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STUDIES ON THE I N VI VO CLEAVAGE AND THE IN VI TRO CULTURE OF NEW ZEALAND
ROMNEY S HEEP OVA
A thesis pr esen t ed in par tial fulfi lment o f the requi r emen t s for the degr e e o f Mas t er of Agr i c ul tural S c i en c e
a t
Mas s ey University
HAR R Y ROBI N TERVI T
A C K N 0 W L E D G E M E N T S
Th e candi da te is indeb t e d to his supervisor Dr M . F. McDonald for the g ui danc e and as s i s tance gi ven d uring the c ours e of this s t udy .
Special thanks are due to Pro fessor A . L . Rae and Dr F.R . Cockrem for advi c e in statis ti cal analys is of data and to Dr R . E . Munford for advi ce in the analys is of data from the bi oassay o f gonado troph ins .
Grat e ful acknowledgement is made to Mr c. Muir for t e chnical
assis tanc e ; to Mr P . H. Whi t ehea d and Mr W . R . Fai rha l l for the care o f animals ; t o Miss M . G . Campbell and th e library s ta f f for pro mptly
pro c uring the reference arti cles us ed in this wo rk ; to Miss D.J . S c o t t and M r G . H . Burns for t h e pho tographic work ; to Mr E . Ormsby and
Mr B . Tha t c h e r for laboratory assis tance ; t o the s ta f f o f the lo cal Freezing Works and Abat toi r ; t o Mrs J . Fisher for taking s pecial care in typing this thes i s ; and t o m y wife who s pent man y hours r eading t his manus c r i pt .
The financial ass i s tan c e o f the Department o f Agr i c ulture and o f a New Z ealand Woo l Board S cholarship is gra t e fully a c know l e dged .
Chapt e r
I
I I
I l l
T A B L E 0 F C 0 N T E N T S
ACKNOWLEDGEMENTS LIST OF TABLES LIST OF FI GURES
INTRODUCTION
REVIEW OF LITERATURE PRODUCTION OF OVA OVA TRANSFER
O VA CULTURE AND S TORAGE OVA CLEAVAGE
OVA ABNORMALITIES
MATERIALS AND METHODS EXPERIMENTAL ANI MALS MANAGEMENT OF ANIMALS EXPERI MENTAL PLAN
HORMONES AND TREATMENTS RECOVERY OF OVA
C ULTURE OF OVA EXAMI NATION OF OVA TRANS FER OF OVA
SLAUGHTER OF EWES AND EXAMI NATION OF R EPRODUCTI VE ORGANS
1
3 3
4 7 9 1 1
13 1 3 13 13
1 7 1 8 1 8 20 21 22
Chapter
IV
V
PRODUCTION OF OVA FOLLOWING HORMONAL STI MULATION OF DONOR EWES
I ND UCTION O F SUPEROVULATION AND MATING O F DONOR EWES
1.
Length o f oes trous cycle following P . M .S . inj ection during the bre eding s eason2 . Ovarian r es pons e R eco very o f o va
4. Ferti lisation o f r ecove r ed ova
PRODUCTION OF OVA FROM HORMONALLY STIMULATED
23
23
23 23 24 26
EWES I N THE EARLY AND LATE ANOESTROUS SEASONS 28
1.
Early anoestrous 282 . Late anoestrous 2 9
CLEAVAGE O F OVA IN VI VO 30
NUMBERS OF CLEAVED , UNCLEAVED , FERTI LISED OR
UNFERTI LISED OVA RECOVERED 30
CLEAVAGE OF FERTI LISED OVA 3 1
1 . Shortes t and long e s t interval a f t er th e ons e t o f o estrus t o t h e recovery o f each
cleavage s tage
3 1
2 . Mean interval a f t er t h e ons et o f o es tr us
t o th e r e covery o f each cleavage s tage 31 3 . Cleavage o f ova r e c overed from th e Fal l o pian
tube unf lushe d a t laparotomy
1,
but flushed a t laparotomy 2 ( Treatment B - Figure 1 ) 32 4 . Cleavage o f ova t rans f e rr ed into one o rboth Fallopian t u b e s a t la parotomy 1 and r e c overed at laparo t o my 2 ( Treatments A
and B - Figure
1�
34Chapt er
VI
VI I
C ULTURE, ABNORMALITIES AND DIMENSI ONS OF OVA C ULTURE OF OVA
1 . Media
2 . Cleavag e o f c ul t ured o va
Reco very o f ova from the c ult ure chambers
ABNORMALITIES OF O VA 1 .
2 .
Abnormali t i es o f ova r e covered a t laparot o my
Abnormali t ies o f ova a f t er cult ure
DIMENSI ONS OF OVA
S TUDIES ON THE SURVI VAL OF TRANS FERRED OVA TRANS FERS I N THE 1 966 BREEDING AND 1 966-67 EARLY ANOESTRO US SEASONS
1 . Control trans fer o f ova
2 . Trans fer o f ova a f t er 24 h o urs cul ture Trans f er of ova a f t er 48 hours culture
TRANSFERS IN THE 1 966- 67 LATE ANOESTROUS AND
36 36 36 36 38
3 9
3 9 40
4 1
43
43 44 45 46
1 967 BREEDING SEASONS 46
1 . Control trans fer o f ova
2 . Trans f e r o f ova a f t er 24 h ours culture Trans fer o f ova a f t er 48 h ours cul ture
FACTORS AFFECTING SURVIVAL OF O VA IN CONTROL
47 48 48
TRANSFERS 49
1 . Number o f corpora l ut ea pres ent in the o vari es o f the recipi en t e w e
2 . Sta t e o f r ecipi ent genita l tract at trans f er
Numl:ier o f ova trans ferred
4 . Cleavage s tage o f trans ferred o va
4 9
4 9 50 50
Chapt er
VI I I
I X
6 .
Synchronisation o f s tage o f development of trans f erred ova and recipien t genital tra c t
Period o va h eld in vitro before trans f er
MEASUREMENT OF EMBRYOS
Measuremen t o f 23-25 day old embryos 2 . Measurement o f 44- 1 01 day old foetus es
DISCUSS ION
PRODUCTION OF OVA FOLLOWING HOR MONAL STIMULATION 50 50
51 5 1 5 1
5 3
OF DONOR EWES 53
THE I N VIVO C LEAVAGE OF FERTILISED OVA 62 CULTUR E , ABNORMALITI ES AND DI MENSIONS OF OVA 65 1 . C ul t ure o f ova
2 . A bnormalities o f ova Dimens ions o f ova
SURVIVAL OF TRANSFERRED OVA
1 . Control trans fers
2 . Trans fer o f culture d ova
SUMMARY
REFERENCES
APPENDIX 1 and 2
65 67 72
73 75 78
90
95
Table
1
2
3 4
5
6
7
8
9
L I S T 0 F T A B L E S
O es trous c y c l e l ength following inj e c tion o f P. M.S . on day 1 2 o r 1 3 o f the cyc l e
Ovarian res pons e o f ewes trea ted with P . M . S . in the bre e ding an d anoes tro us s easons
Ovarian res pons e a f t er trans forma tion
Effect of dos e of P . M . s . on the perc entag e o f ova recovered a n d o n t h e perc en tag e o f recovered ova fert i l i s e d per ewe
Effe c t of time of P. M . s . inje c tion relative to proges tagen wi thdrawal on int erval t o ons et of oes trus and on ovarian respon s e
Ovarian respon s e of ewes trea ted in the la t e ano estro us s eason
Recov ery and fert ilisation of ova in the late ano es trous s eason
Numbers of c l eave d , un c l eaved , fertilised or unfert i lis ed ova r e covered
Shortest and long est int erval a f t er the ons e t o f oes trus to re covery of each cleavag e s tage
1 0 Mean int erval a f t er th e ons et o f oes trus to recovery of each c leavag e s tage
1 1
1 2
1 3
1 4
Eff e c t o f do s e o f P . M . S . on the mean in terval from ons e t of o e s t rus to re c overy o f each c l eavag e s tage Ef fec t o f s eason on th e int erval t o reco very o f 2- c e l l o va
Cleavage rat e of ova in th e experi mental Fal lopian tube
Int erval from th e ons e t of oestrus t o recovery of 6- to 8- c e l l and 8- c ell o va from the ex perimen tal tubes and from t ubes at laparot omy 1
f refers t o following pag e.
f 23
f 23 f 24
f 25
f 28
f 29
f 2 9
f 30
f 31
f 31
f 3 1
f 31
f 32
f 33
Table
1 5
1 6
Effect o f double laparo t omy i n treatment B, and trans fer of ova at laparo tomy 1 be fore rec overy at laparotomy 2, on the percentage of ova recovered per ew e
Contro l c leavage ra t e and c leavage rat e o f ova trans f erred at laparotomy 1 and re covered at laparo to my 2
1 7 Int erval from the onset o f oes trus to recov ery of 6- t o 8 - c el l and 8- c e l l ova a f t er trans fer, and a t laparotomy 1
1 8
1 9
20
2 1
Norma l o va development and develo pment o f a bnormal i t i es re lative t o t h e ini tial c leavag e s tag e o f ova cultured for 24 hours
Comparis on o f th e maxi mum times for c l eavag e o f ova i n vi tro wi th the c l eavage int erval i n vivo Th e types and in c i d e n c e of abnormal ova rec overed at la parotomy
Effec t o f in t erval from ons et of o es trus to
laparo t omy on the inciden c e of a bnormal un ferti lis ed ova
22 Eff e c t of culture length on the inciden c e o f ova a bnormalit i es
23
2 4
Effect of initial c l eavage s tage of ova on t he inc i d e n c e o f abnormalities after c u l t ure
Dimens ions o f o va
25 Mean blastomere volume and total vo lume of ova cyto plasm rela tive to the s tage of o va c leavage 2 6 Suc c ess o f ova trans fer experi ments cond u c t e d in
f 34
f 34
f 35
f 37
f 38
f 39
f 39 f 40
f 40 f 41
f 42
th e breeding and early anoes trous s eas ons f 44 27 Synchronisation of s tag e of d evelopment o f trans ferre d
ova and recipient genital tra c ts f 44
28 S uc c ess o f ova trans fer experiments cond u c t e d in the
la t e anoes trous and bree ding s easons f 47 2 9 S yn c hron isation o f s tage o f development o f t rans ferred
ova and recipi en t g enital tra c ts f 47 30 S yn chronisation of ons e t of oestrus i n donor and
r e ci pi en t ewes and ovarian res pons e o f r e c i pi en t
ewes f 47
Table
31
32
33
34
35
Effect o f number o f recipient corpora lutea on the s u c c ess of trans fer experi ment s
Eff e c t o f s ta t e o f r e c i pi en t g eni tal tra c t on th e s uc c ess of trans f er experiments
Eff e c t of number of ova trans ferred on the s uc c ess of trans fer experiments
Effect o f cl eavag e s tage of the trans ferred ova on the s uc c es s of trans fer experimen ts
Age and crown-rump length o f each embryo
f 49
f 49
f 49
f 49 f 5 1
Figure
1 2
3 4 5
6
7 8
9
1 0
1 1
1 2
1 3
1 4
L I S T 0 F F I G U R E S
Exp erimental plan for 1 966 bre e d ing s eason Experimental plan for 1 966- 67 anoes trous s eason Experi mental plan for 1 967 br e ed ing s eas on Disman t l e d and ass embled dialys i s chamber
Mean perc e ntag e o f ova recovered per ewe relative to the number o f c orpora lut ea in the ovaries o f that ewe
Mean perc entage of ova rec overed per ew e r e lative to the number of corpora l u t ea plus large folli c l es in the ovari es of that ewe
Mean perc entage o f ova recovered per ewe r e lative t o the int e rval from ons e t o f o es t rus t o l aparotomy Mean perc entag e of recovered o va f ertilis e d per ewe relative to th e number of c orpora l u t ea in t h e ovari es o f that ewe
Mean per c en t ag e o f recovered o va fertilis e d per ewe relative to the number of c or pora l ut ea plus large follicles in the ovaries o f that ewe
Mean percentag e o f recovered ova fertilis e d per ewe relative to the interval from ons e t of o es t rus to laparo tomy
Times o f r ec overy of fertilis ed ova at various c leavage s tages in relation to the ons e t o f o e s t rus Inciden c e o f the t ypes of abnormal ova at the
c o mmenc emen t o f and during c ul t ure
Normal 4- c e l l ovum r e c overed after 24 h o urs c ul t ur e . This o vum was fert ilis ed but unc leaved wh en ins ert e d into t h e c u l t ure chamber . (Approx . x 300 )
Fragmenting 2- c e l l ovum r e c overed aft e r 48 h ours c ulture .
This o vum was fertil is e d but unc l eave d when ins e r t e d i n t o t h e c ul t ure chamber . (A pprox . x 400 )
f r e f ers to following page.
1 4 1 5 1 6 f 1 8
f 25
f 25
f 26
f 26
f 27
f 27
f 31
f 40
f 41
f 4 1
Figure
1 5
1 6
1 7
1 8
Fragmenting 4- c e ll o vum re covered after 48 hours cul t ure .
This o vum was a normal 4- c ell when ins ert ed into the cul ture chamber. ( Approx . x 400)
Involut e d , un fert i l i s e d ovum beginning to fragment after 48 hours cul ture . This o vum was involuted when ins erted i n t o the cul t ure chamber.
( Approx . x 400 )
Two 23 day via ble embryos recovered from th e recipient ewe pregnan t a f t er the trans fer of cultured o va. ( Approx. x
12.5)
Mean crown-rump l engths of embryos from 23 to
101
days o f agef 4 1
f 41
f 48
f 52
