1n : R1 = H �=CH3

4 .

(ii) Factors Influencing Phyto-oestrogen Levels

Investigations have shown that there is considerable variation in

the oestrogenic potency of pasture plants and that this is influenced by

both environmental and genetic factors .

GENETIC : Bradbury and White (19 54) tabulated over 50 different

species of plants reported as oestrogenic . There is variation between

species in the ratio of coumestans to isoflavones . The coumestans are

the dominant source of oestrogenicity in lucerne (Bickoff et al . , 19 6 2 ),

ladino clover (Guggolz et al . , 19 61), the annual medics (Bennett et al . ,

196 7b; Francis and Millington, 19 65a; Millington et al . , 19 64a) and

white clover (Wong et al . , 19 71) . The isoflavones account for most of

the oestrogenicity of subterranean clover (Millington et al . , 19 64b) and

red clover (Wong, 196 2 ) .

Wide variations in oestrogen levels are also evident between

varieties and between lines within a species; in lucerne (Francis and

Millington, 19 6 Sa; Stob et al., 19 5 7 ; Stuthman et al. , 19 6 7 ),

subterranean clover (Francis and Millington, 19 64; Davies and Dudzinski,

19 6 5 ) and red clover (Francis et al. , 19 6 7 ) . Francis and Millington

19 6 Sb) treated subterranean clover seed with a mutagenic agent and

produced a number of mutant plants of very low oestrogenicity .

ENVIRONMENTAL: Nutrient deficiencies influence the oestrogen

content of pasture plants . Oestrogenic activity has been shown to

increase significantly when phosphate deficiencies occur in subterranean

clover (Alexander and Rossiter, 19 52) and in red clover (Rossiter and Beck,

19 6 6 b; Butler et al . , 1967) . Concentrations of formononetin were most

affected although diadzein and biochanin A levels also increased (Rossiter

and Beck, 1966b) . Severe deficiencies in sulphur (Rossiter, 1970;

Rossiter and Barrow, 1972), and in nitrogen (Rossiter, 19 6 9) can almost

double formononetin levels .

Pieterse and Andrews (1956) and Kohler and Bickoff (1961) found

that coumestrol levels increased proportionally with maturation of lucerne

5 .

plants. Francis and Millington ( 1965a) demonstrated a similar trend

in annual medics .

Oestrogenicity is reported to fall dramatically after the harvesting

and drying of subterranean clover (Francis and Millington, 1965c), and

of lucerne (Bickoff et al. , 1959 ; 19 60a; Livingstone et al., 196 1) .

Commercial dehydration has a similar effect (Francis and Millington,

19 65a). In contrast, lucerne silage is more potent than the green

lucerne from which it is produced (Pieterse and Andrews, 1956) .

There are conflicting reports on the effects of high temperatures

( Coleman et al . , 1965; Loper and Hanson 19 64; Rossiter and Beck, 196 6a),

season (McLean, 19 67; Squires, 19 66) and location (Millington et al . ,

1964a) , on oestrogen levels of pasture plants .

Bickoff et al . , (1960b) noted that a disease-i�fected sample of

white clover contained considerably more oestrogenic activity than

comparable disease-free samples. Subsequently, a strong correlation

has been reported between pathogenic attack of lucerne and of white

clover, and coumestrol levels (Bennett et al., 19 6 7 a; Wong and Latch,

197 1). Two leaf-spotting organisms shown to be implicated are

Lepto�aerulina briosiana (Loper and Hanson, 19 64), the fungal organism

of 'Pepper spot' disease; and Pseudopeziza medicaginis ( Loper and

Hanson, 19 64), the c2usal organism of 'common leafspot' . Loper et al . ,

(19 6 7 ) found that coumestrol content was very high in the necrotic lesions

of infected leaves, and that selection of plants for disease resistance,

or the use of fungicidal agents, markedly reduced coumestrol levels .

Infestation of lucerne by aphids has also been shown to cause a build-up

of coumestans ( Hanson et al . , 1965; Loper, 1968) .

The accumulation of coumestrol following pathogenic attack may be

implicated in some non-specific disease resistance mechanism . There is

considerable data showing that phenolic compounds are formed in plants

subjected to pathogenic attack or physical damage (Farkas and Kiraly, 19 62).

The 'Phytoalexin Theory' ( Ingham, 1972) proposes that a group of phenolics ·

6 .

theBhY.to-alexins - which exhibit antifungal properties, are produced

by plant cells as a result of pathogenic attack . Coumestans appear not

to have antifungal properties, but they are structurally similar to

Fhyto-alexins. It is suggested that both these compounds arise from a

common biosynthetic pathway (Ingham, 1972) .

In summary, it can be stated that although several environmental

factors may contribute to wide variation in the oestrogenicity of

pasture plants, the presence of pathogenic disease is the single most

influencing factor .

(iii) The Metabolism of Phyto-oestrogens in Sheep .

When considering the biological effects of phyto-oestrogens, of

particular importance are the metabolic changes that they undergo within

the animal .

The presence of a microsomal enzyme system in a number of animal

species, including sheep, which catalyses the demethylation of iso-

flavones has been reported (Nilsson, 196 3 ) . It was demonstrated that the

major reaction occurring to biochanin A and formononetin incubated in

sheep rumen liquor, is demethylation to genistein and diadzein

respectively (Nilsson, 19 61) . There was n o evidence to suggest more

extensive metabolism in vivo than was demonstrated in vitro (Batterham

et al . , (1965) . Subsequent investigations using radioactive isotopes

14 ( C) confirmed that the products of metabolism of formononetin were

diadzein and 7: 4'-dihydroxy isoflavone (equol) (Nilsson,et al . , 19 6 7;

Batterham �. , 19 71) . About 70 percent of the formononetin ingested

by sheep is converted to urinary equol, together with a small amount of

diadzein, desmethylangolensin (Batterham et al . , 1971), 0-methyl equal

and angolensim (Cox and Braden, 1974). The pathways of formononetin

metabolism in sheep are shown in Fig. 1-2.

Formononetin is of negligible oestrogenic potency if administered

to sheep via intramuscular or intravenous pathways, whereas a much greater

oestrogenic activity is apparent if administered intra-rumina�ly

/. I

Flg. 1·2 THE METABOLISM OF FORMONONETIN IN SHEEP

/

CH HO OH 3

Angolensin

CH HO OH 3

HO

Desmethyl Angolensin

HO

0 Formononetin

0 Diad zein

Equal

OH

OH

--•major pathway

c::===:> minor(or possible) pathway

7.

Braden et al., 1 9 67). In contrast, equol proves to be oestrogenically

active when administered intramuscularly (Shutt and Braden, 1 968).

Furthermore high levels of equol are excreted in the urine of sheep

grazing oestrogenic clovers (Shutt et al., 1 9 6 7; 1 9 70). Equol is the

predominant phyto-oestrogen 1n digesta and excretia of sheep grazing

clovers with high formononetin content (Shutt et al., 1 9 70). Formononetin

consequently is now considered to be the most important isoflavone 1n

terms of oestrogenic activity, although it has little oestrogenic

activity itself. Differences in oestrogenicity between strains of

subterranean clover have been related to their formononetin content

(Lindsay and Francis, 1 9 6 8; Davies et al., 1 9 70). 0-methyl equol and

desmethyl angolensin are somewhat less oestrogenic than equol (Micheli

et al., 1 9 6 2 ).

Little is known of the metabolism of coumestans in sheep. The

demethylation of 4'-methoxy coumestrol to coumestrol has been demonstrated

� � (Shutt et al., 1 9 6 9 ) and in vitro ( Adler and Weitzkin-Neiman,

1 9 70).

Braden et al., (1 967) were unable to find any obvious metabolic

products in the urine or plasma of sheep given coumestrol intra-ruminally.

Cayen and Common (1 9 6 5), feeding tritiated coumestrol to chickens, were

unable to detect any radioactive ur1nary equol, suggesting different metabolic

pathways for the coumestans than for the isoflavones.

Less than 1 percent of the daily intake of phyto-oestrogens are

excreted as such in the faeces or urine ( Shutt et al., 1 9 70). The phyto-

oestrogens and their metabolites are absorbed, largely from the rumen,

and circulate in the blood. Plasma of sheep grazing oestrogenic clover

contains water-soluble conjugates of diadzein, genistein and biochanin

A - mainly in the form of glucuronides ( Shutt et al., 1 9 6 7; Lindner,

1 9 6 7; Beck and Knox, 19 7 1 ).

Plasma from sheep fed synthetic coumestrol or medic hay, contains

8 .

water-soluble conjugates of coumestrol and free coumestro� but 4 -methoxy

coumestrol is not present (Shutt et al . , 19 6 9 ) .

Levels o f conjugated phyto-oestrogens, both isoflavones and

coumestans, are considerably higher than levels of free phyto-oestrogens

(Wong and Cox, 19 72). As well as conjugates of glucosiduronates,

relatively high concentrations of sulpho-conjugates have been isolated

from sheep plasma (Wong and Cox, 1971).

(iv) Phyto-oestrogens and Reproductive Failure in Sheep

(a) Isoflavones

CLOVER DISEASE - The classical infertility syndrome associated

with ewes exposed for prolonged periods to oestrogenic subterranean clover

pastures, is a permanent depression in fertility . This infertility

syndrome, which became known as 'clover disease', was first reported by

Bennetts, Underwood and Shier in 194 6 . They described dramatic decreases

in lambing percentages (by up to 90%), a high incidence of dystocia,

uterine prolapse months after parturition or even in unbred ewes, and

copious milk secretion in virgin and non-pregnant ewes (Bennetts et al. ,

194 6; Bennet, 19 6 6 ) . In wethers, enlargement of the teats and induction

of lactation is common; and in ewes, the pathological condition, cystic

glandular hyperplasia, is almost always found in the cervix and uterus,

(Bennetts et al. , 194 6 ; Barrett et al . , 1961) . 'Clover disease' has

a high degree of permanence, persisting for periods of several years, if

not for the lifetime of the ewe, after the ewes are removed from oestrogenic

pastures (Schinckel, 194 8; Underwood and Shier, 1951; Barrett, et al. , 196 5;

Turnbull et al . , 196 6) .

Subsequent investigations have indicated that the main cause of

infertility is the failure of ova to be fertilized (Turnbull et al. , 1966;

Lightfoot et al. , 19 6 7; Kaltenbach and Davies, 1970; Lightfoot et al. ,

1974 ; Wrath and Lightfoot, 1976) . The establishment of a reservoir

of spermatozoa in the cervix immediately after mating, is necessary for

9 .

normal fertility in sheep ( Mattner, 196 6), and sperm movement within

the tract of 'clover disease' effected ewes is impaired . The numbers of

sperm within the cervix (Turnbull et al., 19 6 6 ; Lightfoot et al . , 19 6 7;

Kaltenbach and Davies, 1970; Lightfoot et al . , 1974), within the

fallopian tubes (Turnbull et al . , 1966; Lightfoot et al . , 19 6 7;

Kaltenbach and Davies, 1970), and adhering to the zona pellucida of

recovered ova (Lightfoot et al . , 1974 ; Wrath and Lightfoot, 1976), are

much lower than in 'normal' animals .

The rheological properties of cervical mucus are important in the

establishment and retention of a population of sperm in the cervix

of the ewe ( Gibbons and Mattner, 196 6 ), and the visco-elasticity of

cervical mucus is decreased in ewes affected by 'clover disease' ( Smith,

1971; Adams, 1976a) . Adams (1977) showed that spinbarkheit of cervical

mucus could be related to lowered reproductive performance of ewes

exposed to oestrogenic pasture.

Evidence for a higher than normal incidence of embryonic mortality

(Turnbull et al . , 19 6 6 ; Kaltenbach and Davies, 1970), diminished ovarian

activity ( Lightfoot et al . , 1967; Hearnshaw et al. , 1972; Adams, 1973;

1976 b), anovulatory oestrus (Firth et al . , 1977) and abnormal cycle

lengths (Underwood and

equivocal .

Shier, 1951; Obst and Seamark, 1970) is

TEMPORARY INFERTILITY - When ewes grazed oestrogenic red clover over

the mating period only, a depression in the incidence of multiple births

and in conception rates was evident ( Morley et al . , 1964; Clark, 1965) .

However, fertility and fecundity were restored to normal levels within

five weeks of removal of ewes from oestrogenic pasture (Morley et al. ,

19 6 6 ) . Chang (1963) noted a similar transient loss o f fertility.

There is conflicting evidence as to whether the depression of

multiple births is due to a depression in ovulation rate. Morley et al . ,

( 1964) and Holst and Braden, ( 1972) found no evidence of depressed

ovulation rates . However, recent work has shown ovulation rates to be

depressed in ewes mated on oestrogenic subterranean clover ( Lightfoot

and Wrath, 1974; Wrath and Lightfoot, 19 76) .

( b) Coumestans

In the early 1960's Coop and Clarke first reported a depression in

the lambing percentage of ewes grazing lucerne at the time of mating.

A similar response was reported in ewes mated on white clover ( Sanger

and Bell ( 1959 ) . Coop and Clarke ( 1960) noted a dramatic decrease

in twinning rate and a small, non-significant increase in the incidence

of barrenness .

A number of subsequent trials have confirmed their f indings,

showing reductions in lambing percentages of up to 4 0 percent (Kelly

10 .

et al . , 19 76a; McCutcheon, 1976; Scales and Hoss, 1976; Scales et al . ,

1977). These trials are summarized in Table 1-2.

It was established that the decrease in lambing percentage, was due

to a decrease in the number of multiple births and not to increased

barrenness ( Scales and Moss, 1976; McCutcheon, 1976; Scales et al. ,

1977) . Furthermore, this depression in multiple birth rate was related

to a reduction in ovulation rate and not to increased embryonic mortality

( McCutcheon, 1976; Jagusch et al. , 1977; Scales et al. , 1977).

It is apparent that ewes of a higher inherent ovulation rate are

most affected by coumestans ( See Table 1-2) .

There have been conflicting reports on the ability o f the coumestans

to inhibit or delay oestrus, and to induce anovulatory oestrus or

follicular abnormalities. Kelly et al . , ( 1976a) reported a high

incidence of anovulatory oestrus and prolonged oestrus cycles, when they

administered extremely high levels ( c 1000 ppm) of coumestans . Moderately

TABLE 1-2 OESTROGENIC LUCERNE AND REPRODUCTIVE PERFORMANCE

% DECREASE % DECREASE % DECREASE % INCREASE BREED N IN IN MULTIPLE IN OVULATION IN

LAMBS BORN BIRTHS RATE BARRENNESS

Coop & Clarke (1960) Corriedales 370 6 6 - 0

Border-Corr 24 2 15 -le 1 1 * - 2

Romney 350 6 - - 2

Border-Corr 600 7 * 7 *

Corriedales 610 5 7 - 0

Scales & Moss (1976) Romneys 309 13 13 - 3

Coopworths 539 23 * 32 * - 1

McCutcheon (1976) Border-Romn 80 8 * 2 3 * 2 3 * 2 . 5

Jagusch, Smith & Kelly (1977) - 1000 18 - More 0 Single

Ovul .

Coop (1977) Border-Corr 9 00 - 20 * - 2

Scales, Moss & Kelly (1977) Romneys 309 16.4 13 . 7 - 2 . 7

Coopworths 539 37 . 8 * 32. 3 * - 1.0

Romneys 284 22. 0 21. 5 12 0 . 7

Coopworths 146 27 . 9 * 23 . 6 * 24 * 5 . 1

...... ......

* - Flocks of High Fecundity

12 .

high levels of coumestan,such as occur in field conditions (100-400ppm),

apparently do not induce such abberations (Scales et al . , 1977) .

(B) THE MECHANISM OF ACTION OF OESTROGENS

The current model for oestrogen action in target tissues indicates

that after entering a target cell, the hormone binds with high affinity

to a cytoplasmic protein - the 'oestrogen receptor' - to form a complex .

This complex undergoes rapid translocation to the nucleus, where it

probably interacts with chromatin acceptor sites (Gorski and Gannon,

1 9 76). Considerable evidence suggests that the presence of the receptor-

oestrogen complex in the nucleus is a prerequisite for oestrogen-induced

stimulation (Jenson and De Sombre, 1973 ) . The nature of the oestrogen-

induced biosynthetic events, depends not only on the quantity of receptor

complexes within the nucleus, but also on the length of time the complexes

stay within the nucleus (Anderson et al., 1 975; Lan and Katzenellenbogen,

1976). Thus a single injection of oestriol, a weak oestrogen, can

initiate only early biosynthetic events (0-6 hours) and is ineffective

in stimulating later events (20-4 8 hours) , presumably because of its high

rate of dissociation from the receptor and its rapid clearance (Anderson

et al. , 1975) . However, oestriol can be as effective as oestradiol

in stimulating later responses if high circulating levels are maintained

(Clark et al . , 1977) .

When oestriol is administered in acute doses, it acts as an oestrogen

antagonist . It appears to compete with oestradiol for receptors (Clark

et al. , 1977). Oestrone apparently competes with oestradiol also

(Wise and Payne, 1975).

A number of non-steroidal compounds act as anti-oestrogens, preventing

the full expression of oestrogenic steroids on target tissues. Anti-

oestrogens bind to oestrogen receptors (Capony and Rochefort, 1977) and

induce translocation of receptors into the nucleus in the absence of

Pl asma

oestrogen 5 •

• • molecules

Fig. 1·3. POSTULAT ED MECHANISM OF O ESTROGEN ACTION WITHIN TARGET C ELLS

Target Cel l

� receptor \.molecule

. . �� • • oestrogen

-receptor c omplex

· .chromatin nucleus

•• oestrogen molecules

N'fVV'A R.N.A .

�receptor molecule

oestrogen, while inhibiting the translocation of receptor-oestrogen

complexes ( Ruh and Ruh, 19 74). Receptor synthesis may also be

blocked ( Katzenellenbogen et al . , 19 7 7 ) .

Paradoxically, in the absence of oestrogens, anti-oestrogens can

stimulate some 'oestrogenic ' responses ( Fergusson and Katzenellenbogen,

19 7 7) . The effect of a n anti-oestrogen is not identical in all target

tissues, but differs between uterus, pituitary and hypothalamus

( Luine and McEwen, 19 7 7 ) .

The postulated mechanism of action of oestrogens on target cells is

outlined in Fig . 1- 3 .

( C) EXOGENOUS OESTROGENS AND REPRODUCTION IN THE EWE

( i) The Oestrous Cycle

13 .

Behavioural oestrus can be induced in anoestrous ewes by the

administration of exogenous oestrogens (Moore and Robinson, 19 57; Morley

et al. , 19 6 3 ; Piper and Foote, 19 6 8; Coding et al . , 19 70; Radford

et al . , 19 7 0; Scaramuzzi et al . , 19 7 1a) . The sensitivity of ewes to

exogenous oestrogen is related to plasma progesterone levels (Moore

and Robinson, 1957 ; Scaramuzzi et al . , 19 7lb), and to breeding season

( Reardon and Robinson, 1961; Fletcher and Lindsay, 19 7 1a) . The implication

of oestrogens with behavioural oestrus has been confirmed in experiments

involving the admin�tration of antisera to endogenous oestrogens .

Scaramuzzi ( 19 7 5) and Fairclough et al. , ( 19 7 6) prevented the occurrence

of behavioural oestrus by immunizing sheep against oestradiol . Cox

and Wilson ( 19 7 6) repeated this result with anti-oestradiol and anti­

oestrone sera, but found that immunization against phyto-oestrogens did

not prevent oestrus.

Scaramuzzi et al . , ( 19 7 la) and Land et al. , ( 19 7 2) found a linear

relationship between the dose of exogenous oestrogen and the duration

of the induced oestrus. Land et al . , (197 2) suggested that a breed

14 .

difference in sensitivity to exogenous oestrogens exists; with highly

fecund breeds being more sensitive in relation to this oestrus response .

Scaramuzzi et al . , ( 1972) found a refractoriness to oestrogen, with ewes

showing a decline in response to repeated daily injections of oestradiol

benzoate. Morley et al . , (19 6 3) administered stilboestrol daily to

cycling ewes prior to and during mating, and reported decreased conception

rates, and an increase in the incidence of silent heats .

Oestradiol injections given after day 8 of the oestrous cycle, result

in premature luteolysis in intact ewes (Stormshak et al. , 19 69; Hawk and

Bolt, 1970; Bolt and Hawk, 197 1), but not in hysterectomized ewes

(Stormshak et al . , 19 6 9) . This luteolytic effect of oestrogen is possibly

mediated through the stimulation of uterine prostaglandin F2a

Cox et al., ( 19 74) and Barcikowski et al . , ( 1974) reported increased

PG F2a secretion coinciding with endogenous oestrogen peaks on days 14 - 15

of the oestrous cycle . However, there was no coincidental PGF2a rise with

oestrogen peaks on days 4-5. Increases in PGF2a secretion following the

infusion of physiological doses of oestradiol into uterine plasma, have

been noted (Barcikowski et al . , 1974 ; Ford et al. , 1975).

Obst and Seamark ( 1970) reported that plasma progesterone levels in

sheep grazing oestrogenic clover, fell to 'oestrus' levels earlier than

in sheep grazing pasture (days 14- 15 compared with days 17- 18) . This

supports the findings of Ligttfoot and Wrath (1974) who reported that corpora

lutea of clover-affected ewes weighed less than those of normal ewes.

However, Hearnshaw et al . , ( 1972) and Smith (1975) found clover infertile

sheep to have normal progesterone secretion patterns .

Recently Newsome and Kitts (1977) measured receptor-bound oestrogen

levels in ewes grazing lucerne and found that receptor-bound endogenous

oestrogen levels were lower and more uniform than those of ewes grazing

pasture.

15 .

(ii) Gonadotrophin Secretion

LUTEINIZING HORMONE - A relationship between steroid oestrogens and

the pre-ovulatory LH surge was suggested as early as 19 34 , when Hohlweg

found that oestrogen injections induced ovulation in rats . This

'Hohlweg effect ' is also evident in the ewe (Hammond, 1 94 5).

Moore et al . , (19 6 9) and Cox et al. , (19 71) demonstrated that the

pre-ovulatory release of LH during oestrus in the ewe is primarily a

response to high levels of circulating oestrogens the previous day.

However, although oestradiol injections administered to cyclic ewes on

days 3 -4 of the oestrous cycle induced a pre-ovulatory type LH surge,

injections of a similar dose level on days 10-12 did not (Bolt et al . ,

19 7 1). Much higher dose levels are required at this �ime (Howland

et al . , 1 9 7 1) . These variations in response to exogenous oestrogens

would appear to be related to plasma progesterone levels, however, there

are conflicting reports on the ability of progesterone to block this

oestrogen-induced LH surge (Yuthasastrakosolet al., 19 7 4 ).

This pre-ovulatory-type LH surge can also be induced in anoestrous ewes

(Goding et al . , 19 6 9 ; Radford et al . , 19 7 0; Beck and Reeves, 19 7 3)

and much smaller dose levels of exogenous oestrogens are required

(Howland et al . , 197 1 ) .

Exogenous oestrogens exert a somewhat more complex influence on

ovariectomized ewes . Ovariectomy, and the subsequent decrease in plasma

oestrogen levels, results in an elevation of basal LH levels in anoestrous

ewes (Reeves et al . , 1 9 7 2; Diekman and Malvern, 1 9 7 3) . The administration

of exogenous oestrogen to ovariectomised ewes induces a biphasic response

in circulating LH levels - an initial depression followed by a large

elevation, similar to pre-ovulatory levels (Radford et al. , 196 9; Goding

et al . , 1 9 7 0; Scaramuzzi et al . , 1 9 7 1 b; Howland and Palmer, 1 9 7 3) .

Cen�ral nervous system blockade by sodium pentobarbitone anaesthesia,

prevents only the second phase of the LH response (Radford and Wallace,

1 9 7 4) . This indicates a non-nervous site for the inhibitory action, and a

16 .

CNS site for the stimulatory action on LH release.

Not only is there variation in the sensitivity of ewes to exogenous

oestrogen between the breeding season and the anoestrotts period, but there

is also variation in sensitivity between breeds . Finnish Landrace

ewes are less sensitive than Scottish Blackface ewes ( Land et al . , 19 76)

to the negative-feedback effects of oestrogen, and this is suggested as

a possible reason for variation in ovulation rates between

breeds.

The ingestion of phyto-oestrogens disrupts these responses to

exogenous oestrogens. Hearnshaw et al . , (1972) found that ovariectomized

ewes affected by 'clover disease' failed to produce any LH response to

oestradiol injections. Synthetic Gonadotrophin releasing hormone ( Gn-RH)

does evoke the pre-ovulatory type LH response ( Findlay et al . , 1973) .

This suggests that prolonged exposure to isoflavones impairs hypothalamic

receptors . Hearnshaw et al. , (1977) postulated that 'temporary infertility'

associated with isoflavones may also be related to the disruption of

normal LH secretion patterns . They found that feeding oestrogenic clover

to ovariectomized ewes, caused an initial depression then a rapid

elevation of LH levels . Thus the response to oestrogenic clover paralleled

the response obtained by administration of steroid oestrogens . There

are no reports on the effects of coumestans on LH secretion patterns .

FOLLICLE STIMULATING HORMONE - Unilateral ovariectomy of cyclic

ewes ( Findlay and Cumming, 1977) or bilateral ovariectomy of cyclic or

anoestrous ewes ( Salamonsen et al. , 1973), causes basal FSH levels to rise.

This suggests that basal levels of FSH are regulated by oestrogen through

a negative feedback system. As unilateral ovariectomy invokes a much

greater response in FSH than in LH levels ( Findlay and Cumming, 1977),

it is suggested that FSH is more sensitive to negative feedback effects

from the ovary than is LH - a situation which is also apparent during the

17 .

pre-ovulatory period in the intact ewe ( Salamonsen et al. , 1973; Baird and

Scaramuzzi, 1976 ) .

Infusion of oestradiol-178 into anoestrous ewes causes a dramatic

increase in FSH release, coincident with LH release ( Jonas et al. , 1973;

Salamonsen et al. , 19 73; Reeves et al. , 19 74 ; Pant, 1977) and

in a similar pattern of secretion to that which follows administration of

Gn-RH, except for an increased time lag ( Jonas et al . , 1973).

The addition of oestradiol to ovine pituitary cultures, even at

extremely low concentrations, causes a dramatic decrease in FSH

synthesis ( Miller et al., 1977a; Steinberger and Chowdhury, 19 77).

However, the adminstration of oestradiol in vivo did not alter pituitary

FSH levels ( Steinberger and Chowdhury, 1 977).

There are no reports on the effects of phyto-oestrogens on FSH

secretion patterns.

GONADOTROPHIN RELEASING HORMONE - In 1964 Bognadove postulated that

the variation in LH secretion throughout the oestrous cycle, was due to

a change in pituitary responsiveness to Gn-RH. Gn-RH induces a greater

change in serum LH ( Reeves et al., 1971a) and FSH ( Hooley et al. , 1974 ) during

an eight hour period on day 1, than at any other stage of the cycle .

However, some workers have failed to find the variation in response

( Foster and Crighton, 1974). A strong correlation between the oestradiol:

progesterone ratio and the LH response to Gn-RH has been demonstrated

( Thimonier et al . , 1974 ; Pelletier, 1 976) . When progesterone levels are

high, as in pregnancy, both FSH and LH responses to Gn-RH are reduced

( Chamley � ·, 19 74 a; 1974 b) . Similarly, daily progesterone injections

suppress FSH and LH responses to Gn-RH ( Hooley et al., 1974 ) .

The administration of exogenous steroid oestrogens also affects

pituitary responsiveness. Oestradiol benzoate admi�ration to anoestrous

ewes, increases the LH response to Gn-RH ( Reeves et al . , 1971b). More

precise studies have shown that oestrogens have a biphasic effect on

pituitary responsiveness. When given a few hours (4 -6) prior to Gn-RH,

oestrogens depress the LH response, but an amplified effect of Gn-RH

is observed if the time between oestrogen and Gn-RH treatments is

extended ( Pelletier and Signoret, 19 7 0; Libertum et al . , 19 7 4 ) . From

18 .

changes in the FSH : LH ratio, it would appear that the inhibitory effect

of oestrogen, influences FSH more than LH secretion ( Libertum et al . , 19 7 4 ).

( iii) The Reproductive Tract

CERVICAL MUCUS SECRETION - Copious mucus secretion by the ovine

cervix is generally attributed to oestrogen. Administration of oestradiol

benzoate to ovariectomized ewes increases cervical mucus secretion (Moore

and Robinson, 1957 ) and the response is linear (Allison, 19 7 2 ) . Rexroad

and Barb, ( 19 7 7 ) found that this increase in mucus production was not

accompanied by any change in spinnbarkheit, chloride content or percentage

dry matter .

The ingestion of phyto-oestrogens has also been shown to increase

cervical mucus production for both isoflavones ( Lindsay and Francis,

19 68; Smith, 19 7 1; Kelly et al. , 19 7 6 b) and for coumestans (Kelly et al . ,

197 6a) . Adams ( 1 9 7 6 a) disputed that an increase in the amount of mucus

produced did in fact occur, but that the visco-elasticity of the mucus was

decreased by phyto-oestrogens . He showed that 'clover disease' ewes

had lower spinnbarkheit (Adams, 19 7 7 ). The sensitivity of ewes to

exogenous steroid oestrogens is diminished by prolonged exposure to phyto­

oestrogens (Adams, 19 7 8) .

OVA TRANSPORT AND FERTILIZATION - Exogenous oestrogens induce an

acceleration in ova transport through the fallopian tubes ( Chang, 19 6 6;

Restall, 19 6 6 ) . Holst and Braden ( 19 7 2) found that phyto-oestrogens

produced a similar effect .

1 9 .

The administration of oestradiol increases the ra�e and strength

of uterine contr actions mar kedly (Hawke, 1 9 7 5) and also increases the

number of contraction 'waves ' moving towards the oviduc� (Hawke, 1 9 7 4) .

This improves sperm transpor t within the female tract, increasing the

numbers of sper m found within the cervix , uterus and oviducts (Hawke

and Cooper , 1 9 7 5) . This is in contradict i on to the effects of the

isoflavones on sper m tr anspor t . In sheep affected by clover disease,

the number of sperm entering the cervix is considerably lower than normal

(Turnbull et al . , 1 9 66 ) . Apparently changes in the rheological properties

of cervical mucus which depress sper m transport, outweigh any advantages from

incr eased uter ine motility.

Exogenous oestrogens apparently have no direct effects on fertilization

or on embr yo survival (Hawke and Cooper , 1 9 7 6) .

(iv) Oestrogen Receptor s

Oestr ogen r eceptor levels are r eplenished in the cytoplasm of target

tissues by mechanisms which are par tly dependant on pr otein synthesis.

Oestrogens appear to be the most potent regulator of the dynamics of

oestrogen-r eceptor turnover (Cidlowski and Muldoon, 1 9 7 6; 1 9 7 8) .

Receptor concentr ations in ovine endometrium vary over the oestrous cycle.

The pre-ovulator y surge of oestradiol promotes maximal r eceptor synthesis

(Koligian and Stor mshak, 1 977b) , hence the concentr ation of receptors

is greatest at oestrus (Miller et al., 1 9 7 7 b) . The concentration of

receptors declines after oestrus, being lowest at the time of maximum

progesterone synthesis (Miller et al . , 1 977b) .

A single injection of exogenous oestradiol results in 6 0- 7 5 percent

of cytoplasmic r eceptors being translocated into the nucleus (Sutherland

and Mester, 1 976) , the degree of translocation differing between target

tissues (Morris, 1 9 7 6 ) . Continuous administration of exogenous oestrogen

incr eases the concentration of cytoplasmic oestrogen r eceptors (Koligian

and Stor mshak, 1 9 77a; Murphy et al. , 1 977 ) , but progesterone suppresses

2 0 .

this oestrogen-induced incr ease (Hseuh e t al . , 19 7 6 ) .

Shemesh e t al . , (197 2) d emonstrated that phyto-oestrogens also

bind to oestrogen receptors. Both coumestrol and genistein compete with

oestradiol for receptors. C hamley et al. , (19 74c) found a greater

concentration of oestradiol-receptor complexes in ' clover disease ' ewes.

This suggests that phyto-oestrogens are also able to ind uce the synthesis

of receptors .

( D) FOLLICLE MATURATION IN THE EWE

(i) Patterns of Follicle Growth and Atresia

Follicula r growth is d e scribed as a continuum . It proceeds at

all times, at all ages, uninterrupted by pregnancy, anoestrous or other

periods of non-ovulation (Peters et al . , 19 75; Matton et al . , 19 7 7 ) .

In the ewe, the numbers of follicular growth ' waves ' during the

oestrous cycle have been variously reported as one (Hutchinson and Robertson,

1966), two (Brand and De Jong, 19 7 3), and three or four (Smeaton and

Robertson, 197 1 ) . Recently Turnbull e t al . , (19 7 7 a) disputed the existence

of any ' wave s ' of fol�icular growth, and suggested that follicles are

growing and regressing as�chronously at any given time of the oestrous

cycle .

The initiation of growth of primordial follicles is a continuous

process in ewes (Peters et al. , 19 7 5), and the number of ' initiated '

follicles which finally ovulate is d etermined by the rate of atresia

d uring their growth phase (Hay and Moor, 19 7 5b; Turnbull et al . , 19 7 7 a) .

Turnbull et al. , (1977 a) estimated follicle growth rates by the

mitotic ind ex of granulosa and thecal cells . They found that irrespective

of the stage of the cycle , the growth rate was slow in follicles of up to

0 . 4mm d iameter, after which it accelerated until it reached a maximum in

follicle s of about 0 . 7mm d iameter. This fast rate was maintained until

the follicles r eac hed a diameter of about 2mm and then declined by about

50 per c ent . In sheep, those follicles destined to ovulate would have

a diameter greater than 0.5mm on day 6-7 of the cyc le, although not all

follicles of this diameter would ovulate . In Merinos with an ovulation

rate of 1 to 2, about 3 -4 follicles enter the rapid growth phase each

day (Turnbull et al . , 19 7 7 a) . In single ovulator breeds, follic l es of

a diameter gr eater than lmm have a slower gr owth rate than do follicles

of a similar size range in multiple ovulator breeds (Turnbull et al . ,

21 .

19 7 7 b) . Thus for the same total number of follicles present, the multiple

ovulator will have a greater number capable of ovulation, when compared

with a single ovulator ( Dufour and Matton, 19 7 7 ; Dufour, 19 7 6). The

number of follicles whic h ac tually do ovulate, is apparently determined

within the 3 days prior to oestrus ( Land, 19 7 3 ; Findlay and Cumming,

19 7 7 ; Bherer et al . , 19 7 7).

( ii) Hormonal Contr ol of Follic le Growth and Atresia

GONADOTROPHINS - The initiation of growth of primor dial follicles

appears to be independant of gonadotrophin levels ( Lunenfield et al. ,

19 7 5; Peter s et al., 19 7 5). Two intra-ovarian fac tors have been

demonstr ated to influenc e the initiation of follic ular growth; ( a)

the size of the pool of non-growing follicles - a reduction in pool size

causes fewer follic les to start growing (Krar up et al . , 1 9 69) ( b) the

degeneration of large follicles - large follicles apparently release a

substanc e that suppresses growth initiation ( Peters et al. , 197 3).

There is evidence that the administr ation of gonadotrophins ( PMSG)

to ewes, will increase the number of ' initiated ' follicles entering the

rapid growth phase, prevent atresia in large follicles ( Hay and Moor,

1 9 7 5b; Turnbul l et al. , 19 7 7 a), and inc rease ovulation rates ( Cumming

and McDonald, 19 6 7 ) . Conversely, the reduction of FSH and LH levels by

the administration of antiser a, ac utely prevents the development of

small follicles to Graafian follicles (Welchen and Dullaar� 19 7 6), and

even the temporary absence of LH results in f ollicle atresia (Schwartz,

19 7 4 ).

Although a direct relationship has not been established between

FSH levels and ovulation rate (Findlay and Gumming, 19 7 6a), there is

considerable indirect evidence for such a relationship . Unilateral

ovariectomy results in compensatory fol licular growth and in ovulation

rates, in the remaining ovary (Sundaram and Stob, 19 6 7 ; Mallampati and

Casida, 19 70; Dufour et al . , 197 1a; Land, 19 73; Findlay and Gumming,

19 7 7 ) . This compensatory growth is apparent only if hemicastration is

carried out prior to 3 days before the onset of oestrus. There is a

transient increase in FSH, and to a lesser degree in LH, levels after

hemicastration. which coincides with falling oestradiol levels (Ramir ez

and Sawyer, 19 74; Findlay and Cumming , 19 7 7 ). Oestrogen replacement

therapy suppresses both the rise in FSH levels and compensation in

follicular growth and ovulation following hemicastration (Ramirez and

2 2 .

Sawyer, 19 74). 'Clover disease ' ewes also fail to show expected ovarian

compensatory hypertrophy (Adams, 19 7 6b).

The increase in ovulation rates in ewes fed lupin grain (Lightfoot

et al . � 19 7 6) is also accompanied by increased FSH levels (Brien et al . ,

1 9 7 6 ) .

Trounson et al . , (197 4 ) reported that lambs selected on multiple

birth characteristics of their dam, had a greater number of primordial

follicles than did control lambs . They suggested that this may be related

to higher endogenous gonadotrophin levels . However, Land et al . , (19 7 3)

and Echter nkamp and Laster, (197 6) were unable to find any relationship

between ovulation rates and endogenous LH levels. Bindon et al . , ( 19 7 1)

reported a greater ovarian response to exogenous gonadotrophins by high

fecundity ewes than by low fecundity ewes . Land et al . , (19 7 2, 19 7 6)

and Land ( 19 7 6 ) have shown that high fecundity breeds are less sensitive

to the effects of exogenous oestrogens on gonadotrophin release.

23 .

STEROID HORMONES All follicles, including those in various stages

of atresia, secrete a range of steroids including oestrogens, androgens

and progestins (Seamark et al ., 19 74 ) . The amounts and precise pattern

of steroid secretion of an individual follicle varies according to its

developmental stage . The smaller follicles produce predominantly

androgens, whereas the larger follicles produce both oestrogens and

androgens (Seamark, 19 7 8 ) . Atretic and non-atretic small follicles are

equally active steroidogenically .

Ovarian oestrogen secretion rises from baseline levels on day 13 of

the cycle to a peak on day 15, and then declines rapidly at oestrus

(Moor et al . , 19 7 1; Moor 19 74 ; Edwards, 19 7 4 ) . These high levels of

oestrogens are secreted almost entirely by the largest one or two non­

atretic follicles (Bj ersing et al . , 19 7 2 ; Moor, 19 7 3 ; Baird et al . , 19 7 5 ;

Hay and Moor, 19 7 5 b) . The enzyme 6 5 3 8 hydroxysteroid dehydrogenase

plays a key role in oestrogen synthesis (Moor et al., 197 1) and although

it ' s activity appears to be confined to the thecal cells (Hay and Moor,

19 7 5a), interaction between thecal and gr anulosa cells is apparently

required for oestrogen production (Moor, 19 7 7 ). It is not known what

initiates this surge in oestrogen production, but exogenous gonadotrophin

(PMSG) induces increased activity of this enzyme (Moor et al . , 19 7 1) .

The pre-ovulatory rise in LH terminates this surge in oestrogen production

(Moor, 19 74 ) . The Graafian follicle is extremely sensitive to minor

changes in basal LH concentrations (Baird et al . , 19 7 6 ) and variation in

the secretion of oestradiol- 17 8 probably reflects the response to small

pulses in LH (Wheeler et al . , l 9 7 7 a).

As a consequence of the pre-ovulatory changes in circulating gonado­

trophins, oestrogen production by the large non-atretic follicles is

reduced and replaced by an increase in the release of androgens and

progestins . Twelve to 1 8 hours prior to ovulation, the large follicles

24.

produce little o r no oestrogen, but large amounts of pro gesterone

and 2 0« pregn-4 -en-3-one ( Seamark, 1978) . It has been suggested that

progesterone may have an effect on the ovary independent of its influence

via the hypothalamo-pituitary axis ( Rexroad and Casida, 1977). Du four

et al., ( 19 71b) reported that follicle growth was more rapid in ovaries

that co ntain a corpus lut eum . Inj ection o f progesterone int o one ovary

of anoestrous ewes, with concurrent systemic administration of PMSG,

results in more rapid follicular gro wth, although o estro gen secretion is

unchanged, ( Rexroad and Casida, 1977) and in a greater number of fo llicles

and corpora lutea in the inj ected, than in the co ntrol ovary ( Harned and

Casida, 1971) . Progestero ne may act to alter time-dependent changes in

follicle size, thus enabling some follicles to gro w while o thers undergo

atresia.

HORMONE RECEPTORS - It has been proposed that the selectio n of those

follicles destined to ovulate, is a co nsequence o f both sequential changes

in follicular hormone receptor levels and in circulating pituitary

gonado tro phin levels ( Carson et al., 1978) .

As follicular growth proceeds, FSH receptor levels decrease and LH

recepto r levels increase within granulosa cells ( Channing, 1970; Carson

et a l . , 1978) , but remain unchanged within thecal cells (Weiss and Armstrong ,

1977; Carso n et al . , 19 78) . FSH has been sho wn to increase numbers o f

FSH receptors; o estradiol to increase oestradiol receptors; and FSH

and oestradiol in concert to increase LH receptors, in granulosa cells

( Richards and Midgley, 1976). Termination of follicular growth by

LH-induced luteinizatio n is associated with the loss of receptors for FSH

and oestradiol, and the subsequent increase in LH receptors (Richards

and Midgley, 1976 ) .

2 5.

(E) OVULATION IN THE EWE

(i) Factors Influencing Ovulation Rates

Heape (189 9) first reported that heavy ewes produced more lambs than

light ewes. This is related to a higher production of ova (Wallace, 19 61;

Allen and Lamming, 19 6 1; Killeen, 19 6 7 ; Alli son, 19 68). This correlation

between bodyweight and ovulation rate has been described by Edey (19 68)

who found a linear relationship within a particular weight range.

Ducker and Boyd (19 7 7 ) demon strated that it is the body condition element

of ewe liveweight, not body size, that influences ovulation rates.

However, Cumming (19 7 7 ) showed that liveweight alone was as accurate

a pred i ctor of ovulation rate as both liveweight and bod y condition.

S everal workers have reported evidence of a d ynamic effect of flushing,

independen t of bod ywei ght, on ovulation rate (Coop, 196 6a; 19 6 6b;

Mcinnes and Smi th, 19 6 6; Killeen 19 6 7) although others have found no

effect (Hulet et al. , 19 68; Fletcher, 1 9 7 1 ; Cumming, 19 7 7 ) .

There is consid erable variation i n fecund i ty between breeds and between

strain s within a breed. Ewes of higher fecundity (Trounson _et al.,

19 7 4 ) and their daughters (McDonald and Chang, 1 9 6 6) ovulate a greater

number of follicles, an d a higher proportion of the Graafian follicles

that d evelop (Bradford et al., 19 7 1; Turnbull et al., 19 7 7 b). Fecund ity

and ovulation rates are not related to LH secretion throughout the

cycle (Echternkamp and Laster, 1 9 7 6 ) nor to LH peaks 1 Land et al . , 19 7 3) .

There i s some ind irect evid en ce that FSH levels around days 12 -14 are

important in determining ovulation rates at the subsequent oestrus,

(Cumming and McDonald, 19 6 7 ; Brien et al. , 19 7 6; Findlay and Cumming

19 7 6 b; 19 7 7 ) but there are no reports of a d irect relationship between

FSH levels and fecundi ty. Liveweight or flushing apparently d oes n ot

influence FSH levels (Findlay and Cumming, 19 7 6a).

2 6 .

Wheeler et al., (1 9 7 7 b) showed that mean secretion rates of

oestr adiol- 1 7 S are significantly higher in ewes with high ovulation rates,

and Land et al. , ( 1 9 7 6 ) found such ewes less sensitive to exogenous

oestradiol-induc ed depression of ovulation rates . Ewes of high fec undity

also show a greater r esponse to PMSG-induced superovul ation (Eastwood

and Mc Donald, 1 9 7 5).

It has been suggested that the length of the interval from the onset

of oestrus to the time of ovulation may influenc e ovulation rate.

Land (1 970) and Hanrahan and Quirke ( 1 9 7 5 ) showed a positive relationship

between the duration of oestrus and ovulation rate. Curnrning et al.,

( 1 9 7 3 ) demonstrated a high degree of constancy in the inter val from the

pre-ovulator y LH peak to ovulation. Lindsay et al., ( 1 9 7 5) found that

the presence of rams during oestrus induced ovulation earlier by advancing

the time of the LH surge. There appears to be a consider able variation

in the interval from the onset of oestrus to the LH surge (Lamming

�rs corn . , 1 9 78).

(ii) Postulated Mechanisms of Ovulation

The precise process, or processes, of ovulation r emain obscure

although considerable effort has been put into identifying them. Two

major types of ovulator y mechanisms have been suggested; those

involving ' enzyrnatic breakdown' of follicular tissue and those involving

the 'bur sting' of the follicle due to pressure changes .

There is substantial evidence suggesting that the collagenous

connective tissue of the wall of the Graafian follicle is degraded near

the time of ovulation (Espey, 1974 ) . Collagenolytic enzymes have been

detected within ovarian follicles; both neutral and acid proteases,

capable of degrading collagen, are produced in mature Graafian follicles

(Espey and Coons , 1 976).

2 7 .

Although the growth and maturation of follicles occurs without a

measurable increase in hydrostatic pressure (Ro dbard, 1 9 6 8 ) a follicular

contractile process has been demonstrated. Electron mic roscopy shows

many c ells in the theca externa of mature sheep follicles, with c ytoplasmic

filaments and dense bodies c harac teristic of smooth musc le cells (O ' S hea,

19 7 1) . Strip s of follic ular tissue exhibit classic al contrac tile

activity, (O ' Shea and Phillip s, 19 74) and autonomic innervation, both

adrenergic and cholinergic, is assoc iated with follicles (Bahr et al.,

19 74; Owman et al., 1 9 7 5 ; Walles et al., 19 7 6; 19 7 7 ) . Per fused

ovarian tissue has been shown to be elec tric ally active (Tojo et al.,

19 75).

It has been suggested that prostaglandin Fz� is involved in the

induction of ovulation (Tsafriri et al. , 19 72). Conc entrations of

prostaglandins inc rease within the developing follicle late in the

oestrous cyc le , most dramatic ally in those follic les whic h eventually

ovulate (Yang et al . , 19 7 4 ) . This rise i n prostaglandin levels is

apparently induc ed by the surge in endogenous oestradiol which oc curs

at days 14- 15 of the cycle, but not by earlier oestradiol peaks

(Barcikowski et al., 19 74 ; Cox et al . 19 74) .Infusion of physiologic al doses

of oestradiol into the uterus on days 14-15 will induce comparable

p rostaglandin peaks (Barcikowski et al., 19 7 4 ).

Systemic or intrafollicular injection of prostaglandin Fz� induces

ovulation (Phi et al., 1 9 7 7 ) and similarly p erfusion of follicles ' in

vitro' enhances ovulation (Hamada et al . , 1 9 7 7 ) . This induction of

ovulation is bloc ked by known prostaglandin Fz� antagonists suc h as

p rostaglandin Ez and indomethacin (Carlson et al. , 1 9 7 4 ) .

Y ang et al., ( 1 9 7 4 ) have suggested that prostaglandin Fz« may be

involved in the synthesis, release or ac tivation of collagenolytic

enzymes and Tojo et al., ( 1 9 7 5 ) has shown that prostaglandin F2�

2 8 .

accelerates the electrical activity of perfused ovarian tissue .

Intrafollicular inj ection of LH ( and in some experiments, of FSH)

will induce ovulation ( Jones and Nal bandov, 1972). Most evidence

indicates that LH is the sole agent required to trigger ovulation, while

both FSH and LH are required for the development, maturation and

l uteinization of follicular tissue ( Rondell, 1 974). It has been well

established that LH induces increased synthesis of cyclic AMP and

subsequently, increased protein synthesis ( Robison et al. , 1 971) .

Prostaglandins are capable of increasing cAMP formation ( Marsh, 1971) and

conversely anti-prostaglandin agents block the stimulatory action of LH

on cAMP synthesis (Kuehl et al . , 1970) . Protein synthesis appears to be

an essential part of the metabolic processes supporting ovulation, as

inhibitors of protein synthesis inhibit LH-induced ovulation (Lipner and

Greep, 197 1 ).

( iii) Blockade of Ovulation

It has been well established that indomethacin blocks ovulation in

ewes ( Carlson et al. , 1974; Armstrong et al . , 19 7 4 ; Lewis and Warren,

1975). Indomethacin blocks the LH-induced increase in prostaglandin

F2� levels in ovarian follicles (Armstrong et al. , 1974 ) and inhibits the

oestradiol-induced LH release in anoestrous ewes ( Carlson et al . , 1974).

Similarly the administration of inhibitory analogue s of LH-RH will also

effectively block ovulation ( Phelps et al . , 1977).

This complete blockade of ovulation is not apparent in ewes ingesting

coumestans . Oestrogenic lucerne reduces the number of ovulations, but

seldom prevents ovulation completely .

(F) THE PURPOSE AND SCOPE OF THE INVESTIGATION

29 .

It has been clearly established that the ingestion of oestrogenic

lucerne by ewes, depresses ovulation rates . Because of the important

r ole of the gonadotrophins in the maturation and ultimate ovulatio n of

ovarian follicles , it is possible that the coumestans could alter the

pattern of gonadotrophic secretion . FSH is invol ved in the development

of Graafian follicles, and LH facilitates the final stages of matur ation

and the induction of ovulation. The coumestan-induced depression of

ovulation rate may arise from a decreased pituitary output of these

gonadotrophins, or fr om changes in the pattern of their release, particularly

near the time of oestrus .

The present trials investigated the effects of oestrogenic lucerne,

fed over the oestrous cycle, on LH secretion patterns, and on the timing

of the pre-ovulator y LH-surge in r elation to oestr us and ovulation

(Experiment I) . Ovarian activity was recorded as a measur e of gonadotrophic

function . In Experiment II, the effects of oestrogenic lucerne on

ovarian activity, were studied when lucer ne was fed early or late in

the oestrous cycle, with the view of determining the critical time

involved in the depression of ovulation rates.

C H A P T E R I I

MATERIALS AND METHODS

30.

C H A P T E R I I

MATERIALS AND METHODS

(A) EXPERIMENTAL PLAN

Two separate trials were carried out to investigate the effects

of oestrogenic lucerne on cycling ewes . In the firs t experiment

plas ma gonadotrophin concentrations were measured over a complete

oestrous cycle, and in the second experiment the effects of coumes tan

intake either early or late in the oestrous cycle were recorded. The

experimental procedures and cale nder of events are set out in

FIGS. 2-1 and 2-2.

EXPERIMENT I - 22 mixed aged Romney ewes and 20 mixed aged Border

Leicester x Romney ewes were randomly as signed to two groups with equal

numbers of each breed. At the detection of oestrus following

synchro� ation, each group grazed either oestrogenic lucerne or non­

oestrogenic ryegrass / clover pasture.

Blood samples were collected from all ewes by j ugular venepuncture.

Samples were taken daily for 1 7 days, from day 7 of experimental oestrous

cycle. On predicted day 16 of the cycle, blood samples were taken at

4 -hourly intervals until the detection of oestrus; then at 2-hourly

intervals for 24 hours and at 4 -hourly intervals for the subsequent

1 2 hours.

All ewes underwent laparotomy 3 days post-oestrus at which time

corpora lutea and s urface follicles were recorded.

Over the period of intensive blood sampling, the animals were kept

in s mall holding paddocks and yards adjacent to laboratory facilities

(Mas s ey University Fertility Centre) . Ewes in the lucerne group were

Fig. 2 ·1 EXPERI M ENTAL PLAN Expt . 1

Daily Blood Sampling

n = 21

42 Romne B LX Ewe�Progestagen Sponges

n = 21

L � � -5 b c (/) � gs

� E ..,

(/) 0 - ::J +-' � L e o. t) CO X <lJ Q. (lJ 0 CO

1 0 7 17 20 27

.-�--�-�-��1-----�---- I I -1 0 14 17 24 34 37 44

T I M F (days )

Fig.2·2 EXPERI M ENTAL PLAN

n = 20

ri = 20

L llf)

-5 2 c

�

Expt. 2

L - 1.1) Q;

_£912

1:

a +..J X 1.1) <lJ <lJ 0

81 ror.1ney IProgestagen Sponges & 1

ewes

n = 2 1

n = 2 0

.----0

.--------------,;-------r--- -·� 1 7 20

0 14 17 26 3L1 37

TI M E (days )

31.

f ed f resh lucerne cut daily f rom the crop .

EXPERIMENT II - 81 mixed aged Romney ewes were rando mly assigned to

f o ur groups. At the detection o f oestrus f ollowing synchronizatio n,

the gro ups were assigned to o ne o f four grazing regimes; past ure

thro ughout t he o estro us cycle, lucerne thro ughout the cycle, or these

treatments were interchanged at midcycle. In the lat t er two groups

treatments were changed on day 9 of t he experimental oestro us cycle.

All ewes were slaught ered 3 days po st -oestrus at the lo cal f reezing

works (Bort hwicks - CWS, Longburn) . Repro ductive tracts were recovered

within 1 5 minutes o f slaughter and individually identified.

(B) LUCERNE AND CONTROL PASTURES

The lucerne crop involved in t hese experiments consisted o f t wo

hectares o f Wairau lucerne on a Rangitikei Loamy sand . The paddo ck

was sub-divided into f our plo ts of approximately 0. 5ha . Experiment al

grazing co mmenced eight weeks after the lucerne had been previo usly

harvested, at which time plant heights ranged from 6 0 to 90cms .

Approximately 25 percent o f plants were in flower and there was

moderat ely heavy f ungal infectio n o f plant tissue (more t han 5 lesio ns

per leaf ) .

A three hectare ryegrass/ whit e clover pasture on a Manawatu Fine

Sandy Loam adj acent to the lucerne stand was grazed by contro l animals .

Leaf samples o f lucerne, and o f white clover, were taken weekly

and assayed for coumestan cont ent . This involved random select io n o f

material from 4 0 plants f ro m each plo t .

(C) ANIMALS

The animals utilized were 103 mixed-age Romney ewes and 20 mixed­

aged F1

Border Leicest er X Romney ewes, drawn f rom several f lo cks

maintained on Massey University farms. All ewes were identified by

numbered brass eartags and by midside brands, which were applied at

the commencement of the trial .

32 .

All ewes were weighed prior to, and at the completion of oestrus

synchronization treatment, as well as at the completion of the trial.

Ewes in Experiment I were also weighed two weeks prior to synchronization

treatment . Attempts were made to carry out all liveweight recordings

at the same hour of the day, in order to mi nimise variations in gutfill .

(D) OESTRUS SYNCHRONIZATION

In order to synchronize oestrous cycles, all ewes were subj ected to

1 4 -day treatments with polyurethane intravaginal sponges containing

40mg of methyl-acetoxy-progesterone (M . A . P . ) . Vasectomized rams fitted

with Sire Sine harnesses were used to detect oestrus following pessary

removal .

(E) HORMONE ANALYSES

COUMESTAN S - Lucerne and clover samples were subjected to ethanol

extraction (Kelly, 1 9 7 6) and extracts were forwarded to Dr . R . W . Kelly,

Invermay Agricultural Research Centre, for analysis of coumestan

content.

PLASMA GONADOTROPHINS - Blood samples were assayed for ovine

luteinizing hormone by radioimmunoassay, following the double-antibody

technique of Niswender et. al. ,(19 6 9 ) , modified according to Barrell ( 1 9 7 6 ) .

This assay utilized the following materials: rabbit anti-ovine LH

serum (pool 15 courtesy Dr G . D . Niswender) , NIH-LH-Sl8 as assay

standards, and highly purified ovine LH (LER- 1 374A) for radioiodination.

Assay standards and samples were assayed in triplicate, with hypophysectomized

sheep plasma added to s tanda rd curve tubes . Mean assay s ens i t iv i ty

wa s 0 . 2 ng /ml , and the intra-and inter-assay coef f ic ien t s o f var ia t ion

( CV ) wer e 1 0 . 3 and 23% respec t ively .

At t he t ime o f planning o f these exper iments , a supply of FSH-

an t i s era became ava ilabl e . On the assump t ion tha t this a s say woul d

be i n opera t ion , F S H analyses were to be inc luded i n exper imen tal

proc edur es . However , with t he immuno chemical s ava ilab l e , b inding o f

iod ina t ed FSH wa s inadequa t e , and c ons equen t l y this a s say was omi t ted

from plasma analyses .

(F) CERVICAL MUCUS ANALY SIS

Cerv ica l mucus samples were taken f rom a l l ewes in Exp er imen t I

a t blood col l ec t ion time s over the oestrus pe riod . The rams used

for d e t e c t ion of oes trus were f i t t ed with canvas ' a pron s ' to prevent

penil e in tromiss ion and sub sequent con tamina t ion o f cervical mucus .

3 3 .

Mucus samp l e s were collec ted , wi th the a id o f a speculum , using absorbent

co t ton swab s .

Cerv ica l mucus samples were a s s essed for chlo ride ion con t ent by

the me thod o f Turnbull e t a l . , ( 1 96 7 ) . S i lver chroma t e was prec ip i t a t ed

onto s t rips of Wha tman No . 1 chroma t ogra phy paper , by impregna t ing the

paper , first with 0 . 2 7 5 N s i lver n i t rate and then with 0 . 1 7 5 po t a s s ium

chroma t e . Chl oride ions from the mucus samples produc e whi t e to bu f f -

coloured spo t s , which a r e compared to sodium chloride s tandards . Oes t rus

is a s so c ia t ed wi th a rise in chloride concentrations , and ovula t ion with

a sudden fal l in chloride l evel s .

(G) UTERINE AND OVARIAN WE IGHTS

34 .

Af t er co l l ec t ion a t slaugh t er , u t er i were separa t ed f rom the trac t

by tran s ec t ion immed ia tely an t erior to the c ervix , dried with paper

towe l s and weighed .

Ovaries f rom each ewe were weighed s eparately . Where par t o f the

uterus , or par t of an ovary , wa s l o s t a t s l aughter , tha t animal wa s

omi t t ed from t he analys i s .

(H) FOLL ICLE POPULATIONS

Af t er co l l ec t ion at s laughter , the ovaries wer e preserved in

Bouins f luid un t i l s ec t ioning and a s s e s sment o f fol l i c l e popula t ions .

To a s s e s s the t o ta l ovarian fo l l icular populat ion , a l l ovaries were

sec t ioned in to lmm s l ic e s . Fol l ic l e diame t er wa s c a l c u l a t ed a s the

mean of two measuremen t s taken a t r ight ang les to each o ther . Coun t s

were made o f a l l an tral foll i c l e s with a dimaeter o f 2mm o r greater .

( I ) REPRODUCTIVE TRACT HISTOLOGY

At the t ime of recovery of the reproduc t ive trac t , l cm s ec t ions

of vagina , c ervix , u t eru s and fal lop ian tube were taken and pres erved

in Bouin s Fluid for later his tological examina t ion .

Sec t ions o f 6� thicknes s , wer e taken o f all trac t s pec imens and

s ta ined wi th Ha ema toxyl in and Eo s in . The mean height o f ep i thel ia l

c el l s wa s cal culated fo r a l l sec tion s , by taking 2 5 s epara te mea surements

from each sec t ion .

(J) ANALY S I S OF DATA

Analyses o f d is c r e t e da ta - numb ers o f ovu lations and f o l l i c l e

2 po pulat ions - u til i z ed Chi s ta t is t ic .

Da ta invo lving :

Ewe l ivewe igh t s

LH conc entrat ion i n per ipheral p lasma

Ovar ian weights

Epi theaal c ell height s

were analysed by ana lys i s o f var iance ( Snedeco r and Cochran e , 1 9 6 7 ) .

3 5 .

Da t a from 8 ewe s were excluded from the ana lyses o f LH

conc en tra tions and ovulat ion ra t e s owing to either fa ilure o f o e s t r u s

synchroni za t ion ( 4 ewes) or failure t o detec t the pre-ovula tory LH peak

(4 ewes ) . Da ta f rom 9 ewes wer e excluded f rom Exp t . II due to fa ilure

o f hea t d e t ec t ion for a var iety o f r ea sons ; Sponges lo s t (4 ewes ) ,

bl ind tracts ( 2 ewes ) , s ilent hea t ( 2 ewes ) and pregnancy ( 1 ewe ) .

Data from a fur ther 1 2 ewe s were exc luded from reproduc t ive tract

analyses , due to cys t ic hyperpla s ia ( 1 ewe) , anovula tory o e s t rus (2 ewes ) ,

or incomp l e t e recovery o f the tract a t s laughter ( 9 ewes ) .

C H A P T E R I I I

RESULTS

36 .

C H A P T E R I I I

RESULTS

(A) LUCERNE OESTROGEN IC POTENCY

( i ) Luc erne Coume stan Con t en t

Mean l evels o f the coume s tans , coumes trol and 4 -me thoxycoumes t ro l ,

for each o f the four p l o t s and for c lover samp l e s , are s hown in Tabl e 3 - 1 .

Values are g iven a s par t s per mill ion on a dry ma t t er ba s i s .

TABLE 3- 1 : LUCERNE COUMESTAN CONTENT (ppm)

DATE PLOT A PLOT B PLOT C

6 : 4 : 7 7

Coume s t rol 98 1 5 1 66 4-Methoxycoume s t rol 60 1 06 3 3

1 3 : 4 : 7 7

Coume s t rol 6 9 1 2 2 8 1 4 -Me thoxycoume s t rol 34 7 0 4 1

20 : 4 : 7 7

Coume s trol 88 94 1 4 5 4-Me t hoxycoume s trol 38 59 1 0 7

( i i) Da ily Coumes tan Intake

PLOT D CLOVER

100 < 10 7 7 < 1 0

1 1 0 < 5 6 3 < 5

1 7 2 < 5 1 4 5 < 5

Sheep are capable o f consuming each day, a to tal we ight o f

forage equal t o 3 percent o f their bo dyweigh t . Cons ider ing tha t lucern e ,

during i t s f lower ing s ta ges , i s approxima t ely 2 4 percent dry mat t er ,

then a conserva t ive e s t ima t e o f the daily in take o f lucerne by ewes in

this tria l , would b e 1 kg o f dry ma t t er . The mean daily intake o f

coume s tans , for ewe s i n both exper imen t s , bas ed on a dai ly intake o f

1 kg o f dry ma t t e r , are shown in Tabl e 3-2 .

TABLE 3-2 . THE DAILY COUMESTAN INTAKE (mg/ day) OF EWES

INGEST ING 1 KG ( DM) OF LUCERNE

EXPERIMENTAL STAGE OF OESTROUS CYCLE ( DAY S ) GROUP

0-9 1 0- 1 7 1 8- 2 5

EXPERIMENT I Pas ture 2 2 1 Lucerne 1 1 7 2 5 2 1 7 3

EXPERU1ENT I I Pas ture 1 1 Luc erne 1 9 2 2 1 7 Past-Luc 1 2 1 7 Luc-Pa st 1 9 2 1

The coumes tan intake o f ewes gra z ing ryegras s / c lover pas tu r e , is

based on the as sump t ion tha t whi t e c l over accounts for 10 percent of

daily dry ma t ter in take .

(B ) EWE LIVEWEIGHTS AND OESTRUS

( i ) Ewe Liveweigh t s

Mean l iveweights o f ewes in both exp e rimen t s a r e l i s t ed in

Tab l e 3-3 . There wer e no significant d i f f e rences be tween t r ea tment

groups a t any o f the weight recordings . S imilarly there were no

signi f ican t di f f e renc es in the rate of weight gain b e tween weighings .

3 7 .

TABLE 3-3 :

NUMBER OF EWES

TIME OF WEIGHING :

Pre-Experimen ta1

Sponge Inser t ion

Sponge Withdrawa l

Pos t-Exper imen tal

NUMBER OF EWES

TIME OF WEIGHING

Sponge Ins e r t ion

Sponge Withdrawal

Po s t Exper imen tal

MEAN EWE LIVEWEIGHTS ( kg)

EXPERIMENT I

PASTURE

2 1

5 7 . 00 + 1 . 0

6 1 . 50 + 0 . 95

58 . 50 + l . 0 1

6 1 . 60 + l . 1 9

EXPERIMENT I I

PASTURE LUCERNE

20 2 1

5 7 . 7 0 + l . 6 1 5 6 . 00 + l . 3 2

6 1 . 6 0 + l . 8 6 6 0 . 7 0 + l . 44

6 3 . 8 0 + l . 7 2 6 2 . 00 + l . 30

38 .

LUCERNE

2 1

5 6 . 70 + 1 . 5 3

60 . 4 0 + l . 3 1

5 7 . 90 + l . 3 5

60 . 00 + l . 3 1

PAST-LUCERNE LUCERNE-PAST

20 20

57 . 00 + l . 5 8 58 . 60 + 0 . 99

6 1 . 5 0 + l . 5 3 6 3 . 2 0 + 0 . 9 9

63 . 2 0 + l . 5 1 63 . 90 + l . 1 5

3 9 .

( i i ) Oe s trus Synchroni za t ion

The d i s tribu tion of the onse t of oes trus for bo th the synchronized

and the exper imen t a l oe s trus ar e shown i n FIGS 3-1 and 3-2 .

In Expe r imen t I , 9 3 perce n t of the ewes were d e t ec ted i n oes trus

over a four day per iod fol lowing sponge wi thdrawal . E i gh ty two percent

of the ewe s were de tec ted in heat over a corre s pond ing f ou r day period in

the f o l lowing oes trou s cyc l e .

In Exper imen t II , 83 . 3 percent o f the ewe s were detec ted over a f our

day period for the synchroni zed oes trus and 7 6 . 5 percent in the fo l l owing

oe stru s . Inspe c t ion o f the reproduc t ive trac t s later revea led tha t a

number of ewes we re phy s i o l og i ca l ly unab l e to exhib i t oes trus . Two ewe s had

b l ind r eproductive tra c t s w i th only vag i na l t i s s ue present , and one ewe

was found to be pre gnan t . A fur ther four ewe s had l o s t s po nges dur ing

synchron i za t ion . Only two ewe s exhibi ted ' s i l ent ' hea t s , one in the

pa s ture group and one i n the lucerne-pa s ture group .

Th ere were no s igni f i cant d i f f ere nce s in oes trus synchr ony a t the se cond

oes trous cyc le be tween ewe s gra z i ng l u cerne and ewe s gra z ing pa s ture .

S imi l a rly there we re no d i f f erence s �n mean cyc le length .

( i i i ) Oe s t rus-Ovu l a t ion Int erval

The in terva l b e tween the ons e t of oes trus and the pre-ovu l a tory LH

peak , wa s extreme ly variab l e b e tween anima l s , i n both trea tment group s .

Some anima l s were r e co rded as only enter i ng oes trus , af ter the LH peak

had occurred . The i n terva l length f or ind ividual sheep � s shown i n

FIG 3-3 . Ther e were no s igni f i cant d i f ference s be tween ewes gra z i ng

lucerne and ewes graz ing pas tur e in the mean i n terval dura t ion .

The re were no apparent pat terns shown in the ch loride content o f

cerv i ca l mucus samp l e s co l le c ted . Ch l o r i d e concentr a t ions appeared t o

r i s e and fa l l rand omly . Consequen t ly , the t ime o f ovu l a t ion c ou l d no t

tl) ' :J �I tll QJ 0

"'0 �I u QJ � (!;

0 tll QJ �! -0 L (])

.0 E .:J

Z _

Fig . 3·1 D I STRI BUTION OF THE ON S ET O F O E STRUS - Expt. I

synch ro n i zed oestru s experi mental oestrus

10 .

8 Pa sture 6

Group 4 2

· spon ge withdrawn

-

. 1 I I I I I I I � , I . .

-2 -1 0 1 2 3 4 -2 -1 0 1 2 3 4

10

8 Lucerne 6 Grou p

4 spon ge

w ithdraw n

2 �

-. I I I I I, lf-!J _, I . . '

- 2 -1 0 1 2 3 -2 -1 0 1 2 3 4

Time (days )

Fig. 3·2 D ISTR I BUTION OF TH E O NSET OF OEST RU S - Expt . TI

Cl) 2

+oJ Cl) Q) 0 c u

8

Pasture 6 Group

4 2

8 Lucerne Group 6

4 2

$ 8 � Past/Luc t Group 6 0

-0 L Q)

.0

4

2

E 8 .2_ Luc/Past Group 6

4

2

c - Q) �

rn · ru L _ c u O .c n .......

� �( -

-

-

..

..

...

-

-

-

-

-

..

synchronized oestrus

l 7.

7

I 11 7

.,

L r

.. I

experimental oestrus.

I

I I I I I I I I. 'L , I I I I I rl �2 -1 0 1 2 3 -2 -1 0 1 2 3

Time (days)

I

I

F ig. 3-3 ONSET OF OESTRUS - LH PEAK I NTERVAL

...... LH PEAK . �

Pastu re o--

n = 17 -

�

-......

....... ......

,....

......

-

0 0

•

Lucerne -

n =17 El

•

I I I I I I I I I

0

•

I 24 20 16 12 8 4 0 2 4 6 8

T I M E ( hours )

be e s t ima ted .

( C ) LUTE INIZING HORMONE SECRETION

( i ) Dur ing the Oe s trous Cycle

Mean dai l y concentra t ions o f lu t e in i z i ng hormone i n per ipheral

40 .

p lasma o f ewe s f r om both the pas ture and the lu cerne groups o f Exper iment I

are l i s t ed in Table 3-4 . Analyses o f var i ance b e tween the groups were

made for each samp le day . There we re no s ignif i can t d i f ferenc e s i n LH

secret ion at any s t age of the oe s trous cyc l e . Mean plasma LH c oncent r a t ions

r anged f rom 0 . 5 to 1 . 5 ng /ml over the cyc l e , exc lud i ng t he pre-ovu l a tory

peak . The pa t te rns o f secr e t ion are shown i n F I G . 3-4 .

( i i ) The Pre-Ovula tory LH Peak

Mean concen t r a t ion s of lute ini z i ng hormone in pe r i pher a l p l asma ,

measured a t 2-hourly in terva l s f rom the ons e t of oe s trus , are shown in

Tab le 3-5 . The pre-ovu la tory LH peaks we re inc luded in th i s pe r i od .

Ana l y s es of var i ance be tween the lucerne and the pas ture group s , a t each

sample t ime , f a i led to show any s i gn i f icant d i f f erenc es . The pre-ovu l a tory

LH secr e t ion pa t terns are shown in FIG 3 - 5 .

4 1 .

TABLE 3-4 : MEAN PLASMA LH CONCENTRATIONS (ng/ml ) DURING THE OESTROUS

CYCLE IN EWES GRAZING PAS TURE OR OESTROGENI C LUCERNE

DAY - 7 -6 -5 -4 - 3 -2 - 1 0 + 1

PASTURE 0 . 5 5 9 0 . 4 5 2 0 . 4 35 0 . 4 4 5 0 . 7 5 6 1 0 . 94 9 1 . 0 3 8 7 . 0 5 0 . 5 9 1

(MeaniSE ) +0 . 1 7 +0 . 1 2 +0 . 1 1 +0 . 1 3 +0 . 1 2 +0 . 2 6 +0 . 32 +3 . 9 2 +0 . 1

LUCERNE 0 . 4 0 6 5 0 . 5 7 3 0 . 307 1 0 . 409 0 . 5 7 3

Mean±SE) +0 . 1 3 +0 . 14 +0 . 1 1 +0 . 1 3 +0 . 5

DAY +2 +3 +4 +5 +6

PASTURE 0 . 9 7 5 0 0 . 7 6 5 0 . 859 0 . 7 4 9 0 . 7 4 9

MeaniS E) +0 . 3 6 +0 . 1 7 +0 . 2 +0 . 2 1 +0 . 1 9

LUCERNE 1 . 5648 l . 037 1 . 04 1 0 . 5 7 1 0 . 3 9 9

.(Mean±SE ) +0 . 4 1 +0 . 3 2 +0 . 2 9 +0 . 1 6 +0 . 1 2

1 . 24 7 1 . 1 6 1 9 1 . 1 4 1 . 04

+0 . 3 2 +0 . 2 5 +6 . 5 5 +0 . 1 1

+7 +8 +9 + 1 0

0 . 58 5 0 . 5 34 0 . 4 34 0 . 54 6

+0 . 1 6 +0 . 1 4 +0 . 1 1 +0 . 1 6

0 . 6 7 3 0 . 4 08 0 . 4 3 3 0 . 5 5 6

+0 . 1 2 +0 . 1 1 +0 . 1 4 +0 . 1 3

( Day 0 o f o e s t rous cyc l e co rrespond s t o t ime of pre-ovula tory LH peak)

3.0

2 .5

2 0 ........

E .......... � 1 .5 ..____.

I _j

1 .0

0 .5

_,.,. -

1

Fig. 3 .4 L H S ECRETION OVER O ESTROUS CYC L E

pasture g roup o o

luceme group • •

n = 17

I I I I I I I T--T--uul - 1- ,------.----r-.---r--�--.---T----. -7 - 6 -5 -4 -3 -2 -1 0 +1 + 2 +3 + 4 +5 +6 +7 +8 +9 +10

Ti me fmm Pre - ovulatory LH Pea k (days)

TABLE 3-5 :

PASTURE

LUCERNE

PASTURE

LUCERNE

PASTURE

LUCERNE

PRE-OVULATORY PLASMA LH CONCENTRAT IONS (ng/ml) IN EWES

GRAZING PASTURE OR OESTROGENIC LUCERNE

HOUR- 1 2 - 1 0 -8 - 6 -4 -2 0 +2

1 . 1 3 2 . 4 3 1 . 8 5 6 . 04 7 . 9 9 29 . 20 8 7 . 05 4 0 . 2 6

+0 . 3 7 +0 . 59 +0 . 3 5 +2 . 3 9 +2 . 54 +4 . 84 +3 . 9 2 5 . 8 2

1 . 7 1

+0 . 4

+4

1 . 9 6 3 . 4 1 2 . 5 1 1 8 . 9 1

+0 . 6 7 +0 . 9 9 +0 . 4 8 +3 . 9 3

+6 +8 + 1 0 + 1 2

3 1 . 3 8 1 3 . 3 7 8 . 68 2 . 70 l . 40

39 . 99

6 . 30

+ 1 4

0 . 84

9 1 . 1 4

+6 . 5 5

+ 1 6

0 . 7 5

4 6 . 2 3

+5 . 24

+ 1 8

0 . 9 0

+4 . 1 1 +5 . 8 9 +2 . 6 8 +0 . 5 7 +0 . 2 6 +0 . 1 8 +0 . 1 9 +0 . 38

2 6 . 1 9 1 1 . 6 3 3 . 2 4 2 . 4 0 1 . 1 1 0 . 8 3 0 . 7 2 0 . 5 1

+3 . 7 4 +3 . 5 6 +0 . 3 7 +0 . 2 5 +0 . 2 8 +0 . 1 8 +0 . 2 0 +0 . 08

+20

l. 1 9

+0 . 38

0 . 4 1

+22 +24

0 . 5 1 0 . 2 5

0 . 24 +0 . 06

0 . 44 0 . 6 6

+0 . 06 +0 . 09 +0 . 1 9

4 2 .

............

E ' 0) c

100 .

80

60

'-' 40 I _j

20

Fig. 3·5 PR E -OV U LATORY LH S EC R ETIO N

-12 -8

r ,, ]'1 pa sture groupo-o

I ucerne group �

n = 1 7

(D) OVULAT ION RATE S

( i ) Expe r iment I

Mean ovu l a t ion rates for both t h e p a s ture and the luc erne groups

are shown in Tab le 3-6 . Mean ovu l a t ion rates for the subgroup s wi thin

these t r e a tmen t groups (Border Lei c e s t er x Romney ewe s and Romney ewe s )

are al so g iven . Oe s t rogen ic lucerne d epr e s sed mean ovul a t ion r a t e s f o r

al l group s . The d epress ion of ovu l a t ion r a t e s wa s gr eater f o r ewe s �n

the Bo rder Le i c e s ter x Romney group ( 0 . 7 3 ovu l a t ions per ewe ) than for

ewe s in the Romney group ( 0 . 5 9 ovu l a t ions per ewe) .

TABLE 3- 6 : MEAN OVULAT ION RATE S FOR EWE S GRAZ ING

PASTURE OR OE STROGEN IC LUCERNE -EXPE RIMENT I

Pas ture Lucerne D i f f erence �n Ovul a t ion Ra te

43

(Mean± SE ) (Mean± SE ) n Ovu l /Ewe Percent S ign i f (x 2 )

Bo rder L X

Romney 2 . 50+0 . 1 2 1 . 7 7 +0 . 2 2 0 . 7 3 29 . 2 3 . 66 6 (8) (9)

Romney 2 . 09+0 . 1 6 1 . 50+0 . 1 5 0 . 5 9 2 8 . 3 3 . 2 1 6 (§) (8)

To tal 2 . 2 9 +0 . 1 2 1 . 6 5+0 . 1 3 0 . 6 7 29 . 0 1 1 . 066***

( 1 7 ) ( 1 7 )

F i gure �n perenthe s i s represen t s numb er o f ewe s �n group .

( * * * p <0 . 00 1 )

( i i ) Exp e r imen t I I

Mean ovul a t ion rates for each o f the four trea tmen t grou p s are

shown in Tab l e 3- 7 . Oes trogenic l uc e r ne depre ssed ovul a t ion r a t e s when

compared to the cont r o l group (Pas ture group ) , but only when i t was

inge s ted �n the l a t ter ha l f of the o e s trous cycle .

TABLE 3-7 : MEAN OVULATION RATES FOR EWES GRAZING

PASTURE OR OESTROGENI C LUCERNE-EXPERIMENT I I

TREATMENT OVULATION RATE

(MEAN.±_S E )

Pas ture ( P ) 1 . 7 5 + 0 . 1 3

Luc erne ( L ) l . 3 5 + 0 . 12

Pas ture-Lucerne (PL) 1 . 1 0 + 0 . 1 0

Luc erne-Pas ture (LP ) l . 5 5 + 0 . 12

n .

1 6

1 7

1 0

1 8

Compar isons o f the d i f f er ences in ovulat ion ra t e s b e tween

t r ea tmen t s are shown in Table 3-8 .

TABLE 3-8 : COMPARI S ONS OF THE DIFFERENCES IN OVULATION RATES

BETWEEN TREATMENTS - EXPERIMENT II

P . V L .

P . V PL .

P . V LP .

L . V PL .

LP . v L .

LP . v PL .

( n . s .

Diff er enc es Ovul at ions/ Ewe

0 . 4 0

0 . 6 5

0 . 20

0 . 2 5

0 . 2 0

0 . 4 0

no t s ignif ican t

Percen t x2 Diff erences S ta t i s t ic

2 2 . 9 3 . 34 5 2

37 . 1 8 . 54 6 7

1 1 . 4 0 . 6243

1 8 . 5 2 . 1 09 6

1 2 . 9 1 . 9983

2 9 . 0 5 . 5934

S ignif icance

tt

* *

n . s .

n . s .

n . s .

*

* p < 0 . 05 ** p < O . O l # O . l > p > 0 . 05 )

There we re no s ignif icant d i f f erenc es in ovula t ion ra t e s be tween

the two groups which gra z ed pa s t ure in the lat t er ha l f of the cyc l e

(Pasture and Luc erne-Pas ture) , o r be tween the two gro up s which grazed

44 .

luc erne in the la t t er hal f of the c yc l e ( Lucerne and Pas tur e -Luc erne ) .

However , t her e were d i f f erences in ovula t ion rate b etween the two t r ea t -

men t -in terchange group s .

tf) w � 0:::

z 0 � _J �

6 � <.0 M .rn LL.

1 1 c

l .. I I

I

<D �

I

1 1 c

0 .C\J

1 I

-

CO -1 � L

1 1 I . c

('. � L 1 1 I c

-1 .

1"- I .,...

l l L c l .<I

$2 L -1 1 1 J . c

-1 .

I o. �

_.!

lSVd I :Jn""l

:J nl / lS Vd

_ 3 Nt1 3:Jnl

3 � n1svd

3 N cf3:Jnl _

3 CJ nlS'v'd

I

(\J

1)_ X w

( i ii ) Corpora Lu tea Diameter

The d iameter o f each co rpus lut eum wa s calcula t ed as the mean o f

two measuremen t s taken a t r ight angles a t the widest po in t . The

mean corpus lut eum d iame ter in ewes f rom each trea tmen t group , a r e

shown i n Tabl e 3-9 . There wer e no s ign if icant dif f erences b e tween

trea tmen t groups .

TABLE 3-9 : MEAN CORPORA LUTEA DIAMETER (mm) THREE DAYS

POST-OESTRUS

PASTURE LUCERNE

5 . 9 7 + 0 . 3 7 5 . 59 + 0 . 2 5

( E) FOLLI CLE POPULATIONS

( i ) S ur face Fol l ic l e s

PASTURE-LUCERNE LUCERNE-PAS TURE

6 . 05 + 0 . 3 8 5 . 8 5 + 0 . 2 5

4 5 .

In Exp er imen t I , the number o f unrup tured fo l l ic l es with a d iame t er

grea t er than 3 . 5mm tha t wer e present on t he surface o f the ova r i e s , was

recorded a t laparotomy . Th is wa s comb ined with the number o f co rpora

lutea present to g ive an e s t ima t e o f the to tal number o f large f o l l i c l e s

present a t t h e t ime o f ovula t ion . From this ' es t ima t e ' o f total fol l ic l e

number s , t h e rat io o f the number o f large f o l l icles pr esen t per ovula t ion

is calcul a t ed . The se da ta are shown in Tabl e 3-1 0 .

I n Exper imen t I I , all unrup tured sur fac e fol l i c l e s with a d iamet er

o f grea t er than 2mm were r ecorded on recovery o f the ovaries a t s laugh t er .

Da ta f rom this exper imen t i s shown in Tab l e 3 - 1 1 .

The r emoval of the ova r i es in Exper iment I I enab led sma l l f o l l i c les

to be measu red w i th more accuracy than could be ach ieved in s i tu in

Exper iment I .

TABLE 3 - 1 0 : SURFACE FOLLICLES OF GREATER THAN 3 . 5mrn DIAMETER , CORPORA LUTEA AND FOLLICLES PER OVULATION FOR EWES

GRAZING PASTURE OR OESTROGEN IC LUCERNE-EXPERIMENT I

PASTURE LUCERNE

n . Foll ic l e s C . L . E s t . To tal Fol l / Ovn n · Fo l l ic l es C . L . Es t . To tal Fol l / Ovn

Border Leic, 1 . 8 0 2 . 5 0 4 . 3 0 1 . 7 0 2 . 5 5 1 . 7 7 4 . 33 2 . 8 9 X Romney Ewes 8 +0 . 4 9 +0 . 1 7 +0 . 58 +0 . 1 7 9 +0 . 7 7 +0 . 2 2 +0 . 6 9 +0 . 7 3

- - -

Romney Ewes 9 2 . 09 2 . 09 4 . 1 8 l . 95 l . 6 6 l . 5 0 3 . 1 7 2 . 2 5 +0 . 65 +0 . 1 6 +0 . 7 2 +0 . 3 2 8 +0 . 5 3 +0 . 1 5 +0 . 5 3 +0 . 44 -

To tal 1 7 1 . 9 5 2 . 2 9 4 . 2 4 1 . 8 3 2 . 0 5 l . 62 3 . 6 7 2 . 5 2 +0 . 68 +0 . 1 2 +0 . 4 6 +0 . 1 8 1 7 +0 . 4 4 +0 . 1 3 +0 . 4 4 +0 . 4 0 - - - - -

------

( * p < 0 . 05 )

E s t . To tal ( E s t ima t ed To tal Fo l l ic l e Numbers ) - includes follicles p lus corpora l u t ea and is an e s t ima t e

o f t h e numbers o f f o l l i c l e s present a t t h e instant o f ovul a t ion .

n . = Numbers o f anima l s in each group .

S ignif icanc e o f D if f .

Foll / Ovn

<x z ) 0 . 5 3 9 6

0 . 8 2 6 1

5 . 1 33 2 *

.t:-0'\

TABLE 3- 1 1 : S URFACE FOLLICLES OF GREATER THAN 2 . 0mm DIAMETER , CORPORA LUTEA AND FOLLICLES PER OVULATION FOR EWES GRAZING

PASTURE OF OESTROGENIC LUCERNE - EXPERIMENT I I

PASTURE

Surf Fo llicle Co rpora Lu t ea Est ima ted To tal Fo l l icles /Ovn

5 . 08 + 0 . 46 1 . 7 5 + 0 . 1 3 6 . 8 3 + 0 . 6 1 3 . 9 0 + 0 . 32

(n = 1 6 )

PASTURE-LUCERNE

LUCERNE

Surf Fo l l ic l e Co rpora Lut ea Estima t ed To tal Fo l l ic les/ Ovn

4 . 5 1 + 0 . 3 9 1 . 3 5 + 0 . 1 2 5 . 8 6 + 0 . 53 4 . 34 + 0 . 4 2

( n 1 7 )

LUCERNE-PASTURE

Surf Fol l i c l e Corpora Lu tea E s t imat ed To tal Fol l i c l e s / Ovn I Sur f Fo l l icle Corpora Lu t ea Es t ima t ed Total Foll ic l e s / Ovn

4 . 0 7 + 0 . 4 3 1 . 1 + 0 . 1 5 . 1 7 + 0 . 58 4 . 7 0 + 0 . 52 I 3 . 8 1 + 0 . 3 1 1 . 5 5 + 0 . 1 2 5 . 3 6 + 0 . 55 3 . 4 6 + 0 . 3 9

( n = 10) (n 1 8 )

n = Number o f anima l s i n each group .

.1:'­-..J

4 8 .

( i i ) To tal Foll icle Popula t ions

The total numbers of follicles present , wi th a d iame ter grea ter

than 2rnrn , wer e r ecorded a f t er d i s sec t ion o f the ovar ies . Ther e were

no s ignif icant d i f f e renc e s , nor any apparent trend s , evident in t o t a l

fol l i c l e popula t ions be tween t r ea tmen t s ( Table 3-1 2 ) .

E s t ima t es o f the t o tal numbers o f follicles presen t a t the in s tan t

of ovulat ion wer e mad e , using the same proc edure as wa s u s ed in surface

foll i c l e es t ima t e s . S imilar ly , the total numb er o f f o l l i c l e s presen t

per ovula t ion was cal cula t ed . These da ta are shown in Tab le 3- 1 3

for f o l l icles with a d iame t er o f grea t er t han 3 . 5rnm , and in Table 3 - 1 4

for f o l l icles w i t h a d iamet er o f grea t er t han 2 . 0mrn .

To tal fo l l i c l e popula t ions a r e shown in F IG . 3-7 .

There we re no s ign i f icant d i f ferenc es be tween treatmen t group s , in

the mean numb er of foll icles o f a d iame t er grea ter than 2 . 0mm produced

per gram of ovar ian t i ssue ( Tab le 3- 1 5 ) .

TABLE 3 - 1 5 : FOLLICLES OF GREATER THAN 2 . 0rnm DIAMETER PER GRAM

OF OVARIAN TISSUE

PAS TURE LUCERNE PASTURE-LUCERNE

1 . 9 3 + 0 . 3 1 1 . 8 4 + 0 . 2 6 2 . 2 3 + 0 . 3 4

(F) OVARIAN AND REPRODUCTIVE TRACT RESPON S E

( i ) Ovar ian and U t e r ine Weigh t s

LUCERNE-PASTURE

2 . 0 2 + 0 . 29

Mean ovar ian and u t erine weight s for each trea tmen t gr oup are

shown in Table 3 - 1 6 . Ther e wer e no s ign if icant d i f ferenc e s in organ

weigh t s be tween group s .

TABLE 3 - 1 2 :

Fol l ic l e s 2-3 . 5mrn

3 . 2 5+0 . 48

Fol l i c l e s 2-3 . 5mm

3 . 2 +0 . 4 7

OVARIAN FOLLICLE POPULATIONS OF EWES GRAZ ING PASTURE OR OES TROGEN IC LUCERNE - EXPERIMENT I I

(MEAN + SE)

Fol l i c l e s 3 . 6- 5 . 0

1 . 8 8+0 . 4 1

PASTURE

Fol l ic l es > 5mrn

0 . 1 2+0 . 09

(n = 16 )

PASTURE-LUCERNE

Fol l ic l es 3 . 6- 5 . 0

2 . 00+0 . 3 3

Fo l l ic l e s > 5mrn

0 . 2 0+0 . 1 3

(n = 1 0 )

To tal No. Fo l l ic l e s P er Fo ll ic l e s Gram Ovary

5 . 2 5+0 . 7 6 1 . 9 3+0 . 3 1

To tal No . Fo l l ic l e s Per Fo l l icles Gram Ovary

5 . 4 0+0 . 8 6 2 . 2 3+0 . 34

n = Number of anima l s in each group .

Fo l l ic l es 2-3 . 5mrn

2 . 3 5+0 . 30

Fo l l i c l e s 2 -3 . 5mrn

2 . 3 3+0 . 3 9

Fo l l ic l es 3 . 6-5 . 0

2 . 2 9+0 . 34

Fol l icles 3 . 6-5 . 0

2 . 1 1+0 . 3 3

LUCERNE

Fo l l ic l e s > 5mm

0 . 1 2+0 . 09

�n 17)

LUCERNE-PASTURE

Fo l l ic l e s > 5mrn

0 . 6 7+0 . 2 1

(n = 1 8 )

To tal No . Fol l ic l e s P er Fo l l icles Gram Ovary

4 . 7 6+0 . 7 7 1 . 84+0 . 2 6

To tal No . Fo l l ic l e s Per Fo l l icles Gram Ovary

5 . 1 1+0 . 8 1 2 . 02+0 . 2 9

� \0

TABLE 3- 1 3 : TOTAL NUMBER OF FOLLICLES OF GREATER THAN 3 . 5mm DIAMETER , CORPORA LUTEA AND FOLLICLES PER

OVULATION - EXPERIMENT I I

PASTURE

Foll ic l e s Corpora Lut ea E s t ima ted To tal Fol l ic l e s / Ovn

2 . 00+0 . 48 1 . 7 5+0 . 1 3 3 . 7 5+0 . 4 9 2 . 1 4 +0 . 2 3

( n = 1 6 )

PASTURE-LUCERNE

Fo l l ic les Co rpo ra Lu t ea E s t ima t ed To tal Fol l i c l e s / Ovn

2 . 2 0+0 . 4 6 1 . 1 0+0 . 09 3 . 3 0+0 . 40 3 . 00+0 . 2 6

( n 1 0 )

LUCERNE

Fo ll icles Corpora Lu t ea E s t ima t ed To t a l Fol l ic l e s / Ovn

2 . 4 1+0 . 4 6 1 . 3 5+0 . 1 2 3 . 7 6+0 . 3 7 2 . 7 8+0 . 30

(n = 1 7 )

LUCERNE-PASTURE

Fo l l ic le s Corpo ra Lut ea Est ima ted To tal Fol l icles /Ovn

2 . 78+0 . 3 1 1 . 5 5+0 . 1 2 4 . 3 3+0 . 3 8 2 . 7 9+0 . 3 1

(n 1 8 )

V1 0

TABLE 3- 1 4 : TOTAL NUMBER OF FOLLICLES OF GREATER THAN 2 . 0mm DIAMETER, CORPORA LUTEA AND

FOLLICLES PER OVULATION - EXPERIMENT I I

PASTURE

Fo l l ic l e s Co r po ra Lut ea Es t ima t ed To tal Fo l l ic l e s /Ovn

5 . 2 5+0 . 6 1 1 . 7 5+0 . 1 3 7 . 0o+0 . 8 0 4 . 00+0 . 4 5

( n 1 6 )

PASTURE-LUCERNE

Fo l l ic l e s Corpora Lutea E s t ima t ed To tal Fol l i c l e s /Ovn

5 . 4 0+0 . 6 3 1 . 1 0+0 . 1 0 6 . 50+0 . 4 5 5 . 9 1+0 . 4 8

(n 10)

n = Number of anima l s in each group .

LUCERNE

Fo l l ic l es Corpora Lutea E s t ima t ed To tal Fo l l ic l es /Ovn

4 . 7 6+0 . 6 1 1 . 3 5+0 . 1 2 6 . 1 1+0 . 5 5 4 . 5 �0 . 4 5

(n = 1 7 )

LUCERNE-PASTURE

Fo l l ic l e s Corpora Lu tea E s t ima ted To tal Fol l ic l es / Ovn

5 . 1 1+0 . 59 1 . 55+0 . 1 2 6 . 66±0 . 6 2 3 . 9 8+0 . 3 9

(n = 1 8 )

V1 .......

n = 16

5.0

4 0

3.0

2 .0 w 0::: �

1 .0-1 1-lf) �

Fig. 3·7 PO ST - OVU LAT I O N FOLLICLE POPULAT IONS

n = 17

w z 0::: w u ::) _j

n = 1 0 n = 1 8

u 1-lf) � & _J ........... 1- ......... lf) u � :J

_j I

FOLLIC LES > 2 mm diameter

� Corpora Lutea

n = 16 n = 17

....... w 0:: ::::J f- · lf) �

w

� w 0 � _J

- - - -

n = 1 8

(_) 3 ......... lf) 0

. � ::) _J

FO L L I C LES > 3 · 5 m m d iameter

TABLE 3 - 1 6 :

Ova rian Weight

5 2 .

OVARIAN AND UTERINE WEIGHTS (g) IN EWES GRAZING

PASTURE OR OESTROGEN IC LUCERNE

PASTURE LUCERNE PASTURE-LUG LUCERNE-PAST

(Mean ± SE) 2 . 7 2 + 0 . 1 6 2 . 58 + 0 . 14 2 . 4 2 + 0 . 1 9 2 . 52 + 0 . 1 2

U t erine We ight (Mean ± SE) 5 9 . 06 + 2 . 64 6 3 . 7 2 + 1 . 9 0 60 . 3 8 + 4 . 5 1 6 7 . 74 + 2 . 7 5

( i i ) Reproduc t ive Tra c t H i s to logy

Ther e were no s ignif ican t d i f f erenc es be tween groups in t he height

of epithel ial c el l s , in any of t he trac t t i s sue examined . Mean

ep i the l i a l c e l l heigh t s a r e s hown in Tabl e 3 - 1 7 .

TABLE 3 - 1 7 :

TISSUE

Vagina

Cervix

Uterus

EP ITHELIAL CELL HEIGHT ( � ) OF REPRODUCTIVE TRACT TIS SUE

F ROM EWES GRAZING PASTURE OR OES TROGEN IC LUCERNE

PASTURE

92 . 7 6 + 8 . 54

2 6 . 09 + l . 0 9

2 7 . 3 5 + 0 . 98

LUCERNE PASTURE-LUG LUCERNE-PAST

7 9 . 1 3 + 5 . 6 9 9 9 . 88 + 4 . 1 9 94 . 9 2 + 7 . 7 9

24 . 86 + 1 . 1 1 2 5 . 1 + 1 . 2 5 2 6 . 2 1 + 0 . 83

2 7 . 84 + 0 . 7 8 2 5 . 62 + 1 . 00 2 5 . 6 5 + 0 . 9 9

Fa llop ian Tube 24 . 4 9 ± 0 . 8 9 2 4 . 58 + 0 . 5 7 24 . 4 3 + 0 . 9 6 2 3 . 0 1 + 0 . 99

(G) SUMMARY

5 3 .

1 . The coumes tan l evels in the lucerne were modera t ely high and t h e

daily intake o f t h e ewes would have ea s ily exc e eded tha t required

to induce known oe stro genic respons es .

2 . Oes trogenic luc erne did no t e f f ec t proges tagen-induc ed synchroni­

za t ion of oes trus , or the mean leng th o f oestrous cyc l es .

3 . The length o f the in t erval f rom the on set of o e s t rus to the pre­

ovula tory surge of LH va ried ma rkedly be tween anima l s . The

coumes tans ha d no d e t ec table influenc e .

4 . The mea suremen t of the chlor ide ion con ten t o f c ervical mucus could

not be util i z ed to de t ermine the t ime o f ovul a t ion . Consequen t l y

t h e length o f t h e int e rval f rom the LH peak t o ovulat ion could

not be determin ed .

5 . The inges t ion o f coumes tans d id no t s ign i f icantly a l t er the sec r e t ion

o f lut einizing hormone . Secretion pa t t erns throughout the c yc l e

and over the pr e-ovulatory LH surge were unchanged .

6 . Oestrogen ic luc erne depressed ovulat ion ra tes by up to 2 9 per c en t ,

but only when lucerne wa s consumed for the la tter hal f o f the

o e s t rous cyc l e , wa s the depres sion o f ovulation rate s ta t i s t ic a l l y

s i gnif icant .

7 . The higher the ' na tura l ' ovu la t ion rate o f ewes , the grea t er � s

t h e coumestan-induced d epr e s s ion o f ovul at ion ra t e .

8 . The number s of fol l ic le s present in the ovar i e s were unchanged ,

a l though the number o f fo l l ic l es tha t ovu l a t ed wa s lowered by the

inge s t ion o f coume s tans .

9 . The mean diame t er o f c orpora lutea wa s not a f f e c t ed by the

inges t ion o f coumes tans .

54 .

10. The mean uterine we igh t s and ova r ian weight s o f ewes gra z ing

o e s t rogenic luc e rn e , d id no t d i f f e r s i gnif icantly f rom t ho s e o f

ewes gra z ing non-oe s t rogenic ryegr a s s / clover pa stur e , when

compared three days po s t -oes t rus .

1 1 . The mean h e igh t o f epi thel ial c e l l s in genital t ra c t t i ssue , d id no t

d i f f er be tween ewes gra z ing o e s tro genic lucerne and ewes graz ing

non-o e s t rogenic ryegra s s / c lover pa stur e , when compared three days

post-oes trus .

C H A P T E R I V

DI SCUSS ION

C H A P T E R I V

D I S CUSS ION

(A) OESTROGENIC LUCERNE

S evere infec t ion o f lucerne leaves by the fungi Pseudope z i za

0 med icagin is and Lepto spha erul ina brio s iana (Loper and Hans n , 1 9 64 ) ,

bo th o f which are common fungal pa thogens o f luc erne in New Z ea land

5 5 .

(Close , 1 9 6 7 ) , produc ed ma t er ia l of h igh coumes tan con t en t . A s imilar

degree of oes t rogenic po t ency wa s apparent under similar cond i t ions

with this lucerne stand in a p r evious t r ial (Mc Cutcheon , 1 9 7 6 ) .

The e s t ima tes o f daily in t ake of coumes tans sugges t t ha t p la sma

coumes tan conc en tra t ions woul d be we ll in exc e s s o f the l evels nec essary

to induce o e s t rogenic respon s e s ( Shu t t e t al . , 1 9 6 9 ) . A recen t trial

( Smith et al . , unpubl i shed da ta ) ha s s hown tha t as l i t t l e a s 2 5ppm

coume s t an in the feeds tuff i s suf f ic ient to suppre s s ovula t ion ra t e ,

and t he degree o f suppression i s inc r eased by a n in crease in coumes tan

conten t .

( B ) OESTRUS PHENOMENA

( i ) Oes t rus Synchron iza t ion

P roges t agen trea tment r e sul ted in a h igh propo r t ion o f ewes

s howing oes t ru s over a four day period , following sponge withdrawa l .

Some ewes wer e incapable o f r es ponding to proges tagen t r ea tmen t , owing

to hav ing bl ind trac t s or pregnant u t er i , or to lo s in g s ponges dur ing

synchron i z a t ion . I f t he s e an ima l s are d i sr egarded , t hen 9 3 p er c en t o f

ewes in Experimen t I and 9 1 percent o f ewes i n Exper imen t I I were

detec t ed in hea t over the synchron ized oes t rus . A h igh degree o f

s ynchrony was mai ntained a t the f o l l owing oes trus , wi th 8 2 percent

( Experimen t I) and 85 percent (Exper iment II) o f the ewes �n oes trus

over a comparab le four day per iod . The h igher propor t ion of ewe s

undetec ted in the se cond o e s t rou s cyc le � n Exper iment I , may re f l e c t

a n inadequacy in the per iod o f oes trus d e te c t ion , rather than any

b io logical phenomenon . The skewed d i s tr ibu t ion of the ons e t of

oestrus (FIG . 3- 1 ) sugge s ts tha t some ewe s may have been detec ted i f

rams were introdu ced ear l i er .

Oe s trogenic lucerne d id not a f f e c t the degree of oes trus

synchroni zat ion a t tai ned , or th e inc idence of ' s i l ent heats ' , a l th ough

behav ioura l oes trus ha s been r e l a ted to p l a sma oes trogen leve l s (Piper

56 .

and Foote , 1 9 6 8 ; God ing e t al . , 1 9 7 0 ; Scaramu z z i et al . , l 9 7 lb ) .

Conversely treatment o f cyc l ing ewe s w i th anti sera to endogenou s oes trogens

prevent s the o c currence of behav ioural o e s trus ( Scaramu z z i , 1 9 7 5 ;

Fairc lough e t al . , 1 9 7 6 ) , bu t the adm ini s t r a t ion o f anti-sera to

phy to-oe s trogens does no t prevent oes trus (Cox and Wi l son, 1 9 7 6 ) .

( i i ) The Oes trus-Ovu l a t ion Interva l

The wide var iat ion in the leng th o f the in terval from the ons e t

of oes tru s to the pre-ovu la tory LH peak , be tween indiv idua l ewes , i s

�n agr eement wi th ob servat ions by Lammin g , ( pers . corn . 1 9 7 8 ) . Lamming

sugges ted tha t th is is due to b io l ogical var ia tion ra ther than to

inadequate oes tru s de tec tion . Such wi de var iat ion b e tween ind ividu a l s

wou ld ma sk any d i f f erences induced b y lucerne .

The admi ni s trat ion of exogenous s tero id oe s trogens increases oes trus

dur a t ion ( S caramu z z i et al . , l 9 7 l a ; Land et al . , 197 2 ) . There is a

l i near r e l a t ionship b e tween oes trogen d ose and the durati on o f the i nduced

oes trus , however , ewe s exhib i t a r efrac tor i ne s s to repeated admin i s tra tion

o f oest rogen ( Scaramu z z i e t al . , 1 9 7 2 ) . Land et al . , ( 1 9 7 2 ) sugge s ted that

highly f ecund b reeds were more sen s i t iv e to exo genou s o e s t ro gens in

5 7

r e l a t ion t o thi s o e s t ru s r e s ponse . There i s no ev idence t o sugge s t that

coume s tan s a l ter the durat ion of o e s t ru s .

The f a i l ure of the cerv ical muc u s chloride te s t �n e s t ima t ing the

t ime of ovu l a t ion may hav e b een the r e su l t of poor s amp l ing technique s .

The e rra t i c changes in chl or ide concen trations may have been due to

var iat ion in the quan t i ty of mucus col le ct ed a t each san1p l ing . Turnbu l l

e t a l . , ( 1 9 6 7 ) and Li nd say & Franc i s ( 1 9 6 9 ) sugges t ed that chl o r ide

concentra t ions o f cerv ical mucu s may be u t il ized a s an a s say for phy to -oes t roge

intake in ewes . In this tr ial there were no d if ferenc e s de tec ted �n mucu s

chlor ide content , be tween ewe s in the lucerne and pas ture groups .

In conc lusion, these exper imen t s showed tha t there wa s con s iderab l e

var i a t ion � n the length o f the int erval from the onse t of oes trus t o the

pre-ovula tory LH peak . We were unab l e to determine the l ength of the

in terval f rom the pre-ovu l a tory LH peak to ovul a t ion . However Curnrning

et a l . , ( 1 9 7 3 ) found a high degree of cons tancy in the l ength of the

int erval f rom LH peak to ovul a t ion , in ewe s of wid e l y d i f fer ing age s and

under a number of trea tmen t s . Th i s would sugge s t tha t the re i s cons iderab le

var iat ion be tween anima l s i n the in terval f rom the onse t of o e s trus to

the pre-ovu latory LH peak .

(C ) LUTE IN IZ ING HORMONE SECRET ION

The d epres sion in l eve l s o f receptor-bound endogenou s oes trogens o f

ewe s graz ing lucerne (Newsome and Ki t t s , 1 9 7 7 ) sugge s t s tha t t o t a l endogenous

o e s tro gen conc en tra t ions may al so b e depre s s ed . A r e l a t ionsh ip be tween

s teroid o e s trogens and l u t e in i z ing hormone secret ion ha s c l e a r l y b een

e s tab l i s hed .

Ovar iec tomy , and the sub s equent f a l l in p l asma o e s trogen l evel s , r e su l t s

� n an e l eva t ion o f basal L H l ev e l s ( Reeve s , e t al . , 1 9 7 2 ; D iekrnan and

Ma l vern , 1 9 7 3 ) . The adminis trat ion of exogenous o e s trogens to ovar iec tomized

ewe s induces a biphas ic re sponse ; an init ial depr e s s ion in LH concentra t ions ,

f o l lowed by a large e l eva t ion s im i l ar to pre-ovul a tory l eve l s ( Rad f o rd e t a l . ,

1969 ; God ing et a l . , 1 9 7 0 ; Howl and and Palmer , 1 9 7 3 ) . Feed ing o e s tro genic

58 . c l over , of high i s of l avone content , to ov a r i ec tomi zed ewe s m 1 m 1 c s the

admin istra t ion of exogenous s t eroid oestrog ens on LH secre t ion

(Hearnshaw e t a l . , 1 9 7 7 ) .

In the presen t t r ia l s there were no inc rea ses in basal LH l evel s

in ewes graz ing oestrogenic l uc erne . Presumably , the depre s s ion o f

endogenous o e s t rogen level s , i f i t did occur , was no t o f suf f ic i en t

magni tude t o a f f ec t L H secret ion , o r al ternative ly , t h e coumestans

compensatro for l ower ed endogenous oes tro gen l evel s .

I f the coumes tans d o lower endo genous o e s t rogen l eve l s , i t is

f ea s ible that they may a f f ec t the pre-ovu l a t o ry LH surge as this is

pr imar ily a response to high l ev e l s o f c ircula t ing oes tro gen s o f the

p r evious day (Moore e t a l . , 1 9 69 ; Cox e t a l . , 1 9 7 1 ) . Oes t rad iol

inj ec t ions on days 3 -4 (Bol t e t al . , 1 9 7 1 ) o r on days 1 0- 1 2 ( How land

e t a l . , 1 9 7 1 ) of the oestrous cyc l e , wil l induce ' extra ' pre-ovul a tory

type LH surges in cyc l i c ewes . In the presen t trial such ex tra pre-

ovu l a tory LH p eaks were no t induced by the inge s t ion of coumes t ans ,

no r wa s the ' t rue ' pr e-ovula tory LH peak af fected .

The LH pro f il e s from ewe s graz ing o e s t rogenic l uc erne in this

t r i a l , failed to demon s t ra t e changes in e i t her the nega t ive-feedback

e f f e c ts of oes trogens on ba sal LH l evel s , o r their po s i t ive-f eedback

e f f e c t s on pre-ovulatory LH surges . I t is po ssible that either o r

both o f these o e s t rogenic e f f e c t s d id occu r , b u t were no t appa r en t .

To obta in accura t e quan t i t a t ive secre t ion p a t t erns, the in t erval b e tween

sampl ing mu s t be less than the hal f - l i f e o f the hormone (LH � l i f e is

approximately 2 0 minut es ) . S ampl ing a t such f requent in t erva l s would

demand the use of indwel l ing catheters and would a l so res t r ic t the

numb ers o f animal s used .

With the numbers o f anima l s involved in this tr ial , any maj o r

changes in L H secre tion pat t erns should have been detec ted with 2 -hourly

s amp l ing . Therefore i t i s conc luded tha t i f oes trogen i c lucerne d id

af f e c t secr e t ion pa t terns o f LH , the changes were no t e x tens ive .

(D) DEPRE S S ION OF OVULAT ION RATE S

Ewe l ivewe igh t s were recorded pr ior to , du r ing and a t the

comp l e t ion of the exper imen t s . There were no s i gni f i cant d i f f erence s

in ewe l ivewe igh t s be tween group s a t any of these t ime s , whi ch wou ld

ind i ca t e the abs ence of a ' body condi t i on ' e f f e c t on ovu l a t ion r a te

compar i sons . S imilarly the random a s s i gnment of ewe s to each trea tmen t

group shou l d have minimi sed any gene t i c d i f ferences in ovu l a t ion r a t e s

be twe en group s . Therefore i t may be a s sumed tha t any maj or var ia t ions

59 .

in ovu l a t ion r a t e s were due to the trea tment - the i nge s t ion o f oes t r ogeni c

lucerne .

In bo th experiment s the i nge s t ion o f oe s t rogen ic lucerne depre s s ed

ovu l a t ion rate s . Thi s i s 1 n agr eemen t wi th the ob s erva t i ons o f o ther

worker s ( Sc a l e s and Mo s s , 1 9 7 6 ; McCu tcheo n , 1 9 7 6 ; S c a l e s , et al . , 1 9 7 7 ) .

The mar g inal lev e l s of s i gni f i cance b e twe en groups ref l e c t s the

d i f f i cu l ty in ana l y s i ng d i screte d a ta ( number of ovu l a t i o n s ) wi th

re l a t ively sma l l numbers of anima l s . Th i s i s clearly ev ident f r om

Experiment I , when the depr e s s i on of ovu la t i on rate wi thin the br eed

subgroups (Romney and Border Le i ce s ter x Romney) i s cons idered . Al though

the degree to wh i ch ovu l a t ion r a t e s were de pre s sed in the s e group s ( 29 . 2

percent and 28 . 3 percent respe c t ively) i s comparab l e to the depre s s i o n

of ovu l a t i on r a te when a l l ewe s i n Experiment I ar e cons idered ( 29 percen t )

i t i s no t s ta t i s t i c a l l y s ignif i can t . However , when a l l ewe s are

cons idered , th i s s ame degree o f ovu l a t ion rate depr e s s i on 1s h i ghly

s igni f i can t ( p < O . OO l ) .

60 .

Tho se ewe s of higher f e cund i ty invo lved in the se trial s , were

a f f ec ted mo s t by the ingest ion o f coumes tans . In Exp e r iment I , mean

ovu l a t ion rate o f Bo rder Leice s ter x Romney ewe s is depressed by 0 . 7 3

ovu l a t i ons pe r ewe compared to 0 . 5 ovu l a t ions per ewe for Romney ewe s .

In a sim i lar manner , the ovu l a t ion r a te o f the contro l anima l s (Pas ture

gr oup ) wa s depre ssed une qua l ly b e tween Exper iment I and Expe r iment I I .

The con trol ewe s of Exper iment I had a higher inherent ovu l a t ion rate

than those o f Exper imen t II ( 2 . 29 ovu lat ions per ewe compared to

1 . 7 5 ovu l a t ions per ewe ) , and they were depressed to a greater degree by

oes trogenic luce rne ( 2 9 percent compared to 2 2 percen t ) . Th i s grea ter

sens i t ivi ty of hi ghly f e cund ewes to the coumes tans was also repor t ed

1n ear lier t r i a l s ( Scales and Mo s s , 1 9 7 6 ; S cales e t a l . , 1 9 7 7 ) .

This breed var iation 1n sens i t ivi ty to coume stans wou ld appear to

co rrespond to ob s erved breed d i f f erences 1n sens i t iv i t y to exogenous

s teroid oes trogen s . Th e admini s t rat ion of exog enous s te roi d oes trogens

to cycl ing ewe s increases the dur a t i on o f oes trus (Land , 1 9 7 0 ) and br eed s

of higher fecund i ty d i sp l ay a greater re sponse than d o the less pr o l i f i c

breeds (Land e t a l . , 1 9 7 2 ) . Ewes of high f ecund i ty are however , l e s s

sens i t ive t o exogenous oes tro gen- indu ced L H secret ions ( Land e t al . , 1 9 7 6 ) .

An apparent contradic tion to the higher sens i t iv i ty to coumes tans 1 n the

depr essio n of ovu lat ion r a te , is the repo r t of Land e t aL ( 1 9 7 6 ) of a l ower

sens i t iv i ty to the exogenous oes tradiol- induced depr e s s i on of ovu l a t ion

rate .

From the data co l l e c ted in Exper iment I I , i t is c l early evident

tha t it i s the inges tion o f c oumes tans in the l a t ter ha l f o f the

oes trous cyc le that is invo lved in ovu l a t ion r a te depre s s io n . The

gra z i ng of oes trogenic lucerne early inthe oes trous cyc l e had no e f f e c t

o n ovu l a t i on r a tes provided that the ewe s gra zed non-oes trogenic p as ture

in the l a t ter hal f o f the cyc le . The se f ind ings are con s i s tent wi th

the c la im tha t the number o f f o l l i c l e s whic h are dest ined to ovula t e ,

is d e t e rmined within the three days p r ior to oestrus (Land , 1 9 7 3 ;

Findlay and Cumming , 1 9 7 7 ; Bherer e t al . , 1 97 7 ) .

6 1 .

Hemica s t r a t ion o f ewes p rior to day 1 4 o f the oest rnus cycle , l eads

to a trans ient inc r ease in FSH levels coinc id ing with fal l ing o e s tradiol

levels , and increas ed foll icular growth in the r ema ining ovary over

the f ina l three days o f the cycle ( Ramire z and Sawyer , 1 9 74 ; Findl ay

end Cumming , 1 9 7 7 ) . In exper imen t s where the feeding o f lupin gra in

to ewes inc reased ovula t ion ra t e s , inc r ea ses in FSH l evels were a l so

reported ( Br i en e t a l . , 1 9 7 6) . This st rong . al though ind irec t ,

corr ela t ion b e tween high FSH l evels and incr eased ovulat ion rat e s ,

promp t s t he s ugges t ion tha t t he converse si tua t ion occurs in ewe s

graz ing lucerne . FSH l evels in ewe s inges t ing coumes tans may b e

depressed due to t h e inten s i f ica t ion of t h e negat ive-f eedback inf luence

of oest rogens on gonado trophin relea s e .

(E) FOLLICLE POPULATION S

I t h a s previously been shown t ha t breeds w i t h a high inher ent

ovula t ion ra t e , ovula t e a higher proport ion o f the Graaf ian follic les

tha t develop (Brad ford e t a l . , 1 9 7 1 ; Turnbull e t a l . , 1 9 7 7b ) . Th is

chara c t e ris t ic i s eviden t in the fol l ic l e populat ion data of Experimen t

I . When a l l corpora lutea and unru p tured follicles with a diamet er

o f gre a t er than 3 . 5mm a r e included in an e s t ima t e o f the l a rge f o l l ic l e

popul a t ion , t r end s between t h e number o f fo l l ic l es and ovul a t ion rate

are apparen t . Ewe s o f the Border Lec i s t er x Romney subgroup had

f ewer f o l l i c l e s per ovula t ion t han d id ewes f rom the Romney subgroup .

The l ow numbers o f ewes involved p robab ly p recludes the s e d i f f e r ences

f rom b eing s ta t i s t ically signnican t .

6 2 .

O e s t rogenic l ucerne incr ea s ed the numb er o f fol l i c l e s pre sent per

fo l l ic l e ovu lat ing in the ewes of h igh fecund i t y , a t r end which para l l eled

the correlation b e tween h igh fecund i t y and ovul a t ion ra t e d epress ion .

In Experimen t I I , a l l surface fol l icles o f a d iameter greater than

2mm were incor pora t ed in es t ima tes o f fol l ic l e popul a t ions , and s imilar

trends were shown . The inges t ion of o e s t ro genic lucerne in t he l a t ter

hal f o f the oest rous cyc l e increased the number of fol l ic l e s present

per ovul ation . Expo sure to coumes tan s in the ear ly s tages o f the cyc l e

d id no t a f f ec t f o l l ic l e number s . To tal fol l icle popula t ions , recorded

after s l ic ing t he ovaries , showed t rend s s imilar to tho s e s hown by

su rface f o l l i c l e popul a t ions .

I t would app ear tha t i f the coume s tans do suppress the ma tura t ion

of f o l l icles , then this suppression does no t restric t inc r eases in

fol l ic l e size . Al though fol l ic l e s devel op to a comparab l e s iz e in

ovar ies o f sheep graz ing lucerne , there i s some phy s io logic a l res tric t ion

whic h al lows only the mo st advanc e d fol l i c l es to develop the po t en t ia l

t o ovula te . Al t hough there is no direct ev idence , a d e f i c iency o f FSH

at this s tage o f foll icular developmen t would appear to b e a pos s ib l e

cau s e o f ovula t ion ra te depr e s s ion . Th e hemicas trat ion exp er imen ts

of Ramirez and Sawyer ( 1 9 74 ) and F ind lay and Cumming ( 1 9 7 7 ) give s trong

ind i rec t evidence of the requi rement for FSH . Fur thermore , r epor t s o f

the proper t ies o f exogenous oestro gens i n a l t er ing p i tui tary respons ive­

nes s , could ac coun t for suppre s s io n of FSH l evels by the coume s tans .

The a dm in i s t r a t ion o f o e s tradiol t o ewes a f ew hours ( 4 -6) p r ior to

Gn-RH , depres ses the gonado t rophin response (Pellet ier and S igno re t , 1 9 7 0 ;

Liber tum e t a l . , 1 9 74 ) . I t wou l d al so appear tha t FSH i s much more

sens i t ive to this inhib i tory e f f ec t o f o e s t ro gen than i s LH ( S a l amons en

et a l . , 1 9 7 3 ; Baird an d Scaramuz z i , 1 9 7 6 ; F indlay and Cumming , 1 9 7 7 ) .

6 3 .

I t ha s been established , tha t in sheep tho se follicl es des t ined

to ovula t e would have a d iame ter of grea t er than O . Smm by day 6-7

of the o e s t rous cycle ( Turnbul l e t al . , 1 9 7 7a) . As the ingest ion o f

o e s tr ogenic lucerne i n the f i r s t hal f o f the cycle ( up to day 9 )

does no t inf luence ovu l a t ion ra t e s , t he ' in i t ia t ion ' o f follicular

growth is apparen tly unimpor tant in ovula tion ra te d epression . I t has

been sugge s t ed that large follicles re l ease a subs tanc e that suppresses

follicle ini t ia t ion (Peters et al . , 1 9 7 3 ) . As a consequence o f

lowered ovula t ion ra te , ewes graz ing lucerne will have a grea ter number

o f large f o l l icles presen t early in the next oes trous cyc l e . However ,

even if there is a depression in the number s of follicles ' in i t ia t ed '

i t is unl ikely that this woul d be a factor limi t ing the det ermina t ion

o f ovul a t ion ra tes .

There were no signi f i cant d i f f e r enc e s be tween groups in mean

ovar ian weights nor in the number of follicles present per gram o f

ovar ian t i ssue . Al t hough t hese are l imi t ed observa t ions , this would

sugge s t that the coumes tans do not hav e any maj o r e f fec t s on ovar ian

me tabolism tha t re sul t in weight inc r eases .

( F ) PROGESTERONE SECRETION

Proges t erone may inf luence foll icular growth ind ep end en t l y of i t s

e f f ec t s v i a the hypo t halomo-pitui tary axis . Fo llic ular growth is mor e

rapid i n ova ries that conta in a corpus lut eum ( Dufour e t al . , 1 9 7 1 b ) .

Inj ec t ion o f proge s t e rone into one ovary o f anoestrous ewes , toge ther

with sys t emic adminis t r a t ion o f PMS G , resul t s in mor e rapid f o l l icular

growth and in a grea t e r n umb er o f f o l l icles and corpora lutea in t he

inj ec t ed t han in the con t rol ovary ( Harned and Casiday 1 9 7 1 ) . Thi s

t r ea tmen t does not a l t er oes t rogen secre t ion ( Rexroad and Casida , 1 9 7 7 ) .

Smi th and Robinson ( 1 9 6 9 ) found the d iame t er o f a co rpus lut eum

( an e s t ima t e o f i t s ma s s ) to be h ighly correlated to proges t erone

conc en t r a t ion in the ovar ian vein blood pla sma ( an e s t ima te o f luteal

f unc t ion ) . The inges t ion o f coumes tan s did not inf luence l u t eal

f unc t ion as e s t ima ted by corpora lutea size .

(G) COUMESTAN S AND THE REPRODUCTIVE TRACT

64 .

The ing e s t ion o f coumes tans by overiec tomi zed ewes resul t s in the

expre s s ion o f the ' c lass ical ' o e s t rogenic character i s t i c s in the

r eproduc t ive trac t . The administra t ion o f 7 . 5 to 2 5mg of coumes trol

per day , induces maxima l s t imu l a t ion of u t e r ine growth (Braden e t a l . ,

1 9 6 7 ; Shut t e t al . , 1 9 6 9 ; Kel l y et al . , 1 9 76b) . The coumes tans

also induce corn i f ication o f r ep roduc tive trac t t issue in ovariec tomized

ewes , s imilar to tha t occurring at oes trus in in tac t ewes ( Braden et al . ,

1 9 6 7 ) . S imilarly , the ing e s t ion of o e s t rogenic luc erne causes the

secret ion of c ervical mucus , comparable to l evels found at oes trus ,

in ova r ie c tomized (Kelly , 1 9 7 2 ; Kelly e t al . , 1 9 7 6b ) or in in tact ano e s trous

ewe s (Kel ly et al . , 1 9 7 6a) . Synthe t ic coumes tans have s imilar e f f e c t s

( Shu t t e t al . , 1 9 6 9 ) .

In Exper imen t II reproduc t ive trac t s were examined thr ee days

po s t -oes trus , when t ho se o e s t ro g enic changes which occur at oe strus would

s t il l have b een evident . Ther e were no s ignif icant d if f er ences b e tween

ewes gra z ing pa s ture and ewe s grazing oes tro genic l uc e rne , in the

measurement of those oestro gen ic traits examined - i . e . u t er ine weigh t s

and e p i t hel ial c e l l he igh t s o f r eproduc t ive trac t t i s su e . Apparen t l y

t h e levels of en dogenous o e s t ro gens p resent a t o es t ru s masked t h e e f f ec t s

o f the inges t ed coumestan s .

I t ha s b een d emon s t ra t ed t ha t t he coumes tans compete with

o e s tradiol f o r oes trogen-receptors ( Shemesh � al . , 1 97 2 ) . I t wou l d

appear tha t , hav ing bound to oes tro gen r e c ep tor s , the coumes tan­

rec eptor complex is capab le o f fully exp r e s s ing tho se oestro genic

changes induc ed by the oes trad iol -r eceptor complex , a t l ea s t within

65 .

the reproduct ive trac t . Newsome and K i t t s ( 1 9 7 7 ) recen t ly found tha t

there were r educed l evel s o f o es trad iol-receptor bind ing in ewes

graz ing oes trogen ic lucerne , al though o e s trogenic parame ters of the

r eproduc t ive trac t are unc hanged .

(H) CON CLUS IONS

The l evels of coumes tan intake by ewes grazing luc erne in these

exper imen t s were suf f ic i en t to induce a l l o f i t s o e s trogen ic e f f ec t s .

These oestro genic changes - inc rea s ed u t erine weigh t , inc reased c ervical

mucus produc t ion and corn i f ic a t ion o f r eproduc t ive trac t t i s sue - would

be r ead ily expr e s sed in ovar iec tomized ewes subj ec t ed to s imilar l evels

of coume s tan s . However , in in tac t cyc l i ng ewes these parame t er s d id

no t d i f f er from tho s e shown by ewe s graz ing non-oe s t rogen ic ryegra s s /

clover pa s tur e . The s ingl e chara c t er is tic o f the r eproduc t ive sy s t em

wh ic h was cons i s t en t ly a f f ec t ed by oestrogen ic luc ern e , was a r educ t ion

in the numbers of eggs ovula ted . Al tho ugh not pa r t of th is s tudy ,

observa t ions show tha t the eggs wh ich a r e produced , are capable of

normal f er t il i t y .

I t ha s b een shown tha t ovula t ion ra te i s propo r t iona l to t he

dura t ion o f o e s t rus (Land , 1 9 7 0 ; Hannahan and Qui rk e , 1 9 7 5 ) , whic h

in turn can b e influenced by exog enous o es t rogens ( S caramu z z i e t al . ,

1 9 7 1 a ; Land e t a l . , 1 97 2 ) . The admin i s tra t ion o f exogenous s t ero id

oes trogen s inc r ea s e s the l ength of the o estrus pe riod . However ,

a s ingle admin i s tra t ion o f exogenous o e s trogen is mo s t e f f ec t ive, a s

ewes show r efrac toriness to r ep ea t ed do s es ( S caramu z z i e t al . , 1 9 7 2 ) .

6 6 .

Mor l ey e t al . , ( 1 963) found tha t daily adminis tration of s t ilboes trol

to cyc l ing ewes decrea sed ovulat ion ra tes , a respon s e which pa ra l l e l s

tha t o f the coumes tans . I t may be tha t a s ingl e , appropria t ely- t imed

dose of coumest an s will inc r ea se the dura t ion of oest rus and inc r ea s e

ovu l a t ion ra t e s 1 a s d o e s a si ngle dose o f st ero id o e s t ro gens .

Although Newsome and Ki t t s ( 1 9 7 7 ) fo und tha t t he inge s t ion o f

coume s tans r educ ed the l evel s o f oes trad io l , oestrone and oes trio l

which bound to recep tors , they did no t measure to tal f ree endogenous

o es trogen l evels . Scaramu z z i ( 1 9 7 5 ) and Fa irc lough e t al . , ( 1 9 7 6 )

found tha t the dep r e s s ion o f endog enous o e s t rogen levels , b y the

administrat ion of an t i-sera , p r evented t he occurrence o f behav ioural

o es t r u s . The coume s tans d o not sign if icantly inc r ea s e t he inc id enc e

of ' s il en t ' hea t s . I t is a l s o unl ikely tha t the coumes tans are

involved in the expression of behavioural oestrus and ther efore ma sk

lowered endogenous oes trogen l evel s , as immunizat ion aga in s t phyto­

oes tro gens does no t prevent o es trus ( Cox and Wilson , 1 9 7 6 ) .

The demonst ra tion in Exp er imen t I I , tha t the ing es t ion o f o e s t ro genic

luc erne la t e in the oes trous c yc l e depresses ovulat ion rates , wherea s

ing e s t ion early i n the cyc le ha s no effec t , is ana logous t o t h e e f f ec t s

o f hemica s tration before or a f t er day 1 4 of the cyc le . Un ilateral

ova r i ec tomy before day 1 4 results in a c ompensa tory increase in f o l l icular

growth in t he rema in ing ovary , and cons equen t ly , ovulat ion rates are

unc hanged f rom t ho se o f intac t ewes ( Ramir e z and Sawyer , 1 9 74 ; Findlay

and Gumming , 1 9 7 7 ) . Ther e i s an inc r ea se in FSH l evel s fol lowing

hemicas tra t ion and t he sub s equent fall in endogenous o e s t ro gen l evel s

(Findlay and Gumming , 1 9 7 7 ) . A s imilar relat ionship b e tween c hanges

in FSH level s and foll icular developmen t could be involved in the

6 7 .

coumestan-induced depres s ion o f ovula t ion ra t es . The ing e s t ion o f

o e s trogen ic lucerne may d epre s s F S H leve l s and consequen tly , inhibit the

la t t er s tages of foll icle ma turat ion , so d ecr eas ing the numb er o f

Graa f ian f o l l ic l e s l ikely to ovula t e . Smi th e t al . , ( unpub l i shed data)

recen t ly found t ha t t he administra t ion of Pregnan t Mares S er um

Gonado trophin (PMSG) to ewe s gra zing o e s t rogenic l uc erne , woul d overcome

the coumes t an-induced depression in ovula t ion rates .

A depr e s s ion o f FSH secret ion by the coumes tans could a r i s e f rom

ei ther the direc t ac t ion of the coumes tans on t he p i t u itary , in a manner

s imilar to the n ega t ive-fe edback ac t ion of the endo g enous o e s t ro gens , o r

from a coumes tan-induced inc r ease in endo genous oest rogen l eve l s .

Al though N ewsome and Ki t t s ( 1 9 7 7 ) showed tha t the coumes tans d epressed

the level s o f receptor-bound endo genous oestrogens , they did no t mea sure

leve ls o f ' free ' endo genous o e s t ro gens . The coumes tans may e f f ec t ively

inc r ease pla sma oestrogen level s a c t ing a t the hypo thalamo-p i tu i tary

leve l , by reduc ing the amount o f endo genous oestro gens bo und to

reproduc t ive tract oestro gen recep tors over the per i-oestrus period .

I t ha s been d emonstra ted t ha t the coumes tans compete with endogenous

oes trogens for oes trogen recep tors ( S heme sh et al . , 1 9 7 2 ) .

Al t erna t ive ly , t he coumes tans may l ower pla sma endo genous oes trogen

levels and a f f ec t the p i t u i tary r esponse to Gn -RH . A s t rong correlat ion

be tween t he oes trad iol : progest erone r a t io and the pitui tary r esponse

to Gn-RH ha s been r epor ted ( Chamley e t al . , 1 9 7 4a ; 1 9 74b ; P e l l e t ier ,

1 9 7 6 ) . Coumes tans may a l t er gonad otrophin secret ion pa t t erns by

lowering the oes trad iol : progesterone ra t io and c onsequently , the

response to Gn-RH .

F indlay and Cumming ( 1 9 7 7 ) sugges t ed that FSH secret ion i s much

more s en s i t ive to the nega t ive-f eedback a c t ion o f oes trogen t han is

LH . Such variat ion in s en s i t ivity ha s been d emonstra t ed with the

admin i s t rat ion o f exogenous o e s t rogens ( S alamonsen et a l . , 1 9 7 3 ;

Ba ird and Sca ramuz zi , 1 9 7 6 ) . I f there is such a wid e var ia t ion in

6 8 .

re sponse to coumes tans , a f a l l in FSH l eve l s o f suf f ic i en t magni tude to

in f luence ovu l a t ion ra tes may have occ urred , while the changes in LH

secretion were not large enough to be d e t ec ted by the proc edures used

in this tr ial .

The inge s t ion of coumes tans depresses ovula t ion rates but sel dom

preven t s ovul a t ion f rom occurr ing . How could a depression in FSH

l evel s accoun t for this ? It ha s been shown tha t the pr esence of FSH

l eads to an inc rease in the numb er of FSH r ec ep tors ; tha t FSH and

oest rad io l in concert inc rease LH receptor l eve l s , and tha t LH inc r ea s e s

L H receptor l evels (Richards a n d Midgley , 1 9 7 6 ) . I t is feasible t ha t

t he depress ion of FSH (and the subs equent inc r ease i n LH : FSH ratio )

af ter expo sure t o coumes tans , will r esul t in a depr e s s ion i n FSH

rec ep tor l evels and an inc r ea s e in LH receptor l evel s wi thin d eveloping

fol l ic l es . Presumably , a s FSH l eve l s are l imi ting , the ava ilab l e

FSH wi l l be taken u p preferen t ially b y the fol l ic l e ( o r f o l l ic l e s )

a t t h e highe s t s tage of developmen t , wh ich wil l possess the l a rge s t

numb er of F S H receptor s . Thi s wil l in turn l ead t o an incr ea s e in FSH

receptor l evels and con s equen t l y , to greater FSH up take . This

' ca scading ' e f f ec t wi l l r e su l t in the largest fol l ic l e (or f o l l ic l e s )

developin g to the stage o f po t ential ovul a t ion , a t t b e exp ense o f o ther

Graaf ian f o l l ic l e s .

The inc r ea s ed suscep t ib i l ity o f high fecundity b r eeds to the

coume s tan s may be related to FSH sec r e t io n . Al t hough there is no

69 .

ev idenc e to show tha t ewes o f high fecund i t y have h igh pla sma FSH

l evel s , i t has been shown t ha t s uch ewes are less s ens i t ive to the

nega tive-f eedback ac t ions of exogenous oes trogens ( Land et al . , 1 9 7 2 ;

1 9 7 6 ; Land , 1 9 7 6) . I f these ewes are al so less s ens i t ive to

endogenous oes trogen s , they would presumably have h igher FSH level s ,

a lower LH : FSH ra t io , and a s a cons equence o f these pla sma gonado t ro ph in

l evels , would develop mor e follicles with t he potential to ovula t e .

The mor e follicles with this po t en t ial to ovula te tha t are produced in

normal ewe s , the grea t er i s the pos sible inf luence of the coume s tans .

Dir e c t ev idence in sup por t o f this po s tula t ed a c t ion o f the coumes t an s

awa i t s f u r t her exper imen ta t ion .

POS S IBLE FUTURE TRIALS

Fur t her wo rk needs to be don e to determine the extent to which

FSH is impl ica t ed in the depression o f ovula t ion r a t e s , in ewes grazing

oes trogenic lucerne .

I f a depression in FSH secret ion does occur in ewes graz ing

oes trogenic luc ern e , and FSH is r espons ible for fo l l icular hyp e r t rophy

a f t er hemicas tra tion , t hen if hemica s t rated ewes a r e subj ec t ed to

coumes tan s , fol l icular hype r trophy will be impa ired . Trials involving

hemicas t r a t ion and o e s t ro genic luc erne t rea tmen t s at d i f fer en t t imes

in t he o e s trous cyc l e could d e t ermine thi s . The admini s t ra t ion o f

PMSG to h emicastrated , or intac t ewes , a t d i f f eren t t imes d ur ing the

cyc l e may add fur t her s up po r t to the claim that FSH i s involved in

d e t ermin ing ovula t ion r a t e s .

Fur t her as says o f pla sma gonado trophin l evels in ewes grazing

oestrogenic lucerne , are necessary . These s hould involve inten s ive

blood sampl ing regimes which would enable accura t e quant i ta t ive

7 0 .

secr e t ion r a t e s t o b e calculated . Inves tiga t ions into the mechanisms

which inf luence gonadotrophin s ec r e t io n , i f plasma gonado t rophin levels

are al tered by the coumes tans , could inc lude the e s t ima t ion of plasma

endogenous o e s t rogen l evels and the r e sponses to exo genous Gn- RH

of ewes gra z ing o e s t rogenic luc erne .

PRACTICAL APPLICATIONS OF THIS INVESTIGAT ION

In t ho se area s o f New Zealand wh ich exper ience dry summers , lucerne

often outproduces pas ture in l a t e summer and autumn . Luc erne may be

the only availabl e forage prior to ma t ing , when it is impor tant to

ma inta in breeding ewes a t high livewe igh t s .

The resul t s ob tained in these t r ials sugges t tha t o e s t ro gen ic

lucerne may b e util ized to achieve op t imum pre-ma t ing ewe l iv ewe ight s ,

wi thou t any de trimen tal e f f e c t s on reproduc t ive performanc e i f ewes are

removed from the luc erne eigh t days p r ior to ma t ing . In fac t removal

from lucerne as late as three days pr ior to the ons e t of o e s t rus may be

all that is required to res tore no rmal f ecund i ty . In prac t ical terms ,

i t could b e recommended to remove ewe s from oes trogenic luc erne

immedia t e ly pr ior to ma t ing , as only a small percen t age of ewes would

exhib i t oes trus w i thin this three day perio d . Al terna tively , i t could

be sugge s t ed to r emove ewes a week p r ior t o ma ting .

Fur t her work involving much larger numbers o f ewes , needs t o be

conduc t ed to verify t ha t removal f rom lucerne at mid-cyc l e r estores

normal ovulat ion rates , and henc e lambing rat e s . S imila r l y , the

e f f ec t s of longer t erm gra zing of oes trogenic lucerne prior t o r emoval

( such as f o r 1 -2 mont hs pre-ma t ing) , mus t also be inves t iga t e d before

these f indings are s t rongly advo c a t e d .

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