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Response of Early- and Late-Maturing Commercial Leghorn Pullets to Low Levels of Dietary Phosphorus1
Response of Early- and Late-Maturing Commercial Leghorn Pullets to Low Levels of Dietary Phosphorus1 SfflVARAM K. RAO and DAVID A. ROLAND, SR. Department of Poultry Science and Alabama Agricultural Experiment Station, Auburn University, Alabama 36849 FREDERIC J. HOERR Department of Pathobiology, Auburn University, Alabama 36849 (Received for publication July 25, 1991)
1992 Poultry Science 71:691-699
INTRODUCTION Maturing commercial Leghorn pullets are usually fed layer diets 1 to 2 wk prior to first egg (North, 1984; Roland, 1986; Keshavarz, 1987) or no later than 5% egg production (Roland, 1986). However, in every flock, pullets become sexually mature over several weeks (Dunnington and
Alabama Agricultural Experiment Station Journal Series Number 12-913011P.
Siegel, 1984). The number of days required for early- (EM) and late-maturing (LM) pullets to reach sexual maturity and the proportion of LM pullets may vary from flock to flock. Nevertheless, when pullets are introduced to layer diets based on average flock performance, the LM pullets are inevitably exposed to layer diets for at least 3 to 10 wk prior to reaching sexual maturity. Currently, there is wide variation in dietary P levels and the Ca:P ratio in layer diets used by various companies (Roland,
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ABSTRACT An experiment with a 2 x 2 factorial arrangement of treatments was used to determine differences in responses of early-maturing (EM) and late-maturing (LM) pullets fed low dietary P (.4% total P; LP) and normal dietary P (.7% total P; NP) levels. Six hundred pullets (18-wk-old) were equally and randomly allocated to the LP and NP treatments. Egg production, egg weight, egg specific gravity, and eggshell breaking strength were measured at 26 and 28 wk. When pullets were 28 wk of age, plasma total Ca (TCa), ionic Ca (Ca++), and inorganic P (Pi) concentrations, urine Ca concentrations, and urine pH, bone mineral content (BMC), bone density, total kidney weight, and kidney weight ratio (heavier kidney + lighter kidney) were determined from 15 pullets each from EM-NP, EM-LP, LM-NP, and LM-LP treatments. The pullets with osteoporosis and pullets that died during the experiment were categorized into EM and LM groups. Results showed that LP caused severe adverse effects on LM pullets. The LM pullets fed the LP diet had high plasma Ca++ concentration, low plasma Pj concentration, increased urine Ca concentration, a high incidence of osteoporosis, mild kidney lesions, and elevated mortality compared with pullets subjected to the other treatments. The EM pullets fed the LP diet were also adversely effected by LP, but were less susceptible to osteoporosis and mortality. The LP diet improved eggshell quality, but this beneficial effect was only temporary. The severity of adverse effects of low dietary P was greater for LM than the EM pullets. (Key words: cage-layer fatigue, phosphorus, osteoporosis, maturing pullets, sexual maturity)
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•Hy-Line Indian River Co., Des Moines, IA 50265.
The objective of the present experiment was to determine the effects of low dietary P on plasma concentrations of ionic Ca (Ca ++ ), total Ca (TCa), inorganic P (Pi), urine pH, urine Ca concentration, bone mineral content (BMC), bone density (BD), kidney morphology, and mortality rate of EM and LM pullets.
MATERIALS AND METHODS Pre-Experimental Evaluation Pullets for pre-experimental evaluation were sampled from the same population of pullets used in the experiment. These evaluations were done to determine whether 1) the identification of EM and LM pullets based on secondary sexual characteristics was accurate; and 2) the pullets had normal kidneys prior to the experiment. Eighteen-week-old Single Comb White Leghorn pullets (Hy-Line® W362) were used. As the pullets were housed, 15 EM and LM pullets were selected based on secondary sex characteristics (comb size and color). These pullets were weighed and then killed by cervical dislocation; the comb, oviduct, ovary, and kidneys were removed and weighed. The kidney weight ratio (heavier kidney + lighter kidney) was determined for each pullet. Average live weight of pullets was determined by individually weighing 150 randomly selected pullets.
Experimental Procedure Six hundred pullets (18-wk-old) were housed individually in cages. In remaining cages, 840 pullets were housed three per cage (these 840 pullets were not included in the experiment except for pre-experimental evaluations and for the comparison of mortality). The 600 individually caged pullets were equally and randomly allocated to a low (.4% total P; LP) and a normal P (.7% total P; NP) diet (Table 1). The calculated available P in LP and N P diets were .5 and .2%, respectively. Within each diet, the EM and LM pullets were identified using secondary sexual characteristics and egg production records. The pullets that began egg production before or during 19 wk of age in each
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1989). With such variation in dietary levels of P, it is possible that some LM pullets may be exposed to diets containing low P for a few weeks prior to ovulation. Immature pullets (8-wk-old) exposed to a layer diet containing high Ca and low P for 10 wk prior to first egg had calciurea, urolithiasis, and a high mortality rate (Wideman et ah, 1985). It is possible that LM pullets in a flock fed layer diets containing low P would respond similarly to immature pullets fed a high Ca and low P diet; however, this has been not determined. Severe P deficiency causes rickets and subsequent high mortality rates in pullets and hens (Garlich, 1979), but marginal P level only slightly elevates flock mortality (Garlich, 1979; North, 1984). Slight increases in flock mortality may be attributed to the detrimental effect of low P in combination with layer level of dietary Ca (Ca adequate for laying hens) on LM pullets. Low dietary P could predispose pullets to other stressors (pathogens, inadequate nutrition, high temperature, and inadequate management) through depletion of bone Ca and possibly through renal stress. Increased urine Ca excretion induced by low dietary P may deplete bone Ca reserves, leading to a gradual increase in skeletal problems. Also, with normal and excess dietary Ca levels, low dietary P establishes ideal conditions for solid urate formation in kidney and ureters by elevating urine Ca concentration and p H (Oldroyd and Wideman, 1986). Although low dietary P may have caused kidney or skeletal problems, proper diagnosis in the field would be difficult because subclinical signs or symptoms exhibited by affected pullets may relate to several secondary causes. With various stressors affecting flock mortality, it would be difficult to differentiate the adverse effects of low dietary P on a small proportion of LM pullets in the flock. Therefore, to distinguish low dietary P effects on LM pullets, an experiment using LM and EM pullets would be required rather than using the entire flock.
EFFECT OF DIETARY PHOSPHORUS ON MATURING PULLETS TABLE 1. Composition and calculated analysis of the treatment diets Dietary treatments Ingredients and analysis
NP
LP
(%)
Total Calculated analysis Protein, % ME, kcal/kg Calcium/ % Total phosphorus, % Available phosphorus, % Sodium, % Methionine plus cystine, %
66.62 20.90 8.10 2.18
66.62 20.90 8.99 57
1.00 .37 35 25 .07 26 100.00
1.00 .37 25 25 .07 .98 100.00
16 16 2,809 2,809 3.75 3.75 .7 .4 .5 2 .17 .17 .61 .61
NP = normal phosphorus diet (.7% total); LP = low phosphorus diet (.4% total), respectively. 2 Provided per kilogram of diet: manganese, 65 mg; iodine, 1 mg; iron, 55 mg; copper, 6 mg; zinc, 55 mg; and cobalt, .2 mg. Provided per kilogram of diet: vitamin A, 8,000 IU; cholecalciferol, 2,200 ICU; vitamin E, 8 IU; vitamin B 1 2 , .02 mg; riboflavin, 5.5 mg; D-calcium pantothenic acid, 13 mg; niacin, 36 mg; choline, 500 mg; folic acid, 5 mg; thiamin, 1 mg; pyridoxine, 2.2 mg; biotin, .05 mg; menadione sodium bisulfate complex, .2 mg. 4 Analyzed levels were 3.77 and 3.76% for NP and LP treatments, respectively. 5 Analyzed levels were .68 and .39% for NP and LP treatments, respectively.
dietary P level were considered EM. The last 30 pullets that matured in each dietary P level were considered LM. This categorization resulted in four treatments in the experiment: 1) EM-NP, EM pullets consuming the NP diet; 2) EM-LP; EM pullets
^ o d e l 1011, Instron Corp., Canton, MA 02021. 4 Model NOVA-7, NOVA Biomedicals, Waltham, MA 02256. ^ o d e l DU-70; Beckman Instruments Inc., Fullerton, CA 92634. ^ o d e l 1100 B; Perkin-Elmer Corp., Norwalk, CT 06856. 7 Model 2780; Norland Corp., Fort Atkinson, WI 53538.
consuming the LP diet; 3) LM-NP; LM pullets consuming the NP diet; and 4) LMLP; LM pullets consuming the LP diet. The remaining pullets (the pullets that matured after 19 wk but not the LM) were considered "normal-maturing" and were used in the experiment only for egg production, egg specific gravity, and mortality but not for bone quality criteria, kidney histology, blood parameters, urine Ca concentration, and urine pH. The pullets were exposed to 16 h of continuous light daily (0400 to 2000 h) and feed and water were available for ad libitum consumption. Feed consumption was measured weekly. Egg specific gravity, egg weight, and shell breaking strength3 (probe speed 200 mm/min and probe diameter 15 mm) were determined when pullets were 26 and 28 wk of age. Serum samples from 15 randomly selected pullets within each dietary P level were tested for infectious bronchitis virus (IBV) antibody titers. When approximately 10 nonlaying pullets remained in the treatment LM-NP, blood (anaerobically) and urine samples were collected from 15 pullets in each treatment. The urine collection procedure was as described by Buss et al. (1980). Within each treatment, blood and urine were collected at 8 h postoviposition from the first 15 pullets in each treatment that had oviposition between 0600 and 1100 h. The LM group was limited to pullets that began laying an egg for only 2 or 3 days prior to urine sampling. Within 20 min of blood collection, plasma concentrations of Ca++, TCa, and percentage Ca ++ of the TCa (%Ca++) were determined using an analyzer.4 Plasma Pj concentration was determined using a spectrophotometer5 as described by Goldenberg and Fernandez (1966). The urine pH was determined immediately after collection and urine Ca concentration was determined using an atomic absorption spectrophotometer.6 Following blood and urine sampling, 20 randomly selected pullets within each treatment were weighed and then killed by cervical dislocation; comb, ovary, oviduct, and kidneys were removed and weighed. The kidney weight ratio was calculated. Both legs were removed and bone (tibiae) density and bone mineral content were determined using a Norland Bone Densitometer.7 The experiment was terminated for all criteria except egg production when
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Ground yellow corn Soybean meal (48.5% CP) Ground limestone Dicalcium phosphate Dehydrated alfalfa meal (17% CP) Sodium chloride Mineral prembr Vitamin prembr DL-methionine Sand
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RESULTS AND DISCUSSION pullets were 28 w k of age. Egg production records were until pullets reached 35 wk of age. Pre-Experimental Evaluation At the termination of the experiment, the The average BW of pullets at housing (18 pullets that were sick (lethargic, usually paralyzed, or unable to stand because of leg wk of age) was 1.17 kg. They were consumweakness) or osteoporotic were also killed. ing 60 g of feed daily and none of the pullets During the experiment, deaths were re- were laying. The EM and LM pullets were identified properly after they were housed corded for each dietary treatment. Dead because EM pullets had higher (P<.05) pullets were grouped into either EM or LM weights of comb (2.72 versus .34 g), body based on comb, ovary, and oviduct weights. (1.27 versus 1.04 kg), ovary (7.78 versus .48 The pullets that died during the experiment g), and oviduct (19.5 versus .39 g) than the and all pullets killed at the termination of LM pullets. The kidney weight ratio was the experiment were necropsied. lower than 1.1 in EM (1.01) and LM (1.03) pullets and was not different between the groups. A kidney weight ratio greater than Kidney Histology 1.1 could be considered asymmetric or The kidneys from pullets that were killed abnormal (Wideman and Cowen, 1987). at the beginning and at the termination of the experiment and from the pullets that Difference in Response died during the experiment were preserved of Early- and Late-Maturing Pullets in 10% neutral buffered formalin for histoto Low Dietary Phosphorus logical examination. They were processed for paraffin wax embedding, sectioned, and Irrespective of the onset of sexual maturstained with hematoxylin and eosin and ity, the LP diet caused low plasma Pj concentration, high urine Ca concentration, periodic acid-Schiff. elevated urine pH, kidney asymmetry, low bone mineral content, and low bone density (Table 2). However, the LP diet increased Statistical Analysis plasma Ca + + concentration (Table 2), the The data obtained from t h e p r e - number of sick and dead pullets (Table 3), experimental evaluation for EM and LM and kidney tubule lesions in the LM but not pullets on body, comb, ovary, and oviduct in the EM group. Influence of Low Dietary Phosphorus were subjected to one way ANOVA as outlined in Steel and Torrie (1980). The data on Skeletal System of Late-Maturing obtained for egg specific gravity, egg break- Pullets. Pullets fed the LP diet had higher ing strength, and egg size for the two urinary Ca concentration than the pullets dietary P treatments were also subjected to fed N P diet (Table 2). Increased urinary Ca one-way ANOVA. The data at the termina- concentration in pullets (Wideman, 1987) tion of the experiment for urine p H and and hens (Rao and Roland, 1990) in reurine Ca concentration, plasma Ca + + con- sponse to low dietary P has been reported. Increased urinary Ca excretion in laying centration, TCa concentration and %Ca ++ , hens could result from continuous depleand body, comb, ovary, oviduct, and kidtion of bone Ca reserve, the inability to ney weights for the EM and LM pullets in accrue Ca absorbed from intestine into the both dietary P treatments were subjected to bone, or both. The depletion of bone Ca an ANOVA appropriate for a factorial (DeLuca, 1979) and reduced bone mineraliarrangement of treatments. When signifi- zation (Norman, 1987) u n d e r h y p o cant (P<.05) interaction of dietary P treat- phosphatemia (low plasma Pi concentrament and sexual maturity (EM versus LM) tion) have been documented and in the occurred for any of the dependent vari- present experiment the pullets fed LP diets ables, the least significant difference test as were hypophosphatemic. However, only described in Steel and Torrie (1980) was LM pullets fed LP diets suffered with severe used to separate the means. osteoporosis and death (Tables 2 and 3),
EM LM
EM LM
.4% P .4% P
.7% P .7% P
15 15 ll2 15 15
n
NS
444
*** NS NS
NS
444
NS NS
*** NS ***
NS * NS
9.05 b 7.07b 3.74 57
1.48b 1.28c .03 57
6.93* 6.18b .37 57
21.68 b 28.85* 1.02 57
138b 1.25b
1.50b 1.60* 2.16* 2.16* .16 57
(mmol/L)
Ca 51.75* 6435*
Ca++ (%) 22.52 b 37.44*
Pi
Plasma
- (mmol/L) -
Ca++
6.76* 5.21 b
TCa
NS NS
6.72 b 6.86b .20 57
8.06* 7.83*
pH
Urine
1
Means in a column with no common superscripts are different (P<.05). A kidney weight ratio > 1.1 could be considered asymmetric or abnormal. 2 The pullets that were weak, lethargic, and unable to stand (osteoporosis). These belonged to .4% P dietary P treatment analyzed statistically because urine could not be collected from these pullets. *P<.05. ***P<.001.
a-c
SEM df Source of variation Phosphorus level (P) Maturity (M) P x M
Group
Treatment
TABLE 2. Influence of .4 and .7% (total) dietary P on plasma concentrations of total Ca (TCa), ion percentage Ca++ (%Ca++), urine pH and Ca concentration, kidney weight kidney weight ratio (hea and bone density (BD) in early- (EM) and late- (LM) maturing pullets (2
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RAO ET AL. TABLE 3. Influence of .4 and .7% total dietary phosphorus on pullet mortality rate in the flock from 18 to 28 wk of age Total
Dietarv treatment
Number of pullets dead Early-maturing Late-maturing
Mortality Dead1
In 10 wk
Monthly
(%) .4% total P .7% total P
4 0
22 0
26/300 0/300 2/l,140 1
8.67* 0b .17
3.46 0 .07
a/b
Means in a column with no common superscripts are different (P<.05). d u m b e r dead/ total. The rest of the 840 pullets (placed three pullets per cage) in the house were also fed a .7% (total) dietary P diet, which had the same nutrient composition as .7% (total) P diet used in the experiment. Two pullets out of 840 died [2 + (840 + 300)].
concentration and caused kidney asymmetry (kidney weight ratio > 1.1) (Table 2). In addition, in the LP-LM treatment, three pullets that died during the experiment had one or more kidney lobes missing. However, kidney failure may not be the primary cause of death in LP-LM pullets because only two pullets had solid urates (stones). Moreover, these stones were only partially blocking the ureters. Among the pullets killed at the termination of the experiment, kidneys from 45 pullets (11 from the EM-NP, EM-LP, and LM-LP groups and 12 from the LM-NP group; randomly selected within each treatment) were subjected to a histology study. Only seven kidneys (six from LM-LP and one from EM-LP) had some degree of tubular lesions. Those with mildest lesions had dilation of distal tubules with protein in tubular lesions. More severely affected kidneys had foci in which the distal tubules had necrosis of epithelium accompanied by casts of either protein, inflammatory cells, or detached tubular epithelial cells. In some tubules, urate tophi, represented by flocculent fibrinoid material arranged in radial pattern, were accompanied by necrosis of tubular epithelium and interstitial inflammation. The histologic lesions identified in Influence of Low Dietary Phosphorus the affected kidneys showed definitively on Kidneys of Late-Maturing Pullets. As that renal tubular injury had occurred. The described earlier, immature pullets fed severity of these lesions however was less diets containing layer Ca levels and LP had than that associated with renal failure, such high incidence of urolithiasis and subse- as substantial loss of nephrons with replacequently high mortality rates (Wideman et ment of functional parenchyma by inflamah, 1985). In the present experiment, LP (.4% matory cells and fibrosis. The lesions identitotal P) increased urine pH and urine Ca fied under the conditions of the present
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suggesting that the Ca:P ratio in the diet may be more critical to the maturing rather than the matured pullet. About 2 to 3 wk prior to the onset of egg production, plasma concentration of estrogen and testosterone increases, which in turn induces medullary bone formation in pullets (Kushuara and Schraer, 1982). Adequate dietary Ca or P with the proper ratio of these minerals seem to be more critical for the maturing pullets (because of medullary bone formation) than for matured hens. Medullary bone acts as a reservoir for Ca and P (Hurwitz, 1989) and reserves of these minerals are essential to sustain the high level of Ca metabolism in pullets as they mature (Garlich, 1979). In the present experiment, dietary treatments were introduced to pullets at 18 wk of age. At this age, EM pullets may have, at least partially, developed medullary bone, whereas LM pullets would not. Pullets in the LP-LM treatment only were exposed to a low dietary P situation during medullary bone formation and only in this treatment did several pullets exhibit severe osteoporosis and mortality rates (Tables 2 and 3). However, not all pullets in LP-LM treatment were severely affected, suggesting that pullets vary in their response to LP.
EFFECT OF DIETARY PHOSPHORUS ON MATURING PULLETS
Influence of Dietary Phosphorus on Feed Consumption and Production Parameters There was approximately a 9-wk span between the day of the first egg in the flock and the day the experiment was terminated (approximately 10 nonlaying LM pullets remaining in the LM-NP treatment). The pullets receiving .7% total P diet peaked with 93% production at 27 w k of age and were consuming 94 ± 3 g (x ± SE) of feed per day during peak production. The pullets fed LP diet, however, had delayed and lower peak production (82% peak production in 28th wk) and lower feed consumption (88 ± 4 g) than the pullets fed the NP diet. At 26 wk of age, the pullets consuming the LP diet produced better quality eggshells than pullets consuming the NP diet as indicated by egg specific gravity and egg breaking strength data (Table 4). Improvement in shell quality as a result of low dietary P has been reported previously (Taylor, 1965; Hamilton and Sibbald, 1977;
Miles et ah, 1983) and this could be caused by either one or a combination of the following two metabolic alterations induced by low plasma Pj. First, low plasma Pi stimulates the synthesis of 1,25dihydroxycholecalciferol in the kidney, which in turn influences Ca metabolism (Frost et ah, 1991). Second, low plasma Pj possibly increases Ca source solubilization in the digestive system (Sauveur and Mongin, 1983; Orban et ah, 1990), thereby elevating Ca bioavailability. However, in pullets approaching peak production, the improvement in shell quality as a result of the LP diet was only temporary because immediately following peak production (28 wk) shell quality in the LP treatment was poorer than that of the NP treatment. Pullets consuming the LP diets had lower bone mineral content and bone density at 28 wk of age (Table 2) than the pullets in N P treatments. Therefore, it seems that the pullets in LP treatments may have been using a greater quantity of Ca from bone to form eggshells or may not be adequately mineralizing the bones. Resorption of Ca from bone and inadequate bone mineralization are not considered economical from a nutritional viewpoint (Hurwitz, 1989).
Influence of Dietary Phosphorus on Mortality in Earlyand Late-Maturing Pullets The overall mortality rate for the flock (1,440 pullets) was 1.99%/10 w k [(26 + 2) x 100 + 1,440], or .77%/mo (calculated from Table 3). When the deaths were categorized into EM and LM groups, the highest mortality rate occurred in LM pullets consuming LP diet (Table 3). In addition, some pullets in the LP-LM treatment exhibited various degrees of osteoporosis (Table 2). Also, in the LP-LM treatment, the number of sick pullets and the number of sick pullets that died increased as the production increased and approached peak (data not shown). Soft bones and a slight increase in mortality rate are often observed as flocks approach peak egg production (unpublished data). The primary cause of this skeletal problem and elevated mortality rates in relation to egg production would be difficult to diagnose in the field because
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experiment demonstrated renal injury, but not overt renal failure. Although renal lesions targeted tubular epithelium, it should be recognized that other agents could cause similar lesions. In pullets and hens, severe kidney failure, caused by blocked ureters, would result in sudden death without the animal exhibiting any external signs prior to death (Wideman et ah, 1985). In the present experiment, however, pullets showed typical signs of "cage-layer fatigue" or osteoporosis. They were lethargic, paralyzed, or unable to stand because of weak legs, and subsequently died after several days. The kidney lesions observed in some pullets in LP-LM treatment may have been secondary. Some strains of IBV are known to be nephrotrophic (Wideman and Cowen, 1987). However, in the current experiment, the antibody titer for IBV in the serum was relatively low, indicating low natural exposure (869 ± 431; x ± SD) and was similar for all treatments. Only 7 out of 45 pullets had any degree of kidney lesions and 6 of them were in LP-LM treatment. Therefore, the adverse influence of LP on the kidney could not be completely ruled out.
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TABLE 4. Averages of egg specific gravity, eggshell breaking strength, and egg size for the pullets fed .4 and .7% total P diets
gg specific gravity
26 wk Eggshell breaking strength
Egg weight
1.0878* 1.0869b .0002 597
(kg/cm2) 2.14* 1.99b .04 638
(g) 51.11* 51.07* .4 638
E
Dietary treatment .4% total P .7% total P SEM df
Egg specific gravity b
1.0847 1.0866* .0001 623
28 wk Eggshell breaking strength
Egg weight
(kg/cm2) 1.94b 2.09* .04 675
(g) 51.46* 51.87* .32 675
ab
' Means within a column with no common superscripts are significantly different (P<.05).
ACKNOWLEDGMENTS This work was supported in part by Pitman-Moore Inc., Terre Haute, IN 47808. The authors wish to thank Matilda M. Bryant, Joseph I. Orban, Henry W. Rabon, Jyothi K. Rao, Thomas J. Frost, John S. Kotrola, and Willie J. Hill for their technical assistance.
REFERENCES Buss, E. G., P. Merkur, and R. B. Guyer, 1980. Urinary excretion of calcium in the presence or absence of shell formation by chickens producing thick or thin shells. Poultry Sci. 59:885-887. DeLuca, H. F., 1979. The vitamin D system in the regulation of calcium and phosphorus metabolism. Nutr. Rev. 37:161-366. Dunnington, E. A., and P. B. Siegel, 1984. Age and body weight at sexual maturity in female White Leghorn chickens. Poultry Sci. 63:828-830. Frost, T. J., D. A. Roland, Sr., and D. N. Marple, 1991. The effects of various dietary phosphorus levels on the arcadian patterns of plasma 1,25-dihydroxycholecalciferol, total calcium, ionized calcium, and phosphorus in laying hens. Poultry Sci. 70:1564-1570. Garlich, J. D., 1979. The phosphorus requirement for the laying hens. Pages 104-114 in: Proceedings of the Georgia Nutrition Conference for the Feed Industry. University of Georgia, Athens, GA. Goldenberg, H., and A. Fernandez, 1966. Simplified method for estimation of inorganic phosphorus in body fluids. Clin. Chem. 12:871-882. Hamilton, R.M.G., and I. R Sibbald, 1977. The effects of dietary phosphorus on productive performance and egg quality of ten strains of White Leghorns. Poultry Sci. 56:1221-1228. Hurwitz, S., 1989. Calcium homeostasis in birds. Vitam. Horm. 45:173-221. Keshavarz, K., 1987. Influence of feeding a high calcium diet for various durations in prelaying period on growth and subsequent performance of White Leghorn pullets. Poultry Sci. 66: 1576-158Z Kushuara, S., and H. Schraer, 1982. Cytology and autoradiography of estrogen-induced differentiation of avian endosteal cells. Calcif. Tissue Int. 34-352-358. Miles, R. D., P. T. Costa, and R. H. Harms, 1983. The influence of dietary phosphorus level on laying hen performance, egg shell quality and various blood parameters. Poultry Sci. 62:1033-1037. Norman, A. W., 1987. Studies on the vitamin D endocrine system in the avian. J. Nutr. 117: 797-807.
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only a few pullets exhibit these signs whereas the majority appear normal. Furthermore, diagnosis of the primary cause these problems are complicated by wide variations in body weight, feed consumption, and mortality pattern observed within a flock and among flocks in the field. However, the results of the present experiment demonstrated that the skeletal problems and mortality rates of LM pullets in response to LP diet are similar to observations in commercial flocks. Although diets are formulated to meet the P requirements, some pullets in the flock could be consuming less than 200 mg P / d a y (Roland, 1989). The LP diet used in the present experiment was formulated based on the assumption that some pullets in the field may be consuming P between 100 to 200 m g / d a y . The results demonstrate that low dietary P adversely affects maturing pullets; however, the severity is greater on LM than on EM pullets. These differences in response of LM and EM pullets to dietary P levels imply that flock uniformity in rate of sexual maturation should be a factor in determining P level in pullet diets. A higher margin of safety for dietary P level may be necessary if the flock in question is less uniform.
EFFECT OF DIETARY PHOSPHORUS ON MATURING PULLETS
Sauveur, B., and P. Mongin, 1983. Plasma inorganic phosphorus concentration during egg shell formation, n. Inverse relationship with intestinal calcium content and shell weight. Reprod. Nutr. Dev. 23:755-764. Steel, R.G.D., and J. H. Torrie, 1980. Principles and Procedures of Statistics: A Biometrical Approach. 2nd ed. McGraw Hill Book Co., New York, NY. Taylor, T. G., 1965. Dietary phosphorus and egg shell thickness in the domestic fowl. Br. Poult. Sci. 6: 79-87. Wideman, R. F., Jr., 1987. Renal regulation of avian calcium and phosphorus metabolism. J. Nutr. 117:808-815. Wideman, R. F., Jr., J. A. Qosser, W. B. Roush, and B. S. Cowen, 1985. Urolithiasis in pullets and laying hens: Role of dietary calcium and phosphorus. Poultry Sci. 642300-2307. Wideman, R. F., Jr., and B. S. Cowen, 1987. Effect of dietary acidification on kidney damage induced in immature chickens by excess calcium and infectious bronchitis virus. Poultry Sci. 66: 626-633.
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North, M. O., 1984. Feeding egg-type growing pullets. Pages 496-^516 in: Commercial Chicken Production Manual. AVI Publishing Company, Westport, CT. Orban, J. I., K. S. Rao, and D. A. Roland, Sr., 1990. Influence of phosphorus (P) level on limestone (LMS) solubilization in the digestive tract of Leghorn hens. Poultry Sci. 69(Suppl. 1): 101.(Abstr.) Oldroyd, N. O., and R. F. Wideman, Jr., 1986. Characterization and composition of uroliths from domestic fowl. Poultry Sci. 65:1090-1094. Rao, K. S., and D. A. Roland, Sr., 1990. Influence of dietary calcium and phosphorus on urinary Ca in commercial leghorn hens. Poultry Sci. 69: 1991-1997. Roland, D. A., Sr., 1986. Egg shell quality II. Calcium and phosphorus requirements of commercial leghorns. World's Poult. Sci. J. 42:154-165. Roland, D. A., Sr., 1989. Phosphorus requirement of commercial Leghorns. Pages 120-139 in: Proceedings of Georgia Nutrition Conference for the Feed Industry. University of Georgia, Athens, GA.
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INTRODUCTION Maturing commercial Leghorn pullets are usually fed layer diets 1 to 2 wk prior to first egg (North, 1984; Roland, 1986; Keshavarz, 1987) or no later than 5% egg production (Roland, 1986). However, in every flock, pullets become sexually mature over several weeks (Dunnington and
Alabama Agricultural Experiment Station Journal Series Number 12-913011P.
Siegel, 1984). The number of days required for early- (EM) and late-maturing (LM) pullets to reach sexual maturity and the proportion of LM pullets may vary from flock to flock. Nevertheless, when pullets are introduced to layer diets based on average flock performance, the LM pullets are inevitably exposed to layer diets for at least 3 to 10 wk prior to reaching sexual maturity. Currently, there is wide variation in dietary P levels and the Ca:P ratio in layer diets used by various companies (Roland,
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ABSTRACT An experiment with a 2 x 2 factorial arrangement of treatments was used to determine differences in responses of early-maturing (EM) and late-maturing (LM) pullets fed low dietary P (.4% total P; LP) and normal dietary P (.7% total P; NP) levels. Six hundred pullets (18-wk-old) were equally and randomly allocated to the LP and NP treatments. Egg production, egg weight, egg specific gravity, and eggshell breaking strength were measured at 26 and 28 wk. When pullets were 28 wk of age, plasma total Ca (TCa), ionic Ca (Ca++), and inorganic P (Pi) concentrations, urine Ca concentrations, and urine pH, bone mineral content (BMC), bone density, total kidney weight, and kidney weight ratio (heavier kidney + lighter kidney) were determined from 15 pullets each from EM-NP, EM-LP, LM-NP, and LM-LP treatments. The pullets with osteoporosis and pullets that died during the experiment were categorized into EM and LM groups. Results showed that LP caused severe adverse effects on LM pullets. The LM pullets fed the LP diet had high plasma Ca++ concentration, low plasma Pj concentration, increased urine Ca concentration, a high incidence of osteoporosis, mild kidney lesions, and elevated mortality compared with pullets subjected to the other treatments. The EM pullets fed the LP diet were also adversely effected by LP, but were less susceptible to osteoporosis and mortality. The LP diet improved eggshell quality, but this beneficial effect was only temporary. The severity of adverse effects of low dietary P was greater for LM than the EM pullets. (Key words: cage-layer fatigue, phosphorus, osteoporosis, maturing pullets, sexual maturity)
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•Hy-Line Indian River Co., Des Moines, IA 50265.
The objective of the present experiment was to determine the effects of low dietary P on plasma concentrations of ionic Ca (Ca ++ ), total Ca (TCa), inorganic P (Pi), urine pH, urine Ca concentration, bone mineral content (BMC), bone density (BD), kidney morphology, and mortality rate of EM and LM pullets.
MATERIALS AND METHODS Pre-Experimental Evaluation Pullets for pre-experimental evaluation were sampled from the same population of pullets used in the experiment. These evaluations were done to determine whether 1) the identification of EM and LM pullets based on secondary sexual characteristics was accurate; and 2) the pullets had normal kidneys prior to the experiment. Eighteen-week-old Single Comb White Leghorn pullets (Hy-Line® W362) were used. As the pullets were housed, 15 EM and LM pullets were selected based on secondary sex characteristics (comb size and color). These pullets were weighed and then killed by cervical dislocation; the comb, oviduct, ovary, and kidneys were removed and weighed. The kidney weight ratio (heavier kidney + lighter kidney) was determined for each pullet. Average live weight of pullets was determined by individually weighing 150 randomly selected pullets.
Experimental Procedure Six hundred pullets (18-wk-old) were housed individually in cages. In remaining cages, 840 pullets were housed three per cage (these 840 pullets were not included in the experiment except for pre-experimental evaluations and for the comparison of mortality). The 600 individually caged pullets were equally and randomly allocated to a low (.4% total P; LP) and a normal P (.7% total P; NP) diet (Table 1). The calculated available P in LP and N P diets were .5 and .2%, respectively. Within each diet, the EM and LM pullets were identified using secondary sexual characteristics and egg production records. The pullets that began egg production before or during 19 wk of age in each
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1989). With such variation in dietary levels of P, it is possible that some LM pullets may be exposed to diets containing low P for a few weeks prior to ovulation. Immature pullets (8-wk-old) exposed to a layer diet containing high Ca and low P for 10 wk prior to first egg had calciurea, urolithiasis, and a high mortality rate (Wideman et ah, 1985). It is possible that LM pullets in a flock fed layer diets containing low P would respond similarly to immature pullets fed a high Ca and low P diet; however, this has been not determined. Severe P deficiency causes rickets and subsequent high mortality rates in pullets and hens (Garlich, 1979), but marginal P level only slightly elevates flock mortality (Garlich, 1979; North, 1984). Slight increases in flock mortality may be attributed to the detrimental effect of low P in combination with layer level of dietary Ca (Ca adequate for laying hens) on LM pullets. Low dietary P could predispose pullets to other stressors (pathogens, inadequate nutrition, high temperature, and inadequate management) through depletion of bone Ca and possibly through renal stress. Increased urine Ca excretion induced by low dietary P may deplete bone Ca reserves, leading to a gradual increase in skeletal problems. Also, with normal and excess dietary Ca levels, low dietary P establishes ideal conditions for solid urate formation in kidney and ureters by elevating urine Ca concentration and p H (Oldroyd and Wideman, 1986). Although low dietary P may have caused kidney or skeletal problems, proper diagnosis in the field would be difficult because subclinical signs or symptoms exhibited by affected pullets may relate to several secondary causes. With various stressors affecting flock mortality, it would be difficult to differentiate the adverse effects of low dietary P on a small proportion of LM pullets in the flock. Therefore, to distinguish low dietary P effects on LM pullets, an experiment using LM and EM pullets would be required rather than using the entire flock.
EFFECT OF DIETARY PHOSPHORUS ON MATURING PULLETS TABLE 1. Composition and calculated analysis of the treatment diets Dietary treatments Ingredients and analysis
NP
LP
(%)
Total Calculated analysis Protein, % ME, kcal/kg Calcium/ % Total phosphorus, % Available phosphorus, % Sodium, % Methionine plus cystine, %
66.62 20.90 8.10 2.18
66.62 20.90 8.99 57
1.00 .37 35 25 .07 26 100.00
1.00 .37 25 25 .07 .98 100.00
16 16 2,809 2,809 3.75 3.75 .7 .4 .5 2 .17 .17 .61 .61
NP = normal phosphorus diet (.7% total); LP = low phosphorus diet (.4% total), respectively. 2 Provided per kilogram of diet: manganese, 65 mg; iodine, 1 mg; iron, 55 mg; copper, 6 mg; zinc, 55 mg; and cobalt, .2 mg. Provided per kilogram of diet: vitamin A, 8,000 IU; cholecalciferol, 2,200 ICU; vitamin E, 8 IU; vitamin B 1 2 , .02 mg; riboflavin, 5.5 mg; D-calcium pantothenic acid, 13 mg; niacin, 36 mg; choline, 500 mg; folic acid, 5 mg; thiamin, 1 mg; pyridoxine, 2.2 mg; biotin, .05 mg; menadione sodium bisulfate complex, .2 mg. 4 Analyzed levels were 3.77 and 3.76% for NP and LP treatments, respectively. 5 Analyzed levels were .68 and .39% for NP and LP treatments, respectively.
dietary P level were considered EM. The last 30 pullets that matured in each dietary P level were considered LM. This categorization resulted in four treatments in the experiment: 1) EM-NP, EM pullets consuming the NP diet; 2) EM-LP; EM pullets
^ o d e l 1011, Instron Corp., Canton, MA 02021. 4 Model NOVA-7, NOVA Biomedicals, Waltham, MA 02256. ^ o d e l DU-70; Beckman Instruments Inc., Fullerton, CA 92634. ^ o d e l 1100 B; Perkin-Elmer Corp., Norwalk, CT 06856. 7 Model 2780; Norland Corp., Fort Atkinson, WI 53538.
consuming the LP diet; 3) LM-NP; LM pullets consuming the NP diet; and 4) LMLP; LM pullets consuming the LP diet. The remaining pullets (the pullets that matured after 19 wk but not the LM) were considered "normal-maturing" and were used in the experiment only for egg production, egg specific gravity, and mortality but not for bone quality criteria, kidney histology, blood parameters, urine Ca concentration, and urine pH. The pullets were exposed to 16 h of continuous light daily (0400 to 2000 h) and feed and water were available for ad libitum consumption. Feed consumption was measured weekly. Egg specific gravity, egg weight, and shell breaking strength3 (probe speed 200 mm/min and probe diameter 15 mm) were determined when pullets were 26 and 28 wk of age. Serum samples from 15 randomly selected pullets within each dietary P level were tested for infectious bronchitis virus (IBV) antibody titers. When approximately 10 nonlaying pullets remained in the treatment LM-NP, blood (anaerobically) and urine samples were collected from 15 pullets in each treatment. The urine collection procedure was as described by Buss et al. (1980). Within each treatment, blood and urine were collected at 8 h postoviposition from the first 15 pullets in each treatment that had oviposition between 0600 and 1100 h. The LM group was limited to pullets that began laying an egg for only 2 or 3 days prior to urine sampling. Within 20 min of blood collection, plasma concentrations of Ca++, TCa, and percentage Ca ++ of the TCa (%Ca++) were determined using an analyzer.4 Plasma Pj concentration was determined using a spectrophotometer5 as described by Goldenberg and Fernandez (1966). The urine pH was determined immediately after collection and urine Ca concentration was determined using an atomic absorption spectrophotometer.6 Following blood and urine sampling, 20 randomly selected pullets within each treatment were weighed and then killed by cervical dislocation; comb, ovary, oviduct, and kidneys were removed and weighed. The kidney weight ratio was calculated. Both legs were removed and bone (tibiae) density and bone mineral content were determined using a Norland Bone Densitometer.7 The experiment was terminated for all criteria except egg production when
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Ground yellow corn Soybean meal (48.5% CP) Ground limestone Dicalcium phosphate Dehydrated alfalfa meal (17% CP) Sodium chloride Mineral prembr Vitamin prembr DL-methionine Sand
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RESULTS AND DISCUSSION pullets were 28 w k of age. Egg production records were until pullets reached 35 wk of age. Pre-Experimental Evaluation At the termination of the experiment, the The average BW of pullets at housing (18 pullets that were sick (lethargic, usually paralyzed, or unable to stand because of leg wk of age) was 1.17 kg. They were consumweakness) or osteoporotic were also killed. ing 60 g of feed daily and none of the pullets During the experiment, deaths were re- were laying. The EM and LM pullets were identified properly after they were housed corded for each dietary treatment. Dead because EM pullets had higher (P<.05) pullets were grouped into either EM or LM weights of comb (2.72 versus .34 g), body based on comb, ovary, and oviduct weights. (1.27 versus 1.04 kg), ovary (7.78 versus .48 The pullets that died during the experiment g), and oviduct (19.5 versus .39 g) than the and all pullets killed at the termination of LM pullets. The kidney weight ratio was the experiment were necropsied. lower than 1.1 in EM (1.01) and LM (1.03) pullets and was not different between the groups. A kidney weight ratio greater than Kidney Histology 1.1 could be considered asymmetric or The kidneys from pullets that were killed abnormal (Wideman and Cowen, 1987). at the beginning and at the termination of the experiment and from the pullets that Difference in Response died during the experiment were preserved of Early- and Late-Maturing Pullets in 10% neutral buffered formalin for histoto Low Dietary Phosphorus logical examination. They were processed for paraffin wax embedding, sectioned, and Irrespective of the onset of sexual maturstained with hematoxylin and eosin and ity, the LP diet caused low plasma Pj concentration, high urine Ca concentration, periodic acid-Schiff. elevated urine pH, kidney asymmetry, low bone mineral content, and low bone density (Table 2). However, the LP diet increased Statistical Analysis plasma Ca + + concentration (Table 2), the The data obtained from t h e p r e - number of sick and dead pullets (Table 3), experimental evaluation for EM and LM and kidney tubule lesions in the LM but not pullets on body, comb, ovary, and oviduct in the EM group. Influence of Low Dietary Phosphorus were subjected to one way ANOVA as outlined in Steel and Torrie (1980). The data on Skeletal System of Late-Maturing obtained for egg specific gravity, egg break- Pullets. Pullets fed the LP diet had higher ing strength, and egg size for the two urinary Ca concentration than the pullets dietary P treatments were also subjected to fed N P diet (Table 2). Increased urinary Ca one-way ANOVA. The data at the termina- concentration in pullets (Wideman, 1987) tion of the experiment for urine p H and and hens (Rao and Roland, 1990) in reurine Ca concentration, plasma Ca + + con- sponse to low dietary P has been reported. Increased urinary Ca excretion in laying centration, TCa concentration and %Ca ++ , hens could result from continuous depleand body, comb, ovary, oviduct, and kidtion of bone Ca reserve, the inability to ney weights for the EM and LM pullets in accrue Ca absorbed from intestine into the both dietary P treatments were subjected to bone, or both. The depletion of bone Ca an ANOVA appropriate for a factorial (DeLuca, 1979) and reduced bone mineraliarrangement of treatments. When signifi- zation (Norman, 1987) u n d e r h y p o cant (P<.05) interaction of dietary P treat- phosphatemia (low plasma Pi concentrament and sexual maturity (EM versus LM) tion) have been documented and in the occurred for any of the dependent vari- present experiment the pullets fed LP diets ables, the least significant difference test as were hypophosphatemic. However, only described in Steel and Torrie (1980) was LM pullets fed LP diets suffered with severe used to separate the means. osteoporosis and death (Tables 2 and 3),
EM LM
EM LM
.4% P .4% P
.7% P .7% P
15 15 ll2 15 15
n
NS
444
*** NS NS
NS
444
NS NS
*** NS ***
NS * NS
9.05 b 7.07b 3.74 57
1.48b 1.28c .03 57
6.93* 6.18b .37 57
21.68 b 28.85* 1.02 57
138b 1.25b
1.50b 1.60* 2.16* 2.16* .16 57
(mmol/L)
Ca 51.75* 6435*
Ca++ (%) 22.52 b 37.44*
Pi
Plasma
- (mmol/L) -
Ca++
6.76* 5.21 b
TCa
NS NS
6.72 b 6.86b .20 57
8.06* 7.83*
pH
Urine
1
Means in a column with no common superscripts are different (P<.05). A kidney weight ratio > 1.1 could be considered asymmetric or abnormal. 2 The pullets that were weak, lethargic, and unable to stand (osteoporosis). These belonged to .4% P dietary P treatment analyzed statistically because urine could not be collected from these pullets. *P<.05. ***P<.001.
a-c
SEM df Source of variation Phosphorus level (P) Maturity (M) P x M
Group
Treatment
TABLE 2. Influence of .4 and .7% (total) dietary P on plasma concentrations of total Ca (TCa), ion percentage Ca++ (%Ca++), urine pH and Ca concentration, kidney weight kidney weight ratio (hea and bone density (BD) in early- (EM) and late- (LM) maturing pullets (2
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RAO ET AL. TABLE 3. Influence of .4 and .7% total dietary phosphorus on pullet mortality rate in the flock from 18 to 28 wk of age Total
Dietarv treatment
Number of pullets dead Early-maturing Late-maturing
Mortality Dead1
In 10 wk
Monthly
(%) .4% total P .7% total P
4 0
22 0
26/300 0/300 2/l,140 1
8.67* 0b .17
3.46 0 .07
a/b
Means in a column with no common superscripts are different (P<.05). d u m b e r dead/ total. The rest of the 840 pullets (placed three pullets per cage) in the house were also fed a .7% (total) dietary P diet, which had the same nutrient composition as .7% (total) P diet used in the experiment. Two pullets out of 840 died [2 + (840 + 300)].
concentration and caused kidney asymmetry (kidney weight ratio > 1.1) (Table 2). In addition, in the LP-LM treatment, three pullets that died during the experiment had one or more kidney lobes missing. However, kidney failure may not be the primary cause of death in LP-LM pullets because only two pullets had solid urates (stones). Moreover, these stones were only partially blocking the ureters. Among the pullets killed at the termination of the experiment, kidneys from 45 pullets (11 from the EM-NP, EM-LP, and LM-LP groups and 12 from the LM-NP group; randomly selected within each treatment) were subjected to a histology study. Only seven kidneys (six from LM-LP and one from EM-LP) had some degree of tubular lesions. Those with mildest lesions had dilation of distal tubules with protein in tubular lesions. More severely affected kidneys had foci in which the distal tubules had necrosis of epithelium accompanied by casts of either protein, inflammatory cells, or detached tubular epithelial cells. In some tubules, urate tophi, represented by flocculent fibrinoid material arranged in radial pattern, were accompanied by necrosis of tubular epithelium and interstitial inflammation. The histologic lesions identified in Influence of Low Dietary Phosphorus the affected kidneys showed definitively on Kidneys of Late-Maturing Pullets. As that renal tubular injury had occurred. The described earlier, immature pullets fed severity of these lesions however was less diets containing layer Ca levels and LP had than that associated with renal failure, such high incidence of urolithiasis and subse- as substantial loss of nephrons with replacequently high mortality rates (Wideman et ment of functional parenchyma by inflamah, 1985). In the present experiment, LP (.4% matory cells and fibrosis. The lesions identitotal P) increased urine pH and urine Ca fied under the conditions of the present
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suggesting that the Ca:P ratio in the diet may be more critical to the maturing rather than the matured pullet. About 2 to 3 wk prior to the onset of egg production, plasma concentration of estrogen and testosterone increases, which in turn induces medullary bone formation in pullets (Kushuara and Schraer, 1982). Adequate dietary Ca or P with the proper ratio of these minerals seem to be more critical for the maturing pullets (because of medullary bone formation) than for matured hens. Medullary bone acts as a reservoir for Ca and P (Hurwitz, 1989) and reserves of these minerals are essential to sustain the high level of Ca metabolism in pullets as they mature (Garlich, 1979). In the present experiment, dietary treatments were introduced to pullets at 18 wk of age. At this age, EM pullets may have, at least partially, developed medullary bone, whereas LM pullets would not. Pullets in the LP-LM treatment only were exposed to a low dietary P situation during medullary bone formation and only in this treatment did several pullets exhibit severe osteoporosis and mortality rates (Tables 2 and 3). However, not all pullets in LP-LM treatment were severely affected, suggesting that pullets vary in their response to LP.
EFFECT OF DIETARY PHOSPHORUS ON MATURING PULLETS
Influence of Dietary Phosphorus on Feed Consumption and Production Parameters There was approximately a 9-wk span between the day of the first egg in the flock and the day the experiment was terminated (approximately 10 nonlaying LM pullets remaining in the LM-NP treatment). The pullets receiving .7% total P diet peaked with 93% production at 27 w k of age and were consuming 94 ± 3 g (x ± SE) of feed per day during peak production. The pullets fed LP diet, however, had delayed and lower peak production (82% peak production in 28th wk) and lower feed consumption (88 ± 4 g) than the pullets fed the NP diet. At 26 wk of age, the pullets consuming the LP diet produced better quality eggshells than pullets consuming the NP diet as indicated by egg specific gravity and egg breaking strength data (Table 4). Improvement in shell quality as a result of low dietary P has been reported previously (Taylor, 1965; Hamilton and Sibbald, 1977;
Miles et ah, 1983) and this could be caused by either one or a combination of the following two metabolic alterations induced by low plasma Pj. First, low plasma Pi stimulates the synthesis of 1,25dihydroxycholecalciferol in the kidney, which in turn influences Ca metabolism (Frost et ah, 1991). Second, low plasma Pj possibly increases Ca source solubilization in the digestive system (Sauveur and Mongin, 1983; Orban et ah, 1990), thereby elevating Ca bioavailability. However, in pullets approaching peak production, the improvement in shell quality as a result of the LP diet was only temporary because immediately following peak production (28 wk) shell quality in the LP treatment was poorer than that of the NP treatment. Pullets consuming the LP diets had lower bone mineral content and bone density at 28 wk of age (Table 2) than the pullets in N P treatments. Therefore, it seems that the pullets in LP treatments may have been using a greater quantity of Ca from bone to form eggshells or may not be adequately mineralizing the bones. Resorption of Ca from bone and inadequate bone mineralization are not considered economical from a nutritional viewpoint (Hurwitz, 1989).
Influence of Dietary Phosphorus on Mortality in Earlyand Late-Maturing Pullets The overall mortality rate for the flock (1,440 pullets) was 1.99%/10 w k [(26 + 2) x 100 + 1,440], or .77%/mo (calculated from Table 3). When the deaths were categorized into EM and LM groups, the highest mortality rate occurred in LM pullets consuming LP diet (Table 3). In addition, some pullets in the LP-LM treatment exhibited various degrees of osteoporosis (Table 2). Also, in the LP-LM treatment, the number of sick pullets and the number of sick pullets that died increased as the production increased and approached peak (data not shown). Soft bones and a slight increase in mortality rate are often observed as flocks approach peak egg production (unpublished data). The primary cause of this skeletal problem and elevated mortality rates in relation to egg production would be difficult to diagnose in the field because
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experiment demonstrated renal injury, but not overt renal failure. Although renal lesions targeted tubular epithelium, it should be recognized that other agents could cause similar lesions. In pullets and hens, severe kidney failure, caused by blocked ureters, would result in sudden death without the animal exhibiting any external signs prior to death (Wideman et ah, 1985). In the present experiment, however, pullets showed typical signs of "cage-layer fatigue" or osteoporosis. They were lethargic, paralyzed, or unable to stand because of weak legs, and subsequently died after several days. The kidney lesions observed in some pullets in LP-LM treatment may have been secondary. Some strains of IBV are known to be nephrotrophic (Wideman and Cowen, 1987). However, in the current experiment, the antibody titer for IBV in the serum was relatively low, indicating low natural exposure (869 ± 431; x ± SD) and was similar for all treatments. Only 7 out of 45 pullets had any degree of kidney lesions and 6 of them were in LP-LM treatment. Therefore, the adverse influence of LP on the kidney could not be completely ruled out.
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TABLE 4. Averages of egg specific gravity, eggshell breaking strength, and egg size for the pullets fed .4 and .7% total P diets
gg specific gravity
26 wk Eggshell breaking strength
Egg weight
1.0878* 1.0869b .0002 597
(kg/cm2) 2.14* 1.99b .04 638
(g) 51.11* 51.07* .4 638
E
Dietary treatment .4% total P .7% total P SEM df
Egg specific gravity b
1.0847 1.0866* .0001 623
28 wk Eggshell breaking strength
Egg weight
(kg/cm2) 1.94b 2.09* .04 675
(g) 51.46* 51.87* .32 675
ab
' Means within a column with no common superscripts are significantly different (P<.05).
ACKNOWLEDGMENTS This work was supported in part by Pitman-Moore Inc., Terre Haute, IN 47808. The authors wish to thank Matilda M. Bryant, Joseph I. Orban, Henry W. Rabon, Jyothi K. Rao, Thomas J. Frost, John S. Kotrola, and Willie J. Hill for their technical assistance.
REFERENCES Buss, E. G., P. Merkur, and R. B. Guyer, 1980. Urinary excretion of calcium in the presence or absence of shell formation by chickens producing thick or thin shells. Poultry Sci. 59:885-887. DeLuca, H. F., 1979. The vitamin D system in the regulation of calcium and phosphorus metabolism. Nutr. Rev. 37:161-366. Dunnington, E. A., and P. B. Siegel, 1984. Age and body weight at sexual maturity in female White Leghorn chickens. Poultry Sci. 63:828-830. Frost, T. J., D. A. Roland, Sr., and D. N. Marple, 1991. The effects of various dietary phosphorus levels on the arcadian patterns of plasma 1,25-dihydroxycholecalciferol, total calcium, ionized calcium, and phosphorus in laying hens. Poultry Sci. 70:1564-1570. Garlich, J. D., 1979. The phosphorus requirement for the laying hens. Pages 104-114 in: Proceedings of the Georgia Nutrition Conference for the Feed Industry. University of Georgia, Athens, GA. Goldenberg, H., and A. Fernandez, 1966. Simplified method for estimation of inorganic phosphorus in body fluids. Clin. Chem. 12:871-882. Hamilton, R.M.G., and I. R Sibbald, 1977. The effects of dietary phosphorus on productive performance and egg quality of ten strains of White Leghorns. Poultry Sci. 56:1221-1228. Hurwitz, S., 1989. Calcium homeostasis in birds. Vitam. Horm. 45:173-221. Keshavarz, K., 1987. Influence of feeding a high calcium diet for various durations in prelaying period on growth and subsequent performance of White Leghorn pullets. Poultry Sci. 66: 1576-158Z Kushuara, S., and H. Schraer, 1982. Cytology and autoradiography of estrogen-induced differentiation of avian endosteal cells. Calcif. Tissue Int. 34-352-358. Miles, R. D., P. T. Costa, and R. H. Harms, 1983. The influence of dietary phosphorus level on laying hen performance, egg shell quality and various blood parameters. Poultry Sci. 62:1033-1037. Norman, A. W., 1987. Studies on the vitamin D endocrine system in the avian. J. Nutr. 117: 797-807.
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only a few pullets exhibit these signs whereas the majority appear normal. Furthermore, diagnosis of the primary cause these problems are complicated by wide variations in body weight, feed consumption, and mortality pattern observed within a flock and among flocks in the field. However, the results of the present experiment demonstrated that the skeletal problems and mortality rates of LM pullets in response to LP diet are similar to observations in commercial flocks. Although diets are formulated to meet the P requirements, some pullets in the flock could be consuming less than 200 mg P / d a y (Roland, 1989). The LP diet used in the present experiment was formulated based on the assumption that some pullets in the field may be consuming P between 100 to 200 m g / d a y . The results demonstrate that low dietary P adversely affects maturing pullets; however, the severity is greater on LM than on EM pullets. These differences in response of LM and EM pullets to dietary P levels imply that flock uniformity in rate of sexual maturation should be a factor in determining P level in pullet diets. A higher margin of safety for dietary P level may be necessary if the flock in question is less uniform.
EFFECT OF DIETARY PHOSPHORUS ON MATURING PULLETS
Sauveur, B., and P. Mongin, 1983. Plasma inorganic phosphorus concentration during egg shell formation, n. Inverse relationship with intestinal calcium content and shell weight. Reprod. Nutr. Dev. 23:755-764. Steel, R.G.D., and J. H. Torrie, 1980. Principles and Procedures of Statistics: A Biometrical Approach. 2nd ed. McGraw Hill Book Co., New York, NY. Taylor, T. G., 1965. Dietary phosphorus and egg shell thickness in the domestic fowl. Br. Poult. Sci. 6: 79-87. Wideman, R. F., Jr., 1987. Renal regulation of avian calcium and phosphorus metabolism. J. Nutr. 117:808-815. Wideman, R. F., Jr., J. A. Qosser, W. B. Roush, and B. S. Cowen, 1985. Urolithiasis in pullets and laying hens: Role of dietary calcium and phosphorus. Poultry Sci. 642300-2307. Wideman, R. F., Jr., and B. S. Cowen, 1987. Effect of dietary acidification on kidney damage induced in immature chickens by excess calcium and infectious bronchitis virus. Poultry Sci. 66: 626-633.
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North, M. O., 1984. Feeding egg-type growing pullets. Pages 496-^516 in: Commercial Chicken Production Manual. AVI Publishing Company, Westport, CT. Orban, J. I., K. S. Rao, and D. A. Roland, Sr., 1990. Influence of phosphorus (P) level on limestone (LMS) solubilization in the digestive tract of Leghorn hens. Poultry Sci. 69(Suppl. 1): 101.(Abstr.) Oldroyd, N. O., and R. F. Wideman, Jr., 1986. Characterization and composition of uroliths from domestic fowl. Poultry Sci. 65:1090-1094. Rao, K. S., and D. A. Roland, Sr., 1990. Influence of dietary calcium and phosphorus on urinary Ca in commercial leghorn hens. Poultry Sci. 69: 1991-1997. Roland, D. A., Sr., 1986. Egg shell quality II. Calcium and phosphorus requirements of commercial leghorns. World's Poult. Sci. J. 42:154-165. Roland, D. A., Sr., 1989. Phosphorus requirement of commercial Leghorns. Pages 120-139 in: Proceedings of Georgia Nutrition Conference for the Feed Industry. University of Georgia, Athens, GA.
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