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J. Nutr. 64:587–604. Jackson, M. E., and P. W. Waldrup. 1988. The effect of dietary ... Owings, W. J., S. L. Balloun, W. W. Marion, and J. M. J. Ning. 1967.
Effect of Bird Cage Space and Dietary Metabolizable Energy Level on Production Parameters in Laying Hens1 M. A. Jalal,* S. E. Scheideler,†2 and D. Marx‡ *PO Box 34, Dahiyet El-Amir Rashed, Amman 11831, Jordan; †Department of Animal Science, University of Nebraska, Lincoln 68583-0908; and ‡Department of Statistics, 342C Hardin Hall, University of Nebraska, Lincoln 68583-0963 except for body weight change. Hens housed at 516 cm2/ hen and fed 2,800 kcal of ME/kg exhibited the greatest weight change, which was significantly (P < 0.05) greater than those fed other levels of ME at the same cage space. Hens housed at 690 cm2/hen had significantly (P < 0.05) greater ME efficiency of egg production than hens housed at other cage spaces. Hens fed the diet with 2,900 kcal of ME/kg had significantly (P < 0.001) greater ME digestibility compared with those fed 2,800 or 2,580 kcal of ME/ kg with differences of 107 and 118 kcal of ME/kg, respectively. There were no significant effects of ME levels observed except ME digestibility, and no significant effects of cage space allowance on egg weight, hen weight, bone ash, or maintenance energy intake. It is evident that decreasing the number of birds per cage and increasing cage space allowance per hen had an overall positive effect on performance.

ABSTRACT A study was conducted to assess the effects of varying cage spaces on a commercial laying hen strain fed differing levels of dietary metabolizable energy (ME) for 15 wk. Four cage space allowances (342, 413, 516, and 690 cm2/hen) were combined with 3 levels of dietary ME (2,800, 2,850, and 2,900 kcal of ME/kg) in a 4 × 3 factorial arrangement. Each treatment was assigned to 6 replicate cages for a total of 72 cages in randomized complete block design. Feed intake and metabolizable energy intake were significantly (P < 0.01) greater for hens housed at 690 cm2/hen compared with those housed at 413 and 342 cm2/hen, but not those housed at 516 cm2/hen, across all dietary ME levels. Egg production and egg mass were significantly (P < 0.001) improved for hens housed at 690 cm2/ hen in contrast to other cage spaces and across all energy levels. There were no interaction effects of ME levels on laying hen performance at varying cage space

Key words: cage space, laying hen, metabolizable energy 2006 Poultry Science 85:306–311

has been reported to lower egg production (EP), egg weight (EW), and feed intake (FI), and increase mortality (Marks et al., 1970; Bell, 1981; Roush et al., 1984; Sandoval et al., 1991). Differing diets were not used in these studies to determine whether there was an interaction between diet and caging space. Only a few researchers have investigated the interaction of cage space and diet on performance of laying hens. Jackson and Waldrup (1988) reported that increased dietary nutrient space helped overcome the effects of limited feeder space associated with crowded cages, but the influence was minimal when shallow cages were used. Owings et al. (1967) found that decreased EP caused by decreasing cage space was partially overcome by increased dietary protein, but Brake and Peebles (1992) detected no effects of increased dietary lysine on performance with decreased cage space. Carew et al. (1976, 1980) concluded that increasing the dietary energy level of White Leghorn hens did not reverse the downward trend in EP associated with decreased hen cage space. In 2001, United Egg Producers (Atlanta, GA) put forth new animal welfare guidelines that recommend a cage

INTRODUCTION Modern-day egg producers have attempted to increase net income by utilizing available housing facilities at maximum capacity. Currently, commercial layer operations tend to maximize the number of birds per cage, consequently decreasing cage space allowance per bird (Hester and Wilson, 1986). Producers reduce bird space with the assumption that an increase in total egg production per housing unit increases profit and offsets the negative effects of crowding (Adams and Craig, 1985). This perception, however, started to change in the decade with animal welfare issues receiving more publicity (Anderson et al., 1995). Cage space effects on the performance of commercial laying hens are well documented. Decreased cage space

2006 Poultry Science Association, Inc. Received May 5, 2005. Accepted October 21, 2005. 1 Published with the approval of the Director as Paper Number 14504, Journal Series, Nebraska Agricultural Research Division. 2 Corresponding author: [email protected]

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space of 432 cm2/hen for small White Leghorn hens compared with the current industry practice of 336 to 348 cm2/hen. If cage space changes, then the energy requirement of the laying hen may change. New research needs to be conducted to test the effects of cage on energy needs of the laying hen. The objective of our study was to evaluate the effects of varying cage space and dietary ME levels on energy requirement and production parameters of White Leghorn laying hens.

MATERIALS AND METHODS Experimental Design Four cage space allowances (342, 413, 516, and 690 cm2/ bird) were assigned to Hy-Line W-36 (Hy-Line International, West Des Moines, IA) White Leghorn hens from 20 to 35 wk of age. The hens were beak trimmed at 10 d of age using a precision trimming technique. Each cage space allowance was combined with 3 levels of dietary ME (2800, 2850, and 2900 kcal/kg) in a 3 × 4 factorial arrangement. Each treatment was randomly allotted to 6 replicate cages (total of 72 cages). Individual cages were designated as experimental units and had varying numbers of hens; 3 (690 cm2/hen), 4 (516 cm2/hen), 5 (413 cm2/hen), or 6 (342 cm2/hen) for a total of 324 hens. Experimental cage dimensions were 40.6 × 50.8 cm in a stacked-deck manure belt system (manufactured by Chore-Time, Milford, IN) consisting of the 3 rows (12 cages per row) on each side. Stainless steel feeder troughs with a feeder depth of 13.5 cm were used, providing feeder space of 6.70, 5.0, 4.0, and 3.33 cm/hen for 3, 4, 5, and 6 hens/cage, respectively, and watering equipment consisted of nipple drinkers (1/cage). The experimental design was a randomized complete block design with 6 blocks and 12 cages per block. A block constituted 1 row of 12 cages with 3 blocks on each side of the layer unit. The hens were fed treatment diets for a period of 15 wk from 20 to 35 wk of age.

Diets The experimental diets were formulated according to the recommendations of the breeder’s manual (Hy-Line International) and to meet National Research Council (1994) nutrient requirements of laying hens. The diets were standard corn-soybean meal diets formulated to be isonitrogenous and to contain 4.00% Ca and 0.42% nonphytate phosphorus (Table 1). The intermediate ME diet was mixed by blending equal quantities of high and low ME diets. Dietary samples were collected from each batch of diet formulated, sieved through a 1-mm screen, ground, and stored for analysis of gross energy (GE), Ca, P, Cr, and N. The N content in the diets was multiplied by 6.25 to obtain protein content in the diets. Calcium and P were determined by procedures established by the Association of Official Analytical Chemists (AOAC, 1984). Dietary and fecal Cr were determined according to the

1

Table 1. Diet composition Ingredients

High ME

Low ME (%)

Corn Soybean meal2 Wheat middlings Tallow Limestone Dicalcium phosphate Salt DL-Methionine Lysine Vitamin premix3 Mineral premix4 Calculated nutrient composition5 ME, kcal/kg Protein, % TSAA, % Lysine, % Ca, % Nonphytate P, % Total P, % Analyzed nutrient composition6 ME, kcal/kg Protein, % Ca, % Total P, %

60.53 23.33 — 4.25 9.39 1.70 0.40 0.19 0.06 0.08 0.08

58.71 21.47 4.92 3.00 9.40 1.66 0.40 0.19 0.09 0.08 0.08

2,900 17.20 0.73 0.85 4.00 0.42 0.63

2,800 17.20 0.73 0.85 4.00 0.42 0.63

3,097 15.73 3.97 0.68

2,979 16.14 4.28 0.70

1 Intermediate ME diet was mixed by blending equal quantities of high and low ME diets. 2 Soybean meal incorporated into the diet was high protein (48% CP) soybean meal. 3 Vitamin premix: vitamin A, 6,600 IU; vitamin D3, 2,805 IU; vitamin E, 10 IU; vitamin K, 2 mg; riboflavin, 4.4; pantothenic acid, 6.6 mg; niacin, 24.4 mg; choline, 110 mg; vitamin B7, 8.8 mg/kg. 4 Mineral premix: Mn, 88 mg; Cu, 66 mg; Fe, 8.5 mg; Zn, 88 mg; Se, 0.30 mg/kg. 5 ME values for ingredients used diet formulation were based on values in NRC (1994). 6 Analyzed nutrient composition for intermediate diet was: 2,990 kcal of ME/kg, 15.75% CP, 4.40% Ca, 0.69% total P.

procedure of Williams et al. (1962) using atomic absorption spectrophotometry. Dietary and fecal N were determined using the Kjeldahl method as established by the Association of Official Analytical Chemists (AOAC, 1984). Dietary and fecal GE were determined using a Parr adiabatic oxygen bomb calorimeter.

Parameters Measured Feed intake and EP were recorded daily. Hens were given ad libitum access to feed. Egg production was calculated on a hen-day basis. Egg mass (EM) was calculated by multiplying egg weight by EP. The ME intake (kcal/ hen per d) was calculated by multiplying nitrogen-corrected digestible ME content of the diet by daily feed intake. Egg weight was measured weekly on 1 d of egg production. Hens were weighed individually at the start of the trial and every other week thereafter until the end of the trial at 20, 22, 24, 26 28, 30, 32, 34, and 35 wk of age. Maintenance ME requirements were calculated by subtracting ME requirements for production from ME intake according to the following equation (Peguri and Coon, 1989, 1991):

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maintenance = MEn intake − (2.86 × EM) − 6.00 × BWC + mean BW

main effects of diet, space, and their interaction. The following model statement was used:

where maintenance = maintenance ME (kcal of MEn/kg of BW), BWC = body weight change (g/hen per d), BW = mean BW (g), and MEn = nitrogen-corrected ME intake (kcal/hen per d).The ME efficiency of egg production (MEEP) was calculated according to an equation from Emmans and Charles (1977):

Yijkl = ␮ + Rl + αi + βj + αβij + εijkl where Yijk = measured response, ␮ = overall mean, Rl = block effect, αi = diet effect, βj = cage space effect, αβij = interaction between diet and cage space, and εijkl = residual error.

MEEP = EM × 1.66/MEn

RESULTS

where EM = egg mass output (g/hen per d). The factor 1.66 represents the average energetic equivalent of egg weight as (kcal/g) egg (NRC, 1981). Hen mortality was recorded daily during the course of the experiment. Production parameters such as FI and EP were corrected for hen mortalities.

Cage space had a significant (P < 0.05) effect on FI of laying hens (Table 2). Hens housed at 690 cm2/hen had greater FI than those housed at 342 and 413 cm2/hen, but not those housed at 516 cm2/hen, across all dietary ME levels. A similar effect was observed with ME intake, as hens housed at 690 cm2/hen significantly (P < 0.05) consumed 15.69 and 20.06 more kcal/hen per d than those housed at 342 and 413 cm2/hen, respectively (Table 2). There were no significant effects of dietary ME level on feed or ME intakes. Egg production was significantly (P < 0.05) affected by cage space (Table 2). Hens housed at 690 cm2/hen had greater EP in contrast to those housed at other bird cage spaces across all dietary ME levels. Egg mass followed the same trend as EP. Egg weight was not significantly affected by dietary ME level or cage space (Table 2). Hens fed the highest ME levels and those housed at 690 cm2/hen laid the largest eggs, across dietary ME levels and cage spaces, respectively. There were no significant effects of diet or cage space on average hen weight (Table 2). There was a significant (P < 0.05) diet × cage space interaction on BW change (Table 2). Hens fed the high ME diet and housed at 342 cm2/hen had significantly greater BW change than those housed at 413 cm2/hen, but not those housed at 516 and 690 cm2/hen. Hens fed the intermediate ME diet and housed at 413 cm2/hen had significantly greater BW change than those housed at 342 cm2/hen, but not those housed at other space allowances. Hens fed the low ME diet exhibited the greatest BW change when housed at 516 cm2/hen, and their BW change was significantly greater than those housed at 413 cm2/hen, but not greater than those housed at 342 and 690 cm2/hen. There were no significant effects of dietary ME, cage space, or their interaction on bone ash percentage (Table 2). Maintenance energy intake was not significantly affected by either dietary ME or cage space (Table 3). Reducing the number of birds per cage did not appear to increase ME requirement with extra space available for activity. However, MEEP was significantly (P < 0.05) affected by cage space (Table 3). Hens housed at 690 cm2/ hen had greater MEEP compared with hens housed at other spaces and across all diets. Digestible AMEn was significantly affected by dietary ME level in the diet (Table 3). Hens fed high ME had

ME Digestibility At the end of the trial, chromic oxide (Cr2O3) was added to all diets as an analytical marker for nutrient digestibility at a rate of 0.25% of the diet and fed for 5 d. Representative fecal samples were collected from each pen on the last day of Cr2O3 feeding to determine GE, N, and Cr content of feces. The fecal samples were freeze-dried, sieved through a 1-mm screen to remove feathers, ground, and packed in plastic bags for storage before analysis. The following equation was used for calculation of AME digestibility (Scott et al., 1976): % AME digestibility = 100 − [(dietary Cr/fecal Cr × fecal GE/dietary GE) × 100] The value of AME was corrected for N retention (Hill and Anderson, 1958). The retained n value was multiplied by 8.22 kcal/g and subtracted from AME value. The corrected AME is referred to as AMEn (N-corrected apparent ME).

Bone Ash At 35 wk of age, 6 hens from each treatment were killed by cervical dislocation and their tibias removed. The tibial bones were boiled to remove any traces of flesh. Tibias were solvent-extracted to remove fat, and then dried and ashed at 600°C for 48 h to determine bone ash percentage. Methods used were approved by the institutional animal care and use committee at the University of NebraskaLincoln.

Statistical Analyses Data were analyzed using the mixed model analysis from SAS software (Proc Mixed, 2001; SAS Institute, Inc., Cary, NC) for a randomized complete block design with a 3 × 4 factorial arrangement. The data were tested for

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BIRD CAGE SPACE AND DIETARY METABOLIZABLE ENERGY Table 2. Layer production data: feed intake, ME intake, egg production, egg weight, and egg mass

Diet (kcal/kg ME) High High High High Intermediate Intermediate Intermediate Intermediate Low Low Low Low SEM Main effects ME High Intermediate Low SEM Cage space/hen 690 516 413 342 SEM Statistical probabilities ME Cage space ME × cage space

Cage space (cm2/hen)

Feed intake (g/hen/d)

ME intake (kcal/hen/d)

Egg production (%)

Egg weight (g)

Hen weight1 (kg)

BW change (g/hen/d)

Bone ash (%)

690 516 413 342 690 516 413 342 690 516 413 342

90.54 88.72 83.87 84.99 91.84 90.33 84.03 86.20 92.25 89.49 87.60 84.56 2.095

279.16 274.95 260.46 264.11 271.71 267.56 260.02 254.36 277.26 267.89 260.58 249.47 7.906

81.89 81.18 76.04 75.92 86.91 84.49 76.58 75.44 89.78 79.85 73.48 76.66 1.999

52.16 51.88 52.25 51.15 51.90 49.94 50.94 51.07 51.54 51.93 51.25 50.84 0.605

1.418 1.398 1.386 1.382 1.431 1.370 1.362 1.380 1.403 1.437 1.378 1.396 0.214

1.671dc 1.602dc 1.223c 2.174d 1.853fg 1.888fg 2.332f 1.275g 1.728hi 2.300h 1.196i 1.868h 0.333

59.41 60.41 59.85 60.84 60.01 60.78 60.70 60.31 60.80 60.59 59.77 59.98 0.527

87.04 88.10 88.50 1.119

269.67 263.09 263.41 5.517

78.76 80.85 79.94 1.176

51.85 50.97 51.39 0.303

1.396 1.386 1.403 0.110

1.668 1.773 1.836 0.196

60.13 60.17 60.45 0.374

91.55a 89.52a 85.19b 82.25b 1.259

276.04a 270.13ab 260.35b 255.98b 5.530

86.19a 81.84b 75.37c 76.00c 1.293

51.86 51.25 51.48 51.02 0.350

1.418 1.402 1.375 1.386 0.0125

1.751 1.930 1.583 1.772 0.215

59.92 60.59 60.11 60.38 0.394

NS NS NS

NS NS NS

NS NS 0.02

NS NS NS

NS 0.0002 NS

NS 0.0002 NS

NS 0.0001 NS

Means with no common superscript differ significantly (P < 0.05). High ME means with no common superscript differ significantly (P < 0.05). f,g Intermediate ME means with no common superscript differ significantly (P < 0.05). h,i Low ME means with no common superscript differ significantly (P < 0.05). 1 The average weight of hens at the start of the trial was 1.223 kg/hen and the standard deviation was 0.0474. a–c d,e

significantly greater digestible AMEn than those fed intermediate and low ME, with differences of 107 and 118 kcal of ME/kg, respectively.

DISCUSSION In the present study, an increase in FI was observed when cage space available for hens was increased. Feed intake increased by 6.30 g/hen per d as cage space was increased from 342 to 690 cm2/hen. The effect of increasing cage space on FI is consistent with results reported by Sohail et al. (2001) and Adams and Craig (1985). Our findings disagree with those reported by Anderson and Adams (1992) and Brake and Peebles (1992), who found no effects of cage space on FI. Although our results indicate no significant effect of dietary ME level on FI, previous researchers (Carew et al., 1980; Jackson and Waldrup, 1988) reported a decrease in FI as ME level was elevated. It appears that there was too much variation in FI to detect differences because one may have expected more of a response to a difference of 100 kcal/kg in dietary ME based on previous assumptions. Carew et al. (1976, 1980) used diets with a difference of >150 kcal of ME/kg to elicit a response in FI and detected much less variation compared with our results.

The reduction in EP due to decreased cage space is well cited (Cunningham, 1982; Adams and Craig, 1985; Hester and Wilson, 1986; Craig and Milliken, 1989; Sohail et al., 2001). Our results are in congruence with previous research as EP declined as much as 10.1% (86.19 vs. 76%) as the number of hens per cage was increased from 3 to 6. In our study, increasing dietary energy level did not improve EP as cage space was reduced. Jackson and Waldrup (1988) reported that increasing dietary energy partially alleviated the reduction in EP resulting from decreased cage space. Egg mass exhibited a similar trend to EP with a decline as cage space decreased. Earlier research has shown decreased EM as the number of hens was increased per cage (Cunningham, 1982; Craig and Milliken, 1989). Body weight change was the only parameter exhibiting a significant diet × cage space interaction. There was no consistent change in gain between cage spaces at each ME level, as one would anticipate an increase in gain with increased cage space and ME level. At high ME levels, hens housed at 342 cm2/hen attained the greatest BW change gain at that level, whereas at intermediate and low ME, hens housed at 413 and 516 cm2/hen, respectively, exhibited the greatest BW gain. Carew et al. (1980)

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JALAL ET AL. Table 3. Metabolizable energy data

Diet (kcal of ME/kg) High High High High Intermediate Intermediate Intermediate Intermediate Low Low Low Low SEM Main effects ME High Intermediate Low SEM Cage space/hen1 690 (107) 516 (80) 413 (64) 342 (53) SEM Statistical probabilities ME Cage space ME × cage space

Cage space (cm2/hen)

ME intake (kcal/hen per d)

ME efficiency of egg production (kcal/kcal)

Digestible AMEn (kcal/kg)

690 516 413 342 690 516 413 342 690 516 413 342

98.56 98.68 95.98 96.54 88.05 94.97 94.57 93.68 90.66 89.87 101.16 86.46 5.174

0.263 0.263 0.264 0.255 0.287 0.269 0.261 0.262 0.288 0.269 0.253 0.273 0.00822

3,081.38 3,096.99 3,104.02 3,107.25 2,956.17 2,962.04 3,092.10 2,951.00 3,006.17 2,995.80 2,967.16 2,949.70 43.712

97.44 92.75 92.03 3.020

0.261 0.270 0.271 0.00497

3,097.41a 2,990.33b 2,979.71b 28.364

92.43 94.41 97.24 92.23 3.329

0.280a 0.267b 0.259b 0.263b 0.00542

3,014.57 3,018.28 3,054.43 3,002.65 30.455

NS NS NS

NS 0.013 NS

0.0001 NS NS

Means with no common superscript differ significantly (P < 0.05). Values in parentheses for cage space/hen are in square inches/hen.

ab 1

reported that BW gains improved as ME level increased, thus reversing loss in BW due to reduction in cage space. Maintenance requirement did not decrease as cage space was increased and was not significantly affected by ME level. Reducing the hen’s space in cage has been shown not to alter time allocated to activities such as eating, standing, resting, and preening (Sefton, 1976; Ouart and Adams, 1982). Madrid et al. (1981) reported that maintenance energy requirements increased as the number of hens per cage was increased from 3 to 7, which was in disagreement with our findings. Madrid et al. (1981) reported that crowding of birds was responsible for increasing voluntary activity. Laying hens became energetically more efficient as cage space increased. Hens reared at 690 cm2/hen were energetically more efficient than those housed at other space allowances. Our findings show that as cage space increased, energy intake was increased. This is consistent with the fact that maintenance requirements did not significantly change as the number of birds changed. It is, therefore, possible to assume that hens that consumed more feed were energetically more efficient. Digestible AMEn values were significant for hens fed the high ME diet compared with those fed the intermediate and low ME diets. Cage space did not influence ME digestibility for laying hens and the digestible ME values were very close.

In summary, reducing the number of hens per cage improved feed intake, ME intake, egg production, egg mass, digestible AMEn, and dietary ME efficiency for laying hens. There were no significant effects of dietary ME levels on these response variables. Increasing ME level in the diet did not reverse the negative effects of crowding and decreasing cage space on egg production.

REFERENCES Adams, A. W., and J. V. Craig. 1985. Effects of crowding, and cage shape on productivity and profitability and caged layers: A survey. Poult. Sci. 64:238–242. Anderson, K. E., and A. W. Adams. 1992. Effects of rearing space and feeder and waterer spaces on the productivity and fearful behavior of layers. Poult. Sci. 71:53–58. Anderson, K. E., G. B. Havenstein, and J. Brake. 1995. Effects of strain and rearing dietary regimens on brown-egg pullet growth and strain, rearing dietary regimens, space, and feeder space effects on subsequent laying performance. Poult. Sci. 74:1079–1092. AOAC. 1984. Official Methods of Analysis. 14th ed. Association of Official Analytical Chemists, Washington, DC. Bell, D. 1981. Cage selection and management. Feedstuffs 53:20–24. Brake, J. D., and E. D. Peebles. 1992. Laying hen performance as affected by diet and caging space. Poult. Sci. 71:945–950. Carew, L. B., Jr., D. C. Foss, and D. E. Bee. 1976. Effect of dietary energy concentration on performance of heavy egg-type hens at various space of cages. Poult. Sci. 55:1057–1066.

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Ouart, M. D., and A. W. Adams. 1982. Effects of cage design and bird space on layers. 1. Productivity, feathering and nervousness. Poult. Sci. 61:1606–1613. Owings, W. J., S. L. Balloun, W. W. Marion, and J. M. J. Ning. 1967. The influence of dietary protein level and bird space in cages on egg production and liver fatty acids. Poult. Sci. 46:1303. (Abstr.) Peguri, A., and C. N. Coon. 1989. The efficiency of utilization of dietary energy for layers and the law of diminishing returns. Pages 270–299 in Proc. Minnesota Nutr. Conf. and Heartland Lysine Tech. Symp., Bloomington, MN. Univ. Minnesota, St. Paul. Peguri, A., and C. N. Coon. 1991. Effect of temperature and dietary energy on layer performance. Poult. Sci. 70:126–138. Roush, W. B., M. M. Mashaly, and H. B. Graves. 1984. Effect of increased bird production in a fixed cage area on production and economic responses of Single Comb White Leghorn laying hens. Poult. Sci. 63:45–48. Sandoval, M., R. D. Miles, and R. D. Jacobs. 1991. Cage space and house temperature gradient effects on performance of White Leghorn hens. Poult. Sci. 70(Suppl. 1):103. (Abstr.) Scott, M. L., M. C. Nesheim, and R. J. Young. 1976. Pages 7– 54 in Nutrition of the Chicken. M. L. Scott and Associates, Ithaca, NY. Sefton, A. E. 1976. The interaction of cage size, cage level, social space, fearfulness and production of Single Comb White Leghorns. Poult. Sci. 55:1922–1926. Sohail, S. S., M. M. Bryant, S. K. Rao, and D. A. Roland. 2001. Influence of cage space and prior dietary phosphorus level on phosphorus requirement of commercial Leghorns. Poult. Sci. 80:769–775. United Egg Producers. 2001. Tell United Egg Producers they need more to do to help hens. http://www.unitedegg.org/ links.asp Accessed Dec. 2005. Williams, C. H., D. J. David, and O. Iismaa. 1962. The determination of chromic oxide in fecal samples by atomic absorption spectrometry. J. Agric. Sci. 59:381–385.