(1)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..
(2) ELECTROMYOGRAPHIC STUDIES ON THE OVINE UTERUS. A THESIS PRESENTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF PHILOSOPHY AT MASSEY UNIVERSITY. Li Yuemin _January. 1991.
(3) TABLE O F CONTENTS. ELECTR O MYOGRAPHIC STUDIES ON THE OVINE UTER U S. A b s tra ct . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii Lis t o f Ta b l e s v List o f Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi Acknowl e dg m en ts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v i ii .. I.. .. .. .. .. .. INTR O D U CTION. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 11 . LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 II.1. The Repro d u c t i ve Cyc l e o f S h e e p. . . . . . . . . . . . . ... . ... . . . . 3. Il . l .A . The b reeding seaso n . . . . . . . . . . Il . l . B . Th e o estro u s cycle i n ewes . . . . . Il . l . C. Pregn ancy a n d part u r i t i o n i n ewes Il . l . D .The post- partu m period i n ewes . . . 11 . 2. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. 3 5 7 9. Myo m e tri a l Acti v i ty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3. I I . 2 . A . Stru c t u re a n d o rgan isation o f t h e myom etrial cont r act i l e u n i t . . . . . . . . . . . . . . . . . . . . . . . . . I I . 2 . B . Speci a l featu res o f t h e u l tr as tr ucture o f t h e fu n c t io na l u n i t s o f the m y o m e t rui m . . . . . . . . . . . (a) (b) (c) (d). The c o n tract i l e apparat u s . P lasm a membrane . . . . . . The sarcoplasmic reticu l u m Th e g a p j u nctions . . . . . . .. . . . . . . . . (SR) . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. 13 14. . . . . . . . .. 14 14 15 15. I I . 2 . C . Myo metrial con traction . . . . . . . . . . . . . . . . . . . . . . . . 1 6 II.2.D. E l e c t r o p h y s i o l o g y of myo m e t r i a l con t racti l i ty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 6. 11 .3.. E ffe c t s of Horm o n e s on Emg Activity. 11 . 3 . A . 11 . 3 . B . 11 . 3 . C . 11 . 3 . D . 11 . 3 . E. 11 . 3 . F. 11 .4.. O e strogen . . . . . . . Progesterone . . . . . Oxytoci n . . . . . . . . Prostagl an d i n s . . . . Relaxin . . . . . . . . Vasoactive i n t es t i n al .. . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . pepti d e. . . . . . . . . . . . . . . . . . . . 19. . . . . . . . . . . . . . . . . . . . . . . . . (VIP) .. . . . . . .. . . . . . .. . . . . . .. . . . . . .. . . . . . .. Uteri n e Moti l i ty S t u d i e s with Pa r ticu l ar Refe r e n c e t o t he Ewe .. .. .. •. .. .. •. •. .. .. .. .. .. .. .. .. •. .. .. .. .. •. .. .. .. .. .. .. .. . . . . . .. . . . . . .. . . . . . .. . . . . . .. . . . . . .. . . . . . .. . . . . . .. . . . . . .. . . . . . .. . . . . . .. .. .. .. .. •. .. .. .. .. .. 19 22 24 28 30 30. 31. 11 . 4 .A. Methods o f studying u te r i n e motility . . . . . . . . . . . . . . 3 1 11 . 4 . B . Uterin e motil ity observations recorded from t h e uterus o f t h e i n t act ewe . . . . . . . . . . . . . . . . . . 33 .. ..
(4) 11 . 4 . C . Uter i n e motility observations recorded fro m t h e u terus o f t h e ovariecto m i s e d ewe . . . . . . . . . . . . . Ill.. 11 ! . 1 . 111 . 2 . 11! . 3 . 111 .4. III.5. III.6.. GEN ERAL MATERIALS AND M ETHODS. .. •. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. •. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. M y o m e t r i a l E m g A c t i v i ty i n t h e Inta c t Ewe d u ri n g A n o e s tr u s . . . . . . . . . . . . . . . . . . .. IV .l .A. IV .LB .. IV .l . C . IV .l . D . I V. LE.. 48. 48 48 48 54 54 60. 62. 62. Th e r esting emg of t h e u t e r u s . Th e e ffect o f exogen o u s o xytoci n on restin g e m g o f the u t e r u s d u ri n g anoestru s . . . . . . . . . . . . . Th e e ffect o f exogeno u s o estrogen o n t h e resting emg of t h e uterus d u ri n g anoestrus . . . . . . . . . . . . . Th e e ffect of oxytocin on o estrogen i n d u c ed emg a c t i v i ty during a noestrus . . . . . . . . . . . . . . . . . . . Th e effect of P G F21X on o estrogen i n d u c ed emg a c t i v i ty during anoestrus . . . . . . . . . . . . . . . . . . Th e chan ges in myometr ial activity �hen st i m u l ated by a secon d i n jection o f oestrogen . . . . . . . . . . .. I V. l . F.. IV. 2 .. .. A n i m al . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Electrodes ....................... . ........... . . . . . . Su rgery a n d an aesthesia . . Recordi ngs . . . . . . . . . . . . . . . . . . . . . . . . . . Th e Electromyograms . . . . . . . . . . . . . . . . . . . Q u a n t itative a n alysis o f the Electromyograms . .. IV. R E S U LTS IV. l .. .. 41. 62 the . . . . . . . . . 62 . . . . . . . . . . . . 62 . . . . . . . . . . . . 62. . . . . .. .. . . . . .. .. 66. . . . . . . . . . . . . 66. M y o m e t r i a l Acti v i ty i n t h e O v a r i e c to m i zed Ewe d u ri n g t h e A n o e s tr u s S e a s o n .. . ... . .. . ... . . . .. . . . . . . . . . .. . . . . 7 2. I V. 2 .A.. Specific features of the design o f t h i s experiment . . . . . . . . . . . . . . . . . . . . . 72 I V. 2 . B . The gen eral patterns o f electrical activity . . . . . . . . . . . 73 (a) Resting emg activity a n d t h e e ffects of exogeno u s oxytoci n a n d PGF21X o n it . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 (b) O estrogen i n duced e m g activity i n the ovariectomized e w e and t h e e ffects o f oxytocin a n d P GF21X o n it . . . . . . . . . . . . . . . . . . . . . . . . . 73 (c) Th e e ffects o f progesterone o n 77 oestrogen i n duced e m g activity (d) Th e reduced responsiven ess of t h e uterus i n regime two o f t h e experiment . . . . . . . . . . . . . . . . . . . 83 .. .. .. .. ..
(5) (e) Th e d irection o f propagation o f e m g a ctio n pot e n t i a ls . . . . . . .. .. .. .. .. .. .. .. .. .. . .. .. I V. 2 . C . S o m e q u a n t i t a t iv e aspect s of the e l e c t r o myograms recorded from the ovariectom ized ewes . . . . . . . . . . . . . . . . . . . (a) A m p l i t u de . . . . . . (b ) B u rst frequency . . . . (c) Spikes per burst . . . . . . . . . . . . . . . . . . . ( d ) M o t i l i ty in dex . . . . . . . . . . . . . . . . . . . . . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ·.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. IV.3.. V. V. 2 .. V.3 . V.4 . V.S .. .. 84. . . . . . . . . . . . . . . . . . . . . .. 85 85 85 85 85. .. .. .. .. .. .. .. .. .. .. .. .. .. Th e E m g A c t i v i ty o f the Ute r u s o f the Cycl i n g Ewe .......... 93. DISCU S S ION. .. .. .. .. .. . .. .. .. . . .. .. .. .. .. .. .. . .. .. .. . . .. Emg Activity d u r i n g Anoestrus and i n t h e Ovar i ec t o m ized Ewe . . . . . . Th e D irection o f Pro pagation of t h e Act i o n Poten t i a l s . . . . . . . . . . . . . . . . . . . . . Th e I n fl u en c e o f O estrogen . . . . . . . . . . . Th e I n fl u e n c e o f Progestero n e . . . . . . . U te r in e r e fractoriness . . . . . . . . . . . . . . .. V .2 .. .. .. .. .. .. .. .. .. .. .. 104. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 1 04. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . . . . . . . . . . .. 1 06 1 10 1 12 116. REFER E N C E S ........................................ 1 1 7. A P P E NDIX I. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 130.
(6) ii. A B S TRACT. There a re many repo r t s in t h e l i t er a t u re, u s i n g a variety of t e c h n i qu es , i n dicating t h at h ormonally i n du ced m ovem en t s o f t h e u terus play a m aj o r r o l e i n t h e reproduction process. Th e pu rpose o f t h is study w a s to explore some of t h ese p h enomena by measuring electro-myographical respons es from t h e u terus o f i n t act a n d ovariectom i zed ewes b o t h b e fore a n d during t h ei r n o r m a l b reeding seaso n .. Six h e a l t h y non-pregn a n t Rom n ey ewes o f abou t 5 0 kg body w e i g h t were selected for the i nvestigations which were carried out b etween September 1 987 and Feb r ua ry 1 988. Electrodes, u sually in gro u p s of t h ree, were surg ically impl a n t e d a t pre s elected s i t es (cervix, u t er i n e b o dy, u t erine h o r n , oviduct) on t h e reproductiv e t ract o f t he ewes, le a d s plugged i n t o a u n iversal AC a m p l i fier, a n d the o u t p u t recorded o n a fo ur c h a n n e l i n k wri t i n g c h a r t r ecorder. Four o f t h e six ewes were ovariect omized at the time o f implan t a t i o n o f t h e electrodes.. I n dwelling silastic cat h eters were inserted into t h e ext er n a l. j ug u l ar veins o f t h ese ewes t o e n a b l e oxytocin to be injected wi t h o u t disturb i n g t h e animal. A l l ewes were h o u sed i n dividu ally i n doors i n p e n s t h a t all owed t h em t o moy e freely during reco r d i n g session s .. Recordi n gs b eg an. abo u t 1 0 days after su rgery h ad been carried o u t . Th e electromyographs (emgs) were described u si ng generally accepted ter m s such as t he presence o f s pik es , t h e i r ampl i t u de, t h e presence o f b u rs ts a n d o f episodes o f activity, w h e t h e r el ectr ical activit y was co-ordi nat ed o r u n co ordinat e d , and t h e direction o f propagat i o n o f t h e act ivity. A m o t il i ty i n dex (MI) was derived as a m e t h o d of meas u r ing electrical activity of t h e u te r u s over prolonged periods o f t i m e . D uring t h e anoestro u s p e r i o d i n b o t h i n tact a n d ovariectomized e w e s a n emg pattern o f myo electrical complexes c h aracterised by alternating p h ases o f co ordina t ed b u rst s o f electrical activity a n d q u i escent periods were ob served. Th i s b asic pa ttern of activi ty, and respon s iv e n e ss to u t erine s t im u lan t s s u c h a s oxytocin and Glandin N (a P GF2oc an al og u e), was greater i n t h e i n t ac t.
(7) iii. ewes a s compared to t h e ovariec t o mized ewes a t t h i s t i m e . A l t h o u gh s u ch a d i ffer e n c e c o u l d be anticipated, a clear answer as to w h e t h er i t i s a general p h e n o m e n o n , or whether seaso n al d i ffere n c es exist, cou ld not be det e r m i n ed b ec a u se o f t h e l i m i t ations i n design o f t h i s s t u dy. A d i s t i n c t pattern o f emg activity was also i d e n t i fied d u r i n g d i ffer e n t p h ases of t h e o estro u s cycle in the ewe d u r i n g the n a t u ral b r e e d i n g season .. Th i s. p a t t er n cou l d b e m imicked by a d m i n i s tr a t i o n of th e exogen o u s steroid h o r m o n es oestrogen and proges t ero ne i n the ovariect o m ized ewes. Fou r days a fte r daily oes tradiol- 1 7b i njec t i o n s (50J..Lg s . c . ) t h e emgs of t h e ovariec t o m ized ewes s h owed a marked i n crease in ampl i t u d e and i n b u rs t frequency a n d a c o n s i d erably inc reased respo ns e to oxytocin a n d Gla n d i n N . W h e n t h i s was follo w e d by progestero ne a d m i n i st r a t i o n (50mg. s.c.), even wit h o estrogen i nj e c t i o n s c o n t i n u ing, i n h i b i t ion of activi ty a n d redu ced respon siveness to oxy t o c i n and Glan din N was e q u a l ly m arked. W i t h drawal o f progesteron e , b u t with co n t i n u i ng oest rogen a d m i n is tr at io n , r e s u l t e d i n a recovery fro m t h e i n h i b i t i o n a n d a respo n se t h a t w a s even greater t h an before t he progestero n e h a d been given . Th i s s u ggests both an i n h i b i t i n g and a poten t i a t i n g action o f progestero n e o n u t eri n e electrical ac t ivity, a fin di n g which adds s ome suppo r t to Csapo's c l assical w i t h d r awal of the 'progeste ron e block' as one o f t h e pre-requ isi tes fo r i n i t i at i o n of normal p ar t u r i t io n in sh eep. _. W h i l e o estrogen h as a clear role t o play as a s t i m u l a t o r o f electrical activity it a l so seem s c apable of exh i b i t i n g a b iph asic resp o n s e with a period of depressed a c t ivity occurri n g befo r e the pos i t ive stimu l u s occurs. W h e t h e r t h i s i s a fu n c t io n o f dose o r s o m e o t h er factor c o u l d n o t b e e s t ab l i s h e d i n t h i s s t u dy . I t s poten t i a ti n g e ffec t on t h e action o f both oxytocin a n d G l a n d i n N i n t h e s e experiments adds fu r t h er sign i ficance t o t h e a t t e n t io n t h a t s h o u l d b e p a i d t o t h e reproductive s t a t u s o f t h e a n i m a l when c l i n i c a l u se i s m a d e o f t h es e s u bstances.. Th e d i rection of propagation of action pot e n tials recorded in t h ese s t u di e s d ep e n d ed o n t h e general l evel o f emg activity.. W h e n t h is was l o w t h e. directi o n o f propagation w a s cervico- t u b a l , when h i g h , t h e proportion o f a c t i o n poten ti a l s i s i n both d i rections fro m t h e t u b a l e n d o f t h e u terus. A.
(8) iv. c l e a r u n derst a n d i n g o f such a m e c h a n i sm , a n d i t s sig n i fi c a n c e, m u s t await fu r t h er s t u dy..
(9) V. LIS T O F TAB LES. Tab l e 1 :. Th e e ffect o f oxytocin both o n spo n t an e o u s a n d . . . . . . . 67 oestradiol i n duced e m g activity (Ewe 329) .. Tab l e 11 :. Th e e ffec t of oxytocin on spo n t aneo u s a n d oestradiol i n d u c ed e m g activity 1 3 days a ft er l a s t oest radiol i nject i o n o f ewe 329 (compare w i t h Tab l e I ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69. Tab l e I l l :. Th e d i fferences i n emg activity b etween a n i m a l s du r i n g stage 2 o f regime o n e . . . . . . . . . . . . . . . . . . . . . . 9 1. Tab l e I V:. Th e e ffect o f oxytocin a n d P GF2cx: o n t h e o e s t radiol i n d u c e d electrical act ivity du r i n g stage 2 of regi m e one . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92.
(10) vi. L I S T O F F IGURE S. Fig. 1 :. Th e pos i t i o n s of electrodes s u t u r e d a t pre- s elec t e d sites o n t h e u teru s a m o n g 6 ewes . . . . . . . . . . . . . 50. Fig. 2 :. Active a n d qu iesce n t periods of emg d u r i n g a noestr u s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 6. Fig. 3 :. An e x a m ple o f t h e m e t h o d o f m easu r i n g th e du rat i o n o f bu rsts, th e i n t erval b etween b u rs t s , t h e b u rs t frequ ency, spike frequ ency a n d am p l i t u d e o f t h e spike . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 7. Fig. 4 (a & b): Fig. 5 :. A n example showi n g t h e propagative direc t i o n o f b ursts a n d episodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 8 A n example o f co-o r d in at e d a n d u n co-o r d i n a t e d patterns o f elect rical a c t iv i ty . . . . . . . . . . . . .. Fig. 6 :. An exam ple of calc u l a t i n g t h e m o t i l i ty i n dex. Fig. 7 :. Oxytocin effect o n durin g a n oestrus. .. 59 61. spo n t a n eo u s emg activity . . . . . . . 63. Fig. 8 :. Oestradiol benzoate b o t h i n h i b i t s a n d s t i m u l at es myo m et r i a l activity in ewe 329 d u r i n g a n o e s t r u s . . . . . . . . 64. Fig. 9:. The e ffect o f oxytoci n on oestradiol i n du ce d emg activi ty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65. Fig. 1 0:. Th e effect o f P GF2oc on oestr ad iol i n d u c e d emg activity. during anoes t r us . . . . . . . . . . . . . . . . . . . . . . . . . 68. Fig. 1 1 :. Characteristics o f t h e emg recorded 13 days a ft er the i n i t i a l injec t i o n o f oes trogen . . . . . . . . . . . . . . . . 70. Fig. 1 2 :. Th e e ffects o f oes trogen and oxytocin on ampl i t u de, burst fre q u e n cy and m o t i l i ty i n dex during two expe r i m en t s on i n t a c t anoestro u s ewe 329. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 1. Fig. 1 3 :. Th e patter n o f emg a c t iv i ty i n ovariec t o m i z e d ewe 247 d u r i n g the a n o es t r o u s season . . . . . . . . . . . . . . . . . . 74. Fig. 1 4 :. Th e pattern of emg activity i n d u c e d by o e s t r a d i o l 1 7b (50JLg g i v e n s . c. daily) on ovariec t o m i z e d ewes during the anoestrou s season . . . . . . . . . . . . . . . . . . . . . 75. Fig. 1 5 :. Th e e ffect s o f oxyto c i n (500 m u given i.v.) on t h e oestrogen i n d uced e m g activity o f ovariecto mi z e d ewes d u r i n g t h e anoestrous season . . . . . . . . . . . . . . . . . . 76.
(11) vii. Fig. 1 6a :. Th e d evelopment o f i n hi b it i o n a fter progestero n e a d m i n istration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 8. Fig. 1 6b :. Th e e ffect of oxytoc i n a n d P GF2<x: w h e n t h e uterus i s i n h ibited by progest e r o n e . . . . . . . . . . . . . . 79. Fig. 1 6c :. T h e e m g pattern observed d u r i n g t h e recovery from progesteron e i n h ib it i o n . . . . . . . . . . . . . . . . . . . . . . 8 1. Fig. 1 6d :. Th e pattern of oestrogen i n d u c e d emg i n ewe 1 0 b efor e progesteron e admi n i stration (A) a n d i n t h e sa m e ewe 1 1 5 h after a large dose o f progesterone ( 1 00mg given i . m . ) h ad b e e n g iven when th e ewe h a d recovered from t h e progestero n e tr eatm ent a d m i n istered during reg ime o n e (E) . . . . . . . . . . . . . . . . 82. Fig. 1 7 :. Th e difference i n amplit u d e a n d b u rst frequency b etween 4 electrode positio n s o n t h e reproduct ive . . . . . . . 86 trace. Fig. 1 8 :. Em g amplitu des d u r i n g treatment i n regime o n e .. di fferent. stages. of . . . . . . . 87. Fig. 1 9 :. Di fferences i n bu rst fre q u e n c y at each tr eatm ent stage o f regime one . . . . . . . 88. Fig. 20:. Nu mber of spikes i n a b u r st during the p eriod o f oestradiol i n duced emg activity i n regi me one . . . . . . . 89. Fig . 2 1 :. Th e n u mber of spikes in a bu rst d u r i n g each treatm ent stage of regime one . . . . . . . . . . . . . . . . . . . . . 9 0. Fig. 2 2:. Th e motility i n dex du r i n g stages 2 a n d 4 o f reg i m e o n e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94. Fig. 2 3 :. Th e c h aracteristics of emg act ivity during st ages 2, 3 and 4 of both reg i m es . . . . . .. 95. Fig. 24:. Ewe 661 during dioest r u s. 96. Fig. 2 5 :. Ewe 661 during pro-oest r u s .. Fig. 2 6 :. Ewe 661 during oest r u s . . . . . . . . . . .. Fig. 2 7 :. Ewe 661 du ring metoestru s/early dioestru s. 1 00. Fig. 28:. Ewe 661 during early dioestr u s . . . . . . . . . . . . .. 101. Fig. 29:. Ewe 6 6 1 recorded at m id-oe st r u s 8.4. 88. 102. Fig. 30:. Ewe 6 6 1 recorded at l ate o estr u s 9.4.88. .. . . . . . 97 . . . . . . . . . 98. 103.
(12) viii. ACKNOWLEDGME NTS. I ta ke t h i s opportun ity to t h a n k t h e m an y people who h ave g iv e n me enc o u rage m en t , h elp and advic e d u r i n g my M . P h i l . stu dy . M y s i n c e r e t h an ks g o to m y su pervisors Prof. E . D . Fielden a n d D r . D . H . C a r r for t h ei r u n fa l t e r i n g e n th u siasm a n d g u i da n ce d u r i n g t h e proj e c t . e n c o u r agem e n t ,. e n t h u siasm,. constructive. suggest ions. and. Th eir. assistance. t h r o u gh o u t the project were as important as t h ei r advice on the fi n al draft of t h i s thesis. My t h a n k s a l so go to the Tech n i cal Sta ff o f the Depart ment o f P h ysio logy and Anatomy, pa r t ic u l arly that of Debbie Anthony for h el p i n preparat ion of figu r es, J . C . Pedl ey for making the recor d i n g faci l i t ies u sed in t h is st udy ava i l ab l e t o m e , N . I . B roomfi e l d for givi n g v a l u able advice d u r i n g t h e cou rse o f the exp e r i m e n t s and helping m e i n the u se o f the word processo r , and I r e n e H a l l for a ssist a nc e during the surgical procedures.. I a m also i n debted to Mr Cedric B a r n ett and Rose Law for their w i l l i n g h el p with t h e sh eep, a n d with obtai n i n g t h e drugs a n d disposables u se d . T. Law's assistance w i t h t h e photography of all emg records i n gratefu l ly a c k n owledged.. F i n a l ly I w i l l for ever , a ppreciate t h e su pport and forb earance o f m y fam i ly a n d of Xinj i a n g S h i h ezi Agricu ltu ral College, w i t h o u t whose h elp t h i s s t u dy wo u l d n o t h ave b ee n po ssib le..
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(2) ELECTROMYOGRAPHIC STUDIES ON THE OVINE UTERUS. A THESIS PRESENTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF PHILOSOPHY AT MASSEY UNIVERSITY. Li Yuemin _January. 1991.
(3) TABLE O F CONTENTS. ELECTR O MYOGRAPHIC STUDIES ON THE OVINE UTER U S. A b s tra ct . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii Lis t o f Ta b l e s v List o f Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi Acknowl e dg m en ts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v i ii .. I.. .. .. .. .. .. INTR O D U CTION. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 11 . LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 II.1. The Repro d u c t i ve Cyc l e o f S h e e p. . . . . . . . . . . . . ... . ... . . . . 3. Il . l .A . The b reeding seaso n . . . . . . . . . . Il . l . B . Th e o estro u s cycle i n ewes . . . . . Il . l . C. Pregn ancy a n d part u r i t i o n i n ewes Il . l . D .The post- partu m period i n ewes . . . 11 . 2. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. 3 5 7 9. Myo m e tri a l Acti v i ty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3. I I . 2 . A . Stru c t u re a n d o rgan isation o f t h e myom etrial cont r act i l e u n i t . . . . . . . . . . . . . . . . . . . . . . . . . I I . 2 . B . Speci a l featu res o f t h e u l tr as tr ucture o f t h e fu n c t io na l u n i t s o f the m y o m e t rui m . . . . . . . . . . . (a) (b) (c) (d). The c o n tract i l e apparat u s . P lasm a membrane . . . . . . The sarcoplasmic reticu l u m Th e g a p j u nctions . . . . . . .. . . . . . . . . (SR) . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. 13 14. . . . . . . . .. 14 14 15 15. I I . 2 . C . Myo metrial con traction . . . . . . . . . . . . . . . . . . . . . . . . 1 6 II.2.D. E l e c t r o p h y s i o l o g y of myo m e t r i a l con t racti l i ty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 6. 11 .3.. E ffe c t s of Horm o n e s on Emg Activity. 11 . 3 . A . 11 . 3 . B . 11 . 3 . C . 11 . 3 . D . 11 . 3 . E. 11 . 3 . F. 11 .4.. O e strogen . . . . . . . Progesterone . . . . . Oxytoci n . . . . . . . . Prostagl an d i n s . . . . Relaxin . . . . . . . . Vasoactive i n t es t i n al .. . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . pepti d e. . . . . . . . . . . . . . . . . . . . 19. . . . . . . . . . . . . . . . . . . . . . . . . (VIP) .. . . . . . .. . . . . . .. . . . . . .. . . . . . .. . . . . . .. Uteri n e Moti l i ty S t u d i e s with Pa r ticu l ar Refe r e n c e t o t he Ewe .. .. .. •. .. .. •. •. .. .. .. .. .. .. .. .. •. .. .. .. .. •. .. .. .. .. .. .. .. . . . . . .. . . . . . .. . . . . . .. . . . . . .. . . . . . .. . . . . . .. . . . . . .. . . . . . .. . . . . . .. . . . . . .. .. .. .. .. •. .. .. .. .. .. 19 22 24 28 30 30. 31. 11 . 4 .A. Methods o f studying u te r i n e motility . . . . . . . . . . . . . . 3 1 11 . 4 . B . Uterin e motil ity observations recorded from t h e uterus o f t h e i n t act ewe . . . . . . . . . . . . . . . . . . 33 .. ..
(4) 11 . 4 . C . Uter i n e motility observations recorded fro m t h e u terus o f t h e ovariecto m i s e d ewe . . . . . . . . . . . . . Ill.. 11 ! . 1 . 111 . 2 . 11! . 3 . 111 .4. III.5. III.6.. GEN ERAL MATERIALS AND M ETHODS. .. •. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. •. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. M y o m e t r i a l E m g A c t i v i ty i n t h e Inta c t Ewe d u ri n g A n o e s tr u s . . . . . . . . . . . . . . . . . . .. IV .l .A. IV .LB .. IV .l . C . IV .l . D . I V. LE.. 48. 48 48 48 54 54 60. 62. 62. Th e r esting emg of t h e u t e r u s . Th e e ffect o f exogen o u s o xytoci n on restin g e m g o f the u t e r u s d u ri n g anoestru s . . . . . . . . . . . . . Th e e ffect o f exogeno u s o estrogen o n t h e resting emg of t h e uterus d u ri n g anoestrus . . . . . . . . . . . . . Th e e ffect of oxytocin on o estrogen i n d u c ed emg a c t i v i ty during a noestrus . . . . . . . . . . . . . . . . . . . Th e effect of P G F21X on o estrogen i n d u c ed emg a c t i v i ty during anoestrus . . . . . . . . . . . . . . . . . . Th e chan ges in myometr ial activity �hen st i m u l ated by a secon d i n jection o f oestrogen . . . . . . . . . . .. I V. l . F.. IV. 2 .. .. A n i m al . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Electrodes ....................... . ........... . . . . . . Su rgery a n d an aesthesia . . Recordi ngs . . . . . . . . . . . . . . . . . . . . . . . . . . Th e Electromyograms . . . . . . . . . . . . . . . . . . . Q u a n t itative a n alysis o f the Electromyograms . .. IV. R E S U LTS IV. l .. .. 41. 62 the . . . . . . . . . 62 . . . . . . . . . . . . 62 . . . . . . . . . . . . 62. . . . . .. .. . . . . .. .. 66. . . . . . . . . . . . . 66. M y o m e t r i a l Acti v i ty i n t h e O v a r i e c to m i zed Ewe d u ri n g t h e A n o e s tr u s S e a s o n .. . ... . .. . ... . . . .. . . . . . . . . . .. . . . . 7 2. I V. 2 .A.. Specific features of the design o f t h i s experiment . . . . . . . . . . . . . . . . . . . . . 72 I V. 2 . B . The gen eral patterns o f electrical activity . . . . . . . . . . . 73 (a) Resting emg activity a n d t h e e ffects of exogeno u s oxytoci n a n d PGF21X o n it . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 (b) O estrogen i n duced e m g activity i n the ovariectomized e w e and t h e e ffects o f oxytocin a n d P GF21X o n it . . . . . . . . . . . . . . . . . . . . . . . . . 73 (c) Th e e ffects o f progesterone o n 77 oestrogen i n duced e m g activity (d) Th e reduced responsiven ess of t h e uterus i n regime two o f t h e experiment . . . . . . . . . . . . . . . . . . . 83 .. .. .. .. ..
(5) (e) Th e d irection o f propagation o f e m g a ctio n pot e n t i a ls . . . . . . .. .. .. .. .. .. .. .. .. .. . .. .. I V. 2 . C . S o m e q u a n t i t a t iv e aspect s of the e l e c t r o myograms recorded from the ovariectom ized ewes . . . . . . . . . . . . . . . . . . . (a) A m p l i t u de . . . . . . (b ) B u rst frequency . . . . (c) Spikes per burst . . . . . . . . . . . . . . . . . . . ( d ) M o t i l i ty in dex . . . . . . . . . . . . . . . . . . . . . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ·.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. IV.3.. V. V. 2 .. V.3 . V.4 . V.S .. .. 84. . . . . . . . . . . . . . . . . . . . . .. 85 85 85 85 85. .. .. .. .. .. .. .. .. .. .. .. .. .. Th e E m g A c t i v i ty o f the Ute r u s o f the Cycl i n g Ewe .......... 93. DISCU S S ION. .. .. .. .. .. . .. .. .. . . .. .. .. .. .. .. .. . .. .. .. . . .. Emg Activity d u r i n g Anoestrus and i n t h e Ovar i ec t o m ized Ewe . . . . . . Th e D irection o f Pro pagation of t h e Act i o n Poten t i a l s . . . . . . . . . . . . . . . . . . . . . Th e I n fl u en c e o f O estrogen . . . . . . . . . . . Th e I n fl u e n c e o f Progestero n e . . . . . . . U te r in e r e fractoriness . . . . . . . . . . . . . . .. V .2 .. .. .. .. .. .. .. .. .. .. .. 104. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 1 04. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . . . . . . . . . . .. 1 06 1 10 1 12 116. REFER E N C E S ........................................ 1 1 7. A P P E NDIX I. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 130.
(6) ii. A B S TRACT. There a re many repo r t s in t h e l i t er a t u re, u s i n g a variety of t e c h n i qu es , i n dicating t h at h ormonally i n du ced m ovem en t s o f t h e u terus play a m aj o r r o l e i n t h e reproduction process. Th e pu rpose o f t h is study w a s to explore some of t h ese p h enomena by measuring electro-myographical respons es from t h e u terus o f i n t act a n d ovariectom i zed ewes b o t h b e fore a n d during t h ei r n o r m a l b reeding seaso n .. Six h e a l t h y non-pregn a n t Rom n ey ewes o f abou t 5 0 kg body w e i g h t were selected for the i nvestigations which were carried out b etween September 1 987 and Feb r ua ry 1 988. Electrodes, u sually in gro u p s of t h ree, were surg ically impl a n t e d a t pre s elected s i t es (cervix, u t er i n e b o dy, u t erine h o r n , oviduct) on t h e reproductiv e t ract o f t he ewes, le a d s plugged i n t o a u n iversal AC a m p l i fier, a n d the o u t p u t recorded o n a fo ur c h a n n e l i n k wri t i n g c h a r t r ecorder. Four o f t h e six ewes were ovariect omized at the time o f implan t a t i o n o f t h e electrodes.. I n dwelling silastic cat h eters were inserted into t h e ext er n a l. j ug u l ar veins o f t h ese ewes t o e n a b l e oxytocin to be injected wi t h o u t disturb i n g t h e animal. A l l ewes were h o u sed i n dividu ally i n doors i n p e n s t h a t all owed t h em t o moy e freely during reco r d i n g session s .. Recordi n gs b eg an. abo u t 1 0 days after su rgery h ad been carried o u t . Th e electromyographs (emgs) were described u si ng generally accepted ter m s such as t he presence o f s pik es , t h e i r ampl i t u de, t h e presence o f b u rs ts a n d o f episodes o f activity, w h e t h e r el ectr ical activit y was co-ordi nat ed o r u n co ordinat e d , and t h e direction o f propagat i o n o f t h e act ivity. A m o t il i ty i n dex (MI) was derived as a m e t h o d of meas u r ing electrical activity of t h e u te r u s over prolonged periods o f t i m e . D uring t h e anoestro u s p e r i o d i n b o t h i n tact a n d ovariectomized e w e s a n emg pattern o f myo electrical complexes c h aracterised by alternating p h ases o f co ordina t ed b u rst s o f electrical activity a n d q u i escent periods were ob served. Th i s b asic pa ttern of activi ty, and respon s iv e n e ss to u t erine s t im u lan t s s u c h a s oxytocin and Glandin N (a P GF2oc an al og u e), was greater i n t h e i n t ac t.
(7) iii. ewes a s compared to t h e ovariec t o mized ewes a t t h i s t i m e . A l t h o u gh s u ch a d i ffer e n c e c o u l d be anticipated, a clear answer as to w h e t h er i t i s a general p h e n o m e n o n , or whether seaso n al d i ffere n c es exist, cou ld not be det e r m i n ed b ec a u se o f t h e l i m i t ations i n design o f t h i s s t u dy. A d i s t i n c t pattern o f emg activity was also i d e n t i fied d u r i n g d i ffer e n t p h ases of t h e o estro u s cycle in the ewe d u r i n g the n a t u ral b r e e d i n g season .. Th i s. p a t t er n cou l d b e m imicked by a d m i n i s tr a t i o n of th e exogen o u s steroid h o r m o n es oestrogen and proges t ero ne i n the ovariect o m ized ewes. Fou r days a fte r daily oes tradiol- 1 7b i njec t i o n s (50J..Lg s . c . ) t h e emgs of t h e ovariec t o m ized ewes s h owed a marked i n crease in ampl i t u d e and i n b u rs t frequency a n d a c o n s i d erably inc reased respo ns e to oxytocin a n d Gla n d i n N . W h e n t h i s was follo w e d by progestero ne a d m i n i st r a t i o n (50mg. s.c.), even wit h o estrogen i nj e c t i o n s c o n t i n u ing, i n h i b i t ion of activi ty a n d redu ced respon siveness to oxy t o c i n and Glan din N was e q u a l ly m arked. W i t h drawal o f progesteron e , b u t with co n t i n u i ng oest rogen a d m i n is tr at io n , r e s u l t e d i n a recovery fro m t h e i n h i b i t i o n a n d a respo n se t h a t w a s even greater t h an before t he progestero n e h a d been given . Th i s s u ggests both an i n h i b i t i n g and a poten t i a t i n g action o f progestero n e o n u t eri n e electrical ac t ivity, a fin di n g which adds s ome suppo r t to Csapo's c l assical w i t h d r awal of the 'progeste ron e block' as one o f t h e pre-requ isi tes fo r i n i t i at i o n of normal p ar t u r i t io n in sh eep. _. W h i l e o estrogen h as a clear role t o play as a s t i m u l a t o r o f electrical activity it a l so seem s c apable of exh i b i t i n g a b iph asic resp o n s e with a period of depressed a c t ivity occurri n g befo r e the pos i t ive stimu l u s occurs. W h e t h e r t h i s i s a fu n c t io n o f dose o r s o m e o t h er factor c o u l d n o t b e e s t ab l i s h e d i n t h i s s t u dy . I t s poten t i a ti n g e ffec t on t h e action o f both oxytocin a n d G l a n d i n N i n t h e s e experiments adds fu r t h er sign i ficance t o t h e a t t e n t io n t h a t s h o u l d b e p a i d t o t h e reproductive s t a t u s o f t h e a n i m a l when c l i n i c a l u se i s m a d e o f t h es e s u bstances.. Th e d i rection of propagation of action pot e n tials recorded in t h ese s t u di e s d ep e n d ed o n t h e general l evel o f emg activity.. W h e n t h is was l o w t h e. directi o n o f propagation w a s cervico- t u b a l , when h i g h , t h e proportion o f a c t i o n poten ti a l s i s i n both d i rections fro m t h e t u b a l e n d o f t h e u terus. A.
(8) iv. c l e a r u n derst a n d i n g o f such a m e c h a n i sm , a n d i t s sig n i fi c a n c e, m u s t await fu r t h er s t u dy..
(9) V. LIS T O F TAB LES. Tab l e 1 :. Th e e ffect o f oxytocin both o n spo n t an e o u s a n d . . . . . . . 67 oestradiol i n duced e m g activity (Ewe 329) .. Tab l e 11 :. Th e e ffec t of oxytocin on spo n t aneo u s a n d oestradiol i n d u c ed e m g activity 1 3 days a ft er l a s t oest radiol i nject i o n o f ewe 329 (compare w i t h Tab l e I ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69. Tab l e I l l :. Th e d i fferences i n emg activity b etween a n i m a l s du r i n g stage 2 o f regime o n e . . . . . . . . . . . . . . . . . . . . . . 9 1. Tab l e I V:. Th e e ffect o f oxytocin a n d P GF2cx: o n t h e o e s t radiol i n d u c e d electrical act ivity du r i n g stage 2 of regi m e one . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92.
(10) vi. L I S T O F F IGURE S. Fig. 1 :. Th e pos i t i o n s of electrodes s u t u r e d a t pre- s elec t e d sites o n t h e u teru s a m o n g 6 ewes . . . . . . . . . . . . . 50. Fig. 2 :. Active a n d qu iesce n t periods of emg d u r i n g a noestr u s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 6. Fig. 3 :. An e x a m ple o f t h e m e t h o d o f m easu r i n g th e du rat i o n o f bu rsts, th e i n t erval b etween b u rs t s , t h e b u rs t frequ ency, spike frequ ency a n d am p l i t u d e o f t h e spike . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 7. Fig. 4 (a & b): Fig. 5 :. A n example showi n g t h e propagative direc t i o n o f b ursts a n d episodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 8 A n example o f co-o r d in at e d a n d u n co-o r d i n a t e d patterns o f elect rical a c t iv i ty . . . . . . . . . . . . .. Fig. 6 :. An exam ple of calc u l a t i n g t h e m o t i l i ty i n dex. Fig. 7 :. Oxytocin effect o n durin g a n oestrus. .. 59 61. spo n t a n eo u s emg activity . . . . . . . 63. Fig. 8 :. Oestradiol benzoate b o t h i n h i b i t s a n d s t i m u l at es myo m et r i a l activity in ewe 329 d u r i n g a n o e s t r u s . . . . . . . . 64. Fig. 9:. The e ffect o f oxytoci n on oestradiol i n du ce d emg activi ty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65. Fig. 1 0:. Th e effect o f P GF2oc on oestr ad iol i n d u c e d emg activity. during anoes t r us . . . . . . . . . . . . . . . . . . . . . . . . . 68. Fig. 1 1 :. Characteristics o f t h e emg recorded 13 days a ft er the i n i t i a l injec t i o n o f oes trogen . . . . . . . . . . . . . . . . 70. Fig. 1 2 :. Th e e ffects o f oes trogen and oxytocin on ampl i t u de, burst fre q u e n cy and m o t i l i ty i n dex during two expe r i m en t s on i n t a c t anoestro u s ewe 329. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 1. Fig. 1 3 :. Th e patter n o f emg a c t iv i ty i n ovariec t o m i z e d ewe 247 d u r i n g the a n o es t r o u s season . . . . . . . . . . . . . . . . . . 74. Fig. 1 4 :. Th e pattern of emg activity i n d u c e d by o e s t r a d i o l 1 7b (50JLg g i v e n s . c. daily) on ovariec t o m i z e d ewes during the anoestrou s season . . . . . . . . . . . . . . . . . . . . . 75. Fig. 1 5 :. Th e e ffect s o f oxyto c i n (500 m u given i.v.) on t h e oestrogen i n d uced e m g activity o f ovariecto mi z e d ewes d u r i n g t h e anoestrous season . . . . . . . . . . . . . . . . . . 76.
(11) vii. Fig. 1 6a :. Th e d evelopment o f i n hi b it i o n a fter progestero n e a d m i n istration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 8. Fig. 1 6b :. Th e e ffect of oxytoc i n a n d P GF2<x: w h e n t h e uterus i s i n h ibited by progest e r o n e . . . . . . . . . . . . . . 79. Fig. 1 6c :. T h e e m g pattern observed d u r i n g t h e recovery from progesteron e i n h ib it i o n . . . . . . . . . . . . . . . . . . . . . . 8 1. Fig. 1 6d :. Th e pattern of oestrogen i n d u c e d emg i n ewe 1 0 b efor e progesteron e admi n i stration (A) a n d i n t h e sa m e ewe 1 1 5 h after a large dose o f progesterone ( 1 00mg given i . m . ) h ad b e e n g iven when th e ewe h a d recovered from t h e progestero n e tr eatm ent a d m i n istered during reg ime o n e (E) . . . . . . . . . . . . . . . . 82. Fig. 1 7 :. Th e difference i n amplit u d e a n d b u rst frequency b etween 4 electrode positio n s o n t h e reproduct ive . . . . . . . 86 trace. Fig. 1 8 :. Em g amplitu des d u r i n g treatment i n regime o n e .. di fferent. stages. of . . . . . . . 87. Fig. 1 9 :. Di fferences i n bu rst fre q u e n c y at each tr eatm ent stage o f regime one . . . . . . . 88. Fig. 20:. Nu mber of spikes i n a b u r st during the p eriod o f oestradiol i n duced emg activity i n regi me one . . . . . . . 89. Fig . 2 1 :. Th e n u mber of spikes in a bu rst d u r i n g each treatm ent stage of regime one . . . . . . . . . . . . . . . . . . . . . 9 0. Fig. 2 2:. Th e motility i n dex du r i n g stages 2 a n d 4 o f reg i m e o n e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94. Fig. 2 3 :. Th e c h aracteristics of emg act ivity during st ages 2, 3 and 4 of both reg i m es . . . . . .. 95. Fig. 24:. Ewe 661 during dioest r u s. 96. Fig. 2 5 :. Ewe 661 during pro-oest r u s .. Fig. 2 6 :. Ewe 661 during oest r u s . . . . . . . . . . .. Fig. 2 7 :. Ewe 661 du ring metoestru s/early dioestru s. 1 00. Fig. 28:. Ewe 661 during early dioestr u s . . . . . . . . . . . . .. 101. Fig. 29:. Ewe 6 6 1 recorded at m id-oe st r u s 8.4. 88. 102. Fig. 30:. Ewe 6 6 1 recorded at l ate o estr u s 9.4.88. .. . . . . . 97 . . . . . . . . . 98. 103.
(12) viii. ACKNOWLEDGME NTS. I ta ke t h i s opportun ity to t h a n k t h e m an y people who h ave g iv e n me enc o u rage m en t , h elp and advic e d u r i n g my M . P h i l . stu dy . M y s i n c e r e t h an ks g o to m y su pervisors Prof. E . D . Fielden a n d D r . D . H . C a r r for t h ei r u n fa l t e r i n g e n th u siasm a n d g u i da n ce d u r i n g t h e proj e c t . e n c o u r agem e n t ,. e n t h u siasm,. constructive. suggest ions. and. Th eir. assistance. t h r o u gh o u t the project were as important as t h ei r advice on the fi n al draft of t h i s thesis. My t h a n k s a l so go to the Tech n i cal Sta ff o f the Depart ment o f P h ysio logy and Anatomy, pa r t ic u l arly that of Debbie Anthony for h el p i n preparat ion of figu r es, J . C . Pedl ey for making the recor d i n g faci l i t ies u sed in t h is st udy ava i l ab l e t o m e , N . I . B roomfi e l d for givi n g v a l u able advice d u r i n g t h e cou rse o f the exp e r i m e n t s and helping m e i n the u se o f the word processo r , and I r e n e H a l l for a ssist a nc e during the surgical procedures.. I a m also i n debted to Mr Cedric B a r n ett and Rose Law for their w i l l i n g h el p with t h e sh eep, a n d with obtai n i n g t h e drugs a n d disposables u se d . T. Law's assistance w i t h t h e photography of all emg records i n gratefu l ly a c k n owledged.. F i n a l ly I w i l l for ever , a ppreciate t h e su pport and forb earance o f m y fam i ly a n d of Xinj i a n g S h i h ezi Agricu ltu ral College, w i t h o u t whose h elp t h i s s t u dy wo u l d n o t h ave b ee n po ssib le..
(13)
