A Nutritional Evaluation of Insect Meal as a Sustainable Protein Source for Jumbo Quails: Physiological and Meat Quality Responses

The use of insect meals in place of soybean meal in diets is critical for sustainable intensification of poultry production, but the utility of this alternative dietary protein source is unknown for the Jumbo quail. Therefore, this study investigated the effect of partial substitution of soybean with black soldier fly larvae (BSFL) meal on feed intake, physiological, and meat quality parameters of Jumbo quails. Three hundred and fifteen mixed-gender quails (53.8± 5.19 g live-weight) were reared on five isonitrogenous and isoenergetic experimental diets formulated by replacing soybean products with BSFL meal at 0 (BSFL0), 25 (BSFL25), 50 (BSFL50), 75 (BSFL75), and 100 (BSFL100) g/kg. Feed intake, body weight gain, gain-to-feed ratio (G:F), blood parameters, and carcass and meat quality traits were determined. Quadratic trends were observed for overall feed intake (y = 605 (±17.56) + 2.1 (±0.82)x − 0.02 (±0.008)x2; R2 = 0.20; p = 0.023) and overall body weight gain (y = 155 (±4.70) + 0.57 (±0.22)x − 0.005 (±0.002)x2; R2 = 0.22; p = 0.026) in response to BSFL levels. However, there were neither linear nor quadratic trends for meat quality and blood parameters, except for albumin-to-globulin ratio. Only gizzard size linearly increased (y = 0.05 (±0.004)x + 2.3 (±0.09); R2 = 0.18; p = 0.04) with BSFL levels. We concluded that BSFL meal could replace soybean products in Jumbo quail diets at 54 g/kg without compromising weight gain and meat quality.


Introduction
The production and use of feed in the livestock sector account for approximately 14.5% of total greenhouse gas emissions [1]. An anticipated increase in the demand for poultry products means more arable land will be required for feed grain production. This will inevitably lead to higher levels of atmospheric carbon [2] due to deforestation and land preparation activities. The demand for soybean, the main protein source in poultry diets, has also steadily increased due to competition between animal and human needs [3]. This has led to higher market prices for this vegetable protein thus compromising its contribution to food and nutrition security. Sustainable intensification of poultry production requires the use of feed ingredients whose production requires less water, land, and results in lower greenhouse gas emissions [4]. This approach will ensure environmental, financial, and social sustainability of the poultry industry. The sustainability of the poultry industry also requires diversification of bird species used to supply animal protein. This has seen the introduction of novel bird species such as the quail.

Study Site and Dietary Ingredient Sources
The feeding trial was conducted at the North-West University Molelwane farm (25 • 40.459 S, 26 • 10.563 E), in the North West province of South Africa. The study was carried out during winter season when ambient temperatures ranged between −3 and 25 • C. Infrared lamps were used to provide warmth to the quails. Dried and defatted black soldier fly larvae (BSFL) meal was bought from AgriProtein (PTY) LTD (Cape Town, South Africa), a feed manufacturing company located in Cape Town (Western Cape, South Africa). The BSFL meal was produced on organic waste before undergoing quality control checks. The chemical composition of BSFL meal was quoted by the supplier as 900 g/kg dry matter, 500 g/kg crude protein, 130 g/kg crude fibre and 100 g/kg crude fat. Soybean products (soya oilcake and full-fat soya) and all other ingredients were purchased from Optifeeds (PTY) LTD (Lichtenburg, South Africa).

Diet Formulation
The BSFL meal was milled (2 mm; Polymix PX-MFC 90 D) prior to blending with other dietary ingredients. The experimental diets (Table 1) were formulated to be isonitrogenous and isoenergetic by replacing soybean products with BSFL meal as follows: (1) BSFL0 = commercial grower diet with no BSFL meal inclusion, (2) BSFL25 = commercial grower diet in which 25 g/kg of soybean products was replaced with BSFL meal, (3) BSFL50 = commercial grower diet in which 50 g/kg of soybean products was replaced with BSFL meal, (4) BSFL75 = commercial grower diet in which 75 g/kg of soybean products was replaced with BSFL meal, and (5) BSFL100 = commercial grower diet in which 100 g/kg of soybean products was replaced with BSFL meal. 1 Diets: BSFL0 = a commercial grower diet with no black soldier fly larvae meal inclusion, BSFL25 = a commercial grower diet in which 25 g/kg of soybean products was replaced with black soldier fly larvae meal, BSFL50 = a commercial grower diet in which 50 g/kg of soybean products was replaced with black soldier fly larvae meal, BSFL75 = a commercial grower diet in which 75 g/kg of soybean products was replaced with black soldier fly larvae meal, and BSFL100 = a commercial grower diet in which 100 g/kg of soybean products was replaced with black soldier fly larvae meal.; 2 MDCP = mono-dicalcium phosphate; 3 ME = predicted metabolizable energy.

Experimental Design and Quail Management
The rearing and slaughter procedures of the quails were approved by the Animal Research Ethics Committee of the North-West University (approval no. NWU-00483-18-A5) according to established guidelines for use and care of research animals. A total of 315, day-old mixed-gender, Jumbo quails were purchased from a Quail Breeder (Gauteng, South Africa). The quails were reared using a commercial starter mash from Nutrifeeds (PTY) LTD (Lichtenburg, South Africa), until one week of age. The five dietary treatments were randomly allocated to 35 pens (experimental units), which were replicated seven times per dietary treatment with each replicate pen (100 cm long × 60 cm wide × 30 cm high) carrying nine quails. The pens had wire mesh floor but without bedding. On average, the temperature and humidity inside the poultry house during the experimental period were 30 • C and 40%, respectively. Rearing was done under natural lighting (12 h of daylight). The birds were then adapted to the dietary treatments for one week before the commencement of measurements at two weeks of age. Fresh, clean water and the diets were offered ad libitum during the 42-day feeding trial.

Feed Intake and Growth Performance
At two weeks of age, initial live-weights of the birds were taken and subsequently weighed weekly per pen until they were six weeks of age (final weight). Feed intake per bird was measured by dividing total intake per pen by the total number of quails in the pen. The live weights obtained weekly were used to calculate the average weekly body weight gain (ABWG). The ABWG and AWFI were then used to calculate gain-to-feed ratio (G:F).

Slaughter and Hemato-Biochemical Analyses
Six-week-old quails were transported to Rooigrond poultry abattoir (North West, South Africa) where they were humanely slaughtered. While bleeding the birds, blood was collected into two sets of tubes from two randomly selected birds per pen. For hematological analysis, tubes containing ethylenediaminetetraacetic acid were used during blood collection. Hematological parameters: hemoglobin, white blood cells (WBC), heterophils, eosinophils, monocytes, and lymphocytes were determined using an automated IDEXX LaserCyte Hematology Analyzer (IDEXX Laboratories, Inc., Johannesburg, South Africa). Serum was generated for biochemical analysis according to Washington and van Hoosier [16]. Albumin-to-globulin ratio (ALB/GLOB), total bilirubin, albumin, lipase, alkaline phosphate (ALKP), phosphorus, alanine transaminase (ALT), amylase, creatinine, calcium, globulin, glucose, total protein, and urea were analyzed using an automated IDEXX Vet Test Chemistry Analyzer (IDEXX Laboratories, Inc., Johannesburg, South Africa).

Carcass Traits and Internal Organs
Immediately after slaughter and for each replicate treatment group, the carcasses were labelled and placed in tagged transparent plastic bags. The carcasses were immediately weighed to determine hot carcass weight (HCW). After chilling for 24 h, the carcasses were reweighed to determine cold carcass weight (CCW). Dressing (%) was calculated as the proportion of HCW on slaughter weight. Wings, thighs, drumsticks, and breast, as well as weights of liver, gizzard, heart, proventriculus, and small and large intestine, were weighed and expressed as a proportion of HCW.

Meat Quality
Meat pH and temperature were measured on the pectoralis major muscle using a Corning Model 4 pH-temperature meter (Corning Glass Works, Medfield, MA, USA). A thigh muscle sample was used to determine meat color (L* = lightness, a* = redness, and b* = yellowness) following the guidelines of the Commission Internationale de l'Eclairage [17]. Hue angle and chroma values were calculated as described by Priolo et al. [18].
To determine cooking loss, breast muscle samples were weighed (w1) and cooked in an oven at 130 • C for 20 min. After cooking, meat samples were cooled and re-weighed (w2). The loss in weight was expressed as a proportion of the initial sample weight. Cooked breast samples (~22 g) were then sheared using a Texture Analyzer (TA XT plus, Stable Micro Systems, Surrey, UK) to measure shear force in Newtons.
Water holding capacity (WHC) was determined according to Grau and Hamm [19] using the pectoral major muscle (~10 g), which was placed between plexiglass plates and held under pressure (60 kg of pressure) using dumbbell weights. For drip loss measurements, triplicate breast meat samples from each replicate treatment group were sliced with a knife into strips (~3 g; w1) and thereafter sewed on a flat cardboard using wool and hung on top of the cold room (4 • C) sealing so that the samples did not touch the cardboard. After 72 h, the samples were gently removed and reweighed (w2). The percentage drip loss was calculated by expressing the loss in weight (w1 − w2) of breast meat sample as a proportion of the initial weight of the meat sample (w1).

Statistical Analysis
Data for all measured parameters were analyzed for linear and quadratic effects with the aid of polynomial contrasts. The response of these parameters to incremental levels of BSFL was determined through response surface regression analysis (Proc RSREG; [20]). Weekly measured parameters (feed intake, weight gain, and gain-to-feed ratio) were analyzed using the repeated measures analysis [20] to determine any significant week × diet interaction effects. Data for parameters measured only once such as blood parameters, size of internal organs, carcass characteristics, meat quality, overall feed intake, weight gain, and gain-to-feed ratio were analyzed using the general linear model procedure of statistical analysis system [20] with diet as the only main factor. For all statistical tests, significance was declared at p ≤ 0.05. Treatment means were separated using the probability of the difference option in the LSMEANS statement of SAS [20].

Results
Repeated measures analysis showed a significant diet × week interaction for G:F but not for average weekly feed intake (AWFI) and ABWG. Table 2 shows that there were significant linear trends for G:  1 Diets: BSFL0 = a commercial grower diet with no black soldier fly larvae meal inclusion, BSFL25 = a commercial grower diet in which 25 g/kg of soybean products was replaced with black soldier fly larvae meal, BSFL50 = a commercial grower diet in which 50 g/kg of soybean products was replaced with black soldier fly larvae meal, BSFL75 = a commercial grower diet in which 75 g/kg of soybean products was replaced with black soldier fly larvae meal, and BSFL100 = a commercial grower diet in which 100 g/kg of soybean products was replaced with black soldier fly larvae meal; 2 SEM: standard error of the mean.
No linear trends (p > 0.05) were observed for overall feed intake, overall BWG, and final weight (Table 3). However, there were significant quadratic trends for overall feed intake (y = 605 (±17.56) + 2.1 (±0.82)x − 0.02 (±0.0077)x 2 ; R 2 = 0.20; p = 0.023) and overall BWG (y = 155 (±4.70) + 0.57 (±0.22)x − 0.0049 (±0.002)x 2 ; R 2 = 0.22; p = 0.03), from which an optimum BSFL inclusion level was calculated to be 5.4%. Table 3. Overall feed intake (g/bird), overall body weight gain (g/bird) and slaughter weight (g/bird) of Jumbo quails fed diets containing black soldier fly larvae meal in place of soybean products. 1 Diets: BSFL0 = a commercial grower diet with no black soldier fly larvae meal inclusion, BSFL25 = a commercial grower diet in which 25 g/kg of soybean products was replaced with black soldier fly larvae meal, BSFL50 = a commercial grower diet in which 50 g/kg of soybean products was replaced with black soldier fly larvae meal, BSFL75 = a commercial grower diet in which 75 g/kg of soybean products was replaced with black soldier fly larvae meal, and BSFL100 = a commercial grower diet in which 100 g/kg of soybean products was replaced with black soldier fly larvae meal; 2 SEM: standard error of the mean. Table 4 shows that there were no linear nor quadratic effects for all the blood parameters, except for the ABL/GLOB ratio, which showed a quadratic trend (y = 0.39 (±0.065) + 0.008 (±0.0031)x − 0.00007 (±0.000027)x 2 ; R 2 = 0.337; p = 0.030) in response to BSFL meal inclusion levels. A significant quadratic trend was observed for breast weights (y = 21.9 (±0.637) + 0.054 (±0.0299)x − 0.00058 (±0.000278)x 2 ; R 2 = 0.168; p = 0.046) in response to BSFL levels. However, no significant trends were observed for dressing percentage and any of the other carcass traits (Table 5). 1 Diets: BSFL0 = a commercial grower diet with no black soldier fly larvae meal inclusion, BSFL25 = a commercial grower diet in which 25 g/kg of soybean products was replaced with black soldier fly larvae meal, BSFL50 = a commercial grower diet in which 50 g/kg of soybean products was replaced with black soldier fly larvae meal, BSFL75 = a commercial grower diet in which 75 g/kg of soybean products was replaced with black soldier fly larvae meal, and BSFL100 = a commercial grower diet in which 100 g/kg of soybean products was replaced with black soldier fly larvae meal; 2 Parameters: HCW = hot carcass weight; CCW = cold carcass weight; 3 SEM: standard error of the mean. Table 5 shows that there were linear effects (p < 0.05) for gizzard weight, which linearly increased (y = 0.05 (±0.004)x + 2.3 (±0.085); R 2 = 0.18; p = 0.04) with BSFL levels. There were significant quadratic effects for size of large intestine (y = 1.4 (±0.077) + 0.0074 (±0.0036)x − 0.000073 (±0.0000337)x 2 ; R 2 = 0.16; p = 0.04). There were no dietary effects (p > 0.05) on carcass characteristics, dressing percentage, size of internal organs, and meat quality parameters of Jumbo quails. Similarly, there were neither linear nor quadratic trends (p > 0.05) for meat quality parameters (Table 6). 1 Diets: BSFL0 = a commercial grower diet with no black soldier fly larvae meal inclusion, BSFL25 = a commercial grower diet in which 25 g/kg of soybean products was replaced with black soldier fly larvae meal, BSFL50 = a commercial grower diet in which 50 g/kg of soybean products was replaced with black soldier fly larvae meal, BSFL75 = a commercial grower diet in which 75 g/kg of soybean products was replaced with black soldier fly larvae meal, and BSFL100 = a commercial grower diet in which 100 g/kg of soybean products was replaced with black soldier fly larvae meal; 2 WHC: water holding capacity; 3 SEM: standard error of the mean.

Discussion
Repeated measures analyses showed a significant diet and week interaction effect on G:F, indicating that the efficiency of the birds in converting the dietary treatments into body mass depended on their age. The optimum inclusion level for the BSFL meal was found to be 54 g/kg, suggesting that substituting soybean products with BSFL meal at higher rates depress overall feed intake and body weight gain. Indeed, at three weeks of age, G:F linearly decreased with BSFL meal levels, confirming that higher inclusion levels reduced the feed conversion efficiency. This could be due to the presence of chitin in the larvae, a fibrous substance, which reduces nutrient utilization and digestibility [21]. Indeed, Marono et al. [22] reported a decrease in feed intake in laying hens fed H. illucens larvae meal. However, Cullere et al. [23] reported that overall performance of Japanese quails reared on BSFL meal was comparable to the ones fed de-fatted soybean meal and soybean oil. Similarly, a feeding trial conducted by Maurer et al. [24] with Leghorn laying hens, where SBM was partly substituted with de-fatted BSFL meal at inclusion levels of 50% and 100%, showed no significant dietary effects.
There is a dearth of information on blood indices of Jumbo quails fed BSFL meal. In this study, a quadratic response was observed for the ALB/GLOB ratio, indicating that at higher inclusion levels, the BSFL may compromise autoimmunity of the quails and cause plasma cell disorders. Indeed, Griminger and Scanes [25] reported that a low ALB/GLOB ratio is an indicator of low immune response for the birds. The fact that the ALB/GLOB initially increased and then declined at even higher inclusion levels of BSFL meal confirms that its inclusion must be capped at 54 g/kg to avoid the overproduction of globulins, which may cause autoimmune diseases. Nonetheless, no dietary influences were observed for all the other blood parameters including the liver enzymes (ALKP and ALP), suggesting that partial replacement of soybean products with BSFL meal did not compromise the health status of the birds. Furthermore, the hemato-biochemical values reported in this study fell within the normal ranges reported for quails [3,26,27]. In addition, several studies also revealed no significant dietary effects on blood parameters of broilers [21], laying hens [22], and Barbary partridges [28] when fed with a variety of insect meals.
Breast weights showed a quadratic response to dietary levels of BSFL meal. This trend was not surprising because a quadratic trend was also observed for overall feed intake and overall weight gain, verifying that including BSFL meal beyond 54 g/kg depressed voluntary feed intake and subsequently reduced the development of the pectoralis major muscle. Gizzard weights linearly increased with the inclusion of BSFL meal, indicating that the fibrous nature of chitin present in the larvae meal challenged the birds. As a result, they responded by developing enlarged gizzards as an anatomical adaptation mechanism to physically degrade this structural polysaccharide. A quadratic response was observed for large intestines, which was not expected. For all the other internal organs, there were no significant dietary effects, which is in agreement with several studies that have reported a lack of dietary effects on the size of internal organs of broiler quails fed BSFL meal [23] and broiler chickens fed mealworm [13].
There were no linear and quadratic trends for all the meat quality parameters in response to incremental levels of BSFL meal, suggesting that substituting soybean products with the larvae meal neither negatively nor positively affected the quality of the meat. Similarly, the experimental diets had no effect on meat pH. These findings are in line with those of Secci et al. [29], who observed a lack of dietary effects on meat pH of Barbary partridges. Nonetheless, the pH values observed from these studies were normal for poultry meat. The lack of dietary effects on color is consistent with the findings of Secci et al. [29] and Cullere et al. [30]; however, it was expected that the presence of carotenoids (~2.15 mg/kg) in BSFL meal would improve the yellowness of the meat [29]. In this study, no dietary effects were observed on drip loss, WHC, and shear force. Schiavone et al. [31] also observed similar results on cooking loss, shear force, and drip loss in Barbary partridges and broiler chickens fed BSFL meals.

Conclusions, Limitations and Future Research
It was concluded that BSFL meal could replace soybean products in Jumbo quail diets at an optimal inclusion level of 54 g/kg without compromising their productive performance, health status, and quality of the meat. What may limit the contribution of BSFL to sustainable intensification of the Jumbo quail is the possible reduction in bird performance at inclusion rates beyond 54 g/kg. The antinutritional effects of chitin most likely impose this upper limit in the tolerance level of BSFL.