Effects of Dietary Black Soldier Fly Larvae on Production Performance and Cecal Microbiota of Yunshang Countryside Chickens

Simple Summary

The rapid growth of the global population and livestock industry has significantly increased the demand for meat, while in China, the limited availability of feed resources has become a critical constraint on agricultural development. Conventional protein feed supplies are insufficient to meet current demands, thereby necessitating the exploration of sustainable alternatives. Black Soldier Fly (BSF) larvae contain substantial amounts of protein, lipids, minerals, and trace elements, and their profile of essential amino acids is comparable to that of fishmeal and soybean meal, which positions them as a promising novel protein source for animal feed. The aim of this study was to investigate the effects of dietary supplementation with BSF larvae protein on Yunshang Countryside chickens. The findings indicated that BSF larvae can serve as a high-quality protein ingredient in feed formulations. This study provides empirical support for the development and application of novel feed resources in livestock and poultry production and offers a theoretical foundation for the utilization of BSF larvae as an unconventional feed resource.

Abstract

With the rapid development of the livestock and poultry industry, the availability of feed resources in China has become a critical limiting factor, posing a significant challenge to the sustainable growth of animal husbandry. Black soldier fly (BSF) larvae are rich in protein, lipids, minerals, and trace elements and possess an essential amino acid profile comparable to that of fishmeal and soybean meal, which makes them a promising novel protein source for feed. This study aimed to investigate the effects of dietary BSF larvae protein supplementation on the growth performance, egg production, as well as meat and egg quality, blood biochemical parameters, and cecal microbiota diversity of Yunshang countryside chickens. The results showed that the inclusion of BSF larvae protein in the diet significantly reduced the feed-to-egg ratio and enhanced egg quality. Dietary supplementation with BSF larvae protein also effectively increased the abundance of dominant bacterial phyla and genera in the cecum, with the optimal inclusion level identified as 7.5%. Overall, the results demonstrate that BSF larvae can serve as a high-quality protein source in poultry production, thereby providing a scientific reference for the development and application of new feed resources and offering a theoretical basis for the utilization of BSF larvae as an alternative protein ingredient.

1. Introduction

As the global population grows, the shortage of protein feed resources has become increasingly severe. The rising demand for animal products and feed has created an urgent need to identify novel protein sources to replace traditional soybean meal and fishmeal [1]. Black Soldier Fly (BSF), scientifically named Hermetia illucens, is widely used in entomoremediation due to its ease of rearing, high productivity, high nutritional value, and the ability of its larvae to efficiently utilize a wide range of organic waste materials [2]. A larva hatches from an egg and undergoes five instars as a larva in 13 to 18 days, followed by prepupal and pupal stages before emerging as an adult fly [3]. The larvae and prepupae contain approximately 50% protein and 30% fat, with fat content being the most variable component [4]. BSF larvae contribute to waste degradation and enhance the value of organic residues. Their proteins can be processed into insect meal for animal feed, while lipids can be extracted and transesterified into biodiesel, and also utilized in the production of food and fertilizers [5,6,7,8,9]. With agriculture and land use representing a growing proportion of global greenhouse gas emissions each year, the use of insects as an alternative to conventional animal feed may offer a viable solution to this challenge [10]. In laying hens, a study by Wamai et al. found that feeding diets containing 75% Hermetia illucens larvae meal provided optimal growth performance, reduced feeding costs, increased body weight gain and egg production, and improved the economics of commercial poultry production systems [11]. In broiler chickens, a study by Mlaga et al. showed that the inclusion of larval meal in diets reduced the Atherogenic Index (AI) and Thrombogenic Index (TI)—indices calculated from the fatty acid composition of meat—improved the unsaturation index, and lowered cardiovascular health risks [12]. According to Cullere et al., it is technically feasible to replace soybean oil with the fat of larvae of the black silk worm in the diet of broiler chickens in the later stages of the diet, with replacement levels of up to 100% [13]. However, another study indicated that replacing soybean meal with a high percentage (50–75% or 100%) of full-fat BSF larvae meal throughout the feeding entire feeding period adversely affected broiler growth performance and carcass quality [14]. Previous studies have shown that the lipid profile of insects presents promising potential as dietary supplements in poultry feeds [15], though this remains a subject of considerable debate at present.

Yunshang countryside chicken, a local breed in Yunnan province, is classified as a dual-purpose breed for both eggs and meat. It was selectively bred by the Yunnan Academy of Animal Husbandry and Veterinary Sciences for its stable genetic background, compact body size, firm meat texture, high egg quality, and rich flavor profile. The breed is typically reared in semi-intensive systems that combine cage and free-range housing, which further enhances the quality attributes of both meat and eggs. Given its well-characterized production traits and sensitivity to dietary interventions, Yunshang Countryside Chicken serves as an ideal model for evaluating the effects of novel protein sources on meat and egg quality parameters. We hypothesized that this breed would respond distinctly to dietary BSF protein supplementation, thereby providing clear insights into the nutritional efficacy and safety of BSF as an alternative feed ingredient.

We hypothesized that: (1) dietary supplementation with BSF larvae protein would improve the feed efficiency and product quality (meat and eggs) of Yunshang Countryside chickens without compromising growth performance or health; (2) there would be an optimal inclusion level of BSF protein that maximizes these benefits; and (3) the observed effects would be mediated, in part, through modulations of host metabolism and the cecal microbiota. Therefore, this study aimed to evaluate the effect of different concentrations of BSF feed on slaughter performance, meat quality, egg quality, blood biochemical indices, short-chain fatty acids and gut microorganisms in egg-meat parturition chickens. Based on the documented nutritional value of BSF larvae and its effects in other poultry species.

2. Materials and Methods

2.1. Experimental Materials

The BSF used in this study were supplied by the Institute of Circular Agriculture, Yunnan Academy of Animal Husbandry and Veterinary Science, and the basal rations were produced and provided by Yunnan Yunda Science and Technology Feed Co., Ltd. (Kunming, China) BSF larvae were reared on degreased kitchen waste until reaching the pre-pupal stage. They were then sieved to remove residual substrate, roasted at 105 °C for 10 min to deactivate enzymes and reduce microbial load, and subsequently dried at 65 °C for 48 h to achieve a moisture content below 10%. The resulting dried BSF larvae were used for feed formulation. Subsequently, they were delivered to Yunnan Yunda Science and Technology Feed Co., Ltd. (Kunming, China) for feed preparation. The dried BSF larvae were ground and mixed with corn, soybean meal, and premixes to formulate the experimental diets, which were then stored at low temperature. Nutritional composition of the BSF larvae was analyzed at the Feed Quality Inspection Center of Yunnan Academy of Animal Husbandry and Veterinary Sciences. Dry matter (AOAC 934.01), crude protein (AOAC 984.13), crude fat (AOAC 920.39), calcium (AOAC 968.08), and phosphorus (AOAC 965.17) were determined using standard AOAC methods. Amino acid content was analyzed by hydrolysis followed by high-performance liquid chromatography (AOAC 982.30). Metabolizable energy was calculated based on proximate composition using the predictive equation established in the Chinese Feed Database. The main nutrients composition of BSF larvae were as follows: metabolizable energy 2.591 Mcal/kg, crude protein (CP) content 57.42%, crude fat (18.35%), calcium (Ca) content 2.4%, available phosphorus (AP) content 0.4%, methionine (Met) content 1.2%, lysine (Lys) content 2.5%, tryptophan (Trp) content 0.2%.

2.2. Experimental Design and Chicken Raising

The experiment was conducted at the research facility of the Yunnan Academy of Animal Husbandry and Veterinary Sciences, following approval from the Ethical Committee of Yunnan Academy of Animal Husbandry and Veterinary Sciences (201911004). A total of 225 healthy 120-day-old female Yunshang Countryside Chickens were randomly assigned into five groups, with each group consisting of three replicates of 15 birds. The chickens were housed in three-tiered cages at a stocking density of 4–6 birds per square meter. The control group received a basal diet (0% group), while the experimental groups were fed diets in which 5%, 7.5%, 10% and 12.5% of the corn-soybean meal in the basal diet was replaced with BSF insect meal, respectively. The composition of the experimental diets is presented in

Table 1. The composition of the experimental diets.

Materials	Proportion (Group)
	5%	7.5%	10%	12.5%	0%
Corn	56.50	55.50	54.50	53.50	56.50
Soybean meal 43%	17.00	15.50	14.00	12.50	22.00
Black soldier fly	5.00	7.50	10.00	12.50	0.00
Wheat flour	3.00	3.00	3.00	3.00	3.00
Montmorillonite	1.00	1.00	1.00	1.00	1.00
Glucose	0.50	0.50	0.50	0.50	0.50
Soy lecithin powder	1.00	1.00	1.00	1.00	1.00
Talcum powder	6.00	6.00	6.00	6.00	6.00
5% premix for laying hens	5.00	5.00	5.00	5.00	5.00
60% corn protein powder	5.00	5.00	5.00	5.00	5.00
Total	100.00	100.00	100.00	100.00	100.00

Note: The premix provides per kilogram of ration: Mn 70 mg, Zn 70 mg, Fe 98 mg, Cu 11 mg, I 0.30 mg, Se 0.30 mg, VA 10000 IU, VD 32850 IU, VE 25 IU, VK 3.0 mg, VB 12.0 mg, VB 24 mg, VB 64.2 mg, VB 120.5 mg, D-pantothenic acid 12 mg, niacin 15 mg, biotin 0.2 mg, folic acid 1.5 mg.

, and the nutrient levels are shown in

Table 2. Nutritional levels.

Nutrient Composition	5%	7.5%	10%	12.5%	0%
Dry matter %	90.42	91.12	90.78	90.83	89.85
Crude ash %	12.17	12.53	11.74	11.34	10.84
Crude protein %	18.61	18.19	18.04	18.86	18.26
Crude fat %	5.38	5.07	5.16	6.23	3.47
Total phosphorus %	0.51	0.48	0.46	0.51	0.56

. The trial lasted 70 days and was divided into two phases: a pre-feeding period (days 1–10) and a formal feeding period (days 10–70). During the pre-feeding period, all chickens received the basal diet. In the formal period, the birds were fed at a rate of 110 g per bird per day. Each morning at 08:30, the leftover feed from the previous day was weighed, and the daily feed provision was recorded. Water was provided ad libitum throughout the 60-day formal period. Daily egg production and egg weight were recorded, mortality was monitored, and body weight was measured every two weeks.

2.3. Measurement of Carcass and Meat Quality

At the end of the 70-day feeding trial, 9 chickens per group (3 per replicate, 45 chickens in total) were selected for slaughter and carcass analysis. All chickens underwent pre-slaughter inspection, and only those with no lesions were approved for processing. Birds were fasted for 12 h before slaughter while maintaining moderate water intake, with water withdrawn 2 h prior to slaughter. After passing inspection and completing the pre-slaughter withdrawal period, live weight was measured and recorded. Slaughter was performed using the extracervical bloodletting method: a small number of feathers were removed from the neck, and the common carotid artery behind the mandible was cut with scissors to allow bleeding (3–5 min) until death, keeping the incision as small as possible. Wet plucking was employed: after exsanguination, carcasses were scalded in hot water at 60–62 °C for 60–90 s, followed by manual feather removal, removal of cuticle, toe shells, and beak shells, and rinsing on hooks to drain water. Carcass measurements included: Live weight (LW): Measured before slaughter; Carcass weight (CW): Weight after bleeding, plucking, and removal of head, feet, and viscera (excluding kidneys); Semi-eviscerated weight (SEW): Carcass weight with heart, liver, gizzard, spleen, pancreas, and trachea removed; Eviscerated weight (EW): Carcass weight after removal of all internal organs, including the abdominal fat pad. After chilling at 4 °C for 24 h, the following tissues were dissected and weighed: Breast muscle weight: Both Pectoralis major and minor muscles were completely excised from the carcass and weighed; Abdominal fat weight: The fat depot surrounding the proventriculus and gizzard, extending to the cloaca, was carefully removed and weighed. Meat quality was assessed on the Pectoralis major muscle at 24 h postmortem. pH: Measured using a portable pH meter (Testo Inc., Sparta, NJ, USA) with a penetrating electrode. Meat color: Objective color coordinates (L, a, b*) were measured on the muscle surface using a chroma meter (CR-400, Konica Minolta, Tokyo, Japan) with D65 illuminant. Drip loss: Calculated as the percentage weight loss of a standardized muscle sample approximately 50 g) suspended in a plastic bag at 4 °C for 24 h. Cooking loss: A standardized sample was weighed, cooked in a water bath at 80 °C until an internal temperature of 75 °C was reached, cooled, and re-weighed to calculate percentage weight loss [16].

2.4. Measurement of Egg Quality

At the conclusion of the experiment, 10 eggs from each replicate were selected and analyzed within 24 h. Egg shape index (ratio of transverse to longitudinal diameter), yolk color, egg weight (g), yolk weight (g), eggshell thickness (mm), egg white height (mm), and Hastelloy units were determined. All measurements were conducted using EQM (Egg Quality Measurement, TSS, York, UK) and Instron Mini 55 equipment (Instron, Norwood, MA, USA).

2.5. Measurement of Blood Biochemistry

Prior to slaughter, 3 chickens were randomly selected from each replicate (9 chickens in each group, 45 chickens in total). Approximately 5 mL of blood was collected from the sub-wing vein of each bird. After centrifugation at 3000 r/min for 15 min, serum was separated and stored at −20 °C. Serum samples were subsequently analyzed with an automated analyzer and commercial biodiagnostic kits following the manufacturer’s protocols to determine total protein (TP), albumin (ALB), triglycerides (TG), serum urea nitrogen (BUN), alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST).

2.6. Targeting Fatty Acid Metabolomics and Gut Flora Diversity

After dissection of the experimental animals, cecal contents were collected. Sterile forceps were used to extrude the cecal material, and solid contents (approximately 1 g) were placed directly into sterile Eppendorf tubes. When the intestinal contents were low in solids and high in mucus, the mucus along with a small amount of solid material was rinsed with sterile Phosphate-Buffered Saline, collected into a sterile tube, and approximately 20 mL was retained. The collected samples were grouped and immediately stored at −80 °C. Subsequently, the cecal content samples were sent to Shanghai Meiji Biomedical Technology Co., Ltd. (Shanghai, China) for sample processing and analysis. This included chromatographic and mass spectrometric analyzes, from which test results were obtained and experimental outcomes were evaluated.

2.7. Data Analysis

Experimental data were collated and pre-processed using Microsoft Excel 2020. All datasets were tested for normality (Shapiro–Wilk test) and homogeneity of variances (Brown–Forsythe test). The effect of dietary treatment was evaluated by one-way analysis of variance (ANOVA) using GraphPad Prism (version 8.0.2). When a significant overall effect was detected (p < 0.05), pairwise comparisons between treatment groups were performed with Tukey’s Honestly Significant Difference (HSD) post hoc test. Differences were considered significant at p < 0.05 and highly significant at p < 0.01. For multivariate datasets (metabolomics), principal component analysis (PCA) was applied for exploratory visualization. Differential metabolites were identified based on a combination of univariate ANOVA (p < 0.05) and a variable importance in projection score > 1 derived from orthogonal projection to latent structures discriminant analysis (OPLS-DA). Functional pathway enrichment and topological analysis of differential metabolites were conducted using the ROPLS module in the MetaboAnalyst R package (R version 3.6.2). The experimental unit for growth performance and egg-quality traits was the pen (n = 3 per group). For carcass traits, meat quality, blood biochemistry, cecal microbiota, and metabolomics, the individual bird was the experimental unit (n = 9 per group). ANOVA and post hoc comparisons were applied at the corresponding experimental-unit level. Results are presented as mean ± standard error of the mean (SEM). In tables and figures, means not sharing a common lowercase superscript letter (a, b, c…) differ significantly (p < 0.05); means not sharing a common uppercase letter (A, B, C…) differ highly significantly (p < 0.01). Means labeled with the same letter do not differ significantly (p > 0.05).

3. Results

3.1. Effect of Dietary Supplementation with Black Soldier Fly Larvae on the Performance of Yunshang Countryside Chickens

The effects of BSF protein feed on the performance of parthenogenetic chickens are presented in

Table 3. Effects of different ratios of black soldier fly larvae protein feed on the performance of Yunshang countryside chickens.

Group	Average Weight Gain/kg	Egg Production Rate/%	Feed-to-Egg Ratio
0%	0.56 ± 0.15	0.73 ± 0.1 Aa	3.23 ± 0.28 Aa
5%	0.49 ± 0.28	0.63 ± 0.1 Bc	2.98 ± 0.07 ABab
7.5%	0.63 ± 0.25	0.67 ± 0.1 ABbc	2.77 ± 0.18 ABb
10%	0.71 ± 0.07	0.63 ± 0.09 Bc	2.71 ± 0.07 Bb
12.5%	0.41 ± 0.16	0.69 ± 0.13 ABab	2.84 ± 0.15 ABb

Note: Superscript letters are used to denote statistical significance: the absence of letters, or the same uppercase and lowercase letters, indicates no significant difference (p > 0.05); different lowercase letters indicate a significant difference (p < 0.05); and different uppercase letters indicate a highly significant difference (p < 0.01).

. As shown, the egg laying rate in the 5% and 10% BSF feed groups differed significantly (p < 0.05) from that of the control group (0% group), with both experimental groups showing lower laying rates than the control. Significant differences (p < 0.05) in feed-to-egg ratio were observed between the 7.5% and 12.5% BSF feed groups and the control group, while a highly significant difference (p < 0.01) was found between the 10% BSF feed group and the control. However, no significant differences (p > 0.05) were detected in mean weight gain among the groups.

3.2. Effects of Dietary Supplementation with Black Soldier Fly Larvae on Carcass Traits of Yunshang Countryside Chickens

Table 4. Effects of black soldier fly larvae protein feed with different replacement ratios on carcass traits of Yunshang countryside chickens. (kg).

Group	Live Weight	Slaughter Weight	Half-Eviscerated Weight	Eviscerated Weight	Chest Muscle Weight	Abdominal Fat Weights
0%	1.89 ± 0.27 ab	1.74 ± 0.24 ab	1.46 ± 0.19 ABa	1.17 ± 0.14 ABa	0.16 ± 0.03	0.1 ± 0.1
5%	1.92 ± 0.15 ab	1.74 ± 0.13 ab	1.43 ± 0.15 ABab	1.2 ± 0.24 ABa	0.17 ± 0.02	0.08 ± 0.05
7.5%	1.93 ± 0.23 ab	1.78 ± 0.21 ab	1.51 ± 0.17 ABab	1.21 ± 0.15 ABa	0.20 ± 0.03	0.06 ± 0.03
10%	2.03 ± 0.39 a	1.91 ± 0.37 a	1.64 ± 0.32 Aa	1.26 ± 0.22 Aa	0.19 ± 0.04	0.08 ± 0.04
12.5%	1.72 ± 0.21 b	1.6 ± 0.21 b	1.31 ± 0.2 Bb	0.99 ± 0.16 Bb	0.17 ± 0.04	0.06 ± 0.03

Note: Superscript letters are used to denote statistical significance: the absence of letters, or the same uppercase and lowercase letters, indicates no significant difference (p > 0.05); different lowercase letters indicate a significant difference (p < 0.05); and different uppercase letters indicate a highly significant difference (p < 0.01).

presents the effects of different replacement levels of BSF larval protein on carcass traits of meat-egg type chickens. The results indicate that the 10% group had significantly higher live weight and slaughter weight than the 12.5% group (p < 0.05). Half-eviscerated weight was significantly higher in the 0% group compared to the 12.5% group (p < 0.05) and highly significantly higher in the 10% group compared to the 12.5% group (p < 0.01). Eviscerated weight was significantly lower in the 12.5% group than in the 0%, 5%, and 7.5% groups (p < 0.05) and was highly significantly lower compared to the 10% group (p < 0.01). No significant differences were observed in breast muscle weight or abdominal fat weight with the inclusion of BSF larval protein in the diet.

3.3. Effect of Dietary Supplementation with Black Soldier Fly Larvae on the Meat Quality of Yunshang Countryside Chicken

The effects of different replacement levels of BSF larval protein on meat quality of parthenogenetic chickens are presented in Table 4. As shown, dietary inclusion of BSF protein at 0%, 5%, 7.5%, 10%, and 12.5% had no significant effect (p > 0.05) on meat brightness, redness, yellowness, chroma, or drip loss. However, cooking loss was significantly higher (p < 0.05) in the 7.5% group compared to the 0% group. Regarding pH, the 7.5% group showed significantly lower values than the 0% group (p < 0.05), and the 10% group had significantly lower pH than both the 0% and 5% groups (p < 0.05). No significant differences were observed among the remaining groups (p > 0.05).

3.4. Effect of Dietary Supplementation with Black Soldier Fly Larvae on Egg Quality of Yunshang Countryside Chickens

The effect of different replacement ratios of BSF protein feed on egg quality of parthenogenic hens is shown in Figure 1. The egg shape index in the 12.5% group was significantly higher than that in the 5% and 10% groups (p < 0.05). Egg weight was significantly greater in the 10% group compared with the 0% group (p < 0.05). No significant differences (p > 0.05) were observed in albumen height and Haugh units between any BSF treatment group and the control (0% group), nor among the treatment groups themselves. Yolk weight was significantly higher in the 10% group than in all other groups (p < 0.05). Yolk color in the 5%, 7.5%, 10%, and 12.5% groups was significantly deeper than in the control group (p < 0.05). The yolk percentage was also significantly greater in the 10% group (p < 0.05). Eggshell thickness was significantly lower in the control group (0%) compared with all BSF-fed groups (p < 0.05).

3.5. Effect of Dietary Supplementation with Black Soldier Fly Larvae on Blood Biochemical Indexes of Yunshang Countryside Chickens

The effects of different replacement ratios of BSF protein feed on the blood biochemical indices of parthenogenetic chickens are shown in Figure 2. As shown, the alkaline phosphatase (ALP) content in the 0% group was significantly higher than in all other groups (p < 0.05). Among the treated groups, the 5% group exhibited significantly higher ALP than the 10% and 12.5% groups (p < 0.05). Serum triglycerides (TG) were significantly higher in the 0%, 5%, and 7.5% groups compared to the 10% and 12.5% groups (p < 0.05). Total cholesterol (T-CH) and albumin (ALB) levels were significantly lower in the 0% group than in the 10% and 12.5% groups (p < 0.05). Similarly, total protein (TP) content in the 0% group was significantly lower than in the 5%, 10%, and 12.5% groups (p < 0.05). Urea content was also significantly lower in the 0% group compared to all BSF-treated groups (5%, 7.5%, 10%, and 12.5%; p < 0.05). Aspartate aminotransferase (AST) activity was significantly higher in the 12.5% group than in the 0%, 5%, and 7.5% groups (p < 0.05), and was also significantly higher in the 10% group than in the 5% group (p < 0.05). In contrast, no significant differences were observed in serum alanine aminotransferase (ALT) activity among the groups (p > 0.05).

Figure 1. Effect of BSF larva on egg quality of Yunshang countryside chickens. For data labeled with superscript letters: the absence of letters, or the same lowercase letter, indicates no significant difference (p > 0.05); different lowercase letters indicate a significant difference (p < 0.05).

Figure 1. Effect of BSF larva on egg quality of Yunshang countryside chickens. For data labeled with superscript letters: the absence of letters, or the same lowercase letter, indicates no significant difference (p > 0.05); different lowercase letters indicate a significant difference (p < 0.05).

Figure 2. Effect of BSF larval with different replacement ratios on blood biochemical indexes of Yunshang countryside chickens. For data labeled with superscript letters: the absence of letters, or the same lowercase letter, indicates no significant difference (p > 0.05); different lowercase letters indicate a significant difference (p < 0.05).

Figure 2. Effect of BSF larval with different replacement ratios on blood biochemical indexes of Yunshang countryside chickens. For data labeled with superscript letters: the absence of letters, or the same lowercase letter, indicates no significant difference (p > 0.05); different lowercase letters indicate a significant difference (p < 0.05).

3.6. Effect of Dietary Supplementation with Black Soldier Fly Larvae on the Metabolomics of Targeted Short-Chain Fatty Acids in Yunshang Countryside Chicken

The PCA sample score plot (Figure 3) shows a tight clustering of quality control (QC) samples, indicating reliable experimental design, stable analytical conditions, good reproducibility, and dependable data quality. Cluster analysis of the samples is presented in Figure 4, revealing eight distinct short-chain fatty acid clusters: acetic acid, butanoic acid, propanoic acid, isohexanoic acid, valeric acid, isobutyric acid, isovaleric acid, and hexanoic acid. Multi-group analysis of variance results are displayed in Figure 5. Samples from the 7.5% group contained significantly lower levels of acetic acid compared with the other groups (p < 0.05), and highly significantly lower levels of propanoic acid (p < 0.01). No significant differences were observed in the relative contents of other short-chain fatty acids across groups (p > 0.05). As shown in Figure 6, metabolites from the samples could be classified into seven major categories based on KEGG metabolic pathway mapping. Furthermore, Figure 7 indicates 14 main metabolite enrichment pathways that exhibited significant differences among groups.

3.7. Effect of Dietary Supplementation with Black Soldier Fly Larvae on the Metabolomics of Targeted Medium and Long Chain Fatty Acids in Yunshang Countryside Chicken

The PCA score plot for targeted medium- and long-chain fatty acid metabolomics is presented in Figure 8. Cluster analysis of the samples (Figure 9) revealed a total of 25 distinct clusters corresponding to long-chain fatty acids. Differences in fatty acid profiles among the groups are shown in Figure 10. Samples from the 7.5% group contained significantly lower amounts of lauric acid and tetradecanoic acid compared with the other groups (p < 0.05). No significant differences were observed in the relative levels of other medium- and long-chain fatty acids across groups (p > 0.05). As illustrated in the metabolite heatmap (Figure 11), the relative expression of linoleic acid and tetradecanoic acid in cecal contents was significantly higher in the 12.5% group than in the 10% group (p < 0.01). Additionally, palmitic acid levels were significantly higher in the 12.5% group compared with the 10% group (p < 0.05).

3.8. Effect of Dietary Supplementation with Black Soldier Fly Larvae on Intestinal Microorganisms of Yunshang Countryside Chicken

Figure 12 indicates that both the abundance of cecal microbiota and the homogeneity of the samples were relatively uniform across all groups. As shown in Figure 13, there was a significant difference (p < 0.05) in microbial diversity and richness between the cecal content samples of the 10% BSF feed group and the 7.5% group. Furthermore, the microbial diversity and abundance in the 12.5% BSF feed group differed significantly from those in the 10% group (p < 0.05) and were significantly different from those in the 7.5% group (p < 0.01).

According to Figure 14A, the number of unique and shared bacterial genera in the cecal contents of each group was as follows: the 0% group had 3 unique genera, the 5% group had 4, the 7.5% group had 7, the 10% group had 5, and the 12.5% group had 10. Figure 14B shows that, at the genus level, 31 bacterial genera were identified in the community composition across all groups. Among these, Bacteroides exhibited the highest relative abundance, followed by Rikenellaceae_RC9_gut_group, unclassified o Bacteroidales, and Prevotella. A multi-group comparative analysis of the cecal microbiota is presented in Figure 15. The results reveal that 10 genera showed significant differences in abundance among the groups (p < 0.05). Notably, the abundance of the genus Elusimicrobia differed highly significantly across groups (p < 0.01).

4. Discussion

The application of insect protein in feed production represents a promising strategy to address the growing demand for sustainable protein sources. Compared with conventional animal proteins, insect protein may offer advantages in production efficiency and cost. Insects possess desirable biological traits such as rapid reproduction, short life cycles, and adaptability to various organic substrates [2]. The balanced amino acid profile of insect protein powder could meet the essential amino acid requirements for animal growth. As a strategic feed ingredient, insect protein has the potential to partially replace traditional, costly protein sources like fishmeal, which might reduce feed costs and improve feed conversion efficiency, thereby contributing to more sustainable livestock production systems.

4.1. Effect of BSF Protein Feed on Growth Performance of Yunshang Countryside Chicken

In the present study, dietary BSF larvae meal did not significantly affect the average daily weight gain or feed intake of the dual-purpose chickens (Table 3). However, it significantly reduced the feed-to-egg ratio, indicating improved feed efficiency for egg production. This finding aligns with Moula et al., who reported no significant change in the average daily gain or gain-to-feed ratio of broilers fed 8% fresh BSF larvae [17]. Conversely, other studies have reported variable effects on feed intake and growth in different poultry species and stages [18,19,20]. The discrepancies between studies could be attributed to several factors, including animal species, breed, age, the developmental stage at which BSF larvae are harvested, and subsequent processing methods (e.g., defatting degree). Larvae harvested earlier typically contain higher chitin, which might reduce protein digestibility and intake. The extent of defatting can also influence nutrient digestibility [21]. In this experiment, BSF pre-pupae (at ~10% population) were used without defatting. The results suggest that BSF insect protein can partially replace soybean meal in the diet of Yunshang Countryside chickens, improving feed efficiency for egg production without adversely affecting, and possibly even promoting, growth performance during early lay.

4.2. Effect of BSF Larvae Protein Feed on Meat and Egg Quality of Yunshang Countryside Chickens

The inclusion of BSF larval protein at levels up to 12.5% did not significantly affect most meat quality parameters, including color (L, a, b*), drip loss, and cooking loss (

Table 5. Effects of black soldier fly larvae protein feed with different replacement ratios on meat quality of Yunshang countryside chicken.

Group	L (Lightness)	a (Red-Green)	b (Yellow-Blue)	ΔE	pH	Drip Loss	Cook Loss
0%	40.08 ± 3.4	0.91 ± 0.4	3.05 ± 1.4	56.74 ± 3.3	5.95 ± 0.17 a	2.71 ± 0.76	31.76 ± 2.03 b
5%	41.64 ± 3.4	0.79 ± 0.3	4.96 ± 1.7	55.24 ± 3.4	5.92 ± 0.15 ab	2.63 ± 1.02	32.34 ± 1.54 ab
7.5%	43.64 ± 4.3	0.69 ± 1.0	5.14 ± 1.9	53.27 ± 4.2	5.79 ± 0.12 bc	2.57 ± 0.88	34.7 ± 4.93 a
10%	41.85 ± 4.4	0.66 ± 0.9	4.53 ± 2.1	55.02 ± 4.3	5.76 ± 0.11 c	2.79 ± 0.87	32.11 ± 1.5 ab
12.5%	41.38 ± 2.9	0.40 ± 0.4	4.13 ± 2.8	55.50 ± 2.8	5.88 ± 0.14 abc	2.29 ± 0.78	32.35 ± 2.18 ab

Note: Superscript letters are used to denote statistical significance: the absence of letters, or the same uppercase and lowercase letters, indicates no significant difference (p > 0.05); different lowercase letters indicate a significant difference (p < 0.05).

), which is consistent with findings in broilers [22]. The influence of BSF larval meal on drip loss may be related to the integrity of myofibrillar proteins [23]. However, while Dabbou et al. also observed no significant change in breast muscle pH [22]. However, a significant reduction in pH was observed in the 7.5% and 10% BSF groups compared to the control (Table 5). While the underlying mechanism requires further investigation, it has been suggested that the specific fatty acid profile of full-fat BSF meal, particularly its high lauric acid content, might influence post-mortem muscle metabolism and pH decline [24]. Interestingly, pH in the 12.5% group was not significantly different from other groups, indicating a non-linear relationship that warrants further study.

Regarding egg quality, BSF supplementation had no significant effect on albumen height or Haugh units. However, notable improvements were observed in several key consumer traits (Figure 1). Moreover, BSF protein supplementation improved egg weight, yolk weight, yolk color, and yolk ratio in parthenogenetic chickens. These results are consistent with previous studies by Secci et al. [25], Bovera et al. [26] and Mwaniki et al. [16], who also reported that replacing soybean meal with BSF larvae significantly deepened yolk color. It is suggested that BSF-based diets increase the content of lutein and β-carotene in yolks, which are key pigments responsible for yolk color intensity.

Previous studies on BSF larvae in laying hen diets have shown inconsistent results, particularly concerning eggshell strength and thickness. Bovera et al. [26] observed a linear increase in eggshell thickness and strength with BSF inclusion, whereas Mwaniki et al. [16] found no significant effect. In the present experiment, eggshell thickness was significantly lower in the control group (0%) than in all BSF-fed groups, though no differences were detected among the 5%, 7.5%, 10%, and 12.5% groups. Marono et al. [18] reported that 17% BSF larvae significantly elevated serum calcium levels in laying hens aged 24–45 weeks, proposing that BSF diets may enhance eggshell strength by improving intestinal calcium absorption and metabolism. This effect could be partly attributed to chitin in insects, a type of fiber that promotes hindgut fermentation and thereby increases mineral absorption [27]. Further research is warranted to fully elucidate the mechanism by which BSF insect meal influences eggshell quality.

4.3. Effect of BSF Protein Feed on Carcass Traits of Yunshang Countryside Chickens

The inclusion of BSF larval protein had no significant effect on major carcass traits, including live weight, eviscerated weight, breast muscle weight, and abdominal fat weight (Table 4), except for a reduction in some weight parameters at the 12.5% inclusion level. This aligns with the observation that moderate inclusion levels (e.g., <15%) of BSF meal often do not negatively influence slaughter performance in broilers [28]. Schiavone et al. also reported that highly defatted BSF larval meal can serve as a good source of essential amino acids in Ross 308 broiler rations [21]. The above results confirm that BSF insect protein can effectively replace soybean meal in parthenogenetic chicken diets without adversely affecting carcass traits.

4.4. Effect of Protein Feed of BSF on Blood Biochemical Indexes

Blood biochemical parameters are valuable indicators of metabolic and organ function [29,30]. In this study, dietary BSF protein reduced serum ALP and triglyceride (TG) levels (Figure 2), while alanine aminotransferase (ALT) activity remained unchanged. Total protein (TP), albumin (ALB), and urea levels were generally higher in BSF-fed groups. These modulations suggest an influence of BSF meal on protein and lipid metabolism [28]. In contrast, Marono et al. found that complete substitution of soybean meal with BSF larvae significantly lowered serum levels of the albumin-to-globulin ratio (ALB/GLO), cholesterol (CHOL), and TG in laying hens [18]. Based on these outcomes, it is hypothesized that BSF larvae meal may enhance nutrient absorption in laying hens by supporting liver and kidney function. However, the effects could vary depending on factors such as the degree of defatting, the harvesting stage of the BSF larvae, and their source. Furthermore, whether the chitin and digestible amino acids present in BSF larvae promote hepatic digestion and absorption of proteins, carbohydrates, and lipids remains unclear and warrants further investigation.

4.5. Mechanisms of the Effect of BSF Protein Feed on the Metabolism and Bacterial Diversity of the Cecum

The gut microbiota plays a crucial role in host metabolism and health [29]. Our study demonstrated that BSF protein supplementation significantly modulated the cecal microbial community and its metabolic output.

An untargeted metabolomic analysis of cecal contents from each group revealed eight clusters of short-chain fatty acids (SCFAs) whose levels were significantly altered in parthenogenetic chickens after dietary replacement with BSF insect protein. This profile is consistent with the findings of Opatovsky et al., who reported that the primary fatty acids in BSF larvae are saturated fats, notably lauric, myristic, and palmitic acids [30]. Meanwhile, BSF larvae meal is also rich in various immune-bioactive compounds, such as chitin and antimicrobial peptides. These components contribute to maintaining a healthy gut microbiota in parthenogenetic chickens by regulating bacterial balance and distribution. In particular, the broad-spectrum antimicrobial activity of BSF-derived peptides can positively influence intestinal immune homeostasis [31]. A multi-group comparative analysis at the genus level revealed that 10 genera exhibited significant differences in abundance across the groups. Bacteroides is a key bacterial genus commonly found in the human gut, where it engages in a symbiotic relationship with the host, aiding in food breakdown and the production of essential nutrients and energy [32]. Rikenella, a Gram-negative obligate anaerobe, participates in propionate fermentation, which generates energy and supports gluconeogenesis in normal cells. As a potential probiotic, this genus contributes to maintaining metabolic homeostasis, supporting immune function, and promoting intestinal health [33]. SCFAs, which are primarily influenced by anaerobic microorganisms in the gastrointestinal tract, such as Mycobacterium spp., play a beneficial role in antioxidant, anti-inflammatory, and anti-tumor activities, as well as in regulating gene expression, maintaining gastrointestinal flora balance, and improving gut function [34]. It has been found that short-chain fatty acids can act as signaling molecules to reduce intestinal inflammation by activating extracellular G protein-coupled receptors and inhibiting intracellular histone deacetylases [33].

In summary, dietary BSF protein replacement in Yunshang Countryside chickens not only improved production parameters like feed efficiency and egg quality but also induced significant changes in the cecal microbiome and metabolome. These changes point to potential mechanisms underlying the observed benefits, including enhanced nutrient metabolism, improved gut health, and modulated immune function. The optimal inclusion level of 7.5% identified in this study appears to balance positive production outcomes with favorable microbial modulations.

5. Conclusions

BSF larvae protein is a viable and effective alternative protein source for Yunshang Countryside chickens. Dietary inclusion up to 12.5% did not compromise overall growth or health and significantly improved feed utilization efficiency, as indicated by a reduced feed-to-egg ratio. Supplementation with BSF protein positively influences egg quality by enhancing yolk color, weight, and eggshell thickness, which are key attributes for consumer preference and product value. The improvement in eggshell quality suggests a potential benefit for mineral metabolism. The inclusion of BSF protein did not adversely affect carcass traits or meat quality parameters critical for meat production, confirming its suitability for dual-purpose (meat and egg) poultry systems. The beneficial modulations observed in serum biochemical indices (e.g., reduced triglycerides and alkaline phosphatase) and in the cecal microbiome (increased abundance of beneficial bacterial genera and short-chain fatty acid producers) suggest that BSF protein contributes to improved metabolic health and gut function in chickens. An inclusion level of 7.5% BSF larvae protein is recommended as optimal for Yunshang Countryside chickens, balancing the positive effects on feed efficiency, egg quality, and gut microbiota modulation with economic feasibility. In summary, this study provides foundational evidence that BSF larvae protein can be successfully integrated into the diet of dual-purpose chickens, offering a sustainable strategy to alleviate conventional protein feed shortages while supporting animal productivity and health.