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'I - v ' " .f '- . o-Eibb'i 2K1; 1, nM J, k a: 51‘} '8‘: 5|.3 ‘4' n; j '3 {a z s' r. , ‘ It'l.‘ - 3.3:; . 953v 0~a.‘.,-:. ‘1 ~ I .n.: ..-:.. Ii... ‘ - ‘.- 4 - _ I ,‘i,-'..- . 2" ‘ " ' - . : b a n... ( a'..Ja‘.‘.-~:.: .au \.' .. 14; u .1, . .34 gas L g “10!! I“..- ‘ o'r‘; :' :: "'9?an "o“ {d - .' 8. ~ ‘ . b 4 a - -’ t . ' ‘ ‘ b .‘w' 5-3.3... . d d c .& ’ J. ‘ a \s’ 3 3 n ~_.....- A LIBRARY L Michigan State University THESIS ABSTRACT ENDOCRINE RESPONSES AFTER GONADOTROPIN OR GONADOTROPIN RELEASING HORMONE IN COWS WITH OVARIAN FOLLICULAR CYSTS By Roger John Kittok In a survey designed to monitor serum hormone changes during several phases of reproduction, blood was collected from 91 lactating Holstein cows on days 0 (estrus), 2, 4, 7 and 11 during postpartum estrous cycles. In six cows, ovarian follicular cysts were detected (E = 116 days of lactation, range 37 to 167 days), and blood was taken on days 0, 2, 4, 7 and 11 after injection of 10,000 IU human chorionic gonadotrOpin (HCG) and during post-treatment estrous cycles. Before and after the cystic period, estrous cycles were classified according to length, normal (18 to 23 days) or abnormal (3 to 15 days). Steroids were quantified by radioimmunoassay (estradiol and estrone) or competitive protein binding assay (glucocorticoids and prOgesterone). During normal length cycles in cows with follicular cysts, serum estradiol on days 2 to 7 (11:2 pg/ml) was less than on reSpective days of abnormal length cycles (16:1 pg/ml; P<0.025) in these same animals. Roger John Kittok Serum progesterone in cows which subsequently became cystic increased (P<0.005) from 0.1:0.l to l.2:0.3 ng/ml between days 0 to 2 and days 4‘to ll of normal length cycles. After injection of HCG in cows with follicular cysts, progesterone increased (P<0.05) from 0.8i0.4 ng/ml on days 0 to 2 to 2.8:l.0 ng/ml on days 4 to 11. In these cows, estrous cycles with normal length were typified by higher (P<0.01) progesterone (l.2:0.3 ng/ml) than cycles with abnormal length (0.1:0.1 ng/ml) on days 4 to 11. Corticosterone, cortisol and estrone did not differ between normal and abnormal estrous cycles or after HCG treatment. These data suggest HCG increased serum prOgesterone but did not influence estrOgens or glucocorticoids. Luteinizing hormone (LH) and progesterone were assayed in blood sera collected before and after gonadotrOpin releasing hormone (GnRH) administration (iv) to nine Holstein cows, five of which had ovarian follicular cysts. The four remaining cows were treated on day 10, 11 or 13 of the estrous cycle. GnRH was given in three doses of 100 09 each, at 120 min. intervals. Serum LH (ng/ml) in cows with ovarian follicular cysts was l.2i0.3, 23.8:7.5, l4.3:2.8 and 4.7:1.2 at O, 120, 240 and 360 min. after GnRH admin— istration, respectively, compared to concentrations of 0.5:0.1, 5.9:0.9, 7.2:l.2 and 2.9:0.5, reSpectively, for luteal phase cows (P<0.01). Peak LH occurred at 170 min. Roger John Kittok (33.9:6.6 ng/ml) in cystic cows and 145 min. (17.3i2.4 ng/ml) in luteal cows. Change in LH concentration after each iné jection of GnRH was greater (P<0.01) in cows with follicular cysts. Serum progesterone (ng/ml) increased from 0.9:0.4 at 0 min. to 2.0:l.2 at 20 min. in cystic cows, but this reSponse was due to increased serum prOgesterone in two animals. PrOgesterone increased (P<0.05) from 5.9:0.8 at 0 min. to 10.3:0.9 and 8.2:1.0 at 260 and 360 min., respec- tively, in luteal phase cows. Serum progesterone was 3.6:1.4 on day 11 after GnRH administration in cows with ovarian follicular cysts although only one cow had a palable corpus luteum. All of the cows with follicular cysts exhibited estrus 20 to 24 days after GnRH treatment. Serum estradiol and estrone (pg/ml) prior to GnRH treatment for cows with follicular cysts averaged 9.4:3.1 and 3.8:0.5, respectively, which was not different from means for cows treated during the luteal phase of the estrous cycle (8.8:0.7 and 3.0:0.4, reSpectively). Administration of repeated does of GnRH produced an LH response similar to the preovulatory surge in the bovine and initiated estrous cycles in cows with ovarian follicular cysts. ENDOCRINE RESPONSES AFTER GONADOTROPIN OR GONADOTROPIN RELEASING HORMONE IN COWS WITH OVARIAN FOLLICULAR CYSTS BY Roger John Kittok A THESIS Submitted to Michigan State University in partial fulfillment of the requirements . for the degree of MASTER OF SCIENCE Department of Dairy Science 1974 53 ACKNOWLEDGMENTS The author wishes to gratefully acknowledge the guidance and willing assistance that was given by his major professor, Dr. J.H. Britt. Also he wishes to thank Dr. C.A. Lessiter and the Department of Dairy Science for facilities and financial assistance necessary for his graduate study. The assistance of Drs. E.M. Convey, L.A. Edgerton, H.D. Hafs, H.A. Tucker and R.P. Wettemann in design and implementation of experiments, laboratory techniques and critical review of data was appreciated. And he is grateful for the advice and approval received from his graduate guidance committee, Drs. R.S. Emery and G.D. Riegle. ii TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . INTRODUCTION . . . . . . . . . . . . REVIEW OF THE LITERATURE . . . . . . . . Definition of Ovarian Follicular Cysts . . Incidence of Ovarian Follicular Cysts . . Possible Causes of Ovarian Follicular Cysts . . . . . . . . Enzymatic and Steroidogenic Activity within the Ovarian Follicular Cyst . . Hormone Data and Pituitary Cytology in Cows with Ovarian Follicular Cysts . . Relationship Between Milk Production and Ovarian Follicular Cysts . . . . Treatment and Spontaneous Recovery of Cows with Ovarian Follicular Cysts . . . . Induction of Ovarian Follicular Cysts . . Gonadotropin Releasing Hormone in the Bovine . . . . . . . . . . METHODS AND MATERIALS . . . . . . . . . Experiment 1. . . . . . . . . . . Experiment 2. . . . . . . . . . . RESULTS AND DISCUSSION . . . . . . . . . Experiment 1. . . . . . . . . . . Experiment 2. . . . . . . . . . . SUMMARY AND CONCLUSIONS . . . . . . . . . BIBLIOGRAPHY . . . . . . . . . . . . iii Page iv 0500001“ \l\lU1 10 11 ll 13 13 14 19 19 20 29 33 LIST OF TABLES TABLE Page 1. Serum steroids during estrous cycles and after HCG in cows with ovarian follicular cysts and during estrous cycles in non-cystic herdmates . . . . . 21 2. Serum LH changes after GnRH in cows with follicular cysts or corpora lutea . . . . 23 3. Serum progesterone after GnRH in normal luteal phase cows and cows with ovarian follicular cysts . . . . . . . . . 25 4. Serum progesterone in luteal phase cows after GnRH administration . . . . . . 25 iv LIST OF FIGURES Figure Page 1. Serum LH after GnRH in cows with ovarian follicular cysts or corpora lutea . . . . l6 2. Serum progesterone after GnRH in cows with ovarian follicular cysts . . . . . . . 26 INTRODUCTION Endocrine changes during puberty, estrous cycles, gestation and parturition have been studied in the bovine female. Thus, some understanding of the processes involved in reproduction has arisen. However, such information during aberrant physiolOgical states is not complete. Ovarian follicular cysts cause a significant decrease in, reproductive efficiency in dairy cattle and the etiOIOgy of the condition is lacking. Parameters other than hormonal changes have been studied in cows with ovarian follicular cysts and the conclusions are quite variable. Information is limited on serum hormone concentrations in cows with ovarian follicular cysts and although human chorionic gonadotrOpin (HCG) is an accepted treatment for follicular cysts, there are no reports of serum hormone levels in cows after treatment. Synthesis of gonadotrOpin releasing hormone (GnRH) made it available in quantities that allow study of endocrine reSponses elicited from domestic animals, including the bovine. While cattle of many different physiological states have been subjected to the releasing hormone, re- Sponses obtained from cows with aberrant ovarian function have not been reported. Specifically, the objectives of this work were, (1) to quantify serum steroid hormones in cows before ovarian follicular cysts were detected and after treatment with HCG and (2) to quantify serum luteinizing hormone and prOgesterone responses in cows with ovarian follicular cysts after administration of gonadotrOpin releasing hormone. REVIEW OF THE LITERATURE Although many reports have dealt with ovarian follicular cysts, conflicting observations and lack of research in some critical areas prevent concise summariza- tion of the etiology of this condition. Thus, the cause of this aberrant ovarian function is not fully understood. Perhaps confusion concerning the definition of follicular cysts or existence of differing types of cysts may account for the lack of knowledge concerning the disorder. Definition of Ovarian Follicular Cysts Large Ovarian Follicules (>2.0 cm) were classified ovarian follicular cysts (Albrechtsen, 1917). Garm (1949) used persistent follicules and lack of luteal tissue as criteria. To be considered an ovarian follicular cyst, Casida, McShan and Meyer (1944) Specified a follicle must be larger than 2.5 cm in the absence of palpable luteal tissue. Others Specified both size of follicle and length of time palpable luteal tissue was absent from the ovary: >1.0 cm for more than 7 days (Johnson, Legates and Ulberg, 1966); >l.5 cm for more than 7 days (Johnson and Ulberg, 1967); >2.0 cm for more than 14 days (Marion and Gier, 1968); and >2.5 cm for more than 10 days (Morrow 33 31., 1966). Roberts (1955) and Henricson (1957) diagnosed animals with ovarian follicular cysts after irregular estrous cycles or anestrous was observed, but criteria used was not given. Follicles maintained over 14 days in the absence of palpable luteal tissue, failure to ovulate or undectable luteal tissue were conditions used by Callahan et_a1. (1971). Incidence of Ovarian Follicular Cysts Several researchers have suggested that ovarian follicular cysts occur in 10 to 15 percent of the dairy cattle studied. Wiltbank, Tyler and Casida (1953) observed 31 cases of follicular cysts out of 274 postpartum repro- ductive periods (11.3%) and Morrow et 31. (1966) observed 357 postpartum cycles with an incidence of follicular cysts of 12.3 percent. In a much smaller survey, Callahan 23 El‘ (1971) found 10 out of 65 postpartum cows with follicular cysts (15.4%). Incidence of follicular cysts during the first post- partum estrous cycle was 22.3 percent compared with 6.2 percent during the second cycle (P<0.005) (Morrow 33 31., 1966). Wiltbank et 21. (1953) indicated 12.0 percent of cows had ovarian follicular cysts during 15 to 74 days post— partum, but only 4.6 percent were observed to be cystic from 75 to 300 days (P<0.05). Of 29 follicular cysts that occurred 11 to 78 days postpartum, 24 (83%) were observed 11 to 35 days postpartum (Marion and Gier, 1968). The incidence of ovarian follicular cysts in heifers is low, 4.6 percent (Wiltbank EE.El°' 1953). Roberts (1955) reported that out of 352 cases treated for ovarian follicular cysts, only 3 were heifers. Incidence of ovarian follicular cysts increased with number of times milked per day (Casida and Chapman, 1951). In cows not milked, mostly heifers, 3.4 percent were observed to have ovarian follicular Cysts compared with 6.8 percent for animals milked 2 times per day and 10.6 percent in cows milked more than 2 times per day. Wiltbank gt_gt. (1953) observed 72.6 percent of the annual total of follicular cysts in November and Roberts (1955) treated 48.4 percent of the annual total during the period of December to February. Cows calving from August to December had a 13.4 percent incidence of ovarian folli- cular cysts compared with 8.6 percent of those calving from January to July (Marion and Gier, 1968). In addition, Morrow gt gt. (1966) also commented that follicular cysts occurred more frequently from November to January. Possible Causes of Ovarian Follicular Cysts Albrechtsen (1917) considered that formation and maintenance of follicular cysts were related to infection of the uterus at the last parturition. Although later research (Garm, 1949) refutes this possible cause, Morrow gt gt. (1966) reported that the incidence of follicular cysts in cows normal at calving was 8.3 percent compared with 23.4 percent in cows with abnormal calvings. Cows, abnormal at the last calving, had been afflicted with one or more of a variety of periparturient diseases, such as metritis, milk fever, mastitis or ketosis, or had suffered from abortion, dystocia or retained placenta. Others (Garm, 1949; Henricson, 1957) concluded that follicular cysts involved genetic prediSposition and stress of heavy lactation provoked its incidence. Genetic predis- position is possible, Casida and Chapman (1951), from re- cords in a single Holstein herd, computed a coefficient of heritability of becoming cystic sometime in life of 0.43. Although observing a small number of animals, Wiltbank gt gt. (1953) reported that line breeding increases the incidence (P<0.01) of ovarian follicular cysts. Callahan gt gt. (1971) reported that cows destined to have ovarian follicular cysts exhibit earlier follicular growth (15:1.2 vs 20:2.3 days), delayed ovulation (29:3.9 vs 17:1.2 days), delayed corpus luteum formation (39:3.7 vs 25:1.5 days) and earlier first estrus after parturition (25:3.0 vs 34:3.3 days) compared with normal cows. However, Morrow gt gt. (1966) found the interval between parturition and first estrus longer (P<0.01) than in normal cows (37 vs 20 days). Enzymatic and SteroidOgenic Activity within tHe Ovarian FollicuIartgyst Nakama (1969) reported that luteinized portions of follicular Cysts have enzyme activities similar to luteal cells of functional corpora lutea. Steroid producing cells in follicular Cysts degen- erate (Short, 1962); Cyst fluid contains large amounts of prOgesterone but little or no detectable androstenedione, 17B-estradiol, estrone or 17a—prOgesterone. Short (1962) suggested prOgesterone was produced by luteal tissue within the cyst and estrOgens were produced in the ovary, but not within the follicular Cyst. By histological examination, anovulatory follicles after superovulation with gonadotropins are similar to Spontaneous ovarian follicular cysts (Archbald gt gt., 1973). Hormone Data and Pituitary CytolOgy in Cows with’Ovarian Follicular Cysts In addition to the above mentioned data on steroid hormone concentrations in cyst fluid (Short, 1962), Erb gt gt. (1971) have measured urinary estrOgens, and serum glucocorticoids, prOgesterone and luteinizing hormone postpartum in cows which deve10ped ovarian follicular cysts. No differences were observed in estrOgens and prOgesterone from days 0 to 2 to day 7 of estrous cycles which ocurred befOre day 60 postpartum. And levels of these hormones were not different from normal cows on reSpective days. Garverick gt gt. (1973) measured serum estrOgen, prOgesterone and luteinizing hormone on days 5, 6, 7, 8, 9, 11 and 13 following parturition. Levels of these hormones in cows destined to have ovarian folli- cular Cysts did not differ significantly from normal cows. In cows induced to retain follicles with multiple injections of estrOgen and prOgesterone (Erb gt_gt., 1973), plasma estrOgen and prOgesterone were higher and luteinizing hormone lower during the period prior to Cyst detection than the period in which cysts were present. After the ovarian follicular Cysts were detected, treatment with human chorionic gonadotrOpin increased prOgesterone. Anterior pituitary acidOphils in.cows with folli- cular cysts have increased granulation, with the nucleus being pycnotic (Jubb and McEntee, 1955). Both of the above changes are indicative of inactivity. Jubb and McEntee (1955) also found the baSOphils of these animals had increased activity. This suggests hyperproduction or failure to release gonadotrOpins. The hypothesis of failure to release gonadotrOpins is supported by the report of increased pituitary gonadotrOpin content in animals that retained ovarian follicles after oxytocin treatment (Donaldson and Hansel, 1968). In cows with ovarian follicular cysts that were exhibiting signs of nymphomania, 200 IU ACTH increased serum corticosterone, cortisol and prOgesterone while estradiol and LH decreased (Troconiz gt gt., 1973). In these same animals, dexamethasone administered three days later resulted in decreased serum corticosterone, cortisol, prOgesterone, estradiol and LH. Three of five animals ovulated following dexamethasone treatment. Relationship Between Milk Production and Ovarian FolIicuIar Cysts Relationship between milk production and incidence of ovarian follicular Cysts is not clear. Johnson et al. (1966) reported cows with ovarian follicular Cysts pro- duced more (P<0.01) than herdmates during 305 days of lactation (DHIA, 2X, adjusted to mature equivalent and for days Open). On the basis of previous lactations, it was concluded that cows produced more because follicular cysts were present rather than high production indUCing cysts. Marion and Gier (1968) observed 22.6 percent of cows producing over 30 kg of milk per day had ovarian follicular Cysts, significantly more (P<0.001) than animals producing 22 to 30 kg per day (9.9%). In addition, Henricson (1957) reported a positive correlation between high milk production and incidence of follicular cysts. However, others have found no such relationship (Casida and Chapman, 1951; Wiltbank gt gt., 1953; Morrow gt gt., 1966). 10 Treatment and Spontaneous Recovery of Cows with Ovarian Follicular Cysts AS a means of treatment, rupture of the follicular cyst has been used and Albrechtsen (1917) reported 65.9 percent conception using this method. Using unfractionated pituitary extracts, Casida gt gt. (1944) treated 96 cows and observed corpora lutea in 74 (77.1%) and normal estrus in 71.8 percent within 31 days. Human chorionic gonadotrOpin (HCG) has been admin- istered to cows with follicular cysts. Injected intravenously 5,000 IU produced a 88.2 percent recovery rate in 51 cases (Roberts, 1955). Roberts (1957), using 500 to 1,000 IU HCG injected directly into the Cyst, observed 83.3 percent recovery. And of the 23 cystic animals subjected to 10,000 IU HCG, half intravenously and half intramuscularly, 85.7 percent recovered (Morrow gt gt., 1966). An intravenous treatment of 3,000 IU HCG and 100 mg prOgesterone, admin- istered to 110 cows with follicular Cysts effected 83.6 percent recovery (Trainin and Alder, 1967). Others have administered prOgesterone as treatment for follicular Cysts (Johnson and Ulberg, 1967). PrOges- terone was injected daily for 14 days in doses of 50 and 100 mg and resulted in normal estrus within a few days post—treatment in 61.5 and 62.5 percent of the animals, reSpectively. 11 Four animals (13.3%) conceived out of 30 control cows with ovarian follicular cysts (Johnson and Ulberg, 1967). Morrow gt gt, (1966) treated animals only if ovarian cysts persisted after 60 days postpartum; 48.0 percent of cystic cows recovered without treatment before 60 days postpartum. Induction of Ovarian Follicular Cysts In the bovine, retention of ovarian follicles has been induced by various treatments. Foote gt gt. (1959) removed corpora lutea from 21 heifers in two consecutive cycles, and induced 3 cysts after the first removal and 9 after the second. Anovulatory follicles occur after oxyto- cin treatment on days 4 or 7 of the cycle (Donaldson and Hansel, 1968). Four heifers, treated 2 times each with 250 or 500 mg of 17B—estradiol after corpus luteum removal on day 13 of the cycle resulted in 4 follicular cysts (Whitmore, Ginther and Casida, 1972). Erb gt gt. (1973) induced cystic follicles in 7 of 8 cows receiving twice daily injections of prOgesterone (0.25 mg/day/kg) and 17B- estradiol (0.1 mg/day/kg) for 7 days beginning on day 3 of the estrous cycle. Gonadotropin Releasing Hormone in the Bovine The recent synthesis of gOnadotrOpin releasing hormone (GnRH) (Matsuo gt gt., 1971) has allowed its use 12 for study of endocrine reSponses. In the bovine, reSponses have been elicited from mature bulls (Golter gt gt., 1972; Zolman, Convey and Hafs, 1972; Zolman and Convey, 1973), pubertal bulls (Mongkongpunya gt gt., 1973) and Cycling heifers (Kaltenbach gt gt., 1974; Peck gt gt., 1973; Zolman gt gt., 1973). And ovulation has been observed in reSponse to releasing hormone when administered after with- drawal of a Synchronizing dose of prOgesterone (Mauer and Rippel, 1972; Kaltenbach e_t gt., 1974). METHODS AND MATERIALS Experiment 1 This study was part of a larger survey (Edgerton and Hafs, 1973) designed to monitor serum hormone changes during several phases of reproduction in dairy cattle. During the survey, which involved twice weekly rectal palpations of 91 cows after parturition, six lactating Holstein cows (Efill6 days of lactation, range of 37 to 167 days) retained at least one ovarian follicle (>2.5 cm in diameter) for at least 14 days in the absence of a palpable corpus luteum; then, 10,000 IU human chorionic gonadotrOpin (HCG) (Follutein, E.R. Squibb and Sons, Inc.) was administered, half intravenously and half intramus- cularly. Blood was collected via jugular puncture during estrous cycles before the ovarian follicular Cysts developed (available from the survey of Edgerton and Hafs, 1973), during the period following treatment with HCG and during post-treatment estrous Cycles. Estrous cycles before follicular cysts were detected and after the condition was no longer present were classified as normal length (18 to 23 days) or abnormal length (3 to 15 days). Of the six cows with follicular Cysts, one animal did not have an estrous Cycle of normal length, another animal was 13 14 represented twice in the abnormal cycles and all are repre- sented once in the period following HCG. For all periods, blood was taken on days 0 (estrus or day of HCG administra- tion), 2, 4, 7 and 11. In addition, blood was collected on day 0 and 7 of normal estrous Cycles in four contemporary non-Cystic herdmates. All animals were observed twice daily for signs of estrus. After elution of serum extracts by column chromato- graphy (Sephadex LH-20), apprOpriate fractions were subjected to radioimmunoassay for estradiol and estrone (Wettemann gt gt., 1972) and to competitive protein binding assay for corticosterone, cortisol and prOgesterone (Swanson, Hafs and Morrow, 1972). Statistical differences in serum hormone concen- trations in these animals were determined by t-test if two means had homOgenous variances or approximate t-test if variances were heterOgenous (Sokal and Rohlf, 1969). Experiment 2 GonadotrOpin releasing hormone1 (GnRH) was admin- istered via jugular cannula (three doses of 100 pg each at 120 min. intervals) to each of nine lactating Holstein cows (i=76 days of lactation, range of 41 to 104 days). Iiive cows were included because they had ovarian follicular lGnRH was generously supplied by Dr. R.H. Rippel, zxtfloott Laboratories, North Chicago, Illinois. 15 cysts; as in the first eXperiment, each animal had at least ‘ one ovarian follicle larger than 2.5 cm which was retained in one location for at least 14 days in the absence of a palpable corpus luteum. GnRH was administered after these criteria were satisfied. Prior to treatment, all cows with ovarian follicular cysts were anestrus. The remaining four control cows showed normal length estrous cycles and were treated on day 10 (2 animals), 11 or 13 of the estrous cycle. i”“*firrw Blood was collected to monitor serum hormone levels more frequently immediately after GnRH administration and less frequently as time from administration increased (Figure 1). All animals were observed twice daily for estrus and ovarian activity was monitored twice weekly by rectal palpation. All samples were assayed for serum luteinizing hor- mone (LH) by double antibody radioimmunoassay2 as described by Oxender, Hafs and Edgerton (1972). Pretreatment serum estradiol and estrone were determined by radioimmunoassay (Wettemann gt gt., 1972). Serum prOgesterone in selected samples was quantified by radioimmunoassay as described below. Serum (0.5 ml) was placed in a 15 m1 screw cap culture tube and 3,000 dpm of 3H-l,2—pr0gesterone (New England Nuclear; 34 c/mM; repurified by column chrOmato- graphy) added. After allowing labeled and endOgenous 2NIH-LH-BS standard was supplied by the National Institutes Of Health, Bethesda, Maryland. Purified bovine LH (LER-1072-2) for iodination was supplied by Dr. L.E. Reichert, Emery University, Atlanta, GA. .MCMSH mnomuoo Ho mummo Hmasoflaaom CMHHM>0 sufl3 msou :H mmcw Hmumm ma Ednmmll.a wusmflm T 352.: com OON ocN OCN O_N . . . r p p —::—:-b- 00. p p - 0w. 0! ON. 0a 00 0' ON P b pups-7:». P n b p n P - -::—--pp- p p p p P 112:1 O 01 GT . l .....m"... «.300 among .023 x . 1 Extra .. I I . 1 . i\u--ulu\I.--u| . .u n n u fivfitfififivwwwmw. . .., ...“.nflx. ....“ . .x... .. Lu...” hunk.” . a.” ...w.u.\.x. nuns-.....w. .......... u..~u...vu.u.n.vu“unu.unuu..v.......Hun. ..... I. . fitttfiss...Est”.mfittsflmfifiHESS......wfi..1...m.. .. 5+”. ".....xfin..w.....x: ....Eie: .....u.u.n...u. "u. ....E ...Entsflfinfimxmtwfitx ....wu 1 v an" ...“. .u- I. 1 1 a" .v. . um. Suffix}... ” $1...an .. 1 IL _ gun I III I l I l l l l Ego 3.32:0“. 1 8:95 5.3 $60 1 race 1 0100. 1 O. v. NN ON on cm on Nv 0v (Iw/OU) H‘I 17 steroids to equilibrate for 30 min., serum was extracted with 10 m1 of nanOgrade benzene:hexane (1:2) by gentle inversion for 20 min. Samples were stored at -20 C for at least 1 hr. to freeze the aqueous phase. Solvent extracts were decanted into conical tubes and evaporated under nitrogen to approximately 2.5 m1. A 0.5 m1 aliquant was taken to measure procedural losses and 0.25 and 0.5 ml aliquants were placed in culture tubes (12 x 75 mm) for radioimmunoassay. PrOgesterone (Sigma Chemical Co.) for standards (0, 0.1, 0.5, 1.0, 1.5, 2.0 and 5.0 ng) was dissolved in absolute ethanol. Standards and aliquants of serum extracts were dried under nitrOgen. Antibody3 (Niswender, 1973) (0.2 ml), diluted 1:3,000 in 0.1 M phOSphate buffered saline containing 0.1 percent gelatin (Knox Gelatin, Inc., Johnstown, N.Y.), was added to each tube, mixed for 10 sec. and allowed to incubate at room temperature for 30 min. Two hundred ul of 0.1 percent gelatin in 0.1 M phOSphate buffered saline, containing 45,000 dpm of 3H-1,2-pr0gesterone was added to each tube. The tubes were mixed for 5 sec. and incubated at 4 C for 12 to 18 hr. One ml of 0.025 percent dextran 150 and 0.25 percent carbon decolorizing neutral norit in glass disstilled water was added to each tube to separate bound and free hormone. 3Anti-prOgesterone, generously supplied by Dr. G.D. Niswender, Department of PhySiOlOgy, Colorado State Univer- sity, Fort Collins. The rabbit antiserum (#869) was prepared against 6B—succiny1 prOgesterone conjugated to bovine serum albumin. 18 The contents were mixed, incubated for 10 min. in an ice ' bath and then centrifuged at 2,500 g for 10 min. at 5 C. A 0.5 m1 aliquant of the supernatant was diluted with scintillation fluid (Bray, 1960) for quantification of radioactivity in a liquid scintillation Spectrometer. Significant differences in serum hormone concentra— tions were determined by t—test if two means had homOgenous variances or approximate t-test if variances were hetero- genous (Sokal and Rohlf, 1969). RESULTS AND DISCUSSION Experiment 1 Since previous workers have demonstrated that during normal estrous cycles estrOgen and glucocorticoids are high L at estrus and lower thereafter and prOgesterone is low at i estrus but higher thereafter (Swanson gt gt., 1972; hi Wettemann gt gt., 1972), estrOgen and glucocorticoid data for days 0 or 2 to 7 and prOgesterone data for days 0 to 2 or 4 to 11 were pooled for statistical comparisons. Serum estradiol was lower on days 2 to 7 of normal length estrous cycles (11:2 pg/ml) in cows with follicular cysts than on reSpective days of both abnormal length cycles (16:1 pg/ml; pMU ma on m .mmaomo :umcwa HmEuocnm «mmmp mm on ma .mmaomo numcma HmEnoz .mCOHum>umeo mo H0355: ma mammnucmumm mo Hones: “.m.m H mamsz Q .waco b no o hop :0 omcwmuno mHm3 mmHmEmm .mmumEUHmn oeumSOICOC Homm 1 1 1 Ammo s.ous.e Ammo o.HHm.e imae a.eH>.m s1~ Hemflunoo 1 1 1 Ass m.HHe.m Ame e.mHH.m Ame N.oH~.m e Howeuuoo 1 1 1 Ammo H.oum.o Ammo H.eHm.o AmHe H.eum.o s1m mcoumhmooflunoo 1 1 1 ARC H.0Hm.o Ame H.eHm.o Ame H.oH~.o o mcoumhmooflhuoo rec «.mfls.m rage o.HHm.N Away H.oun.o inns m.on.H HH1e accumummmona lee e.eHe.o ANHV e.on.o teas H.0HH.O lone H.0HH.Q N10 meoumummmoea 1Hs\mcv rev mus AHHV eHeH lees mHeH Ame eHeH e1m mconumm 2: rum 5 @an 13 Gums is 2 0 885mm Ase Hue lame mums Ammo HHGH lens «HHS s1m Heeemunmm rev mum rev eHmH Ase mums mime muss o Hegemuumm 1Hs\mee mmmumfipumm 00m Hmuwd nmaowu bmaowo 00m OHum>01coz Cumcmq HmEHOCQE numcmq HmEuoz Hmumm no CHO>U msouumm mo mhmo mpmmo HCHSOAHHOM swanm>o cufl3 mBOU QCOEHOE .mmumapumn oaumSOIcoc CH mmaomo msounmm mCflHSC Cam mummo HCHSOHHHOw cmfium>o npfi3 mzoo Ca 00m Hmumm Cam mmaomo moonumm mcwusp mofioumum Enummll.a mqmfle 22 Average serum LH reSponse after each dose of GnRH for cows with ovarian follicular Cysts and cows during luteal phase of the estrous cycle are depicted in Figure 1. Pituitary LH release from cows with ovarian follicular cysts appeared more Sporadic, eSpecially following the second dose of GnRH (note standard errors in Figure l) . In contrast, luteal phase cows has a more uniform LH reSponse. The change in serum LH concentration (ALH) after each dose of GnRH . indicates that cows with cystic follicles were more reSpon- sive than cows during luteal phase of the estrous cycle (Table 2). Thus A1, A2, A3 and Agreatest of the cystic cows were significantly greater (P<0.05) than reSpective values for luteal phase cows. Time to peak serum LH reSponse of individual doses Of GnRH and time to peak of greatest LH reSponse did not differ between cows with ovarian follicular cysts and cows treated during the luteal phase of the estrous cycle (Table 2). Peak serum LH in cows with cystic follicles and luteal Cows occurred after the second dose of GnRH. In anestrous eP-Wes (Reeves gt gt., 1972), peak LH reSponse after the first 200 ug intramuscular dose of LH—releasing hormone (LH-RH) Was not different from the reSponse of a second 200‘ug dose administered 4 hr. later. However, 16 intramuscular injec- tions of LH—RH (administered at 6 hr. intervals; 25 liq/in- jeCtion) to prepubertal gilts (Chakraborty gt a_l_., 1972) IE". 23 TABLE 2.—-Serum LH changes after GnRH in cows with follicular cysts or corpora lutea. A1 A2 A3 AGreatest (ng/ml) Cystic 27:7 24:6 11:3 46:11 Luteal 7:2 12:2 5:1 18:2 Time to Peaka (min.) CYstic 81:15 148:8 256:6 148:8 J Luteal 70:20 138:2 261:3 138:2 A1, 2, 3 --Change from immediate pre-injection level of LH to peak reSponse after each injection of 100 pg of GnRH. Individual peak reSponses averaged to obtain ALH. A . . Greatest--Change from time 0 to h1ghest LH response, regardless of time 0 of injections. aTime to peak of ALH figured from time 0 using individual animal peak reSponseS. GnRH administered at time 0, 120 and 240 min. produced LH reSponses from the first and second injections that were significantly greater than subsequent injections, but there was no difference in average pituitary LH content or concentration between treated and control gilts slangh- tered 24 hr. later. The ability of the pituitary to release 1additional LH after three injections of GnRH may diminish, Inowever, data obtained in this study after only three doses czannot be conclusive. 24 Serum progesterone concentration before and after GnRH in cows with ovarian follicular cysts is shown in Table 3. Average serum prOgesterone increased during the first 20 min. following GnRH administration, but this increase is attributable to two cows while the other three exhibited no change in serum progesterone. Cows in the luteal phase of the estrous cycle exhibited significant increases (P<0.05) at 260 and 360 min. compared to prein- jection levels (Table 3). Vi Acute changes in serum progesterone reflect the luteotropic capacity of LH in the bovine (Carlson, Norimoto and Hansel, 1971). Individual differences in‘ amount and activity of luteal tissue present in follicles of cows with ovarian follicular cysts may account for between animal variation in acute progesterone responses to GnRH. In luteal phase cows, presence of a functional corpus luteum, and thus more luteal tissue, would permit the increase in serum progesterone observed. Although a palpable corpus luteum was detected in only one of five cows with ovarian follicular cysts 10 days after treatment, serum progesterone for 3 weeks follow- ing treatment (Figure 2) appear similar to that of the normal bovine estrous cycle (Wettemann gt gt., 1972). All of the cows diagnosed cystic exhibited standing estrus 20 to 24 days after treatment with GnRH. Serum progesterone concen- tration of luteal phase cows during the remaining days of the cycle and cycle lengths are shown in Table 4. 25 TABLE 3.--Serum prOgesterone after GnRH in normal luteal phase cows and cows with ovarian follicular CystS. Time Cows with follicular Cysts Luteal Cows (ng/ml) -24 hr. 0.6:0.3 5.9:0.7 -12 hr. l.3:0.6 4.9:0.8 0 0.9:0.4 5.9:0.8 +20 min. 2.0:1.2 6.1:0.8 +60 min. -—— 6.4:0.9 +120 min. l.4:l.0 7.0:0.3 +140 min. --- 7.7:1.3 +240 min. l.7:l.3 7.9:1.0 +260 min. --- 10.3:0.9 +360 min. 0.4:0.1 8.2:1.0 TABLE 4.--Serum prOgesterone in luteal phase cows after GnRH administration. __.-.11___1,.-. __.....__ ._ . . . . . _ _-- - . -....._. , H 1.. --.__1_1.1. -. . -.. . — .7. -.._- .- -..-.- ._._1-_—_._——._..__..___- ”WA—ll. __--_ . fl - 1, . . ... _ . .. 1. .1 -._.. . ._ .— «e— - --. _____.- .1..- -1, -....-_..__».._ ‘___._. Cow number Day of 847b 1037: / 1048a 1118C Cycle ng/ml) 11 --- 4.0 4.6 --- 15 9.0 9.8 6.5 4.8 18 8.5 7.6 0.5 6.7 19 5.8 10.7 0.6 6.1 20 4.0 6.9 913 3.5 21 1.8 7.9 0.4 22 0.5 6.9 0.5 23 0.3 ttt 911 24 0.3 25 0_._3C1 aTreated on day 10 of the estrous cycle. bTreated on day 11 of the estrous cycle. :Treated on day 13 of the estrous cycle. Underlined value is on the day of estrus. 26 3.3m o- .1 u- .mumwo unasoflaaom smaum>o nufl3 m3oo SH mmco Hmumm mcoumummmoum Eduwm11.~ mhsmflm Im so cub“? w>