Fumigating broiler hatching eggs with lysozyme product (Inovapure) to reduce eggshell microbial load

Fumigating broiler hatching eggs with lysozyme product (Inovapure) to reduce eggshell microbial load Xujie Li,∗,1 Derek Anderson,∗ Bruce Rathgeber,∗ Nancy McLean,† and Janice MacIsaac∗ ∗

Department of Animal Science and Aquaculture, Faculty of Agriculture, Dalhousie University, Truro, NS B2N 5E3, Canada; and † Department of Plant, Food, and Environmental Sciences, Faculty of Agriculture, Dalhousie University, Truro, NS B2N 5E3, Canada 33 d of age. In the microbiological experiments, inoculated eggs fumigated with 3.0% LP and 0.125% QA reduced (P < 0.05) the total amount of E. coli to 11 cfu/egg and 10 cfu/egg, respectively. When eggs were sanitized prior to inoculation, 3.0% LP demonstrated (P < 0.05) ongoing bactericidal action to prevent E. coli penetration. No differences in hatchability, fertility rate or egg weight loss percent were found among sanitation treatments. At hatch, body weight or the ratio of yolk sac weight to yolk-free body weight were not affected by the sanitation treatments. However, the application of sanitizers decreased (P < 0.05) the presence of E. coli in the yolk sac of newly hatched chicks. Feed consumption, body weight and feed conversion ratio were not affected by sanitation treatments. However, average daily body weight gain was lower (P < 0.05) following QA. Overall, 3.0% LP demonstrated acceptable activity against E. coli on eggshells, and provided ongoing bactericidal action to prevent E. coli penetration without negatively affecting growth performance.

ABSTRACT Experiments were conducted to evaluate the effectiveness of a lysozyme product (InovapureTM) (LP) against E. coli penetrating eggshells. In the first microbiological experiment, 60 agar-filled eggs were inoculated with E. coli suspension, then fumigated with distilled water, 1.5% or 3.0% LP or a quaternary ammonium product (QA) at 0.125% for 10 min. In the second microbiological experiment, another 60 agar-filled eggs were fumigated with the same sanitizer treatments first, then inoculated with the E. coli suspension. Eggshells were candled and visual colonies were counted after 48 h incubation. An animal experiment was conducted to evaluate LP applied to the surface of 2080 broiler hatching eggs on hatching and growth performance. Hatching eggs were submerged in an E. coli suspension. After drip drying, eggs were randomly divided into four fumigation treatments, each with four subsets of 150 eggs. Fumigation treatments were the same as in the microbiological experiments. Eggs were incubated in 8 incubators (2 replicate incubators per treatment) and the broilers were grown to

Key words: lysozyme product, sanitation, incubation, hatching performance, Escherichia coli 2018 Poultry Science 0:1–10 http://dx.doi.org/10.3382/ps/pey288

INTRODUCTION

cools down from body temperature. The negative pressure generated by the cooling process can pull bacteria from the shell surface through the shell and its membranes (Bruce and Drysdale, 1994). The effectiveness of traditional hatchery sanitation and pathogen reduction in day-old chicks is limited if the eggs are already heavily contaminated (Coufal et al., 2003). Bailey et al. (1998) have shown that a small percentage of contaminated eggs can spread bacteria from egg to egg within an incubator. With this in mind, the hatching eggs benefit from being sanitized prior to incubation. Eliminating the microbial load on the surface of hatching eggs and preventing cross-contamination during incubation usually involves spraying, fumigating, and washing eggs with chemical sanitizers (Brake and Sheldon, 1990; Scott and Swetnam, 1993; Fasenko et al., 2009; Spickler et al., 2011). Hatching egg sanitizers should be evaluated on their ability to reduce

The transmission of pathogenic bacteria to eggs is a major concern to the poultry industry. Hatching eggs penetrated by pathogenic microorganisms may result in bacterial infection, which could increase the risk of embryo mortality or weaker chicks with poor subsequent growth performance (Brake and Sheldon, 1990; Patterson et al., 1990; Copur et al., 2010). A freshly laid egg is wet, warm, and easily penetrated by microorganisms through the eggshell as the egg  C 2018 Poultry Science Association Inc. Received November 20, 2017. Accepted June 13, 2018. 1 Corresponding author: Department of Animal Science and Aquaculture, Faculty of Agriculture, Dalhousie University, Truro, PO Box 055, NS B2N 5E3, Canada. Tel: +1-(902)-986-9633; E-mail: [email protected] Financial support for this project was provided by the Nova Scotia Department of Agriculture and Chicken Farmers of Nova Scotia.

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microbial load without harming the embryo, while demonstrating cost-effectiveness and safety (Shane and Faust, 1996). Formaldehyde has been a standard disinfectant in the poultry industry due to its bactericidal action and ease of application. Formaldehyde applied by fogging has significantly reduced the total microbial load on eggshells (Williams, 1970). However, the prolonged exposure to formaldehyde has been reported as a health risk to workers. Symptoms include headaches, nausea, respiratory impairment, and kidney damage (Occupational Safety and Health Administration, 1991). Numerous researchers have evaluated different commercial hatching egg sanitizers, including chlorine, hydrogen peroxide, quaternary ammonia compound products, and UV light, respectively (Brake and Shelton 1990; Coufal et al., 2003; Cox et al., 2007; Fasenko et al., 2009; Spickler et al., 2011). An alternative to formaldehyde, which is less hazardous to humans, economical and efficient, is required. Lysozyme, a natural bacteriolytic enzyme, can be isolated from egg white and is widely present in animal tissues and secretions (Salton, 1957). Lysozyme is known as 1,4-β -N-acetylmuramidase. It cleaves the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in bacterial peptidoglycan, which is an important component of bacterial cell walls (Phillips, 1966). Therefore, lysozyme provides protection against bacterial infection. Compared to Gram-positive bacteria, Gram-negative bacteria have an additional outer membrane that consists of lipopolysaccharide and protein. Due to the extra barrier in Gram-negative bacteria, the antibacterial activity of lysozyme is more effective against Gram-positive bacteria than Gram-negative bacteria (Johnson, 1994). Following the addition of substances like EDTA, butylparaben or tripolyphosphate, the activity of lysozyme was enhanced enough to control the growth of Gramnegative bacteria (Durance, 1994; Boland et al., 2003). Several studies have evaluated the antimicrobial activity of lysozyme in vegetable, cheese, and wine production. Hughey et al. (1989) suggested that applying 100 ppm lysozyme with 5 mM EDTA effectively inhibited the growth of Listeria monocytogenes in fresh vegetables and cheese products. According to Makki and Durance (1996), 10 and 50 ppm lysozyme can delay the growth of Gram-positive bacteria in beer production. However, research regarding the use of lysozyme as an alternative sanitizer for hatching eggs is limited. Evaluation of lysozyme as an alternative to commercial hatching egg sanitizers should include measuring its effectiveness against Gram-negative bacteria such as E. coli, which can be problematic for the hatchery industry. Studies to determine the level of lysozyme that is most effective for preventing contamination of hatching eggs with E. coli are needed. The method used to evaluate the effectiveness of a sanitization procedure should reflect the impact of bacteria that are in position to be problematic to a developing chicken embryo. There are numerous

studies that indicate procedures that are adequate to reduce the prevalence of microorganisms on the surface of eggshell (Mellor and Banwart, 1965; Gentry and Quarles, 1972; Berrang et al. 1991). With respect to hatching eggs, it is important to use a method to determine the number of bacteria that penetrate through the eggshell and contaminate the egg contents. An eggshell penetration assay was applied in this study to evaluate the effects of lysozyme as a hatching egg sanitizer. The objectives of the present study included: (1) comparing the effectiveness of a lysozyme product (Inovapure) (LP) and quaternary ammonium (QA) compounds against E. coli penetrating eggshells, (2) establish whether the application of test sanitizers on broiler hatching eggs can reduce the presence of E. coli in newly hatched chicks and improve hatchability, chick quality, and broiler production parameters. This information will be important to the hatchery industry when selecting a sanitizer for hatching eggs.

MATERIALS AND METHODS Eggshell Penetration Assay Egg Preparation A total of 120 fresh, unwashed, infertile eggs weighing 59 ± 1 g were collected from 36-wkold Lohmann LSL-Lite hens housed at the Dalhousie University, Atlantic Poultry Research Center and used within 24 h (storage environment: 11◦ C and 70% relative humidity). This procedure was conducted to minimize the surface area variability among eggs that were being sampled for bacteria penetration. The eggshell penetration assay used was adapted from the method developed by Board and Board (1967). A hole with a diameter of approximately 1 cm was drilled in the large end of each egg using a DeWalt cordless drill (DeWalt industrial tool Co., Baltimore, MD, USA). The drill bit was sanitized using pre-saturated wipes (Kimberly-Clark Professional, Roswell, GA, USA) prior to drilling and between eggs. The egg contents were drained and the shell interior surface was rinsed with distilled water to remove the albumen. After drying at room temperature for 24 h, all eggs were candled to ensure they were free of structural defects (fine cracks). Empty eggshells were filled aseptically with approximately 55 mL of plate count agar medium (Oxoid Ltd., Nepean, ON, Canada) containing 30 mg/L nalidixic acid (NA) (Sigma-Aldrich Co., St. Louis, MO, USA) for exclusion of bacteria without resistance to this antimicrobial at this level and 10 mL/L 1% 2,3,5triphenyltetrazolium chloride (Sigma-Aldrich Co., St. Louis, MO, USA) solution as a colorimetric indicator. After media solidified, the hole was sealed with paraffin wax to prevent contamination through the opening. Preparation of the Sanitizers Sanitizer treatments were prepared the day of treatment using distilled water as the solvent for the concentrated liquid chemicals. A commercially available LP (Inovapure), extracted from hen egg white, was provided by Neova Technologies

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LYSOZYME PRODUCT AS HATCHING EGG SANITIZER

Inc. (Abbotsford, BC, Canada). The LP had an enzymatic activity of 24,000 units/mg. It was commercially prepared as a mixture with EDTA at a ratio of 1:4 (lysozyme:EDTA) to enhance the antimicrobial activity against Gram-negative bacteria. Quaternary ammonium based product, Power Quat (Sani Marc Inc., Victoriaville, QC, Canada), is a general purpose no rinse requirement sanitizer used in foodprocessing, including shell egg cleaning (approved by Canadian Food Inspection Agency in 2004). Quaternary ammonium bears a positive charge, which attaches to the negative charge of the microorganism and can damage the bacterial cell wall resulting in cell death. The concentration (0.125%) used was the level recommended by the manufacturer. NA-resistant E.coli Culture The E. coli isolate was originally collected from the content of a 35-day-old Ross 308 broiler digestive tract and cultured repeatedly with increasing levels of NA to develop resistance to NA levels so that most native bacteria of the chicken digestive tract would not grow in the presence of NA. Well-isolated E. coli were stored at –80 C in 20% glycol until further use. The inoculation suspension was prepared by placing a loop of 30 mg/L NA-resistant E. coli culture into 800 mL of buffer peptone water (BPW, Difco, Detroit, MI) containing 30 mg/L NA and incubated at 37◦ C for 12 h to initiate the exponential growth phase of the NAresistant E. coli. The E. coli culture was mixed with 3 L of sterile BPW prior to inoculation. The concentration of the E. coli suspension was verified by plating dilutions of the final culture on a 3 M Petrifilm (E. coli/coliforms) count plate (3 M Inc., St. Paul, MN). Microbiological Sampling Procedure All agarfilled eggs (eggshell temperature: 22.8–23.2◦ C) were randomly divided into 2 groups of 60 eggs. One group of eggs (post-treated) were submerged in the prepared E. coli suspension (25.6◦ C) by replicates for 1 min and allowed to drip dry in a biosafety cabinet. This procedure provided the time for the bacteria to attach to the surface of the egg. Inoculated eggs were further divided into 4 groups with 3 replicates (5 eggs/replicate/treatment) and fumigated with distilled water as the water control (CON), 1.5% or 3.0% LP solution or 0.125% QA for 10 min in a fumigation room (width × depth × height: 1.25 × 1.85 × 3.02 m). The other 60 agar-filled eggs (pre-treated) were applied with the same sanitizer treatments for 10 min first. After 30 min of air drying, the pre-treated groups of eggs were inoculated with the E. coli suspension for 1 min. For fumigation application, a Drysan-ss 9 MINI (United AGRI Systems Inc., Abbotsford, BC, Canada) was used to generate an aerosol (7 to 10 microns) with ultrasonic technology. The order of sanitizer application within each replicate was randomized to minimize the effects of the duration of sanitation. The temperature of the fumigation solution was 25.4◦ C. The volume applied for each treatment batch was approximately 80 mL. Following the sanitation process, all eggs were handled

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Figure 1. E. coli colony (pink spot) on the interior of an agar-filled eggshell.

aseptically with new clean gloves for each treatment to prevent cross-contamination among eggs. All agar-filled eggs were incubated at 37◦ C before sampling. After 48 h of incubation, all eggs were candled to quantify bacterial penetration. Pink colonies, visible with the use of candling, were counted as E. coli (Figure 1).

Hatching and Production Trial The experimental protocol was carried out in accordance with the Canadian Council of Animal Care guidelines (CCAC, 2009). Incubation and Hatching A total of 2,080 hatching eggs (unwashed) were collected from a 63-wk-old Ross 308 commercial breeder flock. Eggs were stored at 15◦ C and 75% relative humidity for 2 d prior to incubation. A subset of 160 eggs weighing within a narrow weight range (61.0 to 62.0 g) was identified and labeled to evaluate the chick quality and the presence of bacteria in their yolk-sac after hatch. All 2,080 hatching eggs were submerged in a BPW containing 5.7 × 106 cfu/mL NA-resistant E. coli for 1 min. After drip drying, eggs were randomly divided into 4 groups (520 eggs/group) and allocated to the water control, 1.5% LP, 3.0% LP, or 0.125% QA. Each of the 4 treatment groups was further divided into 4 subsets of 130 eggs for the sanitation process. The 4 subsets of eggs from each sanitation treatments were fumigated for 10 min in the same fumigation room as described above. The order of fumigation was randomized as the eggshell penetration assay described above. After sanitation, all treated eggs were distributed into 8 incubators with 2 incubators per treatment. Briefly, 2 subsets of eggs per each sanitation treatment were randomly selected and incubated in 1 of 8 Chick Master G90 incubators (Chick Master,

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Medina, Ohio), the remaining 2 subsets of eggs with the same sanitation treatment were randomly allocated to another incubator. Each incubator, containing 2 subsets of eggs (130 eggs/subset) with the same sanitation treatment, was used as an experimental unit for the hatching performance portion of the research. During the first 18 d of incubation, the temperature was set at 37.5◦ C and at 55% relative humidity. Eggs were turned through 90◦ once every hour. On day 18 of incubation, a subset of 160 eggs were placed into individual labeled plastic pots to evaluate chick quality individually. The remaining eggs were candled and transferred into hatching trays, and incubated in the original incubators. Treated eggs followed the standard procedure of the hatchery until the hatching process was complete. After 512 h of incubation, the number of chicks hatched and their body weight were recorded. Hatchability was calculated based on total egg set and total fertile eggs. All unhatched eggs were broken open to determine fertility status. If fertile, the day of embryonic death was estimated. On day of hatch (21 d plus 8 h), the chicks hatched from the 160 subset eggs were pulled out randomly by incubators and euthanized by cervical dislocation and packaged using aseptic techniques for further microbiological testing. The chick weight, yolk sac weight, and yolk-free body weight (YFBW) were measured using a top pan balance (Thermo Fisher Scientific Inc., Waltham, MA, USA). The yolk sac sample was collected from each chick using aseptic techniques and placed in sterile plastic bags. All yolk sac samples were stored in a –80◦ C until analysis of the presence of NAresistant E. coli. Eosin methylene blue agar (BD Ltd., Mississauga, ON, Canada) containing 30 mg/L NA was poured into sterile petri plates and left to solidify. Each yolk sac sample was placed in a filtered stomacher bag (Mix 2, AES Laboratories, Bruz, France), weighed and diluted 1:10 with BPW. Each yolk sac sample culture (10−1 ) was further diluted to obtain 10−2 and 10−3 in BPW. One milliliter of each diluted culture was individually spread on the surface of the solidified agar plates. All samples were inoculated in duplicate for each dilution. All plates were incubated at 37◦ C. After 24 h, blue-black colonies with a green metallic sheen on eosin methylene blue agar were enumerated as E. coli. Broiler Production A total of 640 male and 640 female day-old Ross 308 broilers were transported to the Atlantic Poultry Research Center and randomly distributed into 32 floor pens, with 40 birds of the same sanitizer treatment and gender in each pen. Each pen (2.13 × 1.40 m) was prepared with new, clean, wood shavings litter at a depth of 4 cm. The stocking density of 40 birds per pen was 0.07 m2 bird−1 . Each pen was used as an experimental unit for the growth performance portion of the research. The diets were formulated to meet or exceed National Research Council (1994) nutrient requirements. All birds were fed the same diets within 3 growth phases. The nutritionally

balanced starter diet in crumble form was supplied from days 0 to 14. The grower and finisher diets in pellet form were supplied from days 15 to 25 and days 26 to 33, respectively. The starter diet was provided on cardboard box lids (width × depth × height: 53.3 × 43.2 × 5.1 cm) during the first 7 d after placement. After day 7 of age, feed was provided from tube feeders. Water was provided ad libitum from 3 nipple drinkers per pen. Birds were group-weighed per pen on day 0, 7, 14, 25, and 33 of age. The feed remaining in the feeders was weighed on each weigh day and as mortality occurred. Mortality was recorded and the dead birds were sent to the veterinary pathologist for necropsy (Animal Health Laboratory, Truro, Canada). Growth performance was evaluated by feed consumption, body weight, body weight gain, and feed conversion ratio.

Statistical Analysis Bacterial penetration data (met assumptions without transformation), hatchability, hatch weight, yolk sac weight, and YFBW were analyzed using the Mixed Procedure of the SAS v.9.4 (SAS Inc., Cary, NC, 2013). Mean differences of the positive presence of 30 mg/L NA-resistant E. coli in yolk sac samples were separated by the Chi-square test pairwise comparison using MINITAB 17 (Minitab Inc., State College, PA, 2010). All broiler body weight, feed consumption, body weight gain, and feed conversion ratio were analyzed as repeated measures by using the Mixed Procedure of the SAS v. 9.4 (SAS Inc., Cary, NC). In repeated measures analysis, 3 covariance structures, compound symmetry, toeplitz, and variance components were compared. The covariance structure, which provided the smallest corrected Akaike Information Criterion and Bayesian Information Criterion numbers, was selected to conduct the ANOVA test. The covariance structure toeplitz was selected. In all cases, if significant effects were found, the Tukey–Kramer test was applied to differentiate the means at 5% level of significance.

RESULTS AND DISCUSSION Eggshell Penetration Assay For inoculated eggs (post-treated), the number of NA-resistant E. coli colonies grown on the interior surface of eggshells was significantly reduced (P < 0.05) by the application of sanitizers (Table 1). Compared to the eggs fumigated with distilled water (20 cfu/egg), 3.0% LP and 0.125% QA treatments significantly (P < 0.05) reduced the population of E. coli (11 and 10 cfu/egg, respectively) penetrated through eggshells. These results support the hypothesis that fumigating contaminated eggs with LP and QA treatments would reduce the risk of E. coli penetration. Several studies have been conducted to evaluate the effectiveness of lysozyme as

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LYSOZYME PRODUCT AS HATCHING EGG SANITIZER Table 1. Effect of applying eggshells with sanitizer treatments after (post-treated) and before (pre-treated) inoculation on mean E. coli counts (cfu egg−1 ). Sanitizer

Distilled water 1.5% LP2 3.0% LP 0.125% QA3 SEM ANOVA P-value

n1

3 3 3 3

E. coli colony per egg Post-treated

Pre-treated

20a 13ab 11b 10b 2 0.0005

22a 14ab 10b 16ab 2 0.0321

1

Number of experimental units. Experimental unit = 1 group of 5 eggs. Lysozyme product (Inovapure). 3 Quaternary ammonium. a,b Means within a column with different letters differ significantly according to Tukey–Kramer test (α = 0.05). 2

an alternative feed ingredient to antibiotics in poultry diet to reduce the digestive tract microbiota. Gong et al. (2017) reported that including 100 ppm dietary lysozyme from day 5 to 14 resulted in reduced E. coli in the ileum of broilers without affecting the production parameters for the duration of the trial. Quaternary ammonium has been regarded as an excellent sanitizer against microorganisms on eggshells. Cox et al. (2007), who performed a study using NA-resistant Salmonella serovar Typhimurium, found that both QA-based sanitizer products with manufacturer recommended concentration (40,000 ppm 4 QA attached to a polymer and 111,000 ppm 2 QA, a biguanide and bronopol attached to a polymer) reduced the population of Salmonella on eggshells by 95% and 100%, respectively. This result supports our findings of a decreased risk for bacterial penetration when eggshells were sanitized with QA. Brake and Sheldon (1990) determined that QA effectively eliminated the total aerobic bacteria count on freshly laid eggshells by 99%, while no significant differences for coliform counts were found between eggshells sprayed with water or QA treatments due to the variable and small amount of coliforms present. The current study indicates that fumigating with QA or LP treatments on the surface of eggshells can reduce E. coli load on the eggshells. When eggs were sanitized prior to inoculation (pre-treated), no significant differences (P > 0.05) were found in the population of E. coli penetrated through eggshell among water control, 1.5% LP and 0.125% QA. However, 3.0% LP reduced (P < 0.05) the number of E. coli colonies penetrating the shell (10 cfu/egg) compared to the distilled water treatment (22 cfu/egg) (Table 1). The result indicated that 3.0% LP provided ongoing protection that prevented E. coli from penetrating eggshells. The research regarding applying disinfectants to broiler hatching eggs before artificial inoculation is limited. A previous study showed that the pre-surface application of a nisinlysozyme treatment effectively prevented the growth of Listeria monocytogenes in turkey bologna for up to 3 wk of storage (Mangalassary et al., 2008). The above result supports our findings that applying lysozyme

on the surface of eggshell reduces the total amount of E. coli on the eggshells and provides continuous bactericidal action to prevent E. coli penetration.

Hatching Trial No significant differences (P > 0.05) were found in percentage of egg weight loss from setting to transfer among treatments (Table 2). It is known that cuticle removal increases water loss during incubation (Peebles et al., 1998). If the cuticle had been partially removed or damaged by the application of sanitizer, it may result in an increased egg weight loss. Our result suggested that fumigating eggs with LP or QA as hatching egg disinfectant does not appear to affect the integrity of the shell cuticle. Egg weight loss is an important parameter for assessing incubation condition and eggshell porosity. Brake and Sheldon (1990) reported that the application of QA on the surface of eggshell did not affect the moisture loss during incubation. In addition, Scott et al. (1993) reported that the hatching eggs treated with sanitizer solutions containing QA had lower moisture loss than those applied with formaldehyde treatment. Egg weight loss is an indirect approach to assessing the deposition of cuticle. Direct assessment of changes to the quality and degree of coverage of the eggshell cuticle before and after sanitation, as those described by Bain et al. (2013), is recommended in future studies. There were no differences (P > 0.05) in the fertility rate among sanitation treatments. No differences (P > 0.05) in hatchability of fertile eggs or total set egg were found among sanitizer treatments (Table 2), indicating that fumigating eggs with LP or QA solution did not negatively affect the hatchability of broiler hatching eggs. The hatchability (total fertile eggs) of artificially inoculated hatching eggs treated with distilled water, 1.5% LP, 3.0% LP, and 0.125% QA solutions were 89.8%, 93.3%, 89.7%, and 89.7%, respectively. Our results agreed with Scott et al. (1993), who found no differences in hatchability between applying QA and formaldehyde as disinfectants for broiler

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LI ET AL. Table 2. Percentage of weight loss (%), hatchability (%), and chick hatch weight (g bird−1 ) of eggs inoculated with E. coli and sanitized. Sanitizer

n1

Egg weight loss percent2

Fertility3

Hatchability4

Hatchability of fertile5

Distilled water 1.5% LP6 3.0% LP 0.125% QA7 SEM

2 2 2 2

9.91 8.54 10.04 10.19 0.31

97.71 97.07 96.66 98.11 0.74

87.68 90.59 86.64 88.05 1.50

89.74 93.32 89.67 89.72 1.29

ANOVA P-value

0.056

0.568

0.405

Hatch weight 48 50 49 49 0.6

0.264

0.268

1

Number of experimental units. Experimental unit = 1 incubator containing 320 eggs (160 egg/subset). Egg weight loss percent = (d18 egg weight – d0 egg weight)/d0 egg weight × 100. 3 Fertility = (number of fertile eggs/number of eggs set) × 100. 4 Hatchability = (number of eggs hatched/number of eggs set) × 100. 5 Hatch of fertile = (number of eggs hatched/number of fertile eggs set) × 100. 6 Lysozyme product (Inovapure). 7 Quaternary ammonium. 2

Table 3. Percentage of embryonic mortality of eggs inoculated with E. coli and fumigated with sanitizers. Sanitizer

n1

Early mortality2

Middle mortality3

Late mortality4

Distilled water 1.5% LP5 3.0% LP 0.125% QA6 SEM

2 2 2 2

6.43 3.63 7.10 5.79 1.02

1.70 1.51 1.30 2.35 0.59

2.13 1.55 1.93 2.14 0.66

0.116

0.620

0.911

ANOVA P-value 1

Number of experimental units. Experimental unit = 1 incubator containing 320 eggs (160 egg/subset). Early mortality = (number of dead embryos between 1 and 7 d of incubation/number of fertile eggs set) × 100. 3 Middle mortality = (number of dead embryos between 8 and 14 d of incubation/number of fertile eggs set) × 100. 4 Late mortality = (number of dead embryos between 15 d of incubation to external pipping/number of fertile eggs set) × 100. 5 Lysozyme product (Inovapure). 6 Quaternary ammonium. 2

hatching eggs. The hatchability of inoculated broiler hatching eggs that were fumigated with LP or QA did not improve (P > 0.05) as compared to the water control eggs. The hypothesis of applying sanitizer can improve hatchability was rejected in this study. However, Brake and Sheldon (1990) reported that the application of 1.5% and 3.0% QA sanitizer improved the hatchability of fertile eggs over 6.0% from a 32-wk-old flock but did not have the same effect when used on the eggs from an older flock (36, 42, 46, or 62 wk of age). The higher hatchability in the eggs from the younger flock (32 wk of age) could be due to the reduction in early embryonic mortality by slightly increased water loss. But the effects of sanitation in embryonic mortality of eggs were not found from older breeder age. The embryonic mortality can be affected by various factors and their combinations including the breeder age, egg size, shell quality, albumen quality, shell porosity (Bains, 1994; Mcloughlin and Gous, 1999; Tona et al., 2001). Chick weight was not significantly affected by the sanitation treatments (Table 2). Chick hatch weight is often used as one of indicators of chick quality (Tona et al., 2004). As with hatchability, the results showed that the application of LP or QA had no negative effects

(P > 0.05) on chick quality. No significant (P > 0.05) reductions in embryonic mortality during the incubation process were found among treatments (Table 3). Therefore, it can be concluded that fumigating broiler hatching eggs with LP or QA did not negatively affect embryo health or development. The weight of the residual yolk sac is an indicator of energy utilization by the embryo during development. There were no differences (P > 0.05) for yolk sac weight and yolk sac weight as a percentage of YFBW (Table 4). In the duck, a reduced yolk sac indicates the embryo development is more advanced (Deeming, 2005). Deeming (2005) also reported that the yolk sac weight and the proportion of yolk to the chick body weight at hatch were variable in the broilers. Wolanski et al. (2004) determined the yolk utilization and chick length as parameters for embryo development for 8 different broiler breeder stains. Chick length is used as parameter to measure embryonic development and as one of the predictors of chick growth potential (Hill, 2001). A significant negative correlation between yolk sac and chick length has been found in the day old chicks. Our results indicated that applying LP or QA did not have a negative effect on the energy utilization by the embryo during development.

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Table 4. Effect of sanitizers’ fumigation on yolk sac residue (g) and the ratio (g/g∗100) between yolk sac weight and yolk-free body weight of chicks hatched from eggs fumigated after being inoculated with E. coli. Sanitizer

n1

Yolk sac residue

Ratio (yolk sac:yolk-free body weight)

Distilled water 1.5% LP2 3.0% LP 0.125% QA3 SEM

2 2 2 2

6.3 6.3 6.2 6.6 0.17

15.3 14.5 14.4 15.5 0.53

ANOVA P-value 1 2 3

0.407

0.218

Number of experimental units. Experimental unit = 1 incubator containing 20 eggs Lysozyme product (Inovapure). Quaternary ammonium.

Table 5. Effect of sanitizers’ fumigation on the NA-resistant E. coli positive in yolk sac samples.

Sanitizer Distilled water 1.5% LP1 3.0% LP 0.125% QA2

NA-resistant E. coli positive sample/total sample (n) 14/35a 0/32b 1/32b 0/29b

1

Lysozyme product (Inovapure). Quaternary ammonium. Means in the sanitizer main effect with no common letters are significantly different according to Chi-square test pairwise comparison (α = 0.05). 2

a,b

In this experiment, the yolk sac samples could only be collected if the egg resulted in a chick. For this reason, the numbers of yolk sac samples varied among treatments. The yolk sac samples collected from eggs fumigated with distilled water, 1.5% LP, 3.0% LP and 0.125% QA treatments, were from 35, 32, 32, and 29 chicks, respectively. The results of NA-resistant E. coli present in the yolk sac sample of newly hatched chicks are shown in Table 5. Fourteen out of 35 yolk sac samples from the eggs treated with distilled water treatment were observed to contain NA-resistant E. coli. The yolk sac samples of chicks hatched from eggs fumigated with 1.5% LP, 3.0% LP, and 0.125% QA were all lower than that amount and not different from each other. When the transovarian route of contamination of a chicken egg occurs, the bacteria are deposited in the yolk from the infected ovary during the egg formation. In the current study, the eggs were artificially inoculated with NA-resistant E. coli (trans-shell contamination) before sanitation. The application of sanitizers decreased the presence of NA-resistant E. coli in yolk sac sample, which can reduce the risk of contamination of the developing ´ embryos during incubation period. Kizerwetter-Swida and Binek (2008) measured the bacterial counts in liver and yolk sac of chicks hatched from eggs without sanitation. E. coli was present in 14 out of 25 yolk sac samples with an average number 1.16 × 106 cfu/g. The penetration of pathogenic bacteria to the yolk leads to infection of the embryo. The avian pathogenic E. coli is

one of the most common bacteria isolated from chicks with yolk sac infection (Dho-Moulin and Fairbrother, 1999). E. coli is present in the normal microflora of the intestinal tract of birds. Only some strains with specific virulence, termed avian pathogenic E. coli, are able to cause invasive infections in poultry (Vidotto et al., 1990; Dho-Moulin and Fairbrother, 1999). The infection of the yolk sac would increase the risk of mortality of young chicks during the first week of the post-hatching period. Fumigating hatching eggs with LP or QA greatly reduced the risk of bacterial infection for broiler chicks during the early stage of their life.

Broiler Production Traits There were no differences (P > 0.05) in final body weight at day 33 among sanitizer treatments (Table 6). On day 33, the body weight of male and female broiler chickens were 2,491±7 and 2,171±7 g, respectively. The expected body weight for male and female Ross 308 broilers at 33 d are 2,075 and 1,838 g, respectively (Aviagen, 2014). The application of LP and QA on the surface of the eggshells did not affect (P > 0.05) the final body weight. Results of the current study agreed with Fasenko et al. (2009), who reported no significant differences in body weight at the end of a 39-d growth period when hatching eggs were treated with electrolyzed oxidizing water. Likewise, Copur et al. (2010) found that that the use of oregano oil and formaldehyde as hatching egg sanitizers did not affect broiler body weight and body weight gain during the production period. There were no differences (P > 0.05) in feed conversion ratio among sanitizer treatments over the entire 33-d production period (Table 7). Body weight gain of the chicks hatched from the eggs fumigated with 0.125% QA was significantly (P < 0.05) lower than those fumigated with distilled water, 1.5% LP and 3.0% LP (Table 7). During the 33-d growth period, 0.125% QA treated chicks gained 64±0.4 g/d, while the other treatments chicks gained 66±0.4 g/d. Hatching eggs treated with LP did not negatively impact (P > 0.05) the body weight gain of broilers during the entire production period. However, the eggs treated with QA had lower body weight gain per day than

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8

LI ET AL. Table 6. Effect of sanitizers applied to hatching eggs on body weight (g bird−1 ) of broiler chickens. n1

Sanitizer

Distilled water 1.5% LP2 3.0% LP 0.125% QA3 SEM

4 4 4 4

Day 0

Day 7

Day 14

Day 25

Day 33

Male

Female

Male

Female

Male

Female

Male

Female

Male

Female

47 48 47 47 1

47 48 46 47 1

193 189 186 182 2

183 186 189 180 2

533 517 527 507 5

486 495 492 487 5

1,546 1,520 1,524 1,505 20

1,326 1,363 1,356 1,343 20

2,537 2,510 2,483 2,434 23

2,171 2,181 2,182 2,149 23

1

Number of experimental units. Experimental unit = 1 pen containing 40 birds. Lysozyme product (Inovapure). 3 Quaternary ammonium. 2

Table 7. Effect of sanitizers applied to hatching eggs on body weight gain (g bird−1 day−1 ), feed consumption (g bird−1 day−1 ), and feed conversion ratio (feed gain−1 ) of broiler chickens. Sanitizer

n1

Body weight gain

Feed consumption

Feed conversion ratio

Distilled water 1.5% LP2 3.0% LP 0.125% QA3 SEM

4 4 4 4

66a 66a 66a 64b 0.4

100 100 100 98 1

1.42 1.43 1.43 1.43 0.01

0.014

0.518

0.839

ANOVA P-value 1

Number of experimental units. Experimental unit = 1 pen containing 40 birds. Lysozyme product (Inovapure). 3 Quaternary ammonium. a,b Means within a column with different letters differ significantly according to Tukey–Kramer test (α = 0.05). 2

Table 8. Effect of sanitizers’ fumigation on mortality (%) of chicks hatched from contaminated eggs. Sanitizer

Distilled water 1.5% LP2 3.0% LP 0.125% QA3 SEM

n1

4 4 4 4

0–33 day Male

Female

Treatment mean

5.0 3.1 3.1 4.4 1.0

1.3 1.3 1.3 0.6 1.0

3.1 2.2 2.2 2.5 0.7

1

Number of experimental units. Experimental unit = 1 pen containing 40 birds. Lysozyme product (Inovapure). 3 Quaternary ammonium. 2

the water control and LP treatments during the whole production period. The growth performance of broiler hatching eggs treatment with QA compound products is limited in other studies. No differences (P > 0.05) in mortality were observed among sanitizer treatments during the 33-d production period (Table 8). Total mortality calculated through the study was 2.5%. Cause of mortality was mainly leg deformities and heart disease. Diseases were not related to bacterial infection. Currently, the relative cost per liter of LP used in the study is higher than that of QA (1.5% LP: 60¢; 3.0% LP: 120¢; 0.125% QA: 0.69¢). However, our expectation is that the LP will be cheaper with a novel extraction method. Considering LP had a better continuous bactericidal action, it can apply to the high-value breeding stock where cost is not the most important consideration.

CONCLUSION AND FUTURE RESEARCH The eggshell assay effectively determined the quantity of E. coli penetrating through the eggshells. Lysozyme is a potential sanitizer for hatching eggs based on the equivalent antimicrobial activity against E. coli as QA. The results of the current study demonstrated that both 0.125% QA and 3.0% LP provided acceptable activity against E. coli on the eggshell. In addition, 3.0% LP provided a more effective continuous bactericidal action to prevent E. coli penetration than 1.5% LP and 0.125% QA. The application of both LP and QA to inoculated hatching eggs nearly eliminated E. coli from the yolk sac of newly hatched chicks. This result may have contributed to a reduction in broiler mortality during the early production period by reducing the risk of bacterial infection in the young birds.

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LYSOZYME PRODUCT AS HATCHING EGG SANITIZER

Lysozyme product did not negatively affect the hatchability, chick weight at hatch or growth performance of broiler chickens during the 33-d production period. The lack of differences in broiler production parameters between the water control group and the QA treatment indicated that E. coli isolated from the broiler digestive tract content was not enough of a challenge to restrict growth or affect the health of these birds (Lan et al., 2005). Further studies should be conducted to examine the level of bacterial infection during the grow-out phase. Based on the results of this study, LP and QA provide effective, safe, and non-toxic means for sanitation of hatching eggs. Comparative studies determining the effectiveness and cost of LP compared with pure lysozyme and other commercial hatching egg sanitizers would be useful.

ACKNOWLEDGMENTS The lysozyme product (Inovapure) was kindly donated by Neova Technologies Inc. (Abbotsford, Canada). The technical assistance of Ron Mekers and Sarah MacPherson of the Atlantic Poultry Research Center is greatly appreciated.

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