NaCl-Acetate Turbidity Test

NaCl-Acetate Turbidity Test A SIMPLE METHOD FOR THE ESTIMATION OF EGG YOLK-LIKE PROTEINS IN THE PLASMA OF LAYING HENS AND OF ESTROGEN-TREATED ROOSTERS KAZUTAKA HOMMA AND KOZO TUCHIDA

Department of Animal Physiology, Faculty of Agriculture, Nagoya University, Anzyo, Japan (Received for publication May 1, 1959)

turbidity tests have been reSEVERAL ported for the clinical diagnosis of the liver dysfunction in man (MacLagan, 1944; Kunkel and Hoagland, 1947; Kunkel et al., 1948). These tests were based upon the reactions between the reagents and the plasma or serum of patients which contained disease specific proteins. By measuring the produced turbidity against a standard, the pathological state of the liver could be classified according to the turbidity units. In addition to the disease specific proteins, most of these reactions were reported to be greatly influenced by the plasma lipids, because the precipitates obtained by centrifugation contained considerable amounts of lipids not different from those usually found in normal plasma. The loss of turbidity by the delipidation of plasma or by the additions of surface active agents, such as Tween-80, also indicated that lipids were essential factors for these turbidity tests. In the laying hen and the estrogen treated-rooster, the appearance of yolklike proteins in the plasma have been shown by means of ultracentrifuge and electrophoresis (Urist el al., 1958; Brandt et al., 1951, 1952; McKinley et al., 1953). The plasma of these birds formed a milky suspension when diluted with water. The particles in this suspension contained proteins and lipids as in the precipitates of other turbidity tests. As it was proven by Hosoda et al. (1955) that the yolk-like proteins are synthesized in the liver, the turbidity of the diluted plasma would in-

dicate the activity of the liver concerning the synthesis of the immediate precursors of egg yolk. Consequently, the turbidity test in laying and estrogen-treated birds, though similar in the chemical properties of the precipitates, has a quite different physiological significance from other turbidity tests devised for the clinical diagnosis of human liver diseases. In a previous paper the first application of a turbidity test to the laying hen was reported (Homma and Tomita, 1959), where the plasma or sera were diluted with water at pH 7.2 (W-test), and the parallelism between the turbidity and serological reactions was investigated in fed and fasted states. In the present experiment, NaCl-acetate buffer (2.34 g. of sodium chloride, 5.48 g. of sodium acetate-3 aq., and 0.55 ml. of glacial acetic acid/1-liter of water. Adjust the pH to 5.4) was used as a diluent of the turbidity test in chicken serum or plasma. When the plasma of roosters and of severely molting hens were diluted with this solution, there was no appreciable turbidity, but the plasma of estrogentreated roosters and of laying hens formed a milky suspension (Homma et al., 1958). The object of the study reported here was to clarify the significance of the NaClacetate turbidity test (Ac-test). MATERIALS AND METHODS

Ten-week old roosters (Rhode Island Red X White Leghorn cross-bred) and 1year old hens (Rhode Island Red X White

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ESTIMATION OF PROTEIN IN PLASMA

Leghorn cross-bred and Nagoya breed) were used. The blood sample to be tested was drawn from the wing vein, and 40 jugof heparin sodium was added to 1 ml. of blood as an anticoagulant. Precipitates for the chemical analysis were usually prepared by centrifuging the milky suspension at 3,000 rpm. for 20 minutes. The nitrogen content was determined by the Kjeldahl-Nessler method (Hoffman and Osgood, 1940) and the phosphorus by the method of Fiske and Subbarow (1925). The Ac-test was performed as in the W-test except for minor alterations. The plasma was diluted 101 times with NaClacetate buffer, and after thorough mixing in a test tube it was immersed into a water bath and kept at 25°C. for several minutes. When room temperature was between 25° and 15°C, the last step could be omitted. The turbidity values were read on the extinction scale of a colorimeter at 655 m/*., in a cuvette of 0.5 cm. deep, using the diluent as the reference control. To avoid the use of figures less than unity, the values read on the scale were multiplied by 1,000. Estrogen preparation used in this study contained 27 mg. of diethylstilbestrol and 3 mg. of its derivative in 1 ml. of aqueous medium. Roosters were injected with 0.25 or 0.50 ml./kg. of this preparation at the base of the skull. Blood samples of these birds were taken 48 hours after the injection.

15 14 13 12

is ~

* Estrogenized rooster

oHen

9

5 8

100

200 Ac-Turbidity

300 400 E(655niu; 0.5cm)x10J

500

FIG. 1. N:P ratio of the precipitates.

to the highest Ac-turbidity found in normal laying hens. However, in an irregularly laying hen, further decrease in the N : P ratio was observed when she stopped laying for several days and showed a very high Ac-turbidity of nearly 300 (Figure 2, C). In this hen, the cessation of laying seemed to be caused by certain disorder in the process of albumen secretion or shell formation rather than of production IIIHMIIIWHHniHIIIHHMMIIIMIIHmHMIlH

RESULTS

/. N:P ratio of the precipitates: The ratio of nitrogen and phosphorus (N:P ratio) was used to estimate the approximate composition of the precipitates. In Figure 1, N : P ratio of the precipitate was plotted co-ordinate to the Ac-turbidity. It was found that an increase in Ac-turbidity caused a reduction in N : P ratio. The lowest N : P ratio in the laying hen was approximately 6, which corresponded

16.V.1958

1.VI.

1.VII.

16.VII.

FIG. 2. Ac-turbidity and egg laying. 0 = Normal egg; ©=Soft shelled egg.

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K . HOMMA AND K . TUCHIDA

of yolk precursors. The Ac-turbidity of the estrogen-treated roosters were distributed over a wide range of nearly zero to 520. In this group, the N : P ratio corresponding to the highest Ac-turbidity level became essentially the same as that of egg yolk being diluted 1,000 times with NaCl-acetate buffer. In brief, there was a tendency that with increasing Ac-turbidity, the chemical properties of precipitates from the plasma approached to those from egg yolk. When the N : P ratio of the precipitate from the estrogen-treated rooster was compared to that from the hen with the same Ac-turbidity, there was no apparent difference between the two. Z. Egg production and Ac-turbidity: The interrelations between Ac-turbidity and rate of laying was investigated in 10 laying hens (5 cross-bred and 5 Nagoya breed) from May 14 to July 16, 1958. One ml. of blood sample was drawn on alternate days at 3 p.m. prior to the afternoon feed. Besides the plasma Ac-turbidity, daily egg production and numbers of molted feathers were also recorded. Three typical patterns are illustrated in Figure 2. Each hen showed a relatively stable turbidity level during laying period. It is evident that the maintenance of optimal concentration of yolk precursors in the blood is the most important factor for good layers (Figure 2, A). Sudden decrease in the Ac-turbidity always followed by severe molting and cessation of laying (Figure 2, B), and marked fluctuations in the Ac-turbidity at a high level, also followed by cessation of laying (Figure 2, C). Gradual decrease in the Acturbidity was observed in moderately molting hens which continued to lay at a rate of one egg per 3 or 4 days. In Table 1, the mean Ac-turbidity (May to July) and the rate of egg laying (number of eggs laid/days from April to

TABLE 1.—Ac-turbidity and rate of laying

Bird No.

Breed of hen

Ac-turbidity /65S m/iA E 1X10 3 \ 0 . 5 cm./

Rate of laying

9 4 3 7 2 10 1 5

C N N C N C N N

155.5 ±13.4* 138.8+ 7.3 112.7 + 25.1 92.2+ 6.9 9 0 . 0 ± 7.3 82.8+ 4.7 65.9 + 10.4 60.2+ 6.4

67.4 63.3 59.0 46.6 23.8 31.9 20.5 38.5

(%)

* = 9 5 % fiducial limits; N=Nagoya breed; C = Cross bred.

October) of hens are summarized. Out of ten hens two were omitted from this table owing to accidental death during the experimental period. In the group of hens showing higher Ac-turbidity (Nos. 9, 4, 3 and 7), the rate of laying was proportional to the Ac-turbidity; therefore it appears that the higher the Ac-turbidity, the higher the rate of laying. However, in the hens of low laying rate (Nos. 2, 10, 1 and 5), this interrelation could not be observed. In the latter group, factors other than the plasma level of the yolk precursors might be the determinants of egg laying. DISCUSSION

The increase in calcium, phosphorus and lipids, and the appearance of new serum phosphoprotein in laying birds have been reviewed by Romanoff and Romanoff (1949) and Sturkie (1954). All these changes are commonly observed in oviparous animals during breeding season, and hence could be attributed to the fact that the precursors of egg yolk must be transported from the liver to the ovary via the blood stream. Though the precise mechanism of yolk deposition in the ovary is not yet clear, it seems reasonable to postulate that the high concentration of yolk precursors in the circulating blood is a necessary factor for the yolk deposition

ESTIMATION OF PROTEIN IN PLASMA

in the ovary. Since the degree of Ac-turbidity could be regarded as an indicator for the approximate concentration of immediate yolk precursors in the plasma, the parallelism between Ac-turbidity and rate of laying, shown in the upper half of Table 1, is not surprising. However, extraordinarily high Ac-turbidity does not mean extraordinarily high egg laying rate of the hen (Figure 2, C). A relatively stable Ac-turbidity level, ranging from 60 to 200, may suggest that the equilibrium between precursors in the plasma and in the ovary is directed to yolk deposition. Low Ac-turbidities less than 60 seem to indicate shortage of available precursors unable to perform yolk deposition in the ovary. The decrease in the N : P ratio with increasing Ac-turbidity may be explained by our recent findings on the fractionated serum that a phospholipoprotein of high density plays a major role in the Ac-test. Other lipoproteins of low density, which are rich in phospholipids and are soluble in the NaCl-acetate buffer, tend to compose insoluble complexes with the dense protein when these two kinds of proteins are mixed together. This coprecipitation may occur in the Ac-test using whole serum or plasma, and hence the large part of phosphorus in the precipitates from the Ac-test can be attributed to the phospholipids contained in the low-density lipoproteins. The rather fixed N : P ratio in the highly estrogenized rooster, as shown in Figure 1, might be the result of the higher concentration of low-density lipoproteins in the plasma exceeding the coprecipitable amounts. SUMMARY

The Ac-turbidity test was devised as an improved method of the W-test reported in the previous paper. The N : P ratio (wt:wt) of the precipitate was the

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same at a certain turbidity level, regardless of the source of the plasma either taken from laying hens or from estrogentreated roosters. The rate of egg laying was found to be proportional to the Acturbidity in good layers, though in bad layers the rate of laying appears to be restricted by factors other than the concentration of yolk precursors in the plasma. Severe molting usually accompanied a sudden drop in the Ac-turbidity. The good layers showed a relatively stable Acturbidity level of about 150 during the laying period. REFERENCES Brandt, L. W., R. E. Clegg and A. C. Andrews, 1951. The effect of age and degree of maturity on the serum proteins of the chicken. J. Biol. Chem. 191: 105-111. Brandt, L. W., H. D. Smith, A. C. Andrews and R. E. Clegg, 1952. Electrophoretic investigation of the serum proteins of certain birds and their hybrids. Arch. Biochem. Biophysics, 36: 11-17. Fiske, C. H., and Y. Subbarow, 1925. The colorimetric determination of phosphorus. J. Biol. Chem. 66: 375-400. Hoffman, W. S., and B. Osgood, 1940. The photoelectric microdetermination of nitrogenous constituents of blood and urine by direct nesslerization. J. Lab. Clin. Med. 25: 856-862. Homma, K., T. Tomita and T. Ohkawa, 1958. Significance of the plasma water turbidity test for egg production in fowls. IV. Effect of salt concentration on the turbidity test. V. Estrogen induced turbidity in young cocks. Jap. J. Animal Reprod. 3: 133-134 and 143-144. Homma, K., and T. Tomita, 1959. Serological and turbidimetric characteristics in the plasma of laying fowls. Poultry Sci. 38: 81-85. Hosoda, T., T . Kaneko, K. Mogi and T. Abe, 1955. Serological studies on egg production in the fowl. 1. On the locus of serum vitellin production. Poultry Sci. 34: 9-15. Zunkel, H. G., and C. L. Hoagland, 1947. Mechanism and significance of the thymol turbidity test for liver disease. J. Clin. Invest. 26: 10601071. Kunkel, H. G., E . H. Ahrens, Jr. and W. J. Eisenmenger, 1948. Application of turbidimetric method for estimation of gamma globulin and

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K. HOMMA AND K . TUCHIDA

total lipid to the study of patients with liver disease. Gastroenterology, 11: 499-507. KcKinley, W. P., W. F. Oliver, W. A. Maw and R. H. Common, 1953. Filter paper electrophoresis of serum proteins of the domestic fowl. Proc. Soc. Exp. Biol. Med. 84: 346-351. MacLagan, N. F., 1944. The turbidity test as an indicator of liver dysfunction. Brit. J. Exper. Path. 25: 234-241.

Romanoff, A. L., and A. J. Romanoff, 1949. The Avian Egg. John Wiley and Sons, Inc., New York. 236-246. Sturkie, P. D., 1954. Avian Physiology. Comstock Pub. Ass., Ithaca, New York. 24-40. Urist, M. R., O. A. Schjeide and F. C. McLean, 1958. The partition and binding of calcium of the laying hen and of the estrogenized rooster. Endocrinology, 63: 570-585.

Calculation of the Nutrient Requirements for Broilers* H. M. EDWARDS, J R . AND R. J. YOUNG

Poultry Department, University of Georgia, Athens, Georgia, and Procter and Gamble Company, Research Division, Cincinnati, Ohio (Received for publication May 4, 1959)

T

HE formulation of high energy diets which are capable of increasinglyimproved feed conversions makes it imperative that all nutrients be present at such a level that they do not limit the potential growth rate of the chick. The problem of expressing the nutrient requirements to ensure adequate levels in the diet is well recognized by nutritionists. However, expressing the nutrient requirements as a percent of the diet is very inadequate, particularly when comparing diets which contain different levels of energy and protein. The following procedure couples the level of dietary nutrients with the feed conversion potential of the diet. The procedure for calculating the nutrients required in broiler rations which differ in energy and protein content was recently discussed by the authors in a feed trade magazine (Edwards and Young, 1957). The authors have since collected additional data and propose that the method can be used to advantage in both

* Journal Paper No. 106 of the College Experiment Station, University of Georgia, College of Agriculture Experiment Stations.

purified and practical diet formulation. This method requires the determination of two factors: (1) the feed-to-gain ratio that can be expected from rations of various calorie and protein content when fed to chickens from day-old to four weeks, and (2) the milligrams of each nutrient required per gram of gain. The data of Donaldson et al. (1956) were used for deriving equations for predicting feed efficiency in rations which vary in energy and protein. From their data the following equations were obtained: Equation l — F/G = 3.497 -0.0290P-0.0012Cy Equation 2 — F/G = 3.497 - 0.0290P - 0.0008Cm Where F/G represents predicted Feedto-gain ratio. P represents percent protein {N X6.25). 1

Values obtained by the use of this equation when compared with values used in constructing the equation have a correlation of 0.949.