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Ultrasound and progesterone monitoring of ovarian follicular cysts in cows treated with GnRH
Br. vet.J. (1994). 150, 489
ULTRASOUND AND PROGESTERONE MONITORING OF OVARIAN FOLLICULAR CYSTS IN COWS TREATED WITH GnRH
A. Y. RIBADU, H. DOBSON and W. R. WARD* Department of Vetelina D, Clinical Science and Animal HusbandTy, University of Livelpool, Leahurst, Neston, South Winal L64 7TE, UK
SUMMARY Five cows, diagnosed as having follicular cysts (by palpation per rectum, ultrasonography and p r o g e s t e r o n e d e t e r m i n a t i o n ) , were treated with 0.5 mg g o n a d o t r o p h i n releasing h o r m o n e (GnRH) 10 days after initial diagnosis. T h e ovaries were scanned w e e n y from the day of treatment (day 0) until clay 35. Mean (+SeM) plasma p r o g e s t e r o n e c o n c e n t r a t i o n on the day o f diagnosis ( d a y - 1 0 ) and day 0 were 0.2_+0.13 ng ml -L and 0.72_+0.27 ng ml -I, respectively. Elevated p r o g e s t e r o n e (5.58_+0.67 ng ml -~) was f o u n d in all cows by day 7 ~ffter treatment. Changes n o t e d during weekly ultrasonography included clouding o f the uniformly n o n e c h o g e n i c (dark) a n t r u m of cysts, luteinization o f the cyst wall, reduction in cyst size (cyst resolution) a n d / o r d e v e l o p m e n t o f 1-4 c o r p o r a lutea in the ovary bearing the cyst or in the contralateral ovary. Oestrus was observed in all cows 18.8_+1.74 days after treatment. It is c o n c l u d e d that ultrasonography and p r o g e s t e r o n e d e t e r m i n a t i o n are useful tools for veterinarians in the m o n i t o r i n g of responses of follicular cysts to treatment. KE'~VORDS: Ultrasound; follicular cyst; cows; GnRH; progesterone.
INTRODUCTION Cystic ovarian d e g e n e r a t i o n is a majo," reproductive disorder of cattle. It is cha,'acterized by the presence of one or m o r e follicular structures larger than 25 mm in diameter for 10 clays o1" more ill the absence of a corpus luteum (CL) (Seguin, 1980). G o n a d o t r o p h i n releasing h o r m o n e (GnRH) is a decapeptide c o m m o n l y used *To whom correspondence should be addressed. 0007/1935/9,1/050,I89-09/$08.00/0
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for the treatment of ovarian cysts. T h e advantages of G n R H are its low price, lack of refractoriness due to antibody formation and the fact that anaphylaxis following G n R H treatment has not been r e p o r t e d in cows (Bosu, 1982; Youngquist, 1986; Nanda et aL, 1989; Drost & Thatcher, 1992). Exogenous G n R H acts on the pituitary gland to cause release of e n d o g e n o u s luteinizing h o r m o n e (LH) (Kittock et aL, 1973; Edquist et al., 1974; Cantley et al., 1975; Garverick et al., 1976; Seguin et al., 1976; Kesler et aL, 1978; Youngquist, 1986). T h e G n R H - i n d u c e d LH release stimulates luteinization of ovarian cysts (Kesler et al., 1977). Response of cows with ovarian cysts appears to va,-y with the dose of G n R H administered (Youngquist, 1986). If sufficient G n R H is given and mature follicles are present, ovulation is induced within 24--30 h (Lee et aL, 1983). T h e integrity o f cyst granuiosa and theca cells may also be an important factor d e t e r m i n i n g the response to t r e a t m e n t (Bierschwal et aL, 1980). Luteal tissue d e v e l o p m e n t following G n R H t r e a t m e n t in cattle may be monitored indirectly by m e a s u r e m e n t of p r o g e s t e r o n e concentrations in plasma or milk. Garverick et aL (1976), using palpation per rectum, described cyst luteinization that was characterized by increased firmness and r e d u c e d cyst size in cows that r e s p o n d e d to GnRH or h u m a n chorionic g o n a d o t r o p h i n (hCG) treatment. Nakao et al. (1983) obtained a correlation of only 30% between palpation per rectum and milk progesterone concentration for assessing iuteinization of follicular cysts 10 days after G n R H treatment. Incorrect assessment of luteinization of follicular cysts by rectal palpation after treatment has been a problem (Whitmore et aL, 1979; Nakao & Kawata, 1980). With the introduction o f ultrasonography, it may be possible to m o n i t o r objectively the d e v e l o p m e n t of luteal tissue in o r d e r to evaluate the effectiveness o f treatment of ovarian cysts. T h e detection o f ovulation within 30 days o f treatment was defined as a positive response (Kesler et al., 1979). Plasma p r o g e s t e r o n e c o n c e n t r a t i o n is elevated 5-9 days following G n R H treatment in most cows that respond positively (Cantley et aL, 1975; Garverick et aL, 1976; Seguin et aL, 1976; Kesler et aL, 1978, 1979) as a result o f luteinization of the cyst or of o t h e r follicles with or without ovulation (Kesler et al., 1976; Youngquist, 1986). M t h o u g h e n d o c r i n e changes following G n R H treatment of ovarian cysts in cattle have been described (Cantley et aL, 1975; Garverick et al., 1976; Segui,a el aL, 1976; Kesler et al., 1978; Nanda et al., 1988), information on ultrasound changes following treatment is scarce. T h e aim of the present study was therefore to monitor ovarian t, ltrasotmd changes and to correlate these with plasma p r o g e s t e r o n e concentration in cows with follicular cysts that had been treated with GnRH.
MATERIAL A N D M E T H O D S
Five lactating Friesian cows on three dairy farnls were diagnosed as having follicular cysts (by palpation per rectum, uitrasonography and p r o g e s t e r o n e determination). T h e diagnosis by rectal palpation was based on the finding of single or multiple follicttlar structures of at least 25 m,n in diameter on oqe or both ovaries in the abse,ace of a co,'pus luteum. By ultraso,aography, a follicular cyst was defined as a tmiformly n o n e c h o g e n i c ovarian structure --> 25 ham in diam-
ULTRASONOGRAPHY OF FOLLICULAR CYSTS AFq'ER GnRFI
491
eter with a wall <3 ram. A plasma p r o g e s t e r o n e concentration of <0.9 ng ml-' was used to classify further the cysts as follicular. T e n days after initial diagnosis (day - 1 0 ) , cows were treated (day 0) with 500 lag synthetic GnRH (gonadorelin, 5 ml Fertagyl; Intervet) and ovarian ultrasonograplay and blood sampling were carried out weekly until day 35 after treatment. Ultrasound examination was p e r f o r m e d using an ultrasound scanner (SSD 210 DXII, Aloka, BCF Technology) e q u i p p e d with a 7.5 MHz rectal transducer (Aloka UST 55111). Representative images were obtained at each examination using an on-line thermal printer (Video Graphic UP 850, Sony). A blood sample was obtained by tail v e n e p u n c t u r e after each examination. Blood was centrifuged at 1000 g and stored at -20°C until analysis for progesterone. Plasma p r o g e s t e r o n e was measured using a validated radioimmunoassay (I~mchev et aL, 1976). T h e c u r r e n t inter and intra-assay coefficient of variations were 11.1 and 4.6% with a sensitivity of 0.05 ng ml -t. T h e r a p e u t i c success was c o n f i r m e d by the d e v e l o p m e n t o f CL a n d / o r luteinization of cysts (characterized by increased cyst wall thickness with granular echogenic a p p e a r a n c e as visualized by ultrasound) and increased plasma progesterone concentration. T h e r a p e u t i c failure was defined as cases in which cysts r e m a i n e d t m c h a n g e d or where new cysts developed without luteal d e v e l o p m e n t and where the cow failed to cycle normally. Mean p r o g e s t e r o n e concentration and cyst size on the day o f treatment were c o m p a r e d with values on day 7 after treatment using Student's t-test.
RESULTS
T h e relationship hetween cyst size and progesterone concentration is given in Figs 1 and 2 while Figs 3-6 are representative ultrasonograms of a follicular cyst (day 0), a luteinized follicular cyst (day 7), luteinized follicular cyst (day 14) and two GnRH-induced c o r p o r a lutea (day 7). Prior to treatnlent, r e c u r r e n t oestrus behaviour was shown by three cows and anoestrus o c c u r r e d ill the o t h e r two. All five cows r e s p o n d e d positively to GnRH treatment. By clay 7 after treatment, lnteinization of cysts (Figs 3 and 4) was n o t e d during ultrasonography in two cows while GnRH-induced CLs were observed in tile remaining three cows (Fig. 5). Changes n o t e d during weekly ultrasonography after treatment inchtded clouding of the uniformly n o n e c h o g e n i c a n t r u m of cysts, luteinization o f cyst walls, and a non significant (P>0.05) reduction in cyst size. Tile d e v e l o p m e n t of 1-4 CLs in tile ovary bearing tile cyst a n d / o r in the contralateral ovary was observed in three cows by clay 7 after treatment. These CLs were an immediate response to GnRH and resulted from ovulations of large follicles present at the time o f treatment. Ovtdations in these cows were not associated with oestrus. A secondary response to GnRH was observed by the d e v e l o p m e n t o f CLs by day 28 ,after treatment in the two cows that initially r e s p o n d e d by cyst luteinization. T h e cysts co-existed with the GnRH-induced CLs in three cows for 2 weeks and following subsequent oestrus and ovulation in four o f five cows by day 28 after treatment. In one cow that initially r e s p o n d e d by cyst luteinization, the cyst was not visible by day 28 (cyst
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Day Fig. 2. Dynamics of follicular cysts that initially responded by corpus luteuna development (n=3). (--VI--), Cavity diameter (cm); ( - - 0 - - ) , cyst diameter (cm); (--M--), progesterone (ng mFI).
ULTRASONOGRAPHY OF FOLLICULAR CYSTS AFTER GnRH
493
Fig.. 3. Ultrasonogram of a follicular cyst. Diameter of cyst cavity=29 mm; diameter of cyst =31 ram. resolution); only a CL was observed d u r i n g ultrasonography. T h e G n R H - i n d u c e d CLs had regressed before s u b s e q u e n t oestrus in the three cows. All five cows were in oestrus 18.8+1.74 days after treatment. Mean (+SEM) plasma p r o g e s t e r o n e c o n c e n t r a t i o n s on day - 1 0 a n d day 0 were 0.2+0.13 ng m1-1 and 0.72+0.27 ng m1-1 respectively. P r o g e s t e r o n e c o n c e n t r a t i o n was significantly (P<0.05) elevated (5.8+0.67 ng ml -I) in all cows by day 7 comp a r e d to p r e - t r e a t m e n t values (day 0). T h e p r o g e s t e r o n e was c o n s i d e r e d to arise f r o m G n R H - i n d u c e d CLs in three cows a n d cyst luteinization in two cows.
DISCUSSION T h e visualization by u l t r a s o n o g r a p h y o f cyst luteinization a n d / o r the p r e s e n c e o f CLs in all cows by day 7 after G n R H shows that in r e s p o n d i n g cases evidence for t h e r a p e u t i c success could be c o n f i r m e d quickly. All five cases in this study
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Fig. 4. Ultrasonogram of a luteinized follicular cyst (7 days after treatment). Diameter of cyst cavity=28 ram; diameter of cyst=35 mm.
responded positively by either cyst luteinization a n d / o r CL formation. Failure to detect luteinization of follicular cysts accurately after GnRI-I by palpation per rectum has led to unnecessary treatment in many cows (Nakao et aL, 1983) and ultrasonography could prevent this problem. It has been reported that GnRH has a success rate of about 80% (i.e. ovulation detected within 30 days post-treatment) in treating ovarian cysts in cattle (Kesler & Garverick, 1982). In the present study, follicular cysts persisted (although with a slight decrease in size) for 3-4 weeks after GnRI-I administration in the presence of a CL; such cysts may not have any functional significance. One cow had only one follicular cyst and three large follicles (15-20 mm) at initial diagnosis (day -10), but three follicular cysts were visualized during ultrasonography at the time of treatment (day 0). This
ULTRASONOGRAPHY OF FOLLICULAR CYSTS AFTER GnRH
495
Fig. 5. Ultrasonogram of a luteinized follicular cyst (14 days after treatment). Diameter of cyst cavity=18 ram; diameter of cyst=33 mm.
supports\ the proposition that ovarian follicular cysts are dynamic structures (Cooke et al., 1990). T h e observation o f an increase in p r o g e s t e r o n e by day 7 after treatment is consistent with previous studies (Candey el aL, 1975; Garverick et al., 1976; Seguin et al., 1976; Kesler et aL, 1978). Progesterone concentration r e m a i n e d low (<0.9 ng ml -~) in all cows from day - 1 0 to day 0. Cantley et aL (1975) f o u n d higher mean pre-treatment concentration o f plasma p r o g e s t e r o n e in cows that r e s p o n d e d to G n R H than in those that did not respond. Kesler et al. (1979), however, did not find any difference in mean pre-treatment plasma progesterone concentration between cows with a positive response and those that did not respond to G n R H treatment.
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Fig. 6. Uhrasonogram of two GnRH-induced corpora lutea (CL) (7 days after treatment). CL diameter=20 nlm.
It is e v i d e n t t h a t u l t r a s o n o g r a p h y a n d p r o g e s t e r o n e m e a s u r e m e n t tools for e v a l u a t i n g t h e r e s p o n s e o f f o l l i c u l a r cysts to G n R H in cows.
a r e useful
REFERENCES BIERS('HW,U.,C.J., ELMORE,R. G., BROWN, E. M. & YOUNGQUXST,R. S. (1980). Patholog3, of the ovary and ovarian disorders and the influence of ovarian abnol-malides on the endometrium, including therapeutic aspects (cow). Proceedings of the 9th International Congress on Animal Reproduction and A.L, Madhd, Spain I, 193-202. Bost:, W. T. K. (1982). The use of GnRH in bovine reproduction. Compendium of Continuing Education for the Practising VeteTinaTqan4, $55-$64. CANTLI:Y,T. C., GARVI-ZRICK,H. A., BIERSCHWAL,C.J., MARTIN,C. E. ~¢ YOUNGQUIST,R. S. (1975). H o r m o n a l responses of dairy cows with ovarian cysts to GnRH. Journal of Animal Science 41, 1666-73.
ULTRASONOGRAPHY OF FOLLICULAR CYSTS AFTER GnRH
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CooK~:, D. L., S~,TH, C. A., P,XRVET,J. R., YOUNGQUIST,R. S., BROWN, E. M. & GARVERICI~,H. A. (1990). Fate and turnover of ovarian follicular structures in dairy cattle. Journal of Reproduction and Fertility90, 37--46. DRosq, M. & THAXCm.:R,W. W. (1992). Application of g o n a d o t r o p h i c h o r m o n e as a therapeutic agent in animal reproduction. Animal Reproduction Science28, 11-19. EDQL'IST,1,. E., EKNAM,L., GUSTAI:SSON,B. & LIffDI.:LL,J. O. (1974). The clinical and hormonal effect of luteinizing hormone-releasing h o r m o n e (LH-RH) in cows with cystic ovaries. Nordish VetetfnaerMedicin 26, 552-6. GARVL.:RI(:K,H. A., Ka:SLER,D.J., Car,q't.EV,T. C., EL,~OR~, R. G., YOUNGQUISr,R. S. & BIERSCHWALL, C . J . (1976). H o r m o n e response of dairy cows with ovarian cysts after treatment with HCG or GnRH. Theriogenology6, 413-25. K..~N(:ltrv, L. N., DOBSON, H., WARD, W. R. & Fn-ZPATRK:K,R.J. (1976). Concentration of steroids in bovine peripheral plasma during the oestrous cycle and the effect of betamethasone treatment.Journal of Reproduaion and Fertility48, 341-5. Ka.:sLza, D.J., ELMORE,R. G., YOVNGQ.Lrlsr,R. S., BRO~, E. M., GARVERICK,H. A. & BIERSCHWAL, C.J. (1977). Ovarian morphology in dairy cows with ovarian cysts following treatment with GnRH.Journal of Animal Science45, Suppl. I, 176 (Abstr.). I~SLER, D.J. & GARVERXCK,H. A. (1982). Ovarian cysts in dairy cattle: a review.Journal of Anintal Science55, 1147-59. Kt:sH.r~, D.J., GARVERZC.K,H. A., CAUDLE,A. B., BtzmCmVAt., C.J., Et.XtORE, R. G. & YOUNGQUIST, R. S. (1978). Clinical and e n d o c r i n e responses of dairy cows with ovarian cysts to GnRH and PGF,., alpha. Journal of Animal Science46, 719-25. KVSLI-R,D.J., G,~RVF:RICK,H. A., EL~tO~, R. G., YOL:Nr.;QUIST,R. S. & BIEP~SCHWAL,C.J. (1979). Reproductive h o r m o n e s associated with the ovarian cyst response to GnRH. The~qogenol0~, 12, 109-14. ~rr()(:K, R.J., BRITr, J. H. & CoNvt:v, E.J. (1973). Endocrine responses after GnRH in luteal phase cows and cows with follictflar cysts.Journal of Animal Science37, 985-9. I.H:, C. N., MAt:RI(:~:,M. S., PI.:NNZGTON,J.A., HOVVMA~,W. F. & BROWN,M. D. (1983). Efficacy of gonadotropin-releasing h o r m o n e administered at the time of artificial insemination of heifcrs, postpartum cows and repeat b r e e d e r dairy cows. AmelJcanJournal of Vetelinmy Research 44, 2160-3. NAtc.\(~,T. & K.\wxr..x, K. (1980). Enzyme immunoassay of progesterone in bovine blood and milk and its application in monitoring the luteinization of ovarian follicular cysts after h o r m o n e treatment. Proceedingsof the 1l th International Congress on Disea.~esof Cattle, Israel, 916--33. N:~..xo, T., SL'C;HL-\SlH, A., SAGa, N., TSUNODA,N., K.~WATA,K. (1983). Use of milk progesterone enzyme immtmoassay for differential diagnosis of follicular cyst, luteal cyst and cystic corpus htteum in cows. A merican.]ournal of Vete~qnmyResearch44, 888-90. NA×DA, A. S., WARD, W. R. & DOBSON, H. (1989). Treatment of cystic ovarian disease in c a t t l e - - a n update. Vete~qnm3,BulMin 59, 537-56. NANt)..X,A. S., ~t."kRI), W. R., WILI.IAMS, P. C. W. & DoBso,',:, H. (1988). Retrospective analysis of different h o r m o n e treatments of cystic ovarian disease in cattle. Veterinmy Record 112, 155-8. Svt,vl×, B. E., Co×vvv, E. M. & OXZXDER,W. D. (1976). Effect of gonadotropin-releasing hormone and hun]an chorionic gonadotropin on cows with ovarian lollicular cysts. Ame~qcan Journal of Veterinmy Research37, 153-7. St.:(;t~x, B. E. (1980). Ovarian cysts in dait-y cattle. In Cunent Therapy in Then?ogenology, 1st Edn, ed. D. A. Morrow, pp. 199-204. Philadelphia: W. B. Saunders Co. Wtlnx~oRF, H. L., HC'RTt;ZN,j . P. & MArFtFR, E. C. (1979). Clinical response of dairy cattle with ovarian cysts to single or repeated treatments of gonadotropin-releasing hormone. Journal of American Vete~4nmyMedicalAssociation 174, 1113-15. YOUN(;QLrlS'I',R. S. (1986). Cystic follicular degeneration in the cow. In Current Therapy in 7"he~4ogenolo~,, 2nd Edn, ed. D. A. Morrow, pp. 243-6. Plailadelphia: W. B. Saunders Co.
(Acceptedfor publication23 December1993)
ULTRASOUND AND PROGESTERONE MONITORING OF OVARIAN FOLLICULAR CYSTS IN COWS TREATED WITH GnRH
A. Y. RIBADU, H. DOBSON and W. R. WARD* Department of Vetelina D, Clinical Science and Animal HusbandTy, University of Livelpool, Leahurst, Neston, South Winal L64 7TE, UK
SUMMARY Five cows, diagnosed as having follicular cysts (by palpation per rectum, ultrasonography and p r o g e s t e r o n e d e t e r m i n a t i o n ) , were treated with 0.5 mg g o n a d o t r o p h i n releasing h o r m o n e (GnRH) 10 days after initial diagnosis. T h e ovaries were scanned w e e n y from the day of treatment (day 0) until clay 35. Mean (+SeM) plasma p r o g e s t e r o n e c o n c e n t r a t i o n on the day o f diagnosis ( d a y - 1 0 ) and day 0 were 0.2_+0.13 ng ml -L and 0.72_+0.27 ng ml -I, respectively. Elevated p r o g e s t e r o n e (5.58_+0.67 ng ml -~) was f o u n d in all cows by day 7 ~ffter treatment. Changes n o t e d during weekly ultrasonography included clouding o f the uniformly n o n e c h o g e n i c (dark) a n t r u m of cysts, luteinization o f the cyst wall, reduction in cyst size (cyst resolution) a n d / o r d e v e l o p m e n t o f 1-4 c o r p o r a lutea in the ovary bearing the cyst or in the contralateral ovary. Oestrus was observed in all cows 18.8_+1.74 days after treatment. It is c o n c l u d e d that ultrasonography and p r o g e s t e r o n e d e t e r m i n a t i o n are useful tools for veterinarians in the m o n i t o r i n g of responses of follicular cysts to treatment. KE'~VORDS: Ultrasound; follicular cyst; cows; GnRH; progesterone.
INTRODUCTION Cystic ovarian d e g e n e r a t i o n is a majo," reproductive disorder of cattle. It is cha,'acterized by the presence of one or m o r e follicular structures larger than 25 mm in diameter for 10 clays o1" more ill the absence of a corpus luteum (CL) (Seguin, 1980). G o n a d o t r o p h i n releasing h o r m o n e (GnRH) is a decapeptide c o m m o n l y used *To whom correspondence should be addressed. 0007/1935/9,1/050,I89-09/$08.00/0
© 1994Bailli/:reTindall
490
BRITISH VETERINARYJOURNAL, 150, 5
for the treatment of ovarian cysts. T h e advantages of G n R H are its low price, lack of refractoriness due to antibody formation and the fact that anaphylaxis following G n R H treatment has not been r e p o r t e d in cows (Bosu, 1982; Youngquist, 1986; Nanda et aL, 1989; Drost & Thatcher, 1992). Exogenous G n R H acts on the pituitary gland to cause release of e n d o g e n o u s luteinizing h o r m o n e (LH) (Kittock et aL, 1973; Edquist et al., 1974; Cantley et al., 1975; Garverick et al., 1976; Seguin et al., 1976; Kesler et aL, 1978; Youngquist, 1986). T h e G n R H - i n d u c e d LH release stimulates luteinization of ovarian cysts (Kesler et al., 1977). Response of cows with ovarian cysts appears to va,-y with the dose of G n R H administered (Youngquist, 1986). If sufficient G n R H is given and mature follicles are present, ovulation is induced within 24--30 h (Lee et aL, 1983). T h e integrity o f cyst granuiosa and theca cells may also be an important factor d e t e r m i n i n g the response to t r e a t m e n t (Bierschwal et aL, 1980). Luteal tissue d e v e l o p m e n t following G n R H t r e a t m e n t in cattle may be monitored indirectly by m e a s u r e m e n t of p r o g e s t e r o n e concentrations in plasma or milk. Garverick et aL (1976), using palpation per rectum, described cyst luteinization that was characterized by increased firmness and r e d u c e d cyst size in cows that r e s p o n d e d to GnRH or h u m a n chorionic g o n a d o t r o p h i n (hCG) treatment. Nakao et al. (1983) obtained a correlation of only 30% between palpation per rectum and milk progesterone concentration for assessing iuteinization of follicular cysts 10 days after G n R H treatment. Incorrect assessment of luteinization of follicular cysts by rectal palpation after treatment has been a problem (Whitmore et aL, 1979; Nakao & Kawata, 1980). With the introduction o f ultrasonography, it may be possible to m o n i t o r objectively the d e v e l o p m e n t of luteal tissue in o r d e r to evaluate the effectiveness o f treatment of ovarian cysts. T h e detection o f ovulation within 30 days o f treatment was defined as a positive response (Kesler et al., 1979). Plasma p r o g e s t e r o n e c o n c e n t r a t i o n is elevated 5-9 days following G n R H treatment in most cows that respond positively (Cantley et aL, 1975; Garverick et aL, 1976; Seguin et aL, 1976; Kesler et aL, 1978, 1979) as a result o f luteinization of the cyst or of o t h e r follicles with or without ovulation (Kesler et al., 1976; Youngquist, 1986). M t h o u g h e n d o c r i n e changes following G n R H treatment of ovarian cysts in cattle have been described (Cantley et aL, 1975; Garverick et al., 1976; Segui,a el aL, 1976; Kesler et al., 1978; Nanda et al., 1988), information on ultrasound changes following treatment is scarce. T h e aim of the present study was therefore to monitor ovarian t, ltrasotmd changes and to correlate these with plasma p r o g e s t e r o n e concentration in cows with follicular cysts that had been treated with GnRH.
MATERIAL A N D M E T H O D S
Five lactating Friesian cows on three dairy farnls were diagnosed as having follicular cysts (by palpation per rectum, uitrasonography and p r o g e s t e r o n e determination). T h e diagnosis by rectal palpation was based on the finding of single or multiple follicttlar structures of at least 25 m,n in diameter on oqe or both ovaries in the abse,ace of a co,'pus luteum. By ultraso,aography, a follicular cyst was defined as a tmiformly n o n e c h o g e n i c ovarian structure --> 25 ham in diam-
ULTRASONOGRAPHY OF FOLLICULAR CYSTS AFq'ER GnRFI
491
eter with a wall <3 ram. A plasma p r o g e s t e r o n e concentration of <0.9 ng ml-' was used to classify further the cysts as follicular. T e n days after initial diagnosis (day - 1 0 ) , cows were treated (day 0) with 500 lag synthetic GnRH (gonadorelin, 5 ml Fertagyl; Intervet) and ovarian ultrasonograplay and blood sampling were carried out weekly until day 35 after treatment. Ultrasound examination was p e r f o r m e d using an ultrasound scanner (SSD 210 DXII, Aloka, BCF Technology) e q u i p p e d with a 7.5 MHz rectal transducer (Aloka UST 55111). Representative images were obtained at each examination using an on-line thermal printer (Video Graphic UP 850, Sony). A blood sample was obtained by tail v e n e p u n c t u r e after each examination. Blood was centrifuged at 1000 g and stored at -20°C until analysis for progesterone. Plasma p r o g e s t e r o n e was measured using a validated radioimmunoassay (I~mchev et aL, 1976). T h e c u r r e n t inter and intra-assay coefficient of variations were 11.1 and 4.6% with a sensitivity of 0.05 ng ml -t. T h e r a p e u t i c success was c o n f i r m e d by the d e v e l o p m e n t o f CL a n d / o r luteinization of cysts (characterized by increased cyst wall thickness with granular echogenic a p p e a r a n c e as visualized by ultrasound) and increased plasma progesterone concentration. T h e r a p e u t i c failure was defined as cases in which cysts r e m a i n e d t m c h a n g e d or where new cysts developed without luteal d e v e l o p m e n t and where the cow failed to cycle normally. Mean p r o g e s t e r o n e concentration and cyst size on the day o f treatment were c o m p a r e d with values on day 7 after treatment using Student's t-test.
RESULTS
T h e relationship hetween cyst size and progesterone concentration is given in Figs 1 and 2 while Figs 3-6 are representative ultrasonograms of a follicular cyst (day 0), a luteinized follicular cyst (day 7), luteinized follicular cyst (day 14) and two GnRH-induced c o r p o r a lutea (day 7). Prior to treatnlent, r e c u r r e n t oestrus behaviour was shown by three cows and anoestrus o c c u r r e d ill the o t h e r two. All five cows r e s p o n d e d positively to GnRH treatment. By clay 7 after treatment, lnteinization of cysts (Figs 3 and 4) was n o t e d during ultrasonography in two cows while GnRH-induced CLs were observed in tile remaining three cows (Fig. 5). Changes n o t e d during weekly ultrasonography after treatment inchtded clouding of the uniformly n o n e c h o g e n i c a n t r u m of cysts, luteinization o f cyst walls, and a non significant (P>0.05) reduction in cyst size. Tile d e v e l o p m e n t of 1-4 CLs in tile ovary bearing tile cyst a n d / o r in the contralateral ovary was observed in three cows by clay 7 after treatment. These CLs were an immediate response to GnRH and resulted from ovulations of large follicles present at the time o f treatment. Ovtdations in these cows were not associated with oestrus. A secondary response to GnRH was observed by the d e v e l o p m e n t o f CLs by day 28 ,after treatment in the two cows that initially r e s p o n d e d by cyst luteinization. T h e cysts co-existed with the GnRH-induced CLs in three cows for 2 weeks and following subsequent oestrus and ovulation in four o f five cows by day 28 after treatment. In one cow that initially r e s p o n d e d by cyst luteinization, the cyst was not visible by day 28 (cyst
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ULTRASONOGRAPHY OF FOLLICULAR CYSTS AFTER GnRH
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Fig.. 3. Ultrasonogram of a follicular cyst. Diameter of cyst cavity=29 mm; diameter of cyst =31 ram. resolution); only a CL was observed d u r i n g ultrasonography. T h e G n R H - i n d u c e d CLs had regressed before s u b s e q u e n t oestrus in the three cows. All five cows were in oestrus 18.8+1.74 days after treatment. Mean (+SEM) plasma p r o g e s t e r o n e c o n c e n t r a t i o n s on day - 1 0 a n d day 0 were 0.2+0.13 ng m1-1 and 0.72+0.27 ng m1-1 respectively. P r o g e s t e r o n e c o n c e n t r a t i o n was significantly (P<0.05) elevated (5.8+0.67 ng ml -I) in all cows by day 7 comp a r e d to p r e - t r e a t m e n t values (day 0). T h e p r o g e s t e r o n e was c o n s i d e r e d to arise f r o m G n R H - i n d u c e d CLs in three cows a n d cyst luteinization in two cows.
DISCUSSION T h e visualization by u l t r a s o n o g r a p h y o f cyst luteinization a n d / o r the p r e s e n c e o f CLs in all cows by day 7 after G n R H shows that in r e s p o n d i n g cases evidence for t h e r a p e u t i c success could be c o n f i r m e d quickly. All five cases in this study
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Fig. 4. Ultrasonogram of a luteinized follicular cyst (7 days after treatment). Diameter of cyst cavity=28 ram; diameter of cyst=35 mm.
responded positively by either cyst luteinization a n d / o r CL formation. Failure to detect luteinization of follicular cysts accurately after GnRI-I by palpation per rectum has led to unnecessary treatment in many cows (Nakao et aL, 1983) and ultrasonography could prevent this problem. It has been reported that GnRH has a success rate of about 80% (i.e. ovulation detected within 30 days post-treatment) in treating ovarian cysts in cattle (Kesler & Garverick, 1982). In the present study, follicular cysts persisted (although with a slight decrease in size) for 3-4 weeks after GnRI-I administration in the presence of a CL; such cysts may not have any functional significance. One cow had only one follicular cyst and three large follicles (15-20 mm) at initial diagnosis (day -10), but three follicular cysts were visualized during ultrasonography at the time of treatment (day 0). This
ULTRASONOGRAPHY OF FOLLICULAR CYSTS AFTER GnRH
495
Fig. 5. Ultrasonogram of a luteinized follicular cyst (14 days after treatment). Diameter of cyst cavity=18 ram; diameter of cyst=33 mm.
supports\ the proposition that ovarian follicular cysts are dynamic structures (Cooke et al., 1990). T h e observation o f an increase in p r o g e s t e r o n e by day 7 after treatment is consistent with previous studies (Candey el aL, 1975; Garverick et al., 1976; Seguin et al., 1976; Kesler et aL, 1978). Progesterone concentration r e m a i n e d low (<0.9 ng ml -~) in all cows from day - 1 0 to day 0. Cantley et aL (1975) f o u n d higher mean pre-treatment concentration o f plasma p r o g e s t e r o n e in cows that r e s p o n d e d to G n R H than in those that did not respond. Kesler et al. (1979), however, did not find any difference in mean pre-treatment plasma progesterone concentration between cows with a positive response and those that did not respond to G n R H treatment.
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Fig. 6. Uhrasonogram of two GnRH-induced corpora lutea (CL) (7 days after treatment). CL diameter=20 nlm.
It is e v i d e n t t h a t u l t r a s o n o g r a p h y a n d p r o g e s t e r o n e m e a s u r e m e n t tools for e v a l u a t i n g t h e r e s p o n s e o f f o l l i c u l a r cysts to G n R H in cows.
a r e useful
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CooK~:, D. L., S~,TH, C. A., P,XRVET,J. R., YOUNGQUIST,R. S., BROWN, E. M. & GARVERICI~,H. A. (1990). Fate and turnover of ovarian follicular structures in dairy cattle. Journal of Reproduction and Fertility90, 37--46. DRosq, M. & THAXCm.:R,W. W. (1992). Application of g o n a d o t r o p h i c h o r m o n e as a therapeutic agent in animal reproduction. Animal Reproduction Science28, 11-19. EDQL'IST,1,. E., EKNAM,L., GUSTAI:SSON,B. & LIffDI.:LL,J. O. (1974). The clinical and hormonal effect of luteinizing hormone-releasing h o r m o n e (LH-RH) in cows with cystic ovaries. Nordish VetetfnaerMedicin 26, 552-6. GARVL.:RI(:K,H. A., Ka:SLER,D.J., Car,q't.EV,T. C., EL,~OR~, R. G., YOUNGQUISr,R. S. & BIERSCHWALL, C . J . (1976). H o r m o n e response of dairy cows with ovarian cysts after treatment with HCG or GnRH. Theriogenology6, 413-25. K..~N(:ltrv, L. N., DOBSON, H., WARD, W. R. & Fn-ZPATRK:K,R.J. (1976). Concentration of steroids in bovine peripheral plasma during the oestrous cycle and the effect of betamethasone treatment.Journal of Reproduaion and Fertility48, 341-5. Ka.:sLza, D.J., ELMORE,R. G., YOVNGQ.Lrlsr,R. S., BRO~, E. M., GARVERICK,H. A. & BIERSCHWAL, C.J. (1977). Ovarian morphology in dairy cows with ovarian cysts following treatment with GnRH.Journal of Animal Science45, Suppl. I, 176 (Abstr.). I~SLER, D.J. & GARVERXCK,H. A. (1982). Ovarian cysts in dairy cattle: a review.Journal of Anintal Science55, 1147-59. Kt:sH.r~, D.J., GARVERZC.K,H. A., CAUDLE,A. B., BtzmCmVAt., C.J., Et.XtORE, R. G. & YOUNGQUIST, R. S. (1978). Clinical and e n d o c r i n e responses of dairy cows with ovarian cysts to GnRH and PGF,., alpha. Journal of Animal Science46, 719-25. KVSLI-R,D.J., G,~RVF:RICK,H. A., EL~tO~, R. G., YOL:Nr.;QUIST,R. S. & BIEP~SCHWAL,C.J. (1979). Reproductive h o r m o n e s associated with the ovarian cyst response to GnRH. The~qogenol0~, 12, 109-14. ~rr()(:K, R.J., BRITr, J. H. & CoNvt:v, E.J. (1973). Endocrine responses after GnRH in luteal phase cows and cows with follictflar cysts.Journal of Animal Science37, 985-9. I.H:, C. N., MAt:RI(:~:,M. S., PI.:NNZGTON,J.A., HOVVMA~,W. F. & BROWN,M. D. (1983). Efficacy of gonadotropin-releasing h o r m o n e administered at the time of artificial insemination of heifcrs, postpartum cows and repeat b r e e d e r dairy cows. AmelJcanJournal of Vetelinmy Research 44, 2160-3. NAtc.\(~,T. & K.\wxr..x, K. (1980). Enzyme immunoassay of progesterone in bovine blood and milk and its application in monitoring the luteinization of ovarian follicular cysts after h o r m o n e treatment. Proceedingsof the 1l th International Congress on Disea.~esof Cattle, Israel, 916--33. N:~..xo, T., SL'C;HL-\SlH, A., SAGa, N., TSUNODA,N., K.~WATA,K. (1983). Use of milk progesterone enzyme immtmoassay for differential diagnosis of follicular cyst, luteal cyst and cystic corpus htteum in cows. A merican.]ournal of Vete~qnmyResearch44, 888-90. NA×DA, A. S., WARD, W. R. & DOBSON, H. (1989). Treatment of cystic ovarian disease in c a t t l e - - a n update. Vete~qnm3,BulMin 59, 537-56. NANt)..X,A. S., ~t."kRI), W. R., WILI.IAMS, P. C. W. & DoBso,',:, H. (1988). Retrospective analysis of different h o r m o n e treatments of cystic ovarian disease in cattle. Veterinmy Record 112, 155-8. Svt,vl×, B. E., Co×vvv, E. M. & OXZXDER,W. D. (1976). Effect of gonadotropin-releasing hormone and hun]an chorionic gonadotropin on cows with ovarian lollicular cysts. Ame~qcan Journal of Veterinmy Research37, 153-7. St.:(;t~x, B. E. (1980). Ovarian cysts in dait-y cattle. In Cunent Therapy in Then?ogenology, 1st Edn, ed. D. A. Morrow, pp. 199-204. Philadelphia: W. B. Saunders Co. Wtlnx~oRF, H. L., HC'RTt;ZN,j . P. & MArFtFR, E. C. (1979). Clinical response of dairy cattle with ovarian cysts to single or repeated treatments of gonadotropin-releasing hormone. Journal of American Vete~4nmyMedicalAssociation 174, 1113-15. YOUN(;QLrlS'I',R. S. (1986). Cystic follicular degeneration in the cow. In Current Therapy in 7"he~4ogenolo~,, 2nd Edn, ed. D. A. Morrow, pp. 243-6. Plailadelphia: W. B. Saunders Co.
(Acceptedfor publication23 December1993)