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Egg shell quality has always been a problem in the layer industry. Economic losses because of poor shell quality are estimated to be greater than $250 million per year (Bell, 1998). Numerous studies have been conducted to solve poor shell quality problems. Many of these studies have focused on macro minerals, especially calcium (Ca) and phosphorus (P) (Keshavarz, 1988; Roush et al.,1986).

A study on different levels of calcium (2.5, 3.5 and 4.5%) has shown that both 3.5 and 4.5% Ca decreased egg deformation and 4.5% Ca also increased shell weight (P<0.05) (Clunies et al., 1992). Wallner-Pendleton and Scheideler (1996) reported improved egg weight, egg production and bone ash when dietary calcium was increased from 3.5 to 5.5% in the diet of older (60+ weeks) laying hens. No significant effects on shell quality were measured. Trace minerals such as manganese (Mn) and zinc (Zn) play an important role in egg shell and shell membrane formation due to the fact that these are co-factor and/or structural components of enzyme systems responsible for carbonate formation and mucopolysaccharide synthesis, respectively (Gomez-Basuri, 1998).

Recent studies by Keshavarz at Cornell (cited by Gomez-Basauri, 1998) and Miles at the University of Florida (1998) on this subject have illustrated that replacement of inorganic manganese and zinc with different levels of zinc and manganese proteinate from Eggshell 49 improved egg shell quality (P<0.05).

One can ask why use organic minerals instead of inorganic? Gill (1997) reported that chelated trace mineral (organic) sources are more biologically available in an animal digestive system than inorganic minerals, perhaps resulting in less mineral pollution of the environment. In a field study conducted by Miles (1998) with 52-61 week old layers, there was an improvement in egg grading when Eggshell 49 was added at a rate of 1 kg/ton. Others have emphasized that the shell to organic membrane relationship is critical to good shell quality. Another factor influencing egg shell quality is hen age; calcium uptake decreases as hens get older (Al-Batshan et al., 1994).

Eggshell 49 has been recommended as a supplement, particularly after week 49 (Klecker et al., 1997).
The mixed results of the studies mentioned indicate that macro and trace mineral form as well as hen age can all have significant effects on egg shell quality. The question asked in the following study was ‘What are the combined effects of organic trace minerals (from Eggshell 49), calcium level and age on egg shell quality?’


Materials and methods

DIETS, MANAGEMENT AND TREATMENTS

During 1997-1998 the University of Nebraska layer nutrition program conducted a long term trial testing effects of supplemental manganese and zinc from Eggshell 49 (Alltech Inc.) and dietary calcium level on egg shell quality and metabolic measurements. Eight diets were assigned to Hy-Line W-36 laying hens beginning at 20 weeks of age and were fed until 60 weeks of age. Two levels of calcium (3.5 and 4.0%) and two levels of Eggshell 49 (0 or 2 lb/ton) were tested.

In order to answer questions about the best time to begin feeding Eggshell 49, the product was offered either not at all, during weeks 20-40 only, during weeks 40-60 only or throughout the experiment (weeks 20-60). This yielded a factorial treatment arrangement. Each treatment was replicated six times with six hens per replicate pen for a total of 48 pens.

The research was conducted at the University of Nebraska (Lincoln) in industry-type caging and stocking densities. Diets were formulated according to the National Research Council (1994) recommendations. After 44 weeks of age diets were reformulated to decrease the diet density as per the Hy-Line Management Guide (1995). Nutrients and ingredient composition of experimental diets are given in Table 1. The diets were presented in mash form and feed and water were consumed ad libitum. Egg production, feed intake and cracked eggs were recorded daily. Egg weights, egg specific gravity, albumen height (Haugh units), dry shell percent and shell strength were measured bi-weekly. Hens were weighed at the start of experiment and at three-week intervals thereafter.


SHELL GLAND CARBONIC ANHYDRASE ACTIVITY

To ascertain potential effects of zinc supplementation on carbonic anhydrase activity, an in vitro assay was conducted with shell gland tissue obtained at 40 and 60 weeks of age. The shell gland was immediately rinsed with ice-cold glass-distilled water and kept in an ice bath. After being blotted dry, the shell gland was cut into two portions longitudinally. One portion was minced finely with scissors into a weighing tube kept on ice and homogenized with ice-cold distilled water. The homogenate was centrifuged at 1500 x g at 4 °C for 10 minutes.


TABLE 1 - Ingredient and nutrient composition of the diets. Click here to see the image


Enzyme activity was determined on the supernatant using modifications (Worthington Enzymes Manual, 1998) of the electrometric method of Wilbur and Anderson (1948) in which the time required for a saturated CO2 solution to lower the pH of 0.02Mtris buffer from 8.3 to 6.3 in the presence of the enzyme was measured at 2°C.


INTESTINAL CALCIUM UPTAKE

To test effects of dietary calcium and Eggshell 49 effects on in vitro calcium utilization, calcium uptake was determined in the duodenum of hens at 40 and 60 weeks of age by a modification of the method of Al-Batshan et al. (1994). Tissues from the same birds killed for shell gland sampling were used to determine calcium uptake in the duodenum. Birds which had a fully calcified egg in the shell gland were selected for the calcium uptake analysis.


Results

20-40 WEEKS OF AGE

Performance and albumen height

Table 2 shows the dietary effects on average hen weights, egg production, feed intake, feed conversion, egg mass, egg weight and Haugh unit for the 20-40 weeks period. Main effect means and interaction (treatment) means are given. In this period four of the eight diets are the same since Eggshell 49 supplementation did not begin until after 40 weeks of age for two of the treatments. Neither Eggshell 49 nor calcium level affected hen weight, egg mass, feed conversion, egg weight or albumen height (Haugh units) during the first period (Table 2).

A significant interaction between calcium level and Eggshell 49 occurred with respect to egg production and egg mass such that when hens were fed 4.0% Ca treatments, Eggshell 49 supplementation decreased egg production by nearly 2%. However, in diets containing 3.5% Ca, Eggshell 49 had a more positive effect on egg production and egg mass. Hens given 4% Ca diets tended to have higher feed intake (P<0.06). Eggshell 49 calcium level treatments resulted in a significant interaction for feed conversion ratio (FCR) in that Eggshell 49 tended to improve feed conversion in birds given 3.5% Ca and have a negative impact on this parameter in birds given 4% Ca.


Egg shell quality, serum calcium, calcium uptake and carbonic anhydrase activity

Average specific gravity, dry shell percent, shell strength, cracked eggs, serum calcium, shell gland carbonic anhydrase activity and calcium uptake results from 20-40 weeks are presented in Table 3. Shell specific gravity was unaffected by treatment (Table 3). Eggshell 49 had a positive impact on dry shell percent, shell strength and percent cracked eggs. These results are in agreement with those reported in the Keshavarz study as well as Miles (1998).

Eggshell 49 supplementation markedly increased activity of shell gland carbonic anhydrase, an enzyme with a potential relationship to shell quality. Increasing calcium level also increased the enzyme activity significantly (P<0.01). Wang et al. (1996) reported carbonic anhydrase activity in hens with high quality eggs to be greater (P<0.01) than activity in hens producing low quality eggs. In this study a positive relationship between enzyme activity and shell quality criteria was evident (Figure 1). Calcium uptake in the duodenumincreased with dietary calcium level (P<0.05) but was unaffected by Eggshell 49 at both 4 and 9 minute measurements. Serum calcium levels were unaffected by treatments. In summary, Eggshell 49 supplementation had a positive impact on most shell criteria during the 20-40 week period.


TABLE 2 - Effects of dietary calcium level, Egshell 49 and hen age on hen weight, egg production, egg weight, egg mass, Haugh unit, feed intake, conversion ratio during 20-40 weeks of age. Click here to see the image



TABLE 3 - Effects of dietary calcium level, Egshell 49 and hen age on specific gravity, dry shell percent, shell strength, cracked eggs, serum calcium, calcium uptake and carbonic anhydrase from 20-40 weeks of age. Click here to see the image

OVERALL RESPONSE: 20-60 WEEKS

Performance and albumen height

The hens receiving Eggshell 49 in both the 20-40 and 40-60 week periods had the lowest weight (Table 4). This can be attributed in part to egg production because these hens, along with those given Eggshell 49 weeks 40-60, had higher egg production. Feed intake was unaffected by treatment for the entire experiment. Significant interaction effects on egg production for the entire period were similar to those noted in the 20-40 week period. Egg production, feed intake, feed conversion and egg mass were unaffected by treatments. Egg weight, Haugh units, and specific gravity were also unaffected. These results are in agreement with Keshavarz (cited by Gomez-Basauri, 1998). Although the difference was not significant, the heaviest egg weight and highest albumen quality was found in the groups receiving Eggshell 49 in both the 20-40 and 40-60 week periods.


Egg shell quality, serum calcium, calcium uptake and carbonic anhydrase

Dry shell percent, shell strength, and percent cracked eggs were influenced significantly by Eggshell 49 supplementation (Table 5). Dry shell percent was highest in hens given diets which had contained Eggshell 49 during the 20-40 week period and 4% Ca and next highest in hens given Eggshell 49 during both periods (Table 5, Figure 2). The lowest percent shell was found in the 3.5% Ca diet without Eggshell 49 during the entire experiment.

Increasing dietary calcium from 3.5 to 4% also improved percent dry shell. These results are in agreement with those reported by Clunies et al. (1992) who reported that 4.5% dietary calcium increased shell weight. Interaction between Eggshell 49 and dietary calcium was also significant (P<0.012). Eggshell 49 had a positive effect on shell strength when given throughout the 20-60 trial period. Eggshell 49 from 20-40 or 40-60 weeks provided intermediary positive effects on shell strength. Both 3.5 and 4.0% Ca without Eggshell 49 for the entire experiment had the poorest shell strength. Klecker et al. (1997) reported that Eggshell 49 improved shell strength, shell weight and shell thickness, in agreement with results of this study. The percentage of cracked eggs was significantly reduced when Eggshell 49 was included from 20-60 weeks of age. Increasing dietary calcium from 3.5 to 4% also tended to decrease (P<0.08) percent cracked eggs.

Shell gland carbonic anhydrase activity was affected by both calcium level and Eggshell 49 (Figure 3). Hens receiving Eggshell 49 in both the 20-40 and 40-60 week periods had the greatest enzyme activity while the hens receiving no Eggshell 49 had the lowest value. Hens receiving 4% Ca tended to have higher carbonic anhydrase activity than those receiving 3.5% Ca. Increasing dietary calcium level from 3.5 to 4% decreased duodenal calcium uptake. The highest calcium uptake was found in the 3.5% Ca diet containing Eggshell 49 from 20-60 weeks.

Calcium uptake tended to increase in response to Eggshell 49 (Table 5). These results are contrary to the 40 week results (Table 3) at which time the 4.0% Ca treatment had greater uptake than 3.5% Ca. We may attribute this change to age as younger layers produce smaller eggs than older hens. As a result, 3.5% Ca is sufficient for young hens, but as hens get older the efficiency of calcium absorption would need to increase in order to meet calcium requirements. Al-Batshan et al. (1994) has reported a decline in egg shell quality as the hen ages which may be attributed in part to reduced intestinal calcium uptake as well as to increased egg size.
There was also a decrease in carbonic anhydrase activity and calcium uptake (Tables 3 and 5) as hens become older. Comparing the 40 week and 60 week results it is evident that egg shell quality is declining. Hence we can postulate that the decline in enzyme activity and calcium uptake as hens age is related to decreased shell quality.

Serum calcium tended to be lower in hens given 3.5% Ca (Table 5). This may explain the increased efficiency of calcium absorption in response to calcium demand. Wang et al. (1996) also noted that serum calcium tended to decrease as egg shell quality decreased. They suggested that serum calcium was lower in birds laying eggs with high quality shells than in hens producing low quality egg shells.


TABLE 4 - Effects of dietary calcium level, Eggshell 49 and hen age on hen weight, egg production, feed intake, egg weight, egg mass, feed conversion ratio, Haugh unit, and specific gravity. Click here to see the image






TABLE 5 - Effects of dietary calcium level, Egshell 49 and hen age on dry shell percent, shell strength, cracked eggs, serum calcium, calcium uptake and carbonic anhydrase activity. Click here to see the image






Summary

From these results we can conclude that Eggshell 49 starting at 20 weeks till 60 weeks of age had a positive impact on all shell quality criteria.

Further, although 3.5% Ca in the diet was found to be adequate for 20-40 weeks, 4.0% Ca should be recommended after 40 weeks.


Acknowledgments

Appreciation is expressed to Alltech, Inc. for support of the study, to Curtis Novak, Lyle Robeson, Tommi Jones, and Minnie Stephens for their help.


References

Al-Batshan, H.L., S.E. Scheideler, B.L. Black, J.D. Garlich and K.E. Anderson. 1994. Duodenal calcium uptake, femur ash, and egg shell quality decline with age and increase following molt. Poultry Sci. 73:1590-1596.

Bell, D. 1998. Egg shell quality: Its impact on production, processing and marketing economics. In: Biotechnology in the Feed Industry, Proceedings of the 14th Annual Symposium (T.P. Lyons and K.A. Jacques, eds). Nottingham University Press, Nottingham, UK. p. 447-466.

Clunies, M., D. Parks and S. Leeson. 1992. Calcium and phosphorus metabolism and egg shell formation of hens fed different amounts of calcium. Poultry Sci. 71:482-489.

Gill, C. 1997. Organic Trace Metal Assays. Feed International. 18(10):18-22.

Gomez-Basauri, J. 1998. Egg shell quality: Overcoming production losses with a novel enzyme activator. In: Biotechnology in the Feed Industry, Proceedings of the 14th Annual Symposium (T.P. Lyons and K.A. Jacques, eds). Nottingham University Press, Nottingham, UK. p. 437-445.

Keshavarz, K. 1988. Calcium nutrition can directly influence shell quality. Feedstuffs. Febr. 20, 1988. p. 23-24.

Klecker,D., L. Zemar, V. Viske, J. Gomez Basauri. 1997. Influence of trace mineral proteinate supplementation on egg shell quality. Poultry Sci. 76(Suppl. 1)131.

Miles, R.D. 1998. The influence of Eggshell 49 on shell quality of hens grouped by their shell quality. Poultry Sci. 77(Suppl. 1):43.

National Research Council. 1994. Nutrient Requirements of Poultry (9th Ed.). National Academy Press, Washington, DC.

Roush, W.B., M. Mylet, J.L. Rosenberger and J. Derr. 1986. Investigation of calcium and available phosphorus requirements for laying hens by response surface methodology. Poultry Sci. 65:964-970.

Wallner-Pendleton, E. and S.E. Scheideler. 1996. The influence of varying levels of calcium and vitamin D3 in the mature laying hen’s diet: effects on egg production, shell quality, bone ash and urolithiasis. The Nebraska Poultry Report, University of Nebr. Coop. Ext. E.C. 96-259A.

Wang, C.W., K. T. Nam, O. E. Olsen and C.W. Carlson. 1996. Relationship between egg shell quality and biochemical parameters of calcium metabolism. AJAS 9(6):715-722.

Wilbur, K., and N. G. Anderson. 1948. Electrometric and calorimetric determination of carbonic anhydrase. J. Biol. Chem. 176:147-154.

Worthington Enzymes Manual. 1998. Carbonic Anhydrase.Worthingtonbiochem. com/C/CA.

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