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In Vivo Studies of Luteinizing Hormone-Releasing Factor in the Chicken Hypothalamus
In Vivo Studies of Luteinizing Hormone-Releasing Factor in the Chicken Hypothalamus H . O P E L 1 AND P . D .
LEPOKE2
United States Department of A griculture (Received for publication October 9, 1971)
POULTRY SCIENCE 51: 1004-1014, 1972
T
H E existence of a luteinizing hormone-releasing factor (LRF) in the avian hypothalamus has not been clearly established. Research in this area has been hindered b y the formidability of collecting hypothalamic tissues in the kilogram quantities needed for isolation and identification of L R F , and b y the lack of a specific and sensitive assay for avian L H . Much of the available evidence is controversial. Early in vitro demonstrations of L R F in the chicken hypothalamus (Jackson and Nalbandov, 1969a; T a n a k a et al., 1969), based on use of the ovarian ascorbic acid depletion (OAAD) assay to measure L H , were quickly invalidated b y the discovery (Jackson and Nalbandov, 1969, b, c; Ishii et al., 1970) t h a t this assay also is sensitive to arginine vasotocin, a hypothalamic agent shown t o be devoid of L H or L R F activity. Follett (1970) used a P 32 u p t a k e assay to show t h a t ex-
1 Animal Science Research Division, A.R.S., Beltsville, Md. 20705. 'Present address: Division of Nutritional Science, Bureau of Veterinary Medicine, F.D.A., Rockville, Md. 20850.
tracts of quail or chicken hypothalami stimulate in vitro release of gonadotrophin from chicken pituitaries, but, since the assay is highly sensitive to both F S H a n d L H , he could not specifically identify the gonadotrophin released. Jackson and Nalbandov (1969a) and Casey et al. (1971) reported t h a t avian hypothalamic extracts stimulate release of L H from the rat pituitary, b u t the value of these findings as evidence for avian L R F is uncertain, particularly since the former authors found t h a t their extracts did not stimulate release of L H from the chicken pituitary. Opel and Lepore (1967) and Clark and Fraps (1967) reported briefly t h a t intrapituitary infusion of chicken hypothalamic extracts evokes premature ovulation in the hen, b u t this evidence for L R F , based on a response of known physiological significance, has not been adequately described. T h e continued lack of pertinent information, and the potential value of the intrapituitary infusion technic for evaluation of the recently synthesized porcine gonadotrophin-releasing hormone (Baba et al., 1971; Schally et al., 1971) and its analogs as releasers of avian
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A BSTRA CT Induction of ovulation was used to evaluate luteinizing hormone-releasing factor (LRF) activity in acidic extracts of chicken hypothalamus infused directly into the anterior pituitary of the hen. LRF activity was found in extracts of stalk-median eminence (SME), posterior pituitary and tuberal hypothalamus, but not in extracts of cerebral cortex. The crude SME extract was highly effective in inducing premature ovulation when infused into the adenohypophysis, but evoked no response when infused into adjacent hypothalamic structures or the systemic circulation. On fractionation of the SME extract on Sephadex G-25, LRF activity emerged in the same region of the column effluent as did oxytocic activity. However, a battery of control procedures indicated that the LRF activity is not attributable to mesotocin or arginine vasotocin. Intrapituitary infusion of dopamine, histamine, acetylcholine, epinephrine, norepinephrine or seratonin gave negative results. From these data, LRF in the chicken appears to be a low molecular weight, peptide-like material distinct from known median eminence hormones.
LUTEINIZING HORMONE-RELEASING FACTOR
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PP fragments and applied to a 2.5X40 cm. column of Sephadex G-25 equilibrated and subsequently eluted with MATERIALS AND METHODS 0.15 M acetic acid at 4°C. Sixty 5-ml. Preparation oj extracts. For tests of fractions were collected, their absorbance crude extracts, stalk-median eminence at 280 mju. monitored, and the peptide (SME), posterior pituitary (PP) and concentration determined by the Folinbrain tissues were obtained from groups Lowry reaction as modified by Lowry of 20-80 adult female chickens decapi- et al. (1951). tated in the laboratory. The tissues were In the first three runs with SME excollected immediately on dry ice and tracts, and the first two runs with PP crude extracts prepared as described by extracts, oxytocic activity in the effluent Campbell et al. (1964) using a total vol. from the Sephadex column was followed of 1 ml. of 0.1 N HC1/150 mg. wet wt. of by the hen and rat uterine assays as detissue. The extracts were stored in sealed scribed by Munsick (1964), using Synglass ampules at — 4°C. and tested within tocinon as the standard. Selected active 5 days. fractions were reassayed after incubation In some experiments, the crude SME with 0.05 M sodium thioglycollate as deextract, or a similarly prepared extract of scribed by Munsick et al. (1960). Concen+ ++ anterior pituitary tissue from the same tration of Na , K+ and Ca were dehens, was heated in a boiling water bath termined by flame photometry. Based on for 10 min. at the end of the extraction the reproducible elution profiles thus procedure. In other experiments, the ex- obtained, appropriate fractions from subtract was hydrolized with twice-crystal- sequent runs were pooled and freeze dried. lized pepsin or trypsin as described by The dried powder was stored at — 20° C. and tested for LRF activity within 1 Munsick et al. (1960). For gel-filtration studies, tissues were week. taken from heads of adult chickens of both Intrapituitary infusions. Experimental sexes obtained at a poultry processing birds were selected from a flock of 960 plant. Since the resident poultry inspec- White Leghorn hens caged individually tors would not permit dissection of the in laying batteries under artificial light heads at the plant, all heads were col- from 6 a.m. through 8 p.m. Patterns of lected on dry ice within 8 min. of death ovulation were determined from hourly and brought to the laboratory. To permit records of lay maintained from 8 a.m. to adequate dissection of the frozen tissues, 4 p.m. Only hens ovulating in recurrent the base of the skull, including the floor sequences of 2 or 3 eggs, with all eggs laid of the sella, was chopped off with a block- during the hours of recording, were used. mounted knife, the bony plate surroundTest substances were infused directly ing the pituitary was partially thawed into the adenohypophysis, median emiand cut free, and the adenohypophysis nence or tuberal hypothalamus via a carefully removed. Under magnification, stereotaxically placed, 27 gauge, platthe PP and SME tissues were excised with inum-irridium cannula (Opel, 1966a). To a pair of fine spring-handle scissors and avoid the median sagittal sinus, all canplaced on dry ice. At the end of each nulae were placed 0.5 mm. to the left of collection day, a crude extract was pre- the midline. To insure insertion of the pared from the yield of 400-500 SME or cannula tip into the main body of the gonadotrophin, prompted us to describe our studies of chicken LRF in detail.
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H. OPEL AND P. D. LEPORE
Test solutions were neutralized with sodium bicarbonate, and sodium chloride added to physiological concentrations, immediately before infusion at a rate of 1.5 /^l./min. over a 90 min. period. All infusions were performed between 3 and 5 p.m. on the day before ovulation of the first (Ci) egg of a sequence. Induced Ci ovulation, premature by 6-9 hours, was detected by the presence of a "plumped" egg on digital palpation of the shell gland at 8:30 a.m. on the next day. During 2:30-4:00 p.m., all hens were infused with an aqueous solution of analine blue dye at the same rate as the test substance had been infused on the previous day. At termination of the infusion, each hen was
decapitated and autopsied. The approximate time of Ci ovulation was verified from the position and stage of formation of the oviducal egg, and the ovary was checked for atretic and freshly ruptured follicles. The base of the skull underlying the pituitary capsule was removed, and the spread of the dye and the position of the pituitary cannula determined macroscopically. Hens in which the intrapituitary infusate reached less than 20% of the adenohypophyseal tissue were eliminated from the experiment. Positions of hypothalamic cannulae were verified histologically by the method of Fox and Eichman (1959). Intrajugular infusions in hypophysectomized hens. To allow adequate testing of the limited supply of gel-filtrated LRF powder, Japanese quail were used. Regularly ovulating hens were kept under conditions similar to those described for chickens. To insure accuracy in the prediction of ovulation (Opel, 1966b), each experiment was performed for effect on the 2nd or 3rd ovulation of a sequence of 6 or more eggs. Spontaneous ovulation was blocked in 50 quail by the intravenous infusion of 0.02-0.03 ml. of Equithesin (JensenSalsbery Laboratories) 14 hours before expected normal ovulation. Twenty hens were hypophysectomized while under the resultant deep anesthesia; 30 served as unoperated controls. All test samples were infused between 11 and 10 hours before the time of normal ovulation. Each hen was reanesthetized with Equithesin and immobilized in the usual backdown position on the hypophysectomy board (Opel, 1969). A wooden cylinder, placed directly behind the headholder, was used to elevate and extend the neck. A 27-gauge needle, with a short length of fine polyethylene tubing attached, was inserted into the left jugular
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adenohypophysis, and to avoid perforation of the ventral surface of the gland or the internal carotid arteries, each pituitary cannula was placed within stereotaxic coordinates A6.s to 7.0 and H0.o to _i.o (van Tienhoven and Juhasz, 1962). Two infusion systems were used. In the tests of crude extracts, infusions were performed in conscious, unrestrained hens within 3-15 days of cannula implantation. Although useful for preliminary work, this method proved too unwieldy for large scale experimentation because some 40% of the hens bearing chronic implants either ceased to lay after one or two eggs, or laid in sequences too variable for adequate prediction of ovulation and thus could not be used in the experiment. Consequently, in subsequent tests of gelfiltrated extracts, synthetic hormones and biogenic amines, cannula placement and infusion were performed in one session in pentobarbital-anesthetized hens. Pentobarbital, during the period administered, has no effect on ovulation (Bastian and Zarrow, 1952). The effects of surgical and infusion procedures were followed in control hens given infusions of neutral saline.
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LUTEINIZING HORMONE-RELEASING FACTOR
RESULTS
Tests of crude extracts. Intrapituitary infusion of 20 mg. equivalents of crude SME extract evoked premature ovulation in 15 of 21 hens (Table 1). Our results with this dosage corroborate those of Clark and Fraps (1967). Infusion of 10 mg. equivalents had a borderline effect, as indicated by forced ovulation in 1 of 6 hens and atresia of the ovulable ovarian follicle in 3 others, while infusion of 5 mg. was ineffective. For reasons unexplained, infusion of 40 mg. of extract consistently prevented ovulation and caused generalized atresia of large follicles. Crude extracts of cerebral cortex showed no detectable LRF activity, whereas intrapituitary infusion of 30 mg. equivalents of tuberal hypothalamus evoked ovulation in 2 of 6 hens (Table 1). Since chicken LH is heat labile (Tanaka et at., 1969; Jackson and Nalbandov, 1969c), heat treatment was used to determine if the ovulation-inducing activity of the SME extract could be accounted for by LH contamination. As shown in Table 1, boiling for 10 min. had no effect on the crude SME extract, but completely destroyed the ovulating potency of a similarly prepared extract of anterior pituitary obtained from the same hens. Other control data supported the prob-
TABLE 1.—Ovulation in the chicken after intrapituitary infusion of crude tissue extracts
Crude extract infused
Mg. equivalent wetwt. of tissue
Stalk-median eminence (SME)
5 10 20 40 SME, boiled 10 min. 20 Anterior pitui5 tary (AP) 5 AP, boiled 10 min. Posterior pitui0.6-1.0 tary (PP) 20 Tuberal 20-30 hypothalamus 20-30 Cerebral cortex
No. of hens
No. ovulating
6 6 21 6 10
0 1 IS 0 6
10 10
8 0
9 9
0 1
12 12
2 0
ability that the SME extract acted directly on the adenohypophysis. Intrajugular infusion of 20-80 mg. of SME extract, i.e., 1-4 times the optimal intrapituitary dose, did not evoke ovulation in any of 20 hens. Infusion of 20 or 40 mg. of extract directly into the median eminence or ventral hypothalamus, at the same anterioposterior and lateromedial coordinates used for intrapituitary infusion, failed to evoke ovulation in 16 hens. To test the possibility that LRF activity in the SME extract might be attributable to the known presence of the PP hormones, oxytocin and arginine vasotocin (AVT), crude extracts of PP tissues were infused into the adenohypophysis at 2 dosages; one (0.6-1.0 mg.) designed to provide the assayable amount of oxytocic activity found in 20 mg. equivalents of SME extract, the other (20 mg.) to provide the equal amount of tissue. Infusion of the low dose had no effect, while infusion of the high dose, which contained 20-30 times the oxytocic activity in 20 mg. of SME extract, evoked ovulation in 1 of 9 hens. Incubation of the SME extract with trypsin, which inactivates
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vein and held in place by a strip of adhesive tape. The free end of the tubing was coupled to the micro-infusion apparatus (Harvard Model 975 infusion pump) and a solution of LRF powder, ovine L H (NIH-S10) or saline was infused in a vol. of 0.2 ml. over a 30 min. period. All hens were killed 9 hours after infusion, the body cavities and oviducts checked for ova and the ovaries examined for freshly ruptured follicles. Completeness of hypophysectomy was verified histologically (Opel, 1969).
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H. OPEL AND P. D. LEPORE TABLE 2.—Effects of peptic or tryptic digestion of crude SME extract
SME incubated with pepsin SME incubated without pepsin
8 8
2 6
SME incubated with trypsin SME incubated without trypsin
8 8
5 5
1 Each hen received 20 mg. equivalents of SME extract.
I
16
).
20
24
.
,
,
,
28
3Z
56
+
,
,
,
40 ' 44
B
«
+
,
92
e
,
t-K)
«
60
-I
FIG. 1. Gel-filtration of7crude^SME extract (4.45 ml.) on Sephadex G-25 (2.5X40 cm.) in 0.15 M acetic acid. Tube vol. was 5 ml. Note that ordinate for oxytocic activity is logarithmic. Broken lines indicate absence of detectable oxytocic activity and show lower limits of assay sensitivity. Using bioassay data as a guide, tubes were combined into fractions A-C for LRF assay.
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AVT, but not oxytocin (Munsick et al., 1960), and which reduced the assayable oxytocic activity by more than 90%, had no effect on LRF activity (Table 2). On the other hand, incubation of the extract with pepsin, which does not inactivate either PP hormone, and which reduced the oxytocic activity by less than 8%, appreciably reduced the LRF activity. Attempts to use thioglycollate inactivation, which destroys the oxytocic activity of both PP hormones (Munsick et al., 1960), failed because the presence of this reducing agent in the infusate disrupted ovulation. Gel-filtration of SME extracts. Fractionation of the acid extract of 400-500 chicken SME fragments on Sephadex G-25 yielded a single zone of oxytocic activity in close association with a peptide peak (Fig. 1, tubes 32-47). Results of hen and rat oxytocic assays, as well as comparisons of rat oxytocic activities with or without 0.5 mM. Mg + + in the bath solution (not shown in the figure), indicated that all tubes from the active zone contained a mixture of oxytocin and AVT, with AVT predominating. Incubation of aliquots of peak fractions with 0.05 M thioglycollate destroyed 98%, while incubation with trypsin destroyed 88% of the oxytocic activity, suggesting that roughly these percentages of the activity were attributable, respectively, to total PP hormones and to AVT. About 84% of the
oxytocic activity was recovered after gelfiltration. Reproducible profiles of oxytocic activity in the first 3 fractionations, and the results of spot assays to define boundaries of activity in subsequent runs, were used as guides to combine the eluates from 14 columns into 3 zones (Fig. 1, A-C). Results of LRF tests of the freeze-dried powder from each zone are shown in Table 3. Significant LRF activity emerged only in the zone containing oxytocic activity. Complete quantitation was prevented by the low yield of dry powder and the relative insensitivity of the ovulation test, but intrapituitary infusion of 0.25 mg. of LRF powder (Zone B), roughly equivalent to 26 mg. wet wt. of SME tissue, induced ovulation in 11 of 20 hens. Infusion of an equivalent dose of the powder from the less strongly retarded Zone A (tubes 1631) did not evoke ovulation, but a 6-fold higher dose induced ovulation in 2 of 10 hens. The trace activity in Zone A may reflect either contamination with LH, or some displacement of LRF activity to the left of tube 32.
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LUTEINIZING HORMONE-RELEASING FACTOR
TABLE 3.—Ovulation in chickens after intrapituitary infusion of gel-filtration zones A-C Amount of dry powder infused (mg.)
No. of hens
No. ovulating
Stalk-median eminence A 0.25-1.5 B 0.25 0.5 - 1 . 0 C 0.25-1.0
16 11 11 6
2 20 21 0
Posterior pituitary A 0.25-1.5 B 0.1 - 2 . 0 C 0.1 - 1 . 0
8 14 5
0 1 0
Zone
TABLE 4.—Intrapituitary infusion of posterior pituitary hormones and biogenic amines ' i n s
6
Arginine vasotocin 20-800 mU.* Oxytocin (Syntocinon) 10-100 mU. Vasotocin 100-200 mU. +oxytocin +10-20 mU. Vasopressin (Pitressin) 20-60 mU. Acetylcholine 5-10 ng. Epinephrine 20-30 jug. Norepinephrine 5-10 jug. Histamine dihydrochloride 1-3 mg. Seratonin creatinine SO« 0.5-1 mg. Dopamine 5-10 /ig.
No. of hens
No._ ovulating
14
0
8 8
0 0
8 14 8 8
0 1 0 0
8
0
8 8
0 0
*Expressed as International Units of rat oxytocic activity per mg.
was tested for LRF activity. As shown in Table 3, ovulation was obtained only in 1 of 14 hens infused with powder from the oxytocic-active Zone B. The effective dose (0.5 mg.) contained about 40 times the assayable oxytocic activity in the most effective dose (0.25 mg.) of LRF powder from the fractionation of SME. Tests oj PP hormones and biogenic amines. The results of intrapituitary infusion of PP hormones, and of some biogenic amines known to occur in the hypothalamus, are shown in Table 4. Infusion of varying levels of oxytocin and AVT, alone or in combination, failed to evoke ovulation. Intrapituitary infusion of acetylcholine chloride (Ach) induced ovulation in 1 of 14 hens. To test the possibility that this isolated ovulation may have resulted from backtracking of the Ach solution along the cannula wall into the hypothalamus, the same amount of Ach solution was infused directly into the median eminence in 6 hens. No premature ovulations were obtained. C\ ovulation in controls. Examination of the records of 124 unoperated hens, selected exactly on the same basis as the
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To achieve greater purification, the oxytocic-active zones from 3 additional runs on the column, representing the oxytocic activity from 1700 SME fragments, were pooled, freeze-dried to a vol. of 20 ml., and recycled through the same column. This time, as indicated by the pattern of ultraviolet absorption and bioassays of aliquots of appropriate tubes, oxytocic activity emerged in tubes 36-44 and was well separated from the larger molecules. Once again LRF activity emerged in the same zone as oxytocic activity. Intrapituitary infusion of 0.75 mg. of the freeze-dried LRF powder evoked ovulation in 3 of 5 hens. Gel-filtration of PP extracts. Fractionation of PP extracts on the 2.5X40 cm. Sephadex column produced profiles of ultraviolet absorption, Folin-Lowry peptide concentration and oxytocic activity quite similar to those obtained by fractionation of equivalent amounts of SME extract, except that peak values of oxytocic activity were many times higher. Since the oxytocic activity was spread throughout the same region, the eluates from fractionation of four PP samples were combined into exactly the same zones used to assess eluates from the fractionation of SME samples (Fig. 1), and the freeze-dried powder from each zone
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H. OPEL AND P. D. LEPORE
TABLE 5.—Ovulation in hypophysectomized or Equithesin-blocked quail after intrajugiilar infusion of LRF powder Treatment
No. No. of ovuquail lating
DISCUSSION 0.25-2.5 mg. 6.0 Mg-
10 10
0 10
0.25 mg. 6.0 Mg.
10 10 10
8 9 0
experimental hens, showed that the predicted Ci ovulation would occur as expected about 96% of the time. No "spontaneous" premature ovulations were observed. Of 20 normally ovulating hens bearing chronic cannula implants and given an intrapituitary infusion 3-15 days following cannula placement, all ovulated at the normally expected time. Of 40 hens given an infusion of saline at 1 to 2 hours after cannula placement, 33 ovulated as normally expected, while 7, or 17%, failed to ovulate. All non-ovulating hens showed massive atresia of the large ovarian follicles on autopsy. The results of the control infusions indicate that the data from all experiments involving the acute preparation (data in Tables 3 and 4) are probably negatively biased. Lack of response to LRF in hypophysectomized quail. Since our LRF-active SME powder was not highly purified, the possibility of a direct action on the ovary had to be examined. As seen in Table 5, intrajugular infusion of 0.25 mg. of the powder evoked ovulation in 8 of 10 Equithesinblocked quail, but infusion of up to 10 times this amount had no effect on hypophysectomized quail. In contrast, infusion of a predetermined minimal effective dose of 6 fig. of LH produced essentially the same proportion of positive responses
The present results show clearly that crude or partially purified extracts of chicken SME evoke premature ovulation on intrapituitary infusion in the hen. Our evidence that the ovulation-inducing substance in the extracts is LRF is best interpreted on the basis of the experiments of Nikitovitch-Winer (1962) and Campbell et al. (1964), who were the first to demonstrate LRF activity in the mammalian hypothalamus. In order to show that their hypothalamic preparations contained a specific mediator that acted directly on adenohypophyseal cells to release LH, these investigators developed a battery of control procedures that allowed them to obviate every known source of falsepositive responses. We have applied their safeguards to our experiments with analogous results. Sources of false-positive responses eliminated or discounted in our experiments include: 1) the occurrence of spontaneous premature ovulation in experimental hens, 2) induction of ovulation by simple mechanical irritation or injury to the hypothalamo-hypophyseal system during implantation of the cannula or infusion of extracts, 3) variations in the ovulatory response due to variations in infusion rate or in the volume of pituitary tissue reached by the infusate, 4) the presence in brain tissue of a "non-specific" substance capable of evoking ovulation by stimulatory or irritative action on the pituitary or ovary, 5) ovulatory discharge of LH secondary to action of the extracts on brain tissues overlying the pituitary gland, 6) discharge of LH resulting from changes in pituitary blood flow produced
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Hypophysectomized infused with LRF powder LH quithesin-blocked fused with LRF powder LH Solvent
Amount of dry powder infused
in hypophysectomized as in Equithesinblocked hens, indicating that the ovaries of the hypophysectomized birds retained their normal sensitivity to LH under our experimental conditions.
LUTEINIZING HORMONE-RELEASING FACTOR
subsequent unpublished work, we found that intrapituitary infusion of 0-80 mM. of K+, added to buffered physiological saline or incorporated into subthreshold doses of SME extract, did not evoke ovulation. It seems unlikely, therefore, that the LRF activity of the SME extracts can be accounted for by their K+ content. Our results provide crude quantitative, as well as qualitative evidence for LRF. The highest effective dose of crude SME extract represented about 4.2 and the lowest effective dose about 2.1 median eminences. Although direct comparisons are prevented by differences in technic, these dosages compare favorably with the dosages of crude rabbit SME extract found by Campbell et al. (1964) to evoke ovulation on intrapituitary infusion in the doe. Surprisingly, intrapituitary infusion of a quantity of SME extract equivalent to about 8.4 median eminences blocked ovulation and caused massive atresia of the large ovarian follicles. This response may indicate a toxic reaction to the peptide material in the extract (Fraps and Neher, 1945) or, alternately, the presence of a specific gonadotrophin-inhibiting substance in the SME. The available data provide few clues to the nature of LRF in the chicken SME. Gel filtration on Sephadex, selected as a simple and repeatable small scale method of proven worth in separating LH from mammalian LRF (Ramirez et al., 1964; Fawcett et al., 1965) suggested that the active material is AVT, oxytocin, or a substance of similar molecular form. Control procedures, however, indicate that LRF is not oxytocin or AVT. Hydrolysis with trypsin, which inactivates AVT, did not reduce the LRF activity of crude SME extract. Intrapituitary infusion of synthetic oxytocin or AVT, alone or in combination, did not evoke ovulation. Infusion of crude or gel-filtrated PP ex-
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by vasoactive substances, such as histamine or adrenaline, known to be present in SME extracts, and 7) the presence of LH or LH-like substances in the extracts. Final proof that significant LH contamination was not present in the extracts was obtained through use of the second in vivo system employing Japanese quail. The fact that intrajugular infusion of 0.25 mg. of the partially purified LRF powder evoked ovulation in 8 of 10 intact quail, but did not evoke ovulation in any of 10 hypophysectomized quail exhibiting normal sensitivity to exogenous LH, established that the ovulation-inducing substance did not act on the ovary, and therefore could not be LH. A potential source of error outside our control was the presence of an ionic imbalance or excessive ionic contamination in the LRF-active preparations. Since we were not able to collect enough SME tissues at any one time to allow the use of volatile extractants, we could not remove the need to neutralize the extracts and thus introduce an ionic imbalance in the final solutions. Further, gel filtration of the crude extract on Sephadex must have resulted in excessive accumulation of ions in the LRF-active fraction. In view of a report by Salmi and Geschwind (1968) that excessive levels of K+ in the incubation medium stimulate in vitro release of LH from rat pituitaries, we determined the levels of K+ in our solutions prepared for intrapituitary infusion. Concentrations of K+ in the optimum doses of crude and partially purified SME extract were 6.3 and 8.9 mM., respectively. The concentration of K+ in the dose of purified SME is about 1.2 times the maximum level of K+ reported for chicken plasma (Sturkie, 1965), but far below the level of 59-60 mM. found to stimulate LH release from the rat pituitary (Salmi and Geschwind, 1968; Wakabayshi et al., 1969). In
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H. OPEL AND P. D. LEPORE
Other control data from this study indicate LRF activity in the SME is not attributable to any of several biogenic amines known to occur in the hypothalamus. Since the LRF material was susceptible to pepsin hydrolysis, it may be a polypeptide. Resistance to boiling suggests that it is heat stable. These results are similar to those obtained in early investigations of mammalian LRF (Ramirez et al., 1964; Fawcett et al, 1965; Nikitovitch-Winer et al., 1965). A biological similarity between avian and mammalian LRF is also implied by the observation that extracts of bovine hypothalamus evoke premature ovulation on intrapituitary infusion in the hen (Clark and Fraps, 1967), while extracts of avian hypothalamus stimulate LH release from rat pituitaries in vitro (Jackson and Nalbandov, 1969a) or in vivo (Casey et al., 1971). In view of these similarities, evaluation of the recently synthesized porcine gonadotrophin-releasing hormone (Baba et al., 1971) and its analogs in avian test systems may provide the means to tentatively
identify avian LRF and answer the now important question of whether in birds, as in mammals (Schally et al., 1971), a single hypothalamic agent controls release of both FSH and LH. The recent development of a radioimmunoassay for chicken LH (Cain and Wilson, 1971) raises the possibility that these problems may now be successfully approached by in vitro technics. Finally, it should be pointed out that assessment of the ovulatory potency of test solutions by intrapituitary infusion is an especially difficult and unwieldy procedure to apply, in the hen. Due to the relative ease with which surgery disrupts ovulation in the hen (Rothchild and Fraps, 1945) and because of the high possibility of severing hypophyseal portal vessels in the dorsal approach to the pituitary, considerable experience and extreme care are required to stereotaxically place the intrapituitary cannula. Despite extensive preliminary work and the use of every known precaution, we were unable to eliminate surgical effects as a complicating factor in our experiments. Attempts to implant the cannula via the parapharyngeal approach were even less successful. A further hinderance to use of the technic is the lack of a completely effective pharmacological block to ovulation in the hen that can be applied late enough to avoid possible interference with follicular maturation. The consequent need to precisely schedule intrapituitary infusions for effect on spontaneous ovulation severely limits the number of experimental hens that can be drawn at any one time from the existing flock. Attempts to employ intrapituitary infusion in the much smaller Japanese quail, in which we found that ovulation could be selectively blocked by injection of Equithesin, were entirely unsuccessful.
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tracts containing either the same or many times the level of oxytocic activity found in an effective dose of SME extract had no effect on ovulation. Since these studies were initiated, several authors (Jackson and Nalbandov, 1969c; Tanaka et al., 1969; Follett, 1970) have concluded from in vitro studies that avian LRF is not a neurohypophyseal hormone. Recently, Acher et al. (1970) have obtained chemical evidence that the neurohypophyseal hormone in birds identified by pharmacological data as oxytocin is, in fact, mesotocin. Since mesotocin would exhibit essentially the same behavior as oxytocin in our purification and pharmacological assay systems, this finding does not alter our conclusion that chicken LRF is not a neurohypophyseal hormone.
LUTEINIZING HORMONE-RELEASING FACTOR ACKNOWLEDGEMENT
We wish to thank Dr. B. Berde, Sandoz Ltd., Basle, for the synthetic arginine vasotocin used in this study. The ovine LH was donated by the Endocrinology Study Section of the National Institutes of Health. We are also grateful to Mr. Millard Sindler, Dover Poultry Products, Inc., Baltimore, for permission to collect chicken heads.
Fraps, R. M., and B. H. Neher, 1945. Interruption of ovulation in the hen by subcutaneously administered non-specific substances. Endocrinology, 37: 407-414. Ishii, S., A. K. Sarkar and H. Kobayashi, 1970. Ovarian ascorbic acid-depleting factor in pigeon median eminence extracts. Gen. Comp. Endocrinol. 14: 461-466. Jackson, G. L., and A. V. Nalbandov, 1969a. Luteinizing hormone releasing activity in the chicken hypothalamus. Endocrinology, 84:12621265. Jackson, G. L., and A. V. Nalbandov, 1969b. A substance resembling arginine vasotocin in the anterior pituitary gland of the cockerel. Endocinology, 84: 1218-1223. Jackson, G. L., and A. V. Nalbandov, 1969c. Ovarian ascorbic acid depleting factors in the chicken hypothalamus. Endocrinology, 85: 113— 120. Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall, 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. Munsick, R. A., 1964. Neurohypophyseal hormones of chickens and turkeys. Endocrinology, 75:104112. Munsick, R. A., W. H. Sawyer and H. B. van Dyke, 1960. Avian neurohypophyseal hormones: pharmacological properties and tentative identification. Endocrinology, 66: 860-861. Nikitovitch-Winer, M. B., 1962. Induction of ovulation in rats by direct intrapituitary infusion of median eminence extracts. Endocrinology, 70: 350-358. Nikitovitch-Winer, M. B., A. H. Pribble and A. D. Winer, 1965. Luteinizing hormone-releasing factor: Partial purification. Am. J. Physiol. 208: 1286-1290. Opel, H., 1966a. A simple cannula for implantation into the chicken brain. Poultry Sci. 45: 856-858. Opel, H., 1966b. The timing of oviposition and ovulation in the quail (Cohtrnix coturnix japonica). Brit. Poultry Sci. 7:29-38. Opel, H., 1969. Transbuccal hypophysectomy in the Japanese quail. Poultry Sci. 48: 722-728. Opel, H., and P. D. Lepore, 1967. Ovulating hormone-releasing factor in the chicken hypothalamus. Poultry Sci. 46:1302. Ramirez, V. D., R. Nallar and S. M. Mc Cann, 1964. Purification of luteinizing hormone-releasing factor from beef hypothalamus. Proc. Soc. Exptl. Biol. Med. 115: 1072-1076. Rothchild, I., and R. M. Fraps, 1945. The relation
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REFERENCES Acher, R., J. Chauvet and M.-T. Chauvet, 1970. Phylogeny of the neuro-hypophysial hormones. Avian active peptides. Eur. J. Biochem. 17:509513. Baba, Y., H. Matsuo and A. V. Schally, 1971. Structure of the porcine LH- and FSH-releasing hormone. II. Confirmation of the proposed structure by conventional sequential analyses. Biochem. Biophys. Res. Comm. 44: 459-464. Bastian, J. W., and M. X. Zarrow, 1952. Failure of nembutal to block ovulation in the hen. Proc. Soc. Exptl. Biol. Med. 79: 249-252. Cain, J. R., and W. O. Wilson, 1971. Development of a radioimmunoassay for chicken LH. Poultry Sci. 50: 1560. Campbell, H. J., G. Feuer and G. W. Harris, 1964. The effect of intrapituitary infusion of median eminence and other brain extracts on anterior pituitary gonadotrophin secretion. J. Physiol. 170: 474-486. Casey, J. M., J. J. Reeves, P. C. Harrison and R. P. Peterson, 1971. Dual bio-radioimmuno detection of LH-RH like activity in hypothalamic tissue of coturnix quail. Poultry Sci. 50: 1562-1563. Clark, C. E., and R. M. Fraps, 1967. Induction of ovulation in the chicken with median eminence extracts. Poultry Sci. 46:1245-1246. Fawcett, C. P., G. W. Harris and M. Reed, 1965. The purification of the luteinizing hormone releasing factor (LRF). In: Proceedings of the 23rd International Congress of Physiological Sciences. Excerpta Medica Int. Congr. Series No. 87, vol. 3:300-305. Follett, B. K., 1970. Gonadotrophin-releasing activity in the quail hypothalamus. Gen. Comp. Endocrinol. 15: 165-179. Fox, C. A., and J. Eichman, 1959. A rapid method for locating intracerebral electrode tracts. Stain Technol. 34: 39-42.
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H. OPEL AND P. D. LEPORE Sturkie, P. D., 1965. Avian Physiology, 2nd Ed., p. 62. Bailliere, Tindal and Cassell, London. Tanaka, K., M. Kamiyoshi and M. Tagami, 1969. In vitro demonstration of luteinizing hormone releasing activity in the hypothalamus of the hen. Poultry Sci. 48: 1985-1987. van Tienhoven, A., and L. P. Juhasz, 1962. The chicken telencephalon, diencephalon and mesencephalon in stereotaxic coordinates. J. Comp. Neurol. 118:185-198. Wakabayashi, K., I. A. Kamberi and S. M. Mc Cann 1969. In vitro response of the rat pituitary to gonadotrophin-releasing factors and to ions. Endocrinology, 85:1046.
Production Traits of Leghorn Pullets in Controlled Temperatures W. O. WILSON, SYUICHI ITOH 1 AND T. D. SIOPES Department of Avian Sciences, University of California, Davis, California 95616 (Received for publication October 12, 1971) ABSTRACT Production traits, including sexual maturity, egg production, egg weight, shell strength, body weight, and feeding efficiency were studied with four strains of Leghorn pullets exposed to temperatures of 10°, 23°, and 36°C. The effect of abruptly changing these temperatures at 4-week intervals and of diurnally cyclic temperatures was investigated in avian bioclimatic chambers. The experiments started when birds were 20 weeks of age, and continued for 24 weeks. Abrupt changes to a temperature of 36°C. resulted in mortality, and susceptibility to heat-stroke increased with age. Egg production was highest in the low temperature for the control groups of birds, whereas feed efficiency tended to be best in the high or cyclic temperature groups. With the petite strain, egg production and feed efficiency were best in the medium temperature. Shifting the birds at intervals of 4 weeks tended to lower the overall egg production. When pullets were shifted from 22° to 34°C. they consistently lost weight, which was regained when the birds were shifted from 34° to 10°C. Egg weight and shell thickness was greatest in the low-temperature group. The percentage of blood spots was very high in two of the strains tested, and tended to be reduced at high temperature. Length of the toe nails varied directly with ambient temperature. POULTRY SCIENCE 51: 1014-1023, 1972
INTRODUCTION
I
T HAS generally been indicated that temperatures within the range of 10° to 18°C. are optimum for egg production, provided all other factors are equal (Warren et al., 1950; Ota et al., 1953). However, Mueller (1961) found that a diurnally cyclic thermal environment
1 Present address: Marugi Hatchery, 1 Chome Yoshisu-cho, Gifu City, Japan.
from 13° to 32°C. improved conversion of feed into eggs by 20%, as compared with 13°C. constant temperature. Research in this area has been summarized by Payne (1966, 1967), who suggested a reappraisal of environmental temperatures and their effect on nutritive allowances for layers. He further called attention to the improved efficiency of food utilization in a constant environment of 30°C. and 50% relative humidity. Studies on the acclima-
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between ovulation frequency and the incidence of follicular atresia following surgical operations in the domestic hen. Endocrinology, 37: 415-430. Salmi, M. H., and I. I. Geschwind, 1968. Some effects of energy-transfer inhibitors and of Ca ++ free or K + -enhanced media on the release of luteinizing hormone (LH) from the rat pituitarygland in vitro. Endocrinology, 82:225-231. Schally, A. V., A. Arimura, A. J. Kastin, H. Matsuo, Y. Baba, T. W. Redding, R. M. G. Nair, L. Debeljuk and W. F. White, 1971. Gonadotrophinreleasing hormone: One polypeptide regulates secretion of luteinizing and follicle-stimulating hormones. Science, 173:1036-1037.
LEPOKE2
United States Department of A griculture (Received for publication October 9, 1971)
POULTRY SCIENCE 51: 1004-1014, 1972
T
H E existence of a luteinizing hormone-releasing factor (LRF) in the avian hypothalamus has not been clearly established. Research in this area has been hindered b y the formidability of collecting hypothalamic tissues in the kilogram quantities needed for isolation and identification of L R F , and b y the lack of a specific and sensitive assay for avian L H . Much of the available evidence is controversial. Early in vitro demonstrations of L R F in the chicken hypothalamus (Jackson and Nalbandov, 1969a; T a n a k a et al., 1969), based on use of the ovarian ascorbic acid depletion (OAAD) assay to measure L H , were quickly invalidated b y the discovery (Jackson and Nalbandov, 1969, b, c; Ishii et al., 1970) t h a t this assay also is sensitive to arginine vasotocin, a hypothalamic agent shown t o be devoid of L H or L R F activity. Follett (1970) used a P 32 u p t a k e assay to show t h a t ex-
1 Animal Science Research Division, A.R.S., Beltsville, Md. 20705. 'Present address: Division of Nutritional Science, Bureau of Veterinary Medicine, F.D.A., Rockville, Md. 20850.
tracts of quail or chicken hypothalami stimulate in vitro release of gonadotrophin from chicken pituitaries, but, since the assay is highly sensitive to both F S H a n d L H , he could not specifically identify the gonadotrophin released. Jackson and Nalbandov (1969a) and Casey et al. (1971) reported t h a t avian hypothalamic extracts stimulate release of L H from the rat pituitary, b u t the value of these findings as evidence for avian L R F is uncertain, particularly since the former authors found t h a t their extracts did not stimulate release of L H from the chicken pituitary. Opel and Lepore (1967) and Clark and Fraps (1967) reported briefly t h a t intrapituitary infusion of chicken hypothalamic extracts evokes premature ovulation in the hen, b u t this evidence for L R F , based on a response of known physiological significance, has not been adequately described. T h e continued lack of pertinent information, and the potential value of the intrapituitary infusion technic for evaluation of the recently synthesized porcine gonadotrophin-releasing hormone (Baba et al., 1971; Schally et al., 1971) and its analogs as releasers of avian
1004
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A BSTRA CT Induction of ovulation was used to evaluate luteinizing hormone-releasing factor (LRF) activity in acidic extracts of chicken hypothalamus infused directly into the anterior pituitary of the hen. LRF activity was found in extracts of stalk-median eminence (SME), posterior pituitary and tuberal hypothalamus, but not in extracts of cerebral cortex. The crude SME extract was highly effective in inducing premature ovulation when infused into the adenohypophysis, but evoked no response when infused into adjacent hypothalamic structures or the systemic circulation. On fractionation of the SME extract on Sephadex G-25, LRF activity emerged in the same region of the column effluent as did oxytocic activity. However, a battery of control procedures indicated that the LRF activity is not attributable to mesotocin or arginine vasotocin. Intrapituitary infusion of dopamine, histamine, acetylcholine, epinephrine, norepinephrine or seratonin gave negative results. From these data, LRF in the chicken appears to be a low molecular weight, peptide-like material distinct from known median eminence hormones.
LUTEINIZING HORMONE-RELEASING FACTOR
1005
PP fragments and applied to a 2.5X40 cm. column of Sephadex G-25 equilibrated and subsequently eluted with MATERIALS AND METHODS 0.15 M acetic acid at 4°C. Sixty 5-ml. Preparation oj extracts. For tests of fractions were collected, their absorbance crude extracts, stalk-median eminence at 280 mju. monitored, and the peptide (SME), posterior pituitary (PP) and concentration determined by the Folinbrain tissues were obtained from groups Lowry reaction as modified by Lowry of 20-80 adult female chickens decapi- et al. (1951). tated in the laboratory. The tissues were In the first three runs with SME excollected immediately on dry ice and tracts, and the first two runs with PP crude extracts prepared as described by extracts, oxytocic activity in the effluent Campbell et al. (1964) using a total vol. from the Sephadex column was followed of 1 ml. of 0.1 N HC1/150 mg. wet wt. of by the hen and rat uterine assays as detissue. The extracts were stored in sealed scribed by Munsick (1964), using Synglass ampules at — 4°C. and tested within tocinon as the standard. Selected active 5 days. fractions were reassayed after incubation In some experiments, the crude SME with 0.05 M sodium thioglycollate as deextract, or a similarly prepared extract of scribed by Munsick et al. (1960). Concen+ ++ anterior pituitary tissue from the same tration of Na , K+ and Ca were dehens, was heated in a boiling water bath termined by flame photometry. Based on for 10 min. at the end of the extraction the reproducible elution profiles thus procedure. In other experiments, the ex- obtained, appropriate fractions from subtract was hydrolized with twice-crystal- sequent runs were pooled and freeze dried. lized pepsin or trypsin as described by The dried powder was stored at — 20° C. and tested for LRF activity within 1 Munsick et al. (1960). For gel-filtration studies, tissues were week. taken from heads of adult chickens of both Intrapituitary infusions. Experimental sexes obtained at a poultry processing birds were selected from a flock of 960 plant. Since the resident poultry inspec- White Leghorn hens caged individually tors would not permit dissection of the in laying batteries under artificial light heads at the plant, all heads were col- from 6 a.m. through 8 p.m. Patterns of lected on dry ice within 8 min. of death ovulation were determined from hourly and brought to the laboratory. To permit records of lay maintained from 8 a.m. to adequate dissection of the frozen tissues, 4 p.m. Only hens ovulating in recurrent the base of the skull, including the floor sequences of 2 or 3 eggs, with all eggs laid of the sella, was chopped off with a block- during the hours of recording, were used. mounted knife, the bony plate surroundTest substances were infused directly ing the pituitary was partially thawed into the adenohypophysis, median emiand cut free, and the adenohypophysis nence or tuberal hypothalamus via a carefully removed. Under magnification, stereotaxically placed, 27 gauge, platthe PP and SME tissues were excised with inum-irridium cannula (Opel, 1966a). To a pair of fine spring-handle scissors and avoid the median sagittal sinus, all canplaced on dry ice. At the end of each nulae were placed 0.5 mm. to the left of collection day, a crude extract was pre- the midline. To insure insertion of the pared from the yield of 400-500 SME or cannula tip into the main body of the gonadotrophin, prompted us to describe our studies of chicken LRF in detail.
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1006
H. OPEL AND P. D. LEPORE
Test solutions were neutralized with sodium bicarbonate, and sodium chloride added to physiological concentrations, immediately before infusion at a rate of 1.5 /^l./min. over a 90 min. period. All infusions were performed between 3 and 5 p.m. on the day before ovulation of the first (Ci) egg of a sequence. Induced Ci ovulation, premature by 6-9 hours, was detected by the presence of a "plumped" egg on digital palpation of the shell gland at 8:30 a.m. on the next day. During 2:30-4:00 p.m., all hens were infused with an aqueous solution of analine blue dye at the same rate as the test substance had been infused on the previous day. At termination of the infusion, each hen was
decapitated and autopsied. The approximate time of Ci ovulation was verified from the position and stage of formation of the oviducal egg, and the ovary was checked for atretic and freshly ruptured follicles. The base of the skull underlying the pituitary capsule was removed, and the spread of the dye and the position of the pituitary cannula determined macroscopically. Hens in which the intrapituitary infusate reached less than 20% of the adenohypophyseal tissue were eliminated from the experiment. Positions of hypothalamic cannulae were verified histologically by the method of Fox and Eichman (1959). Intrajugular infusions in hypophysectomized hens. To allow adequate testing of the limited supply of gel-filtrated LRF powder, Japanese quail were used. Regularly ovulating hens were kept under conditions similar to those described for chickens. To insure accuracy in the prediction of ovulation (Opel, 1966b), each experiment was performed for effect on the 2nd or 3rd ovulation of a sequence of 6 or more eggs. Spontaneous ovulation was blocked in 50 quail by the intravenous infusion of 0.02-0.03 ml. of Equithesin (JensenSalsbery Laboratories) 14 hours before expected normal ovulation. Twenty hens were hypophysectomized while under the resultant deep anesthesia; 30 served as unoperated controls. All test samples were infused between 11 and 10 hours before the time of normal ovulation. Each hen was reanesthetized with Equithesin and immobilized in the usual backdown position on the hypophysectomy board (Opel, 1969). A wooden cylinder, placed directly behind the headholder, was used to elevate and extend the neck. A 27-gauge needle, with a short length of fine polyethylene tubing attached, was inserted into the left jugular
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adenohypophysis, and to avoid perforation of the ventral surface of the gland or the internal carotid arteries, each pituitary cannula was placed within stereotaxic coordinates A6.s to 7.0 and H0.o to _i.o (van Tienhoven and Juhasz, 1962). Two infusion systems were used. In the tests of crude extracts, infusions were performed in conscious, unrestrained hens within 3-15 days of cannula implantation. Although useful for preliminary work, this method proved too unwieldy for large scale experimentation because some 40% of the hens bearing chronic implants either ceased to lay after one or two eggs, or laid in sequences too variable for adequate prediction of ovulation and thus could not be used in the experiment. Consequently, in subsequent tests of gelfiltrated extracts, synthetic hormones and biogenic amines, cannula placement and infusion were performed in one session in pentobarbital-anesthetized hens. Pentobarbital, during the period administered, has no effect on ovulation (Bastian and Zarrow, 1952). The effects of surgical and infusion procedures were followed in control hens given infusions of neutral saline.
1007
LUTEINIZING HORMONE-RELEASING FACTOR
RESULTS
Tests of crude extracts. Intrapituitary infusion of 20 mg. equivalents of crude SME extract evoked premature ovulation in 15 of 21 hens (Table 1). Our results with this dosage corroborate those of Clark and Fraps (1967). Infusion of 10 mg. equivalents had a borderline effect, as indicated by forced ovulation in 1 of 6 hens and atresia of the ovulable ovarian follicle in 3 others, while infusion of 5 mg. was ineffective. For reasons unexplained, infusion of 40 mg. of extract consistently prevented ovulation and caused generalized atresia of large follicles. Crude extracts of cerebral cortex showed no detectable LRF activity, whereas intrapituitary infusion of 30 mg. equivalents of tuberal hypothalamus evoked ovulation in 2 of 6 hens (Table 1). Since chicken LH is heat labile (Tanaka et at., 1969; Jackson and Nalbandov, 1969c), heat treatment was used to determine if the ovulation-inducing activity of the SME extract could be accounted for by LH contamination. As shown in Table 1, boiling for 10 min. had no effect on the crude SME extract, but completely destroyed the ovulating potency of a similarly prepared extract of anterior pituitary obtained from the same hens. Other control data supported the prob-
TABLE 1.—Ovulation in the chicken after intrapituitary infusion of crude tissue extracts
Crude extract infused
Mg. equivalent wetwt. of tissue
Stalk-median eminence (SME)
5 10 20 40 SME, boiled 10 min. 20 Anterior pitui5 tary (AP) 5 AP, boiled 10 min. Posterior pitui0.6-1.0 tary (PP) 20 Tuberal 20-30 hypothalamus 20-30 Cerebral cortex
No. of hens
No. ovulating
6 6 21 6 10
0 1 IS 0 6
10 10
8 0
9 9
0 1
12 12
2 0
ability that the SME extract acted directly on the adenohypophysis. Intrajugular infusion of 20-80 mg. of SME extract, i.e., 1-4 times the optimal intrapituitary dose, did not evoke ovulation in any of 20 hens. Infusion of 20 or 40 mg. of extract directly into the median eminence or ventral hypothalamus, at the same anterioposterior and lateromedial coordinates used for intrapituitary infusion, failed to evoke ovulation in 16 hens. To test the possibility that LRF activity in the SME extract might be attributable to the known presence of the PP hormones, oxytocin and arginine vasotocin (AVT), crude extracts of PP tissues were infused into the adenohypophysis at 2 dosages; one (0.6-1.0 mg.) designed to provide the assayable amount of oxytocic activity found in 20 mg. equivalents of SME extract, the other (20 mg.) to provide the equal amount of tissue. Infusion of the low dose had no effect, while infusion of the high dose, which contained 20-30 times the oxytocic activity in 20 mg. of SME extract, evoked ovulation in 1 of 9 hens. Incubation of the SME extract with trypsin, which inactivates
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vein and held in place by a strip of adhesive tape. The free end of the tubing was coupled to the micro-infusion apparatus (Harvard Model 975 infusion pump) and a solution of LRF powder, ovine L H (NIH-S10) or saline was infused in a vol. of 0.2 ml. over a 30 min. period. All hens were killed 9 hours after infusion, the body cavities and oviducts checked for ova and the ovaries examined for freshly ruptured follicles. Completeness of hypophysectomy was verified histologically (Opel, 1969).
1008
H. OPEL AND P. D. LEPORE TABLE 2.—Effects of peptic or tryptic digestion of crude SME extract
SME incubated with pepsin SME incubated without pepsin
8 8
2 6
SME incubated with trypsin SME incubated without trypsin
8 8
5 5
1 Each hen received 20 mg. equivalents of SME extract.
I
16
).
20
24
.
,
,
,
28
3Z
56
+
,
,
,
40 ' 44
B
«
+
,
92
e
,
t-K)
«
60
-I
FIG. 1. Gel-filtration of7crude^SME extract (4.45 ml.) on Sephadex G-25 (2.5X40 cm.) in 0.15 M acetic acid. Tube vol. was 5 ml. Note that ordinate for oxytocic activity is logarithmic. Broken lines indicate absence of detectable oxytocic activity and show lower limits of assay sensitivity. Using bioassay data as a guide, tubes were combined into fractions A-C for LRF assay.
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AVT, but not oxytocin (Munsick et al., 1960), and which reduced the assayable oxytocic activity by more than 90%, had no effect on LRF activity (Table 2). On the other hand, incubation of the extract with pepsin, which does not inactivate either PP hormone, and which reduced the oxytocic activity by less than 8%, appreciably reduced the LRF activity. Attempts to use thioglycollate inactivation, which destroys the oxytocic activity of both PP hormones (Munsick et al., 1960), failed because the presence of this reducing agent in the infusate disrupted ovulation. Gel-filtration of SME extracts. Fractionation of the acid extract of 400-500 chicken SME fragments on Sephadex G-25 yielded a single zone of oxytocic activity in close association with a peptide peak (Fig. 1, tubes 32-47). Results of hen and rat oxytocic assays, as well as comparisons of rat oxytocic activities with or without 0.5 mM. Mg + + in the bath solution (not shown in the figure), indicated that all tubes from the active zone contained a mixture of oxytocin and AVT, with AVT predominating. Incubation of aliquots of peak fractions with 0.05 M thioglycollate destroyed 98%, while incubation with trypsin destroyed 88% of the oxytocic activity, suggesting that roughly these percentages of the activity were attributable, respectively, to total PP hormones and to AVT. About 84% of the
oxytocic activity was recovered after gelfiltration. Reproducible profiles of oxytocic activity in the first 3 fractionations, and the results of spot assays to define boundaries of activity in subsequent runs, were used as guides to combine the eluates from 14 columns into 3 zones (Fig. 1, A-C). Results of LRF tests of the freeze-dried powder from each zone are shown in Table 3. Significant LRF activity emerged only in the zone containing oxytocic activity. Complete quantitation was prevented by the low yield of dry powder and the relative insensitivity of the ovulation test, but intrapituitary infusion of 0.25 mg. of LRF powder (Zone B), roughly equivalent to 26 mg. wet wt. of SME tissue, induced ovulation in 11 of 20 hens. Infusion of an equivalent dose of the powder from the less strongly retarded Zone A (tubes 1631) did not evoke ovulation, but a 6-fold higher dose induced ovulation in 2 of 10 hens. The trace activity in Zone A may reflect either contamination with LH, or some displacement of LRF activity to the left of tube 32.
1009
LUTEINIZING HORMONE-RELEASING FACTOR
TABLE 3.—Ovulation in chickens after intrapituitary infusion of gel-filtration zones A-C Amount of dry powder infused (mg.)
No. of hens
No. ovulating
Stalk-median eminence A 0.25-1.5 B 0.25 0.5 - 1 . 0 C 0.25-1.0
16 11 11 6
2 20 21 0
Posterior pituitary A 0.25-1.5 B 0.1 - 2 . 0 C 0.1 - 1 . 0
8 14 5
0 1 0
Zone
TABLE 4.—Intrapituitary infusion of posterior pituitary hormones and biogenic amines ' i n s
6
Arginine vasotocin 20-800 mU.* Oxytocin (Syntocinon) 10-100 mU. Vasotocin 100-200 mU. +oxytocin +10-20 mU. Vasopressin (Pitressin) 20-60 mU. Acetylcholine 5-10 ng. Epinephrine 20-30 jug. Norepinephrine 5-10 jug. Histamine dihydrochloride 1-3 mg. Seratonin creatinine SO« 0.5-1 mg. Dopamine 5-10 /ig.
No. of hens
No._ ovulating
14
0
8 8
0 0
8 14 8 8
0 1 0 0
8
0
8 8
0 0
*Expressed as International Units of rat oxytocic activity per mg.
was tested for LRF activity. As shown in Table 3, ovulation was obtained only in 1 of 14 hens infused with powder from the oxytocic-active Zone B. The effective dose (0.5 mg.) contained about 40 times the assayable oxytocic activity in the most effective dose (0.25 mg.) of LRF powder from the fractionation of SME. Tests oj PP hormones and biogenic amines. The results of intrapituitary infusion of PP hormones, and of some biogenic amines known to occur in the hypothalamus, are shown in Table 4. Infusion of varying levels of oxytocin and AVT, alone or in combination, failed to evoke ovulation. Intrapituitary infusion of acetylcholine chloride (Ach) induced ovulation in 1 of 14 hens. To test the possibility that this isolated ovulation may have resulted from backtracking of the Ach solution along the cannula wall into the hypothalamus, the same amount of Ach solution was infused directly into the median eminence in 6 hens. No premature ovulations were obtained. C\ ovulation in controls. Examination of the records of 124 unoperated hens, selected exactly on the same basis as the
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To achieve greater purification, the oxytocic-active zones from 3 additional runs on the column, representing the oxytocic activity from 1700 SME fragments, were pooled, freeze-dried to a vol. of 20 ml., and recycled through the same column. This time, as indicated by the pattern of ultraviolet absorption and bioassays of aliquots of appropriate tubes, oxytocic activity emerged in tubes 36-44 and was well separated from the larger molecules. Once again LRF activity emerged in the same zone as oxytocic activity. Intrapituitary infusion of 0.75 mg. of the freeze-dried LRF powder evoked ovulation in 3 of 5 hens. Gel-filtration of PP extracts. Fractionation of PP extracts on the 2.5X40 cm. Sephadex column produced profiles of ultraviolet absorption, Folin-Lowry peptide concentration and oxytocic activity quite similar to those obtained by fractionation of equivalent amounts of SME extract, except that peak values of oxytocic activity were many times higher. Since the oxytocic activity was spread throughout the same region, the eluates from fractionation of four PP samples were combined into exactly the same zones used to assess eluates from the fractionation of SME samples (Fig. 1), and the freeze-dried powder from each zone
1010
H. OPEL AND P. D. LEPORE
TABLE 5.—Ovulation in hypophysectomized or Equithesin-blocked quail after intrajugiilar infusion of LRF powder Treatment
No. No. of ovuquail lating
DISCUSSION 0.25-2.5 mg. 6.0 Mg-
10 10
0 10
0.25 mg. 6.0 Mg.
10 10 10
8 9 0
experimental hens, showed that the predicted Ci ovulation would occur as expected about 96% of the time. No "spontaneous" premature ovulations were observed. Of 20 normally ovulating hens bearing chronic cannula implants and given an intrapituitary infusion 3-15 days following cannula placement, all ovulated at the normally expected time. Of 40 hens given an infusion of saline at 1 to 2 hours after cannula placement, 33 ovulated as normally expected, while 7, or 17%, failed to ovulate. All non-ovulating hens showed massive atresia of the large ovarian follicles on autopsy. The results of the control infusions indicate that the data from all experiments involving the acute preparation (data in Tables 3 and 4) are probably negatively biased. Lack of response to LRF in hypophysectomized quail. Since our LRF-active SME powder was not highly purified, the possibility of a direct action on the ovary had to be examined. As seen in Table 5, intrajugular infusion of 0.25 mg. of the powder evoked ovulation in 8 of 10 Equithesinblocked quail, but infusion of up to 10 times this amount had no effect on hypophysectomized quail. In contrast, infusion of a predetermined minimal effective dose of 6 fig. of LH produced essentially the same proportion of positive responses
The present results show clearly that crude or partially purified extracts of chicken SME evoke premature ovulation on intrapituitary infusion in the hen. Our evidence that the ovulation-inducing substance in the extracts is LRF is best interpreted on the basis of the experiments of Nikitovitch-Winer (1962) and Campbell et al. (1964), who were the first to demonstrate LRF activity in the mammalian hypothalamus. In order to show that their hypothalamic preparations contained a specific mediator that acted directly on adenohypophyseal cells to release LH, these investigators developed a battery of control procedures that allowed them to obviate every known source of falsepositive responses. We have applied their safeguards to our experiments with analogous results. Sources of false-positive responses eliminated or discounted in our experiments include: 1) the occurrence of spontaneous premature ovulation in experimental hens, 2) induction of ovulation by simple mechanical irritation or injury to the hypothalamo-hypophyseal system during implantation of the cannula or infusion of extracts, 3) variations in the ovulatory response due to variations in infusion rate or in the volume of pituitary tissue reached by the infusate, 4) the presence in brain tissue of a "non-specific" substance capable of evoking ovulation by stimulatory or irritative action on the pituitary or ovary, 5) ovulatory discharge of LH secondary to action of the extracts on brain tissues overlying the pituitary gland, 6) discharge of LH resulting from changes in pituitary blood flow produced
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Hypophysectomized infused with LRF powder LH quithesin-blocked fused with LRF powder LH Solvent
Amount of dry powder infused
in hypophysectomized as in Equithesinblocked hens, indicating that the ovaries of the hypophysectomized birds retained their normal sensitivity to LH under our experimental conditions.
LUTEINIZING HORMONE-RELEASING FACTOR
subsequent unpublished work, we found that intrapituitary infusion of 0-80 mM. of K+, added to buffered physiological saline or incorporated into subthreshold doses of SME extract, did not evoke ovulation. It seems unlikely, therefore, that the LRF activity of the SME extracts can be accounted for by their K+ content. Our results provide crude quantitative, as well as qualitative evidence for LRF. The highest effective dose of crude SME extract represented about 4.2 and the lowest effective dose about 2.1 median eminences. Although direct comparisons are prevented by differences in technic, these dosages compare favorably with the dosages of crude rabbit SME extract found by Campbell et al. (1964) to evoke ovulation on intrapituitary infusion in the doe. Surprisingly, intrapituitary infusion of a quantity of SME extract equivalent to about 8.4 median eminences blocked ovulation and caused massive atresia of the large ovarian follicles. This response may indicate a toxic reaction to the peptide material in the extract (Fraps and Neher, 1945) or, alternately, the presence of a specific gonadotrophin-inhibiting substance in the SME. The available data provide few clues to the nature of LRF in the chicken SME. Gel filtration on Sephadex, selected as a simple and repeatable small scale method of proven worth in separating LH from mammalian LRF (Ramirez et al., 1964; Fawcett et al., 1965) suggested that the active material is AVT, oxytocin, or a substance of similar molecular form. Control procedures, however, indicate that LRF is not oxytocin or AVT. Hydrolysis with trypsin, which inactivates AVT, did not reduce the LRF activity of crude SME extract. Intrapituitary infusion of synthetic oxytocin or AVT, alone or in combination, did not evoke ovulation. Infusion of crude or gel-filtrated PP ex-
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by vasoactive substances, such as histamine or adrenaline, known to be present in SME extracts, and 7) the presence of LH or LH-like substances in the extracts. Final proof that significant LH contamination was not present in the extracts was obtained through use of the second in vivo system employing Japanese quail. The fact that intrajugular infusion of 0.25 mg. of the partially purified LRF powder evoked ovulation in 8 of 10 intact quail, but did not evoke ovulation in any of 10 hypophysectomized quail exhibiting normal sensitivity to exogenous LH, established that the ovulation-inducing substance did not act on the ovary, and therefore could not be LH. A potential source of error outside our control was the presence of an ionic imbalance or excessive ionic contamination in the LRF-active preparations. Since we were not able to collect enough SME tissues at any one time to allow the use of volatile extractants, we could not remove the need to neutralize the extracts and thus introduce an ionic imbalance in the final solutions. Further, gel filtration of the crude extract on Sephadex must have resulted in excessive accumulation of ions in the LRF-active fraction. In view of a report by Salmi and Geschwind (1968) that excessive levels of K+ in the incubation medium stimulate in vitro release of LH from rat pituitaries, we determined the levels of K+ in our solutions prepared for intrapituitary infusion. Concentrations of K+ in the optimum doses of crude and partially purified SME extract were 6.3 and 8.9 mM., respectively. The concentration of K+ in the dose of purified SME is about 1.2 times the maximum level of K+ reported for chicken plasma (Sturkie, 1965), but far below the level of 59-60 mM. found to stimulate LH release from the rat pituitary (Salmi and Geschwind, 1968; Wakabayshi et al., 1969). In
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Other control data from this study indicate LRF activity in the SME is not attributable to any of several biogenic amines known to occur in the hypothalamus. Since the LRF material was susceptible to pepsin hydrolysis, it may be a polypeptide. Resistance to boiling suggests that it is heat stable. These results are similar to those obtained in early investigations of mammalian LRF (Ramirez et al., 1964; Fawcett et al, 1965; Nikitovitch-Winer et al., 1965). A biological similarity between avian and mammalian LRF is also implied by the observation that extracts of bovine hypothalamus evoke premature ovulation on intrapituitary infusion in the hen (Clark and Fraps, 1967), while extracts of avian hypothalamus stimulate LH release from rat pituitaries in vitro (Jackson and Nalbandov, 1969a) or in vivo (Casey et al., 1971). In view of these similarities, evaluation of the recently synthesized porcine gonadotrophin-releasing hormone (Baba et al., 1971) and its analogs in avian test systems may provide the means to tentatively
identify avian LRF and answer the now important question of whether in birds, as in mammals (Schally et al., 1971), a single hypothalamic agent controls release of both FSH and LH. The recent development of a radioimmunoassay for chicken LH (Cain and Wilson, 1971) raises the possibility that these problems may now be successfully approached by in vitro technics. Finally, it should be pointed out that assessment of the ovulatory potency of test solutions by intrapituitary infusion is an especially difficult and unwieldy procedure to apply, in the hen. Due to the relative ease with which surgery disrupts ovulation in the hen (Rothchild and Fraps, 1945) and because of the high possibility of severing hypophyseal portal vessels in the dorsal approach to the pituitary, considerable experience and extreme care are required to stereotaxically place the intrapituitary cannula. Despite extensive preliminary work and the use of every known precaution, we were unable to eliminate surgical effects as a complicating factor in our experiments. Attempts to implant the cannula via the parapharyngeal approach were even less successful. A further hinderance to use of the technic is the lack of a completely effective pharmacological block to ovulation in the hen that can be applied late enough to avoid possible interference with follicular maturation. The consequent need to precisely schedule intrapituitary infusions for effect on spontaneous ovulation severely limits the number of experimental hens that can be drawn at any one time from the existing flock. Attempts to employ intrapituitary infusion in the much smaller Japanese quail, in which we found that ovulation could be selectively blocked by injection of Equithesin, were entirely unsuccessful.
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tracts containing either the same or many times the level of oxytocic activity found in an effective dose of SME extract had no effect on ovulation. Since these studies were initiated, several authors (Jackson and Nalbandov, 1969c; Tanaka et al., 1969; Follett, 1970) have concluded from in vitro studies that avian LRF is not a neurohypophyseal hormone. Recently, Acher et al. (1970) have obtained chemical evidence that the neurohypophyseal hormone in birds identified by pharmacological data as oxytocin is, in fact, mesotocin. Since mesotocin would exhibit essentially the same behavior as oxytocin in our purification and pharmacological assay systems, this finding does not alter our conclusion that chicken LRF is not a neurohypophyseal hormone.
LUTEINIZING HORMONE-RELEASING FACTOR ACKNOWLEDGEMENT
We wish to thank Dr. B. Berde, Sandoz Ltd., Basle, for the synthetic arginine vasotocin used in this study. The ovine LH was donated by the Endocrinology Study Section of the National Institutes of Health. We are also grateful to Mr. Millard Sindler, Dover Poultry Products, Inc., Baltimore, for permission to collect chicken heads.
Fraps, R. M., and B. H. Neher, 1945. Interruption of ovulation in the hen by subcutaneously administered non-specific substances. Endocrinology, 37: 407-414. Ishii, S., A. K. Sarkar and H. Kobayashi, 1970. Ovarian ascorbic acid-depleting factor in pigeon median eminence extracts. Gen. Comp. Endocrinol. 14: 461-466. Jackson, G. L., and A. V. Nalbandov, 1969a. Luteinizing hormone releasing activity in the chicken hypothalamus. Endocrinology, 84:12621265. Jackson, G. L., and A. V. Nalbandov, 1969b. A substance resembling arginine vasotocin in the anterior pituitary gland of the cockerel. Endocinology, 84: 1218-1223. Jackson, G. L., and A. V. Nalbandov, 1969c. Ovarian ascorbic acid depleting factors in the chicken hypothalamus. Endocrinology, 85: 113— 120. Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall, 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. Munsick, R. A., 1964. Neurohypophyseal hormones of chickens and turkeys. Endocrinology, 75:104112. Munsick, R. A., W. H. Sawyer and H. B. van Dyke, 1960. Avian neurohypophyseal hormones: pharmacological properties and tentative identification. Endocrinology, 66: 860-861. Nikitovitch-Winer, M. B., 1962. Induction of ovulation in rats by direct intrapituitary infusion of median eminence extracts. Endocrinology, 70: 350-358. Nikitovitch-Winer, M. B., A. H. Pribble and A. D. Winer, 1965. Luteinizing hormone-releasing factor: Partial purification. Am. J. Physiol. 208: 1286-1290. Opel, H., 1966a. A simple cannula for implantation into the chicken brain. Poultry Sci. 45: 856-858. Opel, H., 1966b. The timing of oviposition and ovulation in the quail (Cohtrnix coturnix japonica). Brit. Poultry Sci. 7:29-38. Opel, H., 1969. Transbuccal hypophysectomy in the Japanese quail. Poultry Sci. 48: 722-728. Opel, H., and P. D. Lepore, 1967. Ovulating hormone-releasing factor in the chicken hypothalamus. Poultry Sci. 46:1302. Ramirez, V. D., R. Nallar and S. M. Mc Cann, 1964. Purification of luteinizing hormone-releasing factor from beef hypothalamus. Proc. Soc. Exptl. Biol. Med. 115: 1072-1076. Rothchild, I., and R. M. Fraps, 1945. The relation
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REFERENCES Acher, R., J. Chauvet and M.-T. Chauvet, 1970. Phylogeny of the neuro-hypophysial hormones. Avian active peptides. Eur. J. Biochem. 17:509513. Baba, Y., H. Matsuo and A. V. Schally, 1971. Structure of the porcine LH- and FSH-releasing hormone. II. Confirmation of the proposed structure by conventional sequential analyses. Biochem. Biophys. Res. Comm. 44: 459-464. Bastian, J. W., and M. X. Zarrow, 1952. Failure of nembutal to block ovulation in the hen. Proc. Soc. Exptl. Biol. Med. 79: 249-252. Cain, J. R., and W. O. Wilson, 1971. Development of a radioimmunoassay for chicken LH. Poultry Sci. 50: 1560. Campbell, H. J., G. Feuer and G. W. Harris, 1964. The effect of intrapituitary infusion of median eminence and other brain extracts on anterior pituitary gonadotrophin secretion. J. Physiol. 170: 474-486. Casey, J. M., J. J. Reeves, P. C. Harrison and R. P. Peterson, 1971. Dual bio-radioimmuno detection of LH-RH like activity in hypothalamic tissue of coturnix quail. Poultry Sci. 50: 1562-1563. Clark, C. E., and R. M. Fraps, 1967. Induction of ovulation in the chicken with median eminence extracts. Poultry Sci. 46:1245-1246. Fawcett, C. P., G. W. Harris and M. Reed, 1965. The purification of the luteinizing hormone releasing factor (LRF). In: Proceedings of the 23rd International Congress of Physiological Sciences. Excerpta Medica Int. Congr. Series No. 87, vol. 3:300-305. Follett, B. K., 1970. Gonadotrophin-releasing activity in the quail hypothalamus. Gen. Comp. Endocrinol. 15: 165-179. Fox, C. A., and J. Eichman, 1959. A rapid method for locating intracerebral electrode tracts. Stain Technol. 34: 39-42.
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H. OPEL AND P. D. LEPORE Sturkie, P. D., 1965. Avian Physiology, 2nd Ed., p. 62. Bailliere, Tindal and Cassell, London. Tanaka, K., M. Kamiyoshi and M. Tagami, 1969. In vitro demonstration of luteinizing hormone releasing activity in the hypothalamus of the hen. Poultry Sci. 48: 1985-1987. van Tienhoven, A., and L. P. Juhasz, 1962. The chicken telencephalon, diencephalon and mesencephalon in stereotaxic coordinates. J. Comp. Neurol. 118:185-198. Wakabayashi, K., I. A. Kamberi and S. M. Mc Cann 1969. In vitro response of the rat pituitary to gonadotrophin-releasing factors and to ions. Endocrinology, 85:1046.
Production Traits of Leghorn Pullets in Controlled Temperatures W. O. WILSON, SYUICHI ITOH 1 AND T. D. SIOPES Department of Avian Sciences, University of California, Davis, California 95616 (Received for publication October 12, 1971) ABSTRACT Production traits, including sexual maturity, egg production, egg weight, shell strength, body weight, and feeding efficiency were studied with four strains of Leghorn pullets exposed to temperatures of 10°, 23°, and 36°C. The effect of abruptly changing these temperatures at 4-week intervals and of diurnally cyclic temperatures was investigated in avian bioclimatic chambers. The experiments started when birds were 20 weeks of age, and continued for 24 weeks. Abrupt changes to a temperature of 36°C. resulted in mortality, and susceptibility to heat-stroke increased with age. Egg production was highest in the low temperature for the control groups of birds, whereas feed efficiency tended to be best in the high or cyclic temperature groups. With the petite strain, egg production and feed efficiency were best in the medium temperature. Shifting the birds at intervals of 4 weeks tended to lower the overall egg production. When pullets were shifted from 22° to 34°C. they consistently lost weight, which was regained when the birds were shifted from 34° to 10°C. Egg weight and shell thickness was greatest in the low-temperature group. The percentage of blood spots was very high in two of the strains tested, and tended to be reduced at high temperature. Length of the toe nails varied directly with ambient temperature. POULTRY SCIENCE 51: 1014-1023, 1972
INTRODUCTION
I
T HAS generally been indicated that temperatures within the range of 10° to 18°C. are optimum for egg production, provided all other factors are equal (Warren et al., 1950; Ota et al., 1953). However, Mueller (1961) found that a diurnally cyclic thermal environment
1 Present address: Marugi Hatchery, 1 Chome Yoshisu-cho, Gifu City, Japan.
from 13° to 32°C. improved conversion of feed into eggs by 20%, as compared with 13°C. constant temperature. Research in this area has been summarized by Payne (1966, 1967), who suggested a reappraisal of environmental temperatures and their effect on nutritive allowances for layers. He further called attention to the improved efficiency of food utilization in a constant environment of 30°C. and 50% relative humidity. Studies on the acclima-
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between ovulation frequency and the incidence of follicular atresia following surgical operations in the domestic hen. Endocrinology, 37: 415-430. Salmi, M. H., and I. I. Geschwind, 1968. Some effects of energy-transfer inhibitors and of Ca ++ free or K + -enhanced media on the release of luteinizing hormone (LH) from the rat pituitarygland in vitro. Endocrinology, 82:225-231. Schally, A. V., A. Arimura, A. J. Kastin, H. Matsuo, Y. Baba, T. W. Redding, R. M. G. Nair, L. Debeljuk and W. F. White, 1971. Gonadotrophinreleasing hormone: One polypeptide regulates secretion of luteinizing and follicle-stimulating hormones. Science, 173:1036-1037.