A study of the ovarian response of New Zealand Romney ewes sequentially superovulated with pregnant mare's serum gonadotrophin : a thesis presented in partial fulfilment of the requirements for the degree of Master of Agricultural Science in Animal Science at Massey University

Copyright is owned by the Author of the thesis. Permission is given for

a copy to be downloaded by an individual for the purpose of research and

private study only. The thesis may not be reproduced elsewhere without

the permission of the Author.

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

R

^{OMNEY 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 the oestrous cycles fol lowing treatment and 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 ovarian surface fol l icles dif fered litt 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 low estimation o f total ovarian fol l i culnr 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 of P,fi1.S. G, had lower numbers of normal nntral fol 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 icular popu l ations may precip 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 fii

a

sscy 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.

Cha

p

^{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&R I �N RESPONSE TC P.M.S.G. I N 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

activity 47

Difference in 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 Observations 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 of the 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 between 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 occurrence 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

5 1

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 super­

ovulated 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 Experim en tal design : �xperiment one and Experiment two

Des ign of

Experiment

^two

2-5

Cal endar of Events for Experim en t ono 2-6

Design of

��xporimcnt

^thr.:::o

2-7 _��-

In vivo m easurement

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 s equential

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 (Expt . 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 sequential P.i'il.S.G. treatmont on

f 45 f 45 f 45

.'1 46

ovulation rate of ewes o�tlnting : transformed data 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 data 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 Effect of sequential P.�:l.S. G. treatment on the number of corpora 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 Comparison of e-.:10s soquon tially 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

great er tha n

1

mm . f

46

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

5-2 Straigh t line and curvilinear equations 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

Fi

g.!:!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.

No

r

mal 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