# Digestibility in H. illucens larvae: resolving faeces collection and ingesta quantification issues

J B Guillaume, W Xu, S Mezdour, F Marion-Poll, C Terrol, P Schmidely

► To cite this version:

J B Guillaume, W Xu, S Mezdour, F Marion-Poll, C Terrol, et al.. Digestibility in H. illucens larvae: resolving faeces collection and ingesta quantification issues. 74. Annual Meeting of the European Federation of Animal Science, Aug 2023, Lyon, France. 2023. [hal-04495298](https://hal.inrae.fr/hal-04495298)

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Book of Abstracts of the 74th
Annual Meeting of the European
Federation of Animal Science

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Book of abstracts No. 29 (2023)

Lyon, France

26 August – 1 September, 2023
## Effect of water-soluble complementary feed on performance in nursery of black soldier fly

L. Schneider¹, M. Brake², A. Heseker², W. Westermeier³ and G. Dusel¹

¹University of Applied Science Bingen, Department of Animal Nutrition, Berlinstraße 109, 55411 Bingen am Rhein, Germany, ²MIAVIT GmbH, Robert-Bosch-Str. 3, 49632 Essen, Germany, ³FarmInsect GmbH, Münchner Str. 10, 85232 Bergkirchen, Germany; l.schneider@th-bingen.de

The starter feed is one of the most important diets of livestock insects and especially for newly hatched black soldier fly (BSF) neonates. Proper nutrition during the starter phase is essential for adequate growth and development, which will ultimately affect the overall performance of the flock. The aim of the study was to evaluate the performance and development of BSF neonates reared in a controlled environment on an iso-nitrogenous and iso-energetic standard diet (wheat bran, chicken feed, water) with either water-soluble mineral complementary feed (WSCF1, 1%, WSCF2, 2%, WSCF3 3%) or a non-water-soluble mineral feed addition for poultry (MFP, 3%) or a control without mineral feed addition (CON). A total of 62 g BSF eggs (1 g eggs = 0.45 g neonates) were placed randomly per unit (6 replicates/ treatment, 60×40×15 cm). At the end of the nursery phase the mean bodyweight of 100 larvae (BW), dry matter (DM) and total weight of 5-day-old larvae output per unit as well as total weight of larval frass was observed. The results showed that the mean BW of WSCF3- and WSCF2-fed (BW, 0.12 g/100 BSFL) larvae were nearly similar and increased by 33% (*P*<0.05) compared to CON-fed larvae (BW, 0.09 g/100 BSFL). Additionally, the DM (%) of WSCF3-fed larvae showed an 8% increase compared to MFP-fed larvae. WSCF2-fed larvae showed a 13% higher (*P*<0.05) total weight of young larvae compared to CON-fed larvae (WSCF2, 678.5 g; CON, 588.4 g). The total weight of the larval frass at the end of the nursery phase of group WSCF2 was 31% lower (*P*<0.05) than that of the CON-group (WSCF2, 1,797.3 g; CON 2,616.6 g). In conclusion, this first study demonstrates the potential of a water-soluble complementary feed on the performance and development of young BSF in the crucial pre-starter phase. Further investigations are necessary to recommend a possible efficient and sustainable preconditioning effect in BSF nursery.

## Digestibility in *H. illucens* larvae: resolving faeces collection and ingesta quantification issues

J.B. Guillaume¹,²,³, S. Mezdour⁴, F. Marion-Poll²,⁵, C. Terrol¹ and P. Schmidely³

¹Agronutris, R&D Department, 35 bld du Libre Echange, 31650 Saint-Orens de Gameville, France, ²CNRS, IRD, Université Paris-Saclay, Laboratoire EGCE, IDEEV, 12 route 128, 91190 Gif-sur-Yvette, France, ³Université Paris-Saclay, INRAE, AgroParisTech, UMR Modélisation Systémique Appliquée aux Ruminants, 22 place de l'Agronomie, 91120 Palaiseau, France, ⁴Université Paris-Saclay, INRAE, AgroParisTech, Sayfood, 22 place de l'Agronomie, 91120 Palaiseau, France, ⁵Université Paris-Saclay, AgroParisTech, 22 place de l'Agronomie, 91120 Palaiseau, France; jeremy.guillaume@agroparistech.fr

Black soldier fly larvae (BSFL; Hermetia illucens) are increasingly studied for their ability to convert organic substrates into body proteins and lipids that can be used for animal nutrition. Although many studies have used BSFL high weight gain to highlight their strong feed conversion efficiency, little is known about the inherent efficiency of each of the four feed conversion stages: ingestion, digestion, absorption, and metabolic utilisation. Assessing digestibility requires quantifying the amount of feed ingested and the associated faeces produced. However, this is challenging in BSFL because they feed and release excreta in the same substrate, which also hosts complex microbiota participating in digestion. This study introduced a new indicator called Estimated Digestibility (ED), defined as the difference between distributed feed and frass macronutrient weight, divided by macronutrient weight in distributed feed. The evolution of ED was assessed with increasing larval density in order to ensure complete feed ingestion and frass free from refused feed. ED was measured on a standard diet with densities from 0 to 29 larvae/cm² for dry matter (DM), starch, nitrogen, ether extract (EE), neutral detergent fibre, acid detergent fibre, acid detergent lignin, ash and energy. The results showed a sigmoidal pattern for ED of all fractions except fibres. Asymptotic ED was 80.3±1.3% (mean ± standard error) for DM, 99.0±2.3% for starch, 78.6±1.1% for nitrogen, 95.3±1.5% for EE, 58.4±1.0% for ash and 80.6±1.2% for energy. Asymptotic ED is the closest estimation of digestibility as defined in other species. It offers perspective on the understanding of BSFL digestive efficiency and could be used for diet formulation.
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# Digestibility in *Hermetia illucens* larvae:
getting over faeces collection and ingesta quantification issues

Guillaume J.B. ¹,²,³, Xu W. ², Mezdour S. ⁴, Marion-Poll F. ²,⁵, Terrol C. ³, Brouzes C. ³, Schmidely P. ¹

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Download in PDF

¹ Université Paris-Saclay, INRAE, AgroParisTech, UMR Modélisation Systémique Appliquée aux Ruminants, Palaiseau, France

² Laboratoire Évolution, Génomes, Comportement et Écologie (EGCE), CNRS, IRD, Université Paris-Saclay, Institut Diversité, Écologie et Évolution du Vivant (IDEEV), Gif-sur-Yvette, France

³ Agronutris, R&D Department, Saint-Orens-de-Gameville, France

⁴ Université Paris-Saclay, SayFood, AgroParisTech, INRAE, Palaiseau, France

⁵ Université Paris-Saclay, AgroParisTech, Palaiseau, France

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Contact: jeremy.guillaume@agroparistech.fr

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## Background

* **Black soldier fly larvae** (BSFL; *Hermetia illucens*) can quickly convert various organic substrates into body proteins and lipids. Due to its high conversion efficiency, this species has gained significant attention in the field of insects as feed and food [1].

* BSFL fed diets with the same crude protein and carbohydrate contents but formulated with different ingredients show various performances [2], possibly due to different **digestive efficiencies**. This highlights the need to obtain digestibility values for accurate diet formulation.

* Digestibility calculation involves a **mass balance** approach based on **ingested feed (ingesta)** and associated faeces (Eq. 1). Accurate ingesta measurement and proper faeces collection in BSFL are challenging because larvae feed and excrete in the same moist substrate.

<figure><img src="image_11.png" /><figcaption>Distributed feed ≠ Ingesta</figcaption></figure>

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Frass =

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Equation 1

$$ \text{Digestibility} = \frac{\text{Mass of ingesta} - \text{Mass of faeces}}{\text{Mass of ingesta}} $$

How can we measure digestibility in BSFL conversion systems?

Two methods (A and B) will be presented

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## Materials and methods

* **Method A:** measuring **Estimated Digestibility** (ED; Eq. 2), calculated through a **mass balance** between **ingested feed** and residual substrate (frass).

* ED of dry mass (DM) was measured at increasing larval densities (0 to 29 larvae/cm²), after a **fixed feeding time**. We hypothesized that **high larval density** would result in **complete ingestion** of discarded feed and that **asymptotic ED** would reflect the total digestion potential of BSFL and their microbiota.

* 7-day old BSFL from Agronutris were fed 420g of fresh substrate. Trials were performed in 17x11x7cm containers in climate-controlled conditions (28°C, 75% RH, L12:D12).

* This approach was performed on chicken feed, discarded potatoes and corn gluten feed.

Equation 2

$$ \text{Estimated Digestibility} = \frac{\text{Mass of distributed feed} - \text{Mass of distributed feed}}{\text{Mass of distributed feed}} $$

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* **Method B:** addition of an **indigestible marker** (chromic oxide, $Cr_2O_3$) in the feed in order to calculate **Approximate Digestibility** (AD; Eq. 3). This method has been extensively used in livestock and other insect species [3].

* 200 eleven-day old BSFL were fed 400g of fresh substrate with 1% $Cr_2O_3$ (%DM). After 3 days, larvae were removed from the substrate, rinsed and put in an **empty container** to let them **defecate** for 24h. Excreta was collected by dilution with distilled water and a pipet, followed by water evaporation. Marker concentration in excreta was measured by **colorimetry** (540nm) after complete oxidation.

* This approach was performed on chicken feed, discarded potatoes, corn gluten feed, wheat bran and wheat distillers grain.

Equation 3

$$ \frac{\text{Approximate Digestibility}}{\text{Marker conc\textsuperscript{a}}} = \frac{\text{Marker conc\textsuperscript{a}} \text{ in excreta}}{\text{Marker conc\textsuperscript{a}} \text{ in ingesta}} $$

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Ingesta

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Excreta

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## Results

* In chicken feed, all containers were sieved after 7 days of feeding. ED of DM increased with larval density following an **asymptotic trend**, up to a maximal value of $80.3\pm1.3\%$ (mean ± standard error).

* Asymptotic ED of **starch** (99.0±2.3%), **nitrogen** (78.6±1.1%), **ether extract** (95.3±1.5%), **ash** (58.4±1.0%) and **energy** (80.6±1.2%) were also assessed. Further details on chicken feed results have been published [4].

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* In discarded potatoes after 7 days of feeding, the frass was too moist and sticky to properly separate it from the larvae. Feeding time was extended to **16 days** to allow proper evolution of frass texture. ED of DM in discarded potatoes increased with density but the asymptote was reached at lower densities than in chicken feed, presumably because longer feeding time allowed low-density containers to achieve similar digestion level as those with more larvae. **Asymptotic ED of DM was 70.4\pm0.7\%**.

* The same issue with frass texture was observed in **corn gluten feed** and feeding time was extended to **19 days**, leading to low ED differences between densities. Slight decrease in ED of DM at high density is probably an artefact resulting from the initial inclusion of more residual frass with starter larvae at the start of the experiment. **Asymptotic ED of DM was 45.6\pm0.5\%**.

* Asymptotic ED of **wheat distillers grain** was also explored, but could not be determined due to **low survival** and **start of pupation** (i.e. end of feeding) before frass texture allowed proper separation of the larvae.

* AD of DM determined with 1% $Cr_2O_3$ was 87.5±1.2% in chicken feed, 77.1±1.1% in discarded potatoes, 64.2±2.6% in corn gluten feed, 57.9±2.1% in wheat bran and 86.1±1.2% in wheat distillers grain.

* AD of nutrients such as starch or proteins could not be determined because **too little excreta** was collected.

<figure><img src="image_23.png" /><figcaption>Excreta left by 200 larvae after 24h (the green color is due to $Cr_2O_3$)</figcaption></figure>

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* **AD of DM (Method B)** was higher than **asymptotic ED of DM (Method A)** in all diets investigated. A possible explanation is that, given their inability to ingest too large particles, BSFL might **exclusively consume the semi-liquid phase** of the diet containing all the marker, leading to an overestimation of AD. This is particularly likely in corn gluten feed which contained large maize pericarp particles.

* The **digestibility order remained the same in both methods**: chicken feed > discarded potatoes > corn gluten feed. These findings are consistent with the notion that chicken feed represents a highly effective formulated diet, discarded potatoes and wheat distillers grain are rich in digestible carbohydrates, while corn gluten feed and wheat bran have higher fiber content.

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## Conclusion

* High quantity of frass is collected, allowing for measurement of ED of various nutrients (DM, starch, nitrogen, specific amino acids or minerals, etc).

* Asymptotic ED measurement requires total ingestion of substrate: **impossible in low-performing diets** (mortality, sticky frass, etc) → Working on composed diets could allow for total ingestion. This approach would require to check additivity of ED.

* Separation of frass and larvae is **time-consuming** at low-density or in low-performing diets.

* Considers overall digestion by **both larvae and microbes** in the substrate.

* In diets requiring extended feeding time to achieve non-sticky frass, **microbial digestion** might continue even after complete ingestion by BSFL, potentially resulting in an overestimation of asymptotic ED. The reliability of comparing asymptotic ED of diets with different feeding times could be questioned.

* Only considers digestion occurring in **larval gut**.

* Easily repeatable on different substrates.

* $Cr_2O_3$ quantification requires **toxic chemicals** → Less toxic indigestible markers could be used. Titanium dioxide has recently been successfully used for AD determination in BSFL [5].

* **Inadapted for heterogenous substrates** or with large particles: risk of feed selection by larvae (marker concentration in ingesta ≠ marker concentration in substrate).

* Excreta collection is **time-consuming**.

* **Small quantity of excreta collected**: difficult to assess AD of various nutrients → The excreta collection procedure could be refined (e.g. using more larvae or longer gut-emptying period). However, these changes may come with new biases such as increased risk of copropagry or microbial degradation of samples, leading to overestimation of AD.

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Two methods have been proposed to assess digestibility in BSFL. These results provide insight into the digestive efficiency of BSFL and lay the ground for diet formulation based on digestible instead of crude nutrient contents.

<footnote>[1] Barragan-Fonseca et al., 2017. Nutritional value of the black soldier fly (Hermetia illucens L.) and its suitability as animal feed - a review. JIFF.</footnote>

<footnote>[2] Gold et al., 2020. Biowaste treatment with black soldier fly larvae: Increasing performance through the formulation of biowastes based on protein and carbohydrates. Waste Manag.</footnote>

<footnote>[3] McGinnis & Kasting, 1964. Comparison of gravimetric and chromic oxide methods for measuring percentage utilization and consumption of food by phytophagous insects. J. of Ins. Phys.</footnote>

<footnote>[4] Guillaume et al., 2013. Asymptotic estimated digestibility, a new indicator of black soldier fly (Hermetia illucens) conversion efficiency in relation to larval density. JIFF.</footnote>

<footnote>[5] Veldkamp, van Wikselaar & van Loon, personal communication.</footnote>