- Email: [email protected]
Relationship of Plasma Estradiol and Progesterone Levels to Egg Productivity in Domestic Chicken Hens
Relationship of Plasma Estradiol and Progesterone Levels to Egg Productivity in Domestic Chicken Hens D. E. LESZCZYNSKI Harlan li. Moore Heart Research
Foundation,
503
South
Sixth Street,
Champaign,
Illinois
61820
R. C. H A G A N Burnsides
Research
Laboratory,
Department
of Food Science,
University
of Illinois,
Urbana, Illinois
61801
J . J . BITGOOD Department
of Poultry
Science,
University
of Wisconsin,
Madison,
Wisconsin
53706
F. A. KUMMEROW Harlan F. Moore Heart Research
Foundation,
503 South
Sixth
Street,
Champaign,
Illinois
61820
(Received for publication March 30, 1984) ABSTRACT Female chickens from eight different genetic stocks, ranging from 13 to 21 m o n t h s of age, and exhibiting various levels of egg p r o d u c t i o n were studied. Plasma samples were collected between 1 7 0 0 and 2 2 0 0 hr to determine basal circulating levels of progesterone ( P 4 ) and 17/3estradiol ( E 2 ) by radioimmunoassay. Linear regression analysis was used t o describe t h e relation between mean h o r m o n e levels ( E 2 , P 4 , and E 2 / P 4 ) and m e a n egg p r o d u c t i o n from these groups, and correlation coefficients were used t o evaluate t h e general usefulness of these h o r m o n e par a m e t e r s for estimating egg p r o d u c t i o n within a g r o u p or flock of hens. Of t h e three h o r m o n e p a r a m e t e r s evaluated, basal circulating E 2 / P 4 was found to be t h e best e s t i m a t o r of egg p r o d u c tivity. (Key words: chickens, egg p r o d u c t i o n , estradiol, progesterone) 1 9 8 5 P o u l t r y Science 6 4 : 5 4 5 - 5 4 9 INTRODUCTION
MATERIALS AND METHODS
Our previous studies of sexually mature White Leghorn hens carrying the sex-linked restricted ovulator (ro) mutation (Jones et al., 1975) indicated that an unusually high basal level of circulating estrogen and an unusually low basal level of progesterone (P 4 ) were associated with the failure to lay eggs (Leszczynski et al., 1983). Low levels of circulating P 4 have also been reported for immature and molting chicken hens (Johnson and van Tienhoven, 1981). In Mallard ducks, cessation of egg-laying induced by stress was associated with low circulating levels of both P 4 and estradiol (E 2 ) (Bluhm et al, 1983). In turkeys, nonlaying hens, and hens with low egg production also have low levels of circulating P 4 (Mashaly and Wentworth, 1974). The purpose of the present study was to measure basal circulating levels of E 2 and P 4 in several varieties of sexually mature female chickens with various levels of egg productivity to test if these parameters can be useful in evaluating egg production within a flock.
Blood samples from mutant ro White Leghorn hens, inbred White Leghorn hens (Babcock strain), and New Hampshire (NH) X Columbian (C) hens were taken at the University of Illinois (Urbana) poultry farm. Blood samples were obtained at the University of Wisconsin (Madison) from hens of several different genetic stocks. These included inbred White Leghorns (ES line), inbred Anconas, carriers of naked neck, Buff Orpingtons, ^i Silkie x Rhode Island Red (RIR), and hens derived from NH x RIR crosses. All hens were individually caged, fed ad libitum a standard corn-soybean based layer ration, and exposed to 14 hr light and 10 hr dark each day with lights on at 0500 hr. All birds, none of which were molting, ranged in age from 13 to 21 months except for three Buff Orpington hens, which were 2.5 years old. Blood samples were collected at least 12 hr before the expected times for ovulation, based on prior observations. Thus, samples were drawn during a period of the day (between
545
546
LESZCZYNSKI ET AL.
1700 and 2200 hr) when both circulating estradiol (E 2 ) and P 4 were expected to be at basal levels. Blood samples were stabilized with .1% tetrasodium-ethylenediaminetetraacetate (EDTA), and plasma was collected after centrifugation and stored at —18 C until analysis. Antisera used for the determination of E 2 and P 4 were purchased from CalbiochemBehring where they were prepared from rabbits against 17|3-estradiol-(6-0-carboxymethyl)-aminebovine serum albumin (BSA) conjugate and progesterone-(l l-hemisuccinate)-BSA. Crossreactivities for estradiol antiserum were: 17/3estradiol (100%), estrone (<.06%), estriol (<.18%), and P 4 (nil). For P 4 antiserum, the crossreactivities were: P 4 (100%), lla-hydroxyprogesterone (<17%), 11/3-hydroxyprogesterone (<16%), 5a-pregnan-3|3-ol-20-one (<42%), A5-pregnan-3j3-ol-20-one, and pregnanolone (<2.4%), all other steroids (<1%). Other chemicals used were: E 2 standard and gamma globulin, Cohn fraction II (Sigma Chemical Co.); Sephadex LH-20 and Dextran T-70 (Pharmacia); and Norit A charcoal (Fisher Chemical Co.). Spectro grade solvents for steroid extraction and column chromatography were purchased from the J. T. Baker Co. These solvents were pretested for residue interference with antibody-antigen binding by drying 4-ml aliquots in tubes; these solvents were used in titer determinations and compared to identical determinations done in tubes without dried solvents. Recovery tracer was equilibrated with plasma by sonication for 5 min followed by room temperature incubation for 2 hr. Estradiol was extracted with four parts ethyl ether, P 4 was extracted with four parts petroleum ether, and tracer recoveries ranged between 80 and 95%. Ether extracts were dried under nitrogen and resuspended in isooctane: benzene: methanol 62:20:18 in the case of E 2 and 90:5:5 in the case of P 4 for Sephadex LH-20 chromatography. For plasma samples that were extremely hyperlipidemic, dried ether extracts were resuspended in 70% methanol, kept at —80 C overnight, and then were centrifuged at —10 C to remove approximately 95% of the fat (mostly triglycerides), after which the supernate was dried under nitrogen and resuspended in the appropriate isooctane: benzene: methanol solvent. This step resulted in an additional 10 to 15% loss of recovery tracer but greatly improved the sensitivity and reliability of the assay. After LH-20 chromatography,
appropriate steroid fractions were dried with nitrogen and resuspended in absolute ethanol, transferred to tubes, and then dried for duplicate radioimmunoassay (RIA) analysis and also for the calculation of recovery. The RIA assays were performed according to procedures given with the P 4 and estrogen Radioimmunoassay Paks supplied by the New England Nuclear Co., with slight modification, based on the methods of Abraham et al. (1971) and Youssefrejadian et al. (1972). Assay radioactivity was converted to picograms of hormone by use of a log-logit plotting procedure described by Wu and Lundy (1971). Assay sensitivity averaged 16.3 pg for P 4 and 3.0 pg for E 2 . Assay precision, measured by calculating within and between assay coefficients of variations (CV) from pooled plasma samples were 3.7 and 11.6%, respectively, for RIA of P 4 and 5.6 and 8.2% for RIA of E 2 . Assay accuracy was tested by recovery of E 2 and P 4 added to pooled plasma; coefficients of correlation (r) were .99 for both P 4 and E 2 . Additional details regarding E 2 and P 4 extraction, RIA assay, and RIA validation have been reported elsewhere (Leszczynski et al, 1984). Values for statistics presented in the text were determined by methods of Steel and Torrie (1960). RESULTS AND DISCUSSION
In female chickens with normal ovulatory function, a broad peak of circulating P 4 precedes ovulation by about 4 to 6 hr (Kappauf and van Tienhoven, 1972; Furr et al, 1973; Lagiie et al, 1975). However, on the last day of a laying clutch in actively laying hens, when there is no ovulation, the preceding P 4 peak is also absent (Haynes et al., 197'3; Etches, 1979) and the circadian profile of circulating P 4 is flat, as is that of mutant nonlayers (Leszczynski et al, 1984). The circadian profile of circulating E 2 in actively laying chicken hens has b.een reported to contain two peaks, one near the time of ovulation and the other approximately 6 hr prior to ovulation (Shodono et al, 1975; Lague et al, 1975). It is clear that sampling time is a critical concern. Therefore, in our present study, blood samples were collected between 1700 and 2200 hr when both circulating E 2 and P 4 were expected to be at basal levels. Basal blood plasma levels of E 2 and P 4 were determined from eight groups of chickens and
PLASMA ESTRADIOL, PROGESTERONE, AND EGG PRODUCTION
547
TABLE 1. Plasma estradiol (E2) and progesterone (P4) levels from eight groups of chicken hens with variable egg productivity1 Group2 of h e n s
Group size
2-Week egg production
E2
Ancona NH X C NH X R I R Silkie X R I R Naked Neck WLH (Bab) WLH (ES) WLH (ro)
6 12 6 6 4 15 6 8
3.5 ± 7.13 4.3 ± 8.2 ± 5.3 ± 10.0 ± 4.8 ± 0
149.3 132.9 140.4 187.1 107.0 113.5 164.2 442.6
1
.22 .33 .48 .63 .71 .48
E2/P4
P< •(pg/ml) ± 23.3 + 12.6 + 15.1 + 16.2 + 13.8 + 11.2 + 19.5 + 105.6
362.9 738.3 445.8 1,953.6 415.5 1,492.0 881.7 453.1
± ± ± ± ± ± ± ±
73.3 77.2 104.4 933.7 69.6 274.1 141.3 141.1
.411 .180 .315 .096 .257 .076 .186 .977
Data presented as means ± standard error.
2
Abbreviations used for breeds and breed crosses: NH, New Hampshire; C, Columbian; RIR, Rhode Island Red; WLH, White Leghorn; Bab, Babcock strain; ES, egg size strain, ro; restricted ovulator strain. 3
Based on egg count from whole flock of 60 hens from which 12 were randomly selected.
are presented in Table 1. The egg production of each group of chickens during a 14-day period prior to bleeding is also presented in the table. The mean levels of circulating E 2 ranged between 107.0 and 187.1 pg/ml in seven actively laying groups; ro White Leghorn hens had hyperestrogenemia, which has been reported previously (Birrenkott et al, 1975; Schjeide et al, 1976; Leszczynski et al, 1984). In contrast to circulating E 2 , the range of mean circulating P 4 in the seven groups of laying hens was larger, between 362.9 and 1,95 3.6 pg/ml. Generally, egg production appears from the table to be associated more strongly with circulating P 4 than with estrogen. In addition to the groups listed in Table 1, samples from three 2.5 — year-old Buff Orpington hens were also collected at Madison. These birds were of particular interest because they represented a group that had not laid any eggs for at least one month prior to bleeding. These birds had a basal mean plasma E 2 of 116.5 pg/ml and a remarkably low P 4 of 69.8 pg/ml, which produced an E 2 / P 4 number greater than 1. In addition to these nonlayers, one of the hens from the naked neck group, which had laid only one egg in 14 days, also had a low circulating P 4 of only 78.0 pg/ml and an E 2 / P 4 ratio greater than 1. Many studies of plasma sex hormone levels in egg-laying chickens use multiple sampling techniques from individual birds to investigate circadian rhythms (Peterson and Common,
1972; Senior 1974; Yu et al, 1974; Tanabe and Nakamura, 1980, Johnson and van Tienhoven, 1981). With this sampling technique, it is possible to separate out, during statistical analysis, the large variation in plasma hormone levels between individual birds, which is characteristic even in tightly controlled experiments. However, in some cases, significant differences between group hormone levels can be demonstrated, especially when groups have basic phenotype differences related to sex physiology (Johnson and van Tienhoven, 1981; Leszczynski et al, 1984). In our analyses of the seven groups that produced eggs, no significant differences (P>.05, two-tailed t test) were detected for either plasma E 2 or plasma P 4 levels between any two groups, but a tendency for the most active layers to have a high basal plasma P 4 levels was noted. Therefore, linear regression coefficients of egg production on plasma E 2 , P 4 , the ratio of estradiol to progesterone (E 2 /P 4 ), and regression lines were calculated. Data from the linear regression analysis of E 2 / P 4 ratio with egg production (a = 9.98; b = 17.56) are presented in Figure 1. The correlation coefficient between the two variables was —.90 (P<.01). No significant correlation coefficient was found between E 2 or P 4 and egg production. In addition to presenting the linear regression line, Figure 1 also presents a "best fit" curve, which was drawn based on the actual data points and reasonable estimates of
548
LESZCZYNSKI ET AL.
\ \ \ c m-
«
X
o »-
7
c
o X, 3 •o S a. o>
*
87-
fi5-
je
a> 0)
3•
CM
4321-
•
1
1
1
1
.1
.2
.3
.4
•
Ratio estradiol: progesterone FIG. 1. Linear regression line of 2-week egg production on the ratio of basal plasma estradiol: progesterone for the seven groups of chicken hens that were actively laying. Also presented is an estimate of a "best fit" curve (dashed line) that is based on the data from die seven groups but also based on estimates of natural limits for egg production and for estradiol: progesterone ratio in both nonlaying hens as well as hens with maximum egg production.
natural limits. The limit of the "best fit" curve in the ordinate direction of Figure 1 is based on an estimate of maximum natural 2-week egg productivity (12) and an estimate of the lower limit for basal E2/P4 value (.05, which approaches values commonly seen during a P4 peak). The limit of the curve in the abscissa direction of Figure 1 is based on an estimate of E 2 /P 4 >1.0, which is based on results from two groups of nonlayers and other individual birds with low egg productivity. These analyses indicate that E2/P4 may be a better parameter than P 4 alone for estimating egg production. Analyses of data in Table 1 revealed that E 2 alone is of no value in estimating egg production. It is now well-established that the plasma P 4 peak in hens that occurs 4-6 hr prior to ovulation is associated primarily with the ability of the Fj follicle to respond to endogeneous gonadotropin stimulation. In particular, this response appears to involve increased P 4 production by the granulosa cells of the follicle
in response to luteinizing hormone (LH) (Hammond et al, 1981). Our general observation of increased basal circulating levels of P 4 in groups with the greatest egg laying activity may be related to similar physiological mechanisms. Because the maturation of follicles in the hen's ovary is associated with an increased mass and number of granulosa cells (Etches, 1984), one might expect a greater basal production of P 4 to be associated with increased granulosa cell mass alone. In addition, there is evidence that indicates the granulosa cells of not only Fi but also F 2 and F 3 follicles have heightened sensitivity to LH stimulus for P 4 synthesis and secretion compared to those from immature follicles (Hammond et al, 1981, Calvo et al, 1981). As it is reasonable to assume that the follicular hierarchy in highly productive layers is physically or biochemically more mature compared to less productive layers, then this overall greater mass or heightened sensitivity of granulosa cells may be sufficient to account for a higher level of basal circulating P 4 . The apparent association of follicular maturation with increased production of P 4 is in contrast to the relationship between estrogen production and follicular maturation. Senior and Furr (1975) have suggested that the majority of the circulating estrogens were derived from immature rather than yolk-filled follicles. Shahabi et al (1975) reported that the estrogen contents in follicular walls was F 3 > F 2 > F i . Huang et al (1979) reported that thecal cells from F 3 and F 2 follicles were capable of producing estrogens when incubated with an aromatizable substrate or granulosa cells, but Fi thecal cells were not. Thus, the apparent tendency of follicles approaching the Fi position in the hierarchy for stimulated P 4 production and reduced estrogen production may offer some insight as to why the basal circulating E 2 / P 4 correlates better with egg production compared to P 4 alone. The finding that E 2 /P 4 may be a better parameter for estimating egg production than the P 4 alone is consistent with the oftentimes antagonistic relationship of E 2 and P 4 in sexual physiology and metabolism (Natrajan et al, 1981; Holt et al, 1983; Leszczynski et al, 1982).
ACKNOWLEDGMENTS
This work was supported by grants from the American Heart Association/Illinois Affiliate
PLASMA ESTRADIOL, PROGESTERONE, AND EGG PRODUCTION
(D. L.), and the Wallace Genetic Foundation (F. K.). The authors recognize the excellent technical assistance of Robert Schafer. REFERENCES Abraham, G. E., R. Swerdloff, D. Tulchinski, and W. D. Odell, 1971. Radioimmunoassay of plasma progesterone. J. Steroid Biochem. 3:893—901. Birrenkott, G. P., W. H. McGibbon, W. H. Burke, and B. C. Wentworth, 1975. A hormonal profile of the genetic restricted ovulator (ro). Poultry Sci. 54:1735. (Abstr.) Bluhm, C. K„ R. E. Philips, and W. H. Burke, 1983. Serum levels of luteinizing hormone, prolactin, estradiol and progesterone in laying and nonlaying mallards (Anas platyrhynchos). Biol. Reprod. 28:295-305. Calvo, F. O., S.-C. Wang, and J. M. Bahr, 1981. LHstimulable adenylyl cyclase activity during the ovulatory cycle in granulosa cells of the three largest follicles and the postovulatory follicle of the domestic hen (Gallus domesticus). Biol. Reprod. 25:805-812. Etches, R. J., 1979. Plasma concentrations of progesterone and corticosterone during the ovulation cycle of the hen (Gallus domesticus). Poultry Sci. 58:211-216. Etches, R. J., 1984. Maturation of ovarian follicles. 17th Br. Poult. Sci. Symp. - Poult. Reprod. (in press). Furr, B.J.A., R. C. Bonney, R. J. England, and F. J. Cunningham, 1973. Luteinizing hormone and progesterone in peripheral blood during the ovulatory cycle of the hen Gallus domesticus. J. Endocrinol. 57:159-169. Hammond, R. W., W. H. Burke, and F. Hertelendy, 1981. Influence of follicular maturation on progesterone release in chicken granulosa cells in response to turkey and ovine gonadotropins. Biol. Reprod. 24:1048-1055. Haynes, N. B., K. J. Cooper, and M. J. Kay, 1973. Plasma progesterone concentration in the hen in relation to the ovulatory cycle. Br. Poult. Sci. 14:349-357. Holt, J. A., J. R. Schreiber, and B. R. Zirkin, 1983. Estradiol-induced changes in rabbit luteal cell progestin production and cholesterol and cholesterol ester content. Biochem. Biophys. Res. Commun. 113:1026-1033. Huang, E.S.U., K. J. Kao, and A. V. Nalbandov, 1979. Synthesis of sex steroids by cellular components of chicken follicles. Biol. Reprod 2 0 : 4 5 4 - 4 6 1 . Johnson, A. L., and A. van Tienhoven, 1981. Pharmacokinetics of progesterone in laying and non-laying hens (Gallus domesticus). J. Endocrinol. 8 9 : 1 - 1 2 . Jones, D. G., W. E. Briles, and O. A. Schjeide, 1975. A mutation restricting ovulation in chickens. Poultry Sci. 54:1780. (Abstr.) Kappauf, B., and A. van Tienhoven, 1972. Progesterone concentrations in peripheral plasma of laying hens in relation to the time of ovulation. Endocrinology. 90:1350-1355. Lagiie, P. C , A. van Tienhoven, and F. J. Cunningham, 1975. Concentration of estrogens, progesterone and LH during the ovulatory cycle of the laying chicken (Gallus domesticus). Biol. Reprod.
549
12:590-598. Leszczynski, D. E., R. C , Hagan, S. E. Rowe, and F. A. Kummerow, 1984. Plasma sex hormone and lipid patterns in normal and restricted-ovulator chicken hens. Gen. Comp. Endocrinol. 55:280— 288. Leszczynski, D. E., J. Pikul, and F. A. Kummerow, 1983. Relationship of circulating estrogen and progesterone to plasma lipids and egg productivity in chicken hens. Poultry Sci. 62:1457. (Abstr.) Leszczynski, D. E., T. Toda, and F. A. Kummerow, 1982. Influence of dietary sex hormones on chick lipid metabolism. Horm. Metab. Res. 14:183-189. Mashaly, M. M., and B. C. Wentworth, 1974. A profile of progesterone in turkey sera. Poultry Sci. 53:2030-2035. Natrajan, P. K., T. G., Muldoon, R. B. Greenblatt, and V. B. Mahesh, 1981. Estradiol and progesterone receptors in estrogen-primed endometrium. Am. J. Obstet. Gynecol. 140:387-392. Peterson, A. J., and R. H. Common, 1972. Estrone and estradiol concentrations in peripheral plasma of laying hens as determined by radioimmunoassay. Can. J. Zool. 50:395-404. Schjeide, O. A., W. E. Briles, S. Holshouser, and D. G. Jones, 1976. Effect of "restricted ovulator" gene on uptake of yolk-precursor protein. Cell Tissue Res. 166:109. Senior, B. E., 1974. Oestradiol concentration in the peripheral plasma of the domestic hen from 7 weeks of age until the time of sexual maturity. J. Reprod. Fertil. 41:107-112. Senior, B. E., and B.J.A. Furr, 1975. A preliminary assessment of the source of oestrogen within the ovary of the domestic fowl (Gallus domesticus). J. Reprod. Fertil. 43:241-249. Shahabi, N. A., H. W. Norton, and A. V. Nalbandov, 1975. Steroid levels in follicles and the plasma of hens during the ovulatory cycle. Endocrinology 96:962-969. Shodono, M., T. Nakamura, Y. Tanabe, and K. Wakabayashi, 1975. Simultaneous determinations of oestradiol-17(3, progesterone and luteinizing hormone in the plasma during the ovulatory cycle of the hen. Acta Endocrinol. 78:565-573. Steel, R.G.D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill Books Co., Inc., New York, NY. Tanabe, Y., and T. Nakamura, 1980. Endocrine mechanism of ovulation in chickens (Gallus domesticus), quail (Coturnix coturnix japonica), and ducks (Anas platyrhynchos domestica). Pages 179—188 in Biological Rhythms in Birds: Neural and Endocrine Aspects. Y. Tanabe et al., ed. Jpn. Sci. Soc. Press, Tokyo/Springer-Verlag, Berlin. Wu, C.-H., and L. E. Lundy, 1971. Radioimmunoassay of plasma estrogens. Steroids 18:91 — 111. Youssefrejadian, E., E. Florensa, W. P. Collins, and I. F. Sommerville, 1972. Radioimmunoassay of plasma progesterone. J. Steroid Biochem. 3: 893-901. Yu, J.Y.-L., P. Yuthasastrakosol, R. R. Marquardt, and W. M. Palmer, 1974. Estrogen levels in peripheral plasma of immature female chickens as determined by radioimmunoassay. Endocrinology. 94:1168-1171.
Foundation,
503
South
Sixth Street,
Champaign,
Illinois
61820
R. C. H A G A N Burnsides
Research
Laboratory,
Department
of Food Science,
University
of Illinois,
Urbana, Illinois
61801
J . J . BITGOOD Department
of Poultry
Science,
University
of Wisconsin,
Madison,
Wisconsin
53706
F. A. KUMMEROW Harlan F. Moore Heart Research
Foundation,
503 South
Sixth
Street,
Champaign,
Illinois
61820
(Received for publication March 30, 1984) ABSTRACT Female chickens from eight different genetic stocks, ranging from 13 to 21 m o n t h s of age, and exhibiting various levels of egg p r o d u c t i o n were studied. Plasma samples were collected between 1 7 0 0 and 2 2 0 0 hr to determine basal circulating levels of progesterone ( P 4 ) and 17/3estradiol ( E 2 ) by radioimmunoassay. Linear regression analysis was used t o describe t h e relation between mean h o r m o n e levels ( E 2 , P 4 , and E 2 / P 4 ) and m e a n egg p r o d u c t i o n from these groups, and correlation coefficients were used t o evaluate t h e general usefulness of these h o r m o n e par a m e t e r s for estimating egg p r o d u c t i o n within a g r o u p or flock of hens. Of t h e three h o r m o n e p a r a m e t e r s evaluated, basal circulating E 2 / P 4 was found to be t h e best e s t i m a t o r of egg p r o d u c tivity. (Key words: chickens, egg p r o d u c t i o n , estradiol, progesterone) 1 9 8 5 P o u l t r y Science 6 4 : 5 4 5 - 5 4 9 INTRODUCTION
MATERIALS AND METHODS
Our previous studies of sexually mature White Leghorn hens carrying the sex-linked restricted ovulator (ro) mutation (Jones et al., 1975) indicated that an unusually high basal level of circulating estrogen and an unusually low basal level of progesterone (P 4 ) were associated with the failure to lay eggs (Leszczynski et al., 1983). Low levels of circulating P 4 have also been reported for immature and molting chicken hens (Johnson and van Tienhoven, 1981). In Mallard ducks, cessation of egg-laying induced by stress was associated with low circulating levels of both P 4 and estradiol (E 2 ) (Bluhm et al, 1983). In turkeys, nonlaying hens, and hens with low egg production also have low levels of circulating P 4 (Mashaly and Wentworth, 1974). The purpose of the present study was to measure basal circulating levels of E 2 and P 4 in several varieties of sexually mature female chickens with various levels of egg productivity to test if these parameters can be useful in evaluating egg production within a flock.
Blood samples from mutant ro White Leghorn hens, inbred White Leghorn hens (Babcock strain), and New Hampshire (NH) X Columbian (C) hens were taken at the University of Illinois (Urbana) poultry farm. Blood samples were obtained at the University of Wisconsin (Madison) from hens of several different genetic stocks. These included inbred White Leghorns (ES line), inbred Anconas, carriers of naked neck, Buff Orpingtons, ^i Silkie x Rhode Island Red (RIR), and hens derived from NH x RIR crosses. All hens were individually caged, fed ad libitum a standard corn-soybean based layer ration, and exposed to 14 hr light and 10 hr dark each day with lights on at 0500 hr. All birds, none of which were molting, ranged in age from 13 to 21 months except for three Buff Orpington hens, which were 2.5 years old. Blood samples were collected at least 12 hr before the expected times for ovulation, based on prior observations. Thus, samples were drawn during a period of the day (between
545
546
LESZCZYNSKI ET AL.
1700 and 2200 hr) when both circulating estradiol (E 2 ) and P 4 were expected to be at basal levels. Blood samples were stabilized with .1% tetrasodium-ethylenediaminetetraacetate (EDTA), and plasma was collected after centrifugation and stored at —18 C until analysis. Antisera used for the determination of E 2 and P 4 were purchased from CalbiochemBehring where they were prepared from rabbits against 17|3-estradiol-(6-0-carboxymethyl)-aminebovine serum albumin (BSA) conjugate and progesterone-(l l-hemisuccinate)-BSA. Crossreactivities for estradiol antiserum were: 17/3estradiol (100%), estrone (<.06%), estriol (<.18%), and P 4 (nil). For P 4 antiserum, the crossreactivities were: P 4 (100%), lla-hydroxyprogesterone (<17%), 11/3-hydroxyprogesterone (<16%), 5a-pregnan-3|3-ol-20-one (<42%), A5-pregnan-3j3-ol-20-one, and pregnanolone (<2.4%), all other steroids (<1%). Other chemicals used were: E 2 standard and gamma globulin, Cohn fraction II (Sigma Chemical Co.); Sephadex LH-20 and Dextran T-70 (Pharmacia); and Norit A charcoal (Fisher Chemical Co.). Spectro grade solvents for steroid extraction and column chromatography were purchased from the J. T. Baker Co. These solvents were pretested for residue interference with antibody-antigen binding by drying 4-ml aliquots in tubes; these solvents were used in titer determinations and compared to identical determinations done in tubes without dried solvents. Recovery tracer was equilibrated with plasma by sonication for 5 min followed by room temperature incubation for 2 hr. Estradiol was extracted with four parts ethyl ether, P 4 was extracted with four parts petroleum ether, and tracer recoveries ranged between 80 and 95%. Ether extracts were dried under nitrogen and resuspended in isooctane: benzene: methanol 62:20:18 in the case of E 2 and 90:5:5 in the case of P 4 for Sephadex LH-20 chromatography. For plasma samples that were extremely hyperlipidemic, dried ether extracts were resuspended in 70% methanol, kept at —80 C overnight, and then were centrifuged at —10 C to remove approximately 95% of the fat (mostly triglycerides), after which the supernate was dried under nitrogen and resuspended in the appropriate isooctane: benzene: methanol solvent. This step resulted in an additional 10 to 15% loss of recovery tracer but greatly improved the sensitivity and reliability of the assay. After LH-20 chromatography,
appropriate steroid fractions were dried with nitrogen and resuspended in absolute ethanol, transferred to tubes, and then dried for duplicate radioimmunoassay (RIA) analysis and also for the calculation of recovery. The RIA assays were performed according to procedures given with the P 4 and estrogen Radioimmunoassay Paks supplied by the New England Nuclear Co., with slight modification, based on the methods of Abraham et al. (1971) and Youssefrejadian et al. (1972). Assay radioactivity was converted to picograms of hormone by use of a log-logit plotting procedure described by Wu and Lundy (1971). Assay sensitivity averaged 16.3 pg for P 4 and 3.0 pg for E 2 . Assay precision, measured by calculating within and between assay coefficients of variations (CV) from pooled plasma samples were 3.7 and 11.6%, respectively, for RIA of P 4 and 5.6 and 8.2% for RIA of E 2 . Assay accuracy was tested by recovery of E 2 and P 4 added to pooled plasma; coefficients of correlation (r) were .99 for both P 4 and E 2 . Additional details regarding E 2 and P 4 extraction, RIA assay, and RIA validation have been reported elsewhere (Leszczynski et al, 1984). Values for statistics presented in the text were determined by methods of Steel and Torrie (1960). RESULTS AND DISCUSSION
In female chickens with normal ovulatory function, a broad peak of circulating P 4 precedes ovulation by about 4 to 6 hr (Kappauf and van Tienhoven, 1972; Furr et al, 1973; Lagiie et al, 1975). However, on the last day of a laying clutch in actively laying hens, when there is no ovulation, the preceding P 4 peak is also absent (Haynes et al., 197'3; Etches, 1979) and the circadian profile of circulating P 4 is flat, as is that of mutant nonlayers (Leszczynski et al, 1984). The circadian profile of circulating E 2 in actively laying chicken hens has b.een reported to contain two peaks, one near the time of ovulation and the other approximately 6 hr prior to ovulation (Shodono et al, 1975; Lague et al, 1975). It is clear that sampling time is a critical concern. Therefore, in our present study, blood samples were collected between 1700 and 2200 hr when both circulating E 2 and P 4 were expected to be at basal levels. Basal blood plasma levels of E 2 and P 4 were determined from eight groups of chickens and
PLASMA ESTRADIOL, PROGESTERONE, AND EGG PRODUCTION
547
TABLE 1. Plasma estradiol (E2) and progesterone (P4) levels from eight groups of chicken hens with variable egg productivity1 Group2 of h e n s
Group size
2-Week egg production
E2
Ancona NH X C NH X R I R Silkie X R I R Naked Neck WLH (Bab) WLH (ES) WLH (ro)
6 12 6 6 4 15 6 8
3.5 ± 7.13 4.3 ± 8.2 ± 5.3 ± 10.0 ± 4.8 ± 0
149.3 132.9 140.4 187.1 107.0 113.5 164.2 442.6
1
.22 .33 .48 .63 .71 .48
E2/P4
P< •(pg/ml) ± 23.3 + 12.6 + 15.1 + 16.2 + 13.8 + 11.2 + 19.5 + 105.6
362.9 738.3 445.8 1,953.6 415.5 1,492.0 881.7 453.1
± ± ± ± ± ± ± ±
73.3 77.2 104.4 933.7 69.6 274.1 141.3 141.1
.411 .180 .315 .096 .257 .076 .186 .977
Data presented as means ± standard error.
2
Abbreviations used for breeds and breed crosses: NH, New Hampshire; C, Columbian; RIR, Rhode Island Red; WLH, White Leghorn; Bab, Babcock strain; ES, egg size strain, ro; restricted ovulator strain. 3
Based on egg count from whole flock of 60 hens from which 12 were randomly selected.
are presented in Table 1. The egg production of each group of chickens during a 14-day period prior to bleeding is also presented in the table. The mean levels of circulating E 2 ranged between 107.0 and 187.1 pg/ml in seven actively laying groups; ro White Leghorn hens had hyperestrogenemia, which has been reported previously (Birrenkott et al, 1975; Schjeide et al, 1976; Leszczynski et al, 1984). In contrast to circulating E 2 , the range of mean circulating P 4 in the seven groups of laying hens was larger, between 362.9 and 1,95 3.6 pg/ml. Generally, egg production appears from the table to be associated more strongly with circulating P 4 than with estrogen. In addition to the groups listed in Table 1, samples from three 2.5 — year-old Buff Orpington hens were also collected at Madison. These birds were of particular interest because they represented a group that had not laid any eggs for at least one month prior to bleeding. These birds had a basal mean plasma E 2 of 116.5 pg/ml and a remarkably low P 4 of 69.8 pg/ml, which produced an E 2 / P 4 number greater than 1. In addition to these nonlayers, one of the hens from the naked neck group, which had laid only one egg in 14 days, also had a low circulating P 4 of only 78.0 pg/ml and an E 2 / P 4 ratio greater than 1. Many studies of plasma sex hormone levels in egg-laying chickens use multiple sampling techniques from individual birds to investigate circadian rhythms (Peterson and Common,
1972; Senior 1974; Yu et al, 1974; Tanabe and Nakamura, 1980, Johnson and van Tienhoven, 1981). With this sampling technique, it is possible to separate out, during statistical analysis, the large variation in plasma hormone levels between individual birds, which is characteristic even in tightly controlled experiments. However, in some cases, significant differences between group hormone levels can be demonstrated, especially when groups have basic phenotype differences related to sex physiology (Johnson and van Tienhoven, 1981; Leszczynski et al, 1984). In our analyses of the seven groups that produced eggs, no significant differences (P>.05, two-tailed t test) were detected for either plasma E 2 or plasma P 4 levels between any two groups, but a tendency for the most active layers to have a high basal plasma P 4 levels was noted. Therefore, linear regression coefficients of egg production on plasma E 2 , P 4 , the ratio of estradiol to progesterone (E 2 /P 4 ), and regression lines were calculated. Data from the linear regression analysis of E 2 / P 4 ratio with egg production (a = 9.98; b = 17.56) are presented in Figure 1. The correlation coefficient between the two variables was —.90 (P<.01). No significant correlation coefficient was found between E 2 or P 4 and egg production. In addition to presenting the linear regression line, Figure 1 also presents a "best fit" curve, which was drawn based on the actual data points and reasonable estimates of
548
LESZCZYNSKI ET AL.
\ \ \ c m-
«
X
o »-
7
c
o X, 3 •o S a. o>
*
87-
fi5-
je
a> 0)
3•
CM
4321-
•
1
1
1
1
.1
.2
.3
.4
•
Ratio estradiol: progesterone FIG. 1. Linear regression line of 2-week egg production on the ratio of basal plasma estradiol: progesterone for the seven groups of chicken hens that were actively laying. Also presented is an estimate of a "best fit" curve (dashed line) that is based on the data from die seven groups but also based on estimates of natural limits for egg production and for estradiol: progesterone ratio in both nonlaying hens as well as hens with maximum egg production.
natural limits. The limit of the "best fit" curve in the ordinate direction of Figure 1 is based on an estimate of maximum natural 2-week egg productivity (12) and an estimate of the lower limit for basal E2/P4 value (.05, which approaches values commonly seen during a P4 peak). The limit of the curve in the abscissa direction of Figure 1 is based on an estimate of E 2 /P 4 >1.0, which is based on results from two groups of nonlayers and other individual birds with low egg productivity. These analyses indicate that E2/P4 may be a better parameter than P 4 alone for estimating egg production. Analyses of data in Table 1 revealed that E 2 alone is of no value in estimating egg production. It is now well-established that the plasma P 4 peak in hens that occurs 4-6 hr prior to ovulation is associated primarily with the ability of the Fj follicle to respond to endogeneous gonadotropin stimulation. In particular, this response appears to involve increased P 4 production by the granulosa cells of the follicle
in response to luteinizing hormone (LH) (Hammond et al, 1981). Our general observation of increased basal circulating levels of P 4 in groups with the greatest egg laying activity may be related to similar physiological mechanisms. Because the maturation of follicles in the hen's ovary is associated with an increased mass and number of granulosa cells (Etches, 1984), one might expect a greater basal production of P 4 to be associated with increased granulosa cell mass alone. In addition, there is evidence that indicates the granulosa cells of not only Fi but also F 2 and F 3 follicles have heightened sensitivity to LH stimulus for P 4 synthesis and secretion compared to those from immature follicles (Hammond et al, 1981, Calvo et al, 1981). As it is reasonable to assume that the follicular hierarchy in highly productive layers is physically or biochemically more mature compared to less productive layers, then this overall greater mass or heightened sensitivity of granulosa cells may be sufficient to account for a higher level of basal circulating P 4 . The apparent association of follicular maturation with increased production of P 4 is in contrast to the relationship between estrogen production and follicular maturation. Senior and Furr (1975) have suggested that the majority of the circulating estrogens were derived from immature rather than yolk-filled follicles. Shahabi et al (1975) reported that the estrogen contents in follicular walls was F 3 > F 2 > F i . Huang et al (1979) reported that thecal cells from F 3 and F 2 follicles were capable of producing estrogens when incubated with an aromatizable substrate or granulosa cells, but Fi thecal cells were not. Thus, the apparent tendency of follicles approaching the Fi position in the hierarchy for stimulated P 4 production and reduced estrogen production may offer some insight as to why the basal circulating E 2 / P 4 correlates better with egg production compared to P 4 alone. The finding that E 2 /P 4 may be a better parameter for estimating egg production than the P 4 alone is consistent with the oftentimes antagonistic relationship of E 2 and P 4 in sexual physiology and metabolism (Natrajan et al, 1981; Holt et al, 1983; Leszczynski et al, 1982).
ACKNOWLEDGMENTS
This work was supported by grants from the American Heart Association/Illinois Affiliate
PLASMA ESTRADIOL, PROGESTERONE, AND EGG PRODUCTION
(D. L.), and the Wallace Genetic Foundation (F. K.). The authors recognize the excellent technical assistance of Robert Schafer. REFERENCES Abraham, G. E., R. Swerdloff, D. Tulchinski, and W. D. Odell, 1971. Radioimmunoassay of plasma progesterone. J. Steroid Biochem. 3:893—901. Birrenkott, G. P., W. H. McGibbon, W. H. Burke, and B. C. Wentworth, 1975. A hormonal profile of the genetic restricted ovulator (ro). Poultry Sci. 54:1735. (Abstr.) Bluhm, C. K„ R. E. Philips, and W. H. Burke, 1983. Serum levels of luteinizing hormone, prolactin, estradiol and progesterone in laying and nonlaying mallards (Anas platyrhynchos). Biol. Reprod. 28:295-305. Calvo, F. O., S.-C. Wang, and J. M. Bahr, 1981. LHstimulable adenylyl cyclase activity during the ovulatory cycle in granulosa cells of the three largest follicles and the postovulatory follicle of the domestic hen (Gallus domesticus). Biol. Reprod. 25:805-812. Etches, R. J., 1979. Plasma concentrations of progesterone and corticosterone during the ovulation cycle of the hen (Gallus domesticus). Poultry Sci. 58:211-216. Etches, R. J., 1984. Maturation of ovarian follicles. 17th Br. Poult. Sci. Symp. - Poult. Reprod. (in press). Furr, B.J.A., R. C. Bonney, R. J. England, and F. J. Cunningham, 1973. Luteinizing hormone and progesterone in peripheral blood during the ovulatory cycle of the hen Gallus domesticus. J. Endocrinol. 57:159-169. Hammond, R. W., W. H. Burke, and F. Hertelendy, 1981. Influence of follicular maturation on progesterone release in chicken granulosa cells in response to turkey and ovine gonadotropins. Biol. Reprod. 24:1048-1055. Haynes, N. B., K. J. Cooper, and M. J. Kay, 1973. Plasma progesterone concentration in the hen in relation to the ovulatory cycle. Br. Poult. Sci. 14:349-357. Holt, J. A., J. R. Schreiber, and B. R. Zirkin, 1983. Estradiol-induced changes in rabbit luteal cell progestin production and cholesterol and cholesterol ester content. Biochem. Biophys. Res. Commun. 113:1026-1033. Huang, E.S.U., K. J. Kao, and A. V. Nalbandov, 1979. Synthesis of sex steroids by cellular components of chicken follicles. Biol. Reprod 2 0 : 4 5 4 - 4 6 1 . Johnson, A. L., and A. van Tienhoven, 1981. Pharmacokinetics of progesterone in laying and non-laying hens (Gallus domesticus). J. Endocrinol. 8 9 : 1 - 1 2 . Jones, D. G., W. E. Briles, and O. A. Schjeide, 1975. A mutation restricting ovulation in chickens. Poultry Sci. 54:1780. (Abstr.) Kappauf, B., and A. van Tienhoven, 1972. Progesterone concentrations in peripheral plasma of laying hens in relation to the time of ovulation. Endocrinology. 90:1350-1355. Lagiie, P. C , A. van Tienhoven, and F. J. Cunningham, 1975. Concentration of estrogens, progesterone and LH during the ovulatory cycle of the laying chicken (Gallus domesticus). Biol. Reprod.
549
12:590-598. Leszczynski, D. E., R. C , Hagan, S. E. Rowe, and F. A. Kummerow, 1984. Plasma sex hormone and lipid patterns in normal and restricted-ovulator chicken hens. Gen. Comp. Endocrinol. 55:280— 288. Leszczynski, D. E., J. Pikul, and F. A. Kummerow, 1983. Relationship of circulating estrogen and progesterone to plasma lipids and egg productivity in chicken hens. Poultry Sci. 62:1457. (Abstr.) Leszczynski, D. E., T. Toda, and F. A. Kummerow, 1982. Influence of dietary sex hormones on chick lipid metabolism. Horm. Metab. Res. 14:183-189. Mashaly, M. M., and B. C. Wentworth, 1974. A profile of progesterone in turkey sera. Poultry Sci. 53:2030-2035. Natrajan, P. K., T. G., Muldoon, R. B. Greenblatt, and V. B. Mahesh, 1981. Estradiol and progesterone receptors in estrogen-primed endometrium. Am. J. Obstet. Gynecol. 140:387-392. Peterson, A. J., and R. H. Common, 1972. Estrone and estradiol concentrations in peripheral plasma of laying hens as determined by radioimmunoassay. Can. J. Zool. 50:395-404. Schjeide, O. A., W. E. Briles, S. Holshouser, and D. G. Jones, 1976. Effect of "restricted ovulator" gene on uptake of yolk-precursor protein. Cell Tissue Res. 166:109. Senior, B. E., 1974. Oestradiol concentration in the peripheral plasma of the domestic hen from 7 weeks of age until the time of sexual maturity. J. Reprod. Fertil. 41:107-112. Senior, B. E., and B.J.A. Furr, 1975. A preliminary assessment of the source of oestrogen within the ovary of the domestic fowl (Gallus domesticus). J. Reprod. Fertil. 43:241-249. Shahabi, N. A., H. W. Norton, and A. V. Nalbandov, 1975. Steroid levels in follicles and the plasma of hens during the ovulatory cycle. Endocrinology 96:962-969. Shodono, M., T. Nakamura, Y. Tanabe, and K. Wakabayashi, 1975. Simultaneous determinations of oestradiol-17(3, progesterone and luteinizing hormone in the plasma during the ovulatory cycle of the hen. Acta Endocrinol. 78:565-573. Steel, R.G.D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill Books Co., Inc., New York, NY. Tanabe, Y., and T. Nakamura, 1980. Endocrine mechanism of ovulation in chickens (Gallus domesticus), quail (Coturnix coturnix japonica), and ducks (Anas platyrhynchos domestica). Pages 179—188 in Biological Rhythms in Birds: Neural and Endocrine Aspects. Y. Tanabe et al., ed. Jpn. Sci. Soc. Press, Tokyo/Springer-Verlag, Berlin. Wu, C.-H., and L. E. Lundy, 1971. Radioimmunoassay of plasma estrogens. Steroids 18:91 — 111. Youssefrejadian, E., E. Florensa, W. P. Collins, and I. F. Sommerville, 1972. Radioimmunoassay of plasma progesterone. J. Steroid Biochem. 3: 893-901. Yu, J.Y.-L., P. Yuthasastrakosol, R. R. Marquardt, and W. M. Palmer, 1974. Estrogen levels in peripheral plasma of immature female chickens as determined by radioimmunoassay. Endocrinology. 94:1168-1171.