Response to egg production, egg quality and fatty acid profile of yolk when feeding ISA Brown laying hen by diets containing full-fat black soldier fly larvae meal

Le Duc Thao, Nguyen Hai Quan and Le Duc Ngoan

University of Agriculture and Forestry, Hue University. 102 Phung Hung, Hue city, Thua Thien Hue, Viet Nam
nhquan@hueuni.edu.vn

Abstract

The study aimed to evaluate the effect of inclusion of 15% and 30% of black soldier fly larvae (BSFL) meal in the diet based on maize-soybean meal on egg performance and quality, and egg yolk fatty acid profile. Total 81 ISA Brown layers at 33-36 weeks of age randomly allocated to one of 3 dietary treatment, including the control diet (CTL) mainly contained maize-soybean meal, and other two diets (L15 and L30) formulated by replacing 15% and 30% of the control diet by BSFL meal. Egg production and weight, Haugh index, and fatty acid profile of yolk were recorded. Results showed that inclusion of BSFL meal did not affect egg production and egg weight but increased egg albumen part and reduced shell part, feed intake and feed conversion to 10 eggs; Albumen height and Haugh value were not affected by replacement 15% and 30% of control diet by BSFL, however the egg’s yolk color and the concentration of fatty acid omega-3 of yolk after 9 days feeding were improved. In conclusion, inclusion of 30% full-fat black soldier larvae meal in maize-soybean based diet for laying hen reduced feed intake but improved efficiency of feed conversion to egg and the yolk color and increased the concentration of the n-3 polyunsaturated fatty acids.

Keywords: cholesterol, Haugh index, omega-3, omega-6, yolk color

Introduction

The insect species with the highest potential for large scale production are the black soldier fly (Hermetia illucens). Specifically, black soldier fly larvae (BSFL) have a high growth rate and an excellent conversion of organic waste to produce a meal with high crude protein and consistent amino acid concentration (Spranghers et al 2017). In addition, Lan et al (2022) reported that BSFL fed by tofu by-products have contained high fatty acids, especially essential fatty acids: linoleic acid (LA, omega-6) and α-linolenic acid (ALA, omega-3). The fatty acid composition of an ingredient has a direct effect on fat utilization or deposition in poultry. Taking advantage of dietary omega-3 fatty acid deposition into yolk, producers are able to create value-added ALA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) enriched eggs (Her et al 2017).

Many studies have investigated the effects of including BSFL in diets to both broilers and laying hens. In broilers, BSFL meal is a good source of apparent metabolizable energy and digestible amino acids (De Marco et al 2015; Schiavone et al 2017) and has been shown to improve growth rate (Oluokun 2000). In laying hens, one study including full-fat dried BSF larvae in the feed resulted in an inferior feed conversion ratio (FCR) and egg production compared to a control treatment that did not receive larvae (Bejaei and Cheng 2018). Kawasaki et al (2019) found an increase in egg weight and egg shell thickness in a group fed dried whole BSF larvae compared to the control. Mwaniki et al (2018) reported that feeding BSFL meal linearly increased yolk color, egg shell-breaking strength and thicknes and concluded that birds fed 7.5% BSFL meal had similar hen-day egg production and egg mass but poor FCR relative to corn–soybean meal diet. On the other hand, inclusion of 24% BSFL meal reduced daily feed intake, body weight and egg production of White Leghorn hens, however, 8 and 16% BSFL meal levels had no negative impact on performance and were not significantly different than the control diet (Patterson et al 2021). The authors also indicated that yolk color was again higher among the meal treatments compared to the control.

To our knowledge, limited studies have been reported on effect of laying hen diets containing full-fat BSFL meal on egg production, particularly chemical composition and fatty acid profile of egg yolk. Therefore, the objective of the present study was to evaluate effects of 15 and 30% inclusion of full-fat BSFL meal in diets fed to laying hens on egg production and quality.

Materials and methods

This experiment was carried out at the Research and Training Centre of the Faculty of Animal Sciences and Veterinary Medicine, Hue University in October-November 2022.

Full-fat black soldier fly larvae meal and diets

Photo 1. Black soldier fly larvae meal

BSFL were fed by tofu by-products at room temperature 26-33^oC and collected at day 7-8th after rearing. Larval meal preparation was followed Lan et al (2022^b). Larvae were washed with water several times to remove all impurities, boiled at 60^oC water for 1 minutes, took out and dried at 55°C for 24 hrs and milled into full-fat BSFL meal (Photo 1).

Photo 2. Birds kept in cages

Control diet was prepared by carefully mixed all ingredients according to their ratio in the diet (Table 1), ground and stored in plastic bags at room temperature prior to use. Two other diets were mixed 85% or 70% of the control diet with 15% or 30% BSFL meal and stored in plastic bags before using. Chemical composition and fatty acid profile of three diets present in Table 2.

Experimental design

Total 81 ISA Brown laying hens at 33 weeks of age kept in cages were randomly allocated to 1 of 3 dietary treatments and 3 replicates per treatment (Photo 2). Three treatments named CTL, L15 and L30, in which, CTL is the control diet mainly contained maize and soybean meal (Table 1), and L15 and L30 are 15% and 30% of the control diet were replaced by full-fat BSFL meal. Birds were fed twice per day at 7.00 and 16.00h and free access to drinking water.

Measurements

Performance: Eggs laid each day were recorded and a sample of eggs were randomly collected and labeled at day 3, 7, 9, 10, 12, 15 and 20 of the experimental periods for quality parameters.

Daily feed offered and refusals were determined in order to calculate daily feed intake (DFI). Daily feed intake and conversion (kg feed/10 eggs) were calculated on an average bird basis within a replicate experimental unit (3 cages of 9 birds each).

Egg quality: Weighted eggs in 3, 7, 9, 10, 12, 15 and 20 after birds fed experimental diets, and then separated white, yolk and shell to determine their proportion (as percentage of total egg weight).

The yolk color of each egg was measured with a Roche yolk color fan (1 to 15 colors scale based on DSM/Roche yolk color fan) (Photo 3). Albumen height was measured (mm) using Palmer.

Photo 3. Yolk color of laying hens fed control diet (CTL) and diets of 15% (L15) and 30% (L30) of full-fat BSFL meal

Haugh index (HU) was calculated as following:

HU = 100 log (H + 7.57 – 1.7 W^0,37)

In which, H: albumen height (mm); W: egg weight (g)

Yolk sampling: Samples of yolks were collected at day 3, 5 and 9 after feeding, vacuum dried and stored for chemical and fatty acid analyses (Photo 4).

Chemical analysis

Samples of diets and BSFL meal were collected for chemical analysis: DM, EE, CP and CF following AOAC (1990) procedures at the Lab of the Faculty of Animal Husbandry and Veterinary Medicine, Hue University.

Photo 4. Vacuum drying egg yolks

Fatty acid compositions of eggs were analyzed by Gas Chromatography of Fatty Acid Methyl Esters Method (ISO 12966-2:2017 & AOAC 996.06). Fatty acid compositions of BSFL and diets were analyzed by Gas Chromatography of Fatty Acid Methyl Esters Method (ISO/TS 17764-1:2002) at the EUROFINS Sac Ky Hai Dang, Thu Duc, Ho Chi Minh City.

Data analysis

Data were statistically processed by analysis of variance (ANOVA) using General Linear Model (GLM) in Minitab v. 16.2 (2010), and presented in the form of the least square mean (M) and standard error of the mean (SEM). The difference between the mean values ​​was determined by the Tukey method at a confidence level of 95%. Statistical model:

Y_ij = µ + T_i + e_ij

Where: µ is the average value; T_i is the effect of diets; e _ij is the experimental error.

Results and discussion

Chemical composition and fatty acid profile of BSFL meal

The analyzed chemical composition and fatty acid profile of full-fat BSFL meal sample are shown in Tables 3. This full-fat BSFL meal contained CP 38.06%, crude fat 47.88%, Ca 3.99% and P 1.17% DM. An estimated gross energy of BSFL meal in this study was very high 7,386 kcal/kg DM. Therefore, including 15% and 30% full-fat BSFL meal in diets increased not only the concentrations of CP and EE but also the energy concentration in diets. However, the metabolizable energy content of the BSFL meal in the recent study was not determined yet, therefore, it needs for methods for estimating this value. In this study, the gross energy concentration of full-fat BSFL meal with 47.88% EE was lower compared to the value of 7,840 kcal/kg oil expressed from BSFL (Patterson et al 2021).

The larval nutritional composition can vary depending on the rearing substrate, but in general BSFL contain around 40% CP and 30% EE on a DM basis (Barragan-Fonseca et al 2017). In addition, crude protein variations in BSFL meal are indications of variable fat and chitin concentrations as well as growth substrates (Liu et al 2017). In this study, the content of CP was comparable to the values of 40 to 44% DM for whole BSFL meal (Spranghers et al 2017) but lower than the values of 58.7% DM reported (Lan et al 2022a). The crude fat content of 47.88% was very much higher than the values of 15 to 35% DM reported for full-fat BFLS meal (Makkar et al 2014; Lan et al 2022a).

In this study, BSFL meal contained high concentrations of lauric acid and omega-3 were much higher than the study report of Lan et al (2022b) (12.95-15.39% and 1.89-2.04% as total fat, respectively), while the content of omega-6 was lower compared to this report (27.57-29.7% as total fat). In addition, the results of this study were comparable to literature review by Ewald et al (2020), who reported that lauric acid, omega-3 and omega-6 concentrations in the larvae fed by different substrates ranged 7.5-51.8%, 1.1-3.6% and 2.6-12.5% DM, respectively.

The differences in fatty acid concentrations of the BSFL not only due to substrates rearing the larvae but also larval weight. Ewald et al (2020) indicated that larval weight was found to be positively correlated to the concentration of lauric acid (R² = 0.8) and total SFA (R ² = 0.7) in the larvae, and negatively correlated to total MUFA (R² = 0.5) and PUFA (R² = 0.7).

The concentration of Ca in this study was lower compared to literature value (5 to 8% DM) whereas concentration of P is comparable (0.6 to 1.5% DM) (Makkar et al 2014; Patterson et al 2021).

Egg production

Data in Table 4 show that inclusion BSFL meal in diets didn’t affect egg production or laying percentage (p>0.05) but affected daily feed intake (DFI) and feed conversion to 10 eggs (p<0.05). DFI and feed conversion to 10 eggs were lower in L30 than in CTL and L15. The hens consumed less feed in L30 (89.05 g/day) than in CTL and L15 (105.98 and 106.7 g/day). It suggests that no palatability issue of diets containing BSFL meal. Probably, low feed intake in high BSFL meal diet (L30) dues to high concentration of metabolizable energy in diets (Table 2).

Maurer et al (2016) fed a BSFL meal to Lohmann hens at 12 and 24% of the diet replacing soybean cake and saw no significant difference in DFI, egg production and FCR compared to the hens fed their control diet. However, Marono et al (2017) fed a dark brown BSFL meal to Lohmann Brown hens from 24 to 45 week of age at 17% of the diet replacing soybean meal and reported that DFI and egg production were significantly reduced by the BSFL meal treatment diet compared to a maize-soybean meal control. In comparison, Patterson et al (2021) reported that inclusion of 24% BSFL meal reduced DFI and egg production compared to the hen fed the control diet and diets of 8 and 16% BSFL meal and the authors suggested that substituting up to 16% BSFL meal would sustain normal hen performance. In this study, egg production maintained and DFI and feed conversion to 10 eggs reduced at the diet replacing 30% maize-soybean meal diet. It suggests that inclusion of BSFL meal in maize-soybean meal diet improves feed efficiency.

Egg quality

The egg weight, albumen height and Haugh value were not significant among treatments (p>0.05) but share of basic part in egg such as albumen part increased and shell one declined (p<0.05) when increasing levels of BSFL meal in diets. The yolk color increased when inclusion of BSFL meal, particularly in diet contained 30% of BSFL meal (p<0.05).

In this study, share values of egg albumen, yolk and shell of 59.77-61.64%, 26.31-27.08% and 12.05-13.15%, respectively were comparable to Kralik et al (2021), who reported that the shares of albumen, yolk and shell in their study were 52.24-62.03, 25.38-28.06 and 12.35-13.74%, respectively.

Patterson et al (2021) found that there was no significant impact of the BSFL meal treatments on egg weight at the highest level of inclusion (24% replacing soybean meal) but the egg yolk color was enhanced by all levels of the BSFL meal. Similarly, Mwaniki et al (2020) substituted 10% and 15% soybean meal by BSFL meal into the diets of Shaver White hens in 28 to 43 week of age and found that the yolk color was increased with greater amounts of BSFL meal, but it had no impact on Haugh index and shell thickness.

In this study, the percentage of egg shell part declined at 30% BSFL meal diet (L30) may due to low Ca intake in L30 compared to CTL. Calculated Ca intake in L30 was 3.56 g/day and in CTL 4.3 g/day. Egg shell is 99% calcium carbonate and daily egg shell formation equate to a removal of 2 to 3 g of Ca equivalent to 10% of the hen body Ca reserve (Gilbert 1983; Etches 1987). About 60 to 75% of Ca in egg shell is derived from diet and 25 to 40% was from the skeletal stores (Comar and Driggers, 1949)

The yolk color is one of the most important aspects for consumers and is determined by the type and profile of carotenoids present in the feed and their intestinal absorption. Carotenoids are a fat-soluble group of yellow, red and orange pigments. The results in this study and many others indicated the yolk color improvement suggested the BSFL meal had pigments that increased intensity of yolk color. In fact, Secci et al (2018) demonstrated that feeding laying hens BSFL meal increased concentration of γ-tocopherol, lutein, β-carotene, and total carotenoids compared with egg yolks from birds fed soybean meal.

Fatty acid profile of yolk

Samples of egg yolks in each replicate (n=3) were collected at day 3, 5 and 9 after feeding for fatty acid analysis. Results in Tables 6 and 7 presented the fatty acid profile of yolks in treatments L15 and L30, respectively. In L15 (Table 6), except for the concentration of monounsaturated fatty acid (MUFA) was lower in day 9 than days 3 and 5 ( p<0.05) and omega-3 was higher in day 5 and 9 than day 3 (p<0.05), all fatty acids were not affected by days of egg collecting.

Similarly, the concentrations of MUFA declined at day 5 to 9 and omega-3 increased after day 5 (p<0.05) when birds fed 30% BSFL meal diet (Table 7). In Table 8, the concentrations of omega-3 were higher in L15 and L30 than in CTL.

In general, the concentrations of SFA, UFA, DHA, omega-6 and 9 and total cholesterol were not affected by inclusion larval meal and day of egg collection but the content of yolk omega-3 fatty acid increased when including either 15% or 30% BSFL meal in diets after 9 days feeding laying hen.

In this study, inclusion oil-rich ingredient to the diet has improved egg yolk omega-3 fatty acid concentration. According to Cherian and Sim (1991), hens readily absorb and transfer omega-3 fatty acids from dietary sources for deposition into the yolk. On average, it takes 2 weeks for a laying hen to adjust to an omega-3 fatty acid enriched diet and reach a transfer plateau of dietary omega-3 fatty acid incorporation into developing ovarian follicles (Cherian and Sim 1991; Nain et al 2012). In addition, birds lack the required Δ¹² - and Δ¹⁵ -desaturase enzymes for de novo endogenous synthesis of metabolites of 22 or more carbons from ALA (Hulbert et al 2002). However, they are capable of biosynthesis of up to 22 or more carbons from ALA and LA through the action of Δ5- and Δ6-desaturase and elongase enzymes (Goyens et al 2006; Holman 1998). Therefore, inclusion of ALA in poultry diets could partially ameliorate the limited but desirable upconversion of ALA to long-chain n-3 PUFA. In this study, the concentrations of ALA in BSFL diets were 0.26% DM in L15 and 0.45% DM in L30 higher than 0.08% DM in CTL.

Conclusion

Replacing 15% and 30% of the control diet based on soybean meal and maize by full-fat black soldier larvae meal:

• Did not affect egg production or laying percentage and reduced daily feed intake and feed conversion per 10 eggs.
  
  
• Did not affect egg weight, albumin height and Haugh value but increased egg’s white weight and reduced egg shell, and improved the egg’s yolk color.
  
  
• Increased the concentration of fatty acid omega-3 of yolk after 9 days feeding.

Acknowledgement

The authors also acknowledge the partial support of the Strong Research Group Program of Hue University (Code: 06/HD-DHH).

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