Broiler breeder feed treatment with a formaldehyde-based sanitizer and its consequences on reproduction, feed and egg contamination, and offspring livability

Rearing

At d 1 of age Ross 708 (Aviagen Group, Huntsville, AL) broiler breeder pullets (n = 508) and cockerel chicks (n = 174; Aviagen Yield Plus males; additional n = 151 were reared later for spiking) were obtained from a primary breeder hatchery. Pullet chicks were randomly distributed to 2 pens (7.3 × 4.6 m²; n = 254 per pen). Cockerel chicks were placed in a separate pen with the same dimensions. All aspects of rearing were maintained as close to industry standards as possible. The facility was light tight, force air heated, and evaporative cooled. Temperature was reduced from 31°C to 21°C during the first 15 d according to bird’s comfort and then maintained close to 21°C through the end of rearing. Birds were fed from a chain feeder line and water was provided free choice by a nipple drinker line. A commercial vaccination program against infectious bronchitis, Newcastle disease, reovirus, and avian encephalomyelitis virus was followed through wk 21. During rearing, birds were exposed to 8 h of light and 16 h of dark. All birds were wing-banded at wk 8.

Cockerels and pullets were fed ad libitum a common starter diet during the first 3 wk of age (18% CP, and 2.85 Mcal per kg). A 2-stage grower diet was fed after wk 3 (Grower I from wk 4 to 10: 15.3% CP, and 2.79 Mcal per kg; Grower II from wk 11 to 22: 14.6% CP, and 2.79 Mcal per kg) using skip-a-day feeding program through wk 21. Feed allowance was adjusted every week according to BW means obtained from BAT2 electronic hanging-platform scale (cat. no. V1.50.2MODBUSU; VEIT Electronics, Moravany, Czech Republic). These data were used to determine mean BW and uniformity expressed as coefficient of variation (%). All birds were individually hand-weighed on an electronic scale during wk 5, 10, 15, and 20 on a day off from feed to collect individual bird weights and uniformity of the entire flock. Mortality was recorded daily by pen through rearing. Management of all birds was similar between pens.

Laying Period

Egg Production and Incubation. Eggs were collected 4 to 5 times a day, recorded, and graded by pen. Egg production per pen was expressed as week egg production % (total week eggs/total hens/7 d × 100%). Settable eggs (n = 90 per pen) were incubated 9 times by pen at wk 29, 33, 37, 41, 45, 49, 54, 57, and 60. Eggs were incubated in Natureform incubators (Natureform Inc., Jacksonville, FL) at 37.5°C (53% relative humidity) and rotated 45° every 2 h during the first 18 d. Eggs from the groups were incubated in separate machines to prevent cross contamination within the machine. Temperature, turning, and relative humidity were checked twice a day during incubation to guarantee equal conditions of the eggs. To determine fertility and early embryo mortality, eggs were candled 11 d after being set, and transferred to a hatcher at d 18 and temperature reduced to 37°C for the remaining 3 d. Hatchability and hatch residue analysis was performed at every hatch.

In ovo vaccination was performed prior to transfer (18 d and 8 h of incubation) against Marek’s disease serotypes 2 and 3 (SB1 + HVT; cat. no. MHSF3175, Boehringer Ingelheim Animal Health Inc., Gainesville, GA). Prior to vaccination, egg surface was sprayed with 70% alcohol, and hole was punched on the blunt end of the egg using a sterile 18 G × 1” needle with a rubber stopper. One frozen vaccine vial (2,000 doses) was thawed for 90 s in a water bath at 27°C and then mixed into a 400 mL sterile diluent bag (Boehringer Ingelheim) using a 10 mL syringe fitted with a 20 G × 1” needle. Mixed vaccine solution was injected at 0.1 mL per egg using a 1 mL syringe fitted with a 1” needle. All syringes, needles and gloves were changed between groups to prevent cross contamination.

Chick Offspring Mortality. Broiler chicks from hens of 45, 49, and 60 wk of age were hatched and placed in pens by maternal treatment. Each maternal pen was represented by 3 broiler pens for a total of n = 18 broiler pens (with 30 chicks per pen). Broilers were fed a common pelleted and crumbled starter feed (21% CP, and 2.97 Mcal per kg of feed) during through 7 d. Chicks had free access to feed by eating from 2 feed trays in each pen. Free access to water was provided using a nipple drinker line. Temperature was decreased from 32°C to 27°C according to bird comfort. Birds had 24 h of light the first day and then 1 h of dark per day through d 7. Mortalities were recorded daily and necropsied to detect visual signs of yolk-sac contamination.

Enumeration of Bacteria and Fungi in Feed and Egg Surface

Feed Sampling. Hen feed was manufactured every 2 wk. A mash basal diet was first mixed for 10 min and separated in different scale-buggies. To mix the CTL diet, basal was mixed with vermiculite carrier (0.4%) for 10 min and then dragged to a bagger. Similarly, the Termin-8 powder (0.4%) was added to a separate basal amount, mixed for 10 min, and bagged. At every mix, n = 10 samples were taken intermittently from the bagger in separate labeled Whirl-Paks (cat. no. 11216-056, VWR, Suwanee, GA). Samples were shipped within 2 d to Antitox Corporation Laboratory (Lawrenceville, GA). Feed samples were evaluated for total presumptive yeast and mold, aerobic bacteria, Enterobacteriaceae, C. perfringens, and qualitative Salmonella. All microbial enumerations were based on standard procedures (Christensen and Meronck, 1976; I added page numbers and corrected editors.; ISO, 2008).

Feed Contamination Analysis. For each sample, 10 g of feed were added to 90 mL of Butterfields Phosphate Diluent (cat. no. FTBFD9060, 3M, Saint Paul, MN). Colony forming units (CFU) were confirmed by serial dilution and spread-plated on respective agar. Standard methods agar (SMA; cat. no. 89406-068, VWR, Suwanee, GA) was utilized for total aerobic bacteria. These plates were incubated aerobically at 35°C for 48 h as described by Maturin and Peeler (2001). Potato dextrose agar plates (PDA; cat. no. 90000-750, VWR, Suwanee, GA) were used to evaluate yeast and mold CFU, and then incubated for 27°C for 120 h as described by Tournas et al. (2001). Enterobacteriaceae were enumerated by using PetriFilm plates (cat. no. 6421, 3M, Saint Paul, MN). To evaluate C. perfringens CFU, 10 g of feed were mixed with 90 mL of 0.1% peptone water. Ten milliliters of this dilution were mixed with 10 mL of tempered tryptose-sulfite-cycloserine agar (TSC; cat. no. 101374-136, VWR, Suwanee, GA) agar in sterile petri dish and allowed to solidify. The solid agar-sample was then overlayed with 5 mL of TSC agar and allowed to solidify. Once plates were solid, they were incubated in a Mitsubishi anaerobic chamber (cat. no. 95061-002, VWR, Suwanee, GA) with system envelopes (AnaeroPack, cat. no. 95060-738, VWR, Suwanee, GA) at 35°C for 24 h. Salmonella in feed identification was performed as described by Wallace et al. (2014) using xylose-lysine-tergitol₄ (XLT4; cat. no. C8033, VWR, Suwanee, GA 30024) and Brilliant green sulfur (BGS; cat. no. 90001-022, VWR, Suwanee, GA) agar plates during the inoculation process.

Termin-8 Concentration Analysis. Each feed sample was evaluated for formaldehyde concentration to confirm the correct amount of formaldehyde was in the treated feed. The CTL samples were also evaluated to confirm no cross contamination of feed treatment occurred throughout the trial. The spectrophotometric method was utilized to determine the formaldehyde concentration (Lugacé and Dumont, 2002). Results of this analysis are shown in Table 1.

Egg Contamination. Egg sampling took place at 31, 36, 45, 51, 54, and 59 wk of age. Clean nest-eggs (n = 10 per pen at each timepoint) were collected with sterile gloves that were changed between pens and stored in sterile plastic containers with labeled plastic egg flats. Containers with eggs were stored in a cooler at 18.3°C for 5 d to mimic industry storage conditions, and then shipped to Antitox Corporation laboratory. Salmonella, total aerobic bacteria, and Enterobacteria recovery on eggshell surface was evaluated at all timepoints and fungal analysis was performed at wk 54 and 58. Additionally, Salmonella presence was performed in crushed eggs containing a mixture of both the eggshell and contents.

Intact eggs were individually placed into sterile plastic Whirl-Pak bags with 10 mL of 1% buffered peptone water (BPW; cat. no. 90000-608, VWR, Suwanee, GA) and gently rubbed for 1 min. This bag BPW-wash was used to determine eggshell bacterial and fungal contamination, and Salmonella presence. All incubations were aerobic. Total aerobic bacteria were evaluated by plating 100 µl of BPW-wash onto nutrient agar (cat. no. 213000, VWR, Suwanee, GA) and incubated at 37°C for 24 h as described by Sanders (2012). Yeast and mold recovery were evaluated by plating 100 µl on PDA and incubating at 25°C for 72 h. For Enterobacteriaceae, 1 mL of the BPW-wash were plated on PetriFilm plates and incubated at 37°C for 24 h. Total number of colonies where then enumerated from each plate postincubation.

For Salmonella presence in eggshell surface, 5 mL of the BPW-wash were added into 2 separate tubes containing either 9 mL of tetrathionate broth (TTB) or 10 mL of Rappaport Vassiliadis broth (RVB; cat. no. 95038-636 and 90003-424, respectively, VWR, Suwanee, GA). Tubes were incubated for 24 h at 37°C for TTB and 42°C for RVB tubes. Those tube solutions were streaked onto BGS and XLT4 plates and incubated again for 37°C for 24 h prior to Salmonella presence recording (+ or −). Also, since Salmonella can be found internally or embedded in the eggshell pores and membrane (Musgrove et al., 2005), the same procedure was used in a crushed egg sample after all BPW-wash samples were taken from the bag. The difference was that 45 mL of BPW were added to each bag and each egg was gently crushed as described by Berrang et al. (1991). Then, 100 µl of BPW and crushed egg mix were added to TTB and RVB tubes and then followed the same procedures described for eggshell surface analysis. All samples were sent to the Georgia Poultry Laboratory Network (Gainesville, GA) for Salmonella presence confirmation via PCR.

Statistical Analysis

Data were analyzed using a generalized linear model (GLM) using Statistical Analysis System (SAS) version 9.4 (SAS Institute Inc., Cary, NC). Means were declared different when P ≤ 0.05 and tendencies were declared when 0.05 < P ≤ 0.10. Hen performance data were analyzed by laying period: early lay (27–45 wk), late lay (46–60 wk), and overall (27–60 wk). All microbial data was log-transformed to obtain a normal distribution prior to the statistical analysis. Both breeder age and the interaction between diet × breeder age were also analyzed as main effects, although their P values will not be shown in the present study to properly focus on the main effect which is feed sanitation treatment.

Hen Reproductive Performance

Hen mortality can be influenced by microbial contamination in feeds and disease challenge (Komnenov et al., 1981; Yegani et al., 2006). Typical U.S. hen mortality for broiler breeder flocks is close 0.25% per week or an approximate cumulative mortality of 9% from wk 25 to 60. Both hen groups had mortality that was less than industry average, which is not unusual for a research facility with an expected lower disease challenge. However, the CTL group mortality was twice as much during early lay (4.5 vs. 2.3%) and overall (8.3 vs. 3.8%) compared to TRT group. Even if the statistical analysis does not denote differences (P ≥ 0.161; Table 2), these results suggest that in a larger study with more hens and replicates we could perhaps see more drastic statistical differences. Flock fertility, hatch and hatch of fertile percentages were not affected by feed sanitation (P > 0.222; Table 2). These breeder reproductive parameters were not negatively affected by formaldehyde feed treatment and agree with other poultry studies where similar doses were used (Spratt, 1987; Anderson and Richardson, 2000; Babar et al., 2001).

Feed Microbial Analysis

The formaldehyde recovered from our feed samples was close to the expected as shown in Table 1. Previous research indicates that formaldehyde-treated feed effectively reduces mold, Salmonella, and Enterobacteria in livestock and poultry feeds (Duncan and Adams, 1972; Spratt, 1987; Sbardella et al., 2014). Likewise, the current study microbial feed analysis showed that treating the feed with formaldehyde-based product reduced total presumptive aerobic bacteria, combined yeasts and molds, Enterobacteriaceae, and C. perfringens, when compared to the CTL feed samples (P < 0.001; Figure 1A). No Salmonella presence was detected in feed (data not shown). Sanitizing broiler breeder feed effectively reduced the microbial populations measured in this study compared to CTL untreated feed.

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Figure 1. Effect of treating feed with a formaldehyde-based sanitized mean bacterial and fungal recovery of broiler breeder hen feed samples (A) and from eggshell surfaces (B). Feed was manufactured every 2 wk through the end of the study (n = 120 total samples per treatment) and samples were taken at the bagger using gloves and sterile sampling Whirl-Pak bags. Significant differences were denoted with (**) when P ≤ 0.05 and tendencies (*) when 0.05 < P ≤ 0.10. Feed analysis results show that treating feed with TRT reduced total presumptive aerobic bacteria, yeast and molds, Enterobacteriaceae, and C. perfringens (P < 0.001). No Salmonella presence was detected in breeder feeds from both groups in feed and eggs (data not shown). Eggs (n = 10 per each hen pen) were collected at wk 31, 36, 44, 50, 54, and 59. Eggshell surfaces from eggs laid by hens consuming TRT-treated feed had reduced presumptive aerobic bacteria (P < 0.001) and tended to have reduced total yeast and mold (P = 0.061).

Eggshell Surface Analysis

Bacteria and mycotoxins can be vertically transmitted from hen to egg via transovarian mechanisms or pass though shell pores and are highly influenced by the sanitary condition of the nests and floor where eggs are laid (Berrang et al., 1991; Bruce and Drysdale, 1994; Thiagarajan et al., 1994; Oliveira et al., 2000). No Salmonella was detected in eggshell surfaces or crushed egg samples (data not shown). The laboratory results indicated that eggs from hens consuming TRT feed had reduced presumptive aerobic bacteria (P < 0.001; Figure 1B) and tended to have reduced total yeast and mold (P = 0.061) compared to those from CTL pens.

Figure 2 shows the egg surface reports analyzed by collection age. Treated feed reduced the total presumptive aerobic bacteria through the entire study compared to those eggs from hens consuming CTL feed (P < 0.006; Figure 2A). Total presumptive Enterobacteriaceae on eggshell surface was not impacted by feed treatment throughout the experiment (P ≥ 0.131; Figure 2B). Eggshell surface total presumptive yeasts and molds were reduced when hens consumed TRT diet only at wk 54 (P < 0.001), but not at wk 59 (P = 0.531; Figure 2C). The seasonal variability in temperature and humidity between the 2 timepoints might explain this inconsistency, given that the second sampling was performed in May at the beginning of the summer as ventilation rates were increased and litter added to pens to address moisture. These results indicate that treating breeder feed can help reduce the total presumptive aerobic bacterial and fungal contamination of egg surfaces when collected from the nests. In commercial breeder operations, it is common to also collect floor and slat eggs, which would potentially add variation to observations like those of this study.

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Figure 2. Effect of treating feed with a formaldehyde-based sanitized on broiler breeder hen eggshell surface total presumptive aerobic bacteria (A), Enterobacteria (B), and yeast and mold (C) by week of collection. Eggs were collected (n = 30 per treatment) at each given timepoint. Significant differences (P ≤ 0.05) are denoted with (**) by week. Treating hen feed with formaldehyde-based sanitizer consistently reduced the presumptive total aerobic bacteria through the experiment (P < 0.006; A). Total presumptive Enterobacteria was not affected by feed treatment through the laying period (P ≥ 0.131; B). Total yeast and mold were only reduced at hen age of 54 wk on eggshell surfaces from eggs laid by hens fed TRT (P < 0.001), but not at wk 59 (P = 0.531).

Incubation

Offspring Mortality

Historically, most of the attention given to formaldehyde-based products for feed are related to its effect on Salmonella eradication in feed and on table eggs (Ricke et al., 2019). Currently, there is no complete set of information regarding the potential use of formaldehyde feed sanitizers and its overall impact on breeder reproduction, variety of microorganisms on feed and egg, and hatchling mortality. In our study, sanitizing the broiler breeder hen feed with a formaldehyde-based product reduced the microbial load in feed and the bacterial and fungal load on eggshell surface. Given the relationship between the degree of contamination of eggs and hatchling health and livability (Barnett et al., 2004; Wales and Davies, 2020), our results show that sanitizing broiler breeder hen feed has positive implications on the offspring hatch quality and hence their livability through 7 d, especially when chicks are obtained from older hens.