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The Effect of Dietary Sinapic Acid (4-Hydroxy-3, 5-Dimethoxy-Cinnamic Acid) on Gastrointestinal Tract Microbial Fermentation, Nutrient Utilization, and Egg Quality in Laying Hens

Department of Animal and Poultry Science, 51 Campus Drive, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5A8, Canada

¹ Corresponding author: hank.classen{at}usask.ca

	ABSTRACT

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Plant-based simple phenolics are natural antimicrobial and antioxidant compounds that may possess biological activity when included in animal diets. Previous work in our lab has shown that sinapic acid (SA) found in canola meal improves nutrient retention and alters gut microbiota when fed at low levels (0 to 0.10%) to broiler chickens. It was of interest to investigate the effect of dietary SA in laying hens that possess a more mature intestinal microbiota. Therefore, 2 experiments were conducted to determine the effects of dietary SA on performance, nutrient digestibility, and egg quality in laying hens. Sinapic acid (Sigma-Aldrich, 98% purity) was included in diets at 0, 0.025, 0.050, and 0.075% in experiment 1, and 0 and 0.5% in experiment 2. Both experiments were conducted for 21 d. Dietary treatment did not affect production traits and gastrointestinal tract volatile fatty acid production in either experiment. Apparent metabolizable energy and protein digestibility were also unaffected by treatment in experiment 1, but in experiment 2, feeding 0.5% SA increased both AME_n and protein digestibility in comparison to the unsupplemented control. Egg weight, albumen weight, yolk weight, egg specific gravity, shell thickness, and Haugh units were not significantly affected by dietary treatments in either of the experiments. Sinapic acid was not detected in eggs from experiment 1 but in experiment 2, low levels of SA were detected in both the yolk and albumen of eggs from hens fed 0.5% SA. Further research is needed to more completely define the relationship between dietary SA level and nutrient utilization as well as the accumulation of SA in egg components.

Key Words: sinapic acid • laying hen • egg quality • nutrient • fermentation

	INTRODUCTION

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Phenolic compounds are important components of plants and include both simple and polymeric forms (Materska and Perucka, 2005). Simple phenolics are biologically active, having antimicrobial and antioxidant properties (Materska and Perucka, 2005; Vuorela et al., 2005), and therefore may have relevance in food and feed. An example of a simple phenolic that might be found in animal feed is sinapic acid (4-hydroxy-3,5-dimethoxy-cinnamic acid; SA), which is the predominant free phenolic acid in canola meal and is most often esterified to choline in a compound known as sinapine at levels as high as 1% (Matthäus, 2002). It is estimated that the free SA content in canola meal may range from 0.05 to 0.4% or higher on a dry matter basis depending on the variety, growing location and conditions, and processing method (Qiao, 2002). Sinapic acid has a bitter and astringent taste and possesses a very low taste-threshold for humans (Rubino et al., 1996). As a consequence, it is often thought to affect the palatability of canola meal as an animal feed.

In contrast to the postulated negative effects of SA on palatability, feeding low levels of SA to broiler chickens resulted in positive effects without any adverse effect on feed intake (Qiao, 2002). Qiao (2002) demonstrated an antimicrobial effect and an altered fermentation pattern in the intestinal tract of broiler chickens when fed SA. This was shown to reflect an altered gastrointestinal microbial profile because the total microbial biomass was not altered. Dietary SA also increased nutrient retention in broiler chickens, and it was speculated that the improvement was due to the change in gut microbiota (Qiao et al., 2008). The authors observed a quadratic response for feed intake and weight gain as SA increased in the diet from 0 to 0.10%. They also demonstrated that the ileal SA digestibility was above 97% for inclusion levels tested while excreta digestibility was 54 to 75%. This demonstrates that a portion of the SA was absorbed before the ileum and then excreted in the urine. As an antimicrobial agent, SA has bactericidal activity against Salmonella enterica subsp. enterica. Extracts from plants belonging to the Brassica family were bactericidal against several Salmonella species (Kassie et al., 1996), and research in our lab has demonstrated specific bactericidal action of pure SA against Salmonella enterica subsp. enterica (T. Dumonceaux, A. G. Van Kessel, and H. L. Classen, University of Saskatchewan, Saskatoon, Canada, unpublished data). The bactericidal action of SA against Salmonella enterica subsp. enterica could be very important; Salmonella enterica subsp. enterica is an important poultry pathogen because it is the most common cause of human foodborne illness (CDC, 2005).

Poultry research on SA has focused on young broiler chickens, but it is of interest to establish if similar effects occur in adult chickens, and in particular laying hens. The microbiota of laying hens would be expected to be more stable and also different than the evolving microbiota of broilers based on the influence of diet and environment. Therefore, effects on intestinal microbiota fermentation and nutrient retention may also be different. Inclusion of simple phenolics in eggs also requires investigation. Research regarding other simple phenolics suggests that inclusion rates are low and only found when the phenolic is included at high concentrations in the diet (Galobart et al., 2001). This requires confirmation for SA because of the potential egg quality implications associated with antioxidant activity and also because of the human nutritional implications. If SA could be included in the egg, it may help to maintain egg quality by inactivating reactive oxygen species (Meluzzi et al., 2000). These reactive oxygen species are responsible for initiating oxidative damage in fatty acids, particularly in the unsaturated fatty acids, which results in off flavors, decreased nutrient quality, and subsequently lower consumer acceptance (Grune et al., 2001; Grobas et al., 2002). Potential positive effects of SA on humans (Kern et al., 2003) could mean that inclusion in the egg would be a beneficial attribute. There is also the potential that dietary SA could attenuate the incidence of Salmonella in eggs by impacting gastrointestinal microbiota or egg contents.

Therefore, the current study was conducted to test the hypothesis that the supplementation of SA into the diets of laying hens would affect gastrointestinal flora, increase nutrient retention and result in SA deposition in the eggs.

	MATERIALS AND METHODS

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Experimental protocol was approved by the Animal Care Committee of the University of Saskatchewan and was performed in accordance with recommendations of the Canadian Council on Animal Care (1993) as specified in the Guide to the Care and Use of Experimental Animals.

Animals and Diets

Two experiments were conducted using Shaver White laying hens individually housed in cages (30.5 cm x 45.7 cm x 40.6 to 45.7 cm). In the first experiment, 4 treatments consisted of maize (Zea mays L.)-soybean meal diets supplemented with graded levels of SA (0, 0.025, 0.05, and 0.075%). Each treatment was replicated 5 times with 2 birds per replication. A total of forty 56-wk-old laying hens were randomly assigned to experimental units. In second experiment, 20 laying hens (60 wk of age) were randomly assigned to 2 treatments containing 0 or 0.50% dietary SA with 2 replication of each treatment having 5 birds per replication. Both experiments were conducted for a 3-wk period.

The hens received 14 h of light per day with light intensity maintained at 5 lx. The housing temperature was maintained at 20°C. The basal diet was formulated to meet or exceed the nutrient requirements of laying hens as recommended by the National Research Council (NRC, 1994). Diet composition and nutrient levels are shown in Table 1. In both experiments, SA was supplemented at the expense of wheat and any naturally occurring SA was not accounted for. Sinapic acid (GC grade; catalog no. D7926) was purchased from Sigma (St. Louis, MO) and was 98% pure. Feed and water were provided for ad libitum consumption.

Hen body weight (in the beginning and end of trial), feed consumption (weekly), and egg production (daily) were recorded in both experiments. Diet samples were collected once for SA analysis. From d 17 to completion of the experiment, 3 eggs per hen were collected for determination of egg quality. Variables measured included whole egg weight, albumen weight, yolk weight, specific gravity, shell thickness, and albumen quality (Haugh units). The albumen and yolk were subsequently frozen separately and stored at –20°C for later analysis of SA content.

Excreta were collected twice daily from d 19 to 21, and all samples were frozen at –20°C until being dried in an oven at 55°C for 3 d. The samples from each replicate were subsequently pooled for grinding using a 1.0-mm screen for lab analysis. On d 21 of experiments, the hens were euthanized using lethal injection (0.40 mL/kg of body weight into the brachial vein) of T-61 Euthanasia Solution (Intervet Canada Ltd., Whitby, Ontario, Canada). Digesta samples were immediately collected from the jejunum, anterior ileum, and cecum, placed into well-sealed plastic tubes and frozen at –20°C for subsequent volatile fatty acid (VFA) analysis.

Laboratory Analysis

Diet, ileal, and excreta samples were analyzed for moisture, crude nitrogen, acid insoluble ash (AIA), and gross energy. Moisture was determined by method 930.15 of the AOAC (1990), whereas gross energy was determined using an oxygen bomb calorimeter (model 1281, Parr Instruments, Moline, IL). Crude nitrogen content was determined by the combustion method (984.13; AOAC International, 1995) using a Leco FP-528 protein analyzer (St. Joseph, MI). The AIA was used as an indigestible marker for the determination of the apparent metabolizable energy and apparent excreta protein digestibility. Acid insoluble ash was analyzed using a modification of the method of Vogtmann et al. (1975) by weighing 0.2 g of sample for excreta and diet, and 0.1 to 0.15 g of ileal sample into 16 x 125 mm disposable borosilicate tubes, and ashing at 500°C for 24 h or until contents were reduced to white ash. Five milliliters of 4 N HCl were slowly added, and then the samples were vortexed, covering the tubes with glass marbles and heating in oven at 120°C for 1 h before centrifuging at 2,500 x g for 10 min. The supernatant was then removed and samples washed repeatedly with 5 mL of water (with vortex and centrifugation steps as described above). Samples were then dried at 80°C overnight, followed by ashing at 500°C overnight. The percent AIA was calculated as (total ashed wt – tube wt)/(original – tube wt).

The status of microbial populations in jejunum, ileum, and cecum was assessed via VFA production. Analyses of VFA (acetic, propionic, isobutyric, isovaleric, and valeric acid) were conducted by gas chromatography according to the method described by Fischer (2003). The samples within a replication were pooled and mixed well before VFA measurement. Excreta AME was determined using the method described by Bartov (1995).

Four eggs per hen were collected from d 17 to 21 of the experiments, weighed, broken, and yolk and albumen were separated and stored at –20°C until subsequent analysis for SA content. Sinapic acid analyses were performed based on the HPLC procedure described by Olkowski et al. (2003) with minor modifications. Briefly, approximately 750 mg of sample (egg yolk or albumen) were extracted with 10 mL of acidified methanol (70:30 vol/vol, methanol:water, 1% HCl, pH = 1.5). These preparations were vortexed thoroughly and centrifuged at 1,000 x g. The supernatants were filtered as required with 0.45 or 0.20 µm syringe filters, and placed in vials for HPLC analyses. An analytical column (reversed phase C18, Gemini, 250 x 4.6mm; Phemomenex, CA) and HPLC system (Agilent 1100 series, Hewlett Packard, Germany) were used. The mobile phase was comprised of HPLC grade methanol and 20 mM dibasic potassium phosphate buffer adjusted to pH 9.35 with 1 N KOH mixed at a ratio 15:85 (vol/vol). The mobile phase was delivered at a rate of 1.2 mL/min and samples (10 µL) were injected onto the column using an autosampler. Detection was performed at 320 nm using a diode array detector. Standards were prepared from a stock solution of SA dissolved in methanol. Analyte recovery, assessed by spiking the sample matrix with a known amount of SA, showed 70 to 80% efficiency, which was deemed satisfactory for the analysis of free/soluble SA in the samples. No further effort to account for conjugated SA in the samples was made. The calibration curves showed linear response, with the regression coefficient being routinely better than 0.999. The lowest limit of detection (defined as 3 x signal:noise ratio) was found to be approximately 0.025 µg/mL. The procedure showed high degree of selectivity, precision, and reproducibility with interday assay coefficients of variation ranging from 2.0 to 5.0.

Statistical Analysis

All data were subjected to 1-way ANOVA according to GLM procedure and the regression analysis (for experiment 1) of SAS program (SAS Institute, 2002). Differences were considered significant when P < 0.05.

	RESULTS

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Dietary SA did not have any significant effect on VFA production in the gastrointestinal tract of laying hens in either of the experiments (Table 2). Regression analysis between digesta VFA content and dietary SA was not significant with the only exception of butyric acid in ileal content in experiment 1 (P = 0.02).

Various egg quality parameters tested in the current study were unaffected by dietary treatment in both experiments (Table 3). Regression analysis between egg specific gravity and dietary SA supplementation was significant (P = 0.03) in experiment 1. The SA was not detected in eggs in experiment 1; however, in experiment 2, yolk and albumen of eggs from hens fed 0.05% SA were found to contain an average of 0.004 and 0.009% SA, respectively (data not shown).

	DISCUSSION

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In the current study, there was no significant effect of dietary SA on VFA production in the intestinal tract of laying hens, regardless of location. The lack of a detectable effect at low dietary levels of SA differs from the results of Qiao (2002), who found an increased VFA content in the small intestine and a decreased VFA content in the ceca when broilers were fed low levels of SA up to 0.1%. In his research, dietary SA increased VFA content in the small intestine by 48% and reduced total VFA content in the ceca by 13.5%. The present research does not permit establishment of the reason for the difference in VFA response of laying hens and broiler chickens, but one can speculate that a mature gastrointestinal tract microbiota may not respond in the same way as the evolving microbiota of the broiler chickens, which is thought to take about 6 wk to be classified as mature (Barnes et al., 1972). Because laying hens are in cages rather than on straw litter, there may also be differences in the nature of the GIT microbiota. It is established that SA does not affect bacteria equally, so a different microbiota composition could impact the response in terms of VFA content. Furthermore the diets of laying hens and broilers are quite different because the laying hen diet has a much higher content of calcium. Considering that SA may bind to amino acids, proteins, and iron (Rubino et al., 1996; Hynes and Coinceanainn, 2002) it is possible that it may also bind to calcium, becoming insoluble, reducing its availability and activity in the GIT.

In experiment 1, there were no detected effects of low dietary SA on excreta AME_n, which was not the case in experiment 2. In experiment 2, the AME_n increased by 8.5% when dietary SA level was 0.5%, which is interesting considering that there were no detectable effects on VFA production in the intestinal tract. Similarly, excreta protein digestibility was not affected in experiment 1 at low dietary SA concentration, whereas in experiment 2 there was a 42% increase when diets were supplemented with 0.5% SA. Qiao (2002) also found that dietary SA increased nutrient retention but at much lower levels of inclusion. Again this suggests that SA in laying hen diets is not as effective as in broilers, with potential reasons outlined above. Although Qiao (2002) did not prove a connection between changes in gastrointestinal microbiota and nutrient retention, he hypothesized that improved nutrient retention was the result of changes in the gastrointestinal microbial community in a fashion similar to the action of antibiotics. In the present study, there was a major increase in AME_n and excreta protein digestibility without significant effect VFA content at the 0.5% dietary SA level. The failure to see a concomitant change in nutrient retention and VFA content does not rule out changes in gastrointestinal microbiota that benefit nutrient retention because the measurement of VFA production is a crude way of determining changes in the intestinal microbial community. In the present study, there is no evidence to connect the 2 response criteria.

There are few available data regarding the deposition of simple phenolic compounds into eggs, but the results of the present study agree with previous findings that high dietary levels are required to detect minute amounts in the eggs (Galobart et al., 2001; Botsoglou et al., 2005). This indicates that the efficacy of deposition of simple phenolic acids into the egg is low and likely passive in nature (i.e., SA is not actively transported into yolk precursor macromolecules in the liver or into albumen in the oviduct).

The lack of an effect of SA on feed intake was surprising considering that the taste-threshold of SA is only 1 mg/kg for some species (Rubino et al., 1996). At the lowest inclusion level the concentration of SA was 250 times greater than the taste-threshold and at the highest inclusion level the SA concentration was 5,000 times greater. It was expected that as dietary SA levels increased feed consumption would decrease. At high dietary levels laying hens may develop an aversion to SA as feed consumption decreased by 12% (P > 0.05). Likewise, body weight change was not affected by dietary SA as hens in all treatments lost weight. It is possible that the loss in body weight could have been greater, but was offset by the increased excreta protein digestibility and AME_n though the increase in both were not enough to compensate for the nutrient intake reduction. These feed intake results are in agreement with Qiao (2002) who found that low dietary SA did not affect feed intake in broilers. In his work, when broilers were fed low levels of SA, growth and body weight were increased. Egg production was not affected by dietary SA in the present study. However, we believe that further research is needed to determine the effect of dietary SA on production characteristics in laying hens in a large scale trial conducted for a longer period of time.

	ACKNOWLEDGMENTS

 
The authors would like to acknowledge the Natural Sciences and Engineering Research Council of Canada and the Saskatchewan Egg Producers for funding this work and the staff at the University of Saskatchewan Poultry Centre for their assistance. Technical assistance of Dawn Abbott (University of Saskatchewan, Saskatoon, Canada) is greatly appreciated.

Received for publication August 21, 2007. Accepted for publication February 5, 2008.

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