In sacco nutrient degradability of silage containing intact and defatted black soldier fly ( Hermetia illucens ) larvae

This study aimed to evaluate the in sacco nutrient degradability of total mixed ration (TMR) silage containing intact and defatted black soldier fly (BSF) larvae. The study evaluated four dietary treatments and six replications. The treatments consisted of control TMR silage without BSF larvae


INTRODUCTION
Animal foods, such as milk and meat, are the key sources of human nutrition. Provision of quality feed to livestock in the right proportion is important since feed as a source of nutrient has a direct impact on livestock productivity. Attempts to enhance livestock productivity usually faces an obstacle due to the tight competition for feed resources. In addition, productivity is also constrained by the high cost of feed. Novel feed ingredients are being explored continuously in order to obtain affordable, easy-to-source and nutrient-rich alternatives. Among these alternative feeds are insects and their nutritional composition are generally rich in protein (Van Huis, 2013;Sogari et al., 2019).
Among the insects, black soldier fly (BSF) larvae or known as maggots have been considered as alternatives to expensive protein sources, and they typically contain more than 40% of crude protein (Barragan-Fonseca et al., 2017). BSF has the capacity to convert organic waste into high protein feed. To date, maggots have been fed as alterative protein sources primarily to monogastric animals such as poultry, i.e., broiler chickens and laying hens (Zotte et al., 2019;Biasato et al., 2020). Only few studies, however, have evaluated the use of maggots as feed ingredients in the diets of ruminants (Mulianda et al., 2020). Furthermore, BSF larvae were able to be used as feed ingredients for ruminants because they have high protein contents and provide high proportion of rumen undegradable or bypass protein (Jayanegara et al., 2017a;2017b).
On the other hand, BSF larvae has a fairly high fat content ranging from 30.1-34.3% (Shumo et al., 2019). This high fat content limits the use of BSF larvae to be given to chickens, pigs and fish, which can only partially replace the protein source commonly fed to these animals (Fonseca et al., 2017). In ruminants, high fat content may negatively affect the rumen fermentation process, decrease nutrient digestibility, and reduce energy intake (Morris et al., 2020). Removal of fat either partially of completely from the BSF larvae is expected to increase the nutrient utilization for ruminants. The objective of this experiment was to determine the in sacco degradability of total mixed ration (TMR) silage containing intact and defatted BSF larvae.

Experimental Animal
This study used two male Ongole cattle with a 3-cm diameter rumen fistula. The cattle were given an ad libitum basal feed and cared for according to the animal welfare standards of Indonesian Research Center for Animal Production (IRCAP).

Sample Preparation and Treatment
Fifteen-day-old BSF larvae were collected from PT Biocycle, Bogor. BSF larvae were defatted mechanically and chemically in the Laboratory of Feed Science and Technology, Faculty of Animal Science, IPB University. The dead larvae were dried in an oven at 50 o C for 24 hours. Mechanical fat removal was performed by using a pressurized fat extractor (Savoire et al., 2013) while chemical fat removal was conducted by using Soxhlet apparatus and hexane as the solvent (Jayanegara et al., 2018). The BSF larvae were included in dietary treatments that consisted of: control TMR silage without BSF larvae (R1), 20% intact BSF larvae + 80% TMR silage (R2), 20% chemically defatted BSF larvae + 80% TMR silage (R3), and 20% mechanically defatted BSF larvae + 80% TMR silage (R4). All materials were mixed homogeneously into 1000 ml lab-scale silos according to the procedure of Kondo et al. (2016). Each dietary treatment was incubated for 30 d in six replicates. Analyses of proximate and Van Soest, i.e., neutral detergent fiber (NDF) and acid detergent fiber (ADF) were performed (Van Soest et al., 1991) in order to determine the chemical composition of the TMR.

In sacco Incubation Procedure
Determination of in sacco feed degrada-tion in the rumen at various incubation times was performed according to Ørskov et al. (1980). The study used 7 cm × 11 cm nylon bags with 60 micron pore holes. The dry weight of each nylon bag was 0.5-1 g. Each bag was marked and three bags of feed ingredients containing 2 mm ground and defatted BSF larvae-based TMR silage were used. Each bag was first dried in the oven at 60°C and then filled with 5 g of the sample, weighed and fastened with rubber. All the nylon bags (containing the samples) were inserted into the rumen at various incubation time intervals.
The incubation was conducted by following a completely randomized design with four dietary treatments and six replications. Samples were put in the rumen for 0, 4, 12, 24, 48 and 72 hours. For the samples at 0 h only, the samples were not put into the rumen but were washed under running water for 5 min to remove dissolved particles and then dried at 60ºC for 24 hours. After incubation, the nylon bags were washed with water and then dried at 60 o C for 24 hours or until the sample weight was stable. The samples were then analysed to determine their chemical composition.

Data Analysis
Data on nutrient degradation were fitted with the following non-linear regression mode: where p is degradability at t time (%), a is dissolved fraction (%), b is degradable fraction (%), c is degradation rate constant (%/hour), e is natural number and t is incubation time (hour). The Neway Excel package program was used to calculate the parameter kinetics of a, b and c. All data were analysed using analysis of variance (ANOVA) by according the completely randomized design. The Tukey's test was used to compare the various treatments. Statistical analysis was carried out by using CoStat statistical program version 6.4. Table 1 indicates the dietary composition of the TMR. The crude protein (CP) contents in all dietary treatments were relatively the same, at around 14% DM. Nitrogen-free extract (NFE) contents were also similar, i.e., approximately 62% in all treatments. The BSF larvae is a potential feed ingredient by providing an alternative protein source and reducing the dependence on soybean meal. In terms of nutritional content, BSF larvae have high CP levels and a balanced composition of amino acids. (Sánchez-Muros et al., 2014;Crosbie et al., 2020). Jayanegara et al. (2017a) stated that BSF larvae have 40-44% CP content. Apart from the protein, BSF larvae also have high ether extract (18-38.8% DM) and ash contents (20-28.4% DM), reflecting the different diets of insects (Makkar et al., 2014). In terms of their fatty acid composition, BSF larvae contain high levels of saturated fatty acids (40%), especially lauric acid and palmitic acid, and relatively low levels of unsaturated fatty acids (3.4-11.6%) (Spranghers et al., 2017;.

RESULTS AND DISCUSSION
The largest part of the insect's exoskeleton is chitin, a polymer of N-acetylglucosamine (Hahn et al., 2020). BSF larvae were reported to contain chitin between 2.9-14.1% DM (Ferrer Llagostera et al., 2019;Brigode et al., 2020). Because chitin contains nitrogen groups, it is recovered as NDICP and ADICP as indicated by the higher values of these components in R2, R3 and R4 than that in R1. The increasing age of BSF larvae is accompanied by the development of the exoskeleton, this increases the proportion of chitin per DM unit (Zhu et al., 2016).
The in sacco nutrient degradability of TMR silage containing intact and defatted BSF larvae incubated for 72 hours is presented in Table 2. The lowest DMD was found in R1 compared to all treatments (p < 0.05), while the highest DMD was found in R2 (p < 0.05). The highest OMD was found in R2 compared to R1, R3 and R4 (p < 0.05). Further, DMD and OMD kinetics of intact and defatted BSF larvae-based TMR silage incubated for 72 hours are presented in Figure 1. The high chitin content in BSF larvae negatively affects DMD and OMD (Jayanegara et al., 2017a). With regard to protein utilisation, the addition of both intact and defatted BSF larvae to TMR increased the CPD as compared to control (p < 0.05). Apparently the effect of chitin on CPD is similar to its deacetylated derivative, i.e., chitosan. Dietary chitosan addition was reported to elevate DMD and CPD (Gandra et al., 2016;2018), although some other reports did not find so (Henry et al., 2015;Seankamsorn et al., 2019). The increase of nutrient digestibility of ruminants due to chitin and chitosan is seemed to be related to the change of microbial population structure in the rumen. The compounds decrease the population and activity of protozoa, and subsequently decrease predation of the fauna on rumen bacteria, and finally enhance the bacteria population for degrading substrates . Further, the compounds may act like tannins that elevate protein utilisation in the digestive tracts of ruminants (Kondo et al., 2014;Jayanegara et al., 2019).
Degradation kinetics of a, b and c for DMD, OMD, CPD and CFD in TMR silage containing BSF larvae are presented in Table 3. Besides having a high protein content, intact BSF larvae have a high fat content as well. The high fat content induced low levels of a, b and c parameters of DMD degradation in R2. Defatted BSF larvae-based TMR silage in R3 and R4 had higher degradability compared to the TMR silage without BSF larvae in R1 (p < 0.05). In a previous study, Mulianda et al. (2020) confirmed that the overall fat content of BSF larvae could reduce DMD and OMD in the rumen. In the rumen, lipids are converted by lipolysis into glyc-  Figure 1. In sacco (a) dry matter degradability (DMD) and (b) organic matter degradability (OMD) absolute mixed ration (TMR) silage kinetics containing BSF (defatted black soldier fly) larvae. The lowest DMD was found in R1, while the highest DMD was found in R2. The highest OMD was found in R2 among all treatments (a) (b) erol and fatty acids by certain microbes particularly Anaerovibrio lipolytica (Granja-Salcedo et al., 2017). After lipolysis, unsaturated fatty acids are biohydrogenated to produce fatty acid isomers with high levels of saturation (Dewanckele et al., 2020). In addition, glycerol is fermented to volatile fatty acids especially propionate in the rumen (Syahniar et al., 2016). Furthermore, lipids can reduce enteric methane emissions by decreasing OM fermentation in rumen, reducing the methanogenic activity of archaea, destroying the protozoa in which some methanogens exist in interdependence with ruminal fauna, and be-having as alternative hydrogen absorbers (Knapp et al., 2014;Broucek, 2018).

CONCLUSION
Inclusion of both intact and defatted BSF larvae into TMR silage up to 20% DM did not have any adverse effects on nutrient profiles and showed a good nutrient degradation values in the rumen. The larvae may be used to replace, at least partially, the commonly used soybean meal as a protein source in the diets of ruminants.