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Determination of Blood Constituents Reference Values in Broilers1
Determination of Blood Constituents Reference Values in Broilers1 ADELE MELUZZI,2 GIUSEPPE PRIMICERI, RAFFAELLA GIORDANI, and GUGLIELMO FABRIS3 Institute of Zooculture, Via San Giacomo 9, 40126 Bologna, Italy (Received for publication April 8, 1991) ABSTRACT Eight hundred broilers were examined to define the reference values of nine blood constituents (total proteins, albumin, total and free cholesterol, triglycerides, aspartate aminotransferase (AST), alkaline phosphatase (ALP), calcium, and phosphorus). The broilers, coming from 20 different commercial poultry operations, were equally divided into different groups according to age (21 and 45 days), strain (Arbor Acres and Hybro), sex, and sampling season (summer or winter). Age and sampling season were the variation sources that most influenced the values of the hematochemical variables examined. The interaction of age with strain influenced total proteins, total and free cholesterol, triglycerides, and ALP. The interaction of age with sampling season influenced all hematochemical constituents except triglycerides. Reference limits defined by .975 and .025 fractiles were computed for each blood constituent according to the significance of different variation sources and their combinations. Reference limits defined herein could be used as indicators of metabolic and health conditions of a poultry farm. (Key words: reference values, blood constituents, age, strain, broilers) 1992 Poultry Science 71:337-345
INTRODUCTION Hematochemistry constitutes an increasingly useful aid in zootechnical and veterinary research. It permits the study of specific pathological alterations of certain blood constituents and recognition, under strictly controlled experimental conditions, of the existence of metabolic alterations of different origin. Many factors can influence the level of a particular blood constituent: genetic type, feeding, microand macro-climate, rearing technique, age, physiological state, and sex, as well as pathological factors. Moreover, methods of sampling and obtaining the biological material and the method of analysis can also influence results.
'This study was supported by the Italian Ministry of Public Instruction. ^To whom correspondence should be addressed. 3 Present address: Industria Vaccini Zootecnici, Vigorea, Padova, Italy.
It would therefore be useful to define a range of values or limits to serve as reference for each blood constituent. The reference limits define an interval and the values within it are considered "normal" (International Federation of Clinical Chemistry, 1978). The reference values can be used to evaluate the state of health of either a single bird or an entire population and would constitute a basic requirement for an indispensable preliminary knowledge of the biological material chosen for scientific research. In avian species, hematochemical studies are nearly always performed for research purposes, in order to evaluate the effect of rearing technique (Meluzzi et al., 1984; Verga et ah, 1984; Cerolini et al., 1986; Giordani et al., 1986; Franchini et al., 1988b), feeding regimens (Franchini et al., 1988a, 1989), environmental conditions, and other parameters. Hematochemical tests are generally not utilized for avian diagnostics, because no reference values
337
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are available. Reference values should be based on a sufficiently large number of samples obtained under all conditions able to influence the level of the variable under study. To date, research on the definition of reference values has been carried out on laying hens of two different commercial hybrids (Cerolini et al., 1987), racing pigeons (Lumeij and Bruijne, 1985; Lumeij and Wolfswinkel, 1988), muscovy ducks (Pad et al, 1988), partridges (Woodward et al, 1983), and broilers (Ross et al, 1976, 1978). The present research, which attempts to extend and integrate the studies performed by Ross et al. (1976,1978), aims at establishing the reference values of certain blood constituents in floor-reared broilers of known strain, age, sex, and sampling season. Moreover, the authors propose to evaluate the influence of different sources of variation on the level of the hematic constituents investigated. MATERIALS AND METHODS A total of 800 broilers were used, randomly chosen from a population of approximately 66,000 birds. The chickens came from 20 farms situated in northern Italy and were reared on the floor at a density of 12 to 13 birds per square meter in open-sided houses similar in design and construction, and under similar environmental conditions. The lighting program was 23 h light:l h dark. The birds had received the same commercial feeds and were clinically healthy. Feed composition of the diets was: first period (1 to 28 days): 23% CP, 3,100 kcal/kg ME; second period (29 to 45 days): 21% CP, 3,100 kcal/kg ME. The production levels of the flocks were similar, corresponding to the standard for the strain. Following the a priori selection criteria (International Federation of Clinical Chemistry, 1984a; Giorgetti and Zappa, 1985), chickens of two commercial strains among the most common in Italy were chosen: Hybro and Arbor Acres. All birds, both male and female, underwent samp-
Boehringer Mannheim, Sanhofer-Strasse, 6800 Mannheim 31, Germany. ^bsystem OY, Pultittie 9, Helsinki, Finland.
ling at the age of 21 and 45 days during the summer (June and July) and winter (November and December) seasons. Each group with the same characteristics (strain, sex, age, and sampling season) was made up of 50 birds from five farms. The blood samples were drawn in the early morning from the wing vein of birds fasted since the previous evening. The blood was collected in heparinized test tubes and centrifuged at 1,800 x g for 15 min: the plasma was subdivided into several parts and preserved at -20 C until the time of analysis. The following variables were measured: total proteins by the biuret method (Weichselbaum, 1946); albumin by the green bromocresol method (Doumas and Biggs, 1972); total and free cholesterol by the cholesterol-oxidase para-aminophenazone (CHOD-PAP) colorimetric enzymatic method (Trinder, 1969); triglycerides by the glycerol-3-phosphate-oxidase paraaminophenazone (GPO-PAP) colorimetric enzymatic method (Trinder, 1969); calcium by the o-cresolphtalein method (Ray Sarckar and Chauhan, 1967); phosphorus by the molybdenum blue method (Peyenet and Pouillot, 1972); aspartate aminotransferase (AST) and alkaline phosphatase (ALP) by the optimized standard method of the German Society of Clinical Chemistry, at a temperature of 30 C (Bergmeyer et al., 1972). Analyses were performed using Biochemia Boehringer4 kits and a Labsystem FP 901 spectrophotometer,5 equipped with a FP 401 incubating unit. The data were analyzed using base SAS® software (SAS Institute, 1986). The normality of the distribution was verified with skewness and kurtosis tests, and the Shapiro and Wilk (1965) test. The values not presenting a Gaussian distribution (triglycerides, AST, ALP, and phosphorus) were normalized by log transformations. Parametric statistical methods were used to calculate the reference limits defined by the fractiles .025 and .975 (International Federation of Clinical Chemistry, 1984b). Subsequently, the raw or transformed data were subjected to ANOVA procedures (General Linear Model) according to the following model:
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BLOOD CONSTITUENTS REFERENCE VALUES
Yijkim = ^ + + + +
+ A£ + B; + C k + Di + (AB)ij (AC)* + (AD) U + (BCW (BD)a + (CD)^ + (ABC)ijk (ABDfy + (ACD)M (BCEOju + (ABCD)ijki + eijkhn,
where [i = population mean; Ai = effect of the sex; Bj = effect of the age (j = 1: 21 days; j = 2: 45 days); C^ = effect of the strain (k = 1: Arbor Acres; k = 2: Hybro); E>i = effect of the sampling season (1 = 1: summer; 1 = 2: winter); (AB)«, (AC)^ (AD)a,
(BO*,
(BDh,
(CD)M,
(ABC)^
(ABDty ( A C D ) ^ (BCD)^ (ABCD)ijld = interactions of the main effects; and Emm = overall error term. The data were then partitioned into subclasses according to the statistical significance established by ANOVA and the reference limits were redefined.
RESULTS AND DISCUSSION Table 1 shows the reference limits defined by the fractiles .025 and .975. The reference intervals were rather wide, particularly that of ALP. This could be due to the effects exerted by the variables chosen for the sampling, i.e., age, sex, strain, and sampling season. Total proteins varied according to age and increased in birds 45 days of age (Table 2). T his effect of age had already been observed in chickens and turkeys by several authors (Sturkie, 1976; Ross et al., 1978; Franchini et al, 1988a, 1990a,b). An increased protein concentration was also
noted in the hot season. In contrast, Deaton et at. (1969) observed a slight, but not significant, decrease of total protein with increasing temperature from 23.9 to 32.2 C. The interactions of age with strain and sampling season were statistically significant (P<.01). Albumin levels did not appear to be influenced by the different sources of variation, with the exception of the sampling season (Table 2). In contrast to the results with total proteins, an increase in albumin was noted during the winter. The interaction between age and season was statistically significant (P<.01). The Hybro chickens had values of total and free cholesterol and triglycerides consistently higher than the Arbor Acres chickens (Table 2). Furthermore, the levels of the three variables were greater in males compared with females, with statistically significant differences only in the case of free and total cholesterol (P<.01). With aging, free cholesterol decreased but triglycerides increased. This same trend was recently noted in meat turkeys (Franchini et al., 1990b,c). For total and free cholesterol, the interactions of age with strain and season were significant (P<.01); in the case of triglycerides, the interactions of strain with sex and age were significant (P<01). The AST values were higher in the older birds and in the birds sampled during the summer months (Table 2). Similar observations were made by Meluzzi et al. (1984) on laying hens, and
TABLE 1. Mathematical transformation in order to normalize the distribution of data, means, confidence intervals of means, and reference limits defined by .025 and .975 fractiles Variable
Units
Samples Mathematical transformation (n)
Total proteins Albumin Total cholesterol Eree cholesterol Triglycerides AST* ALP 2 Calcium Phosphorus
g/100 mL g/100 mL mg/100 mL mg/100 mL mg/100 mL U/L U/L mg/100 mL mg/100 mL
762 783 773 766 780 779 759 788 782
*AST = aspartate aminotransferase. ALP = alkaline phosphatase.
. .. . .. . .. logarithm logarithm logarithm logarithm
References limits
X
238 1.17 87.0 27.0 45.7 70 568 4.70 359
3.90 3.85 1.96 1.93 138 140 73.3 705 88.8 86.6 124 122 2,240 2,130 10.47 10.18 6.40 6.27
to 5.22 to 2.74 to 192 to 119 to 172 to 220 to 8,831 to 16.25 to 11.40
Confidence interval of x to 3.95 to 1.98 to 142 to 76.1 to 90.8 to 127 to 2,353 to 10.76 to 6.53
NS NS NS NS NS <.01 NS NS NS NS NS 14.6008
NS NS <.01 NS NS <.01 NS NS NS NS NS 27.5996
NS NS <.01 NS NS <.01 NS NS NS NS NS 649.4961
140 140
1.87B 2.05A
4.1^ 3.61B NS NS <.01 NS NS <.01 NS NS NS NS NS 479.1162
76^ 70.5B
69.0B 77.3A
134B 146A
1.93 1.98
3.91 3.89
76^ 69.8B
1.94 1.98
3.64B 4.25A
143A 137B 75.6A 70.9B
Free cholesterol
— (mg/100 mL)
Total cholesterol
140 140
1.95 1.96
Albumin
3.86 3.94
Total proteins
•^"Means within main effects with no common superscripts are different (P5.01). ^Only significance levels lower than ,01 were considered. 2 AST = aspartate aminotransferase. 3 ALP = alkaline phosphatase. 4 Error mean square of values transformed in logarithm.
Males Females Age 21 days 45 days Strain Arbor Acres Hybro Season Summer Winter Interactions Sex by age Sex by strain Age by strain Sex by age by strain Sex by season Age by season Sex by age by season Strain by season Sex by strain by season Age by strain by season Age by strain by season by sex Error mean square
Sex
Source of variation
NS <.01 <.01 NS NS NS NS NS NS NS NS .01894
88.5 89.1
83.2B 94.8A
82.6B 97.7A
89.1 885
Triglycerides
NS NS NS NS NS <.01 NS <.01 NS NS NS .01114
13^ 112B
126 123
113B 141 A
124 125
AST2
NS NS NS NS NS
NS
NS NS
.03174
<.01 <.01
<.01
1,832B 2,723A
2,415A 2,080B
3562 A 1,245B
2,427A 2,070B
(U/L)
ALP3
NS NS NS NS NS <.01 NS NS NS NS NS 8.2644
10.08B 10.^
10.36 10.57
10.45 1050
10.41 10.53
Phosphorus
NS NS NS NS NS <.01 NS NS NS NS NS .01114
7.43A 5.46B
6.37 6.41
6.10B 6.79A
6.46 6.34
(mg/100 mL)
Calcium
TABLE 2. Mean values of plasma constituents and results of ANOVA according to sex, age, strain, and sampling season-
Confidence interval of x
X
Confidence interval of x AST, U/L Reference limits
X
Confidence interval of x Phosphorus, mg/100 mL Reference limits
X
Confidence interval of x Caldum, mg/100 mL Reference limits
X
Albumin, g/100 mL Reference limits
Variable and statistic
to 126
98 163 154 to 172
to 271
4.17 to 13.49 7.50 7.06 to 7.94
4.36 to 12.50 7.38 7.00 to 7.78
to 185
4.10 to 14.98 9.54 8.97 to 10.11
4.09 to 17.01 10.55 9.91 to 11.19
79 121 115
1.00 to 2.64 1.82 1.74 to 1.90
1.10 to 2.74 1.92 1.84 to 2.00
21 days
Summer 45 days
69 107 103 to 112
to 167
4.06 to 6.55 5.15 5.04 to 5.27
6.23 to 14.49 10.36 9.98 to 10.74
1.41 to 2.51 1.96 1.91 to 2.01
21 days
69 120 112
to 127
to 207
3.48 to 1058 5.98 5.61 to 6.37
3.85 to 19.67 11.77 10.98 to 12.56
1.38 to 3.02 2.20 2.10 to 2.30
Winter 45 days
79 to 233 136 128 to 143
81 to 250 142 136 to 151
Summer Arbor Acres Hybro
71 to 191 116 110 to 123
66 to 179 109 104 to 114
Winter Arbor Acres Hybro
TABLE 3. Means, confidence intervals of means, and reference limits of albumin, caldum, phosphorus, and aspartate aminotransferase (AST), defined by fractiles .025 and .975, of data partitioned according to the statistical significance established by ANOVA
Total proteins, g/100 mL Reference limits x Confidence interval of x Total cholesterol, mg/100 mL Reference limits x Confidence interval of x Free cholesterol, mg/100 mL Reference limits x Confidence interval of x
Variable and statistic
31.0 to 108 25.9 to 118 683 68.5 64.9 to 71.8 60.8 to 76.6
24.6 to 132 21.8 to 114 81.1 63.9 75.4 to 87 57.6 to 705
24.6 to 117 70.9 66.8 to 75
29.7 to 121 75.6 71.4 to 79.8
2.97 to 525 4.11 3.97 to 4.25
45 days
Winter
230 to 433 331 321 to 3.41
21 days
91.9 to 180 88.8 to 205 136 147 132 to 140 140 to 154
331 to 539 435 421 to 4.46
45 days
Summer
3.01 to 5.05 4.03 3.93 to 4.13
21 days
94.2 to 196 78.4 to 192 145 135 140 to 150 129 to 141
Females
Sex
88.7 to 197 862 to 188 143 137 139 to 147 133 to 141
Males
Season
272 to 112 24.5 to 111 67.7 64.2 633 to 72.2 57.3 to 715
86.6 to 187 78.8 to 180 137 129 132 to 142 123 to 135
3.10 to 5.34 4.22 4.09 to 435
45 days
Arbor Acres
2.49 to 4.93 3.71 339 to 3.82
21 days
374 to 128 22.7 to 119 812 67.1 76.7 to 85.7 603 to 743
985 to 187 89.2 to 207 143 148 138 to 147 142 to 154
3.15 to 539 4.27 4.15 to 439
45 days
Hybro
234 to 4.80 357 3.45 to 3.69
21 days
Strain
TABLE 4. Means, confidence intervals of means, and reference limits of total proteins and free and total cholesterol, defined by fractiles .025 and .975, of data partitioned according to the statistical significance established by ANOVA
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MELUZZI ET AL.
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BLOOD CONSTITUENTS REFERENCE VALUES
TABLE 5. Means, confidence intervals of means, and reference limits of alkaline phosphatase and triglycerides, defined by fractiles .025 and .975, of data partitioned according to the statistical significance established by ANOVA Alkaline phosphatase Source of variation
Reference limits
Summer (21 days) Summer (45 days) Winter (21 days) Winter (45 days) Summer (males) Summer (females) Winter (males) Winter (females) Arbor Acres (21 days) Arbor Acres (45 days) Hybro (21 days) Hybro (45 days) Arbor Acres (males) Arbor Acres (females) Hybro (males) Hybro (females)
1,028 420 2,046 851 485 360 946 853 1,633 491 1,219 538
to to to to to to to to to to to to
9,528 2,432 7,852 2,911 9,036 7,145 8,318 8,222 9,311 2,985 8,551 3,013
X
-(U/L) 3,126 1,009 4,009 1,574 2,094 1,603 2,805 2,649 3,899 1,211 3,228 1,274
by Franchini et al. (1990c) on growing turkeys. Interactions of season with age and strain were significant (P<.01). The level of ALP declined as the age increased (Table 2), probably due to the decrease in osteoblastic activity of chickens with age (Bell and Freeman, 1971; Protais et al., 1982; Franchini et al., 1988a,b). The higher level of the enzyme in males compared with females may have been due to the different growth rates of the two sexes. Similar results have also been observed in growing turkeys (Franchini et al., 1990b,c). The ALP levels were influenced by sampling season and genetic type. The values were higher in the Arbor Acres than in the Hybro chickens (possibly due to the faster growth rates of the Arbor Acres strain) and higher in winter than summer. The interactions of age with strain, age with sampling season, and sex with season were statistically significant (P<01). High summer temperatures had opposite effects on calcium and phosphorus; the levels of the former decreased considerably and those of the latter rose (Table 2). The calcium values were constant at 21 and 45 days, confirming the results of Protais et al. (1982). Phosphorus under-
Confidence interval of x
Triglycerides Reference limits
x hM ~ / i n n
2,793 918 3,758 1,469 1,799 1,371 2,512 2,355 3,589 1,091 2,924 1,161
to 3/489 to 1,109 to 4,276 to 1,687 to 2/438 to 1,875 to 3,162 to 2,979 to 4,236 to 1,343 to 3,565 to 1,394
47.2 43.4 44.8 56.4 44.3 46.8 50.2 44.5
to 142 to 166 to 155 to 214 to 149 to 156 to 189 to 191
Confidence interval of x —T \
81.8 77.6 to 86.3 84.9 785 to 91.8 83.4 783 to 88.7 110 102 to 118 81.1 712. to 86.3 85.3 8 0 2 to 90.8 975 912 to 104 92 8 5 3 to 99.3
went a significant, albeit inconspicuous, increase (from 6.1 to 6.8 mg/100 mL). This is in contrast with the findings of Protais et al. (1982), who observed no variation within the same age interval. For both variables, interactions between age and sampling season were significant (P<.01). Because the sources of variation chosen prior to sampling had different effects on the different plasma constituents studied (Table 2), the calculation of the reference limit for each variable was undertaken according to the ANOVA results. Tables 3 to 5 show the new reference limits. The reference intervals of some variables, such as calcium and ALP, were rather wide, in spite of the subdivision of the data utilizing a significance level of 1%. The reference intervals of free and total cholesterol did not differ greatly when summarized according to variable. Apparently, in spite of their significance from a statistical viewpoint, the effects of the various sources of variation did not possess much biological significance, at least with respect to the definition of reference limits. The number of birds examined (approximately 800) was sufficient to permit a reasonably precise definition of the reference limits of blood constituents in the
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Franchini, A., A. Meluzzi, E. Montemaggi, and G. Manfreda, 1990c. Andamento di taluni parametri ematochimici di tacchini in accrescimento. Zootec. Nutr. Anim. 16:145-151. Giordani, G., A. Meluzzi, and A. Franchini, 1986. Tecniche di allevamento, produttivita, parametri ematochimici e tessutali del broiler e della gallina ovaiola. Avicoltura 55(3):18-21. Giorgetti, A., and A. Zappa, 1985. II profilo metabolico negli animali di interesse agricolo. 1. Contribute alio studio delle metodologie per la definizione dei valori ematici di referimento. Zootec. Nutr. Arum. 11:199-205. International Federation of Clinical Chemistry, 1978. The theory of reference values. Part 1. The concept of reference values. Clin. Chim. Acta 87: 459F-475F. International Federation of Clinical Chemistry, 1984a. The theory of reference values. Part 2. Selection of individuals for the production of reference values. Clin. Chim. Acta 139205F-213F. International Federation of Clinical Chemistry, 1984b. REFERENCES The theory of reference values. Part 5. Statistical treatment of collected references values. Clin. Bell, D. J., and B. M. Freeman, 1971. Physiology and Chim. Acta 13757F-114F. Biochemistry of the Domestic Fowl. Academic Lumeij, J. T., and J. J. de Bruijne, 1985. Blood Press, London, England. chemistry reference values in racing pigeons. Bergmeyer, S. U., H. Burner, and G. Hillmann, 1972. Avian Pathol. 14:401-408. Recommendations of the German Society for Clinical Standardization of methods for the Lumeij, J. T., and J. WolfswinkeL 1988. Blood chemistry reference values for use in colombine estimation of enzyme activities in biological hepatology. Avian Pathol. 17:515-517. fluids. Z. Klin. Chem. Klin. Biochem. 10:182-192. Cerolini, S., A. Baldi, and L. G. Cavalchini, 1987. II Meluzzi, A., G. Giordani, and A. Franchini, 1984. Influenza della tecnica di allevamento su alcuni profilo metabolico nella gallina ovaiola. Pages parametri ematici di galline ovaiole. Zootec. 239-245 in: Atti XXII Simposio Internazionale di Nutr. Anim. 10:481-488. Zootecnia, Milano, Italy. Cerolini, S., L. G. Cavalchini, A. Baldi, and P. Pad, G., I. Romboli, and C. Fedeli Avanzi, 1988. Some haematic parameters in the muscovy Mariani, 1986. Pullets in cage vs floor: effect on drake. Pages 751-752 in: Proceedings XVIH laying performance. Pages 800-804 in: ProceedWorld's Poultry Congress, Nagoya, Japan. ings 7th European Poultry Conference, Paris, Peyenet, J., and J. C. Poufflot, 1972. Etude critique France. d'une dosage du phosphore sans deproteinsaDeaton, J. W., F. N. Reece, and W. J. Tarver, 1969. tion. Page 57 in: Organisation des Laboratoires. Hematocrit, hemoglobin, and plasma-protein Biologie prospective, L'Expansion Scientifique levels of broilers reared under constant temperFrancaise, Paris, France. atures. Poultry Sci. 68:1993-1996. Doumas, B. T., and H. G. Biggs, 1972. Determination Protais, I., M. Bougon, R. L'Hospitalier, T. Lecuyer, P. of serum albumin. Page 175 in: Standard Quemeneur, and M. Le Menec, 1982. Evoluzione Methods of Clinical Chemistry. Vol. 7, Acacomparata dell'eti dei polU, del contenuto di demic Press, New York, NY. sostanze minerali, di calcio e di fosforo delle ossa e nel contenuto di calcio, di fosforo e di Franchini, A., G. Giordani, A. Meluzzi, and G. fosfatasi alcalina nel siero. Zootecnica 22(23): Manfreda, 1990a. High doses of vitamin E in the 2-8. turkey diet. Arch. Gefhigelkd. 54:6-10. Franchini, A., A. Meluzzi, S. Bertuzzi, and G. Ray Sarkar, B. C, and U.P.S. Chauhan, 1967. A new Giordani, 1988a. High doses of vitamin E in the method for determining micro quantities of broiler diets. Arch. Gefltigelkd. 52:12-16. calcium in biological materials. Anal. BioL 20: 155-166. Franchini, A., A. Meluzzi, E. Garella, and E. Montemaggi, 1988b. Parametri ematochimici della Ross, J. D., G. Christie, W. G. Halliday, and R. M. pollastra in rapporto alia tecnica di allevamento. Jones, 1976. Determination of haematology and Avicoltura 57(6)53-57. blood chemistry values in healthy six-week old broiler hybrids. Avian Pathol. 5273-281. Franchini, A., A. Meluzzi, G. Manfreda, and S. Bertuzzi, 1990b. Blood constituents of turkeys Ross, J. G., G. Christie, W. G. Halliday, and R. M. fed high doses of dietary vitamin E. Pages Jones, 1978. Haematological and blood chemis236-239 in: Proceedings VIII European Poultry try "comparison values" for clinical pathology Conference, Barcelona, Spain. in poultry. Vet. Rec. 10229-31. Franchini, A., A. Meluzzi, E. Montemaggi, and G. SAS Institute, 1986. SAS® User's Guide: Statistics. Manfreda, 1989. Profilo metabolico di ovaiole Version 6 Edition. SAS Institute Inc., Cary, NC. alimentate a diverso tenore proteico. Avicoltura Shapiro, S. S., and M. B. Wilk, 1965. An analysis of 58(9):105-107. variance for normality (complete samples). Bi-
broilers examined. However, the results suggest the necessity for more detailed investigations considering other variables in addition to the ones studied in the present. One must also consider that genetic selection induces appreciable differences in blood plasma constituents. According to Table 2, 44% of the variables measured were different due to strain. Therefore, the reported reference limits should be periodically updated. The reference limits defined herein could be exploited as indicators of metabolic and health conditions of a broiler farm, provided an adequate number of birds is examined.
BLOOD CONSTITUENTS REFERENCE VALUES ometrica 52:599-611. SturMe, P. D., 1976. Avian Physiology. 3rd Ed. Springer-Verlag, New York, NY. Trinder, P., 1969. Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. Ann. din. Biochem. 624-27. Verga, M., L. G. Cavalchini, C. Carenzi, and F. Bonetti, 1984. Performance, behavioural reactions and blood plasma parameters of laying hens in cage vs floor. Pages 467-468 in: Proceed-
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ings XVn World's Poultry Congress, Helsinki, Finland. Weichseibaun, T. E., 1946. Accurate and rapid method for determination of proteins in small amounts of blood, serum and plasma. J. Am. din. Pathol. 16:40-49. Woodard, A. E., P. Vohra, and B. Mayeda, 1983. Blood parameters of one-year-old and sevenyear-old partridges (Alectoris chukar). Poultry Sci. 622492-2496.
INTRODUCTION Hematochemistry constitutes an increasingly useful aid in zootechnical and veterinary research. It permits the study of specific pathological alterations of certain blood constituents and recognition, under strictly controlled experimental conditions, of the existence of metabolic alterations of different origin. Many factors can influence the level of a particular blood constituent: genetic type, feeding, microand macro-climate, rearing technique, age, physiological state, and sex, as well as pathological factors. Moreover, methods of sampling and obtaining the biological material and the method of analysis can also influence results.
'This study was supported by the Italian Ministry of Public Instruction. ^To whom correspondence should be addressed. 3 Present address: Industria Vaccini Zootecnici, Vigorea, Padova, Italy.
It would therefore be useful to define a range of values or limits to serve as reference for each blood constituent. The reference limits define an interval and the values within it are considered "normal" (International Federation of Clinical Chemistry, 1978). The reference values can be used to evaluate the state of health of either a single bird or an entire population and would constitute a basic requirement for an indispensable preliminary knowledge of the biological material chosen for scientific research. In avian species, hematochemical studies are nearly always performed for research purposes, in order to evaluate the effect of rearing technique (Meluzzi et al., 1984; Verga et ah, 1984; Cerolini et al., 1986; Giordani et al., 1986; Franchini et al., 1988b), feeding regimens (Franchini et al., 1988a, 1989), environmental conditions, and other parameters. Hematochemical tests are generally not utilized for avian diagnostics, because no reference values
337
338
MELUZZI ET AL.
are available. Reference values should be based on a sufficiently large number of samples obtained under all conditions able to influence the level of the variable under study. To date, research on the definition of reference values has been carried out on laying hens of two different commercial hybrids (Cerolini et al., 1987), racing pigeons (Lumeij and Bruijne, 1985; Lumeij and Wolfswinkel, 1988), muscovy ducks (Pad et al, 1988), partridges (Woodward et al, 1983), and broilers (Ross et al, 1976, 1978). The present research, which attempts to extend and integrate the studies performed by Ross et al. (1976,1978), aims at establishing the reference values of certain blood constituents in floor-reared broilers of known strain, age, sex, and sampling season. Moreover, the authors propose to evaluate the influence of different sources of variation on the level of the hematic constituents investigated. MATERIALS AND METHODS A total of 800 broilers were used, randomly chosen from a population of approximately 66,000 birds. The chickens came from 20 farms situated in northern Italy and were reared on the floor at a density of 12 to 13 birds per square meter in open-sided houses similar in design and construction, and under similar environmental conditions. The lighting program was 23 h light:l h dark. The birds had received the same commercial feeds and were clinically healthy. Feed composition of the diets was: first period (1 to 28 days): 23% CP, 3,100 kcal/kg ME; second period (29 to 45 days): 21% CP, 3,100 kcal/kg ME. The production levels of the flocks were similar, corresponding to the standard for the strain. Following the a priori selection criteria (International Federation of Clinical Chemistry, 1984a; Giorgetti and Zappa, 1985), chickens of two commercial strains among the most common in Italy were chosen: Hybro and Arbor Acres. All birds, both male and female, underwent samp-
Boehringer Mannheim, Sanhofer-Strasse, 6800 Mannheim 31, Germany. ^bsystem OY, Pultittie 9, Helsinki, Finland.
ling at the age of 21 and 45 days during the summer (June and July) and winter (November and December) seasons. Each group with the same characteristics (strain, sex, age, and sampling season) was made up of 50 birds from five farms. The blood samples were drawn in the early morning from the wing vein of birds fasted since the previous evening. The blood was collected in heparinized test tubes and centrifuged at 1,800 x g for 15 min: the plasma was subdivided into several parts and preserved at -20 C until the time of analysis. The following variables were measured: total proteins by the biuret method (Weichselbaum, 1946); albumin by the green bromocresol method (Doumas and Biggs, 1972); total and free cholesterol by the cholesterol-oxidase para-aminophenazone (CHOD-PAP) colorimetric enzymatic method (Trinder, 1969); triglycerides by the glycerol-3-phosphate-oxidase paraaminophenazone (GPO-PAP) colorimetric enzymatic method (Trinder, 1969); calcium by the o-cresolphtalein method (Ray Sarckar and Chauhan, 1967); phosphorus by the molybdenum blue method (Peyenet and Pouillot, 1972); aspartate aminotransferase (AST) and alkaline phosphatase (ALP) by the optimized standard method of the German Society of Clinical Chemistry, at a temperature of 30 C (Bergmeyer et al., 1972). Analyses were performed using Biochemia Boehringer4 kits and a Labsystem FP 901 spectrophotometer,5 equipped with a FP 401 incubating unit. The data were analyzed using base SAS® software (SAS Institute, 1986). The normality of the distribution was verified with skewness and kurtosis tests, and the Shapiro and Wilk (1965) test. The values not presenting a Gaussian distribution (triglycerides, AST, ALP, and phosphorus) were normalized by log transformations. Parametric statistical methods were used to calculate the reference limits defined by the fractiles .025 and .975 (International Federation of Clinical Chemistry, 1984b). Subsequently, the raw or transformed data were subjected to ANOVA procedures (General Linear Model) according to the following model:
339
BLOOD CONSTITUENTS REFERENCE VALUES
Yijkim = ^ + + + +
+ A£ + B; + C k + Di + (AB)ij (AC)* + (AD) U + (BCW (BD)a + (CD)^ + (ABC)ijk (ABDfy + (ACD)M (BCEOju + (ABCD)ijki + eijkhn,
where [i = population mean; Ai = effect of the sex; Bj = effect of the age (j = 1: 21 days; j = 2: 45 days); C^ = effect of the strain (k = 1: Arbor Acres; k = 2: Hybro); E>i = effect of the sampling season (1 = 1: summer; 1 = 2: winter); (AB)«, (AC)^ (AD)a,
(BO*,
(BDh,
(CD)M,
(ABC)^
(ABDty ( A C D ) ^ (BCD)^ (ABCD)ijld = interactions of the main effects; and Emm = overall error term. The data were then partitioned into subclasses according to the statistical significance established by ANOVA and the reference limits were redefined.
RESULTS AND DISCUSSION Table 1 shows the reference limits defined by the fractiles .025 and .975. The reference intervals were rather wide, particularly that of ALP. This could be due to the effects exerted by the variables chosen for the sampling, i.e., age, sex, strain, and sampling season. Total proteins varied according to age and increased in birds 45 days of age (Table 2). T his effect of age had already been observed in chickens and turkeys by several authors (Sturkie, 1976; Ross et al., 1978; Franchini et al, 1988a, 1990a,b). An increased protein concentration was also
noted in the hot season. In contrast, Deaton et at. (1969) observed a slight, but not significant, decrease of total protein with increasing temperature from 23.9 to 32.2 C. The interactions of age with strain and sampling season were statistically significant (P<.01). Albumin levels did not appear to be influenced by the different sources of variation, with the exception of the sampling season (Table 2). In contrast to the results with total proteins, an increase in albumin was noted during the winter. The interaction between age and season was statistically significant (P<.01). The Hybro chickens had values of total and free cholesterol and triglycerides consistently higher than the Arbor Acres chickens (Table 2). Furthermore, the levels of the three variables were greater in males compared with females, with statistically significant differences only in the case of free and total cholesterol (P<.01). With aging, free cholesterol decreased but triglycerides increased. This same trend was recently noted in meat turkeys (Franchini et al., 1990b,c). For total and free cholesterol, the interactions of age with strain and season were significant (P<.01); in the case of triglycerides, the interactions of strain with sex and age were significant (P<01). The AST values were higher in the older birds and in the birds sampled during the summer months (Table 2). Similar observations were made by Meluzzi et al. (1984) on laying hens, and
TABLE 1. Mathematical transformation in order to normalize the distribution of data, means, confidence intervals of means, and reference limits defined by .025 and .975 fractiles Variable
Units
Samples Mathematical transformation (n)
Total proteins Albumin Total cholesterol Eree cholesterol Triglycerides AST* ALP 2 Calcium Phosphorus
g/100 mL g/100 mL mg/100 mL mg/100 mL mg/100 mL U/L U/L mg/100 mL mg/100 mL
762 783 773 766 780 779 759 788 782
*AST = aspartate aminotransferase. ALP = alkaline phosphatase.
. .. . .. . .. logarithm logarithm logarithm logarithm
References limits
X
238 1.17 87.0 27.0 45.7 70 568 4.70 359
3.90 3.85 1.96 1.93 138 140 73.3 705 88.8 86.6 124 122 2,240 2,130 10.47 10.18 6.40 6.27
to 5.22 to 2.74 to 192 to 119 to 172 to 220 to 8,831 to 16.25 to 11.40
Confidence interval of x to 3.95 to 1.98 to 142 to 76.1 to 90.8 to 127 to 2,353 to 10.76 to 6.53
NS NS NS NS NS <.01 NS NS NS NS NS 14.6008
NS NS <.01 NS NS <.01 NS NS NS NS NS 27.5996
NS NS <.01 NS NS <.01 NS NS NS NS NS 649.4961
140 140
1.87B 2.05A
4.1^ 3.61B NS NS <.01 NS NS <.01 NS NS NS NS NS 479.1162
76^ 70.5B
69.0B 77.3A
134B 146A
1.93 1.98
3.91 3.89
76^ 69.8B
1.94 1.98
3.64B 4.25A
143A 137B 75.6A 70.9B
Free cholesterol
— (mg/100 mL)
Total cholesterol
140 140
1.95 1.96
Albumin
3.86 3.94
Total proteins
•^"Means within main effects with no common superscripts are different (P5.01). ^Only significance levels lower than ,01 were considered. 2 AST = aspartate aminotransferase. 3 ALP = alkaline phosphatase. 4 Error mean square of values transformed in logarithm.
Males Females Age 21 days 45 days Strain Arbor Acres Hybro Season Summer Winter Interactions Sex by age Sex by strain Age by strain Sex by age by strain Sex by season Age by season Sex by age by season Strain by season Sex by strain by season Age by strain by season Age by strain by season by sex Error mean square
Sex
Source of variation
NS <.01 <.01 NS NS NS NS NS NS NS NS .01894
88.5 89.1
83.2B 94.8A
82.6B 97.7A
89.1 885
Triglycerides
NS NS NS NS NS <.01 NS <.01 NS NS NS .01114
13^ 112B
126 123
113B 141 A
124 125
AST2
NS NS NS NS NS
NS
NS NS
.03174
<.01 <.01
<.01
1,832B 2,723A
2,415A 2,080B
3562 A 1,245B
2,427A 2,070B
(U/L)
ALP3
NS NS NS NS NS <.01 NS NS NS NS NS 8.2644
10.08B 10.^
10.36 10.57
10.45 1050
10.41 10.53
Phosphorus
NS NS NS NS NS <.01 NS NS NS NS NS .01114
7.43A 5.46B
6.37 6.41
6.10B 6.79A
6.46 6.34
(mg/100 mL)
Calcium
TABLE 2. Mean values of plasma constituents and results of ANOVA according to sex, age, strain, and sampling season-
Confidence interval of x
X
Confidence interval of x AST, U/L Reference limits
X
Confidence interval of x Phosphorus, mg/100 mL Reference limits
X
Confidence interval of x Caldum, mg/100 mL Reference limits
X
Albumin, g/100 mL Reference limits
Variable and statistic
to 126
98 163 154 to 172
to 271
4.17 to 13.49 7.50 7.06 to 7.94
4.36 to 12.50 7.38 7.00 to 7.78
to 185
4.10 to 14.98 9.54 8.97 to 10.11
4.09 to 17.01 10.55 9.91 to 11.19
79 121 115
1.00 to 2.64 1.82 1.74 to 1.90
1.10 to 2.74 1.92 1.84 to 2.00
21 days
Summer 45 days
69 107 103 to 112
to 167
4.06 to 6.55 5.15 5.04 to 5.27
6.23 to 14.49 10.36 9.98 to 10.74
1.41 to 2.51 1.96 1.91 to 2.01
21 days
69 120 112
to 127
to 207
3.48 to 1058 5.98 5.61 to 6.37
3.85 to 19.67 11.77 10.98 to 12.56
1.38 to 3.02 2.20 2.10 to 2.30
Winter 45 days
79 to 233 136 128 to 143
81 to 250 142 136 to 151
Summer Arbor Acres Hybro
71 to 191 116 110 to 123
66 to 179 109 104 to 114
Winter Arbor Acres Hybro
TABLE 3. Means, confidence intervals of means, and reference limits of albumin, caldum, phosphorus, and aspartate aminotransferase (AST), defined by fractiles .025 and .975, of data partitioned according to the statistical significance established by ANOVA
Total proteins, g/100 mL Reference limits x Confidence interval of x Total cholesterol, mg/100 mL Reference limits x Confidence interval of x Free cholesterol, mg/100 mL Reference limits x Confidence interval of x
Variable and statistic
31.0 to 108 25.9 to 118 683 68.5 64.9 to 71.8 60.8 to 76.6
24.6 to 132 21.8 to 114 81.1 63.9 75.4 to 87 57.6 to 705
24.6 to 117 70.9 66.8 to 75
29.7 to 121 75.6 71.4 to 79.8
2.97 to 525 4.11 3.97 to 4.25
45 days
Winter
230 to 433 331 321 to 3.41
21 days
91.9 to 180 88.8 to 205 136 147 132 to 140 140 to 154
331 to 539 435 421 to 4.46
45 days
Summer
3.01 to 5.05 4.03 3.93 to 4.13
21 days
94.2 to 196 78.4 to 192 145 135 140 to 150 129 to 141
Females
Sex
88.7 to 197 862 to 188 143 137 139 to 147 133 to 141
Males
Season
272 to 112 24.5 to 111 67.7 64.2 633 to 72.2 57.3 to 715
86.6 to 187 78.8 to 180 137 129 132 to 142 123 to 135
3.10 to 5.34 4.22 4.09 to 435
45 days
Arbor Acres
2.49 to 4.93 3.71 339 to 3.82
21 days
374 to 128 22.7 to 119 812 67.1 76.7 to 85.7 603 to 743
985 to 187 89.2 to 207 143 148 138 to 147 142 to 154
3.15 to 539 4.27 4.15 to 439
45 days
Hybro
234 to 4.80 357 3.45 to 3.69
21 days
Strain
TABLE 4. Means, confidence intervals of means, and reference limits of total proteins and free and total cholesterol, defined by fractiles .025 and .975, of data partitioned according to the statistical significance established by ANOVA
8
MELUZZI ET AL.
343
BLOOD CONSTITUENTS REFERENCE VALUES
TABLE 5. Means, confidence intervals of means, and reference limits of alkaline phosphatase and triglycerides, defined by fractiles .025 and .975, of data partitioned according to the statistical significance established by ANOVA Alkaline phosphatase Source of variation
Reference limits
Summer (21 days) Summer (45 days) Winter (21 days) Winter (45 days) Summer (males) Summer (females) Winter (males) Winter (females) Arbor Acres (21 days) Arbor Acres (45 days) Hybro (21 days) Hybro (45 days) Arbor Acres (males) Arbor Acres (females) Hybro (males) Hybro (females)
1,028 420 2,046 851 485 360 946 853 1,633 491 1,219 538
to to to to to to to to to to to to
9,528 2,432 7,852 2,911 9,036 7,145 8,318 8,222 9,311 2,985 8,551 3,013
X
-(U/L) 3,126 1,009 4,009 1,574 2,094 1,603 2,805 2,649 3,899 1,211 3,228 1,274
by Franchini et al. (1990c) on growing turkeys. Interactions of season with age and strain were significant (P<.01). The level of ALP declined as the age increased (Table 2), probably due to the decrease in osteoblastic activity of chickens with age (Bell and Freeman, 1971; Protais et al., 1982; Franchini et al., 1988a,b). The higher level of the enzyme in males compared with females may have been due to the different growth rates of the two sexes. Similar results have also been observed in growing turkeys (Franchini et al., 1990b,c). The ALP levels were influenced by sampling season and genetic type. The values were higher in the Arbor Acres than in the Hybro chickens (possibly due to the faster growth rates of the Arbor Acres strain) and higher in winter than summer. The interactions of age with strain, age with sampling season, and sex with season were statistically significant (P<01). High summer temperatures had opposite effects on calcium and phosphorus; the levels of the former decreased considerably and those of the latter rose (Table 2). The calcium values were constant at 21 and 45 days, confirming the results of Protais et al. (1982). Phosphorus under-
Confidence interval of x
Triglycerides Reference limits
x hM ~ / i n n
2,793 918 3,758 1,469 1,799 1,371 2,512 2,355 3,589 1,091 2,924 1,161
to 3/489 to 1,109 to 4,276 to 1,687 to 2/438 to 1,875 to 3,162 to 2,979 to 4,236 to 1,343 to 3,565 to 1,394
47.2 43.4 44.8 56.4 44.3 46.8 50.2 44.5
to 142 to 166 to 155 to 214 to 149 to 156 to 189 to 191
Confidence interval of x —T \
81.8 77.6 to 86.3 84.9 785 to 91.8 83.4 783 to 88.7 110 102 to 118 81.1 712. to 86.3 85.3 8 0 2 to 90.8 975 912 to 104 92 8 5 3 to 99.3
went a significant, albeit inconspicuous, increase (from 6.1 to 6.8 mg/100 mL). This is in contrast with the findings of Protais et al. (1982), who observed no variation within the same age interval. For both variables, interactions between age and sampling season were significant (P<.01). Because the sources of variation chosen prior to sampling had different effects on the different plasma constituents studied (Table 2), the calculation of the reference limit for each variable was undertaken according to the ANOVA results. Tables 3 to 5 show the new reference limits. The reference intervals of some variables, such as calcium and ALP, were rather wide, in spite of the subdivision of the data utilizing a significance level of 1%. The reference intervals of free and total cholesterol did not differ greatly when summarized according to variable. Apparently, in spite of their significance from a statistical viewpoint, the effects of the various sources of variation did not possess much biological significance, at least with respect to the definition of reference limits. The number of birds examined (approximately 800) was sufficient to permit a reasonably precise definition of the reference limits of blood constituents in the
344
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Franchini, A., A. Meluzzi, E. Montemaggi, and G. Manfreda, 1990c. Andamento di taluni parametri ematochimici di tacchini in accrescimento. Zootec. Nutr. Anim. 16:145-151. Giordani, G., A. Meluzzi, and A. Franchini, 1986. Tecniche di allevamento, produttivita, parametri ematochimici e tessutali del broiler e della gallina ovaiola. Avicoltura 55(3):18-21. Giorgetti, A., and A. Zappa, 1985. II profilo metabolico negli animali di interesse agricolo. 1. Contribute alio studio delle metodologie per la definizione dei valori ematici di referimento. Zootec. Nutr. Arum. 11:199-205. International Federation of Clinical Chemistry, 1978. The theory of reference values. Part 1. The concept of reference values. Clin. Chim. Acta 87: 459F-475F. International Federation of Clinical Chemistry, 1984a. The theory of reference values. Part 2. Selection of individuals for the production of reference values. Clin. Chim. Acta 139205F-213F. International Federation of Clinical Chemistry, 1984b. REFERENCES The theory of reference values. Part 5. Statistical treatment of collected references values. Clin. Bell, D. J., and B. M. Freeman, 1971. Physiology and Chim. Acta 13757F-114F. Biochemistry of the Domestic Fowl. Academic Lumeij, J. T., and J. J. de Bruijne, 1985. Blood Press, London, England. chemistry reference values in racing pigeons. Bergmeyer, S. U., H. Burner, and G. Hillmann, 1972. Avian Pathol. 14:401-408. Recommendations of the German Society for Clinical Standardization of methods for the Lumeij, J. T., and J. WolfswinkeL 1988. Blood chemistry reference values for use in colombine estimation of enzyme activities in biological hepatology. Avian Pathol. 17:515-517. fluids. Z. Klin. Chem. Klin. Biochem. 10:182-192. Cerolini, S., A. Baldi, and L. G. Cavalchini, 1987. II Meluzzi, A., G. Giordani, and A. Franchini, 1984. Influenza della tecnica di allevamento su alcuni profilo metabolico nella gallina ovaiola. Pages parametri ematici di galline ovaiole. Zootec. 239-245 in: Atti XXII Simposio Internazionale di Nutr. Anim. 10:481-488. Zootecnia, Milano, Italy. Cerolini, S., L. G. Cavalchini, A. Baldi, and P. Pad, G., I. Romboli, and C. Fedeli Avanzi, 1988. Some haematic parameters in the muscovy Mariani, 1986. Pullets in cage vs floor: effect on drake. Pages 751-752 in: Proceedings XVIH laying performance. Pages 800-804 in: ProceedWorld's Poultry Congress, Nagoya, Japan. ings 7th European Poultry Conference, Paris, Peyenet, J., and J. C. Poufflot, 1972. Etude critique France. d'une dosage du phosphore sans deproteinsaDeaton, J. W., F. N. Reece, and W. J. Tarver, 1969. tion. Page 57 in: Organisation des Laboratoires. Hematocrit, hemoglobin, and plasma-protein Biologie prospective, L'Expansion Scientifique levels of broilers reared under constant temperFrancaise, Paris, France. atures. Poultry Sci. 68:1993-1996. Doumas, B. T., and H. G. Biggs, 1972. Determination Protais, I., M. Bougon, R. L'Hospitalier, T. Lecuyer, P. of serum albumin. Page 175 in: Standard Quemeneur, and M. Le Menec, 1982. Evoluzione Methods of Clinical Chemistry. Vol. 7, Acacomparata dell'eti dei polU, del contenuto di demic Press, New York, NY. sostanze minerali, di calcio e di fosforo delle ossa e nel contenuto di calcio, di fosforo e di Franchini, A., G. Giordani, A. Meluzzi, and G. fosfatasi alcalina nel siero. Zootecnica 22(23): Manfreda, 1990a. High doses of vitamin E in the 2-8. turkey diet. Arch. Gefhigelkd. 54:6-10. Franchini, A., A. Meluzzi, S. Bertuzzi, and G. Ray Sarkar, B. C, and U.P.S. Chauhan, 1967. A new Giordani, 1988a. High doses of vitamin E in the method for determining micro quantities of broiler diets. Arch. Gefltigelkd. 52:12-16. calcium in biological materials. Anal. BioL 20: 155-166. Franchini, A., A. Meluzzi, E. Garella, and E. Montemaggi, 1988b. Parametri ematochimici della Ross, J. D., G. Christie, W. G. Halliday, and R. M. pollastra in rapporto alia tecnica di allevamento. Jones, 1976. Determination of haematology and Avicoltura 57(6)53-57. blood chemistry values in healthy six-week old broiler hybrids. Avian Pathol. 5273-281. Franchini, A., A. Meluzzi, G. Manfreda, and S. Bertuzzi, 1990b. Blood constituents of turkeys Ross, J. G., G. Christie, W. G. Halliday, and R. M. fed high doses of dietary vitamin E. Pages Jones, 1978. Haematological and blood chemis236-239 in: Proceedings VIII European Poultry try "comparison values" for clinical pathology Conference, Barcelona, Spain. in poultry. Vet. Rec. 10229-31. Franchini, A., A. Meluzzi, E. Montemaggi, and G. SAS Institute, 1986. SAS® User's Guide: Statistics. Manfreda, 1989. Profilo metabolico di ovaiole Version 6 Edition. SAS Institute Inc., Cary, NC. alimentate a diverso tenore proteico. Avicoltura Shapiro, S. S., and M. B. Wilk, 1965. An analysis of 58(9):105-107. variance for normality (complete samples). Bi-
broilers examined. However, the results suggest the necessity for more detailed investigations considering other variables in addition to the ones studied in the present. One must also consider that genetic selection induces appreciable differences in blood plasma constituents. According to Table 2, 44% of the variables measured were different due to strain. Therefore, the reported reference limits should be periodically updated. The reference limits defined herein could be exploited as indicators of metabolic and health conditions of a broiler farm, provided an adequate number of birds is examined.
BLOOD CONSTITUENTS REFERENCE VALUES ometrica 52:599-611. SturMe, P. D., 1976. Avian Physiology. 3rd Ed. Springer-Verlag, New York, NY. Trinder, P., 1969. Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. Ann. din. Biochem. 624-27. Verga, M., L. G. Cavalchini, C. Carenzi, and F. Bonetti, 1984. Performance, behavioural reactions and blood plasma parameters of laying hens in cage vs floor. Pages 467-468 in: Proceed-
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ings XVn World's Poultry Congress, Helsinki, Finland. Weichseibaun, T. E., 1946. Accurate and rapid method for determination of proteins in small amounts of blood, serum and plasma. J. Am. din. Pathol. 16:40-49. Woodard, A. E., P. Vohra, and B. Mayeda, 1983. Blood parameters of one-year-old and sevenyear-old partridges (Alectoris chukar). Poultry Sci. 622492-2496.