Improvements in Performance of Nursery Pigs Provided with Supplemental Oil Derived from Black Soldier Fly (Hermetia illucens) Larvae

Simple Summary Black soldier flies are a non-nuisance insect species, and their larvae can be used to efficiently convert low quality byproduct ingredients into valuable nutrient-rich biomass. The lipid fraction obtained from black soldier fly larvae contains high concentrations of lauric acid, which is highly digestible and has antimicrobial properties. The objective of the present study was to determine if black soldier fly larvae oil could improve the growth and efficiency of newly weaned pigs, which are especially compromised due to the stress associated with the weaning process. We conclude that supplemental black soldier fly larvae oil, replacing equal amounts of corn oil, increased average daily gain and improved feed efficiency, resulting in increased final pig body weight after the 40-day test period Abstract The current study evaluated the impact of increasing levels of supplemental black soldier fly larvae (BSFL) oil, a novel and sustainable feed ingredient, on growth performance and blood chemistry indices in nursery pigs. Pigs were weaned at 21 days of age (n = 192; body weight = 6.9 ± 0.15 kg) and randomly allotted within sex and body weight to 1 of 4 dietary treatments, using 48 pens (4 pigs/pen). Treatments contained 0, 2, 4, or 6% supplemental BSFL oil, replacing equivalent quantities of corn oil. A 3-phase feeding program was used with 14, 11, and 15 days for phase 1 to 3, respectively. Supplementation of BSFL oil linearly (p ≤ 0.052) improved pig body weight and daily gain throughout the study and overall. No differences were observed in feed intake; however, feed efficiency was linearly improved with BSFL oil supplementation for phase 1 and 2 (p < 0.05). Serum cholesterol increased linearly (p = 0.002) and platelet count tended to increase linearly (p = 0.082) with increasing BSFL oil. No other differences were detected in blood chemistry and all results were within normal ranges. In conclusion, BSFL oil is a valuable, energy-dense feed ingredient that can promote growth performance of nursery pigs.


Introduction
Products derived from insects can provide a sustainable alternative to traditional feed ingredients used in commercial animal production. Indeed, insects can be reared very efficiently on lower quality feed ingredients, yielding high quality protein, lipids, and energy, while at the same time using less land and water resources compared to other plant and animal sources [1][2][3]. Each insect species has its own distinct nutritional composition based on life stage, environmental factors, and diet, all of which can be controlled when rearing insects commercially [2]. The black soldier fly has great potential as an insect species for successful commercial production. They are relatively easy to grow, the adult fly does not eat and is not a vector for disease, the larval stage is very rich in nutrients, and the larvae are able to efficiently consume and convert organic materials into nutrient-rich biomass that can be recycled back into the food chain. Black soldier fly larvae (BSFL) A total of 192 crossbred barrows and gilts (Smithfield Premium Genetics, Roanoke Rapids, NC, USA; body weight = 6.9 ± 0.15 kg), weaned at 21 days of age, were randomly allotted within body weight and sex to 1 of 4 dietary treatments, using an Excel based allotment program [24]. Pigs were housed in 48 nursery pens (16 pens in each of 3 near identical nursery rooms) with 4 pigs per pen, resulting in 12 blocks per dietary treatment. For block 1 and 2, each pen had 3 barrows and 1 gilt and for blocks 3 to 12, each pen housed 2 barrows and 2 gilts per pen. Each pen of 4 pigs was subsequently evaluated for the presence of littermates. If littermates were present within a particular pen, one of the littermates was exchanged with a pig with the approximate same body weight and of the same sex from another pen within the same weight block. This process ensured that littermates were distributed between treatments (within block), rather than being located within pen. Treatment groups were randomly allocated to the experimental units (pens).
Nursery pens were 0.91 by 1.52 m in dimension with triangular steel flooring (Tri-Bar, Nooyen Manufacturing Inc., Mt. Sterling, KY, USA). Each pen contained 2 nipple water drinkers in the back of the pens and a double space stainless steel feeder located in the front of the pens (Staco, Inc., Schaefferstown, PA, USA). Lighting consisted of fluorescent lights controlled by a timer. There were six light fixtures on each side of the room, each containing two fluorescent bulbs. Lights were turned on from 6:00 a.m. until 8:00 p.m. and turned off from 8:00 p.m. until 6:00 a.m. Temperatures in the nurseries were set at 28 • C for the first 18 days, followed by a drop in temperature of 1 • C every 2 to 4 days until the temperature setting reached a low of 19 • C. Air was mixed using 2 stir fans per room. Rooms had side wall baffle systems to allow for fresh air inflow and ventilation was maintained to exceed minimum air flow requirements [25]. Manure was managed as a flush system and pits were flushed 2 times per day.
Pigs were given unlimited access to feed and water during the 40-day experimental period. Fresh feed was added to the self-feeders as needed to ensure that fresh feed was always available. Feed consumption was calculated weekly from feed added to the feeder minus feed left in the feeder at the end of the feeding phase minus waste feed. Water was provided on ad libitum basis and waterers were checked two times per day to ensure adequate water flow. Temperature and humidity were checked daily to verify proper environmental conditions were maintained. Pigs were observed twice daily for any possible signs of illness. Pigs which looked unthrifty as evidenced by rough hair coat, sunken belly, diarrhea, or weight loss, or pigs that displayed mobility problems, swollen joints, or other signs of illness were closely observed. When deemed necessary, pigs were treated with antibiotics per the advice of a licensed veterinarian. Incidences of medical intervention were recorded. If pigs did not improve after treatment, they were removed from the study. The reason for removal, prior treatments, body weight, and date of the removal were recorded.

Experimental Diets and Manufacturing
Pigs were fed 1 of 4 dietary treatments, consisting of 0, 2, 4, and 6% supplemental BSFL oil (EnviroFlight LLC, Apex, NC, USA), replacing refined edible-grade corn oil (WebstaurantStore, Lancaster, PA, USA) on a 1:1 basis. This approach assumed that the metabolizable energy (ME) content of BSFL oil was similar to corn oil in diet formulation. Diets were formulated using the National Swine Nutrition Guide formulation software [26]. Diets met or exceeded nutrient recommendations established by the NRC [27] and they were fed in 3 phases throughout the nursery (Table 1). Phase 1 diets were fed from day 0 to 14, phase 2 diets from day 14 to 25, and phase 3 diets from day 25 to 40. Diets were manufactured at the North Carolina State University Feed Mill Education Unit. Whole corn was ground using a hammer mill (Model 1522, Roskamp Champion, Waterloo, IA, USA) to pass through a 2.2 mm screen (grind size of 600 to 800 microns). To create the experimental diets, a basal mix was manufactured first, containing only dry ingredients. Dry ingredients were blended in a double ribbon mixer (Model TRDB126-0604, Hayes and Stolz, Fort Worth, TX, USA). This mix was then divided into 4 equal size batches and the appropriate levels and type of oil were then added to make the final dietary treatments. For the addition of lipids, a total of 1% oil was added to the meal first in the mixer. Corn oil was used for this purpose for all diets, except for the diet with BSFL oil replacing all the corn oil, in which case 1% of BSFL oil was added to the meal in the mixer. The appropriate remaining oil (5%) was added directly onto the pellets in a 225 kg capacity mixer for phase 1 and 2 diets (which were offered as pelleted diets) and mixed for 30 s. For the pelleting process for phase 1 and 2 diets, diets were conditioned at approximately 74 • C and pellets were produced with a pellet mill (model PM1112-2, California Pellet Mill Co., Crawfordsville, IN, USA) using a 4.4 × 25 mm die. Pellets were cooled with ambient air in a counter-flow cooler (Model VK09x09KL, Geelen Counterflow USA Inc., Orlando, FL, USA). Phase 3 diets were manufactured in meal form. For these diets, lipid sources were supplemented to the basal phase 3 mixture in an 1800 kg capacity mixer to create the final experimental diets.

Sampling and Measurements
Pigs were weighed individually at the start of the study and on day 7, 14, 21, 25, 33, and 40 to calculate average daily gain (ADG), which was then averaged by pen. Feed additions to the feeders were recorded and leftover feed in the feeders was determined at the end of each period at the same time pigs were weighed. Feed disappearance was calculated from feed added to the feeder minus feed left in the feeder minus feed removed due to spoilage. Average daily feed intake (ADFI) per pen was then calculated from feed consumed during the period divided by the total number of days for pigs within each pen. Feed efficiency was calculated as the ratio of average daily gain for each period (or phase) divided by the average daily feed intake for the period.
Blood samples were collected at the end of the nursery period (day 40) from one median weight pig per pen. Blood was collected via jugular venipuncture in plain vacuum tubes (for serum) and tubes with K 3 -EDTA (for whole blood). Serum was collected following centrifugation of blood at 1000× g for 20 min at 10 • C. Serum samples were analyzed for total protein, albumin, globulin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, γ-glutamyltranspeptidase, urea N, creatinine, glucose, Ca, P, Mg, K, Na, Cl, cholesterol, triglycerides, amylase, lipase, and creatine phosphokinase. Whole blood samples were analyzed for white blood cells, red blood cells, hemoglobin, hematocrit, platelets, neutrophils, lymphocytes, monocytes, eosinophils, and basophils. Analyses were conducted by the Antech Diagnostics laboratory (Cary, NC, USA) using an auto-analyzer (Olympus AU 5400; Olympus America Inc., Melville, NY, USA).
Subsamples of feed were collected during load-out of the feed from the feed mixer (or pellet cooler) to the bagging unit. Ten subsamples were obtained at a port in the auger line at equally spaced intervals between the beginning of load out and the end of load out. Representative samples of the mixture (1000 g) were obtained by splitting the samples from the mixer using a sample splitting device. Samples for phase 1 diets were analyzed for proximate composition, fiber, minerals, and amino acids. Samples for phase 2 diets were analyzed for proximate composition, fiber, minerals, and comprehensive fatty acid composition and samples for phase 3 diets were analyzed for proximate composition, fiber, and comprehensive fatty acid composition. All feed samples were analyzed by Midwest Laboratories (Omaha, NE, USA).

Statistical Analyses
Data were analyzed as a randomized complete block design by analysis of variance (ANOVA) using the General Linear Models procedure of SAS (SAS Inst. Inc., Cary, NC, USA) to examine the effect of BSFL oil inclusion on dependent variables. Pen served as the experimental unit for performance and pig (one pig per pen) was the experimental unit for blood analysis. Orthogonal linear and quadratic contrast comparisons were made to determine the impact of BSFL oil inclusion at 0, 2, 4, and 6% when replacing corn oil. Proper coefficients for linear and quadratic orthogonal contrasts were calculated using Proc IML. Data were expressed as least squares means and differences were deemed to be statistically significant at p ≤ 0.05 and tendencies when 0.05 < p ≤ 0.10.

Ingredient and Feed Analyses
Black soldier fly larvae oil in the present study was obtained from 2 different lots. Lot 1 was used in diets fed during phase 1 and 2. Lot number 2 was used to manufacture diets for phase 3. The analyzed fatty acid composition of both lots was very similar, indicating that variation between lots for fatty acid composition was minor ( Table 2). Total fat content determined after acid hydrolysis was 99.1 and 99.2% for lot 1 and 2, respectively. Therefore, the BSFL oil was relatively pure with very low moisture, impurities, and unsaponifiable compounds (less than 0.9 and 0.8% for lot 1 and 2, respectively).
Black soldier fly larvae oil contained high concentrations of lauric acid (C12:0), averaging 36.9 g/100 g of lipid for the two lots used in the current study. Other fatty acids that were present at high concentrations were linoleic (C18:2), palmitic (C16:0), oleic (C18:1), and myristic (C14:0) acid at concentrations of 17.3, 14.6, 13.1, and 9.8 g/100 g of lipid, respectively. The composition of the BSFL oil was very similar to the values reported previously [15]. Although the composition of BSFL can be manipulated [17][18][19][20], the conditions of rearing and dietary substrate used in commercial production of BSFL are tightly controlled as part of a quality assurance program with the aim of yielding products with a specific and consistent nutrient profile.  When manufacturing diets, we prepared a basal mix within each phase and divided this basal mix into equal portions to manufacture the final treatment diets. Thus, treatment diets within dietary phase were identical in their ingredient make-up and the nutrient composition of diets should be similar between treatments, with the exception of fatty acid composition. The proximate chemical, fiber, mineral, and amino acid composition of dietary treatments was in accordance with target nutrient values and well within expected laboratory variation. Analyzed fatty acid composition for phase 2 and 3 diets (Table 3) confirmed the proper composition of the experimental diets. phase 2 diets contained 0 (not detectable), 0.63, 1.11, and 1.79 g of lauric acid per 100 g of total fatty acids for 0, 2, 4, and 6% supplemental BSFL oil, respectively, whereas phase 3 diets contained 0.01, 0.66, 1.22, and 1.95 g of lauric acid per 100 g of total fatty acids for 0, 2, 4, and 6% supplemental BSFL oil (Table 3), respectively. This clearly demonstrates incremental increases in concentrations of lauric acid as expected, consistent with the incremental increasing levels of BSFL oil added to the experimental diets.

Growth Performance
Two pigs (0% BSFL oil treatment) were removed from the study due to failure to thrive as indicated by progressive weight loss while on test and 1 pig (4% BSFL oil) was removed due to prolapse. Four pigs (1 pig fed 0% BSFL oil and 2 pigs fed 4% BSFL oil) died during the course of the experiment, representing a death loss of 2.1%. A summary of productivity records of the pork industry showed a mortality of 3.62% in nursery pigs for commercial farms in the top 25th percentile (mean was 4.58%) [28]. Some death losses are expected and death losses in university facilities with low stocking density and good health are expected to be modest. None of the pigs in the current study were medically treated with injectable medication or oral antibiotics.  In general, pigs in the present study performed very well as indicated by a mean ADG of 532 g/d, gain:feed ratio of 762 g/kg, and a final body weight of 28.2 kg, reached in 40 days in the nursery (Table 4). This compares to a mean ADG of 444 g/d, a G:F ratio of 671 g/kg, and a final BW of 26.9 kg (after 46 days in the nursery) for the top 25th percentile of a large sample group of pork production companies [28]. Supplementation of BSFL oil, replacing corn oil, generally improved growth performance of nursery pigs. Pig body weight linearly increased with increasing BSFL oil in the diet on day 14 (p = 0.018), day 21 (p = 0.010), day 25 (p = 0.014), day 33 (p = 0.021), and at the end of the study (p = 0.052). This increase in body weight appeared to be due primarily to increased ADG early in the study as evidenced by increased ADG during day 7 to 14 (p = 0.002), phase 1 (day 0 to 14; p = 0.017), and phase 2 (day 14 to 25; p = 0.055), resulting in improved overall ADG (p = 0.048). No significant differences in average ADFI were observed due to BSFL oil inclusion, implying that BSFL oil is a palatable ingredient for nursery pigs. This agrees with studies feeding whole, live BSFL to weaned pigs showing high palatability of BSFL [29,30]. Improvements in ADG without changes in ADFI resulted in improved gain:feed ratio (feed efficiency) during day 7 to 14 (p < 0.001), day 14 to 21 (p = 0.054), phase 1 (day 0 to 14; p < 0.001), and phase 2 (day 14 to 25; p = 0.049). The impact of feeding BSFL oil in pigs has not been previously published. In other species, potential effects of BSFL oil have been reported, showing no benefits when fed to broilers, turkey, quail, and rabbits when BSFL oil partially or totally replaced conventional lipid sources, including coconut oil, corn oil, soybean oil, and palm oil [15]. Recent studies confirm these observations. In laying hens, substitution of 50 or 100% of soybean oil with cold-pressed BSFL oil did not impact productive performance [31]. In a similar study Kieronczyk et al., (2022) reported no effects of cold-pressed BSFL oil replacing 50 or 100% of soybean oil on growth performance of young turkeys [32]. Although no peer-reviewed publications reporting effects of BSFL oil in pigs could be found, studies using full-fat BSFL in diets for pigs may provide some insight into potential impacts of oil derived from BSFL. In nursery pigs, feeding of 1, 2, or 4% full-fat BSFL meal in replacement of fishmeal and soybean oil (up to 1.15%) linearly improved ADG and feed efficiency [33]. In another study [34], no beneficial effects were noted on nursery pig performance when full-fat BSFL were included at up to 19.1%, replacing soybean meal, soy protein concentrate, fish meal, and rapeseed oil (BSFL meal contributed up to 69% of the total dietary fat). The digestible energy (DE) and ME contents in full-fat and defatted BSFL have been shown to be high, with full-fat BSFL providing more DE (4.93 Mcal/kg) and ME (4.57 Mcal/kg) than defatted BSFL (3.94 and 3.40 Mcal/kg for DE and ME, respectively) [11]. Clearly, impacts of additional oil from full-fat BSFL meal on pig performance cannot be distinguished from the potential impact of the protein or chitin fraction of BSFL meal. Collectively, reported data generally show no clear positive effects of BSFL oil on productive performance, which is in contrast with results reported in the present study.
Growth rate of pigs fed BSFL oil in the current study was improved by 18.1% during phase 1, and 5.6% during phase 2, resulting in improved overall ADG and increased body weight at the end of the nursery period. The improved ADG immediately after weaning is especially noteworthy considering that this is an important transition period that correlates with subsequent growth [35,36]. The performance benefits observed in the current study may be related, in part, to the high concentration of lauric acid in BSFL oil. Lauric acid is amongst the medium-chain fatty acids that are generally defined as having chain lengths of 6 to 12 carbon atoms.
Lauric acid has been reported to exhibit antimicrobial activity to bacteria, especially Gram-positive bacteria, and enveloped viruses and has demonstrated immunomodulatory properties [21][22][23]. Medium-chain triglycerides are digested and absorbed more efficiently than long-chain triglycerides and are transported directly to the liver via the portal vein for preferential β-oxidation to produce energy [23]. The rapid absorption of medium-chain fatty acids may preclude sufficient quantities to be present in the gastrointestinal tract to exert antimicrobial effects [37][38][39]. Thus, the use of medium-chain fatty acids bound as part of triglycerides, rather than free fatty acids, may be beneficial, although this ultimately depends on sufficient endogenous lipase activity for the hydrolysis of triglycerides [37,38].

Serological and Hematological Indices
Serum chemistry and complete blood count data were collected to verify normal functioning and health of pigs (Tables 5 and 6). All values were within expected and acceptable ranges [40] and were consistent with our previously published results in growing pigs [41]. Supplementation of BSFL oil during the 40-day experimental period did not significantly alter serum chemistry or complete blood counts, which agrees with studies in poultry [32,42,43]. The only exception was cholesterol. Serum cholesterol concentrations linearly (p = 0.002) increased with increasing BSFL oil in the diet. Cholesterol is naturally present in BSFL oil, whereas corn oil does not contain cholesterol, and may have directly contributed to increased serum cholesterol concentrations. Harris et al., (2003) reported increased plasma cholesterol and low-density lipoprotein cholesterol concentrations in pigs fed tallow or coconut oil compared to corn oil; however, no differences were detected in muscle [44]. In young turkeys, partial or total replacement of soybean oil by BSFL oil increased serum concentrations of cholesterol and low-density lipoprotein cholesterol but did not affect cholesterol concentrations in breast meat or leg meat of turkeys [45]. On the other hand, no differences in serum cholesterol concentrations were detected when soybean oil was partially or totally replaced with BSFL oil in the diets of broilers [32,42,43]. Kim et al., (2022) observed no differences in cholesterol concentration of eggs from hens supplemented with BSFL oil in replacement of soybean oil [31]. The serum cholesterol concentrations reported in the current experiment were within expected published ranges for nursery pigs [40] and are unlikely to impact cholesterol concentrations in final edible products.

Conclusions
Results of the present study indicated that increasing supplemental BSFL oil from 0 to 6% in increments of 2% increased average daily gain and improved feed efficiency of nursery pigs in a linear manner, especially early after weaning, resulting in increased final body weight after the 40-day experimental period. Serum chemistry and total blood count were within normal ranges for young pigs suggesting that health status was not impacted by BSFL oil. Serum cholesterol linearly increased with BSFL oil supplementation, which may be related to the fact that BSFL oil contains some cholesterol, whereas corn oil does not, and BSFL oil contains cholesterogenic fatty acids (similar to coconut oil and palm kernel oil). In conclusion, BSFL oil is a valuable, energy-dense feed ingredient that can be successfully fed to nursery pigs, while improving their growth performance. Informed Consent Statement: Not applicable.

Data Availability Statement:
Data associated with the current study are available from the corresponding author upon reasonable request.