METABOLISM AND NUTRITION

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1 METABOLISM AND NUTRITION The Effect of Maternal Dietary Vitamin D 3 Supplementation on Performance and Tibial Dyschondroplasia of Broiler Chicks J. P. Driver, 1 A. Atencio, G. M. Pesti, H. M. Edwards, Jr., and R. I Bakalli Department of Poultry Science, The University of Georgia, Athens, Georgia ABSTRACT A series of experiments was conducted to in weight at hatch. For the first 2 hatches (wk 39 and 44), investigate the effects of maternal dietary vitamin D 3 supplementation at 4 different times during the laying cycle, on the performance and bone quality of broiler chicks fed a diet that induced tibial dyschondroplasia (TD) or an adequate diet. Ross Ross broiler breeder hens were fed a corn-soy diet with various levels of vitamin D 3 from 24 to 66 wk of age. Eggs were collected at 39, 44, 53, and 64 wk of age and hatched. Chicks from hens fed 250 IU of D 3 /kg (low maternal D 3 or LMD 3 ) and 2,000 IU of D 3 / kg (high maternal D 3 or HMD 3 ) levels were placed in battery brooders and fed the diets from 0 to 16 d. At 16 d, the chicks were weighed and killed; the left tibias were used for bone ash determinations, and the right tibias were used to score the incidence and severity of TD (0, 1, 2, or 3, where 3 is the most severe). Body weight gain and feed intake were significantly lower for the LMD 3 chicks at wk 44 and 64, although there was no difference the LMD 3 and HMD 3 chicks demonstrated high average TD scores (2.03 and 1.57 vs and 1.75 for the LMD 3 vs. HMD 3 chicks, respectively) and high average incidences of severe TD (50 and 35% vs. 45 and 34% for LMD 3 vs. HMD 3 levels, respectively). However, results from the last 2 hatches (wk 53 and 64) showed that HMD 3 chicks, compared with LMD 3 chicks, had reduced average TD scores (1.39 and 1.47 vs and 0.44 for LMD 3 vs. HMD 3 levels, respectively) and severe TD incidence (36 and 40% vs. 17 and 8% for the LMD 3 vs. HMD 3 levels, respectively). In this experiment, as egg production declined toward the end of the laying cycle, hens fed the HMD 3 might have been able to deposit sufficient quantities of vitamin D 3 in the egg to maintain excellent body weight gain at 16 d of age and reduce the incidence and severity of TD. Hens fed the LMD 3 diet were unable to produce similar improvements. Key words: maternal vitamin D 3, tibial dyschondroplasia, chick 2006 Poultry Science 85:39 47 INTRODUCTION The bone disease tibial dyschondroplasia (TD) commonly found in broiler chickens is very sensitive to the vitamin D 3 status of the bird. Tibial dyschondroplasia is characterized by the accumulation of chondrocytes, which fail to mature and differentiate but rather accumulate along the growth plate of long bones such as the tibia (Leach and Nesheim, 1965; Farquharson and Jefferies, 2000). These accumulations may develop into large opaque plugs that eventually lead to bone deformities. Addition of vitamin D 3 metabolites to the diets of chicks with symptoms of TD results in the alleviation of these symptoms by inducing the maturation of immature chondrocytes, which heals the lesions that they form (Edwards et al., 1992; Rennie et al., 1993, 1995; Thorp et al., 1993; Roberson and Edwards, 1996; Elliot and Edwards, 1997; Mitchell et al., 1997a,b; Xu et al., 1997; 2006 Poultry Science Association, Inc. Received December 13, Accepted July 20, Corresponding author: jdriver@jax.org McCormack et al., 2004). In addition to the vitamin D 3 status of the bird, this lack of chondrocyte differentiation is affected by the levels of Ca and P in the diet. Edwards and Veltmann (1983) reported that high levels of P and low levels of Ca induce the disease by yet unknown mechanisms. The vitamin D 3 status of a young chick depends, to some degree, on the level of vitamin D 3 in the breeder hen diet. Hart et al. (1925) first identified variation in the vitamin D 3 content of eggs when it was observed that the antirachitic properties of egg yolks from hens exposed to the radiation from a quartz lamp for 10 min daily was approximately 10 times that of nonirradiated birds fed the same ration. De Vaney et al. (1936), Branion et al. (1934), and Guerrant et al. (1935) showed that the antirachitic potency of the egg depends on the amount of vitamin D 3 fed to the hen. In a more recent study, Mattila et al. (1992) found that the quantity of vitamin D 3 in egg yolks from 12 sources, as determined by HPLC, varied between 1.35 to 1.47 g/100 g of egg yolk. With the same method, Mattila et al. (1999) demonstrated that the vitamin D 3 content of the egg yolk could be increased by increasing the vitamin D 3 content in laying hen diets. 39

2 40 DRIVER ET AL. Table 1. Composition of the tibial dyschondroplasia (TD)-inducing diet and the control diet with NRC (1994) recommended levels of Ca and P TD-inducing Control diet (%) diet (%) Ingredient Ground yellow corn Soybean meal (dehulled) Soybean oil Iodized sodium chloride DL-Methionine Vitamin premix Trace mineral premix Chromic oxide Sand Dicalcium phosphate Limestone Phytase (1,000 phytase units/kg) Calculated composition 3 ME, kcal/kg 3,200 3,200 CP, % Ca, % Total P, % Nonphytate P, % Analyzed composition Ca, % Total P, % Phytate P, % Vitamin mix provided the following (per kg of diet): thiamin-mononitrate, 2.4 mg; nicotinic acid, 44 mg; riboflavin, 4.4 mg; D-Ca pantothenate, 12 mg; vitamin B 12 (cobalamin), 12.0 g; pyridoxine-hcl, 2.7 mg; D- biotin, 0.11 mg; folic acid, 0.55 mg; menadione sodium bisulfate complex, 3.34 mg; choline chloride, 220 mg; cholecalciferol, 1,100 IU; transretinyl acetate, 5,500 IU; all-rac-tocopherol acetate, 11 IU; ethoxyquin, 150 mg. 2 Trace mineral mix provided the following (per kg of diet): manganese (MnSO 4 H 2 O), 60 mg; iron (FeSO 4 7H 2 O), 30 mg; zinc (ZnO), 50 mg; copper (CuSO 4 5H 2 O), 5 mg; iodine (ethylene diamine dihydroiodide), 1.5 mg; selenium, 0.3 mg. 3 Calculated from NRC (1994). The vitamin D 3 content of the egg is an important factor in the Ca metabolism of the developing embryo, and feeding the hen inadequate vitamin D 3 results in reduced hatchability (Bethke et al., 1936). Bethke et al. (1936), using chicks, and Robertson et al. (1941), using turkey poults, also proved that the vitamin D 3 in egg yolk could have a prolonged effect on chick development after hatching in terms of bone ash and growth when the young birds were fed a vitamin D 3 -deficient diet. The amount of vitamin D 3 in the egg may affect the performance and bone mineralization of chicks up until 5 wk of age (Bethke et al., 1936). Not only does vitamin D 3 deposited in the egg depend on the level of vitamin D 3 fed to the breeder hen, but it also appears to increase as the laying cycle progresses. Guerrant et al. (1935) reported that the vitamin D 3 potency of the egg yolk of hens at the end of the laying cycle was greater than that of pullets at the beginning. The authors hypothesized that the lower potency in pullets was due to 1) greater egg production of young birds compared with older birds, 2) less efficient transfer vitamin D 3 into the egg of a pullet compared with an older bird, 3) the possibility that pullets may have a greater requirement for vitamin D 3. The objective of the current work was to investigate the effects of levels of maternal vitamin D 3 (Mat D 3 )on the performance, leg pathology, and bone quality of broiler chicks fed a diet that induced TD or an adequate diet from 0 to 16-d at 4 different hen ages during the course of the laying cycle. MATERIALS AND METHODS General Procedures In a time dependent study, 1-d-old Ross Ross straight-run broiler chicks were hatched and reared from the eggs of dams fed 250 IU/kg (low Mat D 3 ; LMD 3 )or 2,000 IU/kg (high Mat D 3 ; HMD 3 ). The chicks were hatched when the dams were 39, 44, 53, and 64 wk of age (A. Atencio, H. M. Edwards, Jr., G. M. Pesti, and G. O. Ware, University of Georgia, unpublished). Dams were fed the different levels of vitamin D from wk 24 to 66, and hen-day egg production (HDEP) [(number of eggs laid per day/number of hens) 100] was recorded throughout the experiment; average egg weights (EW) were measured for wk 44 and 64. The chicks were housed in wire-floored battery brooders (Petersime, Zulte, Belgium) for 16 d and were provided with water and a diet ad libitum that induced TD or one that would limit TD (control). The number of replicates per treatment varied between 4 and 6, depending on the experiment, with 8 chicks per replicate. Lighting was provided 24 h daily for the entire experimental period. The closed room and shielded lighting used prevented the birds from being exposed to ultraviolet radiation to ensure that they were not able to synthesize their own vitamin D 3. Body weight gain (BWG) and feed intake were measured from 0 to 16 d. At the conclusion of each experiment, birds were killed by CO 2 asphyxiation. Left tibias from all chicks were collected for percentage tibia ash determination on a fat-free dry weight basis (Association of Official Analytical Chemists, 1995), and right tibias were sliced and scored (0, 1, 2, or 3 where 3 is the most severe) for the severity of bone abnormalities including P rickets, Ca rickets, and TD as described by Edwards and Veltmann (1983). Those with a normal growth plate but a lengthened primary spongiosa were classified as having P rickets, whereas those with a normal spongiosa but a lengthened growth plate were classified as having Ca rickets. Tibial dyschondroplasia was characterized by an abnormal mass of cartilage at the proximal end of the tibiotarsus as described by Edwards and Veltmann (1983). In each experiment, excreta samples collected between 14 and 16 d of age, and feed samples were analyzed for phytate P (Latta and Eskin, 1980) and the indigestible marker chromic oxide (Brisson, 1956). The percentage retention of phytate P was calculated according to Edwards and Gillis (1959). Experimental Diets All of the chicks from hens fed 250 IU of D 3 /kg were fed a low Ca corn-soybean meal experimental diet de-

3 EFFECTS OF MATERNAL DIET ON CHICKS 41 Table 2. Effect of maternal vitamin D 3 (Mat D 3 ) supplementation and age of the hens (Age) on body weight gain (BWG), feed intake, feed conversion ratio (FCR), percentage of phytate P retention, and percentage of tibia ash of broiler chicks fed a diet that induced tibial dyschondroplasia (0 to 16 d) 1 Feed Phytate P Tibia BWG intake FCR retention ash Age Mat D 3 Rep 2 (g) (g) (g/g) (%) (%) ± 12 cd 537 ± 15 cd 1.22 ± ± ± 0.4 ab 39 2, ± 10 bcd 569 ± 13 bcd 1.23 ± ± ± 0.6 ab ± 18 d 526 ± 16 d 1.24 ± ± ± 0.2 b 44 2, ± 5 bc 578 ± 7 bc 1.22 ± ± ± 0.6 b ± 13 bc 570 ± 9 bcd 1.22 ± ± ± 0.3 ab 53 2, ± 7 ab 591 ± 7 ab 1.22 ± ± ± 0.5 ab ± 5 bc 570 ± 11 bcd 1.23 ± ± ± 0.6 a 64 2, ± 18 a 622 ± 23 a 1.20 ± ± ± 0.5 ab Main effect ± 8 b ± 11 b 1.23 ± ± ± 0.4 ab ± 13 b ± 13 b 1.23 ± ± ± 0.3 b ± 7 a ± 6 ab 1.22 ± ± ± 0.3 ab ± 15 a ± 17 a 1.21 ± ± ± 0.4 a Mat D ± 8 b ± 8 b 1.22 ± ± ± 0.2 2, ± 7 a ± 8 a 1.22 ± ± ± 0.3 df R Age Mat D Age D Error 30 R (coefficient) Intercept 1 (439.0) (534.0) (1.216) (82.2) (37.8) < < < < < Age 1 ( 6.6) ( 4.2) (0.011) (0.1) ( 2.1) Mat D 3 1 (19.0) (33.0) (0.019) ( 8.2) ( 0.1) Age age 1 (4.0) (4.0) ( 0.003) ( 0.4) (0.5) Age Mat D 3 1 (5.3) (1.5) ( 0.010) (3.2) (0.1) Error 33 a d Values within variables with no common superscripts differ significantly (P < 0.05) when tested with Duncan s multiple range test following. 2 Rep indicates the number of replicate pens used per treatment. Each pen contained 8 chicks. signed to induce TD (Table 1) (Edwards and Veltmann, 1983). The diet was formulated to contain 0.60% Ca and 0.24% nonphytate P (NPP) and was supplemented with 1,000 phytase units/kg Natuphos phytase. Approximately half of the chicks from hens fed 2,000 IU of D 3 / kg were fed the TD-inducing diet. The other half were fed a TD-limiting control diet formulated to contain National Research Council (1994) recommended levels of Ca and P (1.00% Ca and 0.45% NPP; Table 1). Statistical Analysis Data were analyzed by the GLM and R 2 procedures (SAS Institute, 2001), and means were compared by Duncan s new multiple range test (Duncan, 1955). Multiple regression analyses were performed for each variable for chicks fed the TD-inducing diet to test for linear, quadratic, and interaction effects of the level of Mat D 3 fed to the laying hens and the age of the hens when eggs were collected (Tables 2 and 3). Birds fed the control diet were obtained from hens fed the HMD 3 ; therefore, simple regressions involving only age of hens were performed for each variable. The TD score, overall incidence of TD, and the incidence of severe TD were each regressed on HDEP and EW. RESULTS The results in Tables 2 through 5 compare the effect of dietary vitamin D content fed to the breeders when all chicks were fed the TD-inducing diet. The results in Tables 6 and 7 compare the results of chicks from breeders fed 2,000 IU D 3 /kg when chicks were fed the control diet.

4 42 DRIVER ET AL. Table 3. Effect of maternal vitamin D 3 (Mat D 3 ) supplementation and of hen age on leg pathologies of broiler chicks fed a diet that induced tibial dyschondroplasia (TD; 0 to 16 d) 1 P rickets Ca rickets P rickets Ca rickets TD TD3 score 3 score 3 TD score 3 incidence incidence incidence incidence Age Mat D 3 Rep 2 per bird per bird per bird (%) (%) (%) (%) ± ± ± 0.23 ab 30.0 ± ± ± 10.0 ab 17.5 ± 9.4 ab 39 2, ± ± ± 0.23 ab 32.5 ± ± ± 6.1 ab 22.5 ± 10.8 ab ± ± ± 0.30 ab 33.2 ± ± ± 7.6 a 35.3 ± 13.7 a 44 2, ± ± ± 0.21 a 40.6 ± ± ± 7.9 ab 34.4 ± 6.0 ab ± ± ± 0.21 ab 25.3 ± ± ± 7.2 a 36.0 ± 5.8 a 53 2, ± ± ± 0.10 bc 25.9 ± ± ± 4.4 ab 17.3 ± 4.1 ab ± ± ± 0.27 ab 26.7 ± ± ± 11.5 a 40.0 ± 11.5 a 64 2, ± ± ± 0.18 c 20.0 ± ± ± 11.7 b 8.0 ± 4.9 b Main effect ± ± ± 0.16 a 31.3 ± ± ± 5.5 a 20.0 ± ± ± ± 0.17 a 36.9 ± ± ± 5.6 a 34.8 ± ± ± ± 0.12 ab 25.6 ± ± ± 4.3 b 26.6 ± ± ± ± 0.23 b 22.5 ± ± ± 10.3 c 20.0 ± 7.6 Mat D ± ± ± 0.12 a 28.6 ± ± ± ± 4.8 2, ± ± ± 0.14 b 29.0 ± ± ± ± 3.7 df R Age < Mat D Age Mat D Error 30 R (coefficient) Intercept 1 (0.413) ( 0.062) (0.819) (24.9) ( 0.2) (85.0) ( 5.0) < Age 1 (0.009) (0.044) (0.611) (6.7) ( 0.8) ( 2.1) (26.9) Mat D 3 1 (0.003) (0.083) (0.615) (8.3) (4.5) (21.4) (19.6) Age age 1 ( 0.008) ( 0.000) ( 0.124) ( 1.8) (0.6) ( 1.5) ( 4.1) ( 0.006) ( 0.013) ( 0.357) ( 2.9) ( 0.7) ( 11.7) ( 12.5) Error 33 a c Values within variables with no common superscripts differ significantly (P < 0.05) when tested with Duncan s multiple range test following. 2 Rep indicates the number of replicate pens used per treatment. Each pen contained 8 chicks. 3 Rickets and TD were scored 0, 1, 2 or 3, where 3 is the most severe (Edwards and Veltmann, 1983). Chicks from hens fed 2,000 IU/kg were heavier at 16 d of age than those from hens fed 250 IU/kg, although there was no statistical difference in hatch weight between the 2 groups of chicks (Table 8). Hen age and Mat D 3 feeding level were significant contributors to variation in BWG of the progeny (Table 2). The BWG of the chicks at 16 d was greater for older hens and heavier EW compared with younger hens and lower egg EW (Tables 2 and 8). No consistent trends in phytate P retention or percentage of tibia ash were observed, although there was a significant difference in tibia ash between wk 44 and 64. There was a low incidence of Ca rickets and a higher than expected incidence of P rickets in both groups of chicks fed the TD-inducing diet (Table 3). Interactions between Mat D 3 and hen age for TD incidence (P = ) and severity (P = ) indicated that Mat D 3 and hen age could have influenced the development of TD in the progeny; TD decreased as hen age increased at the end of the laying cycle but only at the higher level of Mat D 3. The level of HDEP appeared to influence TD incidence and severity (Table 4) in chicks from hens fed the HMD 3 as did EW (Table 5). When the coefficients of determination (R 2 ) values were calculated for models with only hen age, Mat D 3, HDEP or EW, EW was found to be a better predictor of TD than hen age or HDEP (R 2 = 0.62, 0.36, 0.12, and 0.01 for EW, hen age, Mat D 3 and HDEP, respectively; analyses not included). Addition of hen age or HDEP to the EW model increased the variation accounted for by the model by little more than When chicks hatched from HMD 3 eggs were fed a control diet estimated to be adequate for preventing leg pathologies (Table 1), BWG was better than for those chicks fed the TD-inducing diet (Table 6 vs. 2). Significant differences in BWG were observed for the different

5 EFFECTS OF MATERNAL DIET ON CHICKS 43 Table 4. summary of the effect of hen-day egg production (HDEP 1 ) on tibial dyschondroplasia (TD) score and incidence of chicks from hens fed 250 (LMD 3 ) or 2,000 IU/kg (HMD 3 ) vitamin D (Mat D 3 ) and fed a diet that induced TD TD TD TD3 TD TD TD3 Treatment df score incidence incidence df score incidence incidence R Intercept HDEP HDEP HDEP Mat D HDEP Mat D Error 33 LMD 3 HMD 3 R (coefficient) Intercept 1 ( ) (88.232) (9.913) 1 ( ) ( ) ( ) HDEP 1 ( ) ( 1.179) (1.937) 1 ( ) (17.527) (10.979) HDEP HDEP 1 ( ) (0.025) ( 0.027) 1 ( ) ( 0.125) ( 0.086) Error HDEP = (number of eggs laid per day/number of hens) 100. weeks, but there was no clear trend. Phytate P retention decreased with hen age to 53 wk but then increased P < 0.023; Table 6). Chicks fed the adequate diet (Table 7) had similar incidences of TD to those fed the TD inducing diet (Table 3) but less P rickets. There was a very clear linear trend for the incidence and severity of TD to decrease with hen age (P < 0.04). The incidence of P rickets decreased from 39 to 44 wk but then increased (P < 0.02). DISCUSSION The positive relationship between Mat D 3 and 16-d chick BWG in the current work is consistent with published literature for chickens (Bethke et al., 1936) and turkeys (Robertson et al., 1941; Stadelman et al., 1950; Stevens et al., 1984). Bethke et al. (1936) found that the level of vitamin D 3 in the breeder diet influences chick weight and bone calcification at 5 wk of age when fed a rachitogenic diet. Stadelman et al. (1950) and Stevens et al. (1984) have since shown that growth and calcification in turkey poults fed a vitamin D 3 free diet is directly proportional during the first 4 wk to the vitamin D 3 level in the dam diets. Griminger (1966) reported that chicks from breeder hens fed different levels of vitamin D 3 showed no difference in weight after 1 wk of age. However, an indication of the effect of Mat D 3 was evident at 2 wk and became more pronounced each week during the following 3 wk. For tibia ash, the similarity between the HMD 3 and LMD 3 chicks was unexpected because the percentage bone ash in chicks and turkey poults has been shown to be sensitive to the level of vitamin D 3 in the maternal diet up to 5 wk (Bethke et al., 1936) and 2 wk of age (Robertson et al., 1941), respectively. This lack of response might have been influenced by the addition of the recommended dose of 1,100 IU of vitamin D 3 /kg to the experimental diets, whereas the studies conducted by Bethke et al. (1936) and Robertson et al. (1941) used diets that were deficient in vitamin D 3. The very high phytate P retention values measured from chicks fed the TD-inducing diet (Table 2) were due to the 1,000 phytase units/kg Natuphos phytase in the diet. The relatively low concentrations of P and relatively high concentrations of Ca in the diet might also have increased phytate P retention. Low concentrations of free P in the gut may drive the hydrolysis of labile orthophosphate groups from the phytate molecule into solution, whereas higher P concentrations could slow or reverse this reaction. The TD-inducing diet also contained low levels of Ca (0.60%), which probably contributed to the improved phytate P retention. Calcium at recommended levels in the broiler diet binds a significant portion of the soluble phytate in the gut (Nelson, 1967). The Ca-bound phytate molecule precipitates from solution and remains inaccessible to phytase and phosphatase enzymes that may be present. The low levels of Ca in the TD-inducing diet would have resulted in less Ca-phytate salt formation. The decrease in TD observed over the 4 hen ages for chicks from hens fed the HMD 3 (Table 3) could have resulted from the increased deposition of vitamin D 3 (cholecalciferol) in each egg yolk as the laying cycle progressed. Under normal circumstances, hens transfer cholecalciferol and 25-hydroxycholecalciferol (25- OHD 3 ) to their egg yolks (Fraser and Emtage, 1976). The embryos then convert these compounds to 1,25- dihydroxycholecalciferol [1,25-(OH) 2 D 3 ] during the third week of incubation when it is required for the active transport of Ca from the shell to the embryo across the chorioallantoic membrane (Hart and DeLuca, 1985). Enzymes in the chick embryo are able to convert cholecalciferol to 25-OHD 3 in the liver as early as d 18 (Kubota et al., 1981) and 25-OHD 3 to 1,25-(OH) 2 D 3 in the embry-

6 44 DRIVER ET AL. Table 5. summary of the effect of egg weight (EW) on tibial dyschondroplasia (TD) score and incidence of chicks from hens fed 250 (LMD 3 ) or 2,000 IU/kg (HMD 3 ) vitamin D (Mat D 3 ) TD TD TD3 TD TD TD3 Treatment df score incidence incidence df score incidence incidence R 2 Intercept EW Mat D EW D Error 12 LMD 3 HMD 3 R (coefficient) Intercept 1 (12.2) (1,303.4) ( 474.2) 1 (22.0) (1,015.8) (441.3) EW 1 ( 0.20) ( 19.0) 1 ( 0.31) ( 14.4) ( 6.3) Error 5 7 onic kidney as early as d 14 (Moriuchi and DeLuca, 1974). The development of TD is reduced by the presence of various vitamin D metabolites in the bird, including 25-OHD 3 and 1,25-(OH) 2 D 3 (Edwards et al., 1992; Rennie et al., 1993, 1995; Thorp et al., 1993; Roberson and Edwards, 1996; Elliot and Edwards, 1997; Mitchell et al., 1997a,b; Xu et al., 1997; McCormack et al., 2004). The absence of any decline in TD score or incidence for chicks from hens fed the LMD 3 (Table 3) suggests that these hens did not have sufficient vitamin D 3 reserves to deposit more vitamin D 3 into each egg as the laying cycle progressed. Guerrant et al. (1935) investigated the ability of hens to deposit more vitamin D 3 into the eggs over the course of the laying cycle. These hens had adequate vitamin D 3 reserves like those fed the HMD 3 in the current work. The authors reported that young pullets were found to produce eggs that were less antirachitic than those from mature hens and hypothesized that the lower potency from pullets was due to greater egg production compared with older birds, that pullets might not be able to transfer vitamin D 3 into the egg as efficiently as older birds, or that pullets may have greater requirements for the vitamin. Many of the possible factors that influence the amount of vitamin D 3 that is deposited in the egg are interrelated. It is difficult to evaluate the relative contributions of egg or yolk size, egg production, and hen age to the vitamin Table 6. Effect of age of the hens (Age) on body weight gain (BWG), feed intake, feed conversion ratio (FCR), percentage phytate P retention and percentage tibia ash of broiler chicks fed NRC (1994) recommended levels ofcaandp(0to16d) 1 Maternal BWG Feed FCR Phytate Tibia Age D 3 Rep 2 (g) intake (g) (g/g) P retention (%) ash (%) 39 2, ± 10 b 587 ± 11 ab 1.22 ± ± 1.9 a 39.3 ± , ± 17 b 574 ± 21 b 1.25 ± ± 3.0 ab 37.9 ± , ± 9 a 628 ± 14 a 1.22 ± ± 1.8 c 39.0 ± , ± 5 a 628 ± 11 a 1.23 ± ± 3.6 b 38.0 ± 1.0 df R Age < Error 19 R (coefficient) Intercept 1 (474.4) (574.9) (1.211) (61.6) (39.7) < < < < < Age 1 ( 1.4) (5.9) (0.017) ( 23.7) ( 0.6) Age age 1 (3.1) (2.2) ( 0.004) (3.8) (0.1) Error 20 a c Values within variables with no common superscripts differ significantly (P < 0.05) when tested with Duncan s Multiple Range Test following. 2 Rep indicates the number of replicate pens used per treatment. Each pen contained 8 chicks.

7 EFFECTS OF MATERNAL DIET ON CHICKS 45 Table 7. Effect of hen age on leg pathologies of broiler chicks fed NRC (1994) recommended levels of Ca and P (0 to 16 d) 1 P rickets Maternal score 3 Ca rickets TD score 3 P rickets Ca rickets TD TD3 Age D 3 Rep 2 per bird score 3 per bird per bird incidence (%) incidence (%) incidence (%) incidence (%) 39 2, ± 0.14 a 0.02 ± ± 0.19 a 31.3 ± 9.1 a 1.6 ± ± 7.8 a 23.4 ± 7.3 a 44 2, ± 0.03 b 0.06 ± ± 0.16 b 3.1 ± 3.1 b 3.1 ± ± 8.8 b 9.4 ± 6.0 b 53 2, ± 0.04 b 0.00 ± ± 0.16 b 4.2 ± 2.6 b 0.0 ± ± 9.4 b 8.3 ± 4.2 b 64 2, ± 0.05 b 0.00 ± ± 0.08 b 16.0 ± 4.0 ab 0.0 ± ± 4.0 b 4.0 ± 4.0 b df R Age Error 19 R (coefficient) Intercept 1 (1.072) ( 0.007) (2.088) (74.5) (1.3) (122.3) (38.0) < < Age 1 ( 0.723) (0.039) ( 0.825) ( 53.6) (0.8) ( 45.1) ( 17.3) Age age 1 (0.122) ( 0.010) (0.100) (9.8) ( 0.3) (5.2) (2.3) Error 20 a,b Values within variables with no common superscripts differ significantly (P < 0.05) when tested with Duncan s multiple range test following. 2 Rep indicates the number of replicate pens used per treatment. Each pen contained 8 chick. 3 Rickets and TD were scored 0, 1, 2, or 3, where 3 is the most severe (Edwards and Veltmann, 1983). D 3 status of the chick because as the hen ages, she increases in size, which allows for the production of larger but fewer eggs. However, regression analyses using TD scores and incidences to indicate the vitamin D 3 status of the chick suggest that EW is a more important pre- dictor than HDEP, Mat D 3, and hen age in determining the vitamin D 3 status of the chick (for TD incidence, R 2 = 0.62, 0.36, 0.12, and 0.01 for EW, hen age, Mat D 3, and HDEP, respectively). This is supported by the fact that for the LMD 3 chicks, EW did not change much between Table 8. Effect of maternal vitamin D 3 (Mat D 3 ) supplementation and age of the hens on hen-day egg production (HDEP), egg weight (EW), and hatch weight 1 HDEP 3 Egg Hatch Week Mat D 3 Rep 2 (%) weight (g) weight (g) ± 6.9 ab 41.3 ± , ± 3.8 a 41.2 ± ± 9.5 bc 64.6 ± ± , ± 2.6 abc 64.9 ± ± ± 4.4 abc 44.0 ± , ± 6.4 abc 44.1 ± ± 5.2 d 65.7 ± ± , ± 1.2 c 68.8 ± ± 1.2 Main effect ± 3.7 a 41.3 ± 0.3 b ± 5.1 b 64.8 ± ± 0.5 a ± 3.6 b 44.0 ± 0.5 a ± 6.3 c 67.2 ± ± 0.9 a Mat D ± 5.9 b 65.1 ± ± 0.6 2, ± 3.4 a 66.8 ± ± 0.5 R Age Mat D Age Mat D a d Values within variables with no common superscripts differ significantly (P < 0.05) when tested with Duncan s multiple range test following. 2 Rep indicates the number of replicate pens used per treatment. Each pen contained 8 chicks. 3 HDEP = (number of eggs laid per day/number of hens) A. Atencio, H. M. Edwards, Jr., G. M. Pesti, and G. O. Ware (University of Georgia, unpublished).

8 46 DRIVER ET AL. wk 44 and 64, and the TD scores and incidences measured for chicks hatched at these times remained high (Table 3) even though hen age increased and HDEP decreased (Tables 4, 5, and 8). In addition, TD for the chicks hatched from hens fed the HMD 3 decreased with increased egg size, although there was a concomitant increase in hen age and a decrease in HDEP (Tables 4, 5, and 8). The analyses conducted to determine if HDEP or EW affected TD score (Tables 4 and 5) showed effects only in the chicks from hens fed the higher Mat D 3 level. Our original hypothesis was that as egg numbers declined, more vitamin D 3 could be deposited in each egg and that should be especially important for the LMD 3 chicks. The opposite was observed, and so our hypothesized difference was correct, but the relative data were surprising. The HDEP and EW are negatively related; after egg production peaked, HDEP declined and EW increased. From the present data we could not find a statistical model to determine whether the important factor is HDEP or EW. Further studies are needed to determine if HDEP or EW per se are important predictors of TD in commercial practice, because they are correlated, perhaps using either will suffice. But these data suggest that chicks form older hens with lower HDEP and larger eggs should have reduced incidences of TD. The analyses in Tables 4 and 5 show that hen age, HDEP, or egg size could be used to predict the incidence of TD. The increase in HDEP observed with hens fed the LMD 3 during wk 53 and the significantly greater BWG and feed intakes of their chicks for the same week is inconsistent with the rest of the results obtained (Table 2). Ultraviolet light might have leaked into the breeder house during the summer, which occurred during this period (A. Atencio, H. M. Edwards, Jr., G. M. Pesti, and G. O. Ware, University of Georgia, unpublished). This light leakage would have enabled the hens to produce their own source of vitamin D 3. The high levels of TD as well as P rickets detected in chicks fed the control diet with adequate Ca and P for the first week tested (wk 39) was unexpected and suggests that even the HMD 3 level fed to the hens (2,000 IU/kg) may be insufficient to eliminate all bone pathologies in the progeny, especially during the beginning of the laying cycle when egg production is high and eggs are smaller (Table 7). The severity of both diseases was reduced as the birds became older, which again could have been related to increased deposition of vitamin D metabolites in the egg as the laying cycle progresses. Vitamin D metabolites have been shown to increase phytate P retention in broiler chicks (Edwards, 1993), which would have increased the amount of P available to the bird and decreased the incidence of P rickets. It is difficult to ascertain whether the analyzed NPP values in the TD-inducing diet or adequate control diet are accurate or not. The percentage of NPP analyzed for the TD-inducing diet was 0.1% lower than the calculated 0.26%, whereas NPP in the control diet was approximately 0.1% higher. Both diets were mixed using the same quantities of corn and soybean meal. It seems likely that both diets did in fact contain levels of NPP similar to the predicted levels of NPP and that the discrepancy between the analyzed and predicted values was due to sampling error. In conclusion, results of this study suggest that as egg production declined and egg size increased toward the end of the laying cycle, hens fed adequate levels of vitamin D 3 (HMD 3 ) might have been able to deposit sufficient quantities of vitamin D 3 in the egg to maintain excellent 16-d BWG and reduce the incidence and severity of TD when fed a TD-inducing diet. 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