**1. Introduction**

**Abstract:** We investigated

For decades, soybean meal has been utilized globally in poultry diets, with maize providing the primary source of dietary energy [1]. Yet these feed ingredients are often inaccessible to developing countries for poultry production due to high cost and availability of soybean and corn, with the United States, Brazil, and Argentina being the largest producers and exporters of soybeans [1]. Hence, other protein sources like canola meal, peanut meal, fishmeal, and blood meal are also utilized.

In India, Ghana, and Nigeria, peanut meal is commonly utilized as a protein-rich poultry feed ingredient [2–6], ye<sup>t</sup> in the US, 85% of peanut production is for peanut butter and snacks, with the remaining 15% crushed for oil [7]. Early poultry feeding trials established that peanut meal prepared from conventional normal-oleic peanuts (22–30% protein and 44–56% total fat, with a total fatty acid profile of 52% oleic acid and 27% linoleic acid) is a reasonable poultry feed ingredient [8,9]. Additionally, limited studies have investigated the use of high-oleic peanuts (22–30% protein and 44–56% total fat) with an 80% oleic fatty acid and 2% linoleic fatty acid profile as an alternative poultry ration. Toomer et al. [10] reported that eggs produced from Leghorns in peak egg production (40 weeks of lay) fed high-oleic peanuts had increased β-carotene and oleic fatty acid content, with increased yolk color compared to conventional eggs, with no significant differences in hen performance (with the exception of egg mass) or egg quality between

treatment groups. However, egg weights produced from hens in peak egg production (40 weeks of lay) fed a high-oleic peanut diet were significantly smaller in mass relative to conventional eggs at all time points measured [10]. Hence, the primary aim of this project was to determine the effect of feeding a high-oleic peanut or an oleic acid diet on the size/mass of the eggs produced by older production hens (57 weeks of lay). The establishment of a feeding regimen to reduce the production of oversized eggs commonly seen with increasing production age is of grea<sup>t</sup> commercial interest [11]. Based on our previous findings [10], we conjecture that eggs produced from hens fed a high-oleic peanut or oleic acid diet will have enhanced β-carotene and unsaturated fatty acid content, with smaller egg size (mass), as compared to eggs produced from hens fed a conventional diet. Moreover, we aimed to investigate the effects of a high-oleic peanut diet on the sensory attributes of the eggs produced.

## **2. Materials and Methods**

All animal research procedures used in these feeding trials were approved by the North Carolina State University Institutional Animal Care and Use Committee (IACUC #17-001-A).

#### *2.1. Experimental Design, Animal Husbandry, Dietary Treatments, and Hen Performance*

All experimental diets were formulated in Concept 4 (level 2, version 10.0) software to be isonitrogenous (18% protein) and isocaloric (3080 kcal/kg metabolizable energy), with an estimated particle size between 800 and 1000 μm (Table 1). The control diet (Treatment 1) was prepared as a conventional layer diet with defatted soybean meal and corn, while Treatment 2 was prepared using aflatoxin-free whole non-roasted unblanched high-oleic peanuts. Peanuts were crushed using a Roller Mill to form crumbles, prior to inclusion in Treatment 2. Treatment 3 was prepared by supplementing the control diet with 2.64% (% by weight) of food-grade oleic fatty acid oil (Millipore Sigma, Burlington, MA, USA). Each of the experimental diets were supplemented with vitamin, mineral, and selenium premixes manufactured at the NC State University Feed Mill (Raleigh, NC, USA) to meet and/or exceed poultry requirements for vitamins, minerals, and selenium. All experimental diets were analyzed by the North Carolina Department of Agriculture and Consumer Services and the Food and Drug Protection Division Laboratory (Raleigh, NC, USA) for aflatoxin and microbiological contaminants.

Brown Leghorn hens were selected for use in this study from the University Flock, NC State University (Raleigh, NC, USA). In total, 99 Brown Leghorn hens (57 week of lay) were assigned to three dietary treatment groups for 8 weeks: (1) Conventional diet; (2) HOPN diet; (3) OA diet. There were three replicates per treatment, with hens individually housed in battery cages (each cage measured 12 inches wide × 18 inches deep × 18 inches height) in one room at the Chicken Education Unit, NC State University (Raleigh, NC, USA). Hens were provided feed and water ad libitum and 14 L:D for 8 weeks. Body weights were recorded for each individual hen at week 1 and week 8, with feed weights recorded weekly. Shell eggs were collected, enumerated, and weighed daily. Total number of eggs produced per replicate and per treatment was calculated for each experimental week and for the total 8 week feeding trial. The average feed conversion ratio (FCR) was calculated as total feed consumed over the 8-week feeding (kg)/dozens of eggs produced for each treatment group over the 8-week feeding trial.

#### *2.2. Egg Quality and Grading*

Bi-weekly (0-week, 2-week, 4-week, 6-week, and 8-week), 36 eggs were randomly selected with 12 shell eggs per treatment (4 eggs randomly selected from each replicate) for quality assessment and USDA grading. Fresh shell eggs were collected on the day of quality assessment and USDA grading. Shell eggs were analyzed for DSM yolk color score, vitelline membrane strength, Haugh unit, and shell strength by the Laying Hen and Small Flock Management Lab, Prestage Department Poultry Science, NC State University. Haugh unit values were determined using methods described by Haugh [12] and were recorded with the Technical Services and Supplies (TSS) QCD system (Dunnington, York, United Kingdom). The QCD system was calibrated to the DSM Color Fan, consisting of a series of 15 colored plastic tabs with a range of yolk colors from light yellow to orange red (color index 1 to 15), defined by Vuillemier [13]. In general, a texture analyzer (TA.XTplus) was used to measure the shell strength and vitelline membrane strength by the breaking strength using a 5-kg load cell per the manufacturer's instructions (Stable Micro Systems, Surrey, United Kingdom), with measurements in grams of force. Vitelline membrane strength was determined using methods described by Jones et al. (2005), with a 2 mm/second test speed and 0.0001 kg trigger force [14]. Modified methods of Jones et al. (2002) were used to measure shell strength with a 2 mm/second test speed and a 0.001 kg trigger force [15].


1 Three isocaloric, isonitrogenous (18% protein) formulated diets were fed to Brown Leghorn (57 week of lay) hens for 8 weeks. 2 Treatments: control = conventional soybean meal and corn mash diet, HOPN = unblanched high-oleic peanut crumbles (20%) and corn mash diet, OA = control diet supplemented with 2.64% food-grade oleic fatty acid oil. 3 High-Oleic Peanuts = unblanched raw whole high-oleic peanut crumbles. 4 MYC-Out™ = mycotoxin binder and feed antioxidant manufactured Adisseo (Alpharetta, GA, USA). 5 Mineral premix, manufactured by NCSU FeedMill, supplied the following per kg of diet: manganese, 120 mg; zinc, 120 mg; iron, 80 mg; copper, 10 mg; iodine, 2.5 mg; and cobalt. 6 Vitamin premix, manufactured by NCSU FeedMill, supplied the following per kg of diet: 13,200 IU vitamin A, 4000 IU vitamin D3, 33 IU vitamin E, 0.02 mg vitamin B12, 0.13 mg biotin, 2 mg menadione (K3), 2 mg thiamine, 6.6 mg riboflavin, 11 mg d-pantothenic acid, 4 mg vitamin B6, 55 mg niacin, and 1.1 mg folic acid. 7 Selenium premix, manufactured by NCSU FeedMill, = 1 mg Selenium premix provided 0.2 mg Se (as Na2SeO3) per kg of diet.

#### *2.3. β-Carotene, Lipid Content, and Fatty Acid Analysis*

All experimental diets were analyzed for lipid content, fatty acid, and β-carotene content in triplicate by an external vendor ATC Scientific (Little Rock, AR, USA), using AOAC approved methods. Gross energy analysis of feed samples was performed by ATC Scientific using an adiabatic oxygen bomb calorimeter with standard methods. Biweekly, a total of 45 eggs were randomly selected, with 15 eggs per treatment (5 eggs per replicate) for lipid content, β-carotene, and fatty acid analysis by ATC Scientific using AOAC approved methods. Each egg sample was mixed for homogeneity in a whirl-pak® (Millipore Sigma, St. Louis, MO, USA) bag for 3 min using a Smasher™ Lab Blender (Weber Scientific, Hamilton, NJ, USA). Subsequently, all egg samples were frozen at −20 ◦C and

stored frozen until chemical analysis within 2 weeks of collection. Frozen homogenous egg samples were shipped on dry ice overnight to vendor for analysis within 2 weeks of collection. Lipid (total cholesterol, crude fat) and fatty acid analysis of homogenous egg samples and feed samples were analyzed using direct methylation methods, as described by Toomer et al. [10]. Total cholesterol was measured as mg cholesterol/100 g sample weight (feed or egg), while crude fat was measured as a percentage of gram crude fat/gram sample weight (feed or egg). Fatty acid content was measured as a percentage of gram of fatty acid/gram total lipid content of a sample (feed or egg). Methods used to determine β-carotene content in eggs are detailed in the AOAC 958.05 [16] color of egg yolk method. Egg fat hydrolysis methods were determined using the AOAC method 954.02 [17].

#### *2.4. Cooking Methods and Consumer Acceptance Testing of Scrambled Eggs*

The Sensory Service Center, in the Food, Bioprocessing, and Nutrition Sciences Department, NC State University (Raleigh, NC, USA), performed all sensory testing and data analysis of egg samples. The sensory protocol was reviewed and deemed exempt by the NC State University Institutional Review Board for human subjects. Scrambled egg samples were prepared following safe food handling practices. During preparation and service, all team members wore gloves, hairnets/hats, and lab coats. Whole shell eggs were received and refrigerated at 4 ◦C upon arrival. On the day of testing, preparation of raw eggs was completed on a separate table from the cooking/serving areas of scrambled eggs to prevent cross contamination of any microbial hazards, and gloves were changed and hands were frequently washed during any transition from raw to cooked product.

On the day of the sensory evaluation, approximately 150 shell eggs per treatment were cooked. In total, 10 sets of 15 eggs/treatment were cracked into a bowl and beat together until homogenous. A large non-stick pan was heated over medium heat for approximately 1 min. The homogenous egg mixture was added to the heated pan and stirred slowly with a wooden spatula, bringing in the mixture from the edges of the pan for 3.5 min and subsequently removing the eggs from the pan. Scrambled egg samples from each of the three treatments were placed in labeled aluminum pans, covered with aluminum foil, and held in a heated holding cabinet at 180 ◦F to maintain quality. Scrambled egg portions were dispensed into lidded soufflé cups with three-digit codes to identify treatment for consumer testing.

Self-reported scrambled egg consumers (*n* = 109) were recruited from the NC State University staff and student population. Consumer panelists confirmed no egg food allergies and/or sensitivities prior to participation. Consumer panelists were disqualified if they were younger than 17 years of age, older than 65 years of age, or if they only consumed scrambled eggs once a month. Upon completion of the test, consumer panelists were compensated with a \$5 gift card to a local store. Compusense20 Cloud (Guelph, ON, Canada) was used for data collection and analysis. Samples were presented monadically with a 2-min enforced rest period between egg samples. Consumers evaluated various aroma, flavor, and texture liking attributes using a 9-point hedonic scale, where 1 = dislike extremely and 9 = like extremely. Consumers used a 5-point anchored Just About Right (JAR) scale to evaluate flavor and color attributes. Consumers were provided with spring water and unsalted crackers for palate cleansing.

#### *2.5. Statistical Analysis-Laying Hen Performance and Egg Lipid and Fatty Acid Content*

Each hen served as the experimental unit for all performance data. All performance data was evaluated for significance by one-way analysis of variance (ANOVA) at a significance level of *p* < 0.05 using SAS statistical software (version 9.4). If ANOVA results were significant (*p* < 0.05), Tukey's multiple comparisons t-test was conducted to compare the mean of each treatment group with the mean of every other treatment at *p* < 0.05 significance level. Comparisons were made between body weights (33 birds/treatment), feed intake (33 birds/treatment), feed conversion ratio (33 birds/treatment), and egg weights (total # of eggs collected over the 8-week feeding trial).

In total, 12 eggs per treatment (4 eggs per replicate randomly selected) were statistically analyzed by one-way ANOVA (*p* < 0.05) using SAS. Means were separated by least squares means, with Tukey-Kramer adjustment for multiple comparisons (*p* < 0.05) for treatment differences in egg quality parameters (Haugh unit, vitelline membrane strength, shell strength, yolk color score) at each bi-weekly experimental time-point (0-week, 2-week, 4-week, 6-week, 8-week). Additionally, eggs were statistically analyzed for treatment differences in egg quality parameters in all eggs collected over the 8-week feeding trial (180 total, 60 eggs per treatment). In total, 15 homogenous egg samples (5 per replicate) were statistically analyzed by one-way ANOVA (*p* < 0.05) using SAS. Means were separated by least squares means with the Tukey-Kramer adjustment for multiple comparisons (*p* < 0.05) for treatment differences in egg β-carotene content and egg lipid and fatty acid content (45 total egg samples at each time-point) weekly (2-, 3-, 4-, 5-, 6-, 7-, and 8-week).
