Application of Paracoccus marcusii as a potential feed additive for laying hens

Application of Paracoccus marcusii as a potential feed additive for laying hens Abstract Carotenoids have been used for many years as an added pigment to enhance egg yolk color. One such carotenoid, astaxanthin, has a strong antioxidant activity and is produced by several microorganisms, including the bacterium Paracoccus marcusii, which has shown promise to be used as a feed additive. Therefore, this study investigated the use of P. marcusii as a possible source of pigmentation in layer hen feed to enhance egg yolk color. Paracoccus marcusii was fed to hens in a sucrose solution (10% m/v). The hens were fed daily and all eggs were collected for analysis. Dilutions of egg contents were plated onto selective media to detect the presence of known food pathogens (E. coli, Listeria, and Salmonella). In the feeding trial, there was no negative effect on hen body weight, egg production, or overall egg quality. There was a significant increase (P < 0.05) in yolk color as well as an increase in whole egg and yolk weight. There were also no known food pathogens detected in any of the egg samples. This study has shown promising results in using this bacterium as an effective feed additive for laying hens. INTRODUCTION The chicken (Gallus gallus domesticus) is a domesticated subspecies of the red junglefowl (Gallus gallus). Chickens have become one of the most common domesticated animals and are mainly kept as a source of food for meat and eggs. In 2015, the South African layer flock size was 24.9 million hens and has since increased to 25.05 million hens in 2016 (South African Poultry Association, 2016). Chicken eggs remain one of the largest animal product sectors in the South African agriculture after beef, chicken meat, and milk with a gross turnover of R9.83 billion ($760 million) in 2015 (Department of Agriculture, Forestry and Fisheries (DAFF), South Africa, 2016). For many years carotenoids have been used to manipulate the color of egg yolk to obtain a desired color (Adams, 1985). Poultry can easily absorb carotenoids from their diet (Hudon, 1994). After the hen has ingested the feed, the carotenoids are released by enzymes and absorbed in the small intestines. The free carotenoids are then emulsified to form oil droplets (or portomicrons) and delivered to the liver. These molecules are incorporated into very low density lipoproteins (VLDL) and delivered to the yolk (Surai et al., 2001; Bortolotti et al., 2003). Astaxanthin produced by microorganisms have been commercialized and applied in the pigmentation of cosmetics, beverages, dairy products, and meats (Del Campo et al., 2000; Guerin et al., 2003; Liang et al., 2004; Pulz and Gross, 2004; Chandi and Gill, 2011). There is an increase in demand for naturally derived astaxanthin from microorganisms instead of synthetic astaxanthin, since natural astaxanthin has a higher antioxidant activity when compared to synthetic astaxanthin (Capelli et al., 2013). The yeast, Xanthophyllomyces dendrorhous, and microalga, Haematococcus pluvialis, are currently used for the large-scale cultivation for astaxanthin production. Many studies have used these microorganisms in developing biotechnological processes to produce astaxanthin in large quantities (Lorenz and Cysewski, 2000; Dufosse et al., 2005; Schmidt et al., 2011; Mata-Gómez et al., 2014). In previous studies, whole yeast and microalga cells have been used as a pigmentation source in the diet of trout (Choubert and Heinrich, 1993; Storebakken et al., 2004), salmon (Lorenz and Cysewski, 2000) and laying hens (Johnson et al., 2003). However, yeast and microalga have thicker cell walls compared to bacteria. Therefore, a significant increase in pigmentation was only observed after the partial homogenization, enzymatic digestion or cracking of the cells to increase the release of the available pigments in the digestive tract (Choubert and Heinrich, 1993; Lorenz and Cysewski, 2000; Johnson et al., 2003; Storebakken et al., 2004). However, there was no need to enzymatically digest or homogenize the bacterium, Paracoccus marcusii, when fed to rainbow trout (Oncorhynchus mykiss) for pigmentation effect (De Bruyn, 2013). The results obtained by De Bruyn (2013) showed the promising application of using the whole P. marcusii cell as a pigmentation source, instead of the yeast or microalga. The aim of this study was, therefore, to determine if whole P. marcusii cells can be used as a possible pigmentation source to enhance egg yolk color and possibly improve egg quality without the need to extract the pigment from the bacterial cell. MATERIALS AND METHODS Experimental Layout, Hens and Feed All feeding trials took place at the poultry unit of Mariendahl Experimental Farm, University of Stellenbosch. The facility consisted of a hen house equipped with a 2-tier A-shape battery system. Each row contained 12 individual cages with sliding doors, slightly slanted floors which allowed the eggs to roll out of the cage for easy collection and a feeding tray in front of the cage. All hens had free access to feed and water was provided ad libitum by 2 nipple-type drinkers per cage. Layer hens (Lohmann Brown) were purchased from Quantum Foods, South Africa. Animal experiments were performed based on the Care and Use of Animals for Scientific Purposes of the University of Stellenbosch [SU-ACUM14–0034 (Pilot study) and SU-ACUD15–00088 (Experimental trial)]. Pilot Study A total of 50 hens of 36 wk of age were selected. Ten hens were randomly assigned into a feeding group and caged individually. The hens were allowed to adjust to their new surroundings for 4 d before starting with the experimental diets. After 5 wk of the experimental treatments, the hens were fed a basal diet for 2 wk to determine if the change in pigmentation was because of the bacterium. The basal feed consisted of commercially available layer hen feed (Agrimark, Stellenbosch, South Africa). Experimental Trial A total of 100 hens of 16 wk of age were selected. Twenty hens were randomly assigned into a group and caged individually. For the first 4 wk, all hens were fed pre-lay feed containing white corn before starting with the experimental diets for 8 wk on peaking feed containing either yellow corn or white corn (Table 1). Table 1. Basal feed composition for the experimental trial. Ingredient  Prelay1  Peaking2    %  %  Corn (White or yellow)3  65,806  63,338  Soybean Full Fat  7,088  9,479  Soybean 46  18,790  18,790  DL Methionine  0,109  0,203  L-Threonine  -  0,001  Vitamin and Mineral Premix  0,150  0,150  Limestone  5,975  5,961  Salt (NaCl)  0,262  0,263  Monocalcium Phosphate  1,596  1,593  Sodium bicarbonate  0,225  0,222  Ingredient  Prelay1  Peaking2    %  %  Corn (White or yellow)3  65,806  63,338  Soybean Full Fat  7,088  9,479  Soybean 46  18,790  18,790  DL Methionine  0,109  0,203  L-Threonine  -  0,001  Vitamin and Mineral Premix  0,150  0,150  Limestone  5,975  5,961  Salt (NaCl)  0,262  0,263  Monocalcium Phosphate  1,596  1,593  Sodium bicarbonate  0,225  0,222  1Hens were fed prelay feed containing white corn for the first 4 wk. 2Hens were fed peaking feed containing either white corn or yellow corn for 8 wk, after the prelay feed. 3Only hens in the white control and treatment diet groups were fed feed containing white corn. Only hens in the yellow control diet groups were fed feed containing yellow corn. View Large Experimental Treatments All dosages given every day at each trial is indicated in Table 2. Dosages were given by hand every day in liquid form to ensure that the hens were fed equal amounts of the control or the bacterium. Table 2. Diet groups for all feeding trials and their dosages. Feeding Trial  Diet group name  Dosage  Pilot study  Control  None    Sucrose  1 mL sucrose (10% m/v)    PM-Freezedried  5 mL freeze-dried cells1    PM-1  1 mL live cells2    PM-5  5 mL live cells2  Experimental trial  Yellow Control  None    Yellow Sucrose Control  1 mL sucrose (10% m/v)    White Control  None    White Sucrose Control  1 mL sucrose (10% m/v)    PM-Feed  50 mL live cells2  Feeding Trial  Diet group name  Dosage  Pilot study  Control  None    Sucrose  1 mL sucrose (10% m/v)    PM-Freezedried  5 mL freeze-dried cells1    PM-1  1 mL live cells2    PM-5  5 mL live cells2  Experimental trial  Yellow Control  None    Yellow Sucrose Control  1 mL sucrose (10% m/v)    White Control  None    White Sucrose Control  1 mL sucrose (10% m/v)    PM-Feed  50 mL live cells2  1Cells were lyophilized in sucrose (10% m/v) and resuspended in sterile dH2O before feeding. 2All bacterial dosages contained 2.8 × 109 CFU/mL of Paracoccus marcusii. View Large Pilot Study Five different diets were prepared and randomly assigned to each group of 10 hens. Treatment 1 and 2 served as negative controls which consisted of a basal feed with no additives (Control) and 1 mL sucrose solution (10% m/v) (Sucrose Control). Treatments 3–5 consisted of a basal feed and a dosage of Paracoccus marcusii, either freeze-dried (PM-Freeze-dried) and resuspended in sterile dH2O, or live cells (PM-1 and PM-5), which were dripped onto the feed. Experimental Trial To effectively determine the pigmentation effect of the bacterium to enhance egg yolk color, all experimental diet groups were fed a diet containing white corn, except for the positive control diet groups which were fed a diet containing yellow corn. Five different diets were prepared and randomly assigned to each group of 20 hens. Treatments 1–4 served as the control groups (Yellow Control, Yellow Sucrose Control, White Control and White Sucrose Control). For treatment 5 (PM-Feed), a sucrose solution containing the bacterium, P. marcusii, was dripped onto the feed. Preparation of Paracoccus marcusii In preparation for the feeding trials, P. marcusii was cultured in 2 L Schott bottles containing 1 L of specialised medium (10 g/L bacteriological peptone (Oxoid, Hampshire, United Kingdom), 5 g/L yeast extract (Biolab, Modderfontein, South Africa), 3% NaCl and pH 7–8). Schott bottles were incubated at 26°C for 7 d. The cells were harvested through centrifugation at 10,000 rpm for 10 min and washed once with sterile dH2O. New cells were cultured each wk to ensure viability of cells. Pilot Study Cells were resuspended in equal volumes of sucrose (10% m/v). Aliquots of cells were transferred into separate flasks representing the different diet groups (PM-1 and PM-5). For freeze-drying, 50 mL of cells resuspended in sucrose (10% m/v) were frozen overnight in 250 mL Erlenmeyer flasks at –80°C. The VirTis benchtop K (model: 6KBTEL-85, SP Scientific, Gardiner, NY) was used to freeze-dry the cells until completely dry. Experimental Trial Cells were daily harvested from 1 L of growth medium and resuspended in 20 mL of sucrose (10% m/v) to obtain a high concentration dosage of P. marcusii. Cell concentration fed to each hen was 2.8 × 109 colony forming unit (CFU) per mL. Quantitative Data Measurements Hen Weight and Egg Production The initial and final mean weight was measured for all diet groups in all feeding trials. The egg production rate of each group was calculated using the total number of eggs laid over the duration of the trial:   \begin{eqnarray} &&{ Laying\ rate\ \left( \% \right)}\nonumber\\ && = \left( {\frac{{Total\ number\ of\ eggs\ laid}}{{Number\ of\ hens\, \times Number\ of\ days}}} \right)\ \times \ 100\nonumber\\ \end{eqnarray} (1) Egg Quality Analysis Eggs were collected every d following the 4-d acclimation period. Daily measurements included the following: whole egg weight, shell thickness, shell weight, yolk weight, yolk height, yolk color, and thick albumen height. The Haugh Unit (HU) was calculated using the formula:   \begin{equation}HU = {\rm{\ }}100\log \left( {{\rm{h}} - 1.7{w^{0.37}} + 7.6} \right)\end{equation} (2)where h is the height of the albumen and w is the whole egg weight (Haugh, 1937). The shell thickness was measured using an electronic digital caliper. The height of the albumen and yolk was measured by using a Haugh meter. For measuring the color of the yolk, the yolk was first separated from the albumen and placed into a plastic petri dish (90 mm × 15 mm). The color was then measured using a yolk color fan (YolkFanTM, DSM, Heerlen, Netherlands). The yolk color fan has a blade range between 1 and 15, where 1 is the palest and 15 the darkest. All measurements were taken by placing the petri dish with the yolk on a white background and holding the blades above the yolk. The corresponding blade number was then allocated to each individual yolk. Detection of Bacteria in the Egg Content Eggs were randomly selected during the trials from all diet groups and tested for the presence of potential food pathogens (Salmonella, Listeria, and E. coli) (Gast, 1992), as well as P. marcusii, in the egg content. The surface of the egg was sterilized by rolling the whole egg in 70% ethanol. The internal egg content was separated from the shell and homogenized in a plastic petri dish (90 mm × 15 mm) and a dilution series of 10−1–10−5 was prepared in sterile saline solution (0.9% NaCl). The dilutions were plated in triplicate onto PALCAM-Listeria-selective agar (3 g/L yeast extract, 23 g/L peptone, 5 g/L NaCl, 1 g/L starch, 10 g/L mannitol, 0.8 g/L aesculin, 0.5 g/L glucose, 0.5 g/L ammonium iron(III) citrate, 0.08 g/L phenol-rot, 15 g/L lithiumchloride and 13 g/L agar) (Merck, Darmstadt, Germany), SS agar (10 g/L lactose, 10 g/L peptone, 10 g/L NaCl, 8.5 g/L ox bile dried, 1 g/L ammonium iron(III) citrate, 8.5 g/L sodium thiosulfate, 0.025 g/L neutral red, 0.0003 g/L brilliant green and 12 g/L agar) (Merck, Darmstadt, Germany), LEVINE-EMB-agar (10 g/L lactose, 10 g/L peptone, 0.4 g/L eosin yellow, 0.065 g/L methylene blue, 2 g/L di-potassium hydrogen phosphate and 13.5 g/L agar) (Merck, Darmstadt, Germany) and nutrient agar (2 g/L yeast extract, 5 g/L peptone, 1 g/L meat extract, 8 g/L NaCl and 15 g/L agar) (Biolab, Modderfontein, South Africa). The Listeria, EMB, and SS agar plates were incubated for 2–3 d at 37°C and the nutrient agar plates were incubated for 4 to 5 d at 26°C. Statistical Analysis Statistical analysis was performed using Statistica (version 13.0, Statsoft Inc., Palo Alto, CA). One-way analysis of variance (ANOVA) was used for mean comparisons and Tukey's honest significant difference (HSD) was calculated where P < 0.05. RESULTS Hen Weight Pilot Study The average body weight at the start of the trial was 1.77 ± 0.02 kg between all diet groups. There was no significant increase (P > 0.05) between all diet groups at the end of the trial (Table 3). Table 3. Mean body weight at the start and end of the pilot study. Diet groups1  Weight before (kg)  Weight after (kg)  Control  1.70 ± 0.02  1.76 ± 0.02  Sucrose Control  1.74 ± 0.02  1.77 ± 0.02  PM-Freezedried  1.81 ± 0.02  1.80 ± 0.03  PM-1  1.79 ± 0.02  1.87 ± 0.02  PM-5  1.82 ± 0.03  1.82 ± 0.03  P value  0.44  0.60  Diet groups1  Weight before (kg)  Weight after (kg)  Control  1.70 ± 0.02  1.76 ± 0.02  Sucrose Control  1.74 ± 0.02  1.77 ± 0.02  PM-Freezedried  1.81 ± 0.02  1.80 ± 0.03  PM-1  1.79 ± 0.02  1.87 ± 0.02  PM-5  1.82 ± 0.03  1.82 ± 0.03  P value  0.44  0.60  Data are means ± SEM for 10 replicates per diet group. 1Diet groups are: Control, no dosage of bacteria; Sucrose, 1 mL dosage of sucrose (10% m/v); PM-Freezedried, 5 mL dosage of freeze-dried bacteria resuspended in sterile dH2O; PM-1, 1 mL dosage of bacteria; PM-5, 5 mL dosage of bacteria. View Large Experimental Trial Based on the results of the pilot study, the experimental trial was started with younger hens. Treatments were applied to hens fed on the white corn to minimize the pigmentation effect of the yellow corn. At the start of the experimental trial, the mean body weight was 1.45 ± 0.01 kg between the different diet groups. There was no significant difference (P > 0.05) in hen body weight at the start of the trial. The increase in body weight of the hens were consistent at the end of the trial with a significant difference (P < 0.05) only observed between the different corn groups (white or yellow corn). The yellow corn groups had an end mean weight of 1.97 kg compared to the white corn groups with 1.79 kg (Figure 1). Figure 1. View largeDownload slide Mean body weight of the hens at the start (black) and the end (white) of the experimental trial. Data are expressed as the mean ± SEM for 20 replicates per diet group. Diet groups are: Yellow Control, no dosage of bacteria; Yellow Sucrose Control, 1 mL dosage of sucrose (10% m/v); White Control, no dosage of bacteria; White Sucrose Control, 1 mL dosage of sucrose (10% m/v); PM-Feed, 50 mL dosage of bacteria. a-bLetters above bars indicates a significant difference at a confidence level of 95%, where P < 0.05. Figure 1. View largeDownload slide Mean body weight of the hens at the start (black) and the end (white) of the experimental trial. Data are expressed as the mean ± SEM for 20 replicates per diet group. Diet groups are: Yellow Control, no dosage of bacteria; Yellow Sucrose Control, 1 mL dosage of sucrose (10% m/v); White Control, no dosage of bacteria; White Sucrose Control, 1 mL dosage of sucrose (10% m/v); PM-Feed, 50 mL dosage of bacteria. a-bLetters above bars indicates a significant difference at a confidence level of 95%, where P < 0.05. Egg Production Pilot Study There was a significant difference (P < 0.05) detected in the hen egg production between the Sucrose Control, PM-Freezedried, PM-1 and PM-5 groups compared to the Control group. The Control group had the lowest egg production of 89.35%, followed by PM-Freezedried and PM-5 with 94.52%, Sucrose Control with 96.09% and PM-1 with 96.45% (Figure 2A). Figure 2. View largeDownload slide Mean egg production for (A) pilot study and (B) experimental trial. Data are expressed as the mean ± SEM for (A) 10 and (B) 20 replicates per diet group. Diet groups are (A): Control, no dosage of bacteria; Sucrose, 1 mL dosage of sucrose (10% m/v); PM-Freezedried, 5 mL dosage of freeze-dried bacteria resuspended in sterile dH2O; PM-1, 1 mL dosage of bacteria; PM-5, 5 mL dosage of bacteria; (B) Yellow Control, no dosage of bacteria; Yellow Sucrose Control, 1 mL dosage of sucrose (10% m/v); White Control, no dosage of bacteria; White Sucrose Control, 1 mL dosage of sucrose (10% m/v); PM-Feed, 50 mL dosage of bacteria. a-cLetters above bars indicates a significant difference at a confidence level of 95%, where P < 0.05. Figure 2. View largeDownload slide Mean egg production for (A) pilot study and (B) experimental trial. Data are expressed as the mean ± SEM for (A) 10 and (B) 20 replicates per diet group. Diet groups are (A): Control, no dosage of bacteria; Sucrose, 1 mL dosage of sucrose (10% m/v); PM-Freezedried, 5 mL dosage of freeze-dried bacteria resuspended in sterile dH2O; PM-1, 1 mL dosage of bacteria; PM-5, 5 mL dosage of bacteria; (B) Yellow Control, no dosage of bacteria; Yellow Sucrose Control, 1 mL dosage of sucrose (10% m/v); White Control, no dosage of bacteria; White Sucrose Control, 1 mL dosage of sucrose (10% m/v); PM-Feed, 50 mL dosage of bacteria. a-cLetters above bars indicates a significant difference at a confidence level of 95%, where P < 0.05. Experimental Trial A significant difference (P < 0.05) was detected between the Yellow Control, Yellow Sucrose Control, White Sucrose Control and PM-Feed compared to the White Control. The White Control group had the lowest egg production of 85.06%, followed by PM-Feed with 92.38%, White Sucrose Control with 92.59%, Yellow Control with 95.21% and Yellow Sucrose Control with 96.40% (Figure 2B). Egg Quality Pilot Study There was a significant difference (P < 0.05) detected for all egg quality parameters measured (Table 4). PM-Freezedried and PM-1 had heavier egg weights (58.85 g and 58.65 g, respectively) compared to the control groups (Control and Sucrose Control) and PM-5 (57.23 g, 57.91 g and 56.72 g, respectively). PM-Freeze-dried (15.10 g), PM-1 (15.41 g) and PM-5 (15.02 g) had heavier yolk weights compared to the Control (14.70 g) and Sucrose Control (14.86). Table 4. Egg quality parameters of all feeding trials. Trial  Diet Groups1  Egg size (mm)  Egg Weight (g)  Yolk Height (mm)  Yolk Weight (g)  Yolk Color  Albumen Height (mm)  Haugh Unit  Pilot study  Control  54.65 ± 0.14b,c  57.23 ± 0.27b,c  17.8 ± 0.07a,b  14.70 ± 0.09c  7.0 ± 0.04b  6.6 ± 0.11a,b  83.53 ± 1.12a,b    Sucrose Control  55.29 ± 0.13a  57.91 ± 0.24a,b  17.6 ± 0.06b  14.86 ± 0.08b,c  6.6 ± 0.06c  6.2 ± 0.12b,c  80.79 ± 1.03a,b    PM-Freezedried  55.37 ± 0.11a  58.85 ± 0.35a  17.9 ± 0.07a  15.10 ± 0.10a,b  7.0 ± 0.09b  6.8 ± 0.08a  84.25 ± 0.65a    PM-1  54.91 ± 0.14a,b  58.65 ± 0.24a  17.9 ± 0.08a  15.41 ± 0.09a  7.3 ± 0.05a  6.5 ± 0.11a–c  82.38 ± 0.88a,b    PM-5  54.17 ± 0.14c  56.72 ± 0.34c  17.6 ± 0.07b  15.02 ± 0.10b,c  7.5 ± 0.06a  6.1 ± 0.10c  80.78 ± 0.79b  Experimental trial  Yellow Control  55.03 ± 0.14a  57.96 ± 0.36a  19.0 ± 0.08a  13.95 ± 0.11a  6.2 ± 0.06a  8.7 ± 0.08  93.65 ± 0.38    Yellow Sucrose Control  54.56 ± 0.14a,b  56.93 ± 0.35a,b  18.8 ± 0.06a,b  13.75 ± 0.10a,b  6.1 ± 0.06a  8.7 ± 0.08  93.63 ± 0.39    White Control  54.12 ± 0.16b  54.38 ± 0.44c  18.3 ± 0.07d  13.07 ± 0.14c  1.0 ± 0.0c  8.8 ± 0.09  94.63 ± 0.47    White Sucrose Control  54.56 ± 0.17a,b  54.06 ± 0.39c  18.4 ± 0.06c,d  13.39 ± 0.09b,c  1.0 ± 0.0c  8.9 ± 0.09  95.22 ± 0.46    PM-Feed  54.85 ± 0.18a  55.54 ± 0.37b,c  18.6 ± 0.06b,c  13.67 ± 0.09a,b  3.7 ± 0.07b  8.8 ± 0.10  94.61 ± 0.51  Trial  Diet Groups1  Egg size (mm)  Egg Weight (g)  Yolk Height (mm)  Yolk Weight (g)  Yolk Color  Albumen Height (mm)  Haugh Unit  Pilot study  Control  54.65 ± 0.14b,c  57.23 ± 0.27b,c  17.8 ± 0.07a,b  14.70 ± 0.09c  7.0 ± 0.04b  6.6 ± 0.11a,b  83.53 ± 1.12a,b    Sucrose Control  55.29 ± 0.13a  57.91 ± 0.24a,b  17.6 ± 0.06b  14.86 ± 0.08b,c  6.6 ± 0.06c  6.2 ± 0.12b,c  80.79 ± 1.03a,b    PM-Freezedried  55.37 ± 0.11a  58.85 ± 0.35a  17.9 ± 0.07a  15.10 ± 0.10a,b  7.0 ± 0.09b  6.8 ± 0.08a  84.25 ± 0.65a    PM-1  54.91 ± 0.14a,b  58.65 ± 0.24a  17.9 ± 0.08a  15.41 ± 0.09a  7.3 ± 0.05a  6.5 ± 0.11a–c  82.38 ± 0.88a,b    PM-5  54.17 ± 0.14c  56.72 ± 0.34c  17.6 ± 0.07b  15.02 ± 0.10b,c  7.5 ± 0.06a  6.1 ± 0.10c  80.78 ± 0.79b  Experimental trial  Yellow Control  55.03 ± 0.14a  57.96 ± 0.36a  19.0 ± 0.08a  13.95 ± 0.11a  6.2 ± 0.06a  8.7 ± 0.08  93.65 ± 0.38    Yellow Sucrose Control  54.56 ± 0.14a,b  56.93 ± 0.35a,b  18.8 ± 0.06a,b  13.75 ± 0.10a,b  6.1 ± 0.06a  8.7 ± 0.08  93.63 ± 0.39    White Control  54.12 ± 0.16b  54.38 ± 0.44c  18.3 ± 0.07d  13.07 ± 0.14c  1.0 ± 0.0c  8.8 ± 0.09  94.63 ± 0.47    White Sucrose Control  54.56 ± 0.17a,b  54.06 ± 0.39c  18.4 ± 0.06c,d  13.39 ± 0.09b,c  1.0 ± 0.0c  8.9 ± 0.09  95.22 ± 0.46    PM-Feed  54.85 ± 0.18a  55.54 ± 0.37b,c  18.6 ± 0.06b,c  13.67 ± 0.09a,b  3.7 ± 0.07b  8.8 ± 0.10  94.61 ± 0.51  Data are means ± SEM for 10 and 20 replicates in the pilot study and the experimental trial, respectively. 1Diet groups are: Control, no dosage of bacteria; Sucrose, 1 mL dosage of sucrose (10% m/v); PM-Freezedried, 5 mL dosage of freeze-dried bacteria resuspended in sterile dH2O; PM-1, 1 mL dosage of bacteria; PM-5, 5 mL dosage of bacteria; Yellow Control, no dosage of bacteria; Yellow Sucrose Control, 1 mL dosage of sucrose (10% m/v); White Control, no dosage of bacteria; White Sucrose Control, 1 mL dosage of sucrose (10% m/v); PM-Feed, 50 mL dosage of bacteria. a–dLetters in the same column indicates a significant difference at a confidence level of 95%, where P < 0.05. View Large There was a significant increase (P < 0.05) in yolk color for all bacterial treatments compared to the controls, where PM-5 had the highest yolk color average of 7.5 ± 0.06 (Table 4). The yolk color increased significantly after wk 4 for PM-5 and PM-Freezedried from 7.5 and 7.0, to 8.1 and 7.8, respectively, in wk 5 (Figure 3A). There was a slight increase in yolk color for the control groups, but did not exceed the experimental treatments and seemed to stabilize after 4 wk. After completion of the experimental treatments the color of the yolk decreased for all diet groups. Figure 3. View largeDownload slide Yolk color change over (A) a 7-wk period in the pilot study and (B) an 8-wk period in the experimental trial. Data are expressed as the mean ± SEM for (A) 10 and (B) 20 replicates per diet group per wk. Diet groups are (A): Control, no dosage of bacteria; Sucrose, 1 mL dosage of sucrose (10% m/v); PM-Freezedried, 5 mL dosage of freeze-dried bacteria resuspended in sterile dH2O; PM-1, 1 mL dosage of bacteria; PM-5, 5 mL dosage of bacteria; (B): Yellow Control, no dosage of bacteria; Yellow Sucrose Control, 1 mL dosage of sucrose (10% m/v); White Control, no dosage of bacteria; White Sucrose Control, 1 mL dosage of sucrose (10% m/v); PM-Feed, 50 mL dosage of bacteria. Figure 3. View largeDownload slide Yolk color change over (A) a 7-wk period in the pilot study and (B) an 8-wk period in the experimental trial. Data are expressed as the mean ± SEM for (A) 10 and (B) 20 replicates per diet group per wk. Diet groups are (A): Control, no dosage of bacteria; Sucrose, 1 mL dosage of sucrose (10% m/v); PM-Freezedried, 5 mL dosage of freeze-dried bacteria resuspended in sterile dH2O; PM-1, 1 mL dosage of bacteria; PM-5, 5 mL dosage of bacteria; (B): Yellow Control, no dosage of bacteria; Yellow Sucrose Control, 1 mL dosage of sucrose (10% m/v); White Control, no dosage of bacteria; White Sucrose Control, 1 mL dosage of sucrose (10% m/v); PM-Feed, 50 mL dosage of bacteria. Experimental Trial There was no significant difference (P > 0.05) detected in albumen height and HU (Table 4). The yellow corn controls had heavier egg weights (57. 96 g and 56.93 g) compared to the white corn controls (54.38 g and 54.06 g). However, the PM-Feed had a significantly (P < 0.05) heavier egg weight (55.54 g) compared to the white controls. There was also a significant difference (P < 0.05) in yolk weight between the PM-Feed (13.67 g) compared to the White Control (13.07 g) and White Sucrose Control (13.39 g). PM-Feed and Yellow Sucrose Control had similar yolk weights (13.67 g and 13.75 g, respectively), but the Yellow Control had a significantly heavier yolk weight of 13.95 g. In terms of yolk color, there was a significant difference (P < 0.05) between the yellow corn diet groups compared to the white corn diet groups. A significant difference was also observed between the PM-Feed (3.69) compared to the White Control (1.00) and White Sucrose Control (1.00) (Table 4). Three wk after starting with the experimental treatments the yolk color for the yellow control groups stabilized (Figure 3B). The yolk color for PM-Feed increased significantly after 4 wk to 2.58 and stabilized after 7 wk at 3.78. Detection of Bacteria in the Egg Content None of the potential food pathogens (E. coli, Listeria, and Salmonella) were detected in any of the egg contents plated out. There were also no bacterial colonies typical of Paracoccus marcusii on any of the nutrient agar plates. All the plates were clear at the lowest dilution of 10−1. DISCUSSION The increase in demand for the use of feed additives and colorants in poultry farming to enhance egg yolk color has prompted the application of using a carotenoid producing bacterium. For most consumers, the color of food indicates the quality and freshness of the product (Clydesdale, 1993). Carotenoids have long been used as feed additives to generate good quality food products that meet the demands of the consumer and also hold a health benefit for the animal (Breithaupt, 2007). Plants, algae, fungi and bacteria produce carotenoids, but only a few are of industrial importance (Ambati et al., 2014). Some microorganisms can be used to produce carotenoids economically. One such microorganism, Paracoccus marcusii, produces astaxanthin naturally (Harker et al., 1998). In a previous study, De Bruyn (2013) has shown that P. marcusii can be used as an additive for fish to enhance skin pigmentation. This same principle can be applied in poultry feed to enhance egg yolk color. Hens are not able to synthesize carotenoids and need to consume pigments through their feed (Surai et al., 2001; Bortolotti et al., 2003). In this study we aimed to evaluate the potential whole cell application of P. marcusii to be used as a feed additive for laying hens to enhance egg yolk color. This study has shown that P. marcusii has the potential to be used in the poultry industry as a feed additive. In all of the feeding trials, there were no negative effect on the weight of the hen, the egg production or the overall quality of the egg observed. In some cases, the experimental groups performed better than the control groups, i.e., egg production, egg weight, yolk weight, and color. There seems to be a significant increase in egg production between all the experimental diets compared to their control. In the experimental trial, the white control diets had a significantly lower (P < 0.05) egg production than the yellow control diets and the experimental diet. It is suspected that the white corn used in this study might not have provided enough energy to the hen to produce eggs on a daily basis. Some studies have indicated that a difference in protein, sugar, and starch content between corn varieties can directly influence the layer hens’ performance (Moore, 2007). White and yellow corn are believed to be similar in nutritional composition. However, composition can be affected by corn hybrid type, geographical growing site, harvesting maturity, plant density, and soil nitrogen fertilization (Zeidan et al., 2006; Moore, 2007; Idikut et al., 2009; Raymond et al., 2009). The added bacterium or sucrose might have provided additional energy and, therefore, they performed significantly better than the white control diets. Even in the pilot study where all hens were fed a commercially available feed containing yellow corn, all the experimental diets performed significantly better than the control. These findings are in contrast to a previous study. Walker et al. (2012) fed various concentrations of alga biomass to laying hens to determine the effect on the quality of the egg and yolk color change. They found that the added alga biomass had no effect on the egg production of the hen and all diet groups had a mean egg production above 90% (Walker et al., 2012). There is a definite increase in yolk weight and whole egg weight in all feeding trials where the experimental diet had heavier eggs compared to the control groups. There seems to be no correlation between egg weight and yolk weight. Some diet groups had a lighter egg weight compared to the controls, but had a heavier yolk weight. This can indicate that even if the whole egg is not heavier, the yolk weight increased with dosage of P. marcusii. However, these findings are in contrast to previously reported studies. Some studies have shown that by adding probiotics, antibiotics or bacterial enzymes, such as xylanase, to the feed of hens had no effect on any of the quality parameters, including whole egg and yolk weight (Yörük and Bolat, 2003; Yörük et al., 2004; Mahdavi et al., 2005; Yang et al., 2006). The HU values were not significantly different between the diet groups in the experimental trials. These results are in agreement with previous studies. The supplementation of vitamins C and E (astaxanthin precursors) or alga biomass had no undesirable effect on the HU of different experimental treatments (Franchini et al., 2002; Walker et al., 2012). After the pilot trial, it was clear that a diet free of all pigments was needed to effectively evaluate the pigmentation effect of P. marcusii. In the experimental trial, there was a significant increase in yolk color compared to the white control. A higher dosage of P. marcusii resulted in a higher yolk color change. The intestinal cells of the hen easily absorb natural sources of carotenoids. These pigments are transported to the yolk once it is released from the feed content (Surai et al., 2001; Bortolotti et al., 2003). Different carotenoids have different deposition rates in eggs because of the bioavailability of esterified or free forms of carotenoids (Bowen et al., 2002). To be able to compete with a yellow corn diet a higher dosage of P. marcusii is needed. However, this was not possible in this study because of culturing limitations. The egg quality results indicate that whole P. marcusii cells can be used as a pigmentation source without the need for downstream processing to break the cells. However, higher cell concentrations are still needed to increase yolk color. Some well-known food pathogens associated with chicken egg products are E. coli, Listeria and Salmonella (Gast, 1992). It was, therefore, necessary to determine if these organisms are present in the egg contents. None of the dilution plates had any growth on them. It is possible that the microorganisms were not viable anymore or the colony forming units were too low to detect. However, we did not expect to find any of the pathogens or P. marcusii in the egg content, as previous studies have shown that the internal egg only gets contaminated when it comes into contact with the outer shell where trace amounts of the pathogens might be present and if the pathogens are present in the immediate environment of the hen (De Reu et al., 2005; Mallet et al., 2006; Jones et al., 2011). None of the hens in all the trials were sick and the way the cages are designed prevents the egg from coming into contact with fecal matter on the floor of the house that might contain these pathogens (De Reu et al., 2005; Mallet et al., 2006; Svobodová and Tůmová, 2014). The surroundings of the hen must be kept clean to prevent the potential contamination of food pathogens. The findings in this study demonstrate the potential use of Paracoccus marcusii as a feed additive to enhance yolk color. Paracoccus marcusii significantly increased the yolk color in the experimental trial compared to the white corn diet groups and there is also an increase in whole egg and yolk weight. There was no negative effect on the overall egg quality. Paracoccus marcusii can, therefore, be used as a feed additive to enhance yolk color in laying hens. It is important for future studies to determine the optimum dosage needed and the type of association between the bacterium and the hen. ACKNOWLEDGEMENTS The authors would like to thank Mariendahl Experimental Farm, University of Stellenbosch, for the use of their hen house and the help from the staff. This study was financially supported by the National Research Foundation (NRF), South Africa. REFERENCES Adams C. A. 1985. 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Application of Paracoccus marcusii as a potential feed additive for laying hens

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Abstract

Abstract Carotenoids have been used for many years as an added pigment to enhance egg yolk color. One such carotenoid, astaxanthin, has a strong antioxidant activity and is produced by several microorganisms, including the bacterium Paracoccus marcusii, which has shown promise to be used as a feed additive. Therefore, this study investigated the use of P. marcusii as a possible source of pigmentation in layer hen feed to enhance egg yolk color. Paracoccus marcusii was fed to hens in a sucrose solution (10% m/v). The hens were fed daily and all eggs were collected for analysis. Dilutions of egg contents were plated onto selective media to detect the presence of known food pathogens (E. coli, Listeria, and Salmonella). In the feeding trial, there was no negative effect on hen body weight, egg production, or overall egg quality. There was a significant increase (P < 0.05) in yolk color as well as an increase in whole egg and yolk weight. There were also no known food pathogens detected in any of the egg samples. This study has shown promising results in using this bacterium as an effective feed additive for laying hens. INTRODUCTION The chicken (Gallus gallus domesticus) is a domesticated subspecies of the red junglefowl (Gallus gallus). Chickens have become one of the most common domesticated animals and are mainly kept as a source of food for meat and eggs. In 2015, the South African layer flock size was 24.9 million hens and has since increased to 25.05 million hens in 2016 (South African Poultry Association, 2016). Chicken eggs remain one of the largest animal product sectors in the South African agriculture after beef, chicken meat, and milk with a gross turnover of R9.83 billion ($760 million) in 2015 (Department of Agriculture, Forestry and Fisheries (DAFF), South Africa, 2016). For many years carotenoids have been used to manipulate the color of egg yolk to obtain a desired color (Adams, 1985). Poultry can easily absorb carotenoids from their diet (Hudon, 1994). After the hen has ingested the feed, the carotenoids are released by enzymes and absorbed in the small intestines. The free carotenoids are then emulsified to form oil droplets (or portomicrons) and delivered to the liver. These molecules are incorporated into very low density lipoproteins (VLDL) and delivered to the yolk (Surai et al., 2001; Bortolotti et al., 2003). Astaxanthin produced by microorganisms have been commercialized and applied in the pigmentation of cosmetics, beverages, dairy products, and meats (Del Campo et al., 2000; Guerin et al., 2003; Liang et al., 2004; Pulz and Gross, 2004; Chandi and Gill, 2011). There is an increase in demand for naturally derived astaxanthin from microorganisms instead of synthetic astaxanthin, since natural astaxanthin has a higher antioxidant activity when compared to synthetic astaxanthin (Capelli et al., 2013). The yeast, Xanthophyllomyces dendrorhous, and microalga, Haematococcus pluvialis, are currently used for the large-scale cultivation for astaxanthin production. Many studies have used these microorganisms in developing biotechnological processes to produce astaxanthin in large quantities (Lorenz and Cysewski, 2000; Dufosse et al., 2005; Schmidt et al., 2011; Mata-Gómez et al., 2014). In previous studies, whole yeast and microalga cells have been used as a pigmentation source in the diet of trout (Choubert and Heinrich, 1993; Storebakken et al., 2004), salmon (Lorenz and Cysewski, 2000) and laying hens (Johnson et al., 2003). However, yeast and microalga have thicker cell walls compared to bacteria. Therefore, a significant increase in pigmentation was only observed after the partial homogenization, enzymatic digestion or cracking of the cells to increase the release of the available pigments in the digestive tract (Choubert and Heinrich, 1993; Lorenz and Cysewski, 2000; Johnson et al., 2003; Storebakken et al., 2004). However, there was no need to enzymatically digest or homogenize the bacterium, Paracoccus marcusii, when fed to rainbow trout (Oncorhynchus mykiss) for pigmentation effect (De Bruyn, 2013). The results obtained by De Bruyn (2013) showed the promising application of using the whole P. marcusii cell as a pigmentation source, instead of the yeast or microalga. The aim of this study was, therefore, to determine if whole P. marcusii cells can be used as a possible pigmentation source to enhance egg yolk color and possibly improve egg quality without the need to extract the pigment from the bacterial cell. MATERIALS AND METHODS Experimental Layout, Hens and Feed All feeding trials took place at the poultry unit of Mariendahl Experimental Farm, University of Stellenbosch. The facility consisted of a hen house equipped with a 2-tier A-shape battery system. Each row contained 12 individual cages with sliding doors, slightly slanted floors which allowed the eggs to roll out of the cage for easy collection and a feeding tray in front of the cage. All hens had free access to feed and water was provided ad libitum by 2 nipple-type drinkers per cage. Layer hens (Lohmann Brown) were purchased from Quantum Foods, South Africa. Animal experiments were performed based on the Care and Use of Animals for Scientific Purposes of the University of Stellenbosch [SU-ACUM14–0034 (Pilot study) and SU-ACUD15–00088 (Experimental trial)]. Pilot Study A total of 50 hens of 36 wk of age were selected. Ten hens were randomly assigned into a feeding group and caged individually. The hens were allowed to adjust to their new surroundings for 4 d before starting with the experimental diets. After 5 wk of the experimental treatments, the hens were fed a basal diet for 2 wk to determine if the change in pigmentation was because of the bacterium. The basal feed consisted of commercially available layer hen feed (Agrimark, Stellenbosch, South Africa). Experimental Trial A total of 100 hens of 16 wk of age were selected. Twenty hens were randomly assigned into a group and caged individually. For the first 4 wk, all hens were fed pre-lay feed containing white corn before starting with the experimental diets for 8 wk on peaking feed containing either yellow corn or white corn (Table 1). Table 1. Basal feed composition for the experimental trial. Ingredient  Prelay1  Peaking2    %  %  Corn (White or yellow)3  65,806  63,338  Soybean Full Fat  7,088  9,479  Soybean 46  18,790  18,790  DL Methionine  0,109  0,203  L-Threonine  -  0,001  Vitamin and Mineral Premix  0,150  0,150  Limestone  5,975  5,961  Salt (NaCl)  0,262  0,263  Monocalcium Phosphate  1,596  1,593  Sodium bicarbonate  0,225  0,222  Ingredient  Prelay1  Peaking2    %  %  Corn (White or yellow)3  65,806  63,338  Soybean Full Fat  7,088  9,479  Soybean 46  18,790  18,790  DL Methionine  0,109  0,203  L-Threonine  -  0,001  Vitamin and Mineral Premix  0,150  0,150  Limestone  5,975  5,961  Salt (NaCl)  0,262  0,263  Monocalcium Phosphate  1,596  1,593  Sodium bicarbonate  0,225  0,222  1Hens were fed prelay feed containing white corn for the first 4 wk. 2Hens were fed peaking feed containing either white corn or yellow corn for 8 wk, after the prelay feed. 3Only hens in the white control and treatment diet groups were fed feed containing white corn. Only hens in the yellow control diet groups were fed feed containing yellow corn. View Large Experimental Treatments All dosages given every day at each trial is indicated in Table 2. Dosages were given by hand every day in liquid form to ensure that the hens were fed equal amounts of the control or the bacterium. Table 2. Diet groups for all feeding trials and their dosages. Feeding Trial  Diet group name  Dosage  Pilot study  Control  None    Sucrose  1 mL sucrose (10% m/v)    PM-Freezedried  5 mL freeze-dried cells1    PM-1  1 mL live cells2    PM-5  5 mL live cells2  Experimental trial  Yellow Control  None    Yellow Sucrose Control  1 mL sucrose (10% m/v)    White Control  None    White Sucrose Control  1 mL sucrose (10% m/v)    PM-Feed  50 mL live cells2  Feeding Trial  Diet group name  Dosage  Pilot study  Control  None    Sucrose  1 mL sucrose (10% m/v)    PM-Freezedried  5 mL freeze-dried cells1    PM-1  1 mL live cells2    PM-5  5 mL live cells2  Experimental trial  Yellow Control  None    Yellow Sucrose Control  1 mL sucrose (10% m/v)    White Control  None    White Sucrose Control  1 mL sucrose (10% m/v)    PM-Feed  50 mL live cells2  1Cells were lyophilized in sucrose (10% m/v) and resuspended in sterile dH2O before feeding. 2All bacterial dosages contained 2.8 × 109 CFU/mL of Paracoccus marcusii. View Large Pilot Study Five different diets were prepared and randomly assigned to each group of 10 hens. Treatment 1 and 2 served as negative controls which consisted of a basal feed with no additives (Control) and 1 mL sucrose solution (10% m/v) (Sucrose Control). Treatments 3–5 consisted of a basal feed and a dosage of Paracoccus marcusii, either freeze-dried (PM-Freeze-dried) and resuspended in sterile dH2O, or live cells (PM-1 and PM-5), which were dripped onto the feed. Experimental Trial To effectively determine the pigmentation effect of the bacterium to enhance egg yolk color, all experimental diet groups were fed a diet containing white corn, except for the positive control diet groups which were fed a diet containing yellow corn. Five different diets were prepared and randomly assigned to each group of 20 hens. Treatments 1–4 served as the control groups (Yellow Control, Yellow Sucrose Control, White Control and White Sucrose Control). For treatment 5 (PM-Feed), a sucrose solution containing the bacterium, P. marcusii, was dripped onto the feed. Preparation of Paracoccus marcusii In preparation for the feeding trials, P. marcusii was cultured in 2 L Schott bottles containing 1 L of specialised medium (10 g/L bacteriological peptone (Oxoid, Hampshire, United Kingdom), 5 g/L yeast extract (Biolab, Modderfontein, South Africa), 3% NaCl and pH 7–8). Schott bottles were incubated at 26°C for 7 d. The cells were harvested through centrifugation at 10,000 rpm for 10 min and washed once with sterile dH2O. New cells were cultured each wk to ensure viability of cells. Pilot Study Cells were resuspended in equal volumes of sucrose (10% m/v). Aliquots of cells were transferred into separate flasks representing the different diet groups (PM-1 and PM-5). For freeze-drying, 50 mL of cells resuspended in sucrose (10% m/v) were frozen overnight in 250 mL Erlenmeyer flasks at –80°C. The VirTis benchtop K (model: 6KBTEL-85, SP Scientific, Gardiner, NY) was used to freeze-dry the cells until completely dry. Experimental Trial Cells were daily harvested from 1 L of growth medium and resuspended in 20 mL of sucrose (10% m/v) to obtain a high concentration dosage of P. marcusii. Cell concentration fed to each hen was 2.8 × 109 colony forming unit (CFU) per mL. Quantitative Data Measurements Hen Weight and Egg Production The initial and final mean weight was measured for all diet groups in all feeding trials. The egg production rate of each group was calculated using the total number of eggs laid over the duration of the trial:   \begin{eqnarray} &&{ Laying\ rate\ \left( \% \right)}\nonumber\\ && = \left( {\frac{{Total\ number\ of\ eggs\ laid}}{{Number\ of\ hens\, \times Number\ of\ days}}} \right)\ \times \ 100\nonumber\\ \end{eqnarray} (1) Egg Quality Analysis Eggs were collected every d following the 4-d acclimation period. Daily measurements included the following: whole egg weight, shell thickness, shell weight, yolk weight, yolk height, yolk color, and thick albumen height. The Haugh Unit (HU) was calculated using the formula:   \begin{equation}HU = {\rm{\ }}100\log \left( {{\rm{h}} - 1.7{w^{0.37}} + 7.6} \right)\end{equation} (2)where h is the height of the albumen and w is the whole egg weight (Haugh, 1937). The shell thickness was measured using an electronic digital caliper. The height of the albumen and yolk was measured by using a Haugh meter. For measuring the color of the yolk, the yolk was first separated from the albumen and placed into a plastic petri dish (90 mm × 15 mm). The color was then measured using a yolk color fan (YolkFanTM, DSM, Heerlen, Netherlands). The yolk color fan has a blade range between 1 and 15, where 1 is the palest and 15 the darkest. All measurements were taken by placing the petri dish with the yolk on a white background and holding the blades above the yolk. The corresponding blade number was then allocated to each individual yolk. Detection of Bacteria in the Egg Content Eggs were randomly selected during the trials from all diet groups and tested for the presence of potential food pathogens (Salmonella, Listeria, and E. coli) (Gast, 1992), as well as P. marcusii, in the egg content. The surface of the egg was sterilized by rolling the whole egg in 70% ethanol. The internal egg content was separated from the shell and homogenized in a plastic petri dish (90 mm × 15 mm) and a dilution series of 10−1–10−5 was prepared in sterile saline solution (0.9% NaCl). The dilutions were plated in triplicate onto PALCAM-Listeria-selective agar (3 g/L yeast extract, 23 g/L peptone, 5 g/L NaCl, 1 g/L starch, 10 g/L mannitol, 0.8 g/L aesculin, 0.5 g/L glucose, 0.5 g/L ammonium iron(III) citrate, 0.08 g/L phenol-rot, 15 g/L lithiumchloride and 13 g/L agar) (Merck, Darmstadt, Germany), SS agar (10 g/L lactose, 10 g/L peptone, 10 g/L NaCl, 8.5 g/L ox bile dried, 1 g/L ammonium iron(III) citrate, 8.5 g/L sodium thiosulfate, 0.025 g/L neutral red, 0.0003 g/L brilliant green and 12 g/L agar) (Merck, Darmstadt, Germany), LEVINE-EMB-agar (10 g/L lactose, 10 g/L peptone, 0.4 g/L eosin yellow, 0.065 g/L methylene blue, 2 g/L di-potassium hydrogen phosphate and 13.5 g/L agar) (Merck, Darmstadt, Germany) and nutrient agar (2 g/L yeast extract, 5 g/L peptone, 1 g/L meat extract, 8 g/L NaCl and 15 g/L agar) (Biolab, Modderfontein, South Africa). The Listeria, EMB, and SS agar plates were incubated for 2–3 d at 37°C and the nutrient agar plates were incubated for 4 to 5 d at 26°C. Statistical Analysis Statistical analysis was performed using Statistica (version 13.0, Statsoft Inc., Palo Alto, CA). One-way analysis of variance (ANOVA) was used for mean comparisons and Tukey's honest significant difference (HSD) was calculated where P < 0.05. RESULTS Hen Weight Pilot Study The average body weight at the start of the trial was 1.77 ± 0.02 kg between all diet groups. There was no significant increase (P > 0.05) between all diet groups at the end of the trial (Table 3). Table 3. Mean body weight at the start and end of the pilot study. Diet groups1  Weight before (kg)  Weight after (kg)  Control  1.70 ± 0.02  1.76 ± 0.02  Sucrose Control  1.74 ± 0.02  1.77 ± 0.02  PM-Freezedried  1.81 ± 0.02  1.80 ± 0.03  PM-1  1.79 ± 0.02  1.87 ± 0.02  PM-5  1.82 ± 0.03  1.82 ± 0.03  P value  0.44  0.60  Diet groups1  Weight before (kg)  Weight after (kg)  Control  1.70 ± 0.02  1.76 ± 0.02  Sucrose Control  1.74 ± 0.02  1.77 ± 0.02  PM-Freezedried  1.81 ± 0.02  1.80 ± 0.03  PM-1  1.79 ± 0.02  1.87 ± 0.02  PM-5  1.82 ± 0.03  1.82 ± 0.03  P value  0.44  0.60  Data are means ± SEM for 10 replicates per diet group. 1Diet groups are: Control, no dosage of bacteria; Sucrose, 1 mL dosage of sucrose (10% m/v); PM-Freezedried, 5 mL dosage of freeze-dried bacteria resuspended in sterile dH2O; PM-1, 1 mL dosage of bacteria; PM-5, 5 mL dosage of bacteria. View Large Experimental Trial Based on the results of the pilot study, the experimental trial was started with younger hens. Treatments were applied to hens fed on the white corn to minimize the pigmentation effect of the yellow corn. At the start of the experimental trial, the mean body weight was 1.45 ± 0.01 kg between the different diet groups. There was no significant difference (P > 0.05) in hen body weight at the start of the trial. The increase in body weight of the hens were consistent at the end of the trial with a significant difference (P < 0.05) only observed between the different corn groups (white or yellow corn). The yellow corn groups had an end mean weight of 1.97 kg compared to the white corn groups with 1.79 kg (Figure 1). Figure 1. View largeDownload slide Mean body weight of the hens at the start (black) and the end (white) of the experimental trial. Data are expressed as the mean ± SEM for 20 replicates per diet group. Diet groups are: Yellow Control, no dosage of bacteria; Yellow Sucrose Control, 1 mL dosage of sucrose (10% m/v); White Control, no dosage of bacteria; White Sucrose Control, 1 mL dosage of sucrose (10% m/v); PM-Feed, 50 mL dosage of bacteria. a-bLetters above bars indicates a significant difference at a confidence level of 95%, where P < 0.05. Figure 1. View largeDownload slide Mean body weight of the hens at the start (black) and the end (white) of the experimental trial. Data are expressed as the mean ± SEM for 20 replicates per diet group. Diet groups are: Yellow Control, no dosage of bacteria; Yellow Sucrose Control, 1 mL dosage of sucrose (10% m/v); White Control, no dosage of bacteria; White Sucrose Control, 1 mL dosage of sucrose (10% m/v); PM-Feed, 50 mL dosage of bacteria. a-bLetters above bars indicates a significant difference at a confidence level of 95%, where P < 0.05. Egg Production Pilot Study There was a significant difference (P < 0.05) detected in the hen egg production between the Sucrose Control, PM-Freezedried, PM-1 and PM-5 groups compared to the Control group. The Control group had the lowest egg production of 89.35%, followed by PM-Freezedried and PM-5 with 94.52%, Sucrose Control with 96.09% and PM-1 with 96.45% (Figure 2A). Figure 2. View largeDownload slide Mean egg production for (A) pilot study and (B) experimental trial. Data are expressed as the mean ± SEM for (A) 10 and (B) 20 replicates per diet group. Diet groups are (A): Control, no dosage of bacteria; Sucrose, 1 mL dosage of sucrose (10% m/v); PM-Freezedried, 5 mL dosage of freeze-dried bacteria resuspended in sterile dH2O; PM-1, 1 mL dosage of bacteria; PM-5, 5 mL dosage of bacteria; (B) Yellow Control, no dosage of bacteria; Yellow Sucrose Control, 1 mL dosage of sucrose (10% m/v); White Control, no dosage of bacteria; White Sucrose Control, 1 mL dosage of sucrose (10% m/v); PM-Feed, 50 mL dosage of bacteria. a-cLetters above bars indicates a significant difference at a confidence level of 95%, where P < 0.05. Figure 2. View largeDownload slide Mean egg production for (A) pilot study and (B) experimental trial. Data are expressed as the mean ± SEM for (A) 10 and (B) 20 replicates per diet group. Diet groups are (A): Control, no dosage of bacteria; Sucrose, 1 mL dosage of sucrose (10% m/v); PM-Freezedried, 5 mL dosage of freeze-dried bacteria resuspended in sterile dH2O; PM-1, 1 mL dosage of bacteria; PM-5, 5 mL dosage of bacteria; (B) Yellow Control, no dosage of bacteria; Yellow Sucrose Control, 1 mL dosage of sucrose (10% m/v); White Control, no dosage of bacteria; White Sucrose Control, 1 mL dosage of sucrose (10% m/v); PM-Feed, 50 mL dosage of bacteria. a-cLetters above bars indicates a significant difference at a confidence level of 95%, where P < 0.05. Experimental Trial A significant difference (P < 0.05) was detected between the Yellow Control, Yellow Sucrose Control, White Sucrose Control and PM-Feed compared to the White Control. The White Control group had the lowest egg production of 85.06%, followed by PM-Feed with 92.38%, White Sucrose Control with 92.59%, Yellow Control with 95.21% and Yellow Sucrose Control with 96.40% (Figure 2B). Egg Quality Pilot Study There was a significant difference (P < 0.05) detected for all egg quality parameters measured (Table 4). PM-Freezedried and PM-1 had heavier egg weights (58.85 g and 58.65 g, respectively) compared to the control groups (Control and Sucrose Control) and PM-5 (57.23 g, 57.91 g and 56.72 g, respectively). PM-Freeze-dried (15.10 g), PM-1 (15.41 g) and PM-5 (15.02 g) had heavier yolk weights compared to the Control (14.70 g) and Sucrose Control (14.86). Table 4. Egg quality parameters of all feeding trials. Trial  Diet Groups1  Egg size (mm)  Egg Weight (g)  Yolk Height (mm)  Yolk Weight (g)  Yolk Color  Albumen Height (mm)  Haugh Unit  Pilot study  Control  54.65 ± 0.14b,c  57.23 ± 0.27b,c  17.8 ± 0.07a,b  14.70 ± 0.09c  7.0 ± 0.04b  6.6 ± 0.11a,b  83.53 ± 1.12a,b    Sucrose Control  55.29 ± 0.13a  57.91 ± 0.24a,b  17.6 ± 0.06b  14.86 ± 0.08b,c  6.6 ± 0.06c  6.2 ± 0.12b,c  80.79 ± 1.03a,b    PM-Freezedried  55.37 ± 0.11a  58.85 ± 0.35a  17.9 ± 0.07a  15.10 ± 0.10a,b  7.0 ± 0.09b  6.8 ± 0.08a  84.25 ± 0.65a    PM-1  54.91 ± 0.14a,b  58.65 ± 0.24a  17.9 ± 0.08a  15.41 ± 0.09a  7.3 ± 0.05a  6.5 ± 0.11a–c  82.38 ± 0.88a,b    PM-5  54.17 ± 0.14c  56.72 ± 0.34c  17.6 ± 0.07b  15.02 ± 0.10b,c  7.5 ± 0.06a  6.1 ± 0.10c  80.78 ± 0.79b  Experimental trial  Yellow Control  55.03 ± 0.14a  57.96 ± 0.36a  19.0 ± 0.08a  13.95 ± 0.11a  6.2 ± 0.06a  8.7 ± 0.08  93.65 ± 0.38    Yellow Sucrose Control  54.56 ± 0.14a,b  56.93 ± 0.35a,b  18.8 ± 0.06a,b  13.75 ± 0.10a,b  6.1 ± 0.06a  8.7 ± 0.08  93.63 ± 0.39    White Control  54.12 ± 0.16b  54.38 ± 0.44c  18.3 ± 0.07d  13.07 ± 0.14c  1.0 ± 0.0c  8.8 ± 0.09  94.63 ± 0.47    White Sucrose Control  54.56 ± 0.17a,b  54.06 ± 0.39c  18.4 ± 0.06c,d  13.39 ± 0.09b,c  1.0 ± 0.0c  8.9 ± 0.09  95.22 ± 0.46    PM-Feed  54.85 ± 0.18a  55.54 ± 0.37b,c  18.6 ± 0.06b,c  13.67 ± 0.09a,b  3.7 ± 0.07b  8.8 ± 0.10  94.61 ± 0.51  Trial  Diet Groups1  Egg size (mm)  Egg Weight (g)  Yolk Height (mm)  Yolk Weight (g)  Yolk Color  Albumen Height (mm)  Haugh Unit  Pilot study  Control  54.65 ± 0.14b,c  57.23 ± 0.27b,c  17.8 ± 0.07a,b  14.70 ± 0.09c  7.0 ± 0.04b  6.6 ± 0.11a,b  83.53 ± 1.12a,b    Sucrose Control  55.29 ± 0.13a  57.91 ± 0.24a,b  17.6 ± 0.06b  14.86 ± 0.08b,c  6.6 ± 0.06c  6.2 ± 0.12b,c  80.79 ± 1.03a,b    PM-Freezedried  55.37 ± 0.11a  58.85 ± 0.35a  17.9 ± 0.07a  15.10 ± 0.10a,b  7.0 ± 0.09b  6.8 ± 0.08a  84.25 ± 0.65a    PM-1  54.91 ± 0.14a,b  58.65 ± 0.24a  17.9 ± 0.08a  15.41 ± 0.09a  7.3 ± 0.05a  6.5 ± 0.11a–c  82.38 ± 0.88a,b    PM-5  54.17 ± 0.14c  56.72 ± 0.34c  17.6 ± 0.07b  15.02 ± 0.10b,c  7.5 ± 0.06a  6.1 ± 0.10c  80.78 ± 0.79b  Experimental trial  Yellow Control  55.03 ± 0.14a  57.96 ± 0.36a  19.0 ± 0.08a  13.95 ± 0.11a  6.2 ± 0.06a  8.7 ± 0.08  93.65 ± 0.38    Yellow Sucrose Control  54.56 ± 0.14a,b  56.93 ± 0.35a,b  18.8 ± 0.06a,b  13.75 ± 0.10a,b  6.1 ± 0.06a  8.7 ± 0.08  93.63 ± 0.39    White Control  54.12 ± 0.16b  54.38 ± 0.44c  18.3 ± 0.07d  13.07 ± 0.14c  1.0 ± 0.0c  8.8 ± 0.09  94.63 ± 0.47    White Sucrose Control  54.56 ± 0.17a,b  54.06 ± 0.39c  18.4 ± 0.06c,d  13.39 ± 0.09b,c  1.0 ± 0.0c  8.9 ± 0.09  95.22 ± 0.46    PM-Feed  54.85 ± 0.18a  55.54 ± 0.37b,c  18.6 ± 0.06b,c  13.67 ± 0.09a,b  3.7 ± 0.07b  8.8 ± 0.10  94.61 ± 0.51  Data are means ± SEM for 10 and 20 replicates in the pilot study and the experimental trial, respectively. 1Diet groups are: Control, no dosage of bacteria; Sucrose, 1 mL dosage of sucrose (10% m/v); PM-Freezedried, 5 mL dosage of freeze-dried bacteria resuspended in sterile dH2O; PM-1, 1 mL dosage of bacteria; PM-5, 5 mL dosage of bacteria; Yellow Control, no dosage of bacteria; Yellow Sucrose Control, 1 mL dosage of sucrose (10% m/v); White Control, no dosage of bacteria; White Sucrose Control, 1 mL dosage of sucrose (10% m/v); PM-Feed, 50 mL dosage of bacteria. a–dLetters in the same column indicates a significant difference at a confidence level of 95%, where P < 0.05. View Large There was a significant increase (P < 0.05) in yolk color for all bacterial treatments compared to the controls, where PM-5 had the highest yolk color average of 7.5 ± 0.06 (Table 4). The yolk color increased significantly after wk 4 for PM-5 and PM-Freezedried from 7.5 and 7.0, to 8.1 and 7.8, respectively, in wk 5 (Figure 3A). There was a slight increase in yolk color for the control groups, but did not exceed the experimental treatments and seemed to stabilize after 4 wk. After completion of the experimental treatments the color of the yolk decreased for all diet groups. Figure 3. View largeDownload slide Yolk color change over (A) a 7-wk period in the pilot study and (B) an 8-wk period in the experimental trial. Data are expressed as the mean ± SEM for (A) 10 and (B) 20 replicates per diet group per wk. Diet groups are (A): Control, no dosage of bacteria; Sucrose, 1 mL dosage of sucrose (10% m/v); PM-Freezedried, 5 mL dosage of freeze-dried bacteria resuspended in sterile dH2O; PM-1, 1 mL dosage of bacteria; PM-5, 5 mL dosage of bacteria; (B): Yellow Control, no dosage of bacteria; Yellow Sucrose Control, 1 mL dosage of sucrose (10% m/v); White Control, no dosage of bacteria; White Sucrose Control, 1 mL dosage of sucrose (10% m/v); PM-Feed, 50 mL dosage of bacteria. Figure 3. View largeDownload slide Yolk color change over (A) a 7-wk period in the pilot study and (B) an 8-wk period in the experimental trial. Data are expressed as the mean ± SEM for (A) 10 and (B) 20 replicates per diet group per wk. Diet groups are (A): Control, no dosage of bacteria; Sucrose, 1 mL dosage of sucrose (10% m/v); PM-Freezedried, 5 mL dosage of freeze-dried bacteria resuspended in sterile dH2O; PM-1, 1 mL dosage of bacteria; PM-5, 5 mL dosage of bacteria; (B): Yellow Control, no dosage of bacteria; Yellow Sucrose Control, 1 mL dosage of sucrose (10% m/v); White Control, no dosage of bacteria; White Sucrose Control, 1 mL dosage of sucrose (10% m/v); PM-Feed, 50 mL dosage of bacteria. Experimental Trial There was no significant difference (P > 0.05) detected in albumen height and HU (Table 4). The yellow corn controls had heavier egg weights (57. 96 g and 56.93 g) compared to the white corn controls (54.38 g and 54.06 g). However, the PM-Feed had a significantly (P < 0.05) heavier egg weight (55.54 g) compared to the white controls. There was also a significant difference (P < 0.05) in yolk weight between the PM-Feed (13.67 g) compared to the White Control (13.07 g) and White Sucrose Control (13.39 g). PM-Feed and Yellow Sucrose Control had similar yolk weights (13.67 g and 13.75 g, respectively), but the Yellow Control had a significantly heavier yolk weight of 13.95 g. In terms of yolk color, there was a significant difference (P < 0.05) between the yellow corn diet groups compared to the white corn diet groups. A significant difference was also observed between the PM-Feed (3.69) compared to the White Control (1.00) and White Sucrose Control (1.00) (Table 4). Three wk after starting with the experimental treatments the yolk color for the yellow control groups stabilized (Figure 3B). The yolk color for PM-Feed increased significantly after 4 wk to 2.58 and stabilized after 7 wk at 3.78. Detection of Bacteria in the Egg Content None of the potential food pathogens (E. coli, Listeria, and Salmonella) were detected in any of the egg contents plated out. There were also no bacterial colonies typical of Paracoccus marcusii on any of the nutrient agar plates. All the plates were clear at the lowest dilution of 10−1. DISCUSSION The increase in demand for the use of feed additives and colorants in poultry farming to enhance egg yolk color has prompted the application of using a carotenoid producing bacterium. For most consumers, the color of food indicates the quality and freshness of the product (Clydesdale, 1993). Carotenoids have long been used as feed additives to generate good quality food products that meet the demands of the consumer and also hold a health benefit for the animal (Breithaupt, 2007). Plants, algae, fungi and bacteria produce carotenoids, but only a few are of industrial importance (Ambati et al., 2014). Some microorganisms can be used to produce carotenoids economically. One such microorganism, Paracoccus marcusii, produces astaxanthin naturally (Harker et al., 1998). In a previous study, De Bruyn (2013) has shown that P. marcusii can be used as an additive for fish to enhance skin pigmentation. This same principle can be applied in poultry feed to enhance egg yolk color. Hens are not able to synthesize carotenoids and need to consume pigments through their feed (Surai et al., 2001; Bortolotti et al., 2003). In this study we aimed to evaluate the potential whole cell application of P. marcusii to be used as a feed additive for laying hens to enhance egg yolk color. This study has shown that P. marcusii has the potential to be used in the poultry industry as a feed additive. In all of the feeding trials, there were no negative effect on the weight of the hen, the egg production or the overall quality of the egg observed. In some cases, the experimental groups performed better than the control groups, i.e., egg production, egg weight, yolk weight, and color. There seems to be a significant increase in egg production between all the experimental diets compared to their control. In the experimental trial, the white control diets had a significantly lower (P < 0.05) egg production than the yellow control diets and the experimental diet. It is suspected that the white corn used in this study might not have provided enough energy to the hen to produce eggs on a daily basis. Some studies have indicated that a difference in protein, sugar, and starch content between corn varieties can directly influence the layer hens’ performance (Moore, 2007). White and yellow corn are believed to be similar in nutritional composition. However, composition can be affected by corn hybrid type, geographical growing site, harvesting maturity, plant density, and soil nitrogen fertilization (Zeidan et al., 2006; Moore, 2007; Idikut et al., 2009; Raymond et al., 2009). The added bacterium or sucrose might have provided additional energy and, therefore, they performed significantly better than the white control diets. Even in the pilot study where all hens were fed a commercially available feed containing yellow corn, all the experimental diets performed significantly better than the control. These findings are in contrast to a previous study. Walker et al. (2012) fed various concentrations of alga biomass to laying hens to determine the effect on the quality of the egg and yolk color change. They found that the added alga biomass had no effect on the egg production of the hen and all diet groups had a mean egg production above 90% (Walker et al., 2012). There is a definite increase in yolk weight and whole egg weight in all feeding trials where the experimental diet had heavier eggs compared to the control groups. There seems to be no correlation between egg weight and yolk weight. Some diet groups had a lighter egg weight compared to the controls, but had a heavier yolk weight. This can indicate that even if the whole egg is not heavier, the yolk weight increased with dosage of P. marcusii. However, these findings are in contrast to previously reported studies. Some studies have shown that by adding probiotics, antibiotics or bacterial enzymes, such as xylanase, to the feed of hens had no effect on any of the quality parameters, including whole egg and yolk weight (Yörük and Bolat, 2003; Yörük et al., 2004; Mahdavi et al., 2005; Yang et al., 2006). The HU values were not significantly different between the diet groups in the experimental trials. These results are in agreement with previous studies. The supplementation of vitamins C and E (astaxanthin precursors) or alga biomass had no undesirable effect on the HU of different experimental treatments (Franchini et al., 2002; Walker et al., 2012). After the pilot trial, it was clear that a diet free of all pigments was needed to effectively evaluate the pigmentation effect of P. marcusii. In the experimental trial, there was a significant increase in yolk color compared to the white control. A higher dosage of P. marcusii resulted in a higher yolk color change. The intestinal cells of the hen easily absorb natural sources of carotenoids. These pigments are transported to the yolk once it is released from the feed content (Surai et al., 2001; Bortolotti et al., 2003). Different carotenoids have different deposition rates in eggs because of the bioavailability of esterified or free forms of carotenoids (Bowen et al., 2002). To be able to compete with a yellow corn diet a higher dosage of P. marcusii is needed. However, this was not possible in this study because of culturing limitations. The egg quality results indicate that whole P. marcusii cells can be used as a pigmentation source without the need for downstream processing to break the cells. However, higher cell concentrations are still needed to increase yolk color. Some well-known food pathogens associated with chicken egg products are E. coli, Listeria and Salmonella (Gast, 1992). It was, therefore, necessary to determine if these organisms are present in the egg contents. None of the dilution plates had any growth on them. It is possible that the microorganisms were not viable anymore or the colony forming units were too low to detect. However, we did not expect to find any of the pathogens or P. marcusii in the egg content, as previous studies have shown that the internal egg only gets contaminated when it comes into contact with the outer shell where trace amounts of the pathogens might be present and if the pathogens are present in the immediate environment of the hen (De Reu et al., 2005; Mallet et al., 2006; Jones et al., 2011). None of the hens in all the trials were sick and the way the cages are designed prevents the egg from coming into contact with fecal matter on the floor of the house that might contain these pathogens (De Reu et al., 2005; Mallet et al., 2006; Svobodová and Tůmová, 2014). The surroundings of the hen must be kept clean to prevent the potential contamination of food pathogens. The findings in this study demonstrate the potential use of Paracoccus marcusii as a feed additive to enhance yolk color. Paracoccus marcusii significantly increased the yolk color in the experimental trial compared to the white corn diet groups and there is also an increase in whole egg and yolk weight. There was no negative effect on the overall egg quality. Paracoccus marcusii can, therefore, be used as a feed additive to enhance yolk color in laying hens. It is important for future studies to determine the optimum dosage needed and the type of association between the bacterium and the hen. ACKNOWLEDGEMENTS The authors would like to thank Mariendahl Experimental Farm, University of Stellenbosch, for the use of their hen house and the help from the staff. This study was financially supported by the National Research Foundation (NRF), South Africa. REFERENCES Adams C. A. 1985. 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Poultry ScienceOxford University Press

Published: Mar 1, 2018

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