1. Introduction
In recent years, researchers have paid a great deal of attention to the use of insect-derived products in poultry [
1] and in the nutrition of other monogastric animals, as recently shown in reviews by Sogari et al. [
2] and Gasco et al. [
3]. The main advantage is the low environmental impact of insects, compared to conventional vegetable protein sources, as they require less soil and water, and lead to lower greenhouse gas and ammonia emissions. Indeed, it has been estimated that 1 ha of land can produce less than 1 ton of soybean protein per year compared to 150 tons of insect protein on the same surface [
4]. Similarly, a notable contribution is also given by the limited space requirements of insects, together with their ability to grow on organic wastes, their efficient feed conversion ratio, and their high fecundity [
5]. Some researchers have reported that the nutritional value of insects is adequate to support poultry growth, nutrient digestibility, and health [
6,
7,
8,
9]. However, in other cases, conflicting results have been obtained [
10], thus making a thorough study of adequate dietary inclusion levels necessary.
Among the various insect species, black soldier fly (BSF,
Hermetia illucens) larvae have shown a nutritional composition that is suitable for poultry diets, as it has a high crude protein (CP) content (ranging between 35–57% on a dry matter basis, DM) with a high biological value and an extremely variable ether extract (EE) content (15–49% DM) [
11]. The fatty acid composition of BSF larvae depends on the fatty acid composition of the rearing substrate but, generally, the larvae appear rich in lauric acid (20–40% of total lipids), palmitic acid (11–16% of total lipids), and oleic acid (12–32% of total lipids). BSF larvae are also rich in minerals, particularly Ca (5–8% DM) and P (0.6–1.5% DM) [
11]. BSF larvae also contain chitin, the main constituent of the exoskeleton of arthropods. The chitin content fluctuates during the life cycle of insects, according to their life stage, but the method used to assess it can lead to dramatic differences in the measurements [
12]. The chitin content in BSF larvae ranges from 8.7% [
13] to 5.9% [
14]. It has been reported that chitin has antioxidant and hypocholesterolemic properties for both humans and animals and appears to have a positive effect on the immune system of poultry, as it exhibits prebiotic properties in the large intestine and appears to exhibit a bacteriostatic effect on Gram-negative bacteria [
15,
16,
17,
18].
Although the
in vivo antioxidant effect of chitin has not been investigated in great depth in poultry, it could provide important information about animal welfare. Indeed, the presence of free radicals, especially reactive oxygen species, is associated with several negative biological effects, including deterioration of the DNA, the oxidation of proteins and lipids, and the development of inflammatory disorders [
15,
19,
20]. On the other hand, chitin hypocholesterolemic properties have been mentioned in many studies on broilers and laying hens [
16,
20,
21]. This property could result from the positive charge of this polysaccharide, which binds negatively-charged bile acids and free fatty acids [
22]. Finally, the proper functioning and health of the gastrointestinal tract are crucial for ensuring an adequate growth performance in farm animals [
23,
24]. These aspects are particularly relevant in the poultry industry, where selected birds display an elevated growth potential.
In spite of these interesting biological effects, the use of BSF defatted meal in Muscovy duck (
Cairina moschata domestica) diets has been poorly investigated so far and, to the best of the authors’ knowledge, there is only one paper that shows encouraging results, in terms of growth performance, diet digestibility, and intestinal morphology in ducks [
9], while another paper has assessed the
in vitro digestibility of different insect meals [
25].
Considering this background, the present study has been aimed at investigating the effects of dietary BSF larva meal inclusion on the blood parameters and histological traits of female Muscovy ducks, in order to provide a picture of the animal welfare of Muscovy ducks based on a multidisciplinary approach involving both in vivo and post-mortem parameters.
2. Materials and Methods
2.1. Birds and Experimental Design
The present trial was performed at the poultry facility of the University of Turin (Italy). The experimental protocol (prot. no. 380576, 4th December 2017) was approved by the Bioethical Committee of the University of Turin (Italy).
The experimental design of the present study is reported in Gariglio et al. [
9]. Briefly, a total of 192 female 3-days of age Muscovy ducklings (Canedins R71 L White, Grimaud Freres Selection, France) were divided into four groups, assigned to four different dietary treatments (6 replicates/treatment and 8 birds/replicate) and raised from 3 to 50 days of age. BSF larva meal was included as a substitute for corn gluten meal (substitution 1:1) at increasing levels (0%, 3%, 6%, and 9%; BSF-0, BSF-3, BSF-6, and BSF-9, respectively) in isonitrogenous and isoenergetic diets formulated for three feeding phases: starter (3–17 d), grower (18–38 d), and finisher (39–50 d). In order to evaluate the effects of dietary BSF larva meal inclusion, all the other ingredients were kept constant, with the exception of the synthetic essential amino acids (DL-methionine and L-lysine), as reported in
Table 1. The apparent metabolizable energy (AMEn) of the BSF larva meal has previously been assessed for broiler chickens [
26] and was used to formulate the diets of this experiment. The diets were formulated to meet or exceed the nutritional requirements of female Muscovy ducks, as reported by Pingel et al. [
27].
2.2. Chemical Analysis of the BSF Meal and Experimental Diets
Samples of the experimental diets and BSF larva meal were analyzed for DM (AOAC, #934.01), ash (AOAC, #942.05), CP (AOAC, #984.13), EE (AOAC, #2003.05), neutral detergent fiber (NDF) (AOAC, #2002.04), and acid detergent fiber (ADF) (AOAC, #973.18) [
28,
29]. The method of Finke et al. [
30] was used for the determination of the chitin content of the BSF larva meal using ADF adjusted for its nitrogen content. The chemical composition of the experimental diets is reported in
Table 1. Moreover, the chemical composition of the BSF larva meal (on a DM basis) was as follows: 924.1 g/kg DM, 567.1 g/kg CP, 107.0 g/kg EE, 163.8 g/kg ash, and 64.3 g/kg chitin.
2.3. Growth Performance
The growth performance parameters were evaluated, as previously reported by Gariglio et al. [
9]. The live weight (LW) of the birds was assessed at the beginning and at the end of the trial (at 3 and 50 days of age, respectively), and the average daily gain (ADG), the daily feed intake (DFI), and the feed conversion ratio (FCR) were calculated for the whole experimental period (3–50 days of age). The mortality and health status of birds were monitored on a daily basis.
2.4. Slaughtering Procedures and Sampling
At 50 days of age, 12 ducks per diet (two birds per pen) were selected on the basis of the average LW and identified through a shank ring. Subsequently, after a feed withdrawal period of 12 hours (at 51 days of age), the selected ducks were transferred to a commercial processing plant and slaughtered according to the standard EU regulations.
At slaughtering, blood samples were collected in EDTA tubes and serum-separating tubes, further details of which are provided in
Section 2.5.
Immediately after the completion of the slaughtering phase, spleen, liver, thymus, and bursa of Fabricius samples were collected and fixed in a 10% buffered formalin solution for histochemical staining, further details of which are provided in
Section 2.6.
2.5. Blood Analysis
Blood samples were collected, at slaughtering, from the jugular vein of twelve birds (two animals per pen) per feeding group. An aliquot of 2.5 mL was placed in an EDTA tube and 2.5 mL in a serum-separating tube. A blood smear was prepared from a droplet without any anticoagulant. The total red (erythrocytes) and white (leukocytes) cell counts were determined in an improved Neubauer haemocytometer after mixing with a Natt-Herrick solution in a 1 to 200 ratio, as reported by Natt and Herrick [
31]. The blood smears were stained with May-Grünwald and Giemsa–Romanowski stains. One hundred white blood cells were evaluated per smear to determine the heterophils to lymphocytes (H/L) ratio, while the number of blood cell types was determined according to Campbell [
32].
The serum-separating tubes were left in a standing position, at room temperature, for approximately two hours, until the formation of a blood clot. Subsequently, the tubes were centrifugated at 700×
g for 15 minutes and the obtained serum was immediately frozen at −80 °C. The total protein was quantified using the “biuret method” (Bio Group Medical System kit; Bio Group Medical System, Talamello (RN), Italy); the electrophoretic pattern of the serum was assessed using a semi-automated agarose gel electrophoresis system (Sebia Hydrasys®, Norcross, GA, USA). The alanino-aminotransferase (ALT), aspartate-aminotransferase (AST), gamma glutamyl transferase (GGT), alkaline phosphatase (ALP), triglycerides, cholesterol, Ca, P, Mg, Fe, uric acid, and creatinine serum concentrations were measured using enzymatic methods in a clinical chemistry analyzer (Screen Master Touch, Hospitex diagnostics Srl., Firenze, Italy), as described by Salamano et al. [
33].
In order to obtain plasma, the EDTA tubes were centrifugated at 2000× g for 10 minutes to separate the cell fractions, and the supernatants were immediately frozen at −80 °C and then used to determine the antioxidant status and oxidative metabolites. The blood glutathione peroxidase (GPx, EC 1.11.1.9) and total antioxidant status (TAS) activities of the plasma were determined using a Ransel Enzymatic Kit (RS504, Randox Laboratories, Crumlin, UK) and a TAS Colorimetric Kit (NX2332, Randox Laboratories, Crumlin, UK), respectively, according to the manufacturer’s recommendations.
The enzyme immunoassay for the detection and quantification of methylglyoxal (MG) was performed using an OxiSelectTM Methylglyoxal ELISA Kit (STA-811, Cell Biolabs, San Diego, CA, USA), while an OxiSelectTM MDA Adduct Competitive ELISA Kit (STA-832, Cell Biolabs, San Diego, CA, USA) was used for the malondialdehyde (MDA) quantification. Both tests were performed on plasma samples. The 3-nitrotyrosine plasma concentration was measured by an enzyme-linked immunosorbent assay (ELISA) using an OxiSelectTM Nitrotyrosine ELISA Kit (STA-305, Cell Biolabs, San Diego, CA, USA). All the tests were performed according to the manufacturer’s instructions.
All the analyses were performed in duplicate.
2.6. Histological Investigations
The slaughtered birds (n = 12 per experimental diet, two birds per pen) were submitted to an anatomopathological examination. Spleen (entire organ), liver (left lobe), thymus (left side lobes), and bursa of Fabricius (entire organ) samples were collected (0.5–1.5 g/organ) and fixed in a 10% buffered formalin solution, embedded in paraffin wax blocks, sectioned at a thickness of 5 μm, mounted onto glass slides, and stained with Haematoxylin & Eosin for the histopathological examination [
34]. The following histopathological alterations were evaluated: white pulp hyperplasia and depletion in the spleen, cortical depletion in the thymus, follicular depletion and intrafollicular cysts in the bursa of Fabricius, and hepatocyte degeneration and lymphoid tissue activation in the liver [
6].The observed histopathological alterations were evaluated using a semiquantitative scoring system as follows: absent (score = 0), mild (score = 1), moderate (score = 2), and severe (score = 3). In order to investigate the accumulation of lipids and polysaccharides in the liver, tissue samples of these organs were also stained with Sudan Black and Periodic acid-Schiff (PAS), respectively. The lipid and polysaccharide staining intensity was scored semi-quantitatively as follows: grade 0 for an absence of staining, grade 1 for mild staining, grade 2 for moderate staining, and grade 3 for marked staining. All the slides were blindly evaluated by three different observers and the discordant cases were reviewed using a multi-head microscope until a unanimous consensus had been reached.
2.7. Statistical Analysis
The statistical analysis was performed using the SPSS software package (version 21 for Windows, SPSS Inc., Chicago, IL, USA). The mortality rate was analyzed by means of a Chi-square test, using the BSF0 group as reference. Shapiro-Wilk’s test was used to establish the normality or non-normality of the distributions. The assumption of equal variances was assessed by means of Levene’s homogeneity of variance test. The birds’ pen was identified as the experimental unit to evaluate the growth performance, while the blood traits and histological features were evaluated individually for each duck. The collected data were tested using one-way ANOVA. Polynomial contrasts were used to test the linear and quadratic responses to increased levels of BSF inclusion in the diet. Histopathological scores were analyzed by means of the Kruskal-Wallis test (post-hoc test: Dunn’s Multiple Comparison test). Differences between treatments were considered statistically significant when the p values ≤ 0.05.