Assessment of Novel Protein Ingredient Arthrospira platensis and Soybean Genotype Amino Acid and Oil Selection Improvements on Broiler Performance for a 28–42 d Feeding Period †

: Two experiments were conducted to assess the efficiency of including the novel protein ingredient Arthrospira platensis or improved soybean meal in a broiler diet. The first experiment aimed to determine the feeding value of soybean meal produced from varieties of soybeans bred for increased amino acid content (SBAA) and improved oil content (SBO) compared to a conventional soybean variety in an ANOVA design fed to Cobb 500 female broilers for 28–42 d. The SBAA and SBO soybeans contained overall higher amino acid content and lower oligosaccharide content compared to the conventional soybean variety in addition to improved oil quality. The second experiment assessed the novel protein ingredient microalgae, Arthrospira platensis (algae), and was conducted to evaluate algae and corn distillers’ grain (DDGS) inclusion on broiler performance for a 28–42 d feeding period in Cobb CF05 male broilers with a 2 × 2 factorial treatment array. Prior to the experimental period, all birds were reared on common feeds. In Experiment 1, birds were fed a diet containing 20% inclusion of an experimental soybean source in the form of full-fat soybean meal. In Experiment 2, the four dietary treatments consisted of diets containing algae at inclusion levels of either 0 or 2% and DDGS at inclusion levels of 0% and 8%. Diets were fed to 288 female broilers (Experiment 1) and 384 male broilers (Experiment 2), placed in eight replicate pens of twelve birds, and live performance was assessed from d 28 to 42. At d 42, six birds from each pen were randomly selected and processed for evaluation of carcass traits and incidence of woody breast. For Experiment 1, all performance data were analyzed using a one-way ANOVA using JMP Pro 16 software with diet as the fixed effect and block as a random effect. Statistical significance was considered at p ≤ 0.05. No significant responses were observed for any recorded measurement for live performance, carcass traits, or woody breast. All data in Experiment 2 were analyzed as a full factorial with a mixed model using JMP software with algae, DDGS, and algae × DDGS as fixed effects and block as a random effect. The F-protected Fisher’s LSD test was used to separate means when p ≤ 0.05. No significant responses were observed for the algae, DDGS, and algae × DDGS influences on BWG, FI, and FCR or processing characteristics; the ingredient source did not affect bird performance. Experimental soybean lines developed at the University of Arkansas were able to be incorporated into broiler diets without decreasing performance. Algae has the potential to be a protein-contributing ingredient for broilers.


Introduction
Live production represents half of the broiler integrator cost, with feed approximating three-quarters of live production costs and protein-contributing ingredients representing the highest dietary costs.The high costs associated with ingredients supplying protein provide excellent motivation for researchers to investigate novel and alternative protein-contributing ingredients.Moreover, the global population is predicted to reach 9.7 billion by 2050 [1].Global poultry production has already increased by approximately 700 percent since 1967 (139.2 million tons in 2022) [2] and will continue to increase to nearly 200 million tons by 2050 to keep up with the growing population and consumer demand [3].This will inevitably cause strain on nutritionists to meet the protein needs of poultry as soybean production would also need to increase by 200 percent to meet the feed production requirements of swine and poultry [4].To assist in filling this "protein gap", multiple methods will need to be implemented, including lowering diet crude protein, increasing protein digestibility, and incorporating alternative protein sources in the diet [5][6][7][8].Two experiments were conducted to assess the usefulness of including certain novel or improved protein ingredients in a broiler diet.The first experiment investigated the use of full-fat soybean meal produced from soybeans with genotype selections for improvement in amino acid content (SBAA) and oil quality (SBO), whereas the second experiment assessed the novel protein ingredient Arthrospira platensis (algae).
Soybean meal makes up the largest proportion of protein-contributing ingredients in poultry diets in most countries.The popularity of soybean meal in U.S. poultry diets is likely due to soybean's duality as a protein ingredient and an oil producer, its high quality as a protein ingredient, and its ability to be grown in the United States.Advances in plant breeding and genetic engineering have resulted in many new soybean varieties.Some of these varieties were bred to result in improved amino acid and protein contents [9,10], improved fatty acid and oil quality [11,12], reduced oligosaccharides [13][14][15], and reduced trypsin inhibitors [16][17][18][19].The experimental soybeans used in this study were bred to have improved oil quality (e.g., high oleic, low linoleic) and increased amino acid content.Soybeans with elevated proportions of oleic acid are particularly desirable for food applications because oils from these soybeans have greater oxidative stability, which contributes to improved frying performance [20,21].Moreover, decreased linoleic content is desirable because it does not require hydrogenation [22].Previous studies that investigated the use of improved-oil-quality soybeans in poultry diets used transgenic soybeans [11,12], whereas this study utilized conventionally bred soybeans.Moreover, soybeans used in this study were incorporated into the diet in the form of full-fat soybean meal, which differs from previous work in which the soybeans used were in the form of meal [11] or separated fractions of meal, hulls, and oil [12].Prior research on high-protein soybeans focused on determining the digestibility of genetically modified high-protein soybeans using a cecectomized rooster assay where SBM and excreta were assayed for Kjeldahl nitrogen, energy, amino acid concentrations, and sulfur amino acids [9].However, this research focuses on the use of conventionally bred full-fat soybean meal as a portion of the diet in broiler growth trials.
When investigating novel ingredients, it is important to not only consider protein quality, digestibility, effects on performance, and additional benefits but also interactions with other ingredients.Arthrospira platensis (algae) is a species of blue-green cyanobacteria microalgae that is gaining popularity as a novel protein ingredient.It consists of over 60% crude protein and is considered an excellent source of essential fatty acids [23,24].Typical inclusion levels for algae range from 1 to 10%.Higher inclusion levels of 12 and 15% have been reported to cause a drop in broiler performance, which is likely caused by gelation (increased digesta viscosity) in the gut by indigestible proteins in algae [7,25].In previous research conducted at the University of Arkansas, increased feed conversion (FCR) was observed when algae and distillers' dried grain with solubles (DDGS) were both included in a diet [26].
Therefore, the objective of Experiment 1 was to determine the feeding value of soybean meal produced from varieties of soybeans bred for increased amino acid content (SBAA) and improved oil content (SBO) compared to a conventional soybean variety by incorporating it into diets fed to broiler chickens.The null hypothesis of this experiment was that soybean variety would not impact broiler performance, with the alternative hypothesis being that soybean variety would impact broiler performance.Moreover, the objective of Experiment 2 was to further investigate algae and DDGS interrelationships in practical diets for broilers.The null hypothesis for this experiment was that the protein ingredients would not impact broiler performance, with the alternative hypothesis being that the protein ingredients would impact broiler performance.

Bird Husbandry
In both experiments, broiler chicks were obtained from a commercial hatchery (Siloam Springs, AR) where they received Marek's vaccinations and were vent-sexed on the day of hatch prior to being transported to the University of Arkansas Broiler Research Farm.Upon arrival, chicks were placed in floor pens measuring 3 ′ × 4 ′ at an allocation of 12 birds per pen with 8 replicates for a total of 24 and 32 floor pens for Experiments 1 and 2, respectively.Each pen was equipped with a hanging feeder, a section of a continuous nipple drinker line (5 nipples per pen), and used litter that was top-dressed with new pine shavings.Initial temperature set points were set at 32.2 • C and gradually reduced to 18.3 • C at the conclusion of the experiment (day 42).Water flow rates were set at 21 mL per minute and increased by 7 mL per minute weekly every week, ending at 56 mL per minute.
Lighting schedules differed slightly for the two experiments.In Experiment 1, the lighting schedule was set for 24 L:0 D for day 1, 23 L:1 D from days 2 to 7, and 18 L:6 D from days 8 to 42, with light intensities initially set for 54 lux for days 1 to 7, 32 lux from days 8 to 18, 22 lux from days 18 to 24, and 16 lux from days 24 to 42 using 40-watt lightemitting diode bulbs.In Experiment 2, the lighting schedule was set for 24 L:0 D for days 0 to 21 and 18 L:6 D from days 22 to 42 with light intensities initially set for 54 lux for days 1 to 7, 27 lux from days 8 to 21, and 22 lux from day 22s to 42 using 40-watt light-emitting diode bulbs.Light intensities were verified at the bird level using a light meter (LT300, Extech Instruments, Waltham, MA, USA).
A total of 288 female Cobb 500 broiler chicks and 384 male Cobb CF05 broiler chicks were utilized for Experiments 1 and 2, respectively.The sex of the birds used in the experiments was determined by the availability of chicks at the time of the trial.The use of only one sex in Experiments 1 and 2 was utilized in order to limit variability in responses due to differences in performance between male and female broilers.In Experiment 1, a common starter diet was offered as crumbles from days 0 to 18, and a common grower diet was offered as pellets from days 18 to 28.In Experiment 2, a common starter diet was offered as crumbles from days 0 to 14, and a common grower diet was offered as pellets from days 14 to 28.Birds were fed experimental diets from 28 to 42 d in both experiments.

Experimental Diets
For Experiment 1, two experimental varieties of soybeans developed for amino acid and oil characteristics at the University of Arkansas were planted and grown on the University of Arkansas Department of Agriculture research farm in Stuttgart, Arkansas, in summer 2021.Following harvest, 113 kg of the increased-amino-acid-content soybean variety (SBAA) and 172 kg of the improved-oil-quality soybeans (SBO) were obtained, and whole unprocessed beans were analyzed by NIR in replicates of three.Additionally, the conventional soybeans sourced by Insta-Pro were analyzed by NIR with 10 replicates; these values as well as the NIR values from the experimental soybeans can be found in Table 1.Due to the quantities obtained, it was decided that the soybeans would be processed into full-fat soybean meal to preserve quantity.Soybeans were processed by Insta-Pro International in Grimes, IA, alongside conventional soybeans sourced by Insta-pro.Prior to processing soybean treatments, between 227 and 454 kg of conventional soybeans were sent through processing equipment to warm up the machine to 320 F (160 degrees C) to promote proper antinutrient deactivation and nutrient digestibility.All soybean treatments (SBAA, SBO, and conventional) were processed by high-shear dry extrusion, which is a process whereby beans are subjected to a high temperature (320 F, 160 C) for 3-5 s to limit amino acid damage.Following extrusion, the soybean meal was cooled prior to storage to ensure no further cooking.The final quantities of processed full-fat soybean meal were 91 kg of SBAA, 79 kg of SBO, and 603 kg of conventional soybeans.The resulting experimental full-fat soybean meal, along with other ingredients used in experimental diets including corn, soybean meal, and DDGS, was submitted for total amino acid and proximate analysis prior to formulation (Novus International Inc., St. Charles, MO, USA) (Table 2).Analysis procedures followed AOAC methods 942.05 (ash), 930.15 (dry matter), 920.39 (fat/oil), 990.03 (crude protein), 978.10 (crude fiber), 982.30(amino acids by hydrochloric acid), 994.12 (amino acids by performic acid), and 988.15 (amino acids by sodium hydroxide).
Despite having significantly different values for nutritional components as whole soybeans, once processed into full-fat soybean meal, proximate and AA analysis values were very similar for conventional and experimental FF-Soybean meals.Therefore, diets were formulated based on analysis values from the conventional FF-SBM, and the inclusion amount of experimental FF-SBM was set to 20% in all diets (Table 3).Furthermore, this eliminated the effect of other ingredients on performance results as the inclusion of all other ingredients was also held constant.The decision to set experimental soybean inclusion at 20% was made based on the calculated consumption of diets by birds during the 28-42 d feeding period and the recommended 25% inclusion limit of full-fat soybean meal [27].  The mineral premix supplied (per kg of diet) manganese, 100 mg; zinc, 100 mg; copper, 15 mg; iron, 15 mg; iodide; 1.2 mg; selenium, 0.25 mg. 5 Coccidiostat contributed (per kg of diet) 0.03 g of Salinomycin Sodium provided through BioCox ® . 6Sourced by Insta-Pro International ® .

Measurements
For the determination of diet's effect on live performance results, pen weights were collected at the start and conclusion of each experimental period.Feed intake was recorded for the duration of the experimental periods.Prior to placing the feed, the weight of the empty feeder was recorded.The weight of the feed placed was recorded and any additional feed was also weighed.Mortality was collected twice daily, and the weights of dead birds were recorded.All weights were obtained using a Mosdal Small Cart scale (Mosdal Scale Systems, Inc., Lanesboro, MN, USA).Individual BW gain was recorded by subtracting the initial from final pen weights and dividing by the number of birds.The feed conversion ratio, representing g of feed intake to g of BW gain, was calculated by dividing pen feed intake by the summation of pen BW gain and correcting for mortality weight for each pen.
Following the final pen weigh, six birds per pen were randomly selected and tagged for the determination of carcass traits.On day 43, 144 and 192 broilers were processed from Experiments 1 and 2, respectively.Birds were transported to the University of Arkansas Pilot Processing Plant following an overnight feed withdrawal (10 h).Broilers were individually weighed (GSE Scale Systems Model 460), hung on shackles, electrically stunned (11 V, 11 mA for 11 s), and exsanguinated.Broilers were then scalded (55 C for 2 min), defeathered, and mechanically eviscerated.Carcass fat (fat pads) was collected according to Cabel et al. [28].Hot carcass and fat pad weights were recorded (Mettler Toledo IND236), and carcasses were submerged in plastic containers filled with ice water for a four-hour carcass chill.Carcasses were then deboned on a single debone line to obtain part weights (Mettler Toledo IND236), which included two fillets (pectoralis major), two tenderloins (pectoralis minor), wings, and legs, as shown in Figure 1.Carcass and part yields were calculated using the weight of various cuts divided by the day 43 fasted live BW taken immediately prior to the slaughter of birds, averaged by pen.Following deboning, breast fillets were evaluated for the incidence and severity of woody breast.One individual subjectively scored breast fillets via tactile evaluation on a scale of 0 to 2. Breast fillets with a score of 0 exhibited no hardness in the caudle region of the breast fillet, breast fillets with a score of 1 exhibited hardness in the cranial and caudle regions of the breast fillet but remained flexible in the mid region of the fillet, and breast fillets with a score of 2 exhibited stiffness throughout the fillet including the mid region.

Statistical Analyses
Pens were considered the experimental units, and treatments were assigned to pens in a randomized complete block design with pen location serving as the blocking factor.All treatments were represented by eight replicate pens of 12 birds each.
For Experiment 1, all performance data were analyzed using a one-way ANOVA using JMP Pro 16 software with diet as the fixed effect and block as a random effect.Statistical significance was considered at p ≤ 0.05.Soybean NIR data results were analyzed using a one-way ANOVA with soybean type as a fixed effect.Statistical significance was consid-

Statistical Analyses
Pens were considered the experimental units, and treatments were assigned to pens in a randomized complete block design with pen location serving as the blocking factor.All treatments were represented by eight replicate pens of 12 birds each.
For Experiment 1, all performance data were analyzed using a one-way ANOVA using JMP Pro 16 software with diet as the fixed effect and block as a random effect.Statistical significance was considered at p ≤ 0.05.Soybean NIR data results were analyzed using a one-way ANOVA with soybean type as a fixed effect.Statistical significance was considered at p ≤ 0.05.Means were separated using Tukey's HSD when appropriate.
For Experiment 2, all data were analyzed as a 2 × 2 factorial design with a mixed model using JMP Pro 16 software with algae, DDGS, and algae × DDGS as fixed effects and block as a random effect.Statistical significance was considered at p ≤ 0.05.Means were separated using Tukey's HSD when appropriate.

Soybean Experiment
Analyzed NIR values for whole soybeans reached statistical significance for nearly every analyzed value (Table 1).SBAA and SBO were both significantly higher in moisture, protein, alanine, arginine, glutamic acid, glycine, histidine, isoleucine, leucine, phenylalanine, proline, serine, threonine, valine, and verbascose percentages compared to the conventional soybeans.Moreover, SBAA and SBO were both significantly lower in linoleic acid, fiber, ADF, raffinose, and sucrose percentages compared to the conventional soybeans.Conventional soybeans were highest in oil, linoleic acid, NDF, methionine, and stachyose percentages, while SBAA was the lowest for these values and SBO fell in the middle.SBO was highest in oleic acid and aspartic acid percentages, with conventional soybeans having the lowest percentage of these components and SBAA being intermediate.Conversely, conventional beans were highest in linolenic and ash percentages, with SBO having the lowest percentage and SBAA falling in the middle.SBAA was highest in fructose and glucose percentages, with conventional soybeans having the lowest percentage and SBO falling in the middle.Lastly, conventional and SBAA soybeans were both significantly higher in tryptophan than the SBO soybeans.
Live performance responses are presented in Table 7.No significant differences were observed for soybean variety on live performance.Carcass characteristics and woody breast data are displayed in Tables 8 and 9. Yields represent chilled carcass parts relative to live body weight.Woody breast incidence is the average WB score for the treatment.Woody breast severity is the percentage of each of the scores within a treatment.Consider that woody breast is scored as a 0, 1, or 2. No significant responses were observed for soybean variety on processing yields or the incidence and severity of woody breast.  1 Abbreviation for soybeans bred to have increased amino acid content. 2 Abbreviation for soybeans bred to have improved oil quality. 3Abbreviation for woody breast incidence; WBI is the average woody breast score for the treatment. 4Abbreviation for woody breast severity; WBS is the percentage of each of the scores within a treatment.Woody breast can be scored as a 0 (no hardness), 1 (some amount of hardness in cranial region of breast fillet), or 2 (hardness throughout both cranial and caudal regions of breast fillet).

Algae Experiment
The analyzed total amino acid levels for the experimental diets were in close agreement with calculated total levels and remained similar in all diets.The average BW at day 42 was 3.518 kg, with an average mortality of 1.85% for the experimental period (i.e., 28-42 days).Mortality was not influenced by the main effects of algae, DDGS, or the algae x DDGS interactive effect (p > 0.05).
No significant responses were observed for any recorded measurement for live performance, carcass traits, or woody breast.Live performance responses are presented in Table 10.Carcass characteristics and woody breast data are displayed in Table 11 and Table 12, respectively.

Discussion
As the world population continues to increase, we must continue to improve our agriculture production systems in order to promote sustainability and provide enough product to meet the growing demands of consumers.In this manuscript, two experiments were conducted that assessed novel or alternative protein ingredients.Experiment 1 assessed soybean meal sources produced from varieties of soybean bred to have improved nutritional content.The objective of this experiment was to determine whether soybean meal produced from varieties of soybeans with higher amino acid content could be added to broiler diets and assess their impact on live performance, carcass characteristics, and breast myopathies.Moreover, Experiment 2 investigated the novel protein ingredient Arthrospira platensis (algae).In previous research conducted at the University of Arkansas, increased feed conversion (FCR) was observed when algae and distillers' dried grain with solubles (DDGS) were both included in a diet [26].Therefore, the objective of Experiment 2 was to further investigate algae's interactions with DDGS and assess the impact of both ingredients on broiler live performance, carcass characteristics, and breast myopathies.

Soybean Experiment
Overall, it can be summarized that the SBAA and SBO soybeans contained higher amino acid content and lower fiber and oligosaccharide content compared to the conventional soybeans.Moreover, the SBAA and SBO soybeans were lower in linoleic acid and higher in oleic acid content than the conventional soybeans, with SBO soybeans having a higher oleic acid content than the SBAA.These results demonstrate that the breeding objectives set by soybean geneticists at the University of Arkansas to produce soybeans with higher amino acid content and a better oleic/linoleic ratio were successful.
Birds fed diets containing the improved soybean lines had a decreased (p = 0.183) feed conversion ratio by 13 and 15 points for SBAA and SBO, respectively.The improved soybean meal is from breeding lines, and the increased amino content and decreased fiber and oligosaccharide content of the experimental soybean meals will improve as lines are further developed in the coming years.It is generally accepted that as protein levels increase, FCR decreases.Moreover, besides sucrose, the carbohydrate fraction of SBM is poorly used by poultry due to a lack of endogenous galactosidase and the low fermentative capacity of the gastrointestinal tract [29,30].Galactooligosaccharides (raffinose, stachyose, and verbascose) constitute between 5 and 7% of SBM on a DM basis [31,32] and are poorly digested because monogastric animals do not produce endogenous α-1,6 galactosidase necessary for GAL's hydrolysis into its constituent monosaccharides [29].Moreover, it should be noted that we only incorporated improved lines at a 20% inclusion.It is likely that if these soybean varieties had been processed into defatted soybean meal, either mechanically or through solvent extraction, and included in higher amounts, FCR differences may have occurred.Soybeans are composed of 18-22% oil; when that oil is removed, soybean meal is produced and differences in amino acids and fiber content are increased as concentrations of these components are subsequently increased.
These data demonstrate that overall bird live performance and carcass traits were not affected by soybean variety.Experimental soybean lines developed at the University of Arkansas were able to be incorporated into broiler diets without decreasing performance.Future studies should aim to process experimental soybean varieties produced at the University of Arkansas into defatted soybean meal prior to diet incorporation as this would be more similar and applicable to soybean products used in commercial diets.Moreover, processing into defatted soybean meal would also allow for the experimental soybean ingredients to be incorporated into the diet as the primary protein-contributing ingredient, allowing for a least-cost diet economic analysis.

Algae Experiment
These data demonstrate that overall bird live performance and carcass traits were not affected by the ingredient sources of DDGS or algae.There was no significant effect detected for the influence of algae, DDGS, or an interaction of the two ingredients on broiler live performance, carcass characteristics, or breast myopathies.Therefore, algae has the potential to be a protein-contributing feed ingredient for broilers at inclusion levels of 2% of the diet.Although its inclusion levels are often limited to 10% due to higher inclusion levels of 12 and 15% being reported to cause a drop in broiler performance, these data agree with previous studies' findings that it can be utilized at low inclusion levels without decreasing performance [7,25].

Table 1 .
NIR values for nutritional components of experimental soybean varieties.
Means within a row that share a common superscript cannot be separated by p ≤ 0.05.1NIRvalues of unprocessed soybean seeds.2Abbreviationforsoybeansbred to have increased amino acid content.3Abbreviationfor soybeans bred to have improved oil quality.

Table 2 .
Analyzed proximate and amino acid contents of ingredients (Experiment 1).
1Abbreviation for full-fat soybean meal.2Abbreviation for soybeans bred to have increased amino acid content.3Abbreviationfor soybeans bred to have improved oil quality.

Table 3 .
Diet formulation for experimental diets fed for a 28 to 42 d feeding period (Experiment 1).

Table 4 .
Analyzed nutritional components of experimental diets fed from 28 to 42 d (Experiment 1).
1Abbreviation for soybeans bred to have increased amino acid content.2Abbreviationfor soybeans bred to have improved oil quality.

Table 5 .
Analyzed proximate and amino acid contents of ingredients (Experiment 2).

Table 7 .
Influence of soybean variety on broiler live performance for a 28 to 42 d feeding period.Abbreviation for soybeans bred to have increased amino acid content.2Abbreviationfor soybeans bred to have improved oil quality.

Table 8 .
Influence of soybean variety on carcass yields for a 28 to 42 d feeding period.Abbreviation for soybeans bred to have increased amino acid content.2Abbreviationforsoybeansbred to have improved oil quality.3Yieldsrepresent chilled carcass parts relative to live BW.

Table 9 .
Influence of soybean variety on incidence and severity of woody breast.

Table 10 .
Influence of algae and distillers' dried grain with soluble inclusion on broiler live performance for a 28 to 42 d feeding period.

Table 11 .
Influence of algae and distillers' dried grain with soluble inclusion on carcass yields for a 28-42 d feeding period.
1Yields represent chilled carcass parts relative to live BW.2Abbreviation for distillers' dried grain with solubles.

Table 12 .
Influence of algae and distillers' dried grain with soluble inclusion on incidence and severity of woody breast.