Effects of different organic acids on performance, ileal microflora, and phosphorus utilization in laying hens fed diet deficient in non-phytate phosphorus

Accepted Manuscript Title: Effects of different organic acids on performance, ileal microflora, and phosphorus utilization in laying hens fed a diet deficient in non-phytate phosphorus Author: F. Kazempour R. Jahanian PII: DOI: Reference:

S0377-8401(16)30373-X http://dx.doi.org/doi:10.1016/j.anifeedsci.2016.11.006 ANIFEE 13669

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Please cite this article as: Kazempour, F., Jahanian, R., Effects of different organic acids on performance, ileal microflora, and phosphorus utilization in laying hens fed a diet deficient in non-phytate phosphorus.Animal Feed Science and Technology http://dx.doi.org/10.1016/j.anifeedsci.2016.11.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Effects of different organic acids on performance, ileal microflora, and phosphorus utilization in laying hens fed a diet deficient in non-phytate phosphorus

F. Kazempour a, R. Jahanian b,*

a

Department of Animal Science, Gorgan University of Agricultural Sciences and

Natural Resources, Gorgan 43464-49189, Iran b

Poultry Nutrition Research Center, Bioscitech Research Institute, Isfahan 81398-

67433, Iran

*

Corresponding author. Tel.: +98 31 3442 6808; fax: +98 31 3440 1240; cell-phone:

+98 913 233 3039. E-mail address: [email protected] (R. Jahanian).

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Highlights:  Dietary supplementation of different organic acids increased egg production.  Eggshell thickness of P-deficient hens was increased by organic acid supplementation.  Feeding NPP-deficient diets decreased tibia ash and alkaline phosphatase activity.  Tibia Ca was greater in hens fed citric and fumaric acids-supplemented diets.  The lowest E. coli count was observed for hens supplemented with butyric acid.

ABSTRACT The present study was conducted to investigate the effect of dietary supplementation of organic acids (OA) on performance and phytate phosphorus utilization in laying hens fed diets with different non-phytate phosphorus (NPP) levels. A total of 240 Hy-Line W-36 laying hens, 32-wk-old, were randomly allocated into the 8 dietary treatments with 5 cage replicates of 6 birds each. The main trial period lasted for 10 wk (34-44 wk of age), following a 2 wk adaptation period (32-34 wk of age). Dietary treatments consisted of a 4 × 2 factorial arrangement of treatments, including 4 supplemental OA (control, citric, butyric, or fumaric acids, 5 g/kg of diet) and 2 different NPP levels (60 or 100% of Hy-Line W-36 recommended values). Results showed that dietary OA supplementation increased (P < 0.05) egg production percentage, with the greatest values assigned to the hens fed citric and butyric acids-supplemented diets. Reducing dietary NPP level resulted in a marked decrease in egg production in control hens, while OA supplementation of the NPP-deficient diet could maintain egg production and egg mass, resulted in the significant (P < 0.05) OA × NPP interactions. Although feed intake was not affected by dietary treatments, supplemental OA improved (P < 0.05) feed conversion ratio in NPP-deficient hens (OA × NPP, P < 0.05). Dietary OA supplementation increased (P < 0.05) eggshell thickness, but had no marked impact on

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shell breaking strength. Serum lipid metabolites were influenced by dietary OA supplementation, so that supplemental OA decreased (P < 0.01) serum cholesterol level and increased (P < 0.001) serum high-density lipoproteins. Feeding the NPP-deficient diet decreased (P < 0.01) tibia ash and increased (P < 0.001) serum alkaline phosphatase activity. On the other hand, dietary supplementation of fumaric acid increased (P < 0.01) tibial ash and Ca contents compared to the control hens. Tibia P content was lower (P < 0.05) in hens fed the NPP-sufficient diet. Inclusion of citric acid into the NPP-deficient diet increased tibia P, resulted in a significant (P < 0.001) OA × NPP interaction. Dietary supplementation of OA reduced (P < 0.01) ileal enumeration of Salmonella, and the lowest (P < 0.05) Escherichia coli count was observed for hens supplemented with butyric acid. The present findings indicate that dietary OA supplementation increased egg production, egg mass, and eggshell thickness in hens fed the NPP-deficient diet. The beneficial impact of citric acid on tibia P content of NPPdeficient hens suggests that supplemental OA could improve P utilization even in young laying hens.

Abbreviations: ADFI, average daily feed intake; ALP, alkaline phosphatase; FCR, feed conversion ratio; HDL, high-density lipoproteins; HU, Haugh unit; MEn, metabolizable energy corrected for N retention; OA, organic acids; NPP, non-phytate phosphorus; TG, triglycerides.

Keywords: Bone calcium, Ileal microflora, Laying hens, Non-phytate phosphorus, Organic acids, Reproduction performance

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1. Introduction Phosphorus is an essential mineral and is crucial for growth and development, both structurally and metabolically (Boling et al., 2000). Laying hens require P for egg production, replacement of tissue metabolites such as nucleotides and phospholipids, and to maintain skeletal integrity (Boling et al., 2000). With few exceptions, poultry rations are based largely on cereal grains and oilseed meals. Approximately two thirds of the P in cereal grains and oilseed meals is present in the form of P bound to phytic acid, named as phytate P (Rafacz-Livingston et al., 2005b). Phosphorus in this form is generally unavailable to poultry due to low phytase activity found in the digestive tract of different poultry species (Nelson, 1976). Phytate has been observed to decrease mineral bioavailability as well as nutrient digestibility, it complexes with proteins and inhibits α-amylases, trypsin, tyrosinase, and pepsin activities (Singh and Krikorian, 1982; Deshpande and Cheryan, 1984; Caldwell, 1992). Furthermore, there is a proposition that negatively-charged phytate can form insoluble complexes of less digestibility with positively-charged proteins at the low pH values (Cheryan, 1980; Thomson and Serraino, 1986). This phytate–protein binding might also occur at high pH, mediated by polyvalent cations such as Ca, Mg, or Zn (Anderson, 1985). It is well known that P plays an important role in Ca metabolism. Phosphorus is an expensive element in poultry rations; however, the inefficient use of phytate P can also cause environmental problems. It has been estimated that 250,000 tons of manure P are produced annually and contributes to water pollution (Cromwell, 1992). Several feed additives have been investigated to determine if they can increase P utilization and decrease P excretion by poultry and swine manure. Ravindran et al. (1995) listed numerous factors that influenced phytate P utilization in poultry. Phytase (Edwards, 1983; Biehl et al., 1995; Biehl and Baker, 1996; Gordan and Roland, 1997)

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and vitamin D compounds (Edwards, 1993; Biehl et al., 1995; Angel et al., 2001; Edwards, 2002; Snow et al., 2004) have been shown to be efficacious in improving phytate P utilization in non-ruminant animals. In addition, more recent studies (Boling et al., 2000, 2001; Snow et al., 2004; Rafacz-Livingston et al., 2005b) showed that organic acids (OA) can increase phytate P utilization by poultry. Boling et al. (2000) reported that inclusion of citric acid at the levels of 0 to 60 g/kg to a P-deficient diet caused linear increases in weight gain, final weight, and percentage of tibia ash in broilers. Snow et al. (2004) showed that the effect of citric acid on phytate P utilization is additive with the effects of phytase and 1-α cholecalciferol supplementation. The studies published previously had used bone ash data as a measure of phytate P utilization. Angel et al. (2001) reported an increase in percentage of bone ash and a decrease in feed consumption when citric acid was added to the diet. Another study by Shellem and Angel (2002) suggested that a part of the citric acid effect on bone ash might be confounded by its effect on feed consumption and size of birds. However, Snow et al. (2004) reported that citric acid addition to a P-deficient diet improved tibia ash without reducing weight gain or feed intake. Little to no work has been done to determine if different OA, other than citric acid, will improve phytate P utilization in poultry, especially in laying hens. This study was conducted to clarify the effects of different OA on egg production, egg quality, ileal microflora, and phytate P utilization in laying hens fed diets with different non-phytate phosphorus (NPP) levels.

2. Materials and methods

2.1. Birds and experimental diets

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The study presented here was conducted in the Poultry Research Station of Isfahan University of Technology (Isfahan, Iran). All experimental procedures were approved by the Isfahan University of Technology Animal Care and Use Committee. A total of 240 Hy-Line W-36 laying hens, 32-wk-old, were randomly assigned to 8 experimental groups with 5 replicates of 6 hens each. Dietary treatments consisted of a 4 × 2 factorial arrangement of the treatments, including 4 supplemental OA (control diet, or diets supplemented with 5 g/kg of citric, butyric, or fumaric acids), and 2 NPP levels (60 or 100% of Hy-Line W-36 recommended values). All of the experimental diets were isocaloric and isonitrogenous (Table 1), and were formulated to provide all nutrient specifications according to Hy-Line W-36 recommendations (Hy-Line International, 2007), except for P in the NPP-deficient diets. The hens were 32 wk of age at the beginning, and after a 2 wk adaptation period (32-34 wk of age) the main trial period was started for 70 d (34-44 wk of age). The birds were housed in layer wire-floored cages (total of 40 cages) at a density of 417 cm2 per bird in a thermostatically-controlled windowless house, and had access to artificial light (16L: 8D) throughout the trial period. Feed and drinking water were provided for ad libitum consumption.

2.2. Performance Egg production and egg weight were measured daily. All eggs were weighed and weights were recorded on cage basis. Egg mass was calculated as the percentage of egg production multiplied by the egg weight. Feed consumption and body weight of hens were measured at the beginning and at the end (wk 44 of age) of experimental period. Average daily feed intake (ADFI), egg production percentage, and feed conversion ratio (FCR) were adjusted for mortality.

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2.3. Egg quality measurements To determine the egg quality indices, 5 to 6 eggs produced over the last 2 d of experimental period were collected from each cage. After individual weighing, each egg was broken using a quasistatic compression device to measure eggshell breaking strength. Eggshell thickness was measured at 3 different places (upper and lower ends and middle) by using a micrometer screw gauge. The height of the albumen (average of 4 points), midway between the yolk and the edge of the thick albumen, was measured with a tripod micrometer (Jahanian and Rasouli, 2014). Haugh unit (HU) was calculated using the formula: HU = 100 log (H + 7.57 - 1.7 W0.37), where H is the mean height (mm) of the albumen, and W is the weight (g) of egg (Silversides and Villeneuve, 1994). Yolk index was calculated by dividing the height of the yolk by its diameter. Yolk color was scored using the DSM (DSM Co., Basel, Switzerland) yolk color fan.

2.4. Tibia ash At the end of experimental period, 2 randomly-selected hens per replicate were slaughtered by cutting the jugular vein. The birds were allowed to bleed for approximately 2 min, and then, the right tibia were removed and stored at refrigerator temperature until further analysis for ash, Ca, and P. The bones were defatted, and mineral content was determined according to the methods of AOAC (2002).

2.5. Serum lipid metabolites and enzyme activity Blood samples were collected from the brachial vein of 3 birds per replicate at the end of experimental period. After centrifugation at 4000 × g and 4 ºC for 10 min, the serum samples were collected and stored at –20 °C until the analysis for lipid metabolites could be performed. Serum samples were analyzed for triglycerides (TG),

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cholesterol, and high-density lipoproteins (HDL) concentrations according to the manufacturer’s recommendations using the commercial enzymatic kits (Pars Azmoun, Tehran, Iran) by a biochemical analyzer (ERBA CHEM-5; Beijing Biochemical Instrument Company, Beijing, China). In addition, alkaline phosphatase (ALP) activity was measured using the standard kit (Pars Azmoun, Tehran, Iran).

2.6. Intestinal microbial populations The ileal contents of slaughtered birds (from Meckel’s diverticulum to the ilealcecal-colon junction) were collected directly into the 80 mL sampling cups under CO2, sealed, and put on ice until they were transported to the laboratory for enumeration of bacterial populations.. Immediately, the ileal contents were cultured on specific culture media to enumerate the populations of Escherichia coli (E. coli), Salmonella spp., and Lactobacillus spp., according to the methods described by Jahanian and Golshadi (2015). Lactobacilli was anaerobically assayed using lactobacilli MRS agar and incubated at 37°C for 48 h. Escherichia coli was assayed using Rapid E. coli 2 agar. Ileal population of Salmonella was counted using Salmonella-Shigella agar. All microbiological analyses were performed in duplicate.

2.7. Statistical analysis Data were analyzed using the general linear model procedures of SAS (SAS Institute, 1999) as a 4 × 2 factorial arrangement of treatments, including supplemental OA and dietary NPP level as the main effects and respective interaction. The following model was assumed in the analysis of all parameters: Yijk = μ + Ai + Bj + ABij + eijk, where Yijk = the measured value for each observation (data), μ = overall mean, Ai = supplemental OA, Bj = NPP level, ABij = interaction between supplemental OA and NPP level, and eijk = experimental error. Differences among treatment means were

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compared using the least significant difference test (Steel et al., 1997) at P < 0.05 significant level.

3. Results

3.1. Performance The effects of supplemental OA on performance of laying hens fed diets with different NPP levels are summarized in Table 2. As noted, dietary supplementation of citric acid decreased (P < 0.01) egg weight. Moreover, feeding hens with the NPPdeficient diet decreased (P < 0.01) egg weight. Reducing dietary NPP level decreased egg production and egg mass in control hens, while had no marked effect in OAsupplemented ones, resulted in the significant (P < 0.05) OA × NPP interactions. Although ADFI was not affected by dietary treatments, there was a significant (P < 0.05) OA × NPP interaction for FCR values, so that reducing NPP level increased FCR in control hens and had no marked effect on FCR values of OA-supplemented hens.

3.2. Egg quality As noted in Table 3, reducing dietary NPP level decreased shell thickness only in control hens, resulted in a significant (P < 0.05) OA × NPP interaction. On the other hand, shell breaking strength was not influenced by supplemental OA or dietary NPP level. The finding of interest was that supplementation of fumaric acid into the diets increased (P < 0.05) yolk index compared to the control, citric acid, and butyric acid treatments. Yolk color index and HU were not affected by dietary treatments.

3.3. Serum biochemical parameters

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As presented in Table 4, OA supplementation of diets decreased (P < 0.01) serum cholesterol level at 39 wk of age, and increased (P < 0.001) serum HDL at 44 wk of age. The greatest level of serum TG was allotted to the control (not-supplemented) hens during both 39 and 44 wk of age. Dietary supplementation of butyric acid decreased (P < 0.05) serum TG level during both experimental periods. Serum lipid metabolites were not influenced by dietary NPP level. Although serum cholesterol level was decreased as the result of reducing dietary NPP level in control hens, it was increased in OAsupplemented hens at 44 wk of age, resulted in a significant (P < 0.01) OA × NPP interaction. 2.4. Bone mineral composition and ALP activity Effects of dietary supplementation of OA on tibial contents of ash, Ca, and P are shown in Table 5. As presented, feeding NPP-deficient diets decreased tibia ash (P < 0.01) and Ca (P < 0.05) contents. On the other hand, the lowest (P < 0.01) ash content was seen in tibia of control hens compared with fumaric acid-supplemented hens. Although dietary OA supplementation had no marked impact on bone P content, reducing dietary NPP level resulted in an increase (P < 0.05) in tibia P content. Moreover, supplementation of NPP-deficient diets with citric acid increased tibia P level, resulted in a significant (P < 0.001) OA × NPP interaction. Reducing dietary NPP level had more increasing effect on serum ALP activity in control hens compared with OA-supplemented ones, resulted in an OA × NPP interaction (P < 0.01).

2.5. Ileal micoflora As presented in Table 6, ileal bacterial counts were not affected by dietary NPP level. On the other hand, dietary OA supplementation had marked (P < 0.05) impacts on all microbial populations in ileal contents. Dietary supplementation of butyric acid

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reduced (P < 0.05) ileal E. coli enumeration compared with those fed control and fumaric acid-supplemented diets. In addition, ileal count of Salmonella was reduced (P < 0.01) by all OA. Supplementation of citric and fumaric acids into the diets increased (P < 0.01) ileal Lactobacillus population. There was no OA × NPP interaction for ileal bacterial populations.

4. Discussion Recent attentions to P pollution in the environment have increased concern for the amount of P excreted in poultry and livestock manure. Monogastric animals consume diets composed mostly of oilseed meals and cereal grains that contain high levels of P in the form of phytic acid or phytate. Because of low phytase activity in the digestive tract, avian species could not efficiently use P from plant origins. Therefore, they are strongly dependent on P supplied by inorganic sources such as calcium phosphates, bone meal, meat and bone meal, etc. These P sources are usually expensive, and P is known as the most expensive mineral element in poultry diets. On the other hand, improvement of P use (especially phytate P) has become a primary concern of poultry producers due to increasing concerns about P pollution. In this regard, OA have been shown to have a potential for increasing phytate P utilization in broiler chicks (Rafacz-Livingston et al., 2005a,b; Liem et al., 2008). In the present study, dietary supplementation of citric and butyric acid increased hen-day egg production. In addition, egg mass was greater with all supplemental OA as compared with control hens. The finding of interest was that although reducing dietary NPP level resulted in decreases in egg production and egg mass and impaired FCR value, supplemental OA could maintain optimal performance in hens fed NPP-deficient diets. Consistent with these findings, Dehghani-Tafti and Jahanian (2016) observed that dietary supplementation of citric and butyric acids

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improved FCR values in broiler chicks fed diets with different levels of crude protein. In addition, Rafacz-Livingston et al. (2005b) reported that supplemental OA increased weight gains in male chicks fed a deficient corn-soybean meal-based diet. Improvement of laying performance as the result of dietary OA supplementation can be attributed to increase in ileal nutrient digestibility (Jahanian and Golshadi, 2015), reduction of intestinal pathogenic bacteria (Samik et al., 2007; Fernández-Rubio et al., 2009; Jahanian and Golshadi, 2015), and improvements in morphological indices of absorptive epithelial cells (Samik et al., 2007; Dehghani and Jahanian, 2012; Jahanian and Ajilchi, 2015). Of course, it has been speculated that OA may decrease the pH of intestinal digesta, thereby inhibit phytic acid from chelating minerals and forming insoluble phytate salts that are resistant to hydrolysis by endogenous phytase enzymes (Maenz et al., 1999; Applegate et al., 2003). The observation that some OA increase P utilization while others do not suggests that the effects of OA may not be due simply to pH responses. Boling et al. (2000) hypothesized that some OA, such as citric acid, may competitively chelate Ca and reduce the binding of Ca to phytate, thereby preventing the formation of insoluble Ca-phytate complexes. The latter may result in the dietary phytate being more susceptible to endogenous phytases. Dietary OA supplementation decreased serum cholesterol level and caused increases in serum HDL concentration. The exact mechanism by which OA increase serum HDL is unclear. Of course, the increasing effect of supplemental OA on serum concentration of HDL can be related to beneficial impacts of these feed additives on overall health of an organism. Consistent with the present findings, Dehghani-Tafti and Jahanian (2016) reported that dietary supplementation of citric acid increased serum HDL level in broiler chicks.

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As presented in Table 5, reducing dietary NPP level decreased tibial ash and Ca contents. Unexpectedly, tibia P was greater in hens fed with the NPP-deficient diet. The greater tibia P of the latter group may be related to the fact that hens in low-P diet adopt their absorptive mechanisms to better absorb P from the deficient diet. Among supplemental OA, only citric acid was effective in increasing tibia P in hens fed with the NPP-deficient diet. Consistent with this observation, Rafacz-Livingston et al. (2005a) showed that citric acid was substantially efficacious in improving P utilization in commercial broiler chicks fed P-deficient diets. This greater efficacy of citric acid in comparison with other OA was demonstrated in another study by these researchers (Rafacz-Livingston et al., 2005b). The effects of OA on phytate P utilization might result from a change in the pH of the gastrointestinal tract to a pH more favorable for phytases to hydrolyze phytate (Liem et al., 2008). Although dietary NPP level had no effect on ileal bacterial populations, dietary supplementation of butyric acid caused a marked decrease in ileal E. coli count. Furthermore, all of the used OA reduced Salmonella enumeration compared with unsupplemented diet. Organic acids have a long history of being utilized as food additives and preservatives for preventing food deterioration and extending the shelf life of perishable food ingredients. Specific OA have also been used to control microbial contamination and dissemination of foodborne pathogens in preharvest and postharvest food production and processing (Ricke, 2003). Dietary supplementation of fumaric and citric acids increased intestinal Lactobacillus population. It has been reported that fumaric and citric acids are weak acids (Liem et al., 2008). Therefore, while fumaric acid could diminish intestinal pathogenic bacteria (such as coliforms and Salmonella), it has not a strong inhibitory effect on useful bacteria, including Lactobacillus spp. This is largely because of the pH-

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insusceptibility of Lactobacillus (Ferket et al., 2002) and slow dissociation rate (Skinner et al., 1991; Liem et al., 2008) of fumaric acid. Consistent with our findings, Brzóska et al. (2007) observed that dietary fumaric acid supplementation increased Streptococcus and Lactobacillus counts in the ileal digesta.

5. Conclusions The present findings indicate that dietary supplementation of citric and butyric acids could increase egg production and egg mass in young laying hens. The beneficial impacts of supplemental OA on egg production percentage and subsequent egg mass were seen only with the NPP-deficient diet. Also, reducing NPP level increased FCR value in control hens, while supplemental OA improved feed conversion efficiency. The finding of interest was that OA supplementation of the NPP-deficient diet increased eggshell thickness compared to the control hens, indicating that dietary OA supplementation could nearly compensate for dietary NPP deficiency in laying hens.

Conflict of interest statement The authors declare no conflict of interest.

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Nelson, T.W., 1976. The hydrolysis of phytate phosphorus by chicks and laying hens. Poult. Sci. 55, 2262–2264. Rafacz-Livingston, K.A., Martinez-Amezcua, C., Parsons, C.M., Baker, D.H., Snow, J., 2005a. Citric acid improves phytate phosphorus utilization in crossbred and commercial broiler chicks. Poult. Sci. 84, 1370–1375. Rafacz-Livingston, K.A., Parsons, C.M., Jungk, R.A., 2005b. The effects of various organic acids on phytate phosphorus utilization in chicks. Poult. Sci. 84, 1356– 1362. Ravindran, V., Bryden, W.L., Kornegay, E.T., 1995. Phytates: occurrence, bioavailability and implications in poultry nutrition. Poult. Avian Biol. Rev. 6, 125– 143. Ricke, S.C., 2003. Perspectives on the use of organic acids and short chain fatty acids as antimicrobials. Poult. Sci. 82, 632–639. Samik, K.P., Gobinda, H., Manas, K.M., Gautam, S., 2007. Effect of organic acid salt on the performance and gut health of broiler chicken. Jpn. Poult. Sci. 44, 389–395. SAS Institute, 1999. SAS Statistics User’s Guide. Statistical Analytical System. 5th rev. ed. SAS Institute Inc., Cary, NC, USA. Shellem, T., Angel, R., 2002. Is the effect of citric acid on apparent phosphorus availability mediated primarily through feed consumption changes? Poult. Sci. 81 (Suppl. 1), 94 (Abstr.). Silversides, F.G., Villeneuve, P., 1994. Is the Haugh unit for egg weight valid for eggs stored at room temperature? Poult. Sci. 73, 50–55. Singh, M., Krikorian, A.D., 1982. Inhibition of trypsin activity in vitro by phytate. J. Agric. Food Chem. 30, 799–800.

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Table 1 Ingredients and nutrient composition of basal experimental diets with different non-phytate phosphorus (NPP) levels. Item (g/kg unless stated otherwise)

NPP-sufficient

NPP-deficient

Corn, yellow

493.6

503.5

Soybean meal

228.2

226.6

Barely

70.0

70.0

Wheat bran

30.0

30.0

Alfalfa meal

20.0

20.0

Sunflower oil

21.3

17.4

Limestone

58.8

65.0

Oyster shell

40.0

40.0

Dicalcium phosphate

18.9

8.3

Mineral premixa

2.5

2.5

premixb

2.5

2.5

Salt

2.1

2.1

Sodium sulfate

1.0

1.0

Sodium bicarbonate

2.0

2.0

DL-Met

1.7

1.7

L-Lys·HCl

0.4

0.4

Zeolitec

7.0

7.0

MEn (MJ/kg)

11.09

11.09

CP

152.5

152.5

Ca

40.0

40.0

NPP

4.5

2.7

Na

1.7

1.7

Met

4.3

4.3

Met + Cys

7.0

7.0

Lys

8.1

8.1

Thr

6.3

6.3

Ingredients

Vitamin

Compositiond

a

Mineral premix provided per kilogram of diet: Mn (from MnSO 4·H2O), 80 mg; Zn (from ZnO), 75 mg; Fe (from FeSO4·7H2O), 50 mg; Cu (from CuSO4·5H2O), 8 mg; I (from Ca (IO3)2·H2O), 1.8 mg; Se (from Na selenite), 0.3 mg; Co (from Co2O3), 0.2 mg. b Vitamin premix provided per kilogram of diet: vitamin A (from retinyl acetate), 10,500 IU; cholecalciferol, 2,100 IU; vitamin E (from DL-α-tocopheryl acetate), 22 IU; menadione (from menadione dimethyl-pyrimidinol), 2.5 mg; thiamine, 3.1 mg; riboflavin, 4.4 mg; nicotin amide, 40 mg; calcium pantothenate, 25 mg; pyridoxine, 8 mg; folic acid, 0.8 mg; biotin, 0.1 mg; vitamin B12, 0.06 mg; choline (from choline chloride), 600 mg; ethoxyquin, 100 mg. c Organic acids were added to the basal diets at the expense of equal amount (5 g/kg) of zeolite, so that all of experimental diets were similar in nutrient composition. d ME = metabolizable energy corrected for N retention; CP = crude protein. n

20

Table 2 Effects of dietary supplementation of different organic acids (OA) on performance of laying hens fed diets with different non-phytate phosphorus (NPP) levels. NPP

a

OA

levels

Egg weight

Egg production

Egg mass

Feed Intake

(g/d per hen)

(g/d per hen)

Feed conversion ratio

(g/kg of diet)

(g)

2.7

61.34

85.96c

52.73c

108.1

2.05a

4.5

63.02

88.35ab

55.68a

108.2

1.94c

2.7

61.00

89.42a

54.55b

108.4

1.99b

4.5

61.66

89.89a

55.43ab

108.6

1.96bc

2.7

62.31

89.71a

55.90a

107.9

1.93c

4.5

62.96

88.74ab

55.87a

108.6

1.95c

2.7

62.27

87.51bc

54.49b

108.6

1.99b

4.5

62.25

88.12ab

54.85b

106.3

1.94c

Control

62.18a

87.09

54.15

108.2

2.00

Citric

61.29b

89.65

54.95

108.5

1.97

Butyric

62.64a

89.23

55.79

108.3

1.94

Fumaric

62.25

a

87.76

54.63

107.5

1.96

2.7

61.70b

88.01

54.30

108.3

1.99

4.5

62.53a

88.83

55.54

107.9

1.95

OA

0.002

0.048

0.035

0.278

0.027

NPP level

0.002

0.215

0.043

0.575

0.010

OA × NPP

0.064

0.043

0.020

0.125

0.014

0.38

0.82

0.35

1.3

0.01

Control

Citric

Butyric

Fumaric

OA

NPP levels

(%)

(g feed/ g egg)

Probability

SEM a

Organic acids were supplemented into the diets at the level of 5 g/kg.

abc

Means with no common superscripts within the column of each classification (OA, NPP, or respective interaction) are

significantly (P < 0.05) different.

21

Table 3 Effects of dietary supplementation of different organic acids (OA) on egg quality measurements of laying hens fed diets with different non-phytate phosphorus (NPP) levels. NPP

a

OA

levels

(g/kg of diet)

Control

Citric

Butyric

Fumaric

OA

Eggshell

Shell breaking

thickness

strength

(mm)

(kg/cm2)

Haugh unit

Yolk index

Yolk score

2.7

0.408c

2.35

86.46

0.452

6.65

4.5

0.427

b

2.42

86.79

0.435

6.30

2.7

0.433

ab

2.47

85.66

0.440

6.47

4.5

0.430ab

2.73

87.72

0.441

6.27

2.7

0.436

ab

2.63

85.40

0.444

6.67

4.5

0.435

ab

2.67

86.42

0.441

6.35

2.7

0.437ab

2.30

84.70

0.452

6.67

4.5

0.440a

2.48

87.25

0.456

6.40

Control

0.417

2.39

86.63

0.445b

6.47

Citric

0.432

2.58

86.69

0.440b

6.37

Butyric

0.436

2.65

85.91

0.443b

6.51

a

6.53

Fumaric

0.439

2.39

85.79

0.454

2.7

0.429

2.44

85.55

0.447

6.62

4.5

0.433

2.57

87.03

0.444

6.33

OA

0.014

0.139

0.829

0.014

0.919

NPP level

0.133

0.171

0.066

0.210

0.113

OA × NPP

0.025

0.827

0.743

0.091

0.990

0.004

0.12

0.96

0.004

0.25

NPP levels

color

Probability

SEM a

Organic acids were supplemented into the diets at the level of 5 g/kg.

abc

Means with no common superscripts within the column of each classification (OA or OA × NPP interaction) are

significantly (P < 0.05) different.

22

Table 4 Effects of dietary supplementation of different organic acids (OA) on serum lipid metabolites (mg/dL) of laying hens fed diets with different non-phytate phosphorus (NPP) levels. NPP

OAa

levels

Cholesterol

Triglycerides

High-density lipoproteins

(g/kg of diet)

wk 39

wk 44

wk 39

wk 44

wk 39

wk 44

2.7

214.8

111.0cd

1,445

1,548b

36.0

44.4

188.0

151.7ab

1,437

1,607ab

39.0

44.3

164.3

143.3ab

1,446

1,597ab

42.2

49.3

4.5

145.8

129.8bc

1,406

1,640a

45.8

47.0

2.7

151.3

146.3ab

1,425

1,589ab

42.7

49.8

4.5

146.3

111.0cd

1,406

1,413c

44.8

50.0

161.0

155.5a

1,425

1,571ab

40.4

48.0

4.5

176.5

102.0d

1,4.37

1,574ab

40.7

48.3

Control

199.9a

131.3

1,440a

1,659

37.5

44.3b

Citric

155.0b

134.9

1,435a

1,622

43.8

48.0a

Butyric

148.8b

128.6

1,415b

1,501

43.9

49.9a

Fumaric

168.8b

128.8

1,430ab

1,572

40.5

48.1a

2.7

172.8

140.7

1,434

1,577

40.2

47.6

4.5

165.5

122.3

1,426

1,558

42.6

47.4

OA

0.004

0.881

0.021

0.046

0.388

0.001

NPP level

0.395

0.071

0.126

0.559

0.484

0.516

OA × NPP

0.479

0.005

0.160

0.035

0.985

0.615

12.8

9.7

10

36

4.2

2.9

Control

4.5 Citric

2.7

Butyric

Fumaric

OA

NPP levels

2.7

Probability

SEM a

Organic acids were supplemented into the diets at the level of 5 g/kg.

abcd

Means with no common superscripts within the column of each classification (OA or OA × NPP interaction) are

significantly (P < 0.05) different.

23

Table 5 Effects of dietary supplementation of different organic acids (OA) on tibia mineral contents (%) and serum alkaline phosphatase activity (U/ml) in laying hens fed diets with different nonphytate phosphorus (NPP) levels. NPP

OAa

levels

Alkaline

Tibia ash

Tibia Ca

Tibia P

2.7

57.22bc

33.55

16.10bc

501a

4.5

56.37c

35.37

16.48b

240c

2.7

57.58b

35.86

19.09a

435ab

4.5

57.58b

38.51

15.55c

380b

2.7

56.09c

33.81

16.10bc

433ab

4.5

59.68a

34.56

16.27bc

458ab

2.7

57.69b

36.46

16.31bc

456ab

4.5

59.64a

39.08

16.65b

314c

Control

56.79

34.33b

16.29

370

Citric

57.58

37.38

a

17.07

408

Butyric

57.63

34.13b

16.19

448

Fumaric

58.52

38.01

a

16.50

395

2.7

57.12

34.92b

16.80

456

4.5

58.04

36.88a

16.26

350

OA

0.005

0.001

0.053

0.019

NPP level

0.003

0.016

0.031

0.001

OA × NPP

0.001

0.323

0.001

0.003

0.42

0.65

0.35

35

(g/kg of diet)

Control

Citric

Butyric

Fumaric

OA

NPP levels

phosphatase

Probability

SEM a

Organic acids were supplemented into the diets at the level of 5 g/kg.

abc

Means with no common superscripts within the column of each classification (OA, NPP, or respective

interaction) are significantly (P < 0.05) different.

24

Table 6 Effects of dietary supplementation of different organic acids (OA) on ileal bacterial counts (log 10 cfu/ g fresh digesta) in laying hens fed diets with different non-phytate phosphorus (NPP) levels.

OAa

NPP

levels

Escherichia coli

Salmonella

Lactobacillus spp.

2.7

9.52

5.93

9.32

4.5

9.27

6.15

9.58

2.7

8.53

4.76

10.73

4.5

8.75

4.59

10.51

2.7

8.32

4.14

9.46

4.5

8.06

4.31

10.07

2.7

8.93

5.11

11.05

4.5

9.37

4.86

11.38

Control

9.39a

6.04a

9.45c

Citric

8.64ab

4.67b

10.62ab

Butyric

8.19b

4.22b

9.76bc

Fumaric

9.15a

4.98b

11.21a

2.7

8.82

4.98

10.14

4.5

8.86

4.98

10.38

OA

0.023

0.002

0.005

NPP level

0.895

0.982

0.493

OA × NPP

0.765

0.933

0.870

0.40

0.44

0.52

Control

Citric

Butyric

Fumaric

OA

NPP levels

(g/kg of diet)

Probability

SEM a

Organic acids were supplemented into the diets at the level of 5 g/kg.

abc

Means with no common superscripts within the column of OA are significantly (P < 0.05) different.

25