*3.2. Hen Performance*

There were no mortalities over the 8-week feeding trial, and all laying hens were healthy and exhibited normal behavior. There were no significant treatment differences in body weights at the onset or termination of the 8-week feeding trial (Table 3). Interestingly, while there were no significant treatment differences in average egg weights at 4-weeks or 8 weeks of the study, the 8-week average egg weights produced from hens fed the HOPN dietary treatment were significantly smaller than the controls or OA group (*p* = 0.0004, Table 3). At 2 weeks and 6 weeks, the egg weights from hens fed the HOPN diets produced eggs less than egg weights from the control group, while egg weights were similar between HOPN and OA treatment groups (Table 3). This parallels results by Toomer et al. [10] and Van Elswyk et al. [19], demonstrating a reduction in egg mass when laying hens are fed diets rich in unsaturated fatty acids relative to conventionally produced eggs.


**Table 2.** Lipid content and fatty acid analysis of dietary treatments 1.

1 Treatments: control = conventional soybean meal and corn diet, HOPN = unblanched high-oleic peanut crumbles (20%) and corn diet, OA = control diet supplemented with 2.64% food-grade oleic fatty acid oil. Lipid (crude fat, total cholesterol), fatty acid, and beta-carotene analysis was performed by an AOAC-certified lab, ATC Scientific (Little Rock, AR, USA) food-grade oleic fatty acid oil. 2 Crude Fat content = g crude fat/g total sample weight ∗ 100, Fatty acid content = g of fatty acid/g total lipid content ∗ 100. Total cholesterol (mg/100 g dry weight) and lipid analysis was determined by direct methylation methods. Gross energy analysis was determined using an adiabatic oxygen bomb calorimeter and standard methods. Each value represents the mean ± the standard error for each triplicate sample. a,b,c Means within the same row with differing superscripts are significantly different (*p* < 0.05).



In total, 99 brown Leghorn (57 week of lay) hens were fed one of three isonitrogenous (18% crude protein) diets ad libitum for 8 weeks. Body weights were collected at week 1 and week 8 of the study (33 hens per treatment, 3 replicates, with 11 birds per replicate). 1 Treatments: control = conventional soybean meal and corn mash diet, HOPN = 20% unblanched high-oleic peanut + corn diet, OA = control diet supplemented with 2.64% food-grade oleic fatty acid oil. 2 Weights (g) of eggs were determined daily and weekly for each treatment group. Data represents the bi-weekly (2, 4, 6, and 8 weeks) averages ± standard error for each time point for each treatment group. 3 Feed conversion ratio (FCR) = kg and total feed intake over the 8-week/total dozen eggs produced over 8 weeks for each treatment group. Total number of eggs produced: Control = 1598 eggs, HOPN = 1617 eggs (≈49 eggs/hen), and OA = 1665over the 8-week feeding tria; Each value (body weights and egg weights) represents the mean ± the standard error. a,b,c Means within the same row lacking a common superscript differ significantly (*p* < 0.05).

> Egg size has been shown to be greatly influenced by body weight and age [20], nutrition [21], and pullet managemen<sup>t</sup> [22]. Egg size has been shown to be directly influenced by body weight; for every 45 g of body weight increase, there is a 0.5-g increase in egg size from 18 weeks of age in laying hens [22]. Hence, as the laying hen ages in the production cycle and increases in body weight, there is a proportionate increase in egg size [22]. The United States Department of Agriculture egg classification system [23–25] categorizes eggs by minimum weight per dozen eggs: jumbo 30 ounces (>63 g content per egg), extra-large 27 ounces (63–56 g content per egg), large 24 ounces (56–50 g content per egg), medium 21 ounces (50–44 g content per egg), small 18 ounces (44–38 g content per egg), peewee 15 ounces (<38 g content per egg) [25]. While there were no treatment differences in the total number of eggs produced, with 1598 eggs (≈48 eggs/hen) produced from the

control group, 1617 eggs ( ≈49 eggs/hen) produced from the HOPN group, and 1665 eggs (≈50 eggs/hen) produced from the OA group over the 8-week feeding trial, the control fed hens produced 1246 total jumbo eggs and 352 x-large size eggs, and the OA fed hens produced 1299 jumbo eggs and 366 x-large eggs, respectively. Conversely, hens fed the HOPN diet produced a total of 711 jumbo eggs and 906 x-large eggs over the 8-week feeding trial.

Therefore, in this study, jumbo eggs ( ≈63 g content per egg) were the predominate egg classification for most eggs produced from older production hens (57 week of lay) in all treatment groups. In contrast to our previous high-oleic peanut layer feeding trial with younger age production hens (40 weeks of lay), extra-large eggs were the predominate egg classification for most eggs produced [10], which parallels other studies demonstrating a trend of increased egg size with hen production age [26]. Nonetheless, in this study, eggs produced from hens fed the HOPN diet had significantly smaller egg size/mass relative to the other treatment groups (*p* < 0.001) at 57 weeks of lay, suggesting that a diet rich in unsaturated fats may be an effective commercial feeding regimen to manage the production of oversized eggs in older production hens.

While the feed conversion ratio (calculated as the total kg of feed intake over 8 weeks/total # dozen eggs produced over 8 weeks) of the control group (2.4 kg/dozen eggs) was higher than the other treatment groups (2.2 = HOPN, 2.1 = OA), there was no significant treatment differences between the feed conversion ratio (FCR) between the treatment groups (Table 3). FCR is a very important production parameter within the commercial egg industry, as a predictor of profitability utilizing the cost of kg of feed consumed [27] per total number of eggs produced. Thus, FCR is a measure of how efficiently an animal utilizes incoming dietary feed nutrients to generate the desired product of meat and/or eggs [27]. Typically, within the US commercial egg production industry, the typical FCR for each hen is about 2 kg of feed consumed/kg of egg produced, with each hen producing about 330 eggs per year [28].

#### *3.3. Egg Grading, Quality, and Production*

All eggs produced in this feeding trial were graded as USDA Grade AA of superior quality, with thick, firm egg whites and defect-free egg yolks. Additionally, the shells were clean and without defects. There were minimal numbers of blood spots or meat spots, with no statistical difference at the 95% confidence interval between eggs produced between the treatment groups (data not shown). The Haugh Unit (HU), first defined by Raymond Haugh [12], is commonly used to measure albumen (egg white) quality from the height and thickness of the albumen. Hence, fresher, higher-quality eggs have thicker egg whites and thus higher HU values. In this study, there were no significant differences in the average weekly HU between the treatment groups (Table 4). The vitelline membrane is a twolayer transparent casing enclosing the yolk separating the yolk from the egg albumen [29]. Vitelline membrane strength is an important physical attribute key to processing shell eggs and the separation of egg yolk from the albumen. Vitelline membrane strength is often used as a measurement of freshness since the vitelline membrane strength is time-dependent and reduced with increased time and storage handling conditions [29]. There were no significant differences in the average weekly shell strength or vitelline membrane strength between the treatment groups (Table 4).

However, the average weekly egg yolk color of eggs produced from hens fed the HOPN diet were significantly higher compared to eggs produced from hens fed the control and OA diets (*p* < 0.0001, Table 4). Moreover, upon visual observation, egg yolks from hens fed the HOPN diets were a visibly darker yellow/orange color intensity in comparison to the egg yolks produced from hens fed the control and OA diets (Figure 1).


**Table 4.** Egg quality of eggs produced from laying hens fed a diet of high-oleic peanuts or oleic acid.

In total, 99 brown Leghorn (57 week of lay) hens were fed one of three isonitrogenous (18% crude protein) diets ad libitum for 8 weeks. Egg quality (Haugh unit, yolk color score, vitelline membrane strength, and shell strength) was determined bi-weekly (2, 4, 6, and 8 weeks) using the Technical Services and Supplies (TSS) QCD system, with calibration with the DSM Color Fan for yolk color. 1 Treatments: Control = conventional soybean meal and corn mash diet, HOPN = 20% unblanched high-oleic peanut + corn diet, OA = control diet supplemented with 2.64% food-grade oleic fatty acid oil. Eggs were collected weekly and analyzed for quality. Yolk color = Roche Color Fan color index 1–15 (lightest to darkest color intensity). Each value represents the 8-weekly average ± the standard error with 12 eggs/treatment (3 eggs/replicate), N = 36 total measured bi-weekly for 8 weeks. a,b,c Means on the same row lacking a common superscript differ significantly, (*p* < 0.05).

**Figure 1.** Representative images of yolk color within whole egg samples from each treatment group at week 8 of the feeding trial. Ninety-nine brown Leghorn (57 week of lay) hens were fed one of three isonitrogenous (18% crude protein) diets ad libitum for 8 weeks. At 8-weeks, one whole egg was randomly selected for this photograph as a representative of yolk color observations seen on the day of egg processing with 12 eggs per treatment. This image is not representative of any other egg quality parameters measured. Treatments: Control = conventional soybean meal and corn mash diet, HOPN = 20% unblanched high-oleic peanut + corn diet, OA = control diet supplemented with 2.64% food-grade oleic fatty acid oil.

Similarly, in the yolk color score, the β-carotene content in eggs produced from hens fed the HOPN diet was significantly greater than the β-carotene content in eggs produced from hens fed the control or OA diets at all time points measured (Figure 2; Week 1 *p* < 0.01, Week 2 *p* < 0.01, Week 3 *p* < 0.001, Week 4 *p* < 0.01, Week 5 *p* < 0.0001, Week 6 *p* < 0.01, Week 7 *p* < 0.01, Week 8 *p* < 0.01). β-carotene is a carotenoid, which is a lipid soluble antioxidant found abundantly in plants, is responsible for the rich yellow and deep orange colors in plants, and is a precursor to vitamin A [30]. Conventional commercial eggs are rich in lutein and zeaxanthin [31]. However, yolk lutein and zeaxanthin are highly subjective to oxidation during egg processing, storage, transport, and/or cooking [31].

There were no treatment differences in total cholesterol content between eggs produced from the three treatment groups over the course of the study (Table 5). Similarly, there were no significant treatment differences in total crude fat content in eggs produced from the three treatment groups at weeks 2, 4, 5, 6, 7, or 8 (Table 5). But at week 3 of the 8-week feeding trial, eggs produced from hens fed the OA diet had significantly reduced levels of crude fat relative to the other treatments (*p* < 0.01, Table 5). At week 2 and week 5, there were no significant treatment differences in palmitic acid content between eggs

produced from hens fed the three treatment groups (Table 5). At week 3, 4, 6, 7, and 8, eggs produced from hens fed the HOPN diet had significantly less palmitic acid levels in comparison to eggs produced from the other treatment groups (Table 5). While there were no significant treatment differences in stearic acid levels in eggs produced from the three treatment groups at week 2, eggs produced from hens fed the HOPN and OA diets had significantly lower levels of stearic acid, relative to eggs produced from the control group at experimental weeks 3, 4, 6, 7, and 8 of the study (Table 5). Stearic acid content was similar between eggs produced from hens fed the HOPN and OA diets at weeks 3, 4, 6, 7, and 8 (Table 5). Stearic acid content was lowest in eggs produced from laying hens fed the HOPN treatment group relative to the other treatments only at week 5 of the study (*p* < 0.05).

Monounsaturated OA content was similar between eggs produced from the three treatment groups at week 2 (Table 5). OA content was significantly different between eggs produced from each of the treatment groups, with the highest oleic acid content in eggs produced from hens fed the HOPN diet and lowest in control eggs at week 3 (*p* < 0.0001), week 4 (*p* < 0.0001), week 6 (*p* < 0.001), week 7 (*p* < 0.0001), and week 8 (*p* < 0.0001). At week 5, OA content was significantly greater in eggs produced from hens fed the HOPN diet, while OA levels were similar between eggs produced from hens fed the control and OA diets (*p* < 0.01, Table 5). Total linoleic acid content was significantly greater in eggs produced from control fed hens at all time points (exception of week 4) relative to eggs produced from hens fed the HOPN diet, while levels were similar between eggs produced from hens fed the HOPN and OA diets at week 2, 5, 6, 7, and 8 (Table 6). Regardless, there were no significant treatment differences between the total linoleic content in eggs produced from hens fed the three treatment groups at week 4 only (Table 6).

**Figure 2.** Effect of diet on the β-Carotene content of eggs produced. In total, 99 brown Leghorn (57 weeks of lay) hens were fed one of three isonitrogenous (18% crude protein) diets ad libitum for 8 weeks. Treatments: Control = conventional soybean meal and corn mash diet, HOPN = 20% unblanched high-oleic peanut + corn diet, OA = control diet supplemented with 2.64% food-grade oleic fatty acid oil. Beta-carotene analysis was performed by an AOAC-certified lab, ATC Scientific (Little Rock, AR, USA). β-carotene content in eggs was determined using AOAC 958.05 methods [14]. *p*-values at the various sampling time-points were the following: Week-1 *p* = 0.002, Week-2 *p* = 0.004, Week-3 *p* = 0.0003, Week-4 *p* = 0.006, Week-5 *p* < 0.0001, Week-6 *p* = 0.004, Week-7 *p* = 0.008, Week-8 *p* = 0.001. Each bar column represents the average ± the standard error for each experimental timepoint, with 15 egg samples/treatment group (5 egg samples per replicate, 3 replicates) and a total of 45 eggs analyzed at each time-point. a,b Bar columns with differing superscript are significantly different (*p* < 0.05).


**Table 5.** The effect of feeding laying hens a high-oleic peanut (HOPN) diet or an oleic fatty acid (OA) diet on the fatty acid profile of the eggs produced.

In total, 99 brown Leghorn (57 week of lay) hens were fed one of three isonitrogenous (18% crude protein) diets ad libitum for 8 weeks. Total cholesterol (mg/100 g weight sample) and lipid analysis was determined by direct methylation methods. Egg fat hydrolysis methods were measured using the AOAC method 954.02. 1 Treatments: Control = conventional soybean meal and corn mash diet, HOPN = 20% unblanched high-oleic peanut + corn diet, OA = control diet supplemented with 2.64% food-grade oleic fatty acid oil. Crude fat content = g crude fat/g total sample weight ∗ 100, fatty acid content = g of fatty acid/g total lipid content ∗ 100. Each value represents the mean ± the standard error for each experimental time-point with 15 egg samples/treatment group (5 egg samples per replicate, 3 replicates). In total, 45 eggs were analyzed at each time-point. a,b,c Means within the same row lacking a common superscript differ significantly (*p* < 0.05).

> There were no significant differences in the total linolenic fatty acid content in eggs produced from hens fed the three treatment groups at week 2 (Table 6). Total linolenic fatty acid content was significantly lower in eggs produced from hens fed the HOPN diet in comparison to the total linolenic fatty acid content in eggs produced from hens fed the control and OA treatments at week 3 (*p* < 0.0001), week 4 (*p* < 0.001), week 5 (*p* < 0.01), week 7 (*p* < 0.001), and week 8 (*p* < 0.001). The total linolenic fatty acid content was similar between eggs in the control and OA treatment groups at week 3, 4, 5, and 6. Even so, the average total linolenic acid content in all egg samples from all treatment groups within the study were very low (≤0.2%, Table 6).


**Table 6.** The effect of feeding laying hens a high-oleic peanut (HOPN) diet or an oleic fatty acid (OA) diet on the unsaturated fatty acid profile of the eggs produced.

In total, 99 brown Leghorn (57 week of lay) hens were fed one of three isonitrogenous (18% crude protein) diets ad libitum for 8 weeks. Fatty acid analysis of egg samples was determined using standard direct methylation methods. Egg fat hydrolysis methods were measured using the AOAC method 954.02. 1 Treatments: Control = conventional soybean meal and corn mash diet, HOPN = 20% unblanched high-oleic peanut + corn diet, OA = control diet supplemented with 2.64% food-grade oleic fatty acid oil. Fatty acid content = g of fatty acid/g total lipid ∗ 100. Each value represents the mean ± the standard error for each experimental time-point with 15 egg samples/treatment group (5 egg samples per replicate, 3 replicates). In total, 45 were eggs analyzed at each time-point. ¥ C22:6 n3 = polyunsaturated docosahexaenoic fatty acid. a,b,c Means the same row lacking a common superscript differ significantly (*p* < 0.05).

> There were no significant treatment differences in the total omega 3 content in eggs produced from hens from the three treatment groups at week 2, 3, 4, 5, 6, 7, or 8 (Table 6). There were no significant treatment differences in the total omega 6 content in eggs produced from the three treatment groups at week 2, week 4, or week 7 (Table 6). At week 3, eggs produced from hens fed the control diet had the highest content of total omega 6 fatty acid (*p* < 0.001), while eggs produced from hens fed the OA diet had an intermediate level of total omega 6 fatty acid, and eggs produced from hens fed the HOPN diet had the lowest omega 6 content in comparison to the other treatment groups (Table 6). At week 5, 6, and 8, eggs produced from hens fed the control diet had significantly higher levels of total omega 6 content in comparison to eggs produced from hens fed the HOPN

diet. Nonetheless, there were no significant differences in n3 docosahexaenoic (C22:6 n3) acid content in eggs produced from hens fed the three treatment groups at any of the experimental time points (Table 6). Additionally, eggs were analyzed for the following fatty acid acids: butyric, caproic, caprylic, undecanoic, lauric, tridecanoic, myristic, myristoleic, pentadecylic, pentadecenoic, margaric, margaroleic, arachidic, gadoleic, eicosadienoic, homo-gamma-linolenic, eicosatrienoic, arachiconic, n3 timnodonic, heneicosanic, behenic, erucic, brassic, lignoceric, and nervonic acid, of which no levels were detected (data not shown).

Numerous feeding trials have demonstrated that modification of the fatty acid profile in the diets of food production animals significantly alters the lipid content and fatty acid profile of the meat [32–34] and/or eggs produced [10,35–39]. Similarly, this study demonstrates that eggs produced from older production hens (57 weeks of lay) fed a HOPN or OA diet had significantly reduced saturated fatty acid and trans-fat content with enhanced monounsaturated oleic fatty acid content as compared to conventional eggs.
