Fish meal and soybean meal are the main protein sources for weanling piglet nutrition. However, their price has been fluctuating due to the increase in the human population and the increase in protein sources demand . Therefore, there is a high demand to detect sustainable alternative protein sources to guarantee the livestock industry. Besides, there is annually a total of 1.3 billion tons of wasted food worldwide, which considers around thirty percent of the total food production for humans . The production of insects from organic refuses seems to be an environmentally friendly procedure to introduce a sustainable high-quality protein source. The insect has already been concerned as an appealing protein source for the livestock industry due to the nutritional value and sustainability [3,4] but the limited production rate and high cost of production restricted their use in animal diets.
The mass production, rapid life span, and application of organic side streams are the most important features of insects to be used industrially, and in this case, three types of insects including black soldier fly and mealworm have been reported as suitable feed sources [5,6]. Mealworms can be produced from organic wastes, such as expired feeds and food wastes, and the high protein and trace mineral content of mealworms are comparable to the fish meal and soybean meal . Veldkamp et al.  reported that the mealworm contained high crude protein and fat which ranges from 44% to 69%, and 23% to 47% respectively. In livestock, several studies reported the dietary mealworm affected positively on growth performance, immunity, and digestibility in pig, broiler, and fish [8−10]. This evidences would suggest that the mealworms can consumed as an alternative high-quality protein feed source regionally or selectively in livestock’s diet, and more information about the results using livestock fed mealworm was required for universal use of mealworm as a sustainable protein feed source in responding to food security.
The hypothesis of replacing fish meal with mealworm meal in weanling piglet’s diets should be evaluated by comparing the nutritional assessment as well as the functional effects of fish meal, particularly that there is a lack of information on dietary mealworm in weanling piglet. Therefore, this study is designed to evaluate the influence of Tenebrio molitor (TM) meal in diets on the growth rate, immune response, and intestinal integrity in weanling piglet.
MATERIALS AND METHODS
The experimental design for the current study was permitted by the Committee of Institutional Animal Care and Use of Kangwon National University, Chuncheon 24341, Korea.
Defatted TM meal with approximately 6% crude fat content (as fed) was purchased from a commercial manufacturer (Se-in resource, Anseong, Korea). The TM was fed cereal by-products. The produced larvae were dried by an oven (20 h at 60°C temperature) and ground afterward to prepare the TM meal.
A total of 180 weanling pigs (Landrace × Yorkshire × Duroc) with an average initial bodyweight (BW) of 6.27 ± 0.15 kg (age: 21 d) were distributed between 3 treatments based on the BW. Each treatment included 6 replicates with 10 piglets per replicate. The diets corresponding to treatments consisted of a fish meal diet (control, CON), a fish meal–dried mealworm diet (TM1, 50% replacement of fish meal with TM meal), and a dried mealworm diet (TM2, 100% replacement of fish meal with TM meal). The mash form diets were prepared for pigs in 2 phases including phase 1 (d 0 to14) and phase 2 (d 15 to 28). The diets met the standard of NRC  nutrient requirements (Table 1). The pens were equipped with a partially slatted concrete floor (3.0 m × 3.0 m). The average temperature was adjusted at 30°C at the start of the study and was gradually reduced to 25°C on d 8 onward. The humidity of the barn was ranged from 65% to 70%. The infrared heating lamp, low-pressure nipple drinker, and self-feeder were equipped in all the pens to provide constant light and ad libitum access to water and feed.
1) Supplied per kg diet: 9,600 IU vitamin A, 1,800 IU vitamin D3, 24 mg vitamin E, 1.5 mg vitamin B1, 12 mg vitamin B2, 2.4 mg vitamin B6, 0.045 mg vitamin B12, 1.5 mg vitamin K3, 24 mg pantothenic acid, 45 mg niacin, 0.09 mg biotin, 0.75 mg folic acid, 18 mg ethoxyquin.
2) Supplied per kg diet: 162 mg Fe, 96 mg Cu, 72 mg Zn, 46.49 mg Mn, 0.9 mg I, 0.9 mg Co, 0.3 mg Se.
The BW check was measured for all the pigs at the beginning of the experiment, on day 14, and day 28. Feed intake (ADFI), gain to feed ratio (G:F), and average daily gain (ADG) were measured at the end of each phase. The calculation of ADFI and ADG was performed by dividing the BW and ADFI of the pens by the number of animal days (considering the BW and ADFI of the mortalities). The chromium oxide (2.5 g/kg) was used as an inert marker and mixed with the diets during the last six days in each phase for the measurement of apparent total tract digestibility (ATTD) of nutrients. A 100 g fecal sample was taken in the last 3 days in each phase, from each of the four selected pigs with a BW around the average BW of the cage. An air forced oven was applied to dry the pooled fecal samples for 72 h at 60°C and ground by a mill (Thomas Model 4 Wiley®Mill, Thomas Scientific, Swedesboro, NJ, USA) equipped with a 1-mm screen to prepare the final samples to be used for chemical analysis. Two pigs around the average weight of the pen were selected for blood sampling (jugular vein) using Becton Dickinson anticoagulants-free disposable vacutainer tube (Franklin, NJ, USA). The serum samples were divided after centrifugation for 15 min (3,000×g at 4°C) and kept at −20°C to be stored for the analysis of hematological parameters. Two piglets per cage (around the average BW) were selected to evaluate the treatment effects on small intestinal morphology. The selected pigs were euthanized by electrocution at d 28 and the collected samples from the duodenum, jejunum and ileum of the small intestine were cleaned and washed by physiological saline. After the throughout washing, a fixative solution including 20 g/L paraformaldehyde, 15 g/L acrolein, and 30 g/L glutaraldehyde was used for submerging the samples at pH 7.3 (0.1 M collidine buffer), and then the final samples were used for the morphological integrity evaluation.
The analysis of fece and diet samples were evaluated in triplicate for crude protein (CP, method 990.03; AOAC ) and dry matter (DM, method 930.15; AOAC ). The bomb calorimeter (Model1261, Parr Instrument, Moline, IL, USA) was used for the measurement of gross energy (GE) of feces and diets, and a spectrophotometer (Jasco V-650, Tokyo, Japan) was applied to determine the chromium concentration. The blood samples were analyzed by Hemavet® Hematology System (Drew Scientific, Oxford, CT) to determine the concentrations of leukocytes including white blood cells (WBC), lymphocytes, red blood cells (RBC), and monocytes. Immunoglobin G (IgG), IL-1β, TNF-α, and IL-6 were evaluated using ELISA kit (Cusabio Biotech, Wuhan, China).
The histomorphological evaluation in different sections of intestine including jejunum, duodenum, and ileum was performed as the method suggested by Hosseindoust et al. . In short, azure A and eosin were applied for the cross-sections of jejunum, duodenum, and ileum by standard dehydration procedure through paraffin embedation. A total of ten crypt-villus units in triplicate were used for intestinal cross-sections samples. The villus height (VH) and Crypt depth (CD) were determined from the tip of the villi to the crypt junction and the depth of the invagination between adjacent villi, respectively. An image processing system (Optimus software version 6.5) was used for all intestinal morphological parameters (Media Cybergenetics, North Reading, MA, USA).
In this trial, results were analyzed using the GLM procedure (SAS Institute, Cary, NC, USA). The significant statistical differences were evaluated among the treatments, and were divided by Tukey’s HSD tests. For growth rate, the pen was considered as an experimental unit. The mean of two pigs was considered as an experimental unit for the analysis of nutrient digestibility, blood parameters, and intestinal integrity. The α level of statistical significance was 0.05.
The weanling pigs were healthy and no signs of illness were detected during the whole experiment, and there was no mortality in all the treatments. The result of the growth rate of the weanling pigs is shown in Table 3. Replacing a fish meal with dried mealworm did not affect the ADG and ADFI of weanling pigs (p > 0.05) at phase 1. However, pigs in the TM2 treatment showed a greater G:F ratio compared with the TM1 pigs (p < 0.05). At phase 2, there was no significant difference between three dietary treatments. Throughout the experimental period, ADG of the TM1 pigs was greater than that of the TM2 treatment (p < 0.05).
The nutrient digestibility and fecal score results are presented in Table 4. The ATTD of GE, DM, and CP did not change by the treatments. However, a numerical increase was detected in the TM1 treatment for the ATTD of DM (p = 0.081) and GE (p = 0.078) during the first phase.
1) Fecal scores: 1, hard feces; 2, slightly soft feces; 3, soft and partially-formed feces; 4, loose and semiliquid feces; 5, watery feces.
The WBC, RBC, lymphocyte, and monocyte were not affected (Table 5). However, the IgG concentration was increased in the TM1 and TM2 in phase 1 compared to treatment 1 (p < 0.05).
The cytokine secretion of serum is shown in Table 6. No significant differences were detected in the secretion of serum cytokine among the dietary treatments. However, the secretion of IL-6 was numerically lower (p = 0.052) in the TM1 than in the CON and TM2.
The result of the gut integrity analysis is shown in Table 7. No significant difference was observed in the VH and CD of the small intestine between the three treatments.
The insect meal is now being considered as important protein sources in animal nutrition . Some researchers demonstrated that insect-derived meals have a good potential to be an alternative to fish meal in feeds for animal farming [8,14]. Our results also showed the beneficial effects of replacing fish meal with dried mealworms in weanling pigs. Results of the growth trial indicated that dried mealworm can be replaced at a 50% level of the fish meal in the diet of weanling pig, without any adverse influences on average daily gain. However, the total replacement of fish meal with dried TM meal slightly reduced ADG of weanling pigs. The improved ADG and G:F in the TM1 might be due to the more balanced amino acid composition compared with TM2. However, there are few studies on the influences of TM meal in diet of pigs, using the other high-protein insect meals in pigs and other species diets have been reported by several studies . Jin et al.  found that increasing the level of mealworms meal to 6% in diets linearly improved ADG and G:F of weanling pig.
In the present study, the supplementation of dried mealworms had no effect in the digestibility of nutrients in the piglet. In accordance with the previous report of Biasato et al. , who reported that the dietary insect meal did not affect the nutrient digestibility of piglet. On the other hand, Jin et al.  reported that the inclusion of dried mealworm in diets improved the digestibility of nutrients. There is a close correlation between nutrient digestibility and diet composition. Therefore, these discrepancies may be responded by the variation in the source of insects, dietary inclusion, and processing methods.
The insignificant hematological parameters result in weanling pigs fed TM is in accordance with the report of Biasato et al.  who reported no significant response on the blood profile of weaned piglet fed insect meal. However, immunoglobulin G (IgG) was influenced by the dried mealworm containing diets. In contrast Jin et al.  showed no significant effect in serum IgG concentration when 6% of mealworm was supplemented in the diet. It is well known that the chitin content of commercially farmed insect meals is high and can include from 11.6 to 137.2 g/ton of the meal . Chitin has been known as an immune system stimulator , and its effects may appear as increased blood IgG. In this study, the replacement of fish meal by dried meal up to 5% did not affect the serum cytokines concentrations in weaned pigs. However, the IL-6 concentration tended to be decreased with the supplementation of dried mealworm in phase 1. Similarly, it was reported that dietary supplementation of Hermetia illucens larvae decreased mRNA expression of inflammatory cytokines .
Dietary dried mealworms inclusion showed no effect in the gut integrity of the weaned pigs in this trial, which is consistent with published reports including insect meal in animal diet [10,19]. Biasato et al.  reported that no effect on the gut integrity of weanling pigs fed insect meal. In a previous study, Hosseindoust et al.  reported that the intestinal structure integrity is associated with the maintenance of intestinal nutrition. Dietary inclusion of dried mealworms did not influence the intestinal morphology, it can be hypothesized that the supplementation of insect meal does not have any adverse influence on gut integration or nutrient absorption.