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B . K. OGUNMODEDE
Interrelationships between riboflavin and dietary energy and protein utilisation in growing chicks. Br. J. Nutr. 26: 323-333. Lepkovsky, S., and T. S. Jukes, 1936. The response of rats, chicks and turkey poults to crystalline vitamin G (flavin). J. Nutr. 12: 515-526. Mba, A. U., A. O. Bello, V. A. Oyenuga and F. O. Olubajo, 1974. The effects of varying the levels of proteins and red palm oil on the utilization of poultry feeds by broiler chicks. Niger. Agric. J. 11(2): 174-184. National Research Council, 1971. Nutrient requirements of poultry. National Academy of Sciences.
Ogunmodede, B. K., 1974. Dietary zinc and protein utilization by growing chickens. Nig. J. Anim. Prod. 1(2): 198-203. Peterson, W. J., D. E. Brady and A. O. Shaw, 1943. Fluorometric determination of riboflavin in pork products. Ind. Eng. Chem. Anal. Ed. 15: 634-636. Scott, M. L., M. C. Nesheim and R. J. Young, 1969. Nutrition of the Chicken. M. L. Scott and Associates, Ithaca, New York, p. 49, 428. Wyatt, R. D., H. T. Tung, W. E. Donaldson and P. B. Hamilton, 1973. A new description of riboflavin deficiency syndrome in chickens. Poultry Sci. 52: 237-244.
W . H . BURKE AND E . A. COGGER
Department of Animal Science, University of Minnesota, St. Paul, Minn. 55108 (Received for publication June, 23, 1976)
ABSTRACT Serum L.H. of non-laying turkey hens (NL.) was significantly higher than serum LH of layers (L.). All NL. hens showed a marked increase in serum L.H. within 5 min. of an i.v. injection of 20 or 40 u.g. of L.H.R.H. The L.H. levels of NL. hens were significantly lower at 15 min. after L.H.R.H. injection than at 5 min. The laying turkeys were erratic in their response, with clear cut, small rises in L.H. occurring only in some individuals with hard shelled eggs in utero. Hens with membranous eggs or with empty uteri failed to respond to the L.H.R.H. While F.S.H./L.H. injections caused significant ovarian and oviducal growth, L.H.R.H. given by daily i.m. injection for 7 days or twice daily i.v. injection for 6 days failed to do so. Subcutaneous implants of 1 mg. of L.H.R.H. in polyacrylamide gel led to prolonged elevation in serum L.H.R.H. and increased serum L.H. for about 12 hr. but caused no change in ovarian or oviducal weights. POULTRY SCIENCE 56: 234-242, 1977
T
HE avian hypothalamic factor which causes the release of luteinizing hormone (L.H.) has been shown to be chromatographically and immunologically indistinguishable from synthetic luteinizing hormone releasing hormone (L.H.R.H.) (Jeffcoate et ai, 1974). Synthetic L.H.R.H. induces ovulation in chickens (van Tienhoven and Schally, 1973; Reeves et ai, 1973), elicits the release of gonadotropins from chicken pituitary glands in vitro (Tanaka et ai, 1974),
1. Scientific Journal Series Paper Number 9578, of the Minnesota Agricultural Experiment Station.
causes a rapid rise in serum L.H. when injected into chickens (Furr et ai, 1973; Bonney et ai, 1974) and induces testicular growth in Coturnix quail when they are maintained on a 15 hr. daily light regime and treated with a drug to depress testis growth (El Halawani and Burke, 1975) but not on a 6 hr. light regime (Burke et ai, unpublished observations; Wada, 1975). Reeves et al. (1973) were unable to induce ovarian growth with L.H.R.H. in mature chickens made sexually inactive by reducing the photoperiod from 15 hr. to 6 hr. per day. It was the purpose of this study to determine the effects of L.H.R.H. on serum L.H.
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The Effect of Luteinizing Hormone Releasing Hormone (L.H.R.H.) on Serum L.H. and Ovarian Growth in Turkeys*
L.H.R.H., L.H.
of the turkey (Meleagris gallapavo) and to determine if L.H.R.H. could reactivate ovaries which had undergone normal a seasonal regression in hens maintained on a stimulatory photoperiod. MATERIALS AND METHODS
L.H. Radioimmunoassay. Serum L.H. was determined using a specific radioimmunoassay (RIA.) developed for turkey L.H. as described by Wentworth et al. (1976). For each of the experiments described herein, L.H. determinations were made in a single assay, eliminating interassay variation. Turkey L.H. fraction CGC lot W28BG (Farmer et al., 1975) was used as the reference standard. Progesterone Radioimmunoassay. Progesterone levels were measured by radioimmunoassay using antisera S49#6 (Abraham et al, 1971) obtained from Harbor General Hospital, Torrance, California. Tritium labeled progesterone (1,2,6,7- 3 H progesterone) was obtained from New England Nuclear Corporation and progesterone for the standard curves was from Sigma Chemicals. Serum samples (0.5-1.0 ml.) were incubated for 3 hr. at room temperature with 1000 c.p.m. of tritiated progesterone and then
235
extracted twice with diethyl ether. Ether extraction was carried out by mixing the sample for 30 sec. with 4 ml. of ether on a vortex mixer. The samples were left standing for 3-5 min. to allow the aqueous and ether layers to separate. The aqueous layer was then frozen with liquid nitrogen and the ether layer was decanted. The extraction was repeated and the ether extracts from each sample were pooled, placed in a 45° C. waterbath and dried under a stream of air. The dried extract was solubilized in two successive 0.5 ml. aliquots of hexane:benzene: methanol (85:5:4) and applied to a column of Sephadex LH 20. The column had been prepared by swelling 1.2 gm. of the Sephadex in benzene :methanol (85:15) overnight, pouring the slurry into a glass column, washing the column with 10 ml. of the same solvent mixture, and then washing the column with at least 10 ml. of hexane:benzene:methanol (85:5:4). The resulting column measured 8 mm. x 50 mm. After applying the sample to the column in the hexane:benzene:methanol solvent system, the column was eluted with it. The first 2 ml. eluate from the column was discarded and the 3 to 10 ml. fraction was saved for assay. The column eluate was dried under air and 1 ml. of phosphate buffered saline (0.1 M phosphate, 0.14 M NaCl, pH 7.0) containing 0.01% merthiolate and 0.1% gelatin (PBSG) was added to the vial. After vigorous mixing, the samples were stored overnight at 4° C. to allow the hormone to come into solution. Two 0.2 ml. aliquots of this material were taken to determine the recovery of the tritiated tracer added prior to extraction. These aliquots were placed into 5 ml. of toluene base scintillation fluid containing Liquifluor (New England Nuclear Corporation) as the scintillator. Two 0.2 ml. aliquots were also taken for assay and placed into tubes containing 0.3 ml. PBSG. Two tenths ml. of a 1:10,000 dilution of antisera in PBSG and 0.1 ml. of tritium labeled progesterone (10,000
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All turkeys used in these experiments were of a Large White strain, ranging from 8-10 kg. in weight. They were either in active laying condition (L.) or had undergone a normal spontaneous cessation of ovarian activity after having laid for a period of time (NL.). All birds were maintained on a 15L.:9D photoperiod. Blood samples were taken from the brachial vein by venipuncture. The blood was placed over ice and allowed to clot and then transferred to a 4° C. refrigerator. The following day it was centrifuged and serum was decanted. The serum was frozen and stored at -20° C. until used for the hormone assay.
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W. H. BURKE AND E. A. COGGER
Tubes containing neither antisera nor unlabeled hormone, called non-specific binding tubes (NSB) and tubes containing antisera but no unlabeled hormone (B 0 ) were also run. These were treated in the same manner as tubes containing standards or unknowns. Triplicate total count tubes (TCT) were prepared by placing 0.1 ml. aliquots of the assay tracer into scintillation vials with scintillation fluid and counting them. The average counts for triplicate NSB tubes were subtracted from all other tubes in the assay. The B 0 tubes (after subtraction of NSB) were used as a reference to which all other tubes were compared. The antibody bound radioactivity in the B 0 tubes was called 100% and tubes containing unlabeled hormone (standards or unknowns) were expressed as a percent of the B 0 . Ekins (1974) can be consulted for a general review of the principles
underlying these procedures. The percent bound values obtained for standards were converted to logits and the relationship between logit percent bound vs. the natural log of hormone concentrations was determined using a computerized weighted regression analysis. Potency estimates of unknowns were calculated with the same computer program. L.H.R.H. Radioimmunoassay. Serum L.H.R.H. was assayed using the procedures of Nett et al. (1973). Statistical Methods. Throughout the paper means given with an indication of variability are means ± the standard error of the mean. When appropriate, mean comparisons were made using the Student t test. In cases of multiple mean comparisons, Duncan's new multiple range test was used. In Experiment 4, hormone levels after L.H.R.H. implant were compared to preimplantation levels using student's t test. Experiment 1. Flockmate L. and NL. hens were given a single intravenous injection of 20 or 40 (j.g. of luteinizing hormone releasing hormone (L.H.R.H. Abbot Laboratories) in 1 ml. of 0.15 M NaCl solution. Control hens received 1 ml. of saline. A total of 21 L. and 21 NL. hens were used, seven in each treatment group. Hens were bled before the L.H.R.H. injection and at 5 and 15 min. after injection. Treatments were begun at 0930 hr. and continued until 1530 hr. The hormone treatments were administered to L. and NL. hens in a rotating fashion throughout the day to avoid possible time differences in. response. Laying and NL. hens were chosen based on trap nest records. The NL. hens had not laid any eggs for at least two weeks prior to the experiment. In addition, birds categorized as NL. were physically examined to assure that their ovaries were inactive (i.e.
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c.p.m.) in PBSG were added to each of the assay tubes. The tubes were mixed and incubated at 4° C. for at least 2 hr. After this period, 0.2 ml. of dextran coated charcoal (0.0625 mg. dextran T70, 0.625 mg. Norit A charcoal per 100 ml. of PBSG) was placed in each tube. The tubes were vortexed briefly and left standing for 30 min. at 4-5° C , after which they were centrifuged at 4-5° C. (10 min. x 2200 g). The supernatants were decanted into scintillation vials. Ten ml. of the scintillation fluid described above were added and vials were vortexed. The vials were kept at room temperature overnight to allow antibody bound 3 H-progesterone to be released into the scintillation fluid phase before counting. In each assay, standard curves were established using progesterone levels ranging from 25 to 1000 pg./tube by taking aliquots from stock solutions of progesterone in PBSG (1 ng./ml. and 10 ng./ml.) and diluting to 0.5 ml. with PBSG. Each point on the standard curve was run in triplicate. All assay and recovery samples were counted in a Beckman LS 250 counter with an efficiency for tritium of 60%.
L.H.R.H., L.H.
237
AND OVARIAN GROWTH
inability to evert the oviduct, hard tight pubic bones, small dry cloaca). Laying hens were examined per cloacato determine if they were carrying an egg in the shell gland and if so whether it was hard or soft shelled.
Experiment 3. This experiment was designed to determine if repeated intravenous injections of L.H.R.H. would stimulate ovarian growth in NL. turkeys. Twenty NL. hens were randomly assigned to three groups of seven, seven and six. Hens in one group received injections of 1 ml. of saline twice a day for 6 days (0800 hr. and 1500 hr.). One group received 10 u.g. of L.H.R.H. (Abbot Laboratories) on the same schedule and hens in the third group received 30 u.g. of L.H.R.H. twice a day. All injections were given in 1 ml. of saline and were made into the brachial vein. Hens were killed on the seventh day by cervical dislocation and ovarian, oviducal and anterior pituitary gland weights were taken. Experiment 4. The purposes of this experiment were to determine if L.H.R.H. implanted in polyacrylamide gel would elicit prolonged L.H. release ane gonadal growth in NL. turkeys. Thirteen NL. hens were randomly divided into two groups of seven control and six treated hens. The implant was
RESULTS L.H. Assay Validation. The complete validation of the L.H. R.I.A. is given by Wentworth et al. (1976). Progesterone Assay Validation. The description of the antisera used, its binding properties and specificity are given by Abraham (1971). In the work described here, the starting percent bound in the absence of unlabeled progesterone was 57.4%. A linear standard
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Experiment 2. This experiment was designed to determine if daily intramuscular injections of L.H.R.H. could elicit ovarian growth in NL. female turkeys. Twenty-four NL. hens were randomly assigned to three groups of eight hens each and daily intramuscular injections of either 1 ml. of saline, 50 (xg. of L.H.R.H. in 1 ml. of saline (Spectrum Medical Industries) or a mixture of L.H. and F.S.H. (Armour P.L.H., 0.5 mg. + Armour F.S.H.-P., 1.0 mg./ml. of saline). Injections were given once a day for 7 days. Hens were sacrificed on the eighth day and the ovaries and oviducts were weighed.
made by preparing 10% polyacrylamide cross-linked with methylene bisacrylamide (20% w./w.) in pH 7.6 Tris-HCl buffer. One mg. of L.H.R.H. (Abbot) was dissolved in each ml. of the acrylamide mixture. Polymerization was achieved by adding 5 u,l. of N ^ N ' ,N'-tetramethylamine (TEMED) and 7 mg. of ammonium persulfate to each 10 ml. of the acrylamide-L.H.R.H mixture. One ml. aliquots of this, containing 1 mg. of L.H.R.H., were placed into glass tubing (6 mm. I.D.) and allowed to polymerize. To implant the gels, small skin incisions were made at the base of the skull and the glass tubing containing the gel was inserted under the loose skin. The gel was forced from the glass with a plunger from a 1 ml. disposable syringe. The glass tube was withdrawn and the incision was closed with a wound clip. Control hens received implants with no L.H.R.H. added. Hens were bled prior to and at 2, 4, 8, 16, 20, 24, 48, 72, 96, 120, 144 and 168 hr. after implantation. Serum L.H. levels were measured on blood samples taken at each of these times and progesterone levels were determined in blood samples obtained at 0, 2, 12, 24 and 120 hr. after gel implantation. Hens were sacrificed and ovarian and oviducal weights were obtained immediately after the 168 hr. bleeding. Serum L.H.R.H. levels were determined by RIA according to the procedure of Nett et al. (1973).
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W. H. BURKE AND E. A. COGGER
The extraction procedure used, coupled with the chromatographic procedure described separates progesterone from the 17hydroxyprogesterone and the adrenal corticoids (Nuti et ai, 1974) which are the major cross reacting steroids with this antisera (Abraham, 1971). Experiment 1. Serum L.H. of L. hens averaged 5.8 ± 0.7 ng./ml. of serum prior to L.H.R.H. treatment while NL. hens had an L.H. level of 7.7 ± 0.5 ng./ml. (n = 21 in each group). This difference was significant. Both 20 and 40 |xg. of L.H.R.H. caused rapid increases in the serum L.H. levels of NL. hens from 7-8 ng./ml. to 44-45 ng./ml. by 5 min. after injection (Fig. 1). There was no significant difference in the response to these two levels of L.H.R.H. At fifteen minutes after L.H.R.H. injection, L.H. levels were significantly lower than at 5 min. postinjection, but they were still about five times higher than pre-injection levels. The response of L. hens to the L.H.R.H. injection was smaller than that obtained with NL. hens and was quite variable. Twenty |xg. of the hormone caused mean L.H. levels to rise from 6.1 ± 1.6 ng./ml. to 9.8 ± 2.8 ng. /ml. at 5 min. after injection. The 40 |xg. dose of
l'"
1 0
5
15
0
5
15
0
Time after Injection (minutes)
FIG. 1. Serum L.H. levels of laying (L.) and non-laying (NL.) turkeys following the intravenous injection of 0, 20 or 40 |xg. of L.H.R.H. L.H.R.H. caused a rise from 6.4 ± 1.1 ng./ml. to 9.0 ± 1.2 ng./ml. Only three of the seven laying hens receiving the 20 |xg. dose showed changes in L.H. which exceeded the maximum change seen in any of the saline injected laying hens. One showed no change, one a slight decrease and two others a slight increase in L.H. levels. While six of the seven layers receiving 40 |xg. of L.H.R.H. showed increased L.H. levels 5 min. after injection, only one of these was greater than the greatest change seen in controls. The response to 20 (xg. was not significant but the response to 40 (xg. was. Saline injected laying hens had a mean pre-injection L.H. level of 4.8 ± 1.0 ng./ml. and a level of 5.2 ± 0.8 ng./ml. at 5 min. after injection. Individual control hens had L.H. changes ranging from - 2 . 6 ng. to +4.4 ng. in this period. Non-laying hens injected with saline averaged 7.4 + 0.8 ng. / m l . before injection, 6.9 ± 1.0 ng./ml. 5 min. after injection and 5.9 ± 1.0 ng./ml. 15 min. after saline injection. This change, while small, was consistent and the 15 min. L.H. levels were significantly lower than pre-injection levels. The largest increases in L.H. in response to L.H.R.H. in laying hens (7.1, 8.8 and 10.2 ng./ml.) were obtained with hens that had hard shelled, well calcified eggs in utero at
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curve with a slope of —1.13 was obtained with progesterone doses ranging from 251000 pg./tube (90% bound to 11% bound). Buffer blanks, obtained by assaying extracted, chromatographed PBSG, averaged 8.8 pg./assay tube (96.7% of B 0 ) . This value was subtracted from all unknowns prior to the recovery correction and the potency calculation. Spiking experiments were conducted in which unlabeled progesterone was added to serum and P.B.S.G., then extracted, chromatographed and assayed. In repeated experiments of this type in our laboratory, virtually 100% of the spiked progesterone is accounted for when the material is assayed and corrections are made for recovery of the tritiated tracer progesterone.
239
L.H.R.H., L.H. AND OVARIAN GROWTH
injection time. These responses also happened to be obtained early in the day, between 0930 and 1100 hr. No substantial increment in serum L.H. was obtained in the three laying hens that had uncalcified membranous eggs in utero when injected and only one of five hens with empty uteri showed a substantial response to the L.H.R.H.
Experiment 3. NL. hens receiving twice daily injections of either 10 or 30 |i,g. of TABLE {.—Effect of F.S.H./L.H. and L.H.R.H* on ovary and oviduct weight of non-laying turkeys F.S.H./L.H. Ovary wt. (gm.) Oviduct wt. (gm.)
41.0 ± 13.4a 67.6 ± 9.4a
L.H.R.H.
Saline
8.9 ± 1.6b
6.4 ± 0.5b
13.6 ± 2.1b 9.3 ± 1.1b
* Injected daily for 7 days, 1 x per day intramuscularly. F.S.H./L.H. (1 mg. Armour F.S.H.-P. and 0.5 mg. Armour P.-L.H.) (50 t».g., Spectrum Medical Industries). ab Means within a row having different letters are different at the 1% level of probability.
Experiment 4. Serum L.H. levels of hens implanted with L.H.R.H. rose from a preimplantation level of 6.7 ± 0.4 ng./ml. to a peak level of 22.3 ± 4.7 ng./ml. two hr. later (Table 2). The L.H. levels dropped at each successive bleeding and by 16 hr. after implantation they averaged 8.5 ± 1.0 ng. /ml. which was not significantly different from pre-implantation levels. They remained at these lower levels for the duration of the experiment. Control birds implanted with gel alone showed no significant change in serum L.H. during the experimental period. The L.H. level before receiving the control implant averaged 8.0 ± 0.8 ng./ml; the lowest mean level measured during the experiment
TABLE 2.—Serum L.H. and progesterone levels of turkey hens implanted with
L.H.R.H. implant Progesterone (pg./ml.) L.H. (ng./ml.) SE X SE X
Control implant Hours after implantation 0 2 4 8 12 16 20 24 48 72 96 120 144 168
L.H. (ng./ml.) SE X 8.0 7.3 7.9 8.4 6.3 6.7 8.0 6.8 8.2 7.4 7.8 8.2 9.3 8.2
0.8 0.6 0.8 1.0 0.6 0.5 0.8 0.6 0.6 0.9 0.7 0.9 1.3 1.0
L.H.R.H.
6.7 22.3a 14.2a 10.3a 9.6a 8.5 8.2 7.6 7.7 7.9 7.1 8.0 7.6 8.0
"Means significantly different (P s .05) than time 0.
0.4 4.7 1.4 0.9 1.3 1.0 1.1 0.7 0.7 0.9 0.7 1.0 0.9 1.0
194.7 216.4
— —
202.0
35.8 38.6 48.7
— 220.1 194.6
44.2 36.4
213.6
18.9
— —
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Experiment 2. Daily injections of a mixture of L.H. and F.S.H. for seven days caused significant ovarian and oviducal growth. Daily intramuscular injections of 50 u,g. L.H.R.H. for the same period of time did not elicit significant weight change in either the ovary or oviduct (Table 1).
L.H.R.H. for six days showed no increase in ovary, oviduct or pituitary gland weight when compared to saline injected controls. Ovarian weight of the controls averaged 7.57 ± 1.35 g.; the 10 |xg. L.H.R.H. group averaged 8.83 ± 1.02 g. and the 30 (xg. group averaged 8.21 ± 1.22 g. Average oviduct weights (g.) of the 3 groups were 18.96 ± 5.60, 16.05 ± 3.88 and 16.58 ± 3.22, respectively. Anterior pituitary gland weights averaged 19.17 ± 1.22, 19.96 ± 1.67 and 23.57 ± 2.67 mg. for the 0, 10 and 30 |xg. L.H.R.H. groups, respectively.
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W. H. BURKE AND E. A. COGGER
DISCUSSION The observation that mean serum L.H. of NL. hens is higher than the mean serum L.H. levels of L. hens bled in the morning and early afternoon was unexpected. The individual L.H. values of the L. hens would indicate that only one or two of these birds were bled when L.H. was showing an ovulatory peak. Thus, the L.H. values of L. hens probably represent basal levels. Nevertheless, substantial L.H. was found in NL. hens
with regressed ovaries. It may well be that the lack of ovarian steroid feedback in hens that have been laying and undergone ovarian regression leads to a post-castration like or post-menopausal like rise in L.H. Two pools of sera obtained from many L. and NL. turkeys have verified the higher serum L.H. levels of the NL. hens (Burke, unpublished observations). However, it should be noted that Cogger and Burke (1975) have shown a significant decline in serum L.H. accompanying the shift from an active laying state to a broody state in turkeys. Thus, the elevated serum L.H. of NL. hens seen in this study may follow an initial L.H. decline at the time laying ceases. Imai et al. (1972) also found moulting, non-laying chickens to have higher total serum gonadotropins than layers. The minimal and variable response of L. turkeys to L.H.R.H. appears to be considerably different than the response obtained by Bonney et al. (1974) with laying chickens. While they too found laying birds responded to L.H.R.H. with rather small increments in serum L.H., they obtained significant responses at all times except during that period immediately preceding ovulation. While turkeys are less regular in their laying patterns than chickens are, and even though accurate oviposition-time records were not kept in this study, certain inferences can still be made regarding the stage of the ovulatory cycle that the hens were in at the time of the experiment. Only three laying hens, all carrying heavily calcified eggs (as determined by rectal palpation) showed L.H. responses clearly in excess of the largest change seen in the saline injected L. group. These hens would have been expected to lay their eggs sometime that same day. One of these three birds had a pre-injection L.H. level about 2.7 times greater than the mean level for all L. hens, suggesting she was in the midst of an ovulatory surge. She showed a 10.2 ng./ml. increase in L.H., the largest obtained
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was 6.3 ± 0.6 ng./ml. and the highest was 9.3 ± 1.3 ng./ml. Progesterone levels of the L.H.R.H. implanted hens (Table 2) showed no significant change from pre-implantation levels throughout the course of the experiment. Ovarian weight of the control and L.H.R.H. implanted hens averaged 8.8 ± 1.9 and 9.0 ± 0.9 gm., respectively, at the termination of the experiment. Oviduct weights of the two groups (21.2 ± 10.7 and 11.0 ± 1.9 gm.) did not differ significantly. Relative changes in serum L.H.R.H. levels were determined in a specific R.I.A., using the procedures described byNett etal. (1973). While the assay has not been validated for serum levels of the hormone in turkeys, and the absolute values obtained appeared to be unrealistically high, the pre- and postimplantation levels were, nevertheless, informative. Serum L.H.R.H. levels were elevated in all implanted hens at 2 hr. after implantation. The increases ranged from about 5 fold to more than 30 fold at this bleeding. In all hens, the highest L.H.R.H. levels were found at either the 2, 4 or 8 hr. post-implantation bleeding. Five of the six implanted hens still had elevated L.H.R.H. levels (2.8-4.0 fold higher than pre-implantation values) at 120 hr. Control implanted hens did not show an increase in serum L.H.R.H. On the contrary, their levels seemed to decrease after the first or second bleeding.
L.H.R.H., L.H.
The lack of a difference in response to 20or40(jig. of L.H.R.H. in NL. hens suggests that both doses are on the plateau of the dose response curve. Previous work (Wentworth et al., 1976) has shown a dose response in NL. turkeys using L.H.R.H. at doses of 10 and 50 |xg. Although a strict comparison between these two experiments cannot be made, it appears that around 20 u.g. of L.H.R.H. may elicit a maximal response in NL. turkeys. The ovarian and oviducal growth response obtained by the administration of an F . S . H . / L . H . mixture shows clearly that the ovaries of NL. hens are capable of reactivation if given the proper hormonal stimulus. This agrees with results obtained in chickens when a variety of gonadotropin preparation were administered (Imai et al., 1972; Imai, 1972). The failure of daily i.m. injections of 50 (Ag. of L.H.R.H. or twice daily i.v. injections of 10 or 30 fjLg. to elicit ovarian growth may be due to a number of causes. It is possible that a single i.m. dose of the hormone would not stimulate sufficient gonadotropin release for a long enough period of time to activate the ovary or it may fail to release F.S.H. along with L.H. The latter point has
241
not been directly tested in vivo since until recently it has not been possible to measure circulating levels of F.S.H. in avian blood (Croix et al., 1974). Wentworth et al. (1976) have shown that in NL. turkeys, the response to an i.v. injection of L.H.R.H. is rapid and short lived, L.H. levels peaked within 5-10 min. and fell back to base line levels by 70 min. after injection. A similar time course response was obtained in chickens by Bonney et al. (1974). It might be noted that i.m. injections of L.H.R.H. (1.25 p,g./bird, twice a day for 7 days) does cause sufficient gonadotropin release to stimulate testicular growth in Coturnix housed under a stimulatory photoperiod (El Halawani and Burke, 1975). In Experiment 3 the failure of L.H.R.H. injections to elevate serum progesterone showed further the lack of effectiveness of this type of treatment for reactivating ovarian function. Luteinizing hormone injections do cause rapid increases in serum progesterone in laying chickens (Shahabi et al., 1975) and in laying turkeys (Camper and Burke, 1976). While the L.H.R.H. implants used in Experiment 4 caused a prolonged elevation of serum L.H.R.H., the rather short lived elevation in serum L.H. indicated that the pituitary failed to continuously respond to elevated levels of the releasing hormone. It may require an increase in L.H.R.H. to activate the pituitary release mechanism rather than a high level per se. The prolonged release of L.H.R.H. from the gel implant indicates that this technique may be a useful way to administer peptide hormones and avoid the sudden surges and declines that accompany repeated injection techniques.
ACKNOWLEDGMENTS The authors gratefully acknowledge the splendid technical assistance given by L. A. Ogren in performing the radioimmunoassays for these studies. This work was supported in part by grants
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in any of the L. hens. In addition, three of the hens carrying heavily calcified eggs did not respond to the L.H.R.H. and they could conceivably fall into the pre-ovulatory group of non-responders seen by Bonney et al. (1974). The three birds with a membranous egg in utero (i.e. a few hours after ovulation) did not respond to the L.H.R.H., while only one of four hens with an empty uterus showed a response which was slightly greater than the largest change seen in any of the control hens. Thus, it appears that substantial differences exist in the response of laying chickens and turkeys to this hormone. The striking response of NL. hens is similar to the response obtained in cockerels and immature female chickens (Furr et al., 1973; Bonney et al., 1974).
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from the Minnesota Turkey Growers Association and by grant number HD 07242 from the National Institute of Child Health and Human Development, N.I.H. REFERENCES
JULY 10-13, 1977.
ANNUAL MEETING, CANADIAN SOCIETY OF ANIMAL SCIENCE, UNIVERSITY OF NEW BRUNSWICK, FREDERICTON, NEW BRUNSWICK
JULY 13-16, 1977. INTERNATIONAL CONGRESS OF THE WORLD VETERINARY POULTRY ASSOCIATION, ATLANTA, GEORGIA AUGUST 8-12, 1977. SIXTY-SIXTH ANNUAL MEETING, POULTRY SCIENCE ASSOCIATION, INC., AUBURN UNIVERSITY, AUBURN, ALABAMA
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Abraham, G. E., R. S. Swerdloff, D. Tulchinsky and W. D. Odell, 1971. Radioimmunoassay of plasma progesterone. J. Clin. Endocrinol. 32: 619-624. Bonney, R. C , F. J. Cunningham and B. J. A. Furr, 1974. Effect of synthetic luteinizing hormone releasing hormone on plasma luteinizing hormone in the female domestic fowl, Gallus Domesticus. J. Endocrinol. 63: 539-547. Camper, P. M., and W. H. Burke, 1976. Serum estradiol and progesterone levels of the laying turkey hen following acute treatment with mammalian luteinizing or follicle stimulating hormone. Gen. Comp. Endocr. In press. Cogger, E. A., and W. H. Burke, 1975. Broodiness: behavioral and hormonal relationships. Minnesota Agr. Expt. Sta. Misc. Report, 134: 41-47. Croix, D., J. C. Hendrick, J. Ballhazart and P. Franchimount, 1974. Dosage radioimmunologique de 1'hormone folliculo-stimulant (FSH) hypophysiare de canard a'l'aide d' un systeme de mammiferes. C. R. Soc. Biol. 168: 136-140. Ekins, R. P., 1975. Radioimmunoassay and saturation analysis. Basic principles and theory. Brit. Med. Bull. 30: 3-11. El Halawani, M. E., and W. H. Burke, 1975. Role of catecholamines in photoperiodically-induced gonadal development in Coturnix quail. Biol. Reprod. 13: 603-609. Farmer, S. W., H. Papkoff and P. Licht, 1975. Purification of turkey gonadotropins. Biol. Reprod. 12: 415-422. Furr, B. J. A., G. I. Onuora, R. C. Bonney and F. J. Cunningham, 1973. The effect of synthetic hypothalamic releasing factors on plasma levels of luteinizing hormone in the cockerel. J. Endocrinol. 59: 495-502. Imai, K., 1972. Effects of avian and mammalian
pituitary preparations on follicular growth in hens treated with methallibure or fasting. J. Reprod. Fert. 31: 387-397. Imai, K., M. Tanaka and S. Nakajo, 1972. Gonadotrophic activities of anterior pituitary and of blood plasma and ovarian response to exogenous gonadotrophin in molting hens. J. Reprod. Fert. 30: 433-443. Jeffcoate, S. L., P. J. Sharp, H. M. Fraser, D. T. Holland and A. Gunn, 1974. Immunochemical and chromatographic similarity of rat, rabbit, chicken and synthetic luteinizing hormone releasing hormones. J. Endocrinol. 62: 85-91. Nett, T. M., A. M. Akbar, G. D. Niswender, M. T. Hedlund and W. F. White, 1973. A radioimmunoassay for gonadotropin-releasing hormone (Gn-RH) in serum. J. Clin. Endocr. Metab. 36: 880-885. Nuti, L. C , W. H. McShan and R. K. Meyer, 1974. Effect of ovine FSH and LH on serum steroids and ovulation in hypophysectomized immature female rats. Endocrinology, 95: 682-689. Reeves, J. J., P. C. Harrison and J. M. Casey, 1973. Ovarian development and ovulation in hens treated with synthetic (porcine) luteinizing hormone releasing hormone (LH-RH/FSH-RH). Poultry Sci. 52: 1883-1886. Shahabi, N. A., J. M. Bahr and A. V. Nalbandov, 1975. Effect of LH injection on plasma and follicular steroids in the chicken. Endocrinology, 96: 969-972. Tanaka, K., M. Sakaida and M. Kamiyoshi, 1974. Release of gonadotropins from the chicken pituitary by synthetic LH-RH/FSH-RH. Poultry Sci. 53: 72-75. Van Tienhoven, A. and A. V. Schally, 1973. Mammalian luteinizing hormone-releasing hormone induces ovulation in the domestic fowl. Gen. Comp. Endocr. 19: 594-595. Wada, M., 1975. Cell types in the adenohypophysios of the Japanese quail and effects of injection of luteinizing hormone-releasing hormone. Cell Tiss. Res. 159: 167-178. Wentworth, B. C , W. H. Burke and G. P. Birrenkott, 1976. A radioimmunoassay for turkey luteinizing hormone. Gen. Comp. Endocrinol. 29: 119-127.
B . K. OGUNMODEDE
Interrelationships between riboflavin and dietary energy and protein utilisation in growing chicks. Br. J. Nutr. 26: 323-333. Lepkovsky, S., and T. S. Jukes, 1936. The response of rats, chicks and turkey poults to crystalline vitamin G (flavin). J. Nutr. 12: 515-526. Mba, A. U., A. O. Bello, V. A. Oyenuga and F. O. Olubajo, 1974. The effects of varying the levels of proteins and red palm oil on the utilization of poultry feeds by broiler chicks. Niger. Agric. J. 11(2): 174-184. National Research Council, 1971. Nutrient requirements of poultry. National Academy of Sciences.
Ogunmodede, B. K., 1974. Dietary zinc and protein utilization by growing chickens. Nig. J. Anim. Prod. 1(2): 198-203. Peterson, W. J., D. E. Brady and A. O. Shaw, 1943. Fluorometric determination of riboflavin in pork products. Ind. Eng. Chem. Anal. Ed. 15: 634-636. Scott, M. L., M. C. Nesheim and R. J. Young, 1969. Nutrition of the Chicken. M. L. Scott and Associates, Ithaca, New York, p. 49, 428. Wyatt, R. D., H. T. Tung, W. E. Donaldson and P. B. Hamilton, 1973. A new description of riboflavin deficiency syndrome in chickens. Poultry Sci. 52: 237-244.
W . H . BURKE AND E . A. COGGER
Department of Animal Science, University of Minnesota, St. Paul, Minn. 55108 (Received for publication June, 23, 1976)
ABSTRACT Serum L.H. of non-laying turkey hens (NL.) was significantly higher than serum LH of layers (L.). All NL. hens showed a marked increase in serum L.H. within 5 min. of an i.v. injection of 20 or 40 u.g. of L.H.R.H. The L.H. levels of NL. hens were significantly lower at 15 min. after L.H.R.H. injection than at 5 min. The laying turkeys were erratic in their response, with clear cut, small rises in L.H. occurring only in some individuals with hard shelled eggs in utero. Hens with membranous eggs or with empty uteri failed to respond to the L.H.R.H. While F.S.H./L.H. injections caused significant ovarian and oviducal growth, L.H.R.H. given by daily i.m. injection for 7 days or twice daily i.v. injection for 6 days failed to do so. Subcutaneous implants of 1 mg. of L.H.R.H. in polyacrylamide gel led to prolonged elevation in serum L.H.R.H. and increased serum L.H. for about 12 hr. but caused no change in ovarian or oviducal weights. POULTRY SCIENCE 56: 234-242, 1977
T
HE avian hypothalamic factor which causes the release of luteinizing hormone (L.H.) has been shown to be chromatographically and immunologically indistinguishable from synthetic luteinizing hormone releasing hormone (L.H.R.H.) (Jeffcoate et ai, 1974). Synthetic L.H.R.H. induces ovulation in chickens (van Tienhoven and Schally, 1973; Reeves et ai, 1973), elicits the release of gonadotropins from chicken pituitary glands in vitro (Tanaka et ai, 1974),
1. Scientific Journal Series Paper Number 9578, of the Minnesota Agricultural Experiment Station.
causes a rapid rise in serum L.H. when injected into chickens (Furr et ai, 1973; Bonney et ai, 1974) and induces testicular growth in Coturnix quail when they are maintained on a 15 hr. daily light regime and treated with a drug to depress testis growth (El Halawani and Burke, 1975) but not on a 6 hr. light regime (Burke et ai, unpublished observations; Wada, 1975). Reeves et al. (1973) were unable to induce ovarian growth with L.H.R.H. in mature chickens made sexually inactive by reducing the photoperiod from 15 hr. to 6 hr. per day. It was the purpose of this study to determine the effects of L.H.R.H. on serum L.H.
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The Effect of Luteinizing Hormone Releasing Hormone (L.H.R.H.) on Serum L.H. and Ovarian Growth in Turkeys*
L.H.R.H., L.H.
of the turkey (Meleagris gallapavo) and to determine if L.H.R.H. could reactivate ovaries which had undergone normal a seasonal regression in hens maintained on a stimulatory photoperiod. MATERIALS AND METHODS
L.H. Radioimmunoassay. Serum L.H. was determined using a specific radioimmunoassay (RIA.) developed for turkey L.H. as described by Wentworth et al. (1976). For each of the experiments described herein, L.H. determinations were made in a single assay, eliminating interassay variation. Turkey L.H. fraction CGC lot W28BG (Farmer et al., 1975) was used as the reference standard. Progesterone Radioimmunoassay. Progesterone levels were measured by radioimmunoassay using antisera S49#6 (Abraham et al, 1971) obtained from Harbor General Hospital, Torrance, California. Tritium labeled progesterone (1,2,6,7- 3 H progesterone) was obtained from New England Nuclear Corporation and progesterone for the standard curves was from Sigma Chemicals. Serum samples (0.5-1.0 ml.) were incubated for 3 hr. at room temperature with 1000 c.p.m. of tritiated progesterone and then
235
extracted twice with diethyl ether. Ether extraction was carried out by mixing the sample for 30 sec. with 4 ml. of ether on a vortex mixer. The samples were left standing for 3-5 min. to allow the aqueous and ether layers to separate. The aqueous layer was then frozen with liquid nitrogen and the ether layer was decanted. The extraction was repeated and the ether extracts from each sample were pooled, placed in a 45° C. waterbath and dried under a stream of air. The dried extract was solubilized in two successive 0.5 ml. aliquots of hexane:benzene: methanol (85:5:4) and applied to a column of Sephadex LH 20. The column had been prepared by swelling 1.2 gm. of the Sephadex in benzene :methanol (85:15) overnight, pouring the slurry into a glass column, washing the column with 10 ml. of the same solvent mixture, and then washing the column with at least 10 ml. of hexane:benzene:methanol (85:5:4). The resulting column measured 8 mm. x 50 mm. After applying the sample to the column in the hexane:benzene:methanol solvent system, the column was eluted with it. The first 2 ml. eluate from the column was discarded and the 3 to 10 ml. fraction was saved for assay. The column eluate was dried under air and 1 ml. of phosphate buffered saline (0.1 M phosphate, 0.14 M NaCl, pH 7.0) containing 0.01% merthiolate and 0.1% gelatin (PBSG) was added to the vial. After vigorous mixing, the samples were stored overnight at 4° C. to allow the hormone to come into solution. Two 0.2 ml. aliquots of this material were taken to determine the recovery of the tritiated tracer added prior to extraction. These aliquots were placed into 5 ml. of toluene base scintillation fluid containing Liquifluor (New England Nuclear Corporation) as the scintillator. Two 0.2 ml. aliquots were also taken for assay and placed into tubes containing 0.3 ml. PBSG. Two tenths ml. of a 1:10,000 dilution of antisera in PBSG and 0.1 ml. of tritium labeled progesterone (10,000
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All turkeys used in these experiments were of a Large White strain, ranging from 8-10 kg. in weight. They were either in active laying condition (L.) or had undergone a normal spontaneous cessation of ovarian activity after having laid for a period of time (NL.). All birds were maintained on a 15L.:9D photoperiod. Blood samples were taken from the brachial vein by venipuncture. The blood was placed over ice and allowed to clot and then transferred to a 4° C. refrigerator. The following day it was centrifuged and serum was decanted. The serum was frozen and stored at -20° C. until used for the hormone assay.
AND OVARIAN GROWTH
236
W. H. BURKE AND E. A. COGGER
Tubes containing neither antisera nor unlabeled hormone, called non-specific binding tubes (NSB) and tubes containing antisera but no unlabeled hormone (B 0 ) were also run. These were treated in the same manner as tubes containing standards or unknowns. Triplicate total count tubes (TCT) were prepared by placing 0.1 ml. aliquots of the assay tracer into scintillation vials with scintillation fluid and counting them. The average counts for triplicate NSB tubes were subtracted from all other tubes in the assay. The B 0 tubes (after subtraction of NSB) were used as a reference to which all other tubes were compared. The antibody bound radioactivity in the B 0 tubes was called 100% and tubes containing unlabeled hormone (standards or unknowns) were expressed as a percent of the B 0 . Ekins (1974) can be consulted for a general review of the principles
underlying these procedures. The percent bound values obtained for standards were converted to logits and the relationship between logit percent bound vs. the natural log of hormone concentrations was determined using a computerized weighted regression analysis. Potency estimates of unknowns were calculated with the same computer program. L.H.R.H. Radioimmunoassay. Serum L.H.R.H. was assayed using the procedures of Nett et al. (1973). Statistical Methods. Throughout the paper means given with an indication of variability are means ± the standard error of the mean. When appropriate, mean comparisons were made using the Student t test. In cases of multiple mean comparisons, Duncan's new multiple range test was used. In Experiment 4, hormone levels after L.H.R.H. implant were compared to preimplantation levels using student's t test. Experiment 1. Flockmate L. and NL. hens were given a single intravenous injection of 20 or 40 (j.g. of luteinizing hormone releasing hormone (L.H.R.H. Abbot Laboratories) in 1 ml. of 0.15 M NaCl solution. Control hens received 1 ml. of saline. A total of 21 L. and 21 NL. hens were used, seven in each treatment group. Hens were bled before the L.H.R.H. injection and at 5 and 15 min. after injection. Treatments were begun at 0930 hr. and continued until 1530 hr. The hormone treatments were administered to L. and NL. hens in a rotating fashion throughout the day to avoid possible time differences in. response. Laying and NL. hens were chosen based on trap nest records. The NL. hens had not laid any eggs for at least two weeks prior to the experiment. In addition, birds categorized as NL. were physically examined to assure that their ovaries were inactive (i.e.
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c.p.m.) in PBSG were added to each of the assay tubes. The tubes were mixed and incubated at 4° C. for at least 2 hr. After this period, 0.2 ml. of dextran coated charcoal (0.0625 mg. dextran T70, 0.625 mg. Norit A charcoal per 100 ml. of PBSG) was placed in each tube. The tubes were vortexed briefly and left standing for 30 min. at 4-5° C , after which they were centrifuged at 4-5° C. (10 min. x 2200 g). The supernatants were decanted into scintillation vials. Ten ml. of the scintillation fluid described above were added and vials were vortexed. The vials were kept at room temperature overnight to allow antibody bound 3 H-progesterone to be released into the scintillation fluid phase before counting. In each assay, standard curves were established using progesterone levels ranging from 25 to 1000 pg./tube by taking aliquots from stock solutions of progesterone in PBSG (1 ng./ml. and 10 ng./ml.) and diluting to 0.5 ml. with PBSG. Each point on the standard curve was run in triplicate. All assay and recovery samples were counted in a Beckman LS 250 counter with an efficiency for tritium of 60%.
L.H.R.H., L.H.
237
AND OVARIAN GROWTH
inability to evert the oviduct, hard tight pubic bones, small dry cloaca). Laying hens were examined per cloacato determine if they were carrying an egg in the shell gland and if so whether it was hard or soft shelled.
Experiment 3. This experiment was designed to determine if repeated intravenous injections of L.H.R.H. would stimulate ovarian growth in NL. turkeys. Twenty NL. hens were randomly assigned to three groups of seven, seven and six. Hens in one group received injections of 1 ml. of saline twice a day for 6 days (0800 hr. and 1500 hr.). One group received 10 u.g. of L.H.R.H. (Abbot Laboratories) on the same schedule and hens in the third group received 30 u.g. of L.H.R.H. twice a day. All injections were given in 1 ml. of saline and were made into the brachial vein. Hens were killed on the seventh day by cervical dislocation and ovarian, oviducal and anterior pituitary gland weights were taken. Experiment 4. The purposes of this experiment were to determine if L.H.R.H. implanted in polyacrylamide gel would elicit prolonged L.H. release ane gonadal growth in NL. turkeys. Thirteen NL. hens were randomly divided into two groups of seven control and six treated hens. The implant was
RESULTS L.H. Assay Validation. The complete validation of the L.H. R.I.A. is given by Wentworth et al. (1976). Progesterone Assay Validation. The description of the antisera used, its binding properties and specificity are given by Abraham (1971). In the work described here, the starting percent bound in the absence of unlabeled progesterone was 57.4%. A linear standard
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Experiment 2. This experiment was designed to determine if daily intramuscular injections of L.H.R.H. could elicit ovarian growth in NL. female turkeys. Twenty-four NL. hens were randomly assigned to three groups of eight hens each and daily intramuscular injections of either 1 ml. of saline, 50 (xg. of L.H.R.H. in 1 ml. of saline (Spectrum Medical Industries) or a mixture of L.H. and F.S.H. (Armour P.L.H., 0.5 mg. + Armour F.S.H.-P., 1.0 mg./ml. of saline). Injections were given once a day for 7 days. Hens were sacrificed on the eighth day and the ovaries and oviducts were weighed.
made by preparing 10% polyacrylamide cross-linked with methylene bisacrylamide (20% w./w.) in pH 7.6 Tris-HCl buffer. One mg. of L.H.R.H. (Abbot) was dissolved in each ml. of the acrylamide mixture. Polymerization was achieved by adding 5 u,l. of N ^ N ' ,N'-tetramethylamine (TEMED) and 7 mg. of ammonium persulfate to each 10 ml. of the acrylamide-L.H.R.H mixture. One ml. aliquots of this, containing 1 mg. of L.H.R.H., were placed into glass tubing (6 mm. I.D.) and allowed to polymerize. To implant the gels, small skin incisions were made at the base of the skull and the glass tubing containing the gel was inserted under the loose skin. The gel was forced from the glass with a plunger from a 1 ml. disposable syringe. The glass tube was withdrawn and the incision was closed with a wound clip. Control hens received implants with no L.H.R.H. added. Hens were bled prior to and at 2, 4, 8, 16, 20, 24, 48, 72, 96, 120, 144 and 168 hr. after implantation. Serum L.H. levels were measured on blood samples taken at each of these times and progesterone levels were determined in blood samples obtained at 0, 2, 12, 24 and 120 hr. after gel implantation. Hens were sacrificed and ovarian and oviducal weights were obtained immediately after the 168 hr. bleeding. Serum L.H.R.H. levels were determined by RIA according to the procedure of Nett et al. (1973).
238
W. H. BURKE AND E. A. COGGER
The extraction procedure used, coupled with the chromatographic procedure described separates progesterone from the 17hydroxyprogesterone and the adrenal corticoids (Nuti et ai, 1974) which are the major cross reacting steroids with this antisera (Abraham, 1971). Experiment 1. Serum L.H. of L. hens averaged 5.8 ± 0.7 ng./ml. of serum prior to L.H.R.H. treatment while NL. hens had an L.H. level of 7.7 ± 0.5 ng./ml. (n = 21 in each group). This difference was significant. Both 20 and 40 |xg. of L.H.R.H. caused rapid increases in the serum L.H. levels of NL. hens from 7-8 ng./ml. to 44-45 ng./ml. by 5 min. after injection (Fig. 1). There was no significant difference in the response to these two levels of L.H.R.H. At fifteen minutes after L.H.R.H. injection, L.H. levels were significantly lower than at 5 min. postinjection, but they were still about five times higher than pre-injection levels. The response of L. hens to the L.H.R.H. injection was smaller than that obtained with NL. hens and was quite variable. Twenty |xg. of the hormone caused mean L.H. levels to rise from 6.1 ± 1.6 ng./ml. to 9.8 ± 2.8 ng. /ml. at 5 min. after injection. The 40 |xg. dose of
l'"
1 0
5
15
0
5
15
0
Time after Injection (minutes)
FIG. 1. Serum L.H. levels of laying (L.) and non-laying (NL.) turkeys following the intravenous injection of 0, 20 or 40 |xg. of L.H.R.H. L.H.R.H. caused a rise from 6.4 ± 1.1 ng./ml. to 9.0 ± 1.2 ng./ml. Only three of the seven laying hens receiving the 20 |xg. dose showed changes in L.H. which exceeded the maximum change seen in any of the saline injected laying hens. One showed no change, one a slight decrease and two others a slight increase in L.H. levels. While six of the seven layers receiving 40 |xg. of L.H.R.H. showed increased L.H. levels 5 min. after injection, only one of these was greater than the greatest change seen in controls. The response to 20 (xg. was not significant but the response to 40 (xg. was. Saline injected laying hens had a mean pre-injection L.H. level of 4.8 ± 1.0 ng./ml. and a level of 5.2 ± 0.8 ng./ml. at 5 min. after injection. Individual control hens had L.H. changes ranging from - 2 . 6 ng. to +4.4 ng. in this period. Non-laying hens injected with saline averaged 7.4 + 0.8 ng. / m l . before injection, 6.9 ± 1.0 ng./ml. 5 min. after injection and 5.9 ± 1.0 ng./ml. 15 min. after saline injection. This change, while small, was consistent and the 15 min. L.H. levels were significantly lower than pre-injection levels. The largest increases in L.H. in response to L.H.R.H. in laying hens (7.1, 8.8 and 10.2 ng./ml.) were obtained with hens that had hard shelled, well calcified eggs in utero at
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curve with a slope of —1.13 was obtained with progesterone doses ranging from 251000 pg./tube (90% bound to 11% bound). Buffer blanks, obtained by assaying extracted, chromatographed PBSG, averaged 8.8 pg./assay tube (96.7% of B 0 ) . This value was subtracted from all unknowns prior to the recovery correction and the potency calculation. Spiking experiments were conducted in which unlabeled progesterone was added to serum and P.B.S.G., then extracted, chromatographed and assayed. In repeated experiments of this type in our laboratory, virtually 100% of the spiked progesterone is accounted for when the material is assayed and corrections are made for recovery of the tritiated tracer progesterone.
239
L.H.R.H., L.H. AND OVARIAN GROWTH
injection time. These responses also happened to be obtained early in the day, between 0930 and 1100 hr. No substantial increment in serum L.H. was obtained in the three laying hens that had uncalcified membranous eggs in utero when injected and only one of five hens with empty uteri showed a substantial response to the L.H.R.H.
Experiment 3. NL. hens receiving twice daily injections of either 10 or 30 |i,g. of TABLE {.—Effect of F.S.H./L.H. and L.H.R.H* on ovary and oviduct weight of non-laying turkeys F.S.H./L.H. Ovary wt. (gm.) Oviduct wt. (gm.)
41.0 ± 13.4a 67.6 ± 9.4a
L.H.R.H.
Saline
8.9 ± 1.6b
6.4 ± 0.5b
13.6 ± 2.1b 9.3 ± 1.1b
* Injected daily for 7 days, 1 x per day intramuscularly. F.S.H./L.H. (1 mg. Armour F.S.H.-P. and 0.5 mg. Armour P.-L.H.) (50 t».g., Spectrum Medical Industries). ab Means within a row having different letters are different at the 1% level of probability.
Experiment 4. Serum L.H. levels of hens implanted with L.H.R.H. rose from a preimplantation level of 6.7 ± 0.4 ng./ml. to a peak level of 22.3 ± 4.7 ng./ml. two hr. later (Table 2). The L.H. levels dropped at each successive bleeding and by 16 hr. after implantation they averaged 8.5 ± 1.0 ng. /ml. which was not significantly different from pre-implantation levels. They remained at these lower levels for the duration of the experiment. Control birds implanted with gel alone showed no significant change in serum L.H. during the experimental period. The L.H. level before receiving the control implant averaged 8.0 ± 0.8 ng./ml; the lowest mean level measured during the experiment
TABLE 2.—Serum L.H. and progesterone levels of turkey hens implanted with
L.H.R.H. implant Progesterone (pg./ml.) L.H. (ng./ml.) SE X SE X
Control implant Hours after implantation 0 2 4 8 12 16 20 24 48 72 96 120 144 168
L.H. (ng./ml.) SE X 8.0 7.3 7.9 8.4 6.3 6.7 8.0 6.8 8.2 7.4 7.8 8.2 9.3 8.2
0.8 0.6 0.8 1.0 0.6 0.5 0.8 0.6 0.6 0.9 0.7 0.9 1.3 1.0
L.H.R.H.
6.7 22.3a 14.2a 10.3a 9.6a 8.5 8.2 7.6 7.7 7.9 7.1 8.0 7.6 8.0
"Means significantly different (P s .05) than time 0.
0.4 4.7 1.4 0.9 1.3 1.0 1.1 0.7 0.7 0.9 0.7 1.0 0.9 1.0
194.7 216.4
— —
202.0
35.8 38.6 48.7
— 220.1 194.6
44.2 36.4
213.6
18.9
— —
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Experiment 2. Daily injections of a mixture of L.H. and F.S.H. for seven days caused significant ovarian and oviducal growth. Daily intramuscular injections of 50 u,g. L.H.R.H. for the same period of time did not elicit significant weight change in either the ovary or oviduct (Table 1).
L.H.R.H. for six days showed no increase in ovary, oviduct or pituitary gland weight when compared to saline injected controls. Ovarian weight of the controls averaged 7.57 ± 1.35 g.; the 10 |xg. L.H.R.H. group averaged 8.83 ± 1.02 g. and the 30 (xg. group averaged 8.21 ± 1.22 g. Average oviduct weights (g.) of the 3 groups were 18.96 ± 5.60, 16.05 ± 3.88 and 16.58 ± 3.22, respectively. Anterior pituitary gland weights averaged 19.17 ± 1.22, 19.96 ± 1.67 and 23.57 ± 2.67 mg. for the 0, 10 and 30 |xg. L.H.R.H. groups, respectively.
240
W. H. BURKE AND E. A. COGGER
DISCUSSION The observation that mean serum L.H. of NL. hens is higher than the mean serum L.H. levels of L. hens bled in the morning and early afternoon was unexpected. The individual L.H. values of the L. hens would indicate that only one or two of these birds were bled when L.H. was showing an ovulatory peak. Thus, the L.H. values of L. hens probably represent basal levels. Nevertheless, substantial L.H. was found in NL. hens
with regressed ovaries. It may well be that the lack of ovarian steroid feedback in hens that have been laying and undergone ovarian regression leads to a post-castration like or post-menopausal like rise in L.H. Two pools of sera obtained from many L. and NL. turkeys have verified the higher serum L.H. levels of the NL. hens (Burke, unpublished observations). However, it should be noted that Cogger and Burke (1975) have shown a significant decline in serum L.H. accompanying the shift from an active laying state to a broody state in turkeys. Thus, the elevated serum L.H. of NL. hens seen in this study may follow an initial L.H. decline at the time laying ceases. Imai et al. (1972) also found moulting, non-laying chickens to have higher total serum gonadotropins than layers. The minimal and variable response of L. turkeys to L.H.R.H. appears to be considerably different than the response obtained by Bonney et al. (1974) with laying chickens. While they too found laying birds responded to L.H.R.H. with rather small increments in serum L.H., they obtained significant responses at all times except during that period immediately preceding ovulation. While turkeys are less regular in their laying patterns than chickens are, and even though accurate oviposition-time records were not kept in this study, certain inferences can still be made regarding the stage of the ovulatory cycle that the hens were in at the time of the experiment. Only three laying hens, all carrying heavily calcified eggs (as determined by rectal palpation) showed L.H. responses clearly in excess of the largest change seen in the saline injected L. group. These hens would have been expected to lay their eggs sometime that same day. One of these three birds had a pre-injection L.H. level about 2.7 times greater than the mean level for all L. hens, suggesting she was in the midst of an ovulatory surge. She showed a 10.2 ng./ml. increase in L.H., the largest obtained
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was 6.3 ± 0.6 ng./ml. and the highest was 9.3 ± 1.3 ng./ml. Progesterone levels of the L.H.R.H. implanted hens (Table 2) showed no significant change from pre-implantation levels throughout the course of the experiment. Ovarian weight of the control and L.H.R.H. implanted hens averaged 8.8 ± 1.9 and 9.0 ± 0.9 gm., respectively, at the termination of the experiment. Oviduct weights of the two groups (21.2 ± 10.7 and 11.0 ± 1.9 gm.) did not differ significantly. Relative changes in serum L.H.R.H. levels were determined in a specific R.I.A., using the procedures described byNett etal. (1973). While the assay has not been validated for serum levels of the hormone in turkeys, and the absolute values obtained appeared to be unrealistically high, the pre- and postimplantation levels were, nevertheless, informative. Serum L.H.R.H. levels were elevated in all implanted hens at 2 hr. after implantation. The increases ranged from about 5 fold to more than 30 fold at this bleeding. In all hens, the highest L.H.R.H. levels were found at either the 2, 4 or 8 hr. post-implantation bleeding. Five of the six implanted hens still had elevated L.H.R.H. levels (2.8-4.0 fold higher than pre-implantation values) at 120 hr. Control implanted hens did not show an increase in serum L.H.R.H. On the contrary, their levels seemed to decrease after the first or second bleeding.
L.H.R.H., L.H.
The lack of a difference in response to 20or40(jig. of L.H.R.H. in NL. hens suggests that both doses are on the plateau of the dose response curve. Previous work (Wentworth et al., 1976) has shown a dose response in NL. turkeys using L.H.R.H. at doses of 10 and 50 |xg. Although a strict comparison between these two experiments cannot be made, it appears that around 20 u.g. of L.H.R.H. may elicit a maximal response in NL. turkeys. The ovarian and oviducal growth response obtained by the administration of an F . S . H . / L . H . mixture shows clearly that the ovaries of NL. hens are capable of reactivation if given the proper hormonal stimulus. This agrees with results obtained in chickens when a variety of gonadotropin preparation were administered (Imai et al., 1972; Imai, 1972). The failure of daily i.m. injections of 50 (Ag. of L.H.R.H. or twice daily i.v. injections of 10 or 30 fjLg. to elicit ovarian growth may be due to a number of causes. It is possible that a single i.m. dose of the hormone would not stimulate sufficient gonadotropin release for a long enough period of time to activate the ovary or it may fail to release F.S.H. along with L.H. The latter point has
241
not been directly tested in vivo since until recently it has not been possible to measure circulating levels of F.S.H. in avian blood (Croix et al., 1974). Wentworth et al. (1976) have shown that in NL. turkeys, the response to an i.v. injection of L.H.R.H. is rapid and short lived, L.H. levels peaked within 5-10 min. and fell back to base line levels by 70 min. after injection. A similar time course response was obtained in chickens by Bonney et al. (1974). It might be noted that i.m. injections of L.H.R.H. (1.25 p,g./bird, twice a day for 7 days) does cause sufficient gonadotropin release to stimulate testicular growth in Coturnix housed under a stimulatory photoperiod (El Halawani and Burke, 1975). In Experiment 3 the failure of L.H.R.H. injections to elevate serum progesterone showed further the lack of effectiveness of this type of treatment for reactivating ovarian function. Luteinizing hormone injections do cause rapid increases in serum progesterone in laying chickens (Shahabi et al., 1975) and in laying turkeys (Camper and Burke, 1976). While the L.H.R.H. implants used in Experiment 4 caused a prolonged elevation of serum L.H.R.H., the rather short lived elevation in serum L.H. indicated that the pituitary failed to continuously respond to elevated levels of the releasing hormone. It may require an increase in L.H.R.H. to activate the pituitary release mechanism rather than a high level per se. The prolonged release of L.H.R.H. from the gel implant indicates that this technique may be a useful way to administer peptide hormones and avoid the sudden surges and declines that accompany repeated injection techniques.
ACKNOWLEDGMENTS The authors gratefully acknowledge the splendid technical assistance given by L. A. Ogren in performing the radioimmunoassays for these studies. This work was supported in part by grants
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in any of the L. hens. In addition, three of the hens carrying heavily calcified eggs did not respond to the L.H.R.H. and they could conceivably fall into the pre-ovulatory group of non-responders seen by Bonney et al. (1974). The three birds with a membranous egg in utero (i.e. a few hours after ovulation) did not respond to the L.H.R.H., while only one of four hens with an empty uterus showed a response which was slightly greater than the largest change seen in any of the control hens. Thus, it appears that substantial differences exist in the response of laying chickens and turkeys to this hormone. The striking response of NL. hens is similar to the response obtained in cockerels and immature female chickens (Furr et al., 1973; Bonney et al., 1974).
AND OVARIAN GROWTH
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W. H. BURKE AND E. A. COGGER
from the Minnesota Turkey Growers Association and by grant number HD 07242 from the National Institute of Child Health and Human Development, N.I.H. REFERENCES
JULY 10-13, 1977.
ANNUAL MEETING, CANADIAN SOCIETY OF ANIMAL SCIENCE, UNIVERSITY OF NEW BRUNSWICK, FREDERICTON, NEW BRUNSWICK
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pituitary preparations on follicular growth in hens treated with methallibure or fasting. J. Reprod. Fert. 31: 387-397. Imai, K., M. Tanaka and S. Nakajo, 1972. Gonadotrophic activities of anterior pituitary and of blood plasma and ovarian response to exogenous gonadotrophin in molting hens. J. Reprod. Fert. 30: 433-443. Jeffcoate, S. L., P. J. Sharp, H. M. Fraser, D. T. Holland and A. Gunn, 1974. Immunochemical and chromatographic similarity of rat, rabbit, chicken and synthetic luteinizing hormone releasing hormones. J. Endocrinol. 62: 85-91. Nett, T. M., A. M. Akbar, G. D. Niswender, M. T. Hedlund and W. F. White, 1973. A radioimmunoassay for gonadotropin-releasing hormone (Gn-RH) in serum. J. Clin. Endocr. Metab. 36: 880-885. Nuti, L. C , W. H. McShan and R. K. Meyer, 1974. Effect of ovine FSH and LH on serum steroids and ovulation in hypophysectomized immature female rats. Endocrinology, 95: 682-689. Reeves, J. J., P. C. Harrison and J. M. Casey, 1973. Ovarian development and ovulation in hens treated with synthetic (porcine) luteinizing hormone releasing hormone (LH-RH/FSH-RH). Poultry Sci. 52: 1883-1886. Shahabi, N. A., J. M. Bahr and A. V. Nalbandov, 1975. Effect of LH injection on plasma and follicular steroids in the chicken. Endocrinology, 96: 969-972. Tanaka, K., M. Sakaida and M. Kamiyoshi, 1974. Release of gonadotropins from the chicken pituitary by synthetic LH-RH/FSH-RH. Poultry Sci. 53: 72-75. Van Tienhoven, A. and A. V. Schally, 1973. Mammalian luteinizing hormone-releasing hormone induces ovulation in the domestic fowl. Gen. Comp. Endocr. 19: 594-595. Wada, M., 1975. Cell types in the adenohypophysios of the Japanese quail and effects of injection of luteinizing hormone-releasing hormone. Cell Tiss. Res. 159: 167-178. Wentworth, B. C , W. H. Burke and G. P. Birrenkott, 1976. A radioimmunoassay for turkey luteinizing hormone. Gen. Comp. Endocrinol. 29: 119-127.
