Document Type : Research Paper
Extended Abstract
Introduction
With the continuous growth of the global population and income levels, the demand for animal-derived protein is steadily increasing; however, the production of such protein is associated with considerable environmental challenges. These concerns have shifted scientific attention toward more sustainable protein sources, including edible insects. Many insect species are rich in protein, minerals, and vitamins, and therefore represent promising alternatives to conventional feed ingredients such as soybean meal and fishmeal, which are costly and are expected to become less accessible in the future. The black soldier fly larva (Hermetia illucens) is one of the most promising candidates in this regard due to its suitable chemical composition and its ability to be mass-produced on organic waste substrates. The chemical composition of BSFL typically includes 41.1–43.6% crude protein, 15–34.8% ether extract, 0.7% crude fiber, 14.6–28.4% ash, and approximately 5,278 kcal/kg gross energy on a dry-matter basis. In ruminant nutrition, the extent of protein degradation in the rumen is a major determinant of protein utilization efficiency and the amount of amino-acid–containing protein that reaches the small intestine. Despite its importance, available information on the ruminal degradability of insect-derived protein is limited. On the other hand, tannins—particularly those present in pomegranate peel—can bind to proteins and reduce their rapid degradation in the rumen, allowing a greater proportion of dietary protein to escape ruminal breakdown and become available for intestinal digestion. However, determining the optimal level of tannins is crucial, as excessive amounts can impair microbial activity and fiber digestion. Considering the valuable nutritional characteristics of BSFL and the potential of natural tannins in pomegranate peel, the present study was conducted to evaluate the effects of combining these two resources on rumen fermentation kinetics and fermentation characteristics under in vitro conditions, and to assess the potential synergistic interaction between tannins and insect-derived protein.
Materials and Methods
This study was conducted in two stages to evaluate the effects of combining pomegranate pomace and black soldier fly larvae (BSFL) on ruminal fermentation under in vitro conditions. For material preparation, hatched eggs of Hermetia illucens were obtained from the Iran Larvae Center (Mashhad). Larvae were reared under controlled environmental conditions, including 28°C temperature, 60–70% relative humidity, and a 12:12 h light–dark cycle. After a 12-hour fasting period to empty the digestive tract, the larvae were dried at 55°C and subsequently ground. Fresh pomegranate pomace was sourced from the Kosheh Sorkh Agro-industry (Qain) and shade-dried. Chemical composition, including dry matter, ash, crude protein, and ether extract, was analyzed according to AOAC (2005), and total tannins were determined using the Folin– Ciocalteu method.In the first experiment, BSFL powder and pomegranate pomace were mixed at six inclusion levels (0 to 12.5% pomace). In vitro gas production was assessed following the procedure of Blummel et al. (1997) using 120-mL anaerobic fermentation bottles. Each incubation consisted of 500 mg dry matter and 50 mL of a rumen fluid–buffer mixture (1:2). Gas pressure was recorded at 2 to 120 hours of incubation, while pH, dry matter digestibility, crude protein digestibility, and ammonia nitrogen were measured at specific time points. Rumen fluid was collected from two rumen-fistulated Holstein cows prior to morning feeding, filtered through four layers of cheesecloth, and transported immediately to the laboratory. Dry matter digestibility was determined according to Jahani et al. (2011), and ammonia nitrogen was quantified using the phenol–hypochlorite method.In the second experiment, the optimal mixture identified in Experiment 1 was used as a protein supplement and incorporated into the basal diet at 10%, 20%, and 30% replacement levels for soybean meal. Gas production data were fitted to the Schofield nonlinear model using the NLIN procedure of SAS (Version 9.4). Other parameters were analyzed using the General Linear Model (GLM), and treatment means were compared using Tukey–Kramer’s test at a 5% significance level.
Results and discussion
The results showed that varying tannin levels significantly affected gas production kinetics. The highest potential gas production (V) was observed in treatment 2, while the lowest was in treatment 4. Increasing tannin levels reduced gas volume, and high tannin concentrations exerted an inhibitory effect on fermentation. The highest gas production rate (k) was observed in treatment 3, indicating a faster initial fermentation followed by a more stable process. The g₀.₅ index mirrored potential gas production, suggesting that low tannin levels improved microbial access to substrates, while higher T₀.₅ values indicated slower but more stable fermentation. Regarding dry matter disappearance, significant differences were observed between treatments during early incubation (4–24 h), but no differences were found at 48 h. Low tannin levels (pomegranate peel with black soldier fly larvae powder) improved dry matter and protein digestibility and stabilized rumen pH, whereas high tannin levels limited microbial activity and disrupted normal fermentation. Ammonia nitrogen concentration decreased in the early hours due to tannin–protein complex formation, which inhibited rapid protein degradation; however, over time, partial breakdown of these complexes allowed nitrogen release. Protein digestibility was initially reduced by tannins but increased during mid-to-late incubation as microbial populations adapted and complexes dissociated, reaching levels close to the control. Overall, black soldier fly larvae can partially replace soybean meal in ruminant diets. Adding pomegranate peel at low to moderate levels positively influenced gas production, dry matter and protein digestibility, and pH stability, while high tannin levels had negative effects. The optimal combination of black soldier fly larvae and pomegranate peel enhanced protein utilization, reduced nitrogen losses, and improved the environmental sustainability of ruminant diets, making it a promising, innovative, and eco-friendly protein source.
Author Contributions
Data Availability Statement
The data supporting reported results are collected and used from published articles, to them we have referred in the manuscript.
Acknowledgements
The authors would like to thank the Environmental Stresses Group in Animal Sciences, University of Birjand for financial support.
Ethical considerations
Conflict of interest
The author declares no conflict of interest.
