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f� STUDY OF THE CVi.RIA.t"1 IUSPONSE OF NEV/ ZCA.Lh.ND ROMNEY E-dES

SEQUENTII�LLY S UPEROVUL!'. TED VII TH PREGNi..NT

fii;�RE 18 SERDrii GONADOTROPHIN

A thesis presented in partial fulfilment of the requirements for

the degree of Master of ;�gricul tural Science

in J:'.nimal Science

at

j·,;assey University

If.,IN Ji.JviES CLP.RKE

1973

ABSTP..ACT

A series of experiments investigated the nature and causes of

ovarian refractoriness in ewes sequentially treated with Pregnant

Mare's Serum Gonadotrophin (P.I'vl.S.G. ) . 70 ewes were subjected to

the following treatments over 3 oestrous cycles of the 1972

breeding season:- 1. Injected with P.M.S. G. at each of 3 cycles

(cycles 112 and 3);

2. 1\vo injections of P.M.S.G. (at cycles 1 and 3)

separated by a normal oestrous cycle;

3. Injected with P.M.S.G. at two successivG

cycles (cycles 2 and 3);

4. Injected at 1 cycle only (cycle 3).

These treatments were replicated at 1000 i.u. and 1500 i.u. P.r�i.S.G.

and 9 ewes acted as an uninjected control group. The ev!es were

blood sampled and slaughtered at the end of these treatments and

ovulation data were obtained by recovery of the reproductive

tracts. The terminal ovulation rates showed that ewes were

refractory to a second injection of P.M.S.G. and this condition

persisted. The refractoriness was to some extent alleviated by the

spacing of injections (Treatment 2 above).

Biological Inhibition Tests (using mice) analysed the plasma

of the above ewes for evidence of anti-gonadotrophins. Although

such factors were not detected in the blood of these ewes, the

test did reveal antibody production against P.M.S.G. in the plasma

of a further group of ewes which hcd been chronically treated with

the hormone for 6 weeks. It was concluded that ovarian refractori­

ness, which is rapidly attained in sequentially trented ewes, is

iii

not duo to the development of serol ogical antibodies against the

exogenous gonadotrophin,

Another experiment 1 carried out ea rly in the 1973 breeding

season, investigated ovaric-,n fol l ic l e development in 30 ewes which

V/Gre soquential l y treated with P.M.S.G, fo r up to 3 oestrous cycl es.

Ewes were l aparotomiscd or kil led on Day 10 of t he oestrous cycles

f o l lowing treatment an d measurements on f o l l icl e development wore

tr:,ken. !. group of contro l ev1es were observed nt a s im i l ar timo to

the treated ewes.

Counts on ovar ian surface fol l ic l e s dif fered l i tt l e between

treated and control ev1cs, at each o f the observat ions. However, the

ovaries of s l aughtered ewes were sectioned to al l ow estimation o f

total ovarian fol l i cu l nr popu l ntion s and t o make some a s s es sment o f

fol l icu lar atres ia. Zwcs sl aughtered after 1 injection o f P,fi1.S. G,

had lower numbers of normal nntral f o l l ic l es per ovary then did

control ewes or ewes observed at simi l ar t imes after 2 or 3

injections.

It wes suggested that exhaustion of ovarian fo l l icu l ar

popu l ations may prec i p itate a refracto ry condition but that this

condition pers i st s because of an endogenous hormonal imbal ance.

�urther work shou l d be done to investigate this l atter possibil ity.

A CKNOViLEDGEME N 'rS

The author is specially indebted to his supervisor Dr. M.F.

McDonald for his invaluable guidance and assistance in experinental

work and advice during the preparation of this manuscript.

Special thanks are due to Professor R.E� Munford for his

advice end aid in statistical analysis and computer operation;

to Professors A.L. Rae and D.S. Flux for their helpful discussion

and advice; to Mr. �. Feirhall for the care of the animals and to

�·Jr. c. Muir for technical assistance.

Ackno\'lledgewent is made to Mr. P.H. \'\hitehond and farm staff;

�.;r. l\"1, Birtles and Dr. H . Batten for assistance in histological

preparations; staff of the Small h.nimal Production Unit; staff of

the fiiasscy University Library; staff of the Longburn Freezing ":Iorks;

r'irs. P. Collins for the typing of this manuscript; and all those who

assisted in mouse dissection.

Financial assistance from the New Zealand Wool Board, The George

hlley Trust and The Robcrt Gibson Trust, is gratefully acknowledged.

PHE F / .. CE

This investigation was conducted at the Sheep Production

Centre, part of the :Uepe.rtment of Sheep Husbandry, Masscy University.

The experimental work was carried out between i.pril of 1972 and May

of 1973 and represents original research by the author under

supervision of :Ur. M. F. McDonald, Reader, Sheep Husbandry Department,

Massey University.

Chap te r

I

T A B L E 0 F CONTENTS

ABST�11' .. CT ii

t.CICNOHL�DGEMENTS iv

PREFi' .. CE V

LIST CF T/.BLES

LIST OF FIGtmES

INTRODUCTION 1

R:;;vw�·: CF LITZRI'.TURE 3

Pregnant Mare's S erw:: Gonadotrophin <1

Factors affecting response to P . M.S . G. 6

1 . Breed 6

2. Livcweight nnu Nutrition 7

3. S eason of Ldministration 8

4 . l.ge of Ewe 9

5. Day of Injection 9

6. Nature of P.�. S . G. 9

Mode of t.ction of P.fvi.S.G. 10

Chemical Char�cteristics of P.M.S .G. 1 3

Immunology o f P.l\l.S. G. 1 3

E¥Je Ovarian Veig.'lt Response of P.M.S.G. 16

Assay of Gonadotrophins and r�nti-gonadot rophins 16

aefractoriness of the Ovary wi th S equential

P.M.S .G. Treatment 18

Follicular Growth and Atresia in the Ewe Ovary 23

Purpose and scope of the Investigation 26

Chapter

I I

I ll

27

Sheep and their Management 27

Mice and their Husbandry 29

Experimental Design 29

Experimental Proc�lure 30

Experiment 1 30

Zxperiment 2 33

Experiment 3 35

!;.nalysis of Dato. 38

SYNCHRCNISf.TION i�ND OESTrtOUS :f:HENOMENA 40

Factors affecting the Onset of Oestrus 40

1. P.M.S.G. �dministration 40

2. Injection of Progesterone 40

Effect of Sequential P.M.S.G. Treatment on synchronisation of oestrous cycles subsequent to Progestagen treatment 4 1

1 . Variation in the Time of Onset o f Oestrus 4 1

2. Manifest�tion of oestrus 42

CH/.PT�R SUNiM!li.W 42

I V OV&RI�N RESPONSE TC P.M.S.G. IN SEQUENTI�LLY TP.J!:1'. TED EYIES 4 3

Transformation of Discrete Data 4 3

Ewes Ovulating v 44

Incidence of Cystic Ovaries 44

Ovulation :late 44

1. Corpora Lutea hll Ewes (Expt. 1) 44

Chupte!:

V

2 . Corpor a Lutea ;�wes Ovuluting (Expt, 1) 45

3. Corpora Lutea. • kll Ewes (Expt. 3) 45

Corpora Lutea plus Follicles 46

1. Corpora Lutor. plus Follicles g reater than. 3 mm, : /:...11 Ewes (Expt. 1 ) 4G

2 . Corpora Lutea plus Follicles greater than 3 mm. : Ewes Ovulatin g (Expt. 1) 46

3. CorporQ Lutea plus Follicles greater thon 1 mm. : 1.11 J:wos (Expt. 3) 46

Ovarian \'/eight 47

1. Ovarian i'leight All Ewes (Expt. 1) 47

2. Ovo.rio.n '.'.'oigl1.t Bwes Ovulating (�x�t. 1) 47

3. DopenGonco o f ova ria n weight on ovaria n acti vity 47

Difference i n Ovulation riute betwe�• the Risht

a nd Left Ovaries

Ovulation �ate of Cont rol �wes (Expt. 3) Ef fect of Seas on

CHAPTE� SUNJMi.;.!1Y

DETECT! ON OF i:.NTI -GONf .OOTR.OPHI NS

48

49

49

50

Immature l'Ylouse Uterine Weight Res p onse t o P.M.S.G. 50

Test f or fo.nti�gonuclot r ophins in Plasma of Gwes of Experiment 1 �

Test f or Anti-gonndotrophins in Plasma of 6wes Ch r onically T re ated >lith P�M,S!G, 51

CJlAPTEf:t SUW".RY 52

Chapter

VI 0\'i�hii..N FOLLICUL'..R DEVELOPMENT OF Er!ES SEQUENTI!l.LLY �-t3i:..TI:l2.J!!.T".ti P.M.S.G.

Surface O bservations

Total Ovc.rian i: .. ctivity

Relationships bctwe0n Total Ovsrian Datn and Surface Dnta

CHLPTEi-1 SUM.M/.;W

53

53

55

56

57

VII DISCUSSION 58

Synchronisation and Oestrous Fh�nomena

Effect of Sequential P.M.S.G. Treatment on

Synchronisation

D�welopmont of Ovnrinn P.ofracto riness in Ewce

58

61

Sequentially Treated �ith P.M.S.G. 64

1. Ovarian rtesponse and the Development of

::-tofractoriness to P.M. S. G. 64

2. Development of hnti-gonadotrophins 73

3. Follicul�r Dynamics o f t h e Ovary 78

4 . General Discussion on Uefractor iness to

P.M.S.G.

:REFERENCES

h.PP:::i:NDICES

84

80

Table

1-1

3-1

3-3

3-4

3-5

3-6

3-7

3-8

L I S T 0 F Ti� BLES

Literature reporting refractory conditions of the ova:;:-y after :;:;reGJlnnt IRare' s Serum Gonadotrophin

treatment

Effect of :i?.r!l.S.G. adr.iinistration on the time

interval betwee n progestogen sponge withdra wal and

the onset of oestrus

Effect of a progesterone injection on the time

interval between progestaGen sponge withdrawal

and the onset of oestrus

Effect of �.M.S.G. administration on the occu rrence

of oestrus foll owing progestagen treatment

Effect of a progesterone injection on the

occurrence of oestrus following progest�gen

sponge treatment

Treatment effects on the synchronisation of oestrus

one oestrous cycle from progestagen treatment:­

Zxperiment 1

Treatment effects on the synchronisation of oestrus

two oestrous cycles from progostagcn treatment:­

Experiment 1

Treatment effects on the manifestation of oostrus:­

Experiment 1

Effect of sequential �.M.S. G. treatment on the

manifestation of oestrus

4-1 Effect of sequential P. M.s.G. treatment on the

number of ewes ovulating

4-2

4-3

4-4

4-5

4-6

Effect of sequ ential P.M.S.G. treatment on the

incidence of cystic ovaries

Ovarian response all ewes

Ovarian response ewes ovulating

Comparison of means : ovariun response of all ewes (Experiment 1)

Comparison of means : ovarian response of ewes

ovulating (Experiment 1)

19

f 40

f 40

f 40

f 40

f 41

f 41

f 42

f 42

f 43

f 44

f 45

f 45

f 45

f 45

Table

4-7

4-8

4-9

Eff ect of sequential P,M.S.G. treatr.1ent compared

with untreated eV!os : Corpora Lutea (transf ormed

data).

Effect of sequential F. M, S. G, treatment compared

with untreated ewes : Corpora Lutea plus follicles

greater than 1 mm, (transformed data)

Relationship between ov2rinn weight and ovarian

activity : regression analysis

4- 10 Ovulation rct� of untreated owes Effect o f season

5- 1

6-1

6-2

6-3

6-4

6-5

6-6

6-7

6-8

Comparison of moans after analysis of covarianc e : mean uterine weights of mice given F,M.S.G. alone

or with pl asma of sheep treated with P.M. S.G.

Ovarian surface activity observation one

Ovarian surface o.ctivity observation two

Ovarian surfc.ce activity observation th reG

Ovarian surface activity observation at slaughter

Relationships between ovulation rate and ovarian

activity at Day 10 of the previous oestrous cycle

control ewes

Relationships between o vulation rate and ovarian

activity et Day 10 of thG previous oestrous

cycle : treated ewes

Estimates of total ovarian follicl e population :

numbers of antral follicles per ovary (transf ormed

data)

Estimation of the proportion of normal antral

follicl es per total antral f oll icles

6-9 Estimation of the total normal antral foll icle

population : number of antral follicles per ovary (transformed data)

f refers to f ol lowing page

f 45

f 45

f 48

f 48

f 51

f 53

f 53

f 53

f 54

f 54

f 54

f 55

f 55

f 55

1-1

2- 1

2-2

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

Dose response relations hips for owes super­ovulated with .?resnnnt I�bre' s Scrum Gonadotrophin

Ewe l ivoweights at the beginning and end of Experiment one

Zwe l iveweigh t changes during Experiment three

2-3 Experimen tal design : �xperiment one and Experiment two

Des ign of Experiment two

2-5 Cal endar of Events for Experimen t ono

2-6 Design of ��xporimcnt thr.:::o

2-7 In vivo measurement o:f ovarian foll icl es with ��-

sterile slide c�llipers

2- 8 2 mm. ovarian s lices semi-emb edded in 70'/o al cohol

3- 1 Dis tribu tion of onset of oes trus fol lowing s ponge withdrawal

3 -2 The ef fect of a progesterone inj ection on tlw ons et of oestrus of owes given c,n intravaginal

f 4

f 27

f 27

f 30

f 30

f 32

f 35

't 36

f 36

f 40

progestogen sponge treo.tmont f 4 0

3-3 Effect of repeated gonadotrophic stimul ation on the synchronis ation of oestrous cycles

4-1 Eff ect of sequential F.M.S.G. treatment on th e number of ewes ovu lating

4 -2

4-3

4 -4

4-5

Cystic condition of the ovary

Eff ect of sequential I'.M.S.G. treatmen t on ewe ovulation rate : rnw data

Effect of sequen tial i"'. M.S .G. treatment on ewe ovulation rate : transformed data

Superovulated ov�ries taken f rom a ewe given one inj ection of P.M.s.G. ( Trt.H41 Expt. 1) 36 ovulations

f 41

f 43

f 44

f 45

f 4 5

f 45

Figure

4-6

4-7

4-8

4-9

Inact ive ovaries of a evo in Treatment Hl (Exp t . 1 )

Ovaries observed a t Day 10 of the oestrous cycle

Ovarian slices showing luteinisation of follicles

Effect of sequential ?.l"l.S, G . treatment on ovula tion rate of ewes ovulo.ting : raw data

4-10 Effect of sequen tial P.i'il.S.G. treatmont on

f 45

f 45

f 45

.'1 46

ovulation rate of ewes o�tln ting : transformed da ta f 46

4-11 Effect of sequential �.M.S. G. trea t ment on the number of corpora lu tea pluG follicles grea t0r than 3 nm.

in all ewes : rou dato f 46

4-12 �ffect of sequontiB.l i'.l':i.S.G. treatment on the number of c orpora lu tea plus follicles grea ter than 3 mm. in all eYJes : trnnsformed da ta f 46

4-13 Effect of sequent ial :?. Tvi. s. G. treatment on the number of c orpora lutea plus follicles great er than 3 mm. in ewes ovulating : raw data f 46

4-14 Effec t of sequential P.�:l.S. G. treatment on the number of c orpora lutea plus follicles grea ter

4- 1 5

4-1 6

than 3 rrun. in owes ovulating : t ransformed dat a f 46

Effoct of sequential P.fvl.S.G. treatmen t on ovnrian -.;;eight : all e•.':es f 46

Effect of sequential P.iVl.S. G. treatment on ovarian weight : owes ovulating f 46

4-17 Comparis on of e-.:10s soquon t ially tre:ated with �'.M. S. G.

and c ontrol ev.'os : ovulation rate f 46

4-10 Comparison of ewes sequontinlly treated with �.M.S.G. and control ewes : corpora lutea plus follicles grea t er tha n 1 mm . f 46

5-1 Immat ure mouse ut erine weight response t o P.M.S.G. f 50

5-2 Straigh t line and curvilinear equa tions for imma ture mouse u t erine �eight response t o P.M.S.G. f 50

5-3 l'Yiean uterine weights of mice treated with P.M. s. G. and sh eep plasma : raw data f 51

Fig.!:!Fe

5-4

6- 1

6-2

6-3

6-4

6-5

G-6

6-7

Menn uterine weights of mice treated with P.M.S. G. and sheep plasma : transformed data

Observations on follicle numbers in ewes treated

up to three times wi th P.M.S.G.

Normal follicle sectioned through the ovum

Cross section of u normal tertiar y follicle

Cross section through th e luteinised wall of n cystic follicle

Sarly atresin of a tertiary follicle

�trosia of a tertiary follicle

Late atresia of a tertiary follicle

f refers to following pnge

f 5 1

f 53

f 56

f 56

f 56

f 56

f 56

f 56