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

The poultry industry has turned into a loss-making industry under the pressure of mandatory pricing and is losing jobs at an increasing rate. The number of chicken farms that have gone bankrupt in recent months has been unprecedented


Due to increased demand for broiler chicks, broiler breeder operations are interested in dietary and feeding strategies that can enhance broiler breeder reproduction and improve hatchability. Microbial contamination of fertile breeder eggs can be detrimental to hatchability and broiler chick growth performance. Formaldehyde and organic acids have been used to sanitize layer feed to reduce the contamination of table eggs. Broiler breeder feed sanitation is a practice that needs further exploration as it could improve breeder performance and offspring livability. The objectives of this experiment were to evaluate the effect of a formaldehyde-based feed sanitizer on broiler breeder hen reproduction, feed and eggshell contamination, incubation characteristics, and offspring early livability. Treating broiler breeder feed did not affect overall hen reproduction or hatchability, although it reduced live pip numbers at hatch, and improved the graded quality of hatched chicks. Treated feed showed less presumptive aerobic bacteria, fungus, C. perfringens, and Enterobacteriaceae compared to nontreated (control) feeds. Similar to the feed analysis, the surface of nest eggs from hens consuming treated feed showed reduced presumptive aerobic bacteria and tended to have less fungi contamination. Broiler offspring mortality tended to be reduced when obtained from hens consuming treated feed, especially during late laying periods. Our results indicate that treating broiler breeder hen feed with a formaldehyde-based sanitizer reduced the microbial load of feed and eggs, and positively impacted the quality and livability of the hatched chicks.

Key words

broiler breeder
feed sanitation
egg incubation


The rise in the demand for chicken meat in the United States and globally has caused genetics companies to intensify the selection of broiler chickens in favor of growth performance. Because of this, broiler breeders are feed-restricted to prevent excessive BW gain that favors successful mating behavior and egg production, although achieving high fertility and hatchability has become challenging in recent years. Reports from USDA (2022a) demonstrate that incubated egg numbers in the United States have increased 1% since 2021, although hatchability remains lower than past years. In the United States, the availability of broiler chicks has been affected predominantly by its increasing demand (USDA, 2022b), and the reduction in the reproductive fitness of the parental stock throughout the last decades (Siegel and Dunnington, 1985Pollock, 1999).

Discontinuing the use of feed antibiotic growth promoters has had implications on chicken intestinal bacterial growth rates, diseases, and chick performance (Bedford, 2000). Broiler chick disease challenge can intensify when fertile eggs are exposed to contaminated environments in the breeder house, hatchery, and transportation. Grains and animal feeds can carry microorganisms originating from the harvest locations, exposure to rodents, birds and insects, and unfavorable environmental conditions during transport and storage (Maciorowski et al., 2007). Clostridium perfringens, Salmonella spp., and E. coli are some of the bacteria of major concern in poultry which can be present in feeds. Likewise, poultry feed can also be a source of protozoa parasites of Eimeria genera (causative organism for coccidiosis) which can cause intestinal damage and malabsorption of nutrients (McDougald, 2008). Also, feeds contaminated with fungal genera AspergillusPenicillium, and Fusarium are prone to have mycotoxins. Generally, animals that consume feeds contaminated with high mycotoxin levels can exhibit negative effects on growth, reproduction, induce damage to several organs and cause neurological deterioration (Haque et al., 2020FDA, 2022). Impactful dose and exposure time varies by species, age, and type of mycotoxin.

Breeder feed microbiological contamination can pose risks to hen reproduction and can vertically transmit to fertile eggs and horizontally disseminate to other eggs within a hatchery operation (Wales and Davies, 2020). Several integrated companies have attempted to improve broiler breeder reproduction through nutritional strategies, yet there is limited information pertaining to the positive consequences of sanitizing broiler breeder feed. Sanitation products such as organic acids and formaldehydes have been known to effectively inhibit Salmonella in livestock and poultry feeds (Wales et al., 2009). The FDA (1996) approved the use of formaldehyde as an antimicrobial food additive for poultry feeds. Aldehyde substances such as formaldehydes in poultry feeds serve as fixative agents, giving them antimicrobial and mold-inhibition properties (Spratt, 1987Ricke et al., 2019). Therefore, feed sanitation could reduce the competition in the bird digestive tract between beneficial microbes and those feed borne pathogens (Anderson and Richardson, 2000Ricke et al., 2019). In addition to the challenges associated with breeder reproduction and hatchery efficiency due to genetic selection, controlling breeder feed contamination with formaldehyde-based products is a field that needs further examination. For these reasons, our objectives were to evaluate the impact of broiler breeder feed treatment with a formaldehyde-based feed sanitizer on broiler breeder reproductive performance, feed and egg contamination, and early offspring livability.


All experimental procedures used in this study were approved by The University of Georgia Institutional Animal Care and Use Committee.


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 m2n = 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

Bird Husbandry. After the rearing period, 21-wk old pullets (n = 264 pullets) were designated to laying pens (n = 6 pens; n = 44 birds per pen; 2.4 × 3.6 m2) with each pen having similar mean hen BW and CV obtained from wk 20 weights. Two thirds of the floor space was covered by slats, and the remaining third of the pen was covered with pine shavings. A 6-hole nest section was placed on the slats of each pen. At this age, the photoperiod was changed to 15 h of light and 9 h of dark. At wk 23, n = 3 Aviagen Yield Plus males (closest to 3,300 g of BW and proper fleshing) were included in each hen pen. To maintain an optimal fertility, Yield Plus spike males were grown to 21 wk of age, and one spike male was added to each pen when hens were 52-wk old. Hens were fed using pan feeders (n = 4 per pen) with an exclusion grill to prevent males from accessing the hen feeder. Pan feeders were filled with feed every day and hand-lowered every morning at 6:30 a.m. Males were fed from plastic (4 hole) feeder that was suspended over the shavings area. Birds had free access to water with a nipple drinker line. A sample of hens and roosters were individually hand-weighed weekly (n = 1 pen per treatment) to adjust feed allowance based on BW gain, and egg production. Feed allowance was equally adjusted through lay (80–157 g per bird) for the hens and roosters (91–139 g per male). Prior to lay, all birds were fed a common prelay diet (14.1% CP, 2.83 Mcal per kg, 1.5% Ca) from wk 22 to 25 and then switched to the treatment diets: untreated control (CTL) diet or formaldehyde-based product treatment (TRTTable 1). The product used for TRT diet was Termin-8 Powder containing 18% formaldehyde and 4.7% propionic acid (Antitox Corporation, Lawrenceville, GA). This expected dose of formaldehyde is equivalent to 720 mg per kg of feed (Table 1). Each treatment was represented by 3 pens. A 2-step program was used throughout laying: Breeder I (26–45 wk), and Breeder II (46–60 wk; Table 1).

Table 1. Experimental broiler breeder hen diet composition.

Ingredient Breeder I, % (26–45 wk) Breeder II, % (46–60 wk)
Empty Cell Control Treated Control Treated
Ground corn 62.73 62.73 63.06 63.06
Soybean meal 15.64 15.64 16.00 16.00
Oats, whole 2.00 2.00 3.00 3.00
Wheat middlings 8.00 8.00 6.06 6.06
Soybean oil, crude 1.00 1.00 1.00 1.00
Limestone, fine 3.74 3.74 3.16 3.16
Limestone, coarse 3.74 3.74 4.75 4.75
Mono-dicalcium phosphate 1.59 1.59 1.49 1.49
L-Lysine HCL 0.09 0.09 0.06 0.06
DL-Methionine 0.23 0.23 0.20 0.20
L-Threonine 0.13 0.13 0.11 0.11
Salt 0.26 0.26 0.27 0.27
Sodium sesquicarbonate 0.16 0.16 0.14 0.14
Choline chloride 60% 0.18 0.18 0.17 0.17
Vitamin premix1 0.05 0.05 0.05 0.05
Mineral premix2 0.08 0.08 0.08 0.08
Vermiculite filler 0.40 0.40
Termin-8 dry powder3 0.40 0.40
Total 100.00 100.00 100.00 100.00
Calculated nutrients
Crude protein, % 14.1 14.1 14.0 14.0
Ca, % 3.2 3.2 3.3 3.3
Av. P, % 0.4 0.4 0.3 0.3
ME, Mcal per kg 2.785 2.785 2.785 2.785
Dig. Lysine, % 0.6 0.6 0.6 0.6
Formaldehyde, mg per kg 720 720
Analyzed components
Formaldehyde4, mg per kg 28 702 30 729

Provided per kg of feed: vitamin A, 13,228 IU; vitamin D3, 3,505 IU; vitamin E, 100; vitamin B12, 0.001 mg; menadione, 6.6 mg; riboflavin, 11 mg; pantothenic Acid, 28.6 mg; niacin, 55 mg; folic acid, 4.4 mg; pyridoxine, 6.6 mg; thiamine 2.2 mg; biotin, 0.3 mg.


Provided per kg of feed: Mn, 180 mg as Mn oxide; Zn, 108 mg as Zn sulfate; Fe, 15 mg as ferrous sulfate; Cu, 12 as tribasic Cu chloride; I, 3.5 as ethylenediamine dihydroiodide; Se, 0.3 mg as sodium selenite and Se-yeast.


Premixture of aqueous formaldehyde, propionic acid, and vermiculite as carrier. Guaranteed analysis: 18% total formaldehyde and 4.7% propionic acid.


Analyzed by Antitox Corporation, Lawrenceville, GA as described by Lugacé and Dumont (2002).

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, 1976I 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

Hens from both treatments performed within industry standards peaking at 83% egg production. No differences were observed in early or late egg production between the CTL or TRT pens (P ≥ 0.447; Table 2). These results agree with layer experiments where formaldehyde-based sanitizers were included in hen feed (Anderson and Richardson, 2000). Hens consuming the TRT diet were slightly heavier than those consuming the CTL diet during early lay (P = 0.008; Table 2) but were similar during late lay and overall (P ≥ 0.101). There is no treatment related explanation for this early lay 61 g difference in BW since they were fed equal amounts through lay, and BW and CV were similar at housing in the lay pens at 21 wk of age.

Table 2. Effect of treating laying feed with a formaldehyde-based sanitized on broiler breeder hen reproduction, fertility, and hatchability during early (27–45 wk), late lay (46–60 wk), and overall (27–60 wk).

Empty Cell Hen diet1 Empty Cell Empty Cell
Variable CTL TRT SE P value
Week egg production, %
 Early lay 66.5 66.3 3 0.954
 Late lay 57.8 56.5 1 0.447
 Overall 62.9 62.2 2 0.785
Hen BW, g
 Early lay 3,640a 3,701b 16 0.008
 Late lay 4,063 4,053 19 0.732
 Overall 3,823 3,854 13 0.101
Hen mortality, %
 Early lay 4.5 2.3 1 0.161
 Late lay 3.8 1.5 1 0.255
 Overall 8.3 3.8 2 0.148
Fertility, %
 Early lay 96.9 97.5 0.7 0.549
 Late lay 96.1 95.0 0.8 0.297
 Overall 96.5 96.5 0.5 0.281
Hatchability, %
 Early lay 90.3 90.9 1.1 0.675
 Late lay 86.6 86.7 1.3 0.966
 Overall 88.5 89.3 0.9 0.222
Hatch of fertile, %
 Early lay 93.2 93.3 0.9 0.938
 Late lay 90.1 91.3 1.0 0.297
 Overall 91.6 92.5 0.7 0.529

Hens were fed laying treatment diets starting at wk 26. Hens fed an untreated diet (CTL) were represented by n = 3 pens of initial 44 hens each. Hens fed a formaldehyde-based sanitizer treated diet (TRT) were represented by n = 3 pens of initial 44 hens each.


Means with different superscripts within rows denote significant differences (P ≤ 0.05).

Hen mortality can be influenced by microbial contamination in feeds and disease challenge (Komnenov et al., 1981Yegani 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, 1987Anderson and Richardson, 2000Babar 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, 1972Spratt, 1987Sbardella 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.

Figure 1

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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., 1991Bruce and Drysdale, 1994Thiagarajan et al., 1994Oliveira 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.

Figure 2

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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).


Wales and Davies (2020) explained that persistent microbial contamination on eggshell surface allows transmission to the embryo and subsequent hatchlings, affecting their health and performance. The eggshell is the main barrier against pathogens and become thinner as hens age. Therefore, eggs from older hens are more susceptible to contamination through the shell (Franzo et al., 2020). Also, thinner eggshells allow eggs to dry or lose more moisture during incubation, increasing mid and late embryo mortalities, and reducing hatchability (McDaniel et al., 1979Roque and Soares, 1994). Results from Table 3 indicate that embryonic early, mid, and late mortalities were not affected by maternal feed sanitation (P ≥ 0.153). During hen late lay, live pip in eggshell percent was reduced when obtained from hens fed TRT (P = 0.042). Hatched cull-chick percent tended to be higher during early lay when hens were fed TRT (P = 0.087). The hatched chick quality grade was improved by 4% points during late lay (P = 0.011) and overall, by 1% point (P = 0.002), when hens were fed TRT feed. Breeder operations often depopulate flocks based on their egg production and hatch performance at the end of their productive life. Even if hatch was not improved by feeding breeders TRT diet, these data show that maternal feed sanitation improved the hatching characteristics of chicks especially during late lay.

Table 3. Effect treating laying feed with a formaldehyde-based sanitized on broiler breeder hen residue analysis and percentage of grade “A” chicks during early (27–45 wk), late lay (46–60 wk), and overall (27–60 wk).

Empty Cell Diet Empty Cell Empty Cell
Variable1 CTL TRT SE P value
Early dead, %
 Early lay 3.9 3.1 0.6 0.365
 Late lay 3.1 3.3 0.6 0.789
 Overall 3.5 3.1 0.5 0.939
Mid dead, %
 Early lay 0.0 0.1 0.1 0.153
 Late lay 0.0 0.1 0.1 0.309
 Overall 0.0 0.1 0.0 0.457
Late dead, %
 Early lay 1.9 2.0 0.4 0.891
 Late lay 3.7 3.2 0.5 0.515
 Overall 2.9 2.5 0.4 0.249
Cracked, %
 Early lay 0.3 0.4 0.2 0.765
 Late lay 1.0 0.8 0.3 0.447
 Overall 0.6 0.6 0.2 0.311
Contaminated, %
 Early lay
 Late lay 0.2 0.2 0.1 0.734
 Overall 0.1 0.1 0.1 0.507
Live pip, %
 Early lay 0.2 0.3 0.1 0.722
 Late lay 0.5a 0.1b 0.1 0.042
 Overall 0.4 0.2 0.1 0.507
Dead pip, %
 Early lay 0.1 0.2 0.1 0.657
 Late lay 0.3 0.0 0.1 0.104
 Overall 0.2 0.1 0.1 0.542
Cull chicks, %
 Early lay 0.0y 0.3x 0.1 0.087
 Late lay 0.1 0.0 0.1 0.168
 Overall 0.1 0.2 0.1 0.551
Grade “A” hatched chicks2, %
 Early lay 87.2 87.4 2.0 0.919
 Late lay 91.6b 94.6a 0.8 0.011
 Overall 90.1b 92.2a 0.8 0.002

Hens were fed laying treatment diets starting at wk 26. Hens fed an untreated diet (CTL) were represented by n = 3 pens of initial 44 hens each. Hens fed a formaldehyde-based sanitizer treated diet (TRT) were represented by n = 3 pens of initial 44 hens each.


Grade “A” chicks showed absence of the following visual characteristics: hock abrasion, anatomical defects, injuries, button on navel, open navels, strings on navel, and poor feathering.


Means with different superscripts within rows denote significant differences (P ≤ 0.05).


Means with different superscripts within rows denote tendencies (0.05 < P ≤ 0.10).

Offspring Mortality

Breeder egg contamination and late hen age can negatively influence chick mortality during the grow-out period (Peebles et al., 1999Barnett et al., 2004). The 7-day mortality of broiler offspring obtained from both maternal groups is shown in Table 4. Mortality in chicks from TRT-hens was higher at wk 45 hatch (P = 0.025), although most of these moralities were not found to have signs of yolk-sac contamination (P = 0.022). In contrast, at 60-wk hatch, chick mortality from CTL-hens tended to be higher than chicks from TRT-hens (6.0 vs. 2.3 %; P = 0.094). During this grow-out, mortalities with signs of yolk sac contamination were higher in chicks obtained from CTL-fed hens (P = 0.031). Overall means show that maternal TRT tended to reduce the percentage of contaminated mortalities during our broiler chick grow-outs (1.1 vs. 0.3%; P = 0.099). This effect increased as hens aged, specifically from 60-wk-old hens.

Table 4. Effect of treating breeder hen feed with a formaldehyde-based product on broiler offspring 7-day mortality when eggs were collected and incubated at ages 45, 49, and 60 wk.

Empty Cell Maternal diet1 Empty Cell Empty Cell
Offspring mortality by hatch week CTL TRT SE P value
Wk 45
 Mortality, % 0.4b 3.7a 1.0 0.025
 Contaminated-yolk sac mortalities2, % 0.4 0.7 0.4 0.555
 Noncontaminated mortalities3, % 0.0b 3.0a 0.8 0.022
Wk 49
 Mortality, % 0.7 0.4 0.4 0.555
 Contaminated-yolk sac mortalities2, %
 Noncontaminated mortalities3, % 0.7 0.4 0.4 0.555
Wk 60
 Mortality, % 6.0x 2.3y 1.5 0.094
 Contaminated-yolk sac mortalities2, % 3.0a 0.0b 0.9 0.031
 Noncontaminated mortalities3, % 3.0 2.3 1.3 0.694
Overall data
 Mortality, % 2.4 2.1 0.7 0.805
 Contaminated-yolk sac mortalities2, % 1.1x 0.3y 0.4 0.099
 Noncontaminated mortalities3, % 1.2 1.9 0.5 0.424

Chicks from maternal groups were placed in pens of n = 30 chicks per pen. Chicks were placed by maternal pen (n = 3 broiler pens per maternal pen), and grown for 7 d. Chicks were fed ad libitum a common pelleted and crumbled diet for 7 d.


Contaminated mortalities showed at least one of the following visual signs: dark navels or abdomen; dark internal spots in the abdominal cavity with or without hemorrhages; and signs of infected yolk sac.


Noncontaminated mortalities did not show any signs of infection and were either found flipped on their back or appeared to have been dehydrated.


Means with different superscripts within rows denote significant differences (P ≤ 0.05).


Means with different superscripts within rows denote tendencies (0.05 < P ≤ 0.10).

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., 2004Wales 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.


  • 1.

    Treating breeder hen feed with formaldehyde-based product did not impact hen reproductive performance, fertility, or hatchability.

  • 2.

    Sanitizing broiler breeder hen feed with a formaldehyde-based product effectively reduced the bacterial and fungal load in the feed. Breeder feed sanitation also reduced the aerobic bacterial and fungal load on breeder eggshell surfaces.

  • 3.

    Broiler breeder feed sanitation with formaldehyde-based product improved the quality of hatched chicks and their livability, especially when eggs were collected from older hens.

  • 4.

    Future research should focus on evaluating the impact of formaldehyde-based feed sanitizers on broiler breeder pullet growth performance, mortality, and gastrointestinal functionality.


This work was funded by Antitox Corporation (2021–2022).

L.P.A., K.M.S., J.L.W., and E.M. designed the study, collected, and interpreted the data. C.S. and N.H. analyzed laboratory samples. Manuscript preparation was done by L.P.A., J.L.W., E.M., and C.S.


The authors declare no conflicts of interest.