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Serum Progesterone, Enzymes, and Electrolytes of Hens Laying a Low or High Incidence of Shell-Less Eggs
Serum Progesterone, Enzymes, and Electrolytes of Hens Laying a Low or High Incidence of Shell-Less Eggs D. A. ROLAND, SR.,1 D. N. MARPLE,2 and R. N. BREWER1 Agricultural Experiment
Station
P. A. TEER Department of Pathology and Parasitology, School of Veterinary Medicine F. J.HOERR
(Received for publication June 11, 1981) ABSTRACT Three experiments were conducted to determine possible differences in serum progesterone, enzymes, and electrolytes between hens laying a high or low incidence of shell-less (SL) eggs. Blood was taken at time of oviposition and at 6, 12, 18, and 25 hr later, irrespective of the stage of the sequence. The results indicated no significant differences in average serum progesterone between hens laying a high or low incidence of SL eggs. In general, the patterns of serum progesterone in relation to time of oviposition was similar for each type. No significant differences were observed in serum phosphorus, magnesium, glutamate pyruvate transaminase, or alkaline phosphatase between hens laying a high or low incidence of SL eggs. It was concluded that the production of SL eggs is not related to abnormal serum progesterone, alkaline phosphatase, glutamate pyruvate transaminase, magnesium, or phosphorus levels. (Key words: shell-less eggs, progesterone, enzymes, electrolytes) 1983 Poultry Science 6 2 : 9 1 7 - 9 2 2 INTRODUCTION T h e p r o d u c t i o n of shell-less (SL) eggs appears t o be t h e single most costly shell quality p r o b l e m of t h e commercial egg producer. R e c e n t studies revealed t h a t for every 100 eggs collected, a p p r o x i m a t e l y 7 are n o t collected or recorded as eggs p r o d u c e d ( R o l a n d , 1 9 7 7 ) . A high incidence of SL or a b n o r m a l eggs has also been r e p o r t e d t o occur in m e a t - t y p e hens and is referred t o as t h e erratic oviposition and defective egg s y n d r o m e ( J a p p and Muir, 1 9 6 8 ; R e d d y and Siegel, 1 9 7 6 ; van M i d d e l k o o p , 1 9 7 8 ) . A l t h o u g h t h e p r o d u c t i o n of SL eggs in commercial layers has been k n o w n since 1 8 9 9 (Hargitt), very little is k n o w n a b o u t t h e cause of SL eggs. Hewitt ( 1 9 3 9 ) and Nestor and Bacon ( 1 9 7 2 ) suggested t h a t t h e cause of SL eggs m a y be d u e to violent peristaltic muscle contractions in t h e u t e r u s causing p r e m a t u r e expulsion.
1 2
Department of Poultry Science. Department of Animal and Poultry Science.
Roland et al. ( 1 9 7 5 , 1977) concluded t h a t hens laying SL eggs were able t o absorb calcium from t h e digestive tract, because b l o o d , b o n e , and u t e r u s calcium levels were n o r m a l or above normal. T h e y suggested t h a t SL eggs were caused b y altered calcium t r a n s p o r t or metabolism at t h e actual site of egg shell formation or b y p r e m a t u r e expulsion (oviposition). It has been shown t h a t progesterone can alter t i m e of oviposition and ovulation (Neher and Fraps, 1 9 5 0 ; Etches and Cunningham, 1 9 7 6 ; Wilson and Sharp, 1 9 7 6 ) . Because p r e m a t u r e oviposition has been suggested as a possible cause of SL eggs, t h e following studies were c o n d u c t e d t o d e t e r m i n e if a b n o r m a l serum progesterone levels occurred prior t o oviposition in hens laying SL eggs, S e r u m alkaline phosphatase (Moog and Glazier, 1 9 7 7 ) , serum p h o s p h o r u s (Wolford and Tanaka, 1 9 7 0 ) , serum magnesium (Holder and H u n t l e y , 1 9 7 8 ) , and serum g l u t a m a t e transaminase (Tietz, 1976) have b e e n associated with either shell calcification or steroid h o r m o n e imbalance. Therefore, serum c o n c e n t r a t i o n s of these
917
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Alabama Department of Agriculture and Industries, C. S. Roberts Veterinary Diagnostic Laboratory, Auburn University, Alabama 36849
918 enzymes mined.
ROLAND, SR..ET AL. and
electrolytes
were also deter-
MATERIALS AND METHODS
Serum phosphorus (Woods and Mellon, 1941), magnesium (Gindler and Heth, 1971), alkaline phosphatase (Coleman, 1966), and serum glutamate pyruvate transaminase (Reitman and Frankel, 1957) were also measured. All data were subjected to an analysis of variance, and significant differences were identified using Kramer's (1956) modification of Duncan's new multiple range test. RESULTS AND DISCUSSION No significant differences in average serum progesterone were found between hens laying either a high or low incidence of SL eggs (Table 2). In Experiment 1, hens laying after 1400 hr could have been experiencing preovulatory peaks of progesterone at the time of blood collection. However, mean progesterone values were less than those in Experiment 2, and the variation was consistent with that of the other experiment, suggesting that very
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Leghorn hens used in all experiments were at least 10 months in lay. Blood samples obtained by heart puncture were centrifuged and the serum immediately frozen until analyzed. The hens were individually caged and fed a commercial-type corn-soy diet containing 2830 ME kcal/kg, 16% protein, 3.5% calcium, and .75% phosphorus. A daily photoperiod of 14 hr was used with lights on from 0600 to 2000 hr. Experiment 1. Hens laying either a high (62.5%) or low (0%) incidence of SL eggs during the previous 10 days were used in groups of 12 hens each. The hens laying a high incidence of SL eggs were laying 1.7% normal (HS) eggs for a total production of 64.2%, whereas the control hens had 55% production (HS eggs). Blood was collected at 1400 hr and analyzed for serum progesterone. Experiment 2, Blood taken from hens at time of oviposition of SL and HS eggs and 6, 12, 18, and 25 hr later was analyzed for serum progesterone. The values were reported in relation to oviposition when oviposition was directly followed by ovulation. The number of samples obtained at each time period varied (Table 3). Blood was taken within 5 min of the specified time. Experiment 3. Fifteen hens laying an average of 45.3% SL eggs and 23.3% HS eggs for a total production of 68.6% and 12 hens laying an average of 78.3% HS eggs and no SL eggs during the previous 10 days were used. Blood was obtained at 1400 hr and serum phosphorus, magnesium, alkaline phosphatase, and glutamate pyruvate transaminase were measured. Progesterone Assay. Progesterone antiserum was prepared by immunizing sheep with 4Pregnen-6-j3 -ol -3, 20-dione hemisuccinate BSA (Steraloids, Wilton, NH). Radiolabeled progesterone (2, 6, 7- 3 H-progesterone; New England Nuclear) was purified by thin layer chromatography (chloroform: acetone 95:5) prior to use. Progesterone used for standards and steroids (Sigma Chemical Co.) used in specificity studies were not further purified. Approximately 5000 dpm of ^ - p r o g e s terone were mixed with 1 ml of serum prior to extraction with 9 ml of diethyl ether. The organic fraction was dried, resuspended in 1 ml
of 70% methanol, and frozen to remove neutral lipids (Schiavo et al., 1975). After 12 to 18 hr at —9 C, the lipid phase was removed, dried, and resuspended in 350 [i\ of gel phosphate buffered saline (.1% gelatin, pH 7.6, .01 M PO4, I = .15). Recovery estimates were made by counting 100 /il of the resuspended extract. The mean recovery for the extraction procedure was 82% and the serum estimates were corrected for recovery. Duplicate 100 /il volumes of the same extract in gel-PBS were also taken for assay and placed in 10 x 75 mm culture tubes. Each assay tube then received 400 (A of gel-PBS, 100 ;ul of a 1:3000 dilution of antiserum, and 100 (jl of 3 H-progesterone solution (approximately 50,000 dpm per tube). The assay tubes were incubated for 1 hr at 37 C, followed by 1 hr at 4 C. Dextran-coated charcoal (200 jul of .031% dextran, 31% Norit A in pH 7.6 PBS) was added to each tube to separate bound and free antigen. After centrifugation at 2000 X g for 15 min, a 500/ul aliquot of thesupernate was removed and counted. The results of each assay were determined from a plot of logit Y vs. Log X where Y and X were B/Bo and mass of standard progesterone, respectively (Rodbard and Lewald, 1970). The sensitivity of the assay was 60 pg per tube and the working range of the standard curve was from 125 pg to 4 ng. The specificity of the assay for 21 steroids was evaluated (Table 1). The inter- and intraassay coefficients of variation were 11.7 and 9.2%, respectively.
RESEARCH NOTE
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TABLE 1. Crossreactions of 21 steroids in the radioimmunoassay for progesterone using antiserum Steroids
#1555-3
% Crossreaetion
Cholesterol
0
C 21
Progesterone Pregnenolone 11 -alpha-Hydroxyprogesterone 11-keto-Progesterone 17-alpha-Hydroxyprogesterone Pregnendiol Pregnan-3,20-dione 16-Dehydropregnenolone Corticosterone Hydrocortisone Cortisone Deoxycorticosterone 11-Dehydro-corticosterone Aldosterone
CI9
Testosterone Androstandione Androstendione
<.01 <.01 <.01
C 18
Estrone 17-beta-estradiol Estriol
<.01 <.01 <.01
100.00 48.21 5.30 25.00 5.50 1.05 <.01 <.01 <.01 <.01 <.01 <.01 <.01 <.01
The following chemical names correspond to the above trivial names of steroids tested: cholesterol = ^ 5 cholesten-3-ol; progesterone = &4-Pregnen-3, 20-dione; pregnenolone = AS-Pregnen-3(?-ol-20-one; 11-a-hydroxyprogesterone = & 4 Pregnen-lla-ol-3, 20-dione; 11-ketoprogesterone = A4-Pregnen-3, 11, 20-dione; 17-a-hydroxyprogesterone = A 4 -Pregnen-17a-ol-3, 20-dione; pregnendiol = & 5-Pregnen-3|3, 20(3-diol; pregnen-3, 20-dione = A 5 -pregnan-3, 20-dione; 16-dehydropregnenolone = A 5 , 16-Pregnadien-3(3-ol-20-one; corticosterone = A"-Pregnen11/3, 21-diol-3, 20-dione; hydrocortisone = A 4 -Pregnen-ll/3, 17a, 21-triol-3, 20-dione; cortisone = A "-Pregnen17a, 21-diol-3, 11, 20-trione; deoxycorticosterone = a "-Pregnen-21-ol-3, 20-dione; 11-dehydro-corticosterone = A "-Pregnen-21-ol-3, 11, 20-trione, aldosterone = A "-Pregnen-18, 21-diol-ll/3, 18-epoxy-3, 20-dione; testosterone = A"-androsten-170-ol-3-one, androstandione = 5a-androstan-3,17-dione, androstendione- A "-androsten-3, 17dione; estrone = A l,3,5, (10)-Estratrien-3-ol-17-one; 17(3-estradiol = A l,3,5, (10)-Estratrien-3, 170-diol; estriol = A l,3,5 (10)-Estratrien-3, 16a, 170-triol.
few hens were experiencing a preovulatory peak of progesterone at t h e t i m e b l o o d was collected. When serum progesterone was d e t e r m i n e d in relation t o t i m e of oviposition (Table 3), t h e r e were n o significant differences b e t w e e n hens
laying HS eggs and hens laying a high incidence of SL eggs at 0, 6, 12, 18, or 25 hr postoviposition. Serum progesterone of control hens was significantly greater at t i m e of oviposition
TABLE 2. Average serum1 progesterone from hens laying a low or high incidence of shell-less eggs Control
Experiment 1 Experiment 2
Shell-less
(ng/ml)
(Number samples)
(ng/ml)
1.88*+.26 2.54» + .16
12 96
1.64* ±.48 2.28a+.13
(Number samples) 12 61
Means +. SE followed by different superscripts in the same row differed significantly (P<.05). ' Blood was obtained from hens in Experiment 1 at 1400 hr and from hens in Experiment 2 at various intervals in relation to oviposition.
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C 27
920
ROLAND, SR..ETAL.
TABLE 3. Serum progesterone in relation to time of oviposition from hens laying a low or high incidence of shell-less eggs when oviposition was directly followed by ovulation Time postoviposition
Serum progesterone Control
Shell-less
(hr)
• (ng/ml) a
4.52 ± ,44a (13) 2.29 a ± .28t> (14) 3.37a + .54ab (4) 1.88a + 43b (2) 3.23a ± .38ab, 2 (14)
0 6 12 18 25
3.05a ± 3.3ia± 2.42a + 2.2ia ± 3.12a ±
.57a .59a 1.07a .29a .52a
(5) (3) (2) (7) (7)
t h a n at 6 and 18 hr postoviposition (Table 3). T h e r e were n o significant differences in serum progesterone in relation t o t i m e of oviposition in hens laying a high incidence of SL eggs. In general, serum progesterone levels increase 8 t o 11 h r before ovulation and p e a k a t 4 t o 7 h r before ovulation with levels remaining high until oviposition ( F u r r et al., 1973; Shahabi et al., 1 9 7 5 ) . Progesterone levels in these studies were highest in t h e control hens at t i m e of oviposition, which would be a p p r o x i m a t e l y 30 min before ovulation (Warren and S c o t t , 1 9 3 5 ) . T h e q u a n t i t y of serum progesterone ( 4 . 5 2 ng/ml) at oviposition in control hens when oviposition was directly followed b y ovulation was similar ( 4 . 6 0 n g / m l ) t o that reported b y Furr et al. ( 1 9 7 3 ) at 3 t o 0 hr prior t o ovulation. These studies were designed t o d e t e r m i n e if a b n o r m a l progesterone levels cause p r e m a t u r e oviposition resulting in t h e p r o d u c t i o n of SL eggs. Therefore, s e r u m progesterone was d e t e r m i n e d in relation t o oviposition instead of ovulation.
If a b n o r m a l levels of serum progesterone were causing SL eggs, it was believed t h a t t h e a b n o r m a l levels would occur within approximately 10 h r after ovulation. A b n o r m a l serum progesterone occurring later t h a n Ca. 10 hr after ovulation w o u l d allow t h e egg t o remain in t h e u t e r u s long e n o u g h t o deposit a thin shell. T h u s , an SL egg would n o t be laid. T h e lack of a preovulatory surge of serum progest e r o n e observed at 18 and 2 5 h r after t i m e of lay suggests t h a t m o s t of t h e hens did n o t ovulate a third egg. This was expected d u e t o t h e p o o r rate of lay of t h e old hens and t h e added stress of repeated heart p u n c t u r e . Essentially all steroid h o r m o n e s , b o t h 'the adrenal and sex h o r m o n e s , are at least partially conjugated in t h e liver and secreted i n t o t h e bile. When t h e liver is damaged, these h o r m o n e s frequently accumulate in extensive a m o u n t s in t h e b o d y fluids, causing s y m p t o m s of h o r m o n a l imbalance ( G u y t o n , 1966). Because serum g l u t a m a t e pyruvate transaminase (GPT) is c o m m o n l y used t o test for liver tissue
TABLE 4. Glutamate pyruvate transaminase (GPT), alkaline phosphatase, phosphorus, and magnesium concentrations in serum of hens laying a low or high incidence of shell-less eggs Shell-less
Control GPT (unit) Alkaline phosphatase (IU) Phosphorus (mg%) Magnesium (mg%)
7.47a 186 a 4.4ia 2.49a
+ .43 ±41.0 ± .23 ± .12
5.88a ± .86 174a ±17.0 4.90a± .18 2.7ia ± .06
Means ± SE followed by different letters in the same row are significantly different (P<.05).
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ab ' Means ± SE followed by different letters in the same row are significantly different (P<.05). Means ± SE followed by different letters in the same column are significantly different (P<.05). 1 Number in parentheses represent number of samples. 2 The average interval between oviposition in the control hens was 27.1 hr. It varied from 24 to 30 hr. The average interval between oviposition of the hens laying SL eggs was 26.3 hr. It varied from 20 to 29 hr.
RESEARCH NOTE
REFERENCE Coleman, C. M., 1966. The synthesis of thymolphthalein monophosphate, a new substrate for alkaline phosphatase. Clin. Chim. Acta 13:401-403. Etches, R. J., and F. J. Cunningham, 1976. The interrelationship between progesterone and luteinizing hormone during the ovulation cycle of the hen (Gallus domesticus). J. Endocrinol. 71:51 — 58. 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. Gindler, E. M., and D. A. Heth, 1971. Colorimetric determination with bound "Calmagite" of magnesium in human blood serum. Clin. Chim. Acta. 17:662. Guyton, A. C , 1966. Textbook of Medical Physiology. 3rd ed. W. B. Saunders Co., Philadelphia and London. Hargitt, C. W., 1899. Some interesting egg monstrosities. Zool. Bull. 2:225-229. Hewitt, E. A., 1939. The physiology of the reproductive system of the fowl. J. Am. Vet. Med. Assoc. 95:201-210. Holder, D. P., and D. M. Huntley, 1978. Influence of manganese, magnesium, zinc, and calcium level on eggshell quality. Poultry Sci. 5:1629— 1634. Japp, R. G., and F. V. Muir, 1968. Erratic oviposition and egg defects in broiler-type pullets. Poultry
Sci. 47:417-423. Kramer, C. Y., 1956. Extension of multiple range tests to group means with unequal number of replications. Biometrics 12:307-310. Moog, F., and H. S. Glazier, 1977. Phosphate absorption and alkaline phosphatase activity in the small intestine of the adult mouse and of the chick embryo and hatched chick. Comp. Biochem. Physiol. 42:321-326. Neher, B. H., and R. M. Fraps, 1950. The addition of eggs to the hen's clutch by repeated injections of ovulation inducing hormone. Endocrinology 46:482-488. Nestor, K. E., and O. W. Bacon, 1972. Production of defective eggs by egg and meat type turkey hens. Poultry Sci. 51:1361-1365. Reddy, P.R.K., and P. B. Siegel, 1976. Selection of body weight at eight weeks of age. II. Ovulation and oviposition patterns. Poultry Sci. 55:1518— 1530. Reitman, S., and S. Frankel, 1957. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminase. Am. J. Clin. Pathol. 2 8 : 5 6 - 63. Rodbard, D., and J. E. Lewald, 1970. Complete Analysis of Radioligand Assay and Radioimmunoassay Data. Acta Endocrinol. Suppl. 147:79— 103. Roland, D. A., Sr., 1977. The extent of uncollected eggs due to inadequate shell. Poultry Sci. 56: 1517-1521. Roland, D. A., Sr., D. J. Holcombe, and R. H. Harms, 1977. Further studies with hens producing a high incidence of non-calcified or partially calcified egg shells. Poultry Sci. 56:1232-1236. Roland, D. A., Sr., D. R. Sloan, and R. H. Harms, 1975. Influence of hormonal extracts on hens producing eggs with non-calcified or partially calcified shells and factors associated with this condition. Br. Poultry Sci. 16:423-429. Schiavo, J. J., R. L. Matuszczak, E. B. Oltenacu, and R. H. Foote, 1975. Milk progesterone in post-partum and pregnant cows as a monitor of reproductive status. J. Dairy Sci. 58: 1713 — 1716. Shahabi, N. A., H. W. Norton, and A. V. Nalbandov, 1975. Steroid levels in follicles and the plasma of hens during the ovulating cycle. Endocrinology 96:962-968. Snapir, N., and M. Perek, 1970. Distribution of calcium, carbonic anhydrase and alkaline phosphatase activities in the uterus and isthmus of young and old White Leghorn hens. Poultry Sci. 49:1526-1531. Tietz, N. W., 1976. Fundamentals of Clinical Chemistry. W. B. Saunders Co., Philadelphia and London. van Middelkoop, J. H. 1978. Types of eggs produced in White Plymouth Rock hens. World's Poultry Sci. J. 34:69-80. Warren, D. C , and H. M. Scott, 1935. The time factor in egg formation. Poultry Sci. 14: 195— 207. Wilson, S. C , and P. J. Sharp, 1976. The affects of progesterone on oviposition and ovulation in the domestic fowl (Gallus domesticus). Br. Poultry Sci. 17:163-173.
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damage (Tietz, 1 9 7 6 ) , and because t h e r e was a possibility t h a t a b n o r m a l serum progest e r o n e levels could b e causing hens to lay SL eggs, GPT values were d e t e r m i n e d . T h e results indicated no significant difference in GPT b e t w e e n hens laying a low or high incidence of SL eggs. Alkaline phosphatase is considered to participate in t h e physiological m e c h a n i s m of shell calcification (Snapir and Perek, 1 9 7 0 ) . However, t h e i m p o r t a n c e or exact m e c h a n i s m of action is u n k n o w n . In these studies t h e r e was n o significant difference in serum alkaline p h o s p h a t a s e b e t w e e n hens laying a high or low incidence of SL eggs. T h e r e was also n o significant difference in serum p h o s p h o r u s and magnesium b e t w e e n hens laying a low or high incidence of SL eggs. Because n o significant difference in serum progesterone was observed b e t w e e n c o n t r o l hens and hens laying SL eggs, it appears unlikely t h a t a b n o r m a l progesterone levels cause SL eggs. These d a t a also suggest t h a t t h e p r o d u c t i o n of SL eggs is n o t related t o a b n o r m a l serum alkaline p h o s p h a t a s e , g l u t a m a t e pyruvate transaminase, magnesium, or p h o s p h o r u s levels.
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Wolford, J. H. and K. Tanaka, 1970. Factors influencing egg shell quality. World's Poultry Sci. J. 26:763-780.
Woods, J. T., M. G. Mellon, 1941. The molybdenurn blue reaction: A spectrophotometric study. Ind. Eng. Chem. 13:760-764.
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Station
P. A. TEER Department of Pathology and Parasitology, School of Veterinary Medicine F. J.HOERR
(Received for publication June 11, 1981) ABSTRACT Three experiments were conducted to determine possible differences in serum progesterone, enzymes, and electrolytes between hens laying a high or low incidence of shell-less (SL) eggs. Blood was taken at time of oviposition and at 6, 12, 18, and 25 hr later, irrespective of the stage of the sequence. The results indicated no significant differences in average serum progesterone between hens laying a high or low incidence of SL eggs. In general, the patterns of serum progesterone in relation to time of oviposition was similar for each type. No significant differences were observed in serum phosphorus, magnesium, glutamate pyruvate transaminase, or alkaline phosphatase between hens laying a high or low incidence of SL eggs. It was concluded that the production of SL eggs is not related to abnormal serum progesterone, alkaline phosphatase, glutamate pyruvate transaminase, magnesium, or phosphorus levels. (Key words: shell-less eggs, progesterone, enzymes, electrolytes) 1983 Poultry Science 6 2 : 9 1 7 - 9 2 2 INTRODUCTION T h e p r o d u c t i o n of shell-less (SL) eggs appears t o be t h e single most costly shell quality p r o b l e m of t h e commercial egg producer. R e c e n t studies revealed t h a t for every 100 eggs collected, a p p r o x i m a t e l y 7 are n o t collected or recorded as eggs p r o d u c e d ( R o l a n d , 1 9 7 7 ) . A high incidence of SL or a b n o r m a l eggs has also been r e p o r t e d t o occur in m e a t - t y p e hens and is referred t o as t h e erratic oviposition and defective egg s y n d r o m e ( J a p p and Muir, 1 9 6 8 ; R e d d y and Siegel, 1 9 7 6 ; van M i d d e l k o o p , 1 9 7 8 ) . A l t h o u g h t h e p r o d u c t i o n of SL eggs in commercial layers has been k n o w n since 1 8 9 9 (Hargitt), very little is k n o w n a b o u t t h e cause of SL eggs. Hewitt ( 1 9 3 9 ) and Nestor and Bacon ( 1 9 7 2 ) suggested t h a t t h e cause of SL eggs m a y be d u e to violent peristaltic muscle contractions in t h e u t e r u s causing p r e m a t u r e expulsion.
1 2
Department of Poultry Science. Department of Animal and Poultry Science.
Roland et al. ( 1 9 7 5 , 1977) concluded t h a t hens laying SL eggs were able t o absorb calcium from t h e digestive tract, because b l o o d , b o n e , and u t e r u s calcium levels were n o r m a l or above normal. T h e y suggested t h a t SL eggs were caused b y altered calcium t r a n s p o r t or metabolism at t h e actual site of egg shell formation or b y p r e m a t u r e expulsion (oviposition). It has been shown t h a t progesterone can alter t i m e of oviposition and ovulation (Neher and Fraps, 1 9 5 0 ; Etches and Cunningham, 1 9 7 6 ; Wilson and Sharp, 1 9 7 6 ) . Because p r e m a t u r e oviposition has been suggested as a possible cause of SL eggs, t h e following studies were c o n d u c t e d t o d e t e r m i n e if a b n o r m a l serum progesterone levels occurred prior t o oviposition in hens laying SL eggs, S e r u m alkaline phosphatase (Moog and Glazier, 1 9 7 7 ) , serum p h o s p h o r u s (Wolford and Tanaka, 1 9 7 0 ) , serum magnesium (Holder and H u n t l e y , 1 9 7 8 ) , and serum g l u t a m a t e transaminase (Tietz, 1976) have b e e n associated with either shell calcification or steroid h o r m o n e imbalance. Therefore, serum c o n c e n t r a t i o n s of these
917
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Alabama Department of Agriculture and Industries, C. S. Roberts Veterinary Diagnostic Laboratory, Auburn University, Alabama 36849
918 enzymes mined.
ROLAND, SR..ET AL. and
electrolytes
were also deter-
MATERIALS AND METHODS
Serum phosphorus (Woods and Mellon, 1941), magnesium (Gindler and Heth, 1971), alkaline phosphatase (Coleman, 1966), and serum glutamate pyruvate transaminase (Reitman and Frankel, 1957) were also measured. All data were subjected to an analysis of variance, and significant differences were identified using Kramer's (1956) modification of Duncan's new multiple range test. RESULTS AND DISCUSSION No significant differences in average serum progesterone were found between hens laying either a high or low incidence of SL eggs (Table 2). In Experiment 1, hens laying after 1400 hr could have been experiencing preovulatory peaks of progesterone at the time of blood collection. However, mean progesterone values were less than those in Experiment 2, and the variation was consistent with that of the other experiment, suggesting that very
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Leghorn hens used in all experiments were at least 10 months in lay. Blood samples obtained by heart puncture were centrifuged and the serum immediately frozen until analyzed. The hens were individually caged and fed a commercial-type corn-soy diet containing 2830 ME kcal/kg, 16% protein, 3.5% calcium, and .75% phosphorus. A daily photoperiod of 14 hr was used with lights on from 0600 to 2000 hr. Experiment 1. Hens laying either a high (62.5%) or low (0%) incidence of SL eggs during the previous 10 days were used in groups of 12 hens each. The hens laying a high incidence of SL eggs were laying 1.7% normal (HS) eggs for a total production of 64.2%, whereas the control hens had 55% production (HS eggs). Blood was collected at 1400 hr and analyzed for serum progesterone. Experiment 2, Blood taken from hens at time of oviposition of SL and HS eggs and 6, 12, 18, and 25 hr later was analyzed for serum progesterone. The values were reported in relation to oviposition when oviposition was directly followed by ovulation. The number of samples obtained at each time period varied (Table 3). Blood was taken within 5 min of the specified time. Experiment 3. Fifteen hens laying an average of 45.3% SL eggs and 23.3% HS eggs for a total production of 68.6% and 12 hens laying an average of 78.3% HS eggs and no SL eggs during the previous 10 days were used. Blood was obtained at 1400 hr and serum phosphorus, magnesium, alkaline phosphatase, and glutamate pyruvate transaminase were measured. Progesterone Assay. Progesterone antiserum was prepared by immunizing sheep with 4Pregnen-6-j3 -ol -3, 20-dione hemisuccinate BSA (Steraloids, Wilton, NH). Radiolabeled progesterone (2, 6, 7- 3 H-progesterone; New England Nuclear) was purified by thin layer chromatography (chloroform: acetone 95:5) prior to use. Progesterone used for standards and steroids (Sigma Chemical Co.) used in specificity studies were not further purified. Approximately 5000 dpm of ^ - p r o g e s terone were mixed with 1 ml of serum prior to extraction with 9 ml of diethyl ether. The organic fraction was dried, resuspended in 1 ml
of 70% methanol, and frozen to remove neutral lipids (Schiavo et al., 1975). After 12 to 18 hr at —9 C, the lipid phase was removed, dried, and resuspended in 350 [i\ of gel phosphate buffered saline (.1% gelatin, pH 7.6, .01 M PO4, I = .15). Recovery estimates were made by counting 100 /il of the resuspended extract. The mean recovery for the extraction procedure was 82% and the serum estimates were corrected for recovery. Duplicate 100 /il volumes of the same extract in gel-PBS were also taken for assay and placed in 10 x 75 mm culture tubes. Each assay tube then received 400 (A of gel-PBS, 100 ;ul of a 1:3000 dilution of antiserum, and 100 (jl of 3 H-progesterone solution (approximately 50,000 dpm per tube). The assay tubes were incubated for 1 hr at 37 C, followed by 1 hr at 4 C. Dextran-coated charcoal (200 jul of .031% dextran, 31% Norit A in pH 7.6 PBS) was added to each tube to separate bound and free antigen. After centrifugation at 2000 X g for 15 min, a 500/ul aliquot of thesupernate was removed and counted. The results of each assay were determined from a plot of logit Y vs. Log X where Y and X were B/Bo and mass of standard progesterone, respectively (Rodbard and Lewald, 1970). The sensitivity of the assay was 60 pg per tube and the working range of the standard curve was from 125 pg to 4 ng. The specificity of the assay for 21 steroids was evaluated (Table 1). The inter- and intraassay coefficients of variation were 11.7 and 9.2%, respectively.
RESEARCH NOTE
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TABLE 1. Crossreactions of 21 steroids in the radioimmunoassay for progesterone using antiserum Steroids
#1555-3
% Crossreaetion
Cholesterol
0
C 21
Progesterone Pregnenolone 11 -alpha-Hydroxyprogesterone 11-keto-Progesterone 17-alpha-Hydroxyprogesterone Pregnendiol Pregnan-3,20-dione 16-Dehydropregnenolone Corticosterone Hydrocortisone Cortisone Deoxycorticosterone 11-Dehydro-corticosterone Aldosterone
CI9
Testosterone Androstandione Androstendione
<.01 <.01 <.01
C 18
Estrone 17-beta-estradiol Estriol
<.01 <.01 <.01
100.00 48.21 5.30 25.00 5.50 1.05 <.01 <.01 <.01 <.01 <.01 <.01 <.01 <.01
The following chemical names correspond to the above trivial names of steroids tested: cholesterol = ^ 5 cholesten-3-ol; progesterone = &4-Pregnen-3, 20-dione; pregnenolone = AS-Pregnen-3(?-ol-20-one; 11-a-hydroxyprogesterone = & 4 Pregnen-lla-ol-3, 20-dione; 11-ketoprogesterone = A4-Pregnen-3, 11, 20-dione; 17-a-hydroxyprogesterone = A 4 -Pregnen-17a-ol-3, 20-dione; pregnendiol = & 5-Pregnen-3|3, 20(3-diol; pregnen-3, 20-dione = A 5 -pregnan-3, 20-dione; 16-dehydropregnenolone = A 5 , 16-Pregnadien-3(3-ol-20-one; corticosterone = A"-Pregnen11/3, 21-diol-3, 20-dione; hydrocortisone = A 4 -Pregnen-ll/3, 17a, 21-triol-3, 20-dione; cortisone = A "-Pregnen17a, 21-diol-3, 11, 20-trione; deoxycorticosterone = a "-Pregnen-21-ol-3, 20-dione; 11-dehydro-corticosterone = A "-Pregnen-21-ol-3, 11, 20-trione, aldosterone = A "-Pregnen-18, 21-diol-ll/3, 18-epoxy-3, 20-dione; testosterone = A"-androsten-170-ol-3-one, androstandione = 5a-androstan-3,17-dione, androstendione- A "-androsten-3, 17dione; estrone = A l,3,5, (10)-Estratrien-3-ol-17-one; 17(3-estradiol = A l,3,5, (10)-Estratrien-3, 170-diol; estriol = A l,3,5 (10)-Estratrien-3, 16a, 170-triol.
few hens were experiencing a preovulatory peak of progesterone at t h e t i m e b l o o d was collected. When serum progesterone was d e t e r m i n e d in relation t o t i m e of oviposition (Table 3), t h e r e were n o significant differences b e t w e e n hens
laying HS eggs and hens laying a high incidence of SL eggs at 0, 6, 12, 18, or 25 hr postoviposition. Serum progesterone of control hens was significantly greater at t i m e of oviposition
TABLE 2. Average serum1 progesterone from hens laying a low or high incidence of shell-less eggs Control
Experiment 1 Experiment 2
Shell-less
(ng/ml)
(Number samples)
(ng/ml)
1.88*+.26 2.54» + .16
12 96
1.64* ±.48 2.28a+.13
(Number samples) 12 61
Means +. SE followed by different superscripts in the same row differed significantly (P<.05). ' Blood was obtained from hens in Experiment 1 at 1400 hr and from hens in Experiment 2 at various intervals in relation to oviposition.
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TABLE 3. Serum progesterone in relation to time of oviposition from hens laying a low or high incidence of shell-less eggs when oviposition was directly followed by ovulation Time postoviposition
Serum progesterone Control
Shell-less
(hr)
• (ng/ml) a
4.52 ± ,44a (13) 2.29 a ± .28t> (14) 3.37a + .54ab (4) 1.88a + 43b (2) 3.23a ± .38ab, 2 (14)
0 6 12 18 25
3.05a ± 3.3ia± 2.42a + 2.2ia ± 3.12a ±
.57a .59a 1.07a .29a .52a
(5) (3) (2) (7) (7)
t h a n at 6 and 18 hr postoviposition (Table 3). T h e r e were n o significant differences in serum progesterone in relation t o t i m e of oviposition in hens laying a high incidence of SL eggs. In general, serum progesterone levels increase 8 t o 11 h r before ovulation and p e a k a t 4 t o 7 h r before ovulation with levels remaining high until oviposition ( F u r r et al., 1973; Shahabi et al., 1 9 7 5 ) . Progesterone levels in these studies were highest in t h e control hens at t i m e of oviposition, which would be a p p r o x i m a t e l y 30 min before ovulation (Warren and S c o t t , 1 9 3 5 ) . T h e q u a n t i t y of serum progesterone ( 4 . 5 2 ng/ml) at oviposition in control hens when oviposition was directly followed b y ovulation was similar ( 4 . 6 0 n g / m l ) t o that reported b y Furr et al. ( 1 9 7 3 ) at 3 t o 0 hr prior t o ovulation. These studies were designed t o d e t e r m i n e if a b n o r m a l progesterone levels cause p r e m a t u r e oviposition resulting in t h e p r o d u c t i o n of SL eggs. Therefore, s e r u m progesterone was d e t e r m i n e d in relation t o oviposition instead of ovulation.
If a b n o r m a l levels of serum progesterone were causing SL eggs, it was believed t h a t t h e a b n o r m a l levels would occur within approximately 10 h r after ovulation. A b n o r m a l serum progesterone occurring later t h a n Ca. 10 hr after ovulation w o u l d allow t h e egg t o remain in t h e u t e r u s long e n o u g h t o deposit a thin shell. T h u s , an SL egg would n o t be laid. T h e lack of a preovulatory surge of serum progest e r o n e observed at 18 and 2 5 h r after t i m e of lay suggests t h a t m o s t of t h e hens did n o t ovulate a third egg. This was expected d u e t o t h e p o o r rate of lay of t h e old hens and t h e added stress of repeated heart p u n c t u r e . Essentially all steroid h o r m o n e s , b o t h 'the adrenal and sex h o r m o n e s , are at least partially conjugated in t h e liver and secreted i n t o t h e bile. When t h e liver is damaged, these h o r m o n e s frequently accumulate in extensive a m o u n t s in t h e b o d y fluids, causing s y m p t o m s of h o r m o n a l imbalance ( G u y t o n , 1966). Because serum g l u t a m a t e pyruvate transaminase (GPT) is c o m m o n l y used t o test for liver tissue
TABLE 4. Glutamate pyruvate transaminase (GPT), alkaline phosphatase, phosphorus, and magnesium concentrations in serum of hens laying a low or high incidence of shell-less eggs Shell-less
Control GPT (unit) Alkaline phosphatase (IU) Phosphorus (mg%) Magnesium (mg%)
7.47a 186 a 4.4ia 2.49a
+ .43 ±41.0 ± .23 ± .12
5.88a ± .86 174a ±17.0 4.90a± .18 2.7ia ± .06
Means ± SE followed by different letters in the same row are significantly different (P<.05).
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ab ' Means ± SE followed by different letters in the same row are significantly different (P<.05). Means ± SE followed by different letters in the same column are significantly different (P<.05). 1 Number in parentheses represent number of samples. 2 The average interval between oviposition in the control hens was 27.1 hr. It varied from 24 to 30 hr. The average interval between oviposition of the hens laying SL eggs was 26.3 hr. It varied from 20 to 29 hr.
RESEARCH NOTE
REFERENCE Coleman, C. M., 1966. The synthesis of thymolphthalein monophosphate, a new substrate for alkaline phosphatase. Clin. Chim. Acta 13:401-403. Etches, R. J., and F. J. Cunningham, 1976. The interrelationship between progesterone and luteinizing hormone during the ovulation cycle of the hen (Gallus domesticus). J. Endocrinol. 71:51 — 58. 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. Gindler, E. M., and D. A. Heth, 1971. Colorimetric determination with bound "Calmagite" of magnesium in human blood serum. Clin. Chim. Acta. 17:662. Guyton, A. C , 1966. Textbook of Medical Physiology. 3rd ed. W. B. Saunders Co., Philadelphia and London. Hargitt, C. W., 1899. Some interesting egg monstrosities. Zool. Bull. 2:225-229. Hewitt, E. A., 1939. The physiology of the reproductive system of the fowl. J. Am. Vet. Med. Assoc. 95:201-210. Holder, D. P., and D. M. Huntley, 1978. Influence of manganese, magnesium, zinc, and calcium level on eggshell quality. Poultry Sci. 5:1629— 1634. Japp, R. G., and F. V. Muir, 1968. Erratic oviposition and egg defects in broiler-type pullets. Poultry
Sci. 47:417-423. Kramer, C. Y., 1956. Extension of multiple range tests to group means with unequal number of replications. Biometrics 12:307-310. Moog, F., and H. S. Glazier, 1977. Phosphate absorption and alkaline phosphatase activity in the small intestine of the adult mouse and of the chick embryo and hatched chick. Comp. Biochem. Physiol. 42:321-326. Neher, B. H., and R. M. Fraps, 1950. The addition of eggs to the hen's clutch by repeated injections of ovulation inducing hormone. Endocrinology 46:482-488. Nestor, K. E., and O. W. Bacon, 1972. Production of defective eggs by egg and meat type turkey hens. Poultry Sci. 51:1361-1365. Reddy, P.R.K., and P. B. Siegel, 1976. Selection of body weight at eight weeks of age. II. Ovulation and oviposition patterns. Poultry Sci. 55:1518— 1530. Reitman, S., and S. Frankel, 1957. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminase. Am. J. Clin. Pathol. 2 8 : 5 6 - 63. Rodbard, D., and J. E. Lewald, 1970. Complete Analysis of Radioligand Assay and Radioimmunoassay Data. Acta Endocrinol. Suppl. 147:79— 103. Roland, D. A., Sr., 1977. The extent of uncollected eggs due to inadequate shell. Poultry Sci. 56: 1517-1521. Roland, D. A., Sr., D. J. Holcombe, and R. H. Harms, 1977. Further studies with hens producing a high incidence of non-calcified or partially calcified egg shells. Poultry Sci. 56:1232-1236. Roland, D. A., Sr., D. R. Sloan, and R. H. Harms, 1975. Influence of hormonal extracts on hens producing eggs with non-calcified or partially calcified shells and factors associated with this condition. Br. Poultry Sci. 16:423-429. Schiavo, J. J., R. L. Matuszczak, E. B. Oltenacu, and R. H. Foote, 1975. Milk progesterone in post-partum and pregnant cows as a monitor of reproductive status. J. Dairy Sci. 58: 1713 — 1716. Shahabi, N. A., H. W. Norton, and A. V. Nalbandov, 1975. Steroid levels in follicles and the plasma of hens during the ovulating cycle. Endocrinology 96:962-968. Snapir, N., and M. Perek, 1970. Distribution of calcium, carbonic anhydrase and alkaline phosphatase activities in the uterus and isthmus of young and old White Leghorn hens. Poultry Sci. 49:1526-1531. Tietz, N. W., 1976. Fundamentals of Clinical Chemistry. W. B. Saunders Co., Philadelphia and London. van Middelkoop, J. H. 1978. Types of eggs produced in White Plymouth Rock hens. World's Poultry Sci. J. 34:69-80. Warren, D. C , and H. M. Scott, 1935. The time factor in egg formation. Poultry Sci. 14: 195— 207. Wilson, S. C , and P. J. Sharp, 1976. The affects of progesterone on oviposition and ovulation in the domestic fowl (Gallus domesticus). Br. Poultry Sci. 17:163-173.
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damage (Tietz, 1 9 7 6 ) , and because t h e r e was a possibility t h a t a b n o r m a l serum progest e r o n e levels could b e causing hens to lay SL eggs, GPT values were d e t e r m i n e d . T h e results indicated no significant difference in GPT b e t w e e n hens laying a low or high incidence of SL eggs. Alkaline phosphatase is considered to participate in t h e physiological m e c h a n i s m of shell calcification (Snapir and Perek, 1 9 7 0 ) . However, t h e i m p o r t a n c e or exact m e c h a n i s m of action is u n k n o w n . In these studies t h e r e was n o significant difference in serum alkaline p h o s p h a t a s e b e t w e e n hens laying a high or low incidence of SL eggs. T h e r e was also n o significant difference in serum p h o s p h o r u s and magnesium b e t w e e n hens laying a low or high incidence of SL eggs. Because n o significant difference in serum progesterone was observed b e t w e e n c o n t r o l hens and hens laying SL eggs, it appears unlikely t h a t a b n o r m a l progesterone levels cause SL eggs. These d a t a also suggest t h a t t h e p r o d u c t i o n of SL eggs is n o t related t o a b n o r m a l serum alkaline p h o s p h a t a s e , g l u t a m a t e pyruvate transaminase, magnesium, or p h o s p h o r u s levels.
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Wolford, J. H. and K. Tanaka, 1970. Factors influencing egg shell quality. World's Poultry Sci. J. 26:763-780.
Woods, J. T., M. G. Mellon, 1941. The molybdenurn blue reaction: A spectrophotometric study. Ind. Eng. Chem. 13:760-764.
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