Indian J. Anim.. Res., () : 9 -, AGRICULTURAL RESEARCH COMMUNICATION CENTRE www.arccjournals.com / indianjournals.com BLOOD PROGESTERONE LEVEL DURING ESTROUS CYCLE IN GAROLE EWES AND THE LUTEOLYTIC EFFECT OF OXYTOCIN ADMINISTERED ON DIFFERENT DAYS OF ESTRUS CYCLE, PREGNANCY AND DICLOFENAC SODIUM MEDIATED EXTENDED LUTEAL PHASE Dipyaman Sengupta, Siddhartha Basu and Debasis Mazumdar West Bengal University of Animal and Fishery Science Kshudiram Bose Sarani, Kolkata-7 7, India. Received : -- Accepted : 8-8- ABSTRACT The effect of exogenous oxytocin injected on different days of estrus cycle and pregnancy on uterine PGF a mediated luteal regression in Garole ewes and compared it with physiological pattern of luteal growth and regression was investigated. Three consecutive days progesterone was measured after intravenous injection of oxytocin to non-mated ewes on day (n=), on day 8 (n=), on day (n=), on day 8 to pregnant ewes (n=) and diclofenac treated ewes (days to ) with extended luteal phase. All ewes were detected for heat twice daily by a vasectomized ram. In Garole ewes blood progesterone level starts increasing from day, reaches a maximum level on day 9, stays steady till day and thereafter starts declining from day onwards. Oxytocin injection on day, on day 8 and on day 8 (pregnant) had nonsignificant (P>.) effect on estrus cycle length as well as two successive day progesterone compared to control unlike in ewes injected with oxytocin on day and on day 8 (diclofenac treated). The results indicated that the endometrium of Garole ewes was insensitive to a luteolytic dose of oxytocin during the diestrous period but as the sensitivity was regained on day of estrous, it persisted thereafter. The presence of conceptus rendered the endometrium insensitive to oxytocin during and after the time of normal luteolysis. Key words : Diclofenac sodium, Oxytocin, Luteolysis, Progesterone, Garole ewes. INTRODUCTION In farm animals such as sheep, goat, cow and buffalo, oxytocin plays a vital role in luteolysis by increasing endometrial prostaglandin synthetase activity leading to production of luteolysin PGF á and this is evident from the findings of Fairclough et al. (98) who prolonged the length of estrus cycle in cows and ewes by injection of oxytocin antagonist. In this study It was tried to find out the luteolytic effect of oxytocin in Garole ewes when administered on different days of estrus cycle, early pregnancy and diclofenac sodium (prostaglandin synthetase) mediated extended luteal phase. Functional luteolysis was indicated by a drop in blood progesterone level. Therefore, blood progesterone level after oxytocin administration has been estimated to indicate luteolysis. It was also measured The progesterone in Garole ewes on each day of estrus to assess was measured the normal functional
9 INDIAN JOURNAL OF ANIMAL RESEARCH activity of corpus luteum (CL) during the length of estrous cycle. Thus the physiological pattern of progesterone secretion by the corpus luteum of Garole ewes with conditions where the uterus was sensitized by oxytocin on different periods of estrus cycle was compared. The present work has been performed on Garole ewes. This breed of sheep is native to the Sunderban of West Bengal, the ewes weighing approximately to kg. with average litter size of.8. This is the only Indian breed carrying Fec B mutation similar to Booroola Merinos of Australia (Turner, 98) that is responsible for more than one ovulation (generally to ) per estrus cycle (Piper et al., 98; Montgomery et al., 99). MATERIALS AND METHODS Animals : A total of adult, normal cycling (estrus cycle length of approximately to days) and non-mated Garole ewes of about years of age and approximately - kg body weight were used for the present experiment. All animals used for the present investigation were kept under uniform standard of managemental practices including the schedule of nutrition. Experiments were conducted on ewes those were estrus synchronized by two intramuscular injections of PGF á (Lutalyse, Pfizer India Ltd.) at the rate of mg per ewe 9 days apart. These experiments were conducted at Haringhata Farm, Dist. Nadia, W. Bengal, India. Experiment : Starting from day (day = day of synchronized heat), daily blood samples were collected from six ewes until they were detected in heat. Estrous was detected by a vasectomized ram twice daily. This group (group A) served as the control for all groups in experiments and group H of experiment. Experiment : IU oxytocin dissolved in ml. normal saline solution (Syntocinon inj., Novartis India Ltd.) was injected into the right jugular vein on day to ewes of group B (n=), on day 8 to ewes of group C (n=) and on day to ewes of group D (n=). Experiment : Out of 7 mated ewes, 8 ewes were confirmed pregnant by non-return to heat on day and average day 8 progesterone of. ±. ng/ml. They were then divided into groups. On day 8, one group (group E, n=) was injected intravenously with IU oxytocin dissolved in ml. normal saline solution (Syntocinon Inj., Novartis India Ltd.) into the right jugular vein while the control group (group F, n=) was kept untreated. Experiment : On days to, ewes (n=) were injected intramuscularly with diclofenac sodium (Syntocinon inj., Novartis India Ltd.) at the rate of mg/kg b.wt. twice daily per ewe. They were then divided into two groups viz. G and H. On day 8, group G (n=8) was injected intra-muscularly with IU oxytocin dissolved in ml normal saline solution (Syntocinon Inj.R; Novartis India Limited) into the right jugular vein while group H (n=) was kept untreated. Starting from the day of oxytocin injection in groups B, C, D, E, F, G and H the animals were detected for heat twice daily by a vasectomised ram. Blood was collected for three consecutive days starting from the day of oxytocin injection in groups of experiments and. In experiment, blood samples were collected from group G & H on each day starting from day until the animals returned to heat. On the day of drug injection, blood samples were collected before the injections were made. The collected blood samples were allowed to clot in sterile glass test tubes kept in slanting rack for hour for separation of serum. The serum samples were then packed in properly labeled sterile tubes and stored at - C until use. Progesterone ELISA : Serum s of progesterone were assayed in duplicate by Enzyme- Linked ImmunoSorbent Assay (ELISA) by using ELISA kit (Lilac Medicare) following the manufacture s protocol. The serum samples were diluted with distilled water times to remove any matrix effect before performing the assay. The intra and inter-assay coefficients of variation for this assay
were.8 % and.9 % respectively and the limit of the sensitivity was pg. Statistical Analysis : Length of estrus cycles has been compared by Duncan s test (SPSS, ver.., 997). Progesterone on a particular day has been compared by t-test. Progesterone between two subsequent days has been compared by paired t-test. The change in of progesterone with day was analyzed by regression analysis (SPSS, ver.., 997). RESULTS AND DISCUSSION Garole ewes have an estrus cycle length of.9 ±. day. At estrous the mean progesterone was.8 ng/ml (Figure ). The progesterone rose significantly (P<.) from day till day 9 due to growth and maturation of steroidogenic cells. Schrick et al. Vol., No., 97 (99) detected the presence of new luteal tissue by ultrasonographic technique as late as to days in a group of Suffolk cross ewes. During days 9 to the progesterone remained steady indicating maximum growth of luteal tissue and this is in agreement with Clun Forest ewes (Pant et al., 977) and Suffolk cross ewes (Schrick et al., 99). However, the progesterone fell significantly (P<.) on each day from day onwards with the animals returning to heat on day.8 ±.. Therefore day can be considered to be the day of onset of luteolysis in this breed of sheep. Injection of oxytocin on days and 8 to groups B and C respectively resulted in normal estrous cycle length (Table ) and similar progesterone (P>.) of treated and Table : Estrus cycle length (Mean ± S.E) of ewes injected with oxytocin on different days of estrus cycle. Group n Day of oxytocin injection Day in estrus(day) Significance A.8 ±. B.7 ±. P>. a C 8. ±. P>. b D. ±. P<. c E 8 NR * P>. d F NR G 8 8 9. ±. P<. e H NR NR: Not reported in heat by day, NR: Not reported in heat by day, *: one ewe came to heat on day, a: compared with group A and C, b: compared with group A and B, c: compared with group A, B and C, d: compared with group F, e: compared with group H. Table : Progesterone (Mean ± S.E) of ewes treated with oxytocin on different days of estrus cycle. Day of oxytocin injection n Progesterone Progesterone Progesterone (ng /ml) on day a (ng /ml) on day b (ng /ml) on day c. ±.9.9 ±.. ±. 8. ±.. ±..7 ±..7 ±..7 ±. 8 (pregnant). ±.9. ±.. ±. 8 (diclofenac treated) 8.8 ±.. ±.9.8 ±. a: day = day of oxytocin injection, b: day = day after the oxytocin injection, c: day = Second day after the oxytocin injection.
98 INDIAN JOURNAL OF ANIMAL RESEARCH control group on two consecutive days after oxytocin treatment (figure A and B). However, injection of oxytocin on day (group D) resulted in significantly (P<.) shortening of estrous cycle length compared to groups A, B and C (Table ) as well as significant (P<.) difference in progesterone from control group after oxytocin treatment (Table, Figure C) and significant difference (P<.) in pre and post oxytocin treated progesterone. This indicates that the uterus is refractory to oxytocin on days and 8 and the sensitivity to oxytocin is regained on day resulting in luteolysis. Since oxytocin exerts its effect on the endometrium through oxytocin receptors, it is likely that increased oxytocin receptors expression on day might be responsible for luteolysis. This view is also supported by the findings of Mirando et al. (99) in cows and Sheldrick and Flint (98) in ewes, who found a large rise of oxytocin receptor during the time of luteolysis that was responsible for relative large increase in endometrial PGF a secretion in response to oxytocin. However injection of oxytocin to pregnant ewes of experiment (group E) did not result in luteolysis evident by ewes not returning to heat by day (Table ) and the progesterone did not decline significantly after oxytocin treatment (Fig. D, Table ). The results indicate that the presence of conceptus annuls the luteolytic effect of oxytocin. Interferon tau is secreted by the conceptus from day onwards (Roberts et al., 999 and Bazer, 99) that is responsible for maternal recognition of pregnancy probably by desensitizing the endometrium to oxytocin. Such a view is supported by the work of Spencer and Bazer (99) who found that IFNô suppresses endometrial oxytocin receptor thereby abrogating luteolysis. The findings of this study also proved that this effect of conceptus on endometrium was maintained after the time of normal luteolysis. Fig. : Progesterone on different days of estrus cycle in Garole ewes. P (day -9) =.7 day.9; P<. P (day 9-) = -. day +.7; P>. Progesterone P (day -) =.7 day +.; P>. P (day -) = -.8 day +.7; P<. 7 8 9 Day of estrus
Vol., No., 99 Fig. : Effect of diclofenac treatment (day -) and thereafter oxytocin (day 8) on progesterone of Garole ewes. Line with triangles denotes diclofenac treated (day -) group. Line with squares denote diclofenac (day -) + oxytocin treated (day 8) group. P (day -) = -.7 day + 9.9; P<. progesterone 7 8 9 Day of estrus cycle Whether the sensitivity of uterus to oxytocin retained over the time of luteolysis once it has been regained by day? Abrogation of release of luteolytic dose of PGF a during the time of luteolysis by cyclooxygenase inhibitor diclofenac sodium resulted in prolongation of estrus cycle (not reported in heat by day ) of control ewe in experiment. Similar findings have also been reported by Kraeling et al. (98) in pigs and Lewis and Warren (977) in cows by injection of indomethacin during the time of luteolysis. However, oxytocin treatment to ewes of group G on day 8 brought them to heat by day 9. ±. and the progesterone differed significantly (P<.) from control (Figure E). This indicates that the uterus was sensitive to a luteolytic dose of oxytocin. Thus it could be concluded that, once the uterus regained its sensitivity to oxytocin by day it was retained over the time of luteolysis. If the uterus had regained its sensitivity to oxytocin by day then the fact that it did not release luteolytic dose of PGF a after diclofenac treatment regimen could be due to the fact that prostaglandin synthetase inhibitors might have depressing effect on oxytocin secretion by the neurohypophysis. Such a view is supported by the findings of Knigge et al. () in rats who reported that prostaglandin synthetase inhibitor indomethacin dose-dependently reduced the oxytocin response to histamine or bacterial lipopolysaccharide. Baguma and Nathanielsz (998) reported that administration of nimesulide to ewes lowered sensitivity to oxytocin through down-regulation of oxytocin receptor. Thus administration of diclofenac sodium not only resulted in inhibition of cyclooxygenase activity during days - but also sufficiently suppressed the secretion of oxytocin from neurohypophysis as well as reducing the sensitivity of endometrium to oxytocin that resulted in extension of luteal life span. However, significant decline in progesterone with advancement of day after diclofenac treatment regimen (Figure )
INDIAN JOURNAL OF ANIMAL RESEARCH Fig. : Effect of oxytocin, injected on different days of estrus cycle or pregnancy, on progesterone (ng/ ml) of two successive days in Garole ewes. White bars denote the progesterone of control group. Black bars denote the progesterone of treated group. oxytocin injected on day Oxytocin injected on day 8 (pregnant) Progesterone day day 7 Progesterone 8 day 9 day A C Progesterone oxytocin injected on day 8 day 9 day Progesterone oxytocin injected on day 8 (diclofenac treated) day 9 day B D Progesterone oxytocin injected on day 8 (diclofenac treated) day 9 day E proves that the sensitivity of endometium to oxytocin is slowly regained as the effect of diclofenac waned with day resulting in partial luteolysis. It is conclude from the above discussion that the functional activity of corpus luteum in Garole ewes starts from day onwards, reaches maximum by day 9 and luteolysis starts by day with ewes coming to heat by day. The uterine sensitivity to oxytocin is attained by day of estrous resulting to luteolysis. However, the presence of conceptus is able to abrogate luteolysis by a test dose of oxytocin during and after the time of luteolysis. Once the sensitivity of uterus is
Vol., No., attained by day, it was retained thereafter. Prostaglandin synthetase inhibitors like diclofenac sodium inhibited the release of uterine PGF a during and after the time of luteolysis. REFERENCES Baguma-Nibasheka, M. and Nathanielsz, P. W. (998). J Soc Gynecol Investig, (): 9-99. Bazer, F. W. (99). Proc Soc Exp Biol Med, 99: 7-8. Fairclough, R. J. et al., (98). Journal of Reproduction and Fertility : -9. Knigge, U. et al., (). Journal of Neuroendocrinology, (): 9-9. Kraeling, R. R. et al., (98). Biology of Reproduction, : -8. Lewis, P. E. and Warren, J. E. (977). Journal of Animal Science, : 7-77. Mirando, M. A. et al., (99). Biology of Reproduction, 8: 87-88. Montogomery, G. W. et al., (99) Endocrine Reviews, : 9-8. Pant, H. C. et al., (977). Journal of Endocrinology, 7(): 7-. Piper, L. R. et al., (98). In: Genetics of Reproduction in sheep. (Eds. R. B. Land & D. W. Robinson), Butterworths, London, pp. -. Roberts, R. M. et al., (999). Placenta, : 9-. Schrick, P. N. et al., (99). Biology of Reproduction, 9: -. Sheldrick, E. L. and Flint, A. P. F. (98). Journal of Endocrinology, : 9-8. Spencer, T. E. and Bazer, F. W. (99). Endocrinology, 7: -7. SPSS 7. copyright 997 by SPSS Inc., USA Base 7. Application Guide. Turner, H. N. (98). In: The Booroola Merino (Eds. L. R. Piper, B. M. Bindon, R. D. Nethery), CSIRO, Melbourne, pp. -7.