Piglets can secrete acidic mammalian chitinase from the pre weaning stage

Fishmeal substitutes (such as insect-based feeds) in pig diets can promote sustainable pork production. Insect powders contain chitin, a nitrogen-containing indigestible material, and pigs must have the capacity to secrete chitin-degrading enzymes to benefit from these diets. The chitin-degrading enzyme (acidic mammalian chitinase; AMCase) and its gene expression have been detected in the stomach tissue of approximately 6-month-old fattening pigs; however, it remains unclear from which stage chitin-degrading enzymes are secreted. In the present study, the stomach tissue of piglets was collected from the suckling stage (14 d old) to 56 d to evaluate chitin-degrading enzymes and associated gene expression. AMCase mRNA and protein expression was detected in the stomach tissue of all piglets from days 14 to 56. AMCase secretion might increase with the increase in stomach tissue weight as piglets grow. Insect powders can therefore be used in the diets of pre-weaning piglets. The gastric AMCase level was approximately 30% that of fattening pigs. The appropriate inclusion of insect meals in the diets of pigs at different growth stages still needs to be determined.

Fishmeal substitutes (such as insect-based feeds) in pig diets can promote sustainable pork production. Insect powders contain chitin, a nitrogen-containing indigestible material, and pigs must have the capacity to secrete chitin-degrading enzymes to benefit from these diets. The chitindegrading enzyme (acidic mammalian chitinase; AMCase) and its gene expression have been detected in the stomach tissue of approximately 6-month-old fattening pigs; however, it remains unclear from which stage chitin-degrading enzymes are secreted. In the present study, the stomach tissue of piglets was collected from the suckling stage (14 d old) to 56 d to evaluate chitin-degrading enzymes and associated gene expression. AMCase mRNA and protein expression was detected in the stomach tissue of all piglets from days 14 to 56. AMCase secretion might increase with the increase in stomach tissue weight as piglets grow. Insect powders can therefore be used in the diets of pre-weaning piglets. The gastric AMCase level was approximately 30% that of fattening pigs. The appropriate inclusion of insect meals in the diets of pigs at different growth stages still needs to be determined.
Since the publication of "Edible insects" by the Food and Agriculture Organization in 2013 1 , insects have garnered the attention of researchers as potential feed material globally. Food wastes, which are mostly incinerated or landfilled, can be converted to animal proteins by using them as a feed for insects 2 . In addition, the utilization of insect larvae for recycling resources is consistent with the Sustainable Development Goals, and many firms have been set up worldwide to produce insects as a feed source 3,4 .
Some insects that are being studied for their potential use as animal feeds are the mealworm (Tenebrio molitor) and black soldier fly (BSF, Hermetia illucens) [5][6][7][8][9] . The mealworms have been produced as feed for pets (e.g., birds and reptiles) for a long time 10 , and therefore, their husbandry and breeding methods have been well established 11 . Similarly, the BSF has attracted considerable attention as a potential animal feed and in waste management during recent years 3,7,8,[12][13][14][15] , and its larvae can be cultivated using either food waste or livestock feces; however, the reproductive efficiency of adult BSFs is low 16 .
The amino acid contents of insect powders are similar to those of fishmeal. Thus, defatted powders of such insects could be used as alternatives to fishmeal, whose availability is influenced by natural resource status, as a protein source in pig and poultry diets 13 . In addition, the supplementation of pig diets with insect powder has been reported to enhance growth and nutrient digestibility with no detrimental effects on the immune system in piglets 17,18 .
The insect cuticle consists of chitin, proteins, phenolic compounds, and lipids 19 . Chitin is a nitrogen-containing fiber 20 that can only be degraded by chitinase 21 . Numerous studies have reported the immunomodulatory effects of chitin 22,23 . According to these previous studies, the immunomodulatory effects of chitin implied that chitin is not fully digested and eventually reaches the cecum and colon. The substitution of an easily digestible protein source with insect powder, which is relatively difficult to digest, could decrease the growth rates of piglets that have an immature digestive system; therefore, caution should be exercised when adopting insect powder as a protein source.
Mammals produce two types of chitinase, chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase) 24 . Tabata et al. 24 reported AMCase expression in the stomach tissue of approximately 6-month-old pigs. This suggests that mature pigs have the ability to secrete chitinase. However, it remains unclear from which growth stage chitinase is secreted. Therefore, in the present study, we investigated whether piglets have the capacity to secrete chitinase, from the suckling to growing stages.

AMCase level in the stomach of piglets. AMCase was secreted in the stomach tissue of all piglets. The
AMCase levels per unit stomach weight did not differ significantly among piglets of different ages (P > 0.05, Fig. 2). However, the analysis of variance (ANOVA) showed a significant difference in AMCase level per unit stomach weight between piglet sexes (P < 0.05, Fig. 2).
The AMCase level per whole stomach weight tended to increase with an increase in age (Fig. 3). The AMCase level in the whole stomach of piglets was significantly higher at 56 d than at 14 d (P < 0.05). However, the ANOVA did not reveal significant differences in the AMCase level in the whole stomach between piglet sexes (P > 0.05, Fig. 3).

Discussion
In the present study, we aimed to clarify the stage from which chitin-degrading enzymes are secreted. To this end, the stomach tissue of piglets was collected from the suckling (14 d old) to the growing stages (56 d old) to investigate chitin-degrading enzyme and associated gene expression levels. Piglet AMCase gene expression did not vary among the growth stages, suggesting that the piglets had the ability to secrete AMCase from the suckling stage. Similar to the findings of Tabata et al. 24 , Chit1 mRNA expression in the stomach was low compared  www.nature.com/scientificreports/ to AMCase mRNA expression. In addition, the relative AMCase mRNA expression did not significantly change when the piglets began to wean, and the stress of weaning did not seem to influence the gene expression levels considerably. Although the piglets were allowed to consume only maternal milk and water until weaning, the piglets exhibited interest in commercial feed from around 9 d of age when the feed was placed in the rearing enclosures; they have been reported to poke the feed with their noses or eat it 25 . In the wild, piglets probably begin to eat insects and mushrooms that contain chitin. Thus, pigs may have the ability to secrete AMCase in the pre-weaning stage.
In the present study, the AMCase level did not exhibit an increasing trend with age between the pre-weaning and post-weaning stages; however, it was approximately 28% that in the stomach tissue of fattened pigs 24 . The results suggest that AMCase secretion increases with the growth of pigs. As the weight and area of the gastric mucosa increase with pig growth, even if the AMCase level per unit area of gastric mucosa is the same, the degraded chitin amount would increase with age. This implies that insect powder concentrations in pig diets can be increased with the growth stage of pigs.
Several studies in which the protein source in piglet diets has been substituted with insect powder have been reported; however, in most of these studies, the protein has been substituted with soybean meal, and a few studies have used animal-based proteins 18,[26][27][28] . In addition, it has been reported that soybean meal can be substituted with 10% insect powder 26 ; however, the insect powder used to replace soybean meal was defatted under high pressure and may not contain a considerable amount of chitin. Another study that substituted non-defatted insect powders with animal protein in piglet diets concluded that 2% substitution was possible 29 .
Protein concentrations in pig diets decrease from the pre-weaning stage to the fattening stage, when their diets shift to contain more grains than animal proteins 30 . In addition, based on the composition of the diet, the suckling stage diets contain more animal proteins, but the fattening stage diets contain more grains than animal proteins; therefore, the proportion of animal proteins in the diets of fattening pigs is low 30 . Consequently, animal proteins in the diet of fattening pigs, which are thought to secrete a higher amount of chitin-degrading enzymes than piglets, can be entirely substituted with insect powder. However, as the proportion of animal proteins in the diet during the pre-weaning and early weaning stages are high 30 , it is necessary to consider the chitin level when replacing animal proteins in the diet with insect powder. Otherwise, the chitin nitrogen would be calculated as  www.nature.com/scientificreports/ the amount of nitrogen in the diet, and the amount of piglet digestible nitrogen could be lower than that presumed. Furthermore, inappropriate concentrations of insect powder in diets could inhibit weight gain in piglets. The piglets exhibited the ability to secrete AMCase in the stomach tissue from day 14. Therefore, insect powder could be adopted in the diets of pre-weaning piglets. However, the gastric AMCase level in piglets was approximately 28% that in fattening pigs; this necessitates further investigation to determine the appropriate insect powder concentration in the diets at different growth stages.

Methods
The protocol of this study was approved by the Animal Care and Use Committee of Kagawa University (Permit number: 2019-19,631) and carried out according to the Kagawa University Animal Experimentation Regulation.
Stomach tissue samples from piglets. Forty-two piglets (Landrace × Large white × Duroc: 21 males and 21 females) delivered by six sows (Landrace × Large white) were used. The sows and piglets were reared in farrowing stalls (1.8 m × 2.0 m) until weaning. Weaning was carried out at 28 d. After weaning, out of the 42 piglets, 24 piglets were reared in piglet cages (1.8 m × 1.8 m) in an open-type pig house with an average temperature of 21.8 °C ± 5.5 °C and average humidity of 70.3% ± 6.5%. The piglets were fed commercial feeds (Nosan, Yokohama, Japan) according to the feeding program of the university farm. The feeding program and the feed compositions are shown in Fig. S1 and Table S1, respectively. At 14 d, six piglets (3 males and 3 females) were euthanized by administering 90.0 mg/kg body weight of pentobarbital via the auricular vein and dissected. This process was repeated weekly for 7 weeks with the remaining piglets. Stomach tissue was collected from the gastric body to the pylorus, and then weighed. The collected samples were separated for gene expression analyses and chitinase measurements. The stomach tissue samples for gene expression analyses were stored at 4 °C overnight in RNAlater stabilizer solution (Thermo Fisher Scientific, Waltham, MA, USA), and then frozen at -80 °C until analysis. The samples for chitinase secretion assays were frozen at -80 °C until analysis.
Expression of piglet AMCase. Total RNA was isolated from piglet stomach tissue using TRIzol Reagent Measurement of piglet AMCase. Twenty milligrams of gastric mucosa and 1 ml of RIPA Buffer (Nacalai Tesque, Kyoto, Japan) were added into a 1.5 ml tube. Thereafter, the samples were completely homogenized using a bead crusher (µT-12; TAITEC, Saitama, Japan) by shaking at 3,500 rpm for 20 s, and then cooling on ice for 30 s; this process was repeated three times. Subsequently, the samples were centrifuged at 10,000 × g for 20 min at 4 °C, and the cells were precipitated. The supernatant was transferred to another 1.5 ml tube without disturbing the pellet.
The AMCase level was measured using a fluorometer (Gemini EM; Molecular Devices, San Jose, CA, USA) and the CycLex Acidic Mammalian Chitinase Fluorometric Assay Kit (Medical & Biological Laboratories, Nagoya, Japan), according to the manufacturers' instructions.

Scientific Reports
| (2021) 11:1297 | https://doi.org/10.1038/s41598-020-80368-0 www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.