Abstract

Feeding canola meal to brown-shelled laying hens can result in the production of eggs with a fishy odor. This fishy taint is caused by the accumulation of trimethylamine (TMA) in the yolk. Trimethylamine is produced by the bacterial fermentation of choline in the lower gut. Fishy-egg tainting is caused by a SNP in flavin-containing monooxygenase 3 (FMO3 c.984A > T), rendering the hen unable to metabolize TMA into the nonodorous TMA N-oxide. The purpose of this study was to characterize the inheritance pattern of fishy-egg tainting when hens are fed canola meal at levels reflecting maximum use based on conventional formulation of laying hen diets. Additionally, we wished to examine the effect of choline source (choline chloride vs. canola meal) on egg tainting. In the first of 2 experiments, 6 hens per genotype (AA, AT, and TT) were allocated to each of 5 dietary treatments (0, 6, 12, 18, or 24% canola meal) for 4 wk. Three yolks per hen collected in the last week of the trial were analyzed for TMA concentration. There was a significant linear regression (P < 0.05) between yolk TMA concentration and dietary canola meal level for hens of the TT but not the AA or AT genotypes. In the second experiment, 6 hens of the TT (homozygous tainting) genotype were each assigned to 1 of 9 dietary treatments: the 5 diets used in the first experiment plus 4 diets that used choline chloride to match the total choline concentration of the 6, 12, 18, and 24% canola meal diets, respectively. Three yolks per hen were analyzed for TMA concentration. A significant response in yolk TMA concentration was seen with the canola meal diets but not the choline chloride diets. We conclude that fishy-egg tainting is recessively expressed when hens are fed canola meal at levels from 12 up to 24% inclusion. We also conclude that choline chloride, at levels typical of commercial egg production, will not lead to egg tainting.

INTRODUCTION

It has been well documented that when brown-shelled layers are fed canola meal (CM), some hens within the flock lay eggs with fishy taint. The taint is caused by an accumulation of trimethylamine (TMA) in the yolk (Hobson-Frohock et al., 1973). Fishy-egg tainting is a nutrigenetic condition because both genetic and dietary factors must be present for egg tainting to occur.

Fishy egg tainting is a heritable condition that is caused by a mutation in exon 7 of flavin-containing monooxygenase 3 (FMO3) of brown-shelled laying hens (Bolton et al., 1976; Honkatukia et al., 2005). The causative mutation is an A to T SNP at nucleotide 984 of the coding sequence (FMO3 c.984A > T) that causes a threonine to serine substitution at amino acid 329 (Honkatukia et al., 2005). Hens with this mutation are impaired in their ability to oxidize malodorous TMA into odorless TMA N-oxide (TMAO). The fishy-smelling TMA subsequently accumulates in circulation and is deposited in the developing follicles of affected hens (March and MacMillan, 1979).

Bolton et al. (1976) were the first to study the inheritance of egg tainting and concluded that it was semi-dominant (i.e., additive). After identifying the causative mutation, Honkatukia et al. (2005) found that egg tainting was recessive. Further analysis revealed a significant difference in yolk TMA concentration between AA (homozygous normal) and AT hens, suggesting that egg tainting may indeed be additive (Kretzschmar et al., 2007).

Trimethylamine is produced by bacterial fermentation of choline in the gut (March and MacMillan, 1979). Choline in the form of choline chloride (ChCl) is routinely added to laying hen rations but does not lead to the production of tainted eggs (Goh et al., 1979). Choline chloride is rapidly absorbed in the duodenum and therefore less accessible for fermentation by TMA-producing bacteria located farther down the tract (Budowski et al., 1977; Goh et al., 1979).

The predominant form of choline found in CM is sinapine, an ester of choline and sinapic acid (Pearson et al., 1980). Unlike ChCl, feeding sinapine to susceptible hens does lead to the production of fishy-tainted eggs (Goh et al., 1979). Enteric bacteria must first hydrolyze the ester bond between the choline and sinapic acid before the choline can be absorbed (Goh et al., 1979). Hydrolysis occurs primarily in the distal portions of the digestive tract (Qiao and Classen, 2003) and it is thought that this delay in choline absorption makes choline from sinapine available for fermentation to TMA (Goh et al., 1979).

The glucosinolates present in CM also contribute to the production of fishy-tainted eggs. Goitrin, an antinu-tritional factor formed by the action of myrosinase on glucosinolates, inhibits the oxidation of TMA to TMAO by competing for the active site of FMO3 (Fenwick et al., 1981; Goh et al., 1983). The glucosinolate content of CM has been continually decreasing through successful breeding programs (Hickling, 2001) and as such it is not clear if the levels present in modern CM are high enough to significantly affect TMA oxidation.

Previous studies examining the inheritance of fishy-egg taint have used high levels of ChCl (in addition to 10% CM) to induce tainting (Honkatukia et al., 2005; Kretzschmar et al., 2007) that may not reflect typical commercial production practices. Our first objective was to characterize the inheritance of fishy-egg tainting under typical commercial production conditions by feeding CM. Our second objective was to observe the effects of source and level of choline on fishy egg tainting.

MATERIALS AND METHODS

All experiments were performed in accordance with the Guide to the Care and Use of Experimental Animals (Canadian Council on Animal Care, 1993). Protocols were approved by the University of Saskatchewan Animal Care Committee.

Experiment 1

Hens and roosters of a commercial brown-shelled line were bred via artificial insemination to produce progeny to be used for this experiment. The progeny were genotyped for FMO3 c.984A > T, and at 43 wk of age, 6 hens per genotype were allocated to 1 of 5 dietary treatments (n = 90). The treatments were 0, 6, 12, 18, or 24% CM, and the diets were formulated to meet or exceed NRC (1994) requirements (Table 1, Table 2). Individually housed hens were fed the diets for a total of 4 wk, consisting of a 3-wk adaptation period followed immediately by a 1-wk collection period. Three eggs were collected per hen for TMA analysis

Mean yolk TMA concentration was analyzed in a 3 (genotype) × 5 (CM level) factorial design using the MIXED procedure of SAS version 9.1 (SAS Institute Inc., Cary, NC) to examine the main effects. Means were separated using the least significant difference method. Regression analysis of the effects of CM level on mean yolk TMA concentration was performed separately for each genotype. Individual hens were the experimental unit and P < 0.05 was considered significant.

Experiment 2

The purpose of this experiment was to examine the effects of source and level of choline on fishy-egg tainting. The experiment was performed concurrently and designed to overlap with experiment 1. Only hens of the TT genotype were used because they would display the greatest response in yolk TMA concentration to the treatments. Six hens were assigned to each of 9 dietary treatments (n = 54). The treatments were a control (0% CM, 0% ChCl) and 4 levels of total choline (0.158, 0.187, 0.215, and 0.243%) from either CM or ChCl (Table 1). These choline levels correspond to the total choline concentrations of the diets from experiment 1. The choline concentrations of the wheat, soybean meal, and CM (0.09, 0.44, and 0.79%, respectively) were used to calculate the choline concentrations of the diets (contributions from the other ingredients were negligible).

The diets were again formulated to meet or exceed NRC (1994) requirements (Table 2). The diets were fed for the same 4-wk period as experiment 1. The results of the control and CM diets are the same as those presented for the TT hens in experiment 1. As in experiment 1, three eggs were collected per hen and analyzed for yolk TMA concentration.

Mean yolk TMA concentration was analyzed as a 2 (choline source) × 5 (choline level) factorial arrangement using the MIXED procedure of SAS. The control treatment was pseudo-replicated for analysis. Means were separated using the least significant difference method. Regression analysis was also performed for each choline source to examine the effect of increasing choline level on yolk TMA concentration. As in experiment 1, each hen was an experimental unit and P < 0.05 was considered significant.

Genotyping

At 1 d of age, each chick was dubbed to collect a sample of comb tissue for DNA extraction. The tissue was digested with proteinase K (final concentration of 0.20 mg/mL) followed by ammonium acetate-isopropanol precipitation (Sambrook et al., 1989).

All birds were genotyped for FMO3 c.984A > T. A PCR-RFLP assay was developed with the aid of Sequencher version 4.1 (Genecodes, Ann Arbor, MI) and the reported genomic FMO3 sequence for Gallus gallus (GenBank accession number AH012591).

The forward and reverse primers (forward: 5′-GCT CAT CAC CCG CTT CTG G-3′; reverse: 5′-GCC TCG TTG TTC TTG CTT TCG-3′) were developed using OligoAnalyzer version 3.1 (Integrated DNA Technologies, Coralville, IA) and were obtained from Operon Biotechnologies Inc. (Huntsville, AL).

Each PCR reaction consisted of 1 μL of DNA template and 20 μL of PCR cocktail [0.2 pmol of forward primer, 0.2 pmol of reverse primer, 0.2 mM deoxynucleoside triphosphate mix (Fermentas, Burlington, Ontario, Canada), 10% 10× PCR buffer (Fermentas), 1.9 mM MgCl2 (Fermentas), 0.5 units of Taq polymerase (Fermentas), and double-distilled H2O]. Thermocycling was performed using a Robocycler Gradient 96 thermocycler (Stratagene, La Jolla, CA). The reaction began with a 2-min denaturation period at 94°C. This was followed by 35 cycles of 50 s at 94°C, 50 s at 63°C, and 50 s at 72°C. The last step was a 4-min final extension period at 72°C.

The resulting 462-bp PCR product was then digested with the restriction endonuclease BsrI (Fermentas) and incubated for 4 h at 65°C. The A allele of FMO3 c.984A > T is cleaved into fragments of 62 and 400 bp. The digest products were then electrophorized on a 2% aga-rose gel (Figure 1).

TMA Yolk Analysis

The procedures for determination of TMA concentration in serum and egg yolk were modified from those reported by Reese et al. (2004). Eggs were collected on the day of lay and stored for up to 1 wk at 4°C. Whole egg weight and yolk weight were recorded. In a 50-mL centrifuge tube, the yolk was mixed with 10% trichloracetic acid (Sigma-Aldrich, St. Louis, MO) at a concentration of 1 mL per gram of yolk until homogenized. The mixture was incubated overnight at room temperature. The solution was filtered through a folded sheet of 11 cm #2 filter paper (Whatman International Ltd., Maidstone, UK) and the filtrate was stored for up to 4 wk at 4°C.

A 2-mL aliquot of the filtrate was transferred to a 16 × 100 glass culture tube and 1.5 mL of potassium hydroxide (VWR, Edmonton, Alberta, Canada) was added to separate TMA from its salts. The tube was vortexed briefly and 0.5 mL of neutral buffered formalin (Sigma-Aldrich) was added. The tube was briefly vortexed and 5 mL of toluene (Sigma-Aldrich) was added. The tube was capped with a polypropylene stopper and shaken at 250 rpm in an orbital shaker for 2 h at room temperature.

After shaking, a 2.5-mL aliquot of the toluene phase was transferred to a new 16 × 100 tube and 2.5 mL of 0.02% picric acid (Sigma-Aldrich) in toluene was added, forming a yellow picrate complex with the nitrogen of the TMA. Approximately 1.5 mL was transferred to a plastic cuvette and placed in a Spectronic 601 (Milton Roy, Rochester, NY) spectrophotometer and the absorbance at 410 nm was recorded. A standard curve was produced from 14 TMA standard solutions (ranging in concentration from 0 to 29.3 μg/mL of TMA-nitrogen) to calculate the TMA-nitrogen concentration of each yolk sample (R2 = 0.9974). The TMA concentration was then calculated from the TMA-nitrogen concentration.

Choline Analysis

Samples of wheat, soybean meal, and CM from the second-generation diets were analyzed for free choline concentration. Extraction was performed using the procedures developed by Menten and Pesti (1998). In a Goldfisch beaker (Labconco, Kansas City, MO), 2 g of finely ground feed ingredient was combined with 25 mL of extractant (0.5 M potassium hydroxide in methanol). Boiling beads were added before simmering for 2 h on a Goldfisch apparatus. When the solution had cooled to room temperature, 30 mL of distilled water was added and the pH was adjusted to between 6.0 and 6.5 with HCl (Sigma-Aldrich). The solution was filtered through one 11-cm sheet of Whatman #2 filter paper into a 100-mL volumetric flask and the volume of the filtrate was increased to 100 mL with distilled water. This solution was then used for choline quantification.

The Choline/Acetylcholine Quantification Kit from BioVision Research Products (Mountain View, CA) was used to determine the choline concentration of the filtrate (http://www.biovision.com/pdf/K615.pdf). The kit oxidizes free choline to betaine and this reaction yields products that react with the supplied Choline Probe and fluoresce at 587 nm when excited at 535 nm. Standards and samples were prepared in a 96-well microtiter plate. The standards (50 μL each of 0, 2, 4, 6, 8, and 10 pmol/μL of choline) were prepared using the choline standard supplied in the kit. Samples were prepared by adding 1 μL of filtrate (from the extraction) to 49 μL of Choline Assay Buffer (supplied in the kit).

To each well was added 48 μL of a reaction mix, which consisted of 44 μL of Choline Assay Buffer, 2 μL of Choline Probe (dissolved in anhydrous dimethyl sulfoxide), and 2 μL of Enzyme Mix (dissolved in Choline Assay Buffer). The fluorescence was read in a Fluoroskan Ascent FL fluorometer (Thermo Fisher Scientific, Waltham, MA) at excitation/emission = 535/590 nm. The background reading (from the 0 pmol/μL standard) was subtracted from all of the fluorescence readings and a standard curve was used to calculate the concentration of choline in the filtrate. Dilution factors were then used to calculate the concentration of free choline in the samples.

RESULTS

Experiment 1

The effects of genotype, diet, and their interaction on yolk TMA concentration were all significant (P < 0.05). In all of the treatments, some eggs were produced with yolk TMA concentration greater than the estimated human detection threshold of 4 μg/g (Table 3, Griffiths et al., 1979). The mean yolk TMA concentration remained below the human detection threshold for both the AA and AT genotypes across all of the diets (Figure 2). For the TT genotype, however, the mean rose above the detection threshold for the 12, 18, and 24% CM diets.

A significant linear regression between yolk TMA concentration and CM level was found for hens of the TT genotype (P < 0.05, R2 = 0.3599, y = 0.186x + 2.613), but not of the AA or AT genotypes, indicating that only hens of the TT genotype respond with increasing yolk TMA concentrations to CM inclusion.

Experiment 2

The effects of choline source, level, and their interaction on yolk TMA concentration were all significant (P < 0.05). Mean yolk TMA concentrations remained below the detection threshold (4 μg/g) for all of the ChCl diets (Figure 3). In contrast, the mean yolk TMA concentration for the CM diets was above the human detection threshold and significantly greater than that of the ChCl diets at the 3 highest levels of inclusion. There was a significant linear relationship between yolk TMA concentration and choline level for the CM (P < 0.05, R2 = 0.3608, y = 39.411x – 2.513) but not the ChCl diets.

DISCUSSION

The human detection threshold of TMA in whole-egg homogenate was estimated to be 1 μg/g (Griffiths et al., 1979). Given that TMA is almost exclusively concentrated in the yolk (Hobson-Frohock et al., 1973) and that the yolk makes up approximately 25% of the mass of the total egg contents (Suk and Park, 2001), the human detection threshold can be estimated to be 4 μg/g in the yolk. However, there is variability in the ability of individuals to detect TMA (Griffiths et al., 1979), and even in the AA genotype 0% CM group, 12% of the eggs analyzed had a yolk TMA concentration greater than 4 μg/g (Table 3). Although 4 μg/g is an appropriate benchmark, it is possible that eggs with yolk TMA concentrations above that would be acceptable to consumers.

The presence of TMA in the yolks of AA hens fed the control diet was expected and is in accordance with previous reports (Honkatukia et al., 2005; Kretzschmar et al., 2007). Though the control diet did not contain any CM or ChCl, it still contained 0.130% choline, primarily from the soybean meal and wheat. Though FMO3 is very efficient at oxidizing TMA to TMAO, the serum half-life of TMA (in nontainting hens) is 46 min (Emmanuel et al., 1984). Thus, some TMA is able to accumulate in the developing follicles of AA hens.

Honkatukia et al. (2005) found that when hens were fed diets containing high levels of ChCl (6,000 mg/kg) only hens of the TT genotype produced fishy-tainted eggs, suggesting that the trait is recessively inherited. Upon reanalyzing that data, Kretzschmar et al. (2007) found a significant difference between the yolk TMA concentrations of AA and AT hens, introducing the possibility that egg-tainting is additive. Due to these conflicting results, combined with the fact that the production of tainted eggs is highly dependant upon dietary factors, the inheritance pattern of fishy-egg tainting under commercial production conditions (i.e., when hens are fed CM) was not clear.

Our data definitively shows that fishy-egg tainting is recessive when hens are fed CM at levels of inclusion from 12 to 24%. This is in agreement with the inheritance of fishy odor syndrome in humans and fish-tainted milk in Swedish Red and White dairy cattle, both of which are inherited recessively through FMO3 (Dolphin et al., 1997; Lundén et al., 2002). The presence of a significant regression between CM level and yolk TMA concentration for hens of the TT but not the AA or AT genotypes is definitive evidence that egg tainting is a recessive condition. Additionally, the lack of a significant difference in yolk TMA concentration between hens of the AA and AT genotypes for each dietary treatment (Figure 2) gives further support to this conclusion. The recessive nature of the trait is highly advantageous for commercial breeding companies wishing to market hens that do not produce tainted eggs. They only need to remove the T allele from one, rather than both, parental lines in their crossbreeding scheme. The results of Kretzschmar et al. (2007) suggest that when excessive ChCl is fed, at levels much higher than would be seen in commercial production, fishy-egg tainting may be expressed in an additive or semidominant fashion.

Choline is an essential nutrient for laying hens. Deficiency can lead to perosis in chicks and increased liver weight (Budowski et al., 1977). Unlike chicks, hens are able to synthesize a large portion of their choline requirements (Crawford et al., 1969). The NRC (1994) choline requirements for brown-shelled layers are 0.1225% for chicks, 0.047% for prelay pullets, and 0.1050% for laying hens. All of our experimental diets (Table 2) exceeded NRC (1994) choline requirements. Even the control diet used in experiment 2, which was not supplemented with ChCl, contained 0.130% total choline. This raises the question as to whether laying hen diets really need to be supplemented with choline.

Our results are the first to confirm that genotypically predisposed (FMO3 c.984 TT) hens fed ChCl at and above levels of inclusion that reflect industry recommendations (NRC, 1994) do not produce fishy-tainted eggs (Figure 2). This confirms the results of Goh et al. (1979), who reported that when taint-producing hens were fed 0.05% ChCl, no tainted eggs were produced, but when the same hens were fed 0.14% sinapine bisulfate (the equivalent of 10% CM), tainted eggs were produced. Budowski et al. (1977) found that when 0.101% ChCl (in a casein-glucose diet) was fed to 30-d-old chicks, over 90% was absorbed by the duodenum and upper jejunum. This leaves very little, if any, choline available for fermentation to TMA by bacteria located in the lower intestine and ceca (Emmanuel et al., 1984; Qiao and Classen, 2003).

As previously stated, Honkatukia et al. (2005) and Kretzschmar et al. (2007) were able to induce egg tainting in TT hens by feeding high concentrations of ChCl (0.60% total choline). Choline is transported across the intestinal brush-border membrane via facilitated diffusion, a mechanism that can become saturated (Hegazy and Schwenk, 1984; Saitoh et al., 1992). It is possible that the very high level of inclusion used by Honkatukia et al. (2005) and Kretzschmar et al. (2007) overwhelmed the absorptive mechanisms in the distal small intestine, allowing more ChCl to reach, and thus be utilized by, TMA-producing bacteria.

We conclude that fishy-egg tainting is recessively expressed when hens are fed CM from 12 to 24%. Choline chloride does not lead to the production of fishy-tainted eggs at typical commercial levels of inclusion.

Table 1

Ingredient formulations of the experimental diets1

  Canola meal (% choline) Choline chloride (% choline) 
Ingredient (%) Control 0.158 0.187 0.215 0.243 0.158 0.187 0.215 0.243 
1Ingredients are presented as a percentage on an as-fed basis. 
2Celite Corporation, Lompoc, CA. 
3Supplied per kilogram of diet: vitamin A, 8,000 IU; vitamin D3, 3,000 IU; vitamin E, 25 IU; menadione, 1.5 mg; riboflavin, 5 mg; pantothenic acid, 8 mg; vitamin B12, 0.012 mg; pyridoxine, 1.5 mg; thiamine, 1.5 mg; folic acid, 0.5 mg; niacin, 30 mg; biotin, 0.06 mg; iodine, 0.8 mg; copper, 10 mg; iron, 80 mg; selenium, 0.3 mg; manganese, 80 mg; zinc, 80 mg; quinguard M6S (Novus International, St. Louis, IL), 0.625 mg, and calcium carbonate, 500 mg. 
4Avizyme 1302, Danisco Animal Nutrition, Marlborough, UK. 
Wheat 69.74 66.79 63.87 60.96 58.03 69.685 69.63 69.575 69.52 
Canola meal 0.00 6.00 12.00 18.00 24.00 0.00 0.00 0.00 0.00 
Soybean meal 15.20 11.51 7.81 4.11 0.41 15.20 15.20 15.20 15.20 
Canola oil 1.76 2.49 3.22 3.95 4.68 1.76 1.76 1.76 1.76 
Celite2 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 
Dicalcium phosphate 0.71 0.67 0.62 0.57 0.53 0.71 0.71 0.71 0.71 
Limestone 10.17 10.13 10.08 10.03 9.99 10.17 10.17 10.17 10.17 
NaCl 0.24 0.24 0.24 0.23 0.23 0.24 0.24 0.24 0.24 
Choline chloride 0.00 0.00 0.00 0.00 0.00 0.055 0.11 0.165 0.220 
Vitamin-mineral premix3 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 
Wheat enzyme4 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 
dl-Methionine 0.13 0.12 0.11 0.10 0.08 0.13 0.13 0.13 0.13 
  Canola meal (% choline) Choline chloride (% choline) 
Ingredient (%) Control 0.158 0.187 0.215 0.243 0.158 0.187 0.215 0.243 
1Ingredients are presented as a percentage on an as-fed basis. 
2Celite Corporation, Lompoc, CA. 
3Supplied per kilogram of diet: vitamin A, 8,000 IU; vitamin D3, 3,000 IU; vitamin E, 25 IU; menadione, 1.5 mg; riboflavin, 5 mg; pantothenic acid, 8 mg; vitamin B12, 0.012 mg; pyridoxine, 1.5 mg; thiamine, 1.5 mg; folic acid, 0.5 mg; niacin, 30 mg; biotin, 0.06 mg; iodine, 0.8 mg; copper, 10 mg; iron, 80 mg; selenium, 0.3 mg; manganese, 80 mg; zinc, 80 mg; quinguard M6S (Novus International, St. Louis, IL), 0.625 mg, and calcium carbonate, 500 mg. 
4Avizyme 1302, Danisco Animal Nutrition, Marlborough, UK. 
Wheat 69.74 66.79 63.87 60.96 58.03 69.685 69.63 69.575 69.52 
Canola meal 0.00 6.00 12.00 18.00 24.00 0.00 0.00 0.00 0.00 
Soybean meal 15.20 11.51 7.81 4.11 0.41 15.20 15.20 15.20 15.20 
Canola oil 1.76 2.49 3.22 3.95 4.68 1.76 1.76 1.76 1.76 
Celite2 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 
Dicalcium phosphate 0.71 0.67 0.62 0.57 0.53 0.71 0.71 0.71 0.71 
Limestone 10.17 10.13 10.08 10.03 9.99 10.17 10.17 10.17 10.17 
NaCl 0.24 0.24 0.24 0.23 0.23 0.24 0.24 0.24 0.24 
Choline chloride 0.00 0.00 0.00 0.00 0.00 0.055 0.11 0.165 0.220 
Vitamin-mineral premix3 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 
Wheat enzyme4 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 
dl-Methionine 0.13 0.12 0.11 0.10 0.08 0.13 0.13 0.13 0.13 
Table 2

Calculated nutrient composition of the experimental diets1

 Canola meal (% choline) Choline chloride (% choline) 
Nutrient (%) Control 0.158 0.187 0.215 0.243 0.158 0.187 0.215 0.243 
1Values are listed as a percentage unless otherwise noted. 
AME (kcal/kg) 2,770 2,770 2,770 2,770 2,770 2,770 2,770 2,770 2,770 
CP 16.20 16.20 16.20 16.20 16.20 16.20 16.20 16.20 16.20 
Choline 0.130 0.158 0.187 0.215 0.243 0.158 0.187 0.215 0.243 
Calcium 4.10 4.10 4.10 4.10 4.10 4.10 4.10 4.10 4.10 
Nonphytate P 0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.33 
Lysine 0.78 0.79 0.81 0.82 0.83 0.78 0.78 0.78 0.78 
Methionine 0.37 0.37 0.37 0.37 0.37 0.37 0.37 0.37 0.37 
TSAA 0.61 0.62 0.62 0.63 0.63 0.61 0.61 0.61 0.61 
 Canola meal (% choline) Choline chloride (% choline) 
Nutrient (%) Control 0.158 0.187 0.215 0.243 0.158 0.187 0.215 0.243 
1Values are listed as a percentage unless otherwise noted. 
AME (kcal/kg) 2,770 2,770 2,770 2,770 2,770 2,770 2,770 2,770 2,770 
CP 16.20 16.20 16.20 16.20 16.20 16.20 16.20 16.20 16.20 
Choline 0.130 0.158 0.187 0.215 0.243 0.158 0.187 0.215 0.243 
Calcium 4.10 4.10 4.10 4.10 4.10 4.10 4.10 4.10 4.10 
Nonphytate P 0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.33 
Lysine 0.78 0.79 0.81 0.82 0.83 0.78 0.78 0.78 0.78 
Methionine 0.37 0.37 0.37 0.37 0.37 0.37 0.37 0.37 0.37 
TSAA 0.61 0.62 0.62 0.63 0.63 0.61 0.61 0.61 0.61 
Table 3

Effects of canola meal and FMO3 c.984A > T genotype on yolk trimethylamine (TMA) concentration

Diet1 Genotype Minimum Maximum Mean SEM % Above 4 μg/g2 
1CM = canola meal. 
2Percentage of eggs per treatment with yolk TMA concentration >4 μg/g. 
0% CM AA 0.78 7.95 2.23 0.67 12 
 AT 0.73 6.08 2.76 0.67 33 
 TT 0.66 16.18 3.12 0.67 31 
6% CM AA 1.24 6.87 2.68 0.67 22 
 AT 1.04 6.54 2.36 0.74 13 
 TT 1.01 5.73 2.84 0.67 31 
12% CM AA 0.96 5.40 1.99 0.67 17 
 AT 1.02 6.69 2.51 0.67 22 
 TT 1.21 14.03 5.39 0.67 50 
18% CM AA 0.86 9.17 1.54 0.67 12 
 AT 0.45 6.63 2.07 0.67 13 
 TT 1.87 18.16 5.53 0.67 56 
24% CM AA 1.16 10.01 3.25 0.67 18 
 AT 0.45 9.00 3.53 0.67 35 
 TT 3.22 14.66 7.34 0.67 88 
Diet1 Genotype Minimum Maximum Mean SEM % Above 4 μg/g2 
1CM = canola meal. 
2Percentage of eggs per treatment with yolk TMA concentration >4 μg/g. 
0% CM AA 0.78 7.95 2.23 0.67 12 
 AT 0.73 6.08 2.76 0.67 33 
 TT 0.66 16.18 3.12 0.67 31 
6% CM AA 1.24 6.87 2.68 0.67 22 
 AT 1.04 6.54 2.36 0.74 13 
 TT 1.01 5.73 2.84 0.67 31 
12% CM AA 0.96 5.40 1.99 0.67 17 
 AT 1.02 6.69 2.51 0.67 22 
 TT 1.21 14.03 5.39 0.67 50 
18% CM AA 0.86 9.17 1.54 0.67 12 
 AT 0.45 6.63 2.07 0.67 13 
 TT 1.87 18.16 5.53 0.67 56 
24% CM AA 1.16 10.01 3.25 0.67 18 
 AT 0.45 9.00 3.53 0.67 35 
 TT 3.22 14.66 7.34 0.67 88 
Figure 1

Representative gel for the FMO3 c.984A > T PCR-RFLP. Lane 1 is a kb1+ ladder, lanes 2 and 6 are TT, lanes 3 and 5 are AT, and lanes 4 and 7 are AA chickens, respectively.

Figure 1

Representative gel for the FMO3 c.984A > T PCR-RFLP. Lane 1 is a kb1+ ladder, lanes 2 and 6 are TT, lanes 3 and 5 are AT, and lanes 4 and 7 are AA chickens, respectively.

Figure 2

Effects of FMO3 c.984A > T genotype and canola meal on yolk trimethylamine concentration (TMA; μg/g) in experiment 1. Means sharing the same letter are not significantly different (P < 0.05). Error bars represent SEM.

Figure 2

Effects of FMO3 c.984A > T genotype and canola meal on yolk trimethylamine concentration (TMA; μg/g) in experiment 1. Means sharing the same letter are not significantly different (P < 0.05). Error bars represent SEM.

Figure 3

Effects of choline source (canola meal or choline chloride) and level on yolk trimethylamine concentration (TMA; μg/g) of TT hens in experiment 2. The light gray bars represent the canola meal (CM) diets, and the dark gray bars represent the choline chloride (ChCl) diets. The white bar represents the control diet (0% CM and 0% ChCl), which was pseudo-replicated. Means sharing the same letter are not significantly different (P < 0.05). Error bars represent SEM.

Figure 3

Effects of choline source (canola meal or choline chloride) and level on yolk trimethylamine concentration (TMA; μg/g) of TT hens in experiment 2. The light gray bars represent the canola meal (CM) diets, and the dark gray bars represent the choline chloride (ChCl) diets. The white bar represents the control diet (0% CM and 0% ChCl), which was pseudo-replicated. Means sharing the same letter are not significantly different (P < 0.05). Error bars represent SEM.

We thank the Saskatchewan Canola Development Commission (Saskatoon, Saskatchewan, Canada) and the Canadian International Grains Institute (Winnipeg, Manitoba, Canada) for funding this project. We would also like to acknowledge Dawn Abbott and Pamela Karcha (University of Saskatchewan) for performing all of the TMA and choline analyses. We would like to acknowledge HyLine International (Des Moines, IA) for providing the parents of the hens used in this trial as well as for the DNA extraction procedure.

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