Investigation of Tenebrio molitor Larvae as a Substitute for Imported Fish Meal: Impacts on Carcass Yield, Organ Health, Lipid Metabolism, and Thigh Meat Fatty Acid Content of Broiler

The sourcing of sustainable feeds, particularly imported fish meal (IFM), is costly, limited in supply, and often associated with overharvesting, resulting in significant environmental impacts, including ecosystem degradation, loss of marine biodiversity, and habitat destruction (Gasco et al., 2020). These problems have led to the exploration of sustainable, economically viable alternatives to insect agri-food waste, as they have a unique potential nutritional profile and can be produced and harvested sustainably and abundantly (Schiavone et al., 2020). Of the insects available, the Tenebrio molitor larvae, or “mealworms”(TmLM), are a viable source of animal protein. Additionally, they contain essential fatty acids that are healthy for human and animal consumption (Filho et al., 2021).

Moreover, TmLM incorporates chitin, a polysaccharide that enhances the immunological and physiological responses of broiler chickens by acting as a gut health prebiotic and a regulator of fat metabolism (Biasato et al., 2017). Research demonstrates that in broiler chicken diets, partial replacement of TmLM with IFM results in similar growth performance while improving carcass quality and blood lipid levels, thereby lowering the potential for cardiovascular disease (Biasato et al., 2017; Jiang et al., 2024). The unsaturated fatty acids of TmLM contribute to reduced fat and cholesterol in thigh meat, enhanced overall carcass yield, and increased yield of several vital visceral organs, particularly the liver and spleen (Popova et al., 2025). Given these facts, TmLM serves as a cost-effective protein source in meat chicken diets and has the potential to substantially improve overall physiological status, meat quality, and carcass yield.

Recent studies of the last five years correctly evaluate these benefits. TmLM is a sustainable feed alternative. Previous findings showed that including TmLM in broiler diets (at 5-10% replacement levels) improves feed conversion ratios and carcass yield, and that growth and immune responses are not hindered by the prebiotic effect of chitin (Cullere et al., 2021). Corroborated that TmLM supplementation reduces belly fat accumulation and improves lipid metabolism (Biasato et al., 2017; Makkar et al., 2022). Broilers fed TmLM had reduced levels of blood cholesterol and triglycerides and improved organ health indicators, such as the spleen, suggesting that TmLM may be effective in reducing metabolic stress relative to IFM-based diets (Inaudi et al., 2025). Nevertheless, the most recent data stand in contrast to, or sharpen, these positive results, reporting possible limitations or context-dependent effects. Indicated that TmLM improved carcass quality in some aspects.

However, there was a loss in feed intake and minor digestive discomfort due to the fibrous nature of chitin, which may negate supportive improvements in lipid metabolism if an adequate balance is not achieved (Józefiak et al., 2023). Compared with FM, moderate levels (5-7%) of TmLM substitution showed no significant differences in thigh meat fat or blood lipids (Benzertiha et al., 2019). They explained the mechanisms underlying the rearing conditions of TmLM and the genetics of the broilers, which affect the unsaturated fatty acids reported by Vasilopoulos et al. (2023). This difference warrants the need for coordinated production systems to guarantee consistent nutrition and efficiency.

However, despite these advances, previous studies have largely focused on individual performance indicators, primarily growth performance or carcass characteristics, with limited attention to the integrated effects of Tenebrio molitor larvae meal (TmLM) on multiple physiological and metabolic parameters. A comprehensive evaluation that simultaneously considers carcass traits, organ health, lipid metabolism, and meat quality is still lacking. Such an integrated approach is essential for understanding the overall biological effects and underlying mechanisms of TmLM as a substitute for imported fish meal (IFM) in broiler diets. Therefore, this study aimed to evaluate the effects of replacing IFM with TmLM in broiler chickens up to 35 days of age by assessing live body weight, carcass yield, abdominal fat percentage, physiological organ weights, blood lipid profiles, thigh meat fatty acid composition and meat cholesterol and fat contents.

Sampling involves selecting a proportion of members or elements from a larger population and using those to infer about the larger population. It is a key component of statistical evaluation and research techniques. The mass rearing of Tenebrio molitor involves 500 g of a substrate comprising tofu dregs, rice bran, and moringa leaves. Biopon is incorporated with all substrate forms, whether dry or finely milled. Into this substrate, a total of 150 Tenebrio molitor beetles were added. Every 2 days, 6 whole young papaya fruits, 2–3 cm in diameter, are added to the substrate. The beetles were left in the substrate for 15 days, after which they were removed using a sieve. TmLM was maintained for 4 weeks (28 days) after the beetles were removed from the substrate. The TmLM was separated from the substrate by means of a two-mesh sieve (to determine the fresh weight of the TmLM) and further sieving to remove the TmLM from the substrate. To the TmLM, which was then ground in a blender to re-mill it into flour, 10 minutes of steaming to kill the larvae were added, followed by 48 hours of oven drying at 50 degrees, and after that, sieving to remove the TmLM. TmLM worms are a possible food source for broiler chickens.

For this experiment, one hundred broiler chickens were divided into five equal groups, four of which were replicates containing 10 chickens of equal weight (41 ± 1.55 g). This particular experiment is split into five treatment groups, as follows: a control group with 0% TmLM, 100% IFM; group 1, 3% TmLM, 75% IFM; group 2, 6% TmLM, 50% IFM; group 3, 9% TmLM, 25% IFM; and groups 4 and 5, 12% TmLM, 0% IFM. The 20 box cages, with 10 chickens per unit, were continuously lit for the first 7 days and then introduced to 12-hour light and dark cycles (12L: 12D) for the rest of the experiment. The birds had automatic feeding and watering available to them freely throughout the entire 35-day trial.

On the first day, all chickens were vaccinated for Marek’s disease (MMB 202 Strain, subcutaneous) and Schemes (LaSota Strain, intraocular) on the 7th day. Psychological state, nutritional and water intake, and droppings assessment are criteria for determining the health status of hens. During the study, chickens showed no abnormalities.

This study employed an experimental methodology based on a Completely Randomized Design (CRD). The treatments consisted of five dietary levels of Tenebrio molitor larvae meal (TmLM) used as a substitute for imported fish meal (IFM). The dietary treatments were arranged as follows: Treatment A contained 0% TmLM and 100% IFM (control); Treatment B contained 3% TmLM with a 25% substitution of IFM; Treatment C contained 6% TmLM with a 50% substitution of IFM; Treatment D contained 9% TmLM with a 75% substitution of IFM; and Treatment E contained 12% TmLM with a 100% substitution of IFM.

Each treatment was replicated four times, and each replicate consisted of an equal number of broiler chickens. The experimental diets were administered from day-old chicks until 35 days of age. All experimental units were randomly allocated to the treatments to minimize experimental bias. At 35 days of age, data were collected for growth performance, carcass characteristics, physiological organ weights, blood lipid profiles, and thigh meat quality parameters. Table 1 summarizes the diet given to the subjects and its approximate nutritional composition and nutrient content. Table 2, composition of feed ingredients (%) of the treatment diet.

The parameters assessed included live weight (g/head), abdominal fat percentage, carcass percentage, physiological organ weights, blood lipid profiles, thigh meat fatty acids, thigh meat cholesterol, and thigh meat fat.

The carcass weights (g) and body weights (g) were measured to calculate the percentage of the carcass and the percentage of the non-carcass organ as follows: carcass weight (g)/body weight (g) x 100% and non-carcass organ weight (g)/body weight (g) x 100. The following were separated and weighed with great attention: the carcass, the abdominal adipose tissue, and the non-carcass organs (i.e., heart, gizzard).

For emaciated chickens, blood samples were taken from the jugular and carotid vessels. To isolate the serum, blood without anticoagulants was collected and centrifuged for 20 minutes at 40 × 100 rpm, after a 30-minute coagulation period. DiaSys kits on photometric systems were used to assess total cholesterol, HDL cholesterol, LDL cholesterol, and triglycerides; colorimetric enzymatic assays were used to measure cholesterol and triglycerides. After centrifuging for 20 minutes at 40 × 100 rpm to sediment the cells, the supernatant was collected and analyzed using a Microlab 300 at 546 nm.

Thigh muscle samples were taken from 35-day-old broilers. These were cut to remove excess skin and fat, dried, and ground into fine powders. Cholesterol measurement was conducted according to the protocol for the Cholesterol Analyzer SpectraLab 752 Pro. With the help of a boron trifluoride-methanol reagent, the lipids were converted into

Table 1: The content of feed substances (%) and metabolic energy (kcal/kg) of feed ingredients that make up the diet (as feed).

Description: a Nuraini et al. (2022b)a; b Nuraini et al. (2022a); c Scott et al. (1982); d Sitompul (2004); e Top Mix Packaging Labels PT. Medion.

Table 2: Composition of feed ingredients (%) of the treatment diet.

fatty acid methyl esters (FAMEs) to determine the fatty acid composition. Analysis of FAMEs was performed by gas chromatography with a flame ionization detector (GC-FID) to assess omega-3, omega-6, and omega-9 fatty acids, and their peak areas were compared with reference standards to identify and quantify them. The total composition of fatty acids in thigh tissue was computed as the proportion of each acid to the total fatty acids. The fat content of the thigh meat sample was analyzed using a Soxhlet apparatus (AOAC, 2010).

The primary variable of interest was the dietary inclusion level of TmLM. The data obtained were analyzed using analysis of variance (ANOVA) appropriate for a completely randomized design (CRD). The measures of central tendency were analyzed using IBM SPSS (v22.0) software. All results are presented as mean values, with p < 0.05 as the cutoff for statistical significance.

The effect of different levels of inclusion (0, 3, 6, 9, 12%) of TmLM on the live weight, percentage of carcass, fat deposition, and the weight of some physiological organs (liver, heart, gizzard) of broilers are presented in Table 3.

Table 3: Effect of TmLM treatment on live weight, percentage of carcass abdominal fat and physiological organs.

No meaningful changes were recorded on the variables considered (P > 0.05). In the study, some individual broilers weighed 1502 g and others weighed 1533.6 g. Unchanged carcass yields of the broilers were recorded from 75.4% to 75.96%. Regardless of TmLM inclusion, the organ weights remained constant.Recent research suggests that TmLM has a very slight, statistically insignificant impact on live weight and carcass yield. This supports the perspective of the reviewed study on insect meals, specifically the calories of Tenebrio molitor, as a potential alternative protein source to fish meal with no negative impact on growth performance in broiler diets (Biasato et al., 2022; Mao et al., 2020). Biasato et al. (2022), reported the same growth and carcass yield in broilers as supported by this study. Other research suggests minimal differences in development are related to the insect meal utilized and the diet formulation. This indicates that the influencing factor may be the nutritional quality and digestibility of the diet, which may explain the differences in performance (Schiavone et al., 2020).

There was some fluctuation in abdominal fat content without a consistent pattern, suggesting that the inclusion of TmLM does not influence fat deposition in broilers. Therefore, insect meal substitutions appear not to have a substantial impact on this parameter. Similar findings have been reported by Bovera et al. (2020). Losses in body and visceral fat were attributed to the incorporation of insect meal and were explained by changes in fatty acid profile and chitin (Cullere et al., 2019; Toral et al., 2025). A lack of statistical significance suggests that the level or timing of TmLM inclusion might not be sufficient to influence lipid metabolism in a meaningful way.

Table 4: Effect of TmLM treatment on total blood cholesterol, triglycerides, HDL, LDL and thigh meat cholesterol.

No statistically significant differences in the relative weights of the liver, heart, and gizzard were observed among treatments, indicating that substituting TmLM up to 12% does not have detrimental effects on the health and function of these organs. This is in line with Gasco et al. (2019), who showed that the inclusion of insect meals was not associated with negative histopathological changes or organ hypertrophy. Concerns raised in the past that organ overload might result from anti-nutritional factors or allergens linked to insect protein were not confirmed in this study (Henry et al., 2021). The close agreement in organ weights suggests that the interventions were successful in providing a balanced substrate that allowed efficient nutrient metabolism and the maintenance of proper dynamic equilibrium.

Including Tenebrio molitor larvae meal as a protein replacement in broiler feeds aligns with sustainable animal production trends over the last five years (Van Huis et al., 2021). The rearing substrate and processing of TmLM can affect the fatty acid profile and nutritional composition variables that influence lipid metabolism outcomes and meat quality (Montalbán et al., 2023). The small sample size and non-significant differences in carcass and organ parameters of broilers in this study suggest that TmLM can be included in the diet without adverse effects, at least under these confined conditions.

The effect of different levels of inclusion (0, 3, 6, 9, 12%) of TmLM (TmLM) on the total blood cholesterol, triglycerides, HDL, LDL and thigh meat cholesterol of broilers are presented in Table 4.

The difference in total blood cholesterol level is highly significant (p<0.05), from 134.43 mg/dL (TmLM 0) to 116.85 mg/dL (TmLM 12), with a 17.58 mg/dL decrease in overall blood cholesterol level (BCL). Continued positive blood lipid-lowering results are precise, with basal triglyceride (TG) levels decreasing from 39.03 mg/dL to 28.18 mg/dL. The (Lipid-lowering) Treatment is confirmed positive-blood: Cardiovascular and lipids. Healthy HCL is identified and increased from 38.93 mg/dL to 51.60 mg/dL (TmLM 0 to 12). Low-Density Lipids (LDL) (high cardiovascular risk). The remaining cholesterol, 81.68 mg/dL, decreased by 27.63 mg/dL to 53.35 mg/dL, suggesting a reduction in cardiovascular risk. The thigh meat cholesterol (health nutrient quality of meat) is reduced from 120.45 mg/100g to a positive 100.10 mg/100g cholesterol (TmLM 12). The evidence confirms the treatment’s improvement, and high-nutrient-quality meat has a moderate overall positive cardiovascular effect. The therapy now significantly improves overall blood lipid profile from (Lipid lowering) to still cardiovascular positive overall blood cholesterol, high to low (restrictive).

Blood lipid profile was significantly improved due to the high and moderate inclusion of Tenebrio molitor larva meal (TmLM) in the diets administered to the chickens. Total blood cholesterol was reduced by 14.59 mg/dL, from 134.44 mg/dL to 116.85 mg/dL, and triglycerides by 10.85 mg/dL, from 39.03 mg/dL to 28.18 mg/dL. In comparison, high-density lipoprotein (HDL) cholesterol increased from 38.93 mg/dL to 51.60 mg/dL, and low-density lipoprotein (LDL) cholesterol decreased from 81.68 mg/dL to 53.35 mg/dL. This alteration in the blood lipid profile reduced cardiovascular disease risk by increasing the HDL/LDL ratio.

In addition, there was a reduction in the cholesterol level of thigh meat from 120.45 mg/100 g to 100.10 mg/100 g in the TmLM 12 group (12% TmLM), and this was a potential improvement in the nutritional quality of the meat due to the health benefits derived from the substitutive traditional meat and animal protein. In addition, the TmLM supplement reduced cholesterol levels in thigh meat and improved its nutritional quality, benefiting consumers’ health who replace traditional animal protein. The potential health benefits to humans from improved lipid metabolism stemmed from the unbalanced high-protein, low-saturated fat, and high-fiber content of TmLM.

The use of TmLM at levels of 6-12% has been reported to reduce total cholesterol and LDL by 15–20%, and to increase the HDL/LDL ratio, without any negative effects on the growth performance of broiler chickens (Mao et al., 2020). Makkar et al. (2022) assessed the antihyperlipidemic effects of TmLM, attributed to omega-3 fatty acids and other antioxidants, and confirmed a 25% reduction in triglycerides in animals. In TmLM, Cullere et al. (2021), reported increases in triglycerides and LDL above 20%, likely due to synergistic effects with other dietary constituents. Van Huis (2022), documented the range of TmLM functionality across various animal species, nutritional formulations, and other climatic conditions, and suggested the need for further research to optimize these.

The effect of different levels of inclusion (0, 3, 6, 9, 12%) of TmLM on the on omega-3, -6, -9, DHA, EPA and fat of thigh meat. of broilers are presented in Table 5.

However, at TmLM 12, there is a significant percentage increase (p < 0.05) to 0.91 mg/dL, meaning that the additions or treatments of 12 TmLM are beneficial to increasing the Omega-3 fatty acids concentration. There is consistency in omega-6 fatty acid percentages from TmLM 0 to TmLM 9, and from TmLM 0 to TmLM 9 and 17.18 mg/dL to 17.39 mg/dL, and then at TmLM 12, there is a significant increase to 18.65 mg/dL, indicating that this level of treatment increases omega-6 levels. Omega-9 fatty acids percentage consistently increases from TmLM 0 (40.56 mg/dL) to TmLM 12 (42.14 mg/dL). There are significant percentages at each treatment level, indicating that this is significantly facilitated and determined to be a function of omega-9, with increasing percentages of fatty acids. There is a correlation with the proportions of TmLM treatments. The fat content at each TmLM level is minimal, ranging from 8.40% in TmLM 0 to 8.33% in TmLM 12.

Table 5 shows that the addition of TmLM to the diet of broiler chickens improved the omega-3 fatty acid profile of the thigh meat. There was a 0.82 mg/dL to 0.85 mg/dL stabilization at TmLM levels between 0 and 9, followed by a significant elevation to 0.91 mg/dL at 12% TmLM. There were positive effects on the production or deposition of omega-3 fatty acids up to 12% levels of TmLM in TmLM therapy. This may be attributed to the relatively high nutritional profile of TmLM, consisting of preeminent unsaturated fatty acids and high-quality proteins known to optimize lipolysis. According to Cullere et al. (2021), dietary supplementation with mealworm (Tenebrio molitor) larva meal resulted in a 15–20% increase in omega-3 fatty acid content in chicken meat. We attribute this phenomenon to higher levels of desaturase-activating enzymes that drive omega-3 synthesis, thus explaining the heightened levels observed in this study at higher TmLM.

A similar pattern was observed with omega-6 fatty acids; however, the levels remained essentially constant from TmLM 0 to TmLM 9 at around 17.18-17.39 mg/dL, before increasing to 18.65 mg/dL at TmLM 12. This positive increase indicates that at this level, TmLM enhanced omega-6 content, presumably due to the exercise of TmLM lipids and antioxidants on omega-6 fatty acids’ metabolic routes. This is contrary to the findings of Schiavone et al. (2020), who claimed that the supplementation of insect meal in hens’ diets did not consistently lead to an increase in omega-6 levels. The findings were contingent on the insect species used and particularities of the basal diet formulation, demonstrating the variability due to the specific details of the study.

The omega-9 fatty acids showed a gradual, consistent increase from TmLM level 0 (40.56 mg/dL) to TmLM level 12 (42.14 mg/dL). Each treatment level showed a significant difference, demonstrating a direct relationship to the strength of the TmLM treatment. Increased monounsaturated fatty acids in TmLM, and the greater increase, likely explain why fatty acids are absorbed and stored in chicken muscle tissue. As noted by Gasco et al. (2020), mealworm meal can increase omega-9 fatty acids through increased lipogenic activity, which explains the increase observed in the study. Contrary to Gasco et al. (2020), research carried out by Józefiak et al. (2023) suggests that the impact of TmLM on omega-9. Across chicken strains, the difference is not significant. Variations in the increase were due to genetics, suggesting that other variables need to be considered.

The fat content of thigh meat at TmLM levels of 0 (8.40%) and 12% (8.33%) was not directly influenced by the inclusion of TmLM in the diet. The observed differences are likely attributable to interactions between TmLM and other dietary ingredients, as well as overall diet balance, which can affect fat deposition. Biasato et al. (2017) reported that Tenebrio molitor larvae meal does not consistently increase total fat content and may, in certain

Table 5: Effect of TmLM treatment on omega-3, -6, -9, DHA, EPA and fat of thigh meat.

Note: a,b,c = Superscripts that differ in each row showing a significant difference (p< 0.05). ns = non-significant (p> 0.05).

cases, decrease fat deposition due to the anti-obesogenic properties of insect-based feeds, with observed variability likely associated with dose and genetic differences among broilers. The results of this study indicate that including TmLM up to 12% improves the fatty acid composition of broiler thigh meat, with changes observed across several fatty acid types.

This study demonstrates that dietary inclusion of Tenebrio molitor larvae meal (TmLM) up to 12% can effectively replace imported fish meal in broiler diets without adversely affecting growth performance, carcass yield, abdominal fat deposition, or the relative weights of major physiological organs. The absence of significant differences in carcass and organ parameters indicates that TmLM maintains normal physiological function and productive performance in broiler chickens. Furthermore, supplementation with TmLM significantly improved blood lipid profiles by reducing total cholesterol, triglycerides, and low-density lipoprotein (LDL) concentrations while increasing high-density lipoprotein (HDL) levels. In addition, TmLM reduced thigh meat cholesterol content and favorably modified the fatty acid composition, thereby enhancing the nutritional quality of broiler meat without increasing total fat content. These findings support the potential of TmLM as a sustainable and nutritionally valuable alternative to imported fish meal in broiler production. Further studies are recommended to evaluate the long-term effects of TmLM supplementation, investigate its application in different broiler genotypes, assess the influence of rearing substrates and processing methods on its nutritional composition, and conduct economic analyses and large-scale field trials to facilitate its commercial adoption in poultry production systems.

The authors would like to express their sincere gratitude to Universitas Andalas which funded this research with Klaster Riset Publikasi Guru Besar No.: T/20/UN16.17/PP.Pangan/PDU-KRP1GB-Unand/2022. The authors also thank the Dean and the Chancellor for providing the opportunity and facilities to conduct this research.

This study provides a comprehensive evaluation of Tenebrio molitor larvae meal (TmLM) as a complete substitute for imported fish meal in broiler diets by simultaneously assessing carcass yield, physiological organ health, blood lipid metabolism, and thigh meat fatty acid composition. Unlike previous studies that primarily focused on growth performance or carcass traits alone, this research integrates multiple health and meat quality indicators, demonstrating that TmLM inclusion up to 12% can fully replace imported fish meal without adverse effects while significantly improving lipid profiles and enriching beneficial fatty acids in broiler thigh meat.

Nuraini conceived and designed the study, supervised the experiment, analyzed the data, and drafted and revised the manuscript. YSN contributed to data analysis. RA and YCS contributed to assisted in animal management, data curation and formal analysis. FM contributed to data collection, statistical analysis and manuscript revision. MA contributed to laboratory work, result interpretation, and manuscript revision. AT participated in data validation, literature review, and final manuscript editing. All authors read and approved the final manuscript.

All experimental protocols were sanctioned by the Animal Care Committee of Universitas Andalas (2019). All investigations were executed in accordance with the International Guiding Principles for Animal Research and Welfare.

The authors declare that no generative AI and AI assisted technology was used in the creation of this manuscript.

The authors have declared no conflict of interest.