Calcium and Phosphorus Metabolism and Eggshell Formation of Hens Fed Different Amounts of Calcium

METABOLISM AND NUTRITION Calcium and Phosphorus Metabolism and Eggshell Formation of Hens Fed Different Amounts of Calcium M CLUNIES, D. PARKS, and S. LEESON Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, NIG 2W2, Canada (Received for publication February 25, 1991)

1992 Poultry Science 71:482-489

INTRODUCTION A large amount of research has been done on the effect of feeding various amounts of Ca to laying hens (Hurwitz and Bar, 1965; Ousterhout, 1980; Gilbert et ah, 1981). Some variability in results is due to laying hens having different demands for Ca on days when an eggshell is being formed (Mongin and Sauveur, 1974), and the fact that, for birds fed complete diets, feed consumption is greater on shellforming (SF) days compared with days on which no shell formation occurs (NSF) (Morris and Taylor, 1967; Taylor, 1970). Taylor and Kirkley (1967) investigated Ca and P metabolism and found an increase in Ca and P retention on SF days. Hurwitz and Bar (1969) reported differences in Ca absorption due to differing levels of die-

tary Ca, in response to the process of shell formation. The more rapid stage of shell secretion partially occurs during the dark period, when the digestive tract is relatively low in Ca reserves (Roland et ah, 1973). Miles et ah (1984) measured plasma P in laying hens actively secreting a shell and found plasma levels of P were evaluated during late shell secretion for eggs subsequently laid in the morning. Because Ca is incorporated into bone as hydroxyapatite, a Pcontaining compound, the mobilization of bone mineral Ca for shell formation leads to elevated levels of plasma P (Miles et ah, 1984). This is due to the high Ca:P ratio of eggshell compared with hydroxyapatite of bone. Common (1932), Tyler (1940), and Hurwitz and Bar (1965) reported that large amounts of P are excreted during the

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ABSTRACT Twenty-seven 42-wk-old Single Comb White Leghorn hens housed in separate cages were fed either 2.5, 3.5, or 4.5% Ca diets, each providing .45% available P. Birds were allowed a 7-day adaption period followed by an 8-day collection period. Feed and water were available for ad libitum consumption with feed intake recorded daily. Eggs and excreta were collected daily for mineral analysis. Feed, Ca, and P intake of hens increased significantly (P<.05) on shell-forming (SF) days compared with days on which shell formation did not take place (NSF). Dietary Ca level had a significant (P<.05) effect on feed and Ca intake of hens. On SF days, hens retained more dietary Ca, both as a percentage and per gram Ca basis, compared with NSF days. As dietary Ca increased, the percentage Ca retained decreased (P<05) and per gram Ca retained increased (P<.05). Dietary Ca had no effect (P>.05) on egg weight or egg production. Increasing dietary Ca significantly (P<.05) decreased shell deformation and increased (P<.05) shell weight and grams of shell Ca, although there was no significant (P>.05) effect on percentage shell Ca. Calcium retention increased linearly (P<.05) as Ca intake increased, and shell weight increased quadratically (P<.05). There was a diminishing response of shell weight to Ca intake at higher levels. (Key words: feed intake, calcium intake, calcium retention, phosphorus retention, eggshell formation)

483

CALCIUM METABOLISM OF LAYING HENS TABLE 1. Composition of experimental diets

Ingredients and composition

53.77 16.00 1950 550 1.66 1.00 35 .75 .30 123 .04 2^18 15.9 .80 32 15 .45

Dietary calcium 45% 35% <%) — 55.49 12.00 20.00 8.13 1.68 \3S 25 .75 30

. . .

.05

2^05 15.8 .80 31 35 .46

5650 5.99 22.00 10.75 1.70 2.75 35 .75 50 . . . 2309 16.0 .82 30 45 .46

2.46 4.44 353 .75 .76 .73 1 Provides per kilogram of diet: vitamin A, 80,000 IU; cholecalciferoL 1,600 IU; vitamin E, 11 mg; riboflavin, 7.0 mg; pantothenic acid, 7.0 mg; vitamin Bj^ 8 ug; niacin, 20 mg; choline, 900 mg; vitamin K, 15 mg; folic acid, 15 mg; biotin, 35 mg; ethoxyquin (antioxidant), 125 mg; manganese, 50 mg; zinc, 50 mg; copper, 5.0 mg; iron 30 mg. ^Assumes 50% of the plant phosphorus is available for absorption (Summers and Leeson, 1984).

process of shell formation. In this context, P balance would provide a valuable indication of bone mineral balance in hens fed diets containing different amounts of Ca. The following experiments were designed to examine the effect of dietary Ca level on Ca and P retention of hens of SF and NSF days and to determine, using P retention as an indicator, whether bone mineral metabolism is influenced by the amount of dietary Ca. MATERIALS AND METHODS

8-day period for collection of data. Birds were housed in individual cages with separate feeders and aluminum foil paper placed below cages for collection of excreta. A total of nine replicate birds were used per dietary treatment. Hens were subjected to a 14 h light:10 h dark (0800 to 2200 h) photoperiod. Feed and water were provided for ad libitum consumption with feed weigh-back recorded. Excreta samples were collected at 0900 h. Eggs were collected twice daily, at 1100 and 1500 h. Egg production was recorded, all eggs weighed, and shell deformation determined (Summers et ah, 1976).

Animal Experiments Single Comb White Leghorn hens, 42 wk of age, were offered one of three diets differing in Ca level: 2.5, 3.5, and 4.5% Ca (Table 1). All diets were formulated to approximately the same level of available P (.45%) and chromium as chromic oxide at .3% inclusion. Birds were fed respective diets for a total of 15 days with the latter

Chemical Analyses Samples of diet, excreta, and shells were oven dried at 65 C in a forced-fan oven, cooled, weighed, and ground prior to ashing. Approximately 1-g samples of diet and .5-g samples of shell and excreta were ashed in a muffle furnace at 500 C for 24 h, and digested in 10 mL concentrated HC1

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Corn Barley Soybean meal (48% CP) Limestone Calcium phosphate (20% P) Fat (animal-vegetable blend) Salt (iodized) Vitamin and mineral mix Chromic oxide Cellulose DL-methionine Calculated analysis ME, kcal/g Crude protein Lysine Methionine Calcium Available phosphorus2 Chemical analysis Calcium Phosphorus

25%

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CLUNKS ET4L.

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different levels of dietary Ca (Table 2). Hens fed the 2.5% Ca diet consumed the least quantity of feed but the highest feed intake was observed with hens fed the 3.5% Ca diet. On SF days there were significant (P<.05) differences in feed and Ca and P intake of hens fed different levels of dietary Ca. Birds fed the 2.5% Ca diet had the lowest feed intake, and those fed 3.5% Ca exhibited the highest (P<.05) level of feed intake. Hens fed the 4.5% Ca diet had feed intakes that were no different (P>.05) than those fed the 3.5% Ca diet. The results of Ca intake are consistent each day regardless of whether eggshells are formed or not, with Ca intake increasing (P<-05) as dietary Ca level increased. The pattern of total P intake on SF days was consistent with that of feed intake. Dietary Ca content had no significant (P>.05) effect on absolute Ca and P retention on NSF days (Table 3). On SF days as dietary Ca level increased, perStatistical Analyses centage Ca retained decreased and grams Data were subjected to ANOVA using Ca retained increased (P<.05). On SF days, the General Linear Models procedures of hens fed the 2.5% Ca diet retained a SAS® (SAS Institute, 1979) according to the significantly (P<05) greater proportion of method of Snedecor and Cochran (1980). dietary Ca compared with those offered This method was used due to unequal 3.5 and 4.5% Ca. There was no significant replication of SF and NSF days. Analysis of difference (P>.05) in percentage Ca and P variance was carried out on data for SF and retained by hens consuming 3.5 or 4.5% NSF days separately. Planned mean con- Ca diet on SF days. Although hens fed the trasts were made using least square mean 3.5 and 4.5% Ca diet did not differ (P>.05) differences (Cochran and Cox, 1957). Re- in grams P retained, those hens fed 2.5% gression and correlation analyses were Ca retained significantly (P<.05) less used to determine the relation between grams P on SF days. Calcium level had no significant (P>.05) shell weight and feed intake, mineral intake, and mineral retention according to effect on egg production or egg weight the methods of Snedecor and Cochran (Table 4). Hens consuming the 2.5% Ca diet produced eggs with significantly (1980). (P<.05) greater shell deformation compared with those fed 3.5 or 4.5% Ca diets. RESULTS There was no significant (P>.05) difference There were significant (P<.05) differ- in eggshell deformation of hens fed 3.5 or ences in feed, Ca, and P intake of hens on 4.5% Ca diets. As dietary Ca level was NSF days between groups of hens fed increased there was a significant (P<.05) increase in shell weight and grams shell Ca. Percentage shell Ca did not differ significantly (P>.05) between dietary treat^Tectron Model AA4, Varian Canada Inc., George- ments. town, ON, L7G 2J4, Canada. Regression analysis showed there was a 2 Model AA2, Technicon Instrument Corp., Tar- significant (P<.05) positive linear relationrytown, NY 10591. 3 Vye Unicam SP6-500, Anachemia Sciences Inc., ship between Ca intake and grams Ca retained (Figure 1). However, the relationMississauga, ON, L5L 4X4, Canada. and 30 mL double-distilled water. Calcium was determined by atomic absorption spectrophotometry1 and P using an autoanalyzer 2 as described by Association Official Analytical Chemists (1980). Similarly, 1-g samples of feed and excreta were ashed and digested in 20 mL of H 2 0:H2S0 4 : HCIO4 (4:2:2) solution. Chromium content of feed and excreta were determined colorimetrically used a spectrophotometer3 at 400 nm (Fenton and Fenton, 1980). Daily mineral retention, defined as the proportion of dietary mineral not appearing in the excreta, was determined for SF days and NSF days. An SF day is defined as the day prior to which an egg was laid and an NSF day preceded a day on which no egg was laid. Apparent Ca balance, defined as dietary Ca retained minus that in the eggshell, was determined for SF days, NSF days, and on an average daily basis for the 8-day study.

485

CALCIUM METABOLISM OF LAYING HENS TABLE 2. Feed, calcium, and phosphorus intake of hens Calcium intake

Phosphorus intake

88.1b 108.1* 94.6*b

250° 3.78b 457*

(mg/day) 669b 843* 737*b

4*

4

Shell formation

Calcium level

Feed intake

NSFday

(% diet) 23 3.5 4.5

- (g/day)

Significance SD

6.1

Significance SD

67 718° 857* 813b

2.6 29 .08 ~ Means within columns and days with no common superscripts are significantly different (P<.05). •'NSF = day on which shell formation does not occur; SF = shell-forming day. *P<05. **P<.01. a c

ships between Ca intake or Ca retention (Figures 2 and 3) and shell weight was quadratic (P<.01). Regression analysis showed that the hens' Ca intake and grams Ca retained had the most significant (P<.05) effect upon predicting shell weight of the eggs they produced. Regression analysis indicated a quadratic relationship between shell weight and both Ca intake and grams Ca retained (P<05).

Percentage P retention increased linearly as Ca intake increased (P<.05, Figure 4). Phosphorus intake, percentage and grams P retained were not significant (P>.05) parameters in predicting the shell weight of eggs produced by hens fed different levels of dietary Ca. On SF days, hens fed 2.5 and 3.5% Ca diets deposited more Ca as eggshell than was retained from the diet (Table 5).

TABLE 3. Calcium and phosphorus retention of hens Shell formation1

Calcium level

Calcium retention2

NSFday

(% diet) 25 35 45

58.6* 38.4b 365 b

Significance SD c

Calcium retention

Phosphorus retention

55

31.3 325 25.9 NS 6.6

(g/day) 155 1.49 153 NS 50

(mg/day) 190 250 170 NS 40

625* 51.4b 505 b

115 b 23.8* 21.9*

1.45° 1.94b 235*

80b 180* 160*

4*

44

4*

44

<%)

4*

Significance SD SF day

Phosphorus retention2

25 3.5 45

1.9

253

.07

19

*~ Means within columns and days with no common superscripts are significantly different (P<.05). *NSF = day on which shell formation does not occur; SF = shell-forming day. Due to some missing excreta values, these data do not correspond exactly with actual intake data shown in Table 2. »P<.01.

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94.7b 110.0* 1045*

25 35 45

till

SFday

.19

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CLUNIES ET AL. TABLE 4. Egg production, egg weight; shell deformation, shell weight, and shell calcium of hens fed diets varying in calcium concentration

Dietary calcium

Egg production im\

25 35 4.5 Significance SD

91.7 91.4 89.1 NS 5.8

Egg weight

Egg deformation

Shell weight

(g) 57.6 56.6 60.0 NS 2.8

(um) 31.4* 23.6b 245 b

(g) 4.99° 5.48b 5.79* <** .07

4.8

Shell calcium (%) 36.7 385 375 NS 1.9

(g) 1.83b 2.11* Z17* ** .01

a-c

However, hens fed the 4.5% Ca diet retained more Ca from the diet than was deposited as shell. Dietary Ca had no significant (P>.05) effect on the overall average daily Ca balance of hens.

NSF days, results can be compared, as the same trends were observed for SF and NSF days in the present study. Although Roland et al. (1985) indicated that hens fed low levels of Ca would overconsume energy in order to obtain Ca needed for shell secretion, Roush et al. (1986) reported DISCUSSION that for hens fed lower concentrations of Results from the present experiment Ca, increasing the amount of available P suggest that high-producing hens fed a decreased feed intake. Also, for any given low level of Ca do not increase their feed level of P, when hens were fed diets low intake on SF days in order to compensate in Ca, feed intake was depressed (Roush et for low dietary Ca (Table 2). These results al., 1986). This decrease in feed intake may are different from those of Roland et al. also be related to the elevated concentra(1985), who reported a significant (P<.05) tions of P in plasma reported by Miles et inverse linear relationship between level al. (1984). Although plasma P was not of dietary Ca and feed intake of hens. measured, apparent P retention (Table 3) Although the data of Roland et al. (1985) indicates that hens fed 2.5% Ca retained did not differentiate between SF days and less grams total P on SF days compared

• * • * • *

•

s

CALCIUM INTAKE (GRAMS)

FIGURE 1. Effect of calcium intake on calcium retention of hens on shell-forming days. Y = 326(± .036, SE) X ± .720(± .138); R2 = .320; residual standard deviation = 561; where Y = calcium retention in grams, and X = calcium intake in grams.

a

CALCIUM INTAKE (GRAMS)

FIGURE 2. Effect of calcium on shell weight of hens on shell-forming days. Y = -.146(± .037, SE)X2 + 1.364(± .274)X + 2529(± .479); R2 = .249; residual standard deviation = ± .6%; where Y = shell weight in grams, and X = calcium intake in grams.

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Means within columns with no common superscripts are significantly different (F<.05). **P<.01.

487

CALCIUM METABOLISM OF LAYING HENS

tained. Despite the higher efficiency of Ca retention of hens offered low Ca diets, those fed higher levels of Ca retained 1 f, Ca balance more total Ca on SF days. These results level SF day NSF day Overall are in keeping with data of Hurwitz and Bar (1969). As shown in Figure 1, Ca (% of diet) (g) retention increased linearly as Ca intake 25 -.38 b 1.25 -.107 increased, with no plateau apparent at 3.5 ~.17b 1.49 -.022 higher levels of intake. Hurwitz and Bar 45 +.18* 153 +.163 Significance * NS NS (1969) also reported no apparent plateau in grams Ca retained for hens consuming SP .U .20 .04 ^"Within columns means with no common from .6 to 3.96 g Ca/day. These results are somewhat surprising, as it has been assuperscripts are significantly different (P<.05). 1 NSF = day on which shell formation does not sumed that the hen would adjust its retention of Ca to compensate for higher occur; SF = shell-forming day. dietary Ca concentration by absorbing less *P<05. Ca. If P retention can be used as an indication of bone mineral metabolism, it with hens fed higher levels of Ca. This appears that on SF days, birds fed 2.5% Ca lower P retention is probably due to mobilized more bone mineral in response increased P excretion in response to to the process of shell formation (Table 3). elevated concentrations of this mineral in Regression analysis indicated that on SF plasma during shell formation as a result days P retention increased linearly (P<.05) of bone Ca resorption. Differences (P<.05) as Ca intake increased; consequently Ca in Ca intake on NSF days can be explained retention increased (Figure 3). These results are in agreement with Farmer et al. by the differences in dietary concentration (1986) showing that as dietary Ca of this mineral. decreased a greater proportion of shell Ca Differences (P<.05) in percentage Ca was derived from bone mineral. Tyler retained on SF and NSF days can be (1946) suggested that during shell formaattributed to a compensatory response of tion Ca absorbed from the digestive tract hens to differences in dietary Ca level. is not able to meet the requirements for Similarly, Hurwitz and Bar (1969) re- shell calcification. It was postulated that ported that as dietary Ca concentration any difference was made up by bone decreased, a greater proportion was re- mineral resorption (Tyler, 1940; Taylor, TABLE 5. Average daily calcium balance of hens fed three levels of dietary calcium

FIGURE 3. Effect of calcium retention on shell weight of hens on shell-forming days. Y = -.159(± .049 SE)X2 + 1.184(± .248)X + 3.747(± 302); R2 = .177; residual standard deviation = ±.737; where Y = shell weight in grams, and X = calcium retention in grams.

CALCIUM INTAKE (GRAMS)

FIGURE 4. Effect of calcium intake on phosphorus retention of hens on shell-forming days. Y = .049(± .009 SE)X; R2 = .145; residual standard deviation = ± .137; where Y = phosphorus retention in grams, and X = calcium intake in grams.

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CALCIUM RETENTION (GRAMS)

488

CLUNIES ET AL.

retention but may be the shell gland's ability to further increase the rate of shell secretion. It should be emphasized that in the present study the respective diets were only fed for a short time period. With more prolonged feeding birds could adjust their shell Ca output to balance that retained from the digestive tract or vice versa. Results of a simulation using equations generated from regression analysis suggest that when hens consumed 3.96 g Ca on SF days the amount of Ca retained (2.08 g Ca) was equal to that deposited as shell, and the net Ca balance for a SF day would be zero. Dietary Ca level had a significant (P<.05) effect on Ca balance of hens only on SF days. Birds fed low Ca diets appeared to rely more on bone Ca for shell secretion compared with those fed higher Ca levels. The average daily Ca balance for the entire 8-day study showed that hens fed the lowest level of Ca (2.5%) had a slight negative Ca balance, but hens fed 4.5% Ca were in a slight positive Ca balance, although these were not significantly (P>.05) different. In summary, although hens showed the capacity to adapt Ca intake within dietary treatment in response to whether shell formation was taking place or not, they were unable to compensate completely for differences in Ca content of the diet. Hens fed lower Ca diets were not able to increase their efficiency of Ca retention from the diet to compensate for differences in intake. Differences in P retention indicate that when Ca intake decreased, hens relied more on the mobilization of bone mineral to supply Ca for shell secretion. Results from the present experiment show that in order to increase shell weight and shell quality, Ca intake must be increased, though there is a diminishing response of shell weight at higher levels of Ca intake. ACKNOWLEDGMENTS

This work was supported by the Natural Sciences and Engineering Research Council of Canada and the Ontario Ministry of Agriculture and Food.

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1970). There appeared to be no difference (P>.05) in bone mineral metabolism (as indicated by P retention) on SF days of hens fed 3.5 or 4.5% Ca (Table 3). Taylor (1970) suggested that bone mineral used during shell formation would be subsequently replenished during the period when no shell was being calcified. This period would include both the time between SF on consecutive days and NSF days. There was, however, no significant (P>.05) effect of dietary treatment on P retention during NSF days, though hens offered 3.5% Ca had a numerically higher (P>.05) P retention compared with hens fed either 2.5 or 4.5% Ca diets. It was anticipated that birds that had to mobilize more bone Ca to compensate for the lower level of Ca in the diet would replenish more bone mineral on NSF days in an effort to maintain bone mass. Although hens fed 4.5% Ca retained more grams Ca on NSF days, P retention data suggests that this may not have been deposited as bone mineral. The fate of this retained Ca cannot be explained due to the fact that grams P retained by these birds was no different to that retained by birds offered 3.5% Ca. There appeared to be little difference in the accuracy of predicting shell weight using either Ca intake or grams Ca retained as indicated by regression analysis. Due to low correlation between shell weight and other mineral parameters measured (Table 5), the body weight of hens was included in regression analysis. Hen body weight had a significant (P<.05) effect upon the prediction of shell weight but resulted in no change in correlation coefficients. As a result, analysis including hen body weights are not reported here. As Ca intake increased, Ca retention increased linearly (Figure 1), and shell weight increased quadratically (Figure 2). Thus, in order to improve shell quality Ca intake must be increased; however, mere is a diminishing response of shell weight at higher Ca intakes (Figure 2). Although at higher levels of mineral intake more Ca was apparently absorbed from the digestive tract, it appears this was not transferred to the shell. The data suggest that the limit to improving shell quality may not be totally dependent on increasing Ca

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REFERENCES

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