*Article* **Effects of Non-Heated and Heat Processed Krill and Squid Meal-Based Diet on Growth Performance and Biochemical Composition in Juvenile Pacific Bluefin Tuna** *Thunnus orientalis*

**Jeong-Hyeon Cho 1,†, Takayuki Kurimoto 1,‡, Yutaka Haga 1,\*, Yuji Kamimura 2, Akira Itoh <sup>3</sup> and Shuichi Satoh 1,§**


**Abstract:** This study investigated the effects of krill and squid meal and their heat processing on the growth performance and biochemical composition of juvenile Pacific bluefin tuna (PBT) *Thunnus orientalis*. An experiment using a 2 × 2 factorial design examined the effects of two dietary protein sources (squid and krill meal) and heat treatment (heated and non-heated). Prey fish were provided to a reference group. Fish with an initial mean weight of 74.1 mg were fed one of the five diets. After six days of the feeding trial, the fish fed with krill meal and non-heated diets showed improved growth compared to those fed with the squid meal and heated diets. Fish fed the non-heated diets showed significantly higher whole-body crude protein and crude lipid contents than fish fed the heated diets. These results suggest that nutrient availability could be improved by using krill meal and the non-heated treatment to improve the growth performance of juvenile PBT.

**Keywords:** Pacific bluefin tuna; squid meal; krill meal; heat treatment; growth performance

#### **1. Introduction**

Tuna aquaculture is one of the most important aquacultures worldwide. In 2017, the global production of tuna species was approximately 7.89 million tonnes [1]. Japan is not only one of the countries with the highest consumption of tuna species in the world, but total tuna production was as high as 195,200 metric tons in 2018 [2]. In particular, the total production of Pacific bluefin tuna (PBT) in Japan gradually increased from 2,000 metric tons in 2000 to 17,600 metric tons in 2018 [2,3]. This species is a top predator, grows quickly, and requires high dietary protein. Aquafeed is typically based on fish meal which is made from forage fish, such as sardine *Sardinops melanostictus* and anchovy *Engraulis ringens*; the major producers and exporters are Peru and Chile [4]. However, the rapid rise in global demand for aquafeed has reflected soaring prices [5]. In addition, fish meal production relies on natural fish resources. The dependence of the aquaculture industry on high dietary fish meal consumption is a serious concern regarding its sustainability. To overcome these limitations, considerable research efforts are being made to reduce the dependency of aquafeed manufacturers on fish meal [6]. In the last few decades, several studies have focused on plant proteins that can be utilized as fish meal alternatives [7–10]. However,

**Citation:** Cho, J.-H.; Kurimoto, T.; Haga, Y.; Kamimura, Y.; Itoh, A.; Satoh, S. Effects of Non-Heated and Heat Processed Krill and Squid Meal-Based Diet on Growth Performance and Biochemical Composition in Juvenile Pacific Bluefin Tuna *Thunnus orientalis*. *Fishes* **2022**, *7*, 83. https://doi.org/ 10.3390/fishes7020083

Academic Editor: Geneviève Corraze

Received: 8 March 2022 Accepted: 30 March 2022 Published: 5 April 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

plant protein sources have relatively imbalanced amino acid profiles, low palatability, low nutrient digestibility, and anti-nutritional factors are present [6,9,11–15].

In terms of marine resources, Antarctic krill *Euphausia superba* is one of the most promising resources because of large biomass levels unparalleled anywhere else in the world's oceans. Furthermore, it has been commercially harvested since the 1960s, and today it is targeted by active fisheries of several nations [16,17]. Compared to conventional animal proteins, krill has several advantages such as similar amino acid and fatty acid composition with those of marine farmed fish [18,19] and higher contents of carotenoids, chitin, nucleotides, phospholipids, taurine, and vitamins [20–23]. Squid has been another popular aquafeed ingredient since the 1980s. Squid meal has high protein and strong palatability, is rich in taurine and polar lipids, and excellent feed ingredient [24–27]. Furthermore, improvement of egg quality and fish growth by squid meal based-diet were reported [24,28]. Although krill and squid meals are expensive as the main protein source due to their high unit price, they are ideal as supplemental proteins for low fishmeal diet that can effectively provide the nutrients that are lacking in plant ingredients.

Free amino acids and peptides are considered solubilized dietary proteins because they are water soluble and easily absorbed [29]. Most of the raw protein sources are denatured during heat cooking process, which reduces utility of amino acid and peptide from it. The krill and squid meals available in the market are heat processed, and therefore, they have low nutritive value because of decreased levels of free amino acids and peptides. Therefore, if squid and krill meal without heat processing are available, they may be an effective protein source for tuna. Previously, we showed that higher feed consumption and growth of red sea bream *Pagrus major* fed non-heated krill and squid meal-based diets [28]. However, there are few studies investigate utility of squid and krill meal without heat processing as dietary ingredients for tuna.

Therefore, this study aimed to evaluate the effect of krill and squid-based diets and their heat treatment on the biochemical composition and growth performance of PBT juveniles.

#### **2. Materials and Methods**

#### *2.1. Experimental Diets*

The test diets were formulated with 51.4% of four different animal protein sources (heated squid meal, HS; non-heated squid meal, NHS; heated krill meal, HK; non-heated krill meal, NHK). Raw squid and krill were pulverized using a centrifugal milling devise equipped with sieve (2.5 mm diameter, ZM500, Retsch Co, Clifton, NJ, USA). The resultant pulverized was kept under −30 ◦C. The frozen samples were then lyophilized by an automatic vacuum freeze drying devise (RLE-206II, Kyowa Vacuum Eng., Co, Saitama, Japan). The freeze-dried squid or krill meal were sieved using a 500 μm sieve. To make the HK and HS, the freeze-dried meals were kept for 12 h under 105 ◦C. Porcine blood meal, defatted horse mackerel meal, albumin from hen egg, DHA70E, fish oil, vitamin E, α-starch, taurine, calcium phosphate, choline chloride, sodium ascorbyl phosphate, mineral and vitamin premix, and bonito peptide were included in the test diets (Table 1). These ingredients were mixed and pelletized by fluidized bed granulation which is suspending particles in an air stream and liquid binder such as carboxy methyl cellulose is sprayed from the top of the system down onto the fluidized bed. The resultant pellets (ca. Ø 750 μm) were sieved. The moisture of diets (sinking pellets) was removed in the freeze dryer for 12 h and then kept under −30 ◦C until use. Spangled emperor *Lethrinus nebulosus* larvae which is popular prey for PBT larvae, was used as control (prey fish, PF). Fertilized eggs obtained from spangled emperor were introduced and hatched in 200 L aquaria.


**Table 1.** Formula, proximate composition, and water-soluble and -insoluble protein content in the test diets.

<sup>1</sup> HS: heated squid meal; NHS: non-heated squid meal; HK: heated krill meal; NHK: non-heated krill meal; PF: prey fish, Spangled emperor larvae *Lethrinus nebulosus*. <sup>2</sup> Squid meal (CP: 67.6%, CL: 13.5%); Krill meal (CP: 67.7%, CL: 10.7%); Porcine blood meal (CP: 71.9%, CL: 1.3%); Defatted horse mackerel meal (CP: 78.6%, CL: 2.9%); Egg albumin (CP: 82.1%, CL: 0.1%). <sup>3</sup> DHA70E (Harima Foods Co., Osaka, Japan). <sup>4</sup> Cod liver oil (Kanematsu Shintoa Foods Co., Tokyo, Japan). <sup>5</sup> Mineral mixture (mg/kg diet): Na (as NaCl) 197; Mg (as MgSO4·7H2O) 735; Fe (as FeC6H5O7·5H2O) 258; Zn (as ZnSO4·7H2O) 40; Mn (as MnSO4·5H2O) 18; Cu (as CuSO4·5H2O) 3.9; Al (as AlCl3·6H2O) 0.56; Co (as CoCl2·6H2O) 0.15; I (as KIO3) 0.89; <sup>α</sup>-cellulose carrier. <sup>6</sup> Vitamin mixture (amount/kg diet): thiamine hydrochloride, 60 mg; riboflavin, 100 mg; pyridoxine hydrochloride, 40 mg; cyanocobalamin, 0.1 mg; ascorbic acid, 5000 mg; niacin, 400 mg; calcium pantothenate, 100 mg; inositol, 2000 mg; biotin, 6 mg; folic acid 15 mg; *<sup>p</sup>*-aminobenzoic acid, 50 mg; vitamin K3, 50 mg; vitamin A acetate, 9000 IU; vitamin D3, 9000 IU. <sup>7</sup> Feeding stimulants. Values of proximate composition are presented as means of triplication.

Moisture content in the formulated diets ranged from 6.3–7.6%, and in the PF group moisture content was 91.8%. The formulated diets were isonitrogenous (57%) and isolipidic (22%) (Table 1). Crude protein and lipid levels in the control were 63.7 and 22.4%, respectively. The NHS and NHK diets contained higher water-soluble protein than the heated meal diets (the protein contents of NHS and NHK were 7.8% and 4.9% compared to 4.1% and 3.3% for HS and HK, respectively). Total and free amino acid content in the non-heated meal diets were greater than those of the heated meal diets (Tables 2 and 3). The most abundantly observed free amino acids in the krill-based diets (HK and NHK) were arginine and glycine, whereas the NHS diet contained a significant portion of proline. The control diet PF had the highest gross free amino acid level (5.69 g/100 g) compared to the other diets (2.55~4.57 g/100 g) (Table 3). Docosahexaenoic acid (DHA) level of essential fatty acids was highest in the PF group (25.0% of total fatty acid), followed by the squid-based diets (15.0~18.3%) and the krill-based diets (11.5~11.7%), whereas eicosapentaenoic acid (EPA) was highest in the krill-based diets (11.5~11.7%), followed by the squid-based diets (8.2~10.7%) and the PF group (6.3%) (Table 4).


**Table 2.** Total amino acid content of the test diets (g/100 g, dry-weight).

Values are presented as means of triplication. HS, heated squid meal; NHS, non-heated squid meal; HK, heated krill meal; NHK, non-heated krill meal; PF, prey fish; Spangled emperor larvae, *Lethrinus nebulosus*. N/D: not detected (detection limit: 0.01 g/100 g, dry-weight).

**Table 3.** Free amino acid content of the test diets (g/100 g, dry-weight).


Values are presented as means of triplication. N/D: not detected (detection limit: 0.01 g/100 g, dry-weight). HS, heated squid meal; NHS, non-heated squid meal; HK, heated krill meal; NHK, non-heated krill meal; PF, prey fish; Spangled emperor larvae, *Lethrinus nebulosus*.


**Table 4.** Fatty acid composition (area% of total lipid) of the test diets.

Values are presented as means of triplication. HS, heated squid meal; NHS, non-heated squid meal; HK, heated krill meal; NHK, non-heated krill meal; PF, prey fish; Spangled emperor larvae, *Lethrinus nebulosus*. <sup>1</sup> LC−PUFA, long chain poly unsaturated fatty acids; Σn−3 LC−PUFA: 18:4n−3, 20:4n−3, 20:5n−3, 22:5n−3, 22:6n−3.

#### *2.2. Feeding Experiment and Sampling Schedule*

Fertilized eggs were collected natural spawning of Pacific bluefin tuna (PBT) reared at Amami Fish Farm Co, Kagoshima, Japan. The rotifers *Brachionus rotundiformis* and *Artemia* nauplii were offered to larval PBT before they became 10 mm of total length prior to start of the feeding experiment. The larval PBT were fed larvae of spangled emperor *Lethrinus nebulosus* with yolk-sac until the PBT reached 20 mm in total length. Pacific bluefin tuna larvae with total length of 20.5 ± 0.2 mm (19 days post hatching, dph) were introduced into ten 500-L circular polycarbonate aquaria. The aquaria had walls carrying black tape in a checkered pattern to prevent collision of fish against aquarium walls [30] (Table 5).

**Table 5.** Rearing conditions for feeding trial of Pacific bluefin tuna juveniles.


<sup>1</sup> Mean ± standard deviation (SD) (*<sup>n</sup>* = 100). <sup>2</sup> Mean ± SD (*<sup>n</sup>* = 10).

The initial three days were designated as acclimation period for the formulated diet (19–21 dph), and the following six days were allocated for feeding with only the test diets for the juveniles (22–28 dph). A commercial diet for marine fish (CP: 58.1%, CL: 19.4%, dry weight) was provided to all treatment groups hourly between 7:00 and 19:00 during the weaning period. Moreover, PF was offered to all groups (three times a day at 7:00, 13:00, and 19:00). The PF offered was reduced continuously; PF: PBT larvae (ind.: ind.) = 150:1, 120:1, and 90:1, so as to promote acclimation of PBT larvae to the experimental diets. PBT juveniles were fed only one of the experimental diets after a three-day weaning period. Fish in the PF group were subjected to satiation feeding of PF for whole period of the feeding trial. Dietary treatment was carried out in duplicate. Rearing of fish was finished after the sixth day from the beginning of the trial to have enough fish for chemical composition analysis. The daily seawater exchange rate was 1200% of the capacity of an aquarium. The average of dissolved oxygen concentration and water temperature were 11.0 ± 1.7 mg/L and 27.8 ± 0.6 ◦C, respectively (Table 5).

Prior to each sampling, fish were starved for 12 h. In this case, 50 fish were collected just before the beginning of the feeding trial and kept at −80 ◦C. PBT were also collected at 4, 8, and 10 days after feeding to evaluate fish growth. Ten fish were measured at four and eight days, and 120 fish were measured at ten days, for fork and total length. The whole body was weighed to obtain wet weight for calculation of the condition factor (CF). The carcasses were kept under −80 ◦C.

The bottom of aquaria was cleaned every day with a syphon, and survival of fish was estimated by counting the number of dead fish. Number of dead fish was subtracted from the fish number introduced at the start of the feeding trial for calculating survival rate. CF, specific growth rate (SGR), thermal growth coefficient (TGC), and weight gain (WG) were calculated by the formulae:

WG (%) = [wet weight of the final fish (g) − wet weight of the initial fish (g)]/wet weight of the initial fish (g) × 100

SGR (%) = [ln wet weight of the final fish (g) − ln wet weight of the initial fish (g)] × 100/time (days)

TGC = (wet weight of the final fish (g)1/3 - wet weight of the initial fish (g)1/3) <sup>×</sup> (sum day-degrees Celsius)−<sup>1</sup> <sup>×</sup> <sup>1000</sup>

CF = wet weight (g)/[fork length (cm)]3 <sup>×</sup> <sup>100</sup>

#### *2.3. Chemical Analyses*

Diets and carcass were analyzed using standard methods for dry and wet matter, crude protein, and ash [31]. Chemical analysis was conducted in triplicate for each sample at the Laboratory of Fish Nutrition, Tokyo University of Marine Science and Technology, Minato, Tokyo, Japan, and the data was averaged. Moisture content was determined gravimetrically by drying the sample in a dry oven at 105 ◦C until achieving a constant weight. The samples were incinerated under 650 ◦C for 8 h by a muffle furnace (FO200, Yamato Co., Tokyo, Japan) for ash content determination. The Kjeldahl method with using an automatic titlator (Kjeltec 2400, FOSS Japan Co, Tokyo, Japan) and conversion index of 6.25 were employed for crude protein analysis. Dietary water-soluble protein content was determined according to de Schrijyer and Ollevier (2000) [32]. Chloroform and methanol mixture (2:1) was used for crude lipids extraction [33]. Preparation of fatty acid methyl ester (FAME) was followed the previously reported methods [34]. FAME was analyzed by a gas chromatograph (GC-2025, Shimadzu, Tokyo, Japan) installed with a fiberglass capillary column (30 m × 0.32 mm, i. d., SUPERCO-WAX10, Sigma-Aldrich Co, St. Louis, MO, USA) and peak area of FAME was measured by a recorder (C-R8A Chromatopac; Shimadzu). Temperature of the column oven was elevated from 170 ◦C at 2 ◦C/min for 40 min and kept under 250 ◦C. An automatic amino acid analyzer (JLC-500/v; JEOL Co., Tokyo, Japan) was used for the total and free amino acid analysis as previously described [35]. Samples were hydrolyzed with 10 mL 2% sulfosalicylic acid (*w/v*, Wako Fujifilm Co, Tokyo, Japan), or 4M methanesulfonic acid (Sigma-Aldridge Co, St. Louis, MO, USA) for free and total amino acid analysis, respectively. The digesta was homogenized for 1.5 min and centrifuged at 1610× *g* under 4 ◦C for 15 min twice by a devise (SRX-201; Tommy Co., Tokyo, Japan); the upper layers were pooled and filtered with a membrane filter (0.45 μm, Millipore Co, Darmstadt, Germany). The filtrate was analyzed for amino acid. Constitutional amino acid content was calculated by subtracting the free amino acid from the total amino acid.

#### *2.4. Statistical Analyses*

Data on fish growth, initial and final carcass composition were analyzed by a one-way analysis of variance (ANOVA) followed by Tukey's multiple range tests. The main effects of dietary protein source and the heat processing were tested using a two-way ANOVA. Statistical significance was accepted when probability was below 95%. IBM SPSS 19 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis.

#### **3. Results**

#### *3.1. Survival and Growth of PBT Juveniles*

The survival of juvenile PBT fed the treatment and control diets during the test period is presented in Figure 1. There were no significant differences in survival during the period from one to five days (including the weaning period of one to three days) in all treatment groups. However, significant differences in survival were observed thereafter until the final day. The highest survival of the final fish was found in the PF group (75.3%), followed by the NHK, HK, NHS, and HS groups (52.6%, 51.9%, 45.9%, and 25.8%, respectively). There were no significant differences among the HK, NHK, and NHS groups on the final day, but these groups differed significantly from the HS group. Survival in the krill-based diet groups (HK and NHK) was similar during the rearing period (Figure 1). Survival was not affected by the heat treatment. Effects were significant for protein sources during the rearing period; however, no interactive effects of two factors were detected (Table 6).

**Figure 1.** Survival of Pacific bluefin tuna juveniles fed different test diets during the rearing period. Different superscript letters indicate significant difference among the dietary groups (Tukey's test, *p* < 0.05). HS, heated squid meal; NHS, non-heated squid meal; HK, heated krill meal; NHK, nonheated krill meal; PF, prey fish; Spangled emperor larvae, *Lethrinus nebulosus*.


**Table 6.** Growth performance, proximate composition and biological indices of Pacific bluefin tuna fed the test diets <sup>1</sup> for 9 days.

Values of total length, body depth, body weight and CF (condition factor) are means ± SD of 100 (19 dph) or 120 (28 dph) fish. Values are means ± SD of 2 groups of fish (*n* = 2; WG, SGR, TGC and survival rate) or 3 groups of fish (*n* = 3; proximate composition). Values in a same row with different letter are significantly different (Tukey's test, *p* < 0.05). I, ingredients (squid meal and krill meal); H, heated and non-heated treatment; ns, no significant difference (two-way ANOVA, *p* > 0.05); \*, *p* < 0.05. WG, weight gain; SGR, specific growth rate; TGC, thermal growth coefficient. <sup>1</sup> HS, heated squid meal; NHS, non-heated squid meal; HK, heated krill meal; NHK, non-heated krill meal; PF, prey fish; Spangled emperor larvae, *Lethrinus nebulosus*.

The growth performances of juvenile PBT during all test periods are shown in Table 6, Figures 2–4. The best growth was found in the PF group, and its difference was significant among all groups (*p* < 0.05). The PBT juveniles in non-heated diet groups grew significantly better than those in the heated treatment diet groups (*p* < 0.05), and the fish in krill mealbased diet groups grew significantly better than those in the squid meal-based diet groups (*p* < 0.05) (Figures 2 and 3). The WG, SGR, and TGC of fish were affected by both protein sources and heat treatments, but there were no interactive effects of either factor (Figure 4).

**Figure 2.** Final total length of Pacific bluefin tuna juveniles fed different test diets during the rearing period. Different superscript letters indicate significant difference among the dietary groups (Tukey's test, *p* < 0.05; 1st day, *n* = 50; 4th and 8th day, *n* = 10; 10th day, *n* = 120). HS, heated squid meal; NHS, non-heated squid meal; HK, heated krill meal; NHK, non-heated krill meal; PF, prey fish; Spangled emperor larvae, *Lethrinus nebulosus*.

**Figure 3.** Final body weight of Pacific bluefin tuna juveniles fed different diets during the rearing period. Different superscript letters indicate significant difference among the dietary groups (Tukey's test, *p* < 0.05; 1st day, *n* = 50; 4th and 8th day, *n* = 10; 10th day, *n* = 120). HS, heated squid meal; NHS, non-heated squid meal; HK, heated krill meal; NHK, non-heated krill meal; PF, prey fish; Spangled emperor larvae, *Lethrinus nebulosus*.

**Figure 4.** Means of WG (g, **upper-left**), SGR (%, **upper-right**), TGC (**lower-left**), and survival (%, **lower-right**) of juvenile Pacific bluefin tuna after rearing period for the main effects of ingredient and heat treatment. Data was expressed as means ± SE. WG, weight gain; SGR, specific growth rate; TGC, thermal growth coefficient. The asterisks indicate significant differences by two-way ANOVA analysis (*p* < 0.05).

#### *3.2. Chemical Property of PBT*

The highest moisture content was noted in the HK group, followed by the HS, PF, NHK, and NHS groups (*p* < 0.05). The crude protein contents in the PF and NHK groups were significantly higher than those in the other groups, whereas the crude lipid content in the NHS group was significantly higher than in the other groups (*p* < 0.05). No significant differences were observed in the crude ash among all the groups (*p* > 0.05). The moisture, crude protein, and crude lipid of the carcasses were affected by both protein source and heat treatment, but an interactive effect between both factors was detected only for crude lipids. By the final day, the CF observed in the NHK group was significantly higher than those of the PF, NHS, and HK groups, which were significantly higher than that of the HS group (*p* < 0.05).

Significant differences between the PF and the other groups were observed in all constitutional amino acid levels except valine and tryptophan. Specifically, most of the amino acid levels were higher in the PF group than in the other treatment groups. Constitutional amino acids were affected by both the protein source and heat treatment, while the free amino acids tended to be affected by the interaction. Only free histidine was affected by both factors (Table 7).

**Table 7.** Constitutional and free amino acid content of Pacific bluefin tuna fed the test diets <sup>1</sup> for 9 days (g/100 g, dry-weight).


Values are presented as means of triplication. Values in a same row with different superscript letters are significantly different (Tukey's test, *p* < 0.05). I, ingredients (squid meal and krill meal); H, heated and non-heated treatment; ns, no significant difference (two-way ANOVA, *p* > 0.05); \*, *p* < 0.05. <sup>1</sup> HS, heated squid meal; NHS, non-heated squid meal; HK, heated krill meal; NHK, non-heated krill meal; PF, prey fish; Spangled emperor larvae, *Lethrinus nebulosus*.

The major fatty acids in the carcass were 16:0 (palmitic), 18:0 (stearic), 18:1n−9 (oleic), 20:5n−3 (eicosapentaenoic, EPA), and 22:6n−3 (docosahexaenoic, DHA) acids (Table 8). The sum of these major fatty acids accounted for more than 66.2%, 70.0%, 69.9%, 68.4%, and 77.4% of the total fatty acids in the HS, NHS, HK, NHS, and PF treatment groups, respectively. The major saturated fatty acid (SAFA) was 16:0 in all groups. Regarding polyunsaturated fatty acids (PUFAs), bluefin tuna is considered to be a good source of n−3 fatty acids, particularly DHA, which exhibited the highest levels in the PF group. DHA occurred in a higher proportion than EPA in all treatment groups. The sum of DHA

and EPA reached 28.9%, 34.6%, 32.7%, 30.5%, and 35.5% for HS, NHS, HK, NHK, and PF, respectively.


**Table 8.** Fatty acid composition (area% of total lipid) of whole fish body of Pacific bluefin tuna for 9 days.

<sup>1</sup> LC−PUFA, long chain poly unsaturated fatty acids; <sup>Σ</sup>n−3 LC−PUFA: 18:4n−3, 20:4n−3, 20:5n−3, 22:5n−3, 22:6n−3. Values are presented as means of triplication. Values in a same row with different superscript letters are significantly different (Tukey's test, *p* < 0.05). I, ingredients (squid meal and krill meal); H, heated and non-heated treatment; ns, no significant difference (two-way ANOVA, *p* > 0.05); \*, *p* < 0.05. HS, heated squid meal; NHS, non-heated squid meal; HK, heated krill meal; NHK, non-heated krill meal; PF, prey fish; Spangled emperor larvae, *Lethrinus nebulosus*.

#### **4. Discussion**

Kvåle et al. (2009) [36] reported that different feeding practices greatly affect fish performance, and the weaning period and method of introducing formulated diets are important. In the present study, based on the findings of Haga et al. (2010) [37], Cho et al. (2016) [38] and Cho et al. (2022) [39], we fed yolk-sac larvae of spangled emperor and a commercial diet on the first day, but the frequency of feeding yolk-sac larvae was gradually reduced over the subsequent two days due to acclimation to the formulated diet. According to Cho et al. (2016) [38], early larvae of PBT is able to weaned to formulated diets once they successfully accepted and ingested a suitable formulated diet. Similarly, in the present study, no significant differences were observed in fish growth and survival immediately after the weaning period, but we were able to observe significant differences in growth performance after changing their diet (Figures 1–3).

In the present study, there were significant differences in the contents of water-soluble proteins and free amino acids between the heated and non-heated diets (Tables 1 and 3). Considering an extruded pelleting process which is subjected to heat treatment, protein denaturation of the non-heated meal could be expected. To avoid protein denaturation of the non-heated meal, an extruder was not used to prepare the test diet in the present study. According to Cho et al. (2018) [28], in a study comparing the effects of heat treatment of ingredients on the growth performance of juvenile red sea bream *Pagrus major*, the growth of fish fed a non-heated diet was significantly better than that of fish fed a heated diet. Cho et al. attributed this difference in growth performance to the high water-soluble protein and free amino acid content in the feed. Watanabe (1982) [40] reported the incorporation of water-soluble proteins by pinocytosis in rectal epithelium cells in larval and juvenile teleosts. In the present study, it is suggested that the highly water-soluble protein contained in the non-heated diets has been effectively absorbed and assimilated in the early stages of

PBT. Furthermore, higher utilization of free amino acids was demonstrated in the larvae and juvenile of fish [41,42]. Current result showed that approximately 1.7 times higher gross free amino acid contained in the non-heated diets than the heated treatment diets, suggesting that they were utilized more effectively for growth.

Satoh (2005) [43] reported the in vitro digestibility of fish meal and raw fish by pepsin and trypsin from yellowtail *Seriola quinqueradiata*. Both pepsin and trypsin activity of fish fed raw fish were much higher than in those fed fish meal, and these enzymes worked better on protein from raw fish compared to that from fish meal. Furthermore, Seoka et al. (2010) [27] reported that the growth and survival (at 30 days post hatching) of PBT juvenile fed non-heated Toyama squid *Watasenia scintillans* meal-based diet were better than that fed a commercial diet. Yellowtail and tuna species feed on live fish in natural ecosystems; therefore, it is thought that the proteins of non-heated treatment without denaturation are highly digestible; therefore, using a non-heated protein source in an aquaculture setting is likely to be superior in terms of digestibility. It was reported that PBT juveniles exhibited higher gustatory responses to alanine, leucine, valine, methionine, isoleucine, and proline [44]. Here we demonstrated that the gross levels of these amino acids in non-heated diets (1.90 and 1.16 g/100 g) were higher than those in the heated diets (0.85 and 0.64 g/100 g). These results suggest that a diet containing high levels of water-soluble proteinous component such as free amino acids that are effective for fish growth can be produced without heating the ingredients and such a diet will promote the feed intake of PBT juveniles. Furthermore, Marubeni Nisshin Feed Co, Tokyo, Japan, developed a commercial formulated diet (Magokoro) based on enzyme-treated Chilean fish meal called BioCP from LANDES Co., Talcahuano, Chile, for PBT juveniles. Higher water solubility after enzyme treatment has been well documented for fish protein [45], and 60% of protein in BioCP accounts for soluble protein. However, the production of BioCP is expected to decline in the near future. When fish meal is in short supply, non-heated proteins can be more competitive depending on the availability of soluble proteins in the market.

The effects of feed ingredient on nutritional status were investigated in the present study. As a result, nutritional status indices, including growth performance, whole body composition, constitutional amino acid, and fatty acid altered. Fish fed krill meal-based diet (HK and NHK) had a positive WG, SGR, TGC, and survival. It was suggested that krill meal diet promotes digestive ability of yellowtail by regulating the digestive enzyme secretion in intestine [43].

Compared to squid meal, krill meal is considered to be a more effective ingredient for marine fish, including for the early life stage of PBT. The crude protein in krill contains 40% of the extractable nitrogen-containing components that are not contained in the protein itself [46]. Since the extractable component contains free amino acids and peptides and does not require decomposition by proteases, it might be easily absorbed. In the present study, the content of water-soluble proteins in the krill meal was not high. This could be because the extract component was dissolved and lost when it thawed. The essential amino acid contents, except tryptophan and valine, of the whole fish body were affected by the feed ingredients. This finding suggests that the amino acid balance of krill meal makes it more suitable than squid meal for PBT juveniles.

Krill is rich in astaxanthin, which has antioxidant activity, whereas squid meal is low in astaxanthin [47]. Although the content of astaxanthin was not measured in the present study, it is expected that the krill meal-based diet contains astaxanthin, which is considered to be effective in preventing the oxidation of long-chain polyunsaturated fatty acids. The fatty acid 18:1n−9 was identified as the major monounsaturated fatty acid (MUFA) and was significantly higher in the heated treatment (HS and HK) and PF groups. The most abundant saturated fatty acid (SAFA) in the present study was 16:0, which is considered to be a predominant source of potential metabolic energy in fish during growth [48], and it is the predominant SAFA in the main live feed source of cultured tuna [48,49]. The ratio of DHA/EPA in raw feed for PBT was reported to exceed 2.0 [50], and the DHA/EPA ratio of PF used in the present study was 4.0. Further, Seoka et al. (2008) [51] reported that PBT juveniles fed yolk-sac larvae of striped beakfish *Oplegnathus fasciatus* with a DHA/EPA ratio of 3.6 show better growth performance than those fed a formulated diet with a DHA/EPA ratio of 1.7. It has also been considered that the dietary DHA/EPA ratio affects the growth performance of marine fish larvae and juveniles, and a ratio of at least 1.0 is appropriate for normal growth [52,53]. Although the DHA/EPA ratio of the test diet used in the present study exceeded 1.0, the growth in the test group was lower than that of the PF group (4.0).

Although the present study clearly demonstrated that non-heated meal is a suitable protein source for formulated diet for PBT juveniles, overall survival of the PBT fed the formulated diets was not very high (25.8–52.6%). Considering high mortality of hatcheryraised PBT juvenile is the one of the biggest bottleneck of mass production of the PBT juveniles, further improvement of the feed performance of the formulated diet for PBT is desired.

#### **5. Conclusions**

In conclusion, it is important to understand whether the selection of a feed protein source and its heat treatment would improve the nutritional status and thus growth of PBT juveniles. The present study showed that heat treatment of the protein source in feed adversely affected growth performance and survival of PBT juveniles. Furthermore, the influence of heat treatment was more remarkable in squid meal than in krill meal. Taken together, the results of the present study suggest that protein source as well as heat treatment may influence the nutritional status of cultured juvenile PBT, and this should be taken into consideration for the management of seed production.

**Author Contributions:** J.-H.C.: conceptualization, methodology, data curation, writing—original draft, and visualization. T.K.: conceptualization, methodology, investigation, validation. Y.H.: Conceptualization, supervision, project administration, funding acquisition, writing, review, and editing. Y.K.: Investigation, validation, resources. A.I.: Conceptualization, funding acquisition, project administration. S.S.: Conceptualization, funding acquisition, project administration. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Maruha Nichiro Co. and Fisheries Agency Japan.

**Institutional Review Board Statement:** All activities related to animal ethical considerations, such as anesthesia, dissection, and euthanasia, were conducted according to Handling Rules for Animal Experiments, etc., Tokyo University of Marine Science and Technology (13 March 2020, TUMSAT Regulations No. 8) based on Basic Guidelines for Conducting Animal Experiments at Research Institutes, etc. (Ministry of Education, Culture, Sports, Science and Technology), Act on Welfare and Management of Animals (Act No. 105), Guidelines for the Proper Implementation of Animal Experiments (Science Council of Japan), and Guidelines on How to Dispose of Animals (Prime Minister's Office).

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The expense of this work was partly supported part by a project on the development of production techniques for healthy PBT juveniles from Maruha Nichiro Co. and a grant for Technological Development for Selection and Secure Stock of Brood stock for Culture of Bluefin Tuna from the Fisheries Agency.

**Conflicts of Interest:** The authors have no declaration of interest.

#### **References**


## *Review* **Success of Aquaculture Industry with New Insights of Using Insects as Feed: A Review**

**Amna Hameed 1, Waqar Majeed 1,\*, Muhammad Naveed 1, Uzma Ramzan 2,\*, Matteo Bordiga 3, Maryam Hameed 1, Saud Ur Rehman <sup>4</sup> and Naureen Rana <sup>1</sup>**


**Abstract:** Most of world's fish and seafood are produced by aquaculture, which is one of the biggest contributors to the world's food security. The substantial increase in prices of conventional feed ingredients and the over-exploitation of natural resources are some of the biggest constraints to aquaculture production. To overcome this stress, different approaches and techniques are used, among which the use of non-conventional feed ingredients in the aquaculture sector is the most recent approach. Different non-conventional feed ingredients such as plant-based products, algae (both micro and macroalgae), single-cell protein (bacteria and yeast), and insect meal are currently used in aquaculture for sustainable food production. Amongst all these novel ingredients, insects have greater potential to replace fishmeal. The existence of about 1.3 billion tons of food and agriculture waste from the food chain supply poses a serious environmental threat. Insects are tiny creatures that can thrive on organic waste and thus can convert the waste to wealth by the bioconversion and nutritional upcycling of organic waste. Insects have the potential to recover nutrients from waste aquaculture products, and many fish species feed on insects naturally. Therefore, employing insects in the aquaculture sector to replace fishmeal is an eco-friendly approach. The present review briefly highlights emerging non-conventional feed ingredients, with special attention given to insects. The current review also focuses on the nutritional value of insects, factors affecting the nutritional value of insects, potential insects that can be employed in the aquaculture sector, the physiological response of fish when fed with insect meal, techno-functional properties of insect meal, and emerging approaches for addressing possible downsides of employing insect meal in fish diets. Finally, it suggests avenues for further research into these inventive fishmeal replacements.

**Keywords:** feed; insects; aquaculture; innovation; sustainability

#### **1. Background**

The largest sector of food production is aquaculture, which has the potential to contribute to sustainable food production in the future. In 2018, the aquaculture sector, which is expanding, produced 114.5 million metric tons of live weight [1]. By supplying the world's expanding population with high-quality food, aquaculture is essential to ensuring food security [2]. Making sure that everyone has access to enough food at all times is known as food security. While the global population is growing by 1.6 percent per year, the demand for seafood is rising at a rate of 3.1% annually. At a rate of 2.1 percent per year, the aquaculture industry is expanding more quickly than other areas of animal production, such as livestock [1]. This might be because the aquaculture and fisheries sectors generate more economy through production, selling, and marketing [3,4].

Despite the fact that aquaculture has many advantages for the availability of food, its sustainability depends on a variety of factors, including knowledge, experience, the

**Citation:** Hameed, A.; Majeed, W.; Naveed, M.; Ramzan, U.; Bordiga, M.; Hameed, M.; Ur Rehman, S.; Rana, N. Success of Aquaculture Industry with New Insights of Using Insects as Feed: A Review. *Fishes* **2022**, *7*, 395. https://doi.org/10.3390/ fishes7060395

Academic Editors: Marina Paolucci and Shunsuke Koshio

Received: 1 November 2022 Accepted: 8 December 2022 Published: 17 December 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

regulatory environment, the ecological conditions for the growth of cultured species, and the presence of a sizable market for the sale of aquaculture products [5]. Currently, 50% of world fish production is generated from aquaculture, and it is one of the biggest suppliers of high-quality protein in terms of quantity and quality [6]. Organisms from terrestrial and aquatic environments provide about 43% of the world's protein and play a key role in preventing malnutrition, especially in developing nations [1]. Aquaculture production, like other types of terrestrial farming, is fully dependent on the availability of nutrients as feed [6].

Fishmeal has frequently been replaced in aquaculture with plant proteins. Previous research has shown that plant-based diets are unfavorable for fish growth and health, and this could be because of the presence of anti-nutritional substances (ANFs) [7–9]. Among plant-based diets, soybean is considered the best to replace fishmeal due to its costeffectiveness and nutritional value [10]. However, the addition of high quantities of soybean meal in the fish diet has been shown to negatively impact growth, gut and liver integrity, intestinal microbiota composition, and immunological response in various carnivorous fish species [11–13]. When the salmonids were fed with SBM-based diets, severe gut health impairment with enteritis and increased inflammatory influx was observed in fish [13]. The researchers were therefore forced to create new aquafeeds with innovative feed ingredients that can replace fishmeal and minimize the detrimental effect caused by using vegetable protein because they were aware of the negative effects of utilizing a plant-based diet, even at a low inclusion level [14–16].

#### *1.1. Emergent Non-Conventional Feed Ingredients*

The aquaculture sector is one of the major contributors to food security worldwide [2], but the increased food demand has compelled scientists to look for novel and non-conventional feed ingredients such as algae, land animal by-products, single-cell proteins, and insects [17].

Bacteria and yeast have the highest potential to replace fishmeal due to their good nutritional value. Bacteria and yeast have good AA profiles comparable to fishmeal and the protein content ranges from 50–80% and 45–55%, respectively. They also have great potential to be utilized as raw materials and functional feed additives [18]. Microalgae have good nutritional profiles, which have emerged as good candidates for use in aquaculture [19,20]. Algae can promote growth, stimulate the immune system, and enhance fish color, in addition to utilizing waste-derived nutrients such as nitrate, nitrite, and TAN (total ammonia nitrogen) [21]. Yadav et al. [22] conducted a study to evaluate the effect of dried chlorella species on the survival and growth of common carp (*Cyprinus carpio*). The fish fed with the microalgal biomass showed a great improvement in weight gain, lower FCR, and protein efficiency ratio [22]. A cost/benefit analysis showed a significant reduction in the cost of formulated feed compared to commercial feed, which can enhance the profit margin of fish farmers. This research highlights the importance of algae as a novel non-conventional feed ingredient. Using this strategy, the waste carbon dioxide can be further utilized to formulate alternative feed supplements.

Biofloc production is a reasonable strategy to reduce environmental impacts by contributing low emissions. The Biofloc production and efficiency depend upon different factors, such as the cultured species [23,24]. Debbarma et al. [25] reported Biofloc production's importance for managing aquaculture nitrogen wastes. The Biofloc production system showed the best performance in terms of growth, condition factor, digestive enzyme activity, and survival. The production cost is one of the biggest causes of the limitation of the applicability of SCP and other newly emerged non-conventional feed ingredients in aquafeed [26,27].

Designing the new, highly effective aquafeed diets by combining plant- and animalbased diets (insect meal/poultry by-product meal) may be a revolutionary technique [12]. Poultry by-product meal (PBM) is a cost-effective and sustainable protein source that is rich in essential amino acids and has high nutrient digestibility [28]. Different studies

have been conducted to replace the vegetable-protein-based diet with an animal-based diet (insect meal/poultry by-product meal). Intestinal histological changes and inflammatory responses were reduced in gilthead seabream due to this novel strategy when the amount of vegetable protein in the fish diet was decreased. Incorporating insect meal into the diet of seabream had a favorable impact on molecular inflammatory markers [12]. Poultry by-product meal positively improved lipid absorption in the seabream diet. Zarantoniello et al. [29] examined the effect of conventional and novel feed additives, which included Louisiana red claw crayfish (*Procambarus clarkii*) meal (RCM) and dried microbial biomass from *Tetraselmis suecica* (TS) and *Artrhospira platensis* (AP), on rainbow trout (*Oncorhynchus mykiss*). The fish's development was unaffected by the vegetable protein sources, including 0.25 percent of conventional feed additives, and AP-based diets, but their severely compromised gut health condition marginally improved. The fish's general welfare and intestinal integrity were preserved by RCM and TS-based diets, but the fish's growth performance was poor. This might be because these diets contain certain biogenic amines, which could negatively affect nutrient uptake. Gaudioso et al. [11] evaluated the effect of poultry byproduct meal and *H. illucens* meal on gut microbiota inflammatory and immune markers of fish when substituted with vegetal protein. When poultry by-product meal was added to vegetal protein meal (VM), the growth performance and microbiota composition were maintained as in FM. Meals made from insect and poultry by-products demonstrated better fish health and were recommended as the ideal replacement for plant-based proteins. In the case of insect meal, the aquaculture industry faced some cost-effectiveness issues. Therefore, it was recommended to use a combination of insect meal and poultry by-product meal for better fish growth performance. Another study conducted by Pulido-Rodriguez et al. [30] showed the positive effect on growth performance of red swamp crayfish when replacing vegetal protein with insect meal by-product. However, *Tisochrisis lutea* and *Tetraselmis suecica* (marine algae) were added at a low inclusion level, which badly affected the fish growth performance and increased the FCR value.

A feed with balanced nutrients is vital for improving fish growth and development. By 2020, fishmeal production was expected to rise to 70,969 thousand tons globally, a nearly 10-fold increase from 1995 [31]. The fishmeal ingredients used to prepare feeds are under a lot of stress as a result of these expanding numbers. According to studies, insects may be a feasible alternative to fishmeal in the near future, as they are a good source of macromolecules and micromolecules [32–35]. The substitution of fishmeal with insect meal is a unique concept in aquaculture. However, aquaculture practices currently employ a diverse range of insect species. This category of insects includes maggots (*Musca domestica*), black soldier flies (*H. illucens*), silkworm pupae (*Bombyx mori*), grasshoppers, and mealworms (*Tenebrio molitor*).

A significant effect has been observed in the growth performance of fish when fish are fed an insect-based meal. Tran et al. [36] reported that when aquatic animals were fed with *Tenebrio molitor* and pupal full-fat silkworm *Bombyx mori* they showed a substantial increase in Hedges' g value for SGR with the reduction in FCR. The growth performance of aquatic animals fed with insects vary according to the stage and species of both the insects and aquatic animals. It is documented in many studies that when fishmeal is substituted with more than 30% insect meal, a negative effect on growth performance is observed [37,38].

The nutritional profile of insects has a major role in deciding the substitution level of fishmeal. The protein part of insects' diets ranges from 50–82% (dry matter basis), comparable to fishmeal ranging from 60–72% by weight. Insect meals are rich with essential amino acids, but different species have observed minor variations depending upon their orders. Hydroxyproline and taurine are unique ingredients in insects, absent in a plantbased diet but present in fishmeal [39]. The insect species are enriched with saturated fatty acids and less in the concentration of PUFAs. On average, the n-6/n-3 ratios in terrestrial insects are three times higher than in aquatic insects [40,41]. Fishmeal and fish oil are the main components of fish feed.

To preserve the sustainability of food, new protein substitutes must be used in replacement of fishmeal and fish oil due to their scarcity [42]. Although there is literature on the nutritional value of insects, there is a lack of information for a thorough assessment of the factors influencing that nutritional value. The challenges with employing insects as a feed for aquaculture to address fishmeal scarcity are discussed in this review.

#### *1.2. Ecological Advantages of Using Insects in Fishmeal Diet*

Environmental issues such as the overuse of natural resources, loss of biodiversity, and rising pollution levels directly affect global climatic conditions. This fact is evident from the scientific reports and data published in the last decades [43]. Different strategies and approaches have been applied to combating environmental problems, among which the concept of bioeconomy is the most prominent. The concept of bioeconomy focuses on utilizing food waste or algae [44].

As the world population increases rapidly, there is a need for a constant supply of protein with reduced environmental impacts [45]. Recent research suggested that insect-based meals could positively impact the environment by selecting a suitable diet. Incorporating yellow mealworms into the diet of rainbow trout decreased the net primary production use. Still, it did not lower the land use, global warming potential (GWP), eutrophication, and energy demand [46]. However, different results have been achieved in the case of *H. illucens*. When *H. illucens* is added to the arctic char diet, it reduces the environmental impacts by reducing abiotic depletion, GWP, acidification, and land use [47].

#### *1.3. Waste to Wealth Concept*

Agro-industrial waste's continuous production and proper disposal are major environmental issues. Still, the improved nutritional value of these agro-industrial wastes is paving the way for these wastes to be valorized in animal/fish feed [48]. Jayan et al. [49] proposed this concept by valorizing castor cake or meal by standard processes. The growth and nutrient utilization indices of *L. rohita* were significantly improved when fed with castor oil protein isolates. The biodegradation of plastic is also one of the biggest environmental problems. Using insects in the biodegradation of plastic is a novel approach. *Tenebrio molitor* has ability to biodegrade plastic waste. Both forms of *Tenebrio molitor* larvae and imago are plastic eaters. Bulak et al., 2021, also reported that *Tenebrio molitor* could actively degrade plastic waste.

Aquafeed has a finite shelf-life and is easily subjected to rancidity due to the peroxidation of lipids [50]. The oxidated lipids have a detrimental effect on the feed quality, reducing the palatability and nutritional value of feed [51]. Several insect species have shown the potential to recover the nutrients from expired fish feed. The black soldier fly has great potential to recover nutrients from expired fish feed [52].

Moreover, the black soldier fly is also considered an ideal candidate for converting waste into protein-rich biomass that can be used as animal feed [53,54]. This method relies on the black soldier fly larvae to feed on the wastes, which will reduce it to 50–80% (wet weight) residue and yield useable and high-quality biomass at a rate of 20–30% (solid basis) [55]. Black soldier fly larvae fed on waste are a good source of protein and are used as feed for fish [39], and the residue can be used as fertilizer for soil [56].

Insects are simple to grow and can be produced in large quantities with little need for water or land [39,57]. They grow quickly due to their fast reproduction rate [53,58] and have high feed conversion efficiency [57,58]. Insects can be grown on organic food waste, and they can convert this waste into protein and fat-rich biomass [59]. Additionally, this approach reduces environmental problems related to the reuse of food waste [53].

#### **2. Nutritional Value of Insects**

There are around one million insect species around the globe, and they are essential to the integrity of food chains and ecosystems. Although the majority of Europeans still view insects as harmful pests, several insect species are used as food in 100 different countries [33,60]. Insects are the largest class of arthropods and have been used as a food source for humans throughout history. Numerous insect species can be used as food, but only a few have been domesticated, such as silkworms and honeybees [61]. Insects, a high protein source, are also thought to be an excellent substitute for fishmeal for many freshwater and marine fish as aquaculture production rises day by day. Due to their adequate protein content, amino acid composition, and digestibility, animal by-product meals (AM) can be used in aquaculture instead of fishmeal [62].

Among the plant proteins, soybean meal is one of the most logical alternatives for high-quality fishmeal due to its high protein value, healthy amino acid profile, and low price [63]. Plant-based protein diets have been extensively studied in the past few years as a protein source for aquafeed formulations [64–66]. Plant-based diets are more economical as compared to animal proteins, but they have many limitations that make their substitution with fishmeal questionable, such as the absence of some vital amino acids, low palatability, the presence of anti-nutritional factors, and competitive prices with other food production sectors [66–68]. Additionally, essential components such as taurine and hydroxyproline which are rich in fishmeal are lacking in plant-based substances [69,70], whereas a variety of insect species contain high levels of taurine [71]. Taurine is the essential ingredient for the growth of brood stock and juvenile fish. A taurine-deficient diet results in increased demand for vitamin E and C, particularly in the larvae of marine water fish [72]. The insufficiency of taurine may cause serious psychological abnormalities, green liver disorder, and stunted growth [73]. Fish's principal physiological and behavioral responses are associated with taurine availability in the diet, including the survival rate [74], growth, immune response, and antioxidant activity [71].

As a result, interest in insect meal (IM) as a feasible feed alternative in fish aquaculture has developed dramatically. Insects are a rich source of energy due to macronutrients and micronutrients in sufficient amounts, making them the best suitable fish food [57,75]. It has been discovered that insects contain an adequate level of crude protein similar to soybean meal but slightly less than fishmeal by comparing the nutritional value of several insect orders, soybean meal, and fishmeal. The quality of protein depends upon the presence of essential amino acids, so fishmeal is used as an aquatic feed due to its healthy amino acid profile. Fishmeal is considered a source of high-quality protein because it contains a high amount of digestible essential amino acids, including lysine and methionine, which are thought to be absent in grains, the primary source of animal feed [76]. The amino acid composition of many insect species is currently unknown. With an adequate amount of methionine and lysine, Diptera's amino acid profile is comparable to fishmeal. Some insect species even have a higher amount of EAA than fishmeal, such as histidine and threonine in Diptera and leucine in Coleoptera and Orthopterans. Insects have a high level of polyunsaturated fatty acid n-6, lower than soybean meal but higher than fishmeal [77,78]. One critical point is that nutritional composition varies among the species. Their nutritional composition also varies according to order, diet, and developmental stage [79], as shown in Figure 1.


**Figure 1.** Nutritional value of potential insects used as fish feed. Reproduce from sources [80,81] (L = larvae; A = adult).

#### *2.1. Proteins*

Insects are an excellent source of protein since they contain a good proportion of amino acids; their levels of protein range from 25 to 75 percent (DM) [82–85]. A protein factor (Kp) of 6.2 can be used to calculate crude protein content by multiplying nitrogen content by protein. In order to fulfill fish nutritional demands, we advise using Kp modification based on the quantity of amino acids when calculating insect-containing practical feed compositions [86]. Hung et al. [87] studied that increased protein content shown among the treatments does not seem to be important for the observed growth performance improvement. The erroneous measurement of amino acids and amino acid loss due to methodological difficulties such as hydrolysis might result in incorrect calculations of crude protein [88]. The nitrogen of chitin is the main factor in the over-estimation of protein [89]. When insect protein is compared with plant and meat proteins, it is found that insects contain a substantial amount of good-quality protein due to balanced amino acid proportions [90,91]. Protein content varies amongst different orders due to different factors, and Orthoptera has the maximum protein values, ranging from 60 to 77% [92–94]. The crude protein ranges from 40 to 50% in Diptera, such as in the case of larvae of *Musca domestica* where CP was 46.9% and in house fly where it was 37–57% [92,95]. *Holotrichia parallela* is an edible beetle with a protein content of about 66% and an amino acid score of 87 and 100, with threonine as a limiting amino acid [96]. The protein level of *T. molitor* larvae is 46% [97], *H. illucens* 39% [98], *A. domesticus* 64–71% [99], *L. migratoria* 48–52% [100], *B. mori* 48–55% [101], termites 20–43% [102,103], beetle species 26–50% [102,104], and grasshoppers 26–45% [102,103].

It has been found from recent studies that usually an animal-based diet contains 40% of essential amino acids, but the range of essential amino acids in edible insects is about 46–96% [105]. House cricket meal contains some essential amino acids even at a higher concentration than plant- and other animal-based meals. House cricket meal also meets all the amino acid requirements recommended by the WHO (World Health Organization) [106–108]. Leucine content in Orthoptera is similar to animal-based protein but greater than soybean [106]. Another important amino acid found in greater quantities in insect proteins than in plant proteins is lysine [105]. Beef and eggs are good sources of methionine, serine, and tryptophan, which cannot be found in adequate amounts in some edible insects such as house crickets [107]. The silkworm is an edible insect containing 17 amino acids, with all essential amino acids. The content of essential amino acids is 47% in silkworm pupal protein, according to the recommendation of the WHO. In both male and female silkworm pupae, lysine was the amino acid that was most prevalent [109]. Numerous research studies have indicated that protein and AAs act as mediators of the trade-off between growth and immune protection. They are considered growth-limiting nutrients, particularly in those insects that feed on plant material.

#### *2.2. Carbohydrates*

Chitin makes up between 5% and 20% of the dry weight of insects [110,111]. It is a polysaccharide that is present in the exoskeleton of many insects in varying amounts and makes up 6.71–15.98% of carbohydrates [112]. Chitin is impermeable in a liquid medium and is the most common carbohydrate in biomass after cellulose. Carbohydrates are a rich source of nitrogen and carbon [113,114]. Generally, carbohydrates computed as nitrogen-free extract are found in minor levels in insects [83,85]. Some of the carbohydrate levels of edible insect species are described in the following; yellow mealworm larvae have a carbohydrate content that ranges from 1–7% [115], *Rhynchophorus phoenicis* larvae 5.53% [116], *R phoenicis* 20.23%, *Heteroligus meles* 20.10% [104], *Gynanisa maja* larvae 10.70%, *Ruspolia differens* 8.40%, *Macrotermes falciger* 32.80% [103], *H. illucens* 10–20% [98], *Locusta migratoria* 4–5.5% [100], and *Bombyx mori* 23% [101]. However, these levels are probably due to food left in the gastrointestinal tract.

#### *2.3. Lipids*

Researchers have realized that certain insects contain a lot of lipids. Insects can therefore be used to increase lipid intake. When 20% ground yellow mealworm larvae were used with wheat flour to generate extruded cereals, the lipid content of the final product increased from 0.9 to 5.4% [117]. Insects contain a significant concentration of unsaturated fatty acids, which must be examined during the preparation and preservation of insect meals [118]. Triacylglycerols account for approximately 80% of lipids, which is a reservoir of energy during periods of heavy energy consumption. Orthoptera, Lepidoptera, Blattodea, Isoptera, and Coleoptera have average lipid contents of 13%, 28%, 29%, 32%, and 33% dry weight, respectively [119]. Insects at larval and pupal stages have more lipids than adults [111]. The lipid content of *T. molitor* larvae is 33% [97], *H. illucens* larvae 38% [98], *L. migratoria* 19–20% [100], crickets 12–25% [120], termites 22–43% [102,103], beetle species 21–32% [102,104], *A. domesticus* 18–22% [99], *B. mori* 19% [101], and grasshoppers 3–49% [102,103].

Phospholipids are commonly found in nature and are responsible for maintaining the structure of membranes [121]. The phospholipid concentration of lipid content is about 20%; however, it changes with life stage, insect order, and species [121,122]. In insects, lipid content comprises a comparatively high concentration of C18 fatty acids, including oleic, linoleic, and linolenic acid [121]. The palmitic acid level is slightly higher than other lipids, and the lipid profile is greatly influenced by the diet fed by insects [90].

Insects are rich in n-6 fatty acids but deficient in n-3 fatty acid contents. However, this n-6/n-3 ratio is unsuitable for human health [123]. Therefore, different strategies are being employed to improve the fatty acid profile of insects. The content of n-3 fatty acids can be increased by using a suitable substrate; thus, the nutritional value of insects can be improved. Different factors also affect the fatty acid profile of insects and substrates, such as the rearing environment. This fact was also supported by Ruschioni et al. [124], who reported that levels of linoleic (C18:2n6) and alpha-linolenic (C18:3n3) acids varied amongst the insects even when they were reared on the same substrate. PUFAs are abundant in edible insects, commonly known as linolenic and linoleic acids [125]. However, the insects

do not contain eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), which are necessary for the health of human beings [125]. Lauric acids are one of the most important medium-chain saturated fatty acids in most insects. HI contains a substantial amount of lauric acid, which has antimicrobial properties and thus can modulate the intestinal health of host organism [126].

#### *2.4. Vitamins*

Insects have a range of vitamins that are either water-soluble or lipophilic [127,128]. A variety of insects having thiamine were listed by [90]. Its concentration varies between 0.1–4 mg per 100 g (DM). Riboflavin, also known as vitamin B2, is found in edible insects and plays an important role in metabolic function. The yellow mealworm and the house cricket *Acheta domesticus* have high contents of vitamin B12. Many other species that have been studied possess only trace quantities of this vitamin [90]. Insects often have low quantities of retinol but high levels of biotin, pantothenic acid, and riboflavin. Another important vitamin, folic acid, is identified in significant concentrations in different insects [119]. This trade-off is thought to focus on dietary nutrients, a significant idea in studying nutritional immunology [129,130].

#### *2.5. Minerals*

Edible insects are a good source of minerals such as zinc, sodium, potassium, iron, magnesium, copper, calcium, manganese, and phosphorous [131,132]. A substantial amount of iron is found in the moth *G. belina* (31–77 mg per 100 g of dry matter) and *Locusta migratoria* (8–20 mg per 100 g of dry matter) [133]. Similarly, a good quantity of zinc is found in caterpillars of mopane (14 mg per 100 g of dry matter) and *R. phoenicis* (26.5 mg per 100 g of dry matter) [90]. Hyun et al. [134] reported that edible insects such as the grasshopper *Oxya chinensis* contain very little heavy metal that is safe for human consumption. The solubility of iron was found in the following order in different insects: cricket > mealworms > grasshopper = buffalo worms > sirloin beef. Grasshoppers, crickets, and mealworms are reported to have high solubilities for Cu, Mn, and Zn. Similarly, the high solubility of Ca and Mg were reported in mealworms [135].

Koseˇcková et al. [136] examined the mineral content of the house cricket, yellow mealworm, desert locust, and super worm. All these insect species were reported to be good sources of iron. They reported that a good quantity of Zn, Cu, and P was found in house crickets, yellow mealworms, and desert locusts, even greater than the DRV (dietary recommended values). These insect species are all very low in Cd and Pb content, indicating that eating them poses no risk. Eating insects can increase zinc, sodium, phosphorus, potassium, calcium, iron, copper, manganese, and magnesium availability in terms of mineral nutrition [137]. Mopani or mopane, a caterpillar of moths, contains an iron concentration of 31–77 mg per 100 g DM. The ranges of different minerals (mg/kg) in *T. molitor* are Ca (349.2), P (5600–5700), Mg (1290–1416), Zn (95–101), Fe (60–64), Cu (11.4), Mn (7.0), B (20.3–24.2), and Mo (0.6), and in *B. mori* Ca (987.2), P (8550–8860), Mg (2400–2600), Zn (130–151), Fe (49–50), Cu (9.3–9.5), Mn (16.2–17.6), B (10–18.8), and Mo (0.2) [101].

#### *2.6. Functional Properties of Insect Meals*

Insect proteins have many favorable characteristics that make them fit to be used as food and feed. However, it is very important to evaluate the functional properties of insect protein before their substitution in any food and feed [138]. Depending upon insect protein's techno-functional properties, which include solubility, water and oil holding capacity, gelling, and emulsification, different food processing methods can be applied to improve the quality of insect-based food [139]. Several studies have been conducted to investigate the improvement in the functional properties of insect protein by applying suitable processing strategies [138]. Drying, defatting, and extraction are the different processing methods commonly used to improve the functional properties of edible insect proteins [140]. By enhancing the protein content, fractionation processing enhances the functionality of insect proteins [138,141–143]. Kim et al. [142] reported that the functional properties of *T. molitor*, *P. brevitarsis*, and *Allomyrina dichotoma* increased with protein content. Another important method for increasing protein functionality is enzymatic hydrolysis [138,144]. Purschke et al. [145] examined the effect of enzymatic hydrolysis on the functional properties of *L. migratoria*. They observed the different changes in functional properties of *L. migratoria* by applying hydrolysis conditions. The functional properties of *L. migratoria* (solubility, gelling, foaming, and water and oil holding capacity) were increased with the decrease in the molecular weight of protein.

There was no difference in the protein functionality, but a reduction in emulsifying activity was observed with the application of enzymatic hydrolysis [146]. Mintah et al. [143] examined the enzymatic hydrolysis of soldier flies that improved the dispersibility, turbidity, and particle size by changing the protein secondary structure. Lipids and chitin removal also improve foaming and emulsifying properties, respectively.

#### **3. Factors Influencing the Nutritional Value of Insects**

#### *3.1. Insect's Species*

An insect's species and order affect its nutritional value. The majority of the insects studied have a significant amount of protein [123], almost similar to soybean meal but inferior to fishmeal [147]. Usually, Orthoptera has a higher CP, between 50 to 55 g/ 100 g [148], Diptera amino acid content is analogous to fishmeal [92], and Coleoptera has a better amino acid profile than soy meal [149], although not comparable to fishmeal. The ratio of amino acids varies widely amongst the various insect orders, plant meals, and fishmeals. Histidine and threonine are more deficient in insect meals than in fishmeals out of all the essential amino acids. However, insect meals contain a sufficient level of tyrosine, lysine, and methionine compared to soy meals. Diptera has a similar amount of histidine, lysine, and threonine as fishmeal. Diptera contains a higher level of phenylalanine than soybean meal and fishmeal, but the methionine level is comparable to fishmeal. Leucine is abundant in the orders Orthoptera and Coleoptera, but deficient in the order Diptera. The percentages of tyrosine and valine are higher in Orthoptera, Coleoptera, and Diptera than in fishmeal [150,151]. Some of the values of the amino acids in different edible insects are presented in Figure 2 and Table S1.

**Figure 2.** Patterns of amino acids (mg/g CP) of six commonly used edible insect species. Sources [80,82,152–155].

#### *3.2. The Developmental Stage of Insects*

An insect's developmental stage may also have an impact on how nutritious it is. With advanced developmental stages, it has been demonstrated that the protein content is generally increased. On the other hand, lipid content is the reverse. The orthopteran species is a high-protein species in its adult form. In Diptera, CP levels range from 40 to 50% in larvae and 50–62% in pupae in several species [150]. In general, larval stages have higher lipid contents than adults. At various phases of development, the lipid percentage in *A. domestica* varies between 14% and 22% [152]. Five-day-old larvae of the black soldier fly contain a maximum percentage of crude protein which is about 61%, and as the insect becomes older its crude protein content gradually declines [156].

The pupae of most insects contain a sufficient amount of saturated fatty acid but a lower amount of unsaturated fatty acid, however, adults behave differently [157]. Ademolu et al. [158] investigated differences in the nutritional composition of distinct post-embryonic developmental stages of the grasshopper *Zonoceros variegatus* nymphs and adults. There is an increase in protein content but a decrease in lipids from nymphs to adults. Similar to this, several Blattodea species show a correlation between developmental stages and higher protein and lower lipid levels [159]. One of the most significant features of insect development is the distinction between larvae and adult forms. Larvae and adults show diverse amino acid profiles; for example, yellow mealworm (*Tenebrio molitor*) adults are rich in protein and chitin, whereas larvae are rich in lipids [160,161].

#### *3.3. Diet*

Diet appears to affect the lipid composition of insects. Larvae and adults may consume a diverse range of diets, nonetheless, pupae usually do not, explaining the differences in amino acid, lipid, and mineral contents [162]. For example, in male bees amino acid, protein, and mineral contents vary with the development stage [157]. The orthopteran breeds in the wild have an 8.2% lipid content, while orthopterans bred in captivity have greater contents.

According to Barroso et al. [163], saturated fatty acids are abundant in terrestrial insects but they are deficient in polyunsaturated fatty acids, limiting their use as feed. In this study, the diet composition of BSFL was altered to improve lipid composition. This study suggests that diet-fed larvae contained three times more n-3 fatty acids and a reduction in the n-6: n-3 ratio compared to the control diet group. Dietary manipulation could quickly affect the lipid composition of the experimental larvae group. Van Huis et al. [164] prepared experimental diets from different organic products. Three types of edible mealworms were fed these diets to examine the effects of the experimental diet on the nutritional composition of the insects. Larval growth and survival rates as well as the protein and carbohydrate contents of experimental meals differed. The manipulated diet improved the FCE, crude protein, lipid composition, weight gain, and survival. Zarantoniello et al. [165] studied the rearing of BSF on coffee and *Schizochytrium* sp., and they found significant results in improving the FA profile of zebrafish larvae. The nutritional addition of BSF meal significantly impacted the fatty acid classes of the zebrafish larvae fed on the various diets. For example, the proportion of SFAs increased when insect meal was added to the diets, but the percentages of MUFAs and PUFAs frequently reduced as BSF inclusion levels increased. The higher inclusion of the BSFL in the diet reduces the n-3 and increases the n-6 FA. Chemello et al. [166] explained the effect of BSF mixed with *Schizochytrium* and stated that oocyte maturation stages, fish stress responses, and spawning and hatching success were all negatively impacted by higher replacement levels (BSF 75% and BSF 100%).

#### *3.4. Processing of Insects*

The ability of insect meal to replace fishmeal depends not only on the nutrition of insects but also on the bioavailability of nutrients. Many insect species have very high lipid levels. Different processing techniques are applied, including mechanical pressure for the defatting of insects by using different organic solvents such as petroleum ether. These processing methods can improve the protein content of IM further by generating

various combinations of free amino acids (AAs) and decreasing the amount of undesirable lipids [167].

Insect meals have low palatability, which can be attributed to various factors, including chemical or biological contamination, anti-nutritional factors such as flavonoids and terpenoids in insect feedstuffs, and high monounsaturated fatty acid [92]. All these factors make insect meals susceptible to oxidation and subsequent rancidity problems. Drying, hydrolyzing, ensiling, and defatting are the different processing methods that can improve fish nutrition by improving the nutrient availability, digestibility, palatability, and composition of insect meal [168]. The processing technique of edible insects can affect their nutritional content. Toasting and solar drying of grasshoppers decreased their protein digestibility and niacin content, but winged termites retained their riboflavin and retinol content [169]. The processing step directly affects the protein quality and content of insect extracts. The nutritional composition of the mopane caterpillar was improved in terms of digestibility and crude protein after degutting, but the roasting resulted in decreased crude protein level and digestibility of the insect [170]. Thus, exploring and developing efficient processing procedures to enhance insect meal commercialization and consumer acceptability is necessary [131].

Fermentation, drying, extraction, enzymatic modifications, and thermal processing are the most common methods that improve insect meal quality, flavor, and texture. It is interesting to note that different insects react to processing methods differently, demonstrating various outcomes for in vitro digestibility [171]. Pre-processing technologies are the first steps in the processing of edible insects. These initial steps are normally performed before food production and are stated as pre-processing phases. Insect degutting, drying, and defatting are pre-processing steps [172,173]. Protein extractability is frequently improved with defatting. This pre-processing stage is completed in some studies following measurements of other protein-related parameters impacted by defatting [142,174]. Additional innovative processes, including pulsed electric field, ultrasound, cold plasma, and high-pressure processing, are extensively applied to replace standard operations and can be used as a pre-processing step [175]. These techniques improve the product's quality, digestibility, and preservation capacity [138,176]. At the industrial level, protein extraction is performed by utilizing different enzymes and organic and inorganic solvents. Newly emerging processing techniques are currently utilized to extract chitin, protein, and lipids in edible insects. The extraction rate varies among the insect species and can affect the extraction yield and insects' physical, chemical, functional, and bioactive properties [174]. A layout of the processing techniques is illustrated in Figure S1.

#### 3.4.1. Enzymatic Hydrolysis

Enzymatic hydrolysis is principally performed to understand the bioactive properties of edible insect hydrolysate after stimulation of digestion in the gastrointestinal tract [177], specifically angiotensin-converting enzyme inhibition [178] and antioxidant capacities [179]. Techno-functional properties of enzyme hydrolysates also change during the processing, and this area needs to be investigated further. In the case of the locust *L. migratoria* L. [145], techno-functionality was increased by utilizing different food-grade proteases alone or in combination with varied enzyme–substrate ratios.

#### 3.4.2. Drying and Thermal Processing

Drying is one of the best processing techniques that increases the shelf-life of insect meals. This processing technique ranges from conventional methods such as sun drying to contemporary methods, including microwave-assisted drying and freeze-drying. Drying stops food spoilage by reducing the water content, inhibiting the enzymatic and microbial reactions. Microbial growth depends upon the availability of water content; when the water content is low, microorganisms stop growing [172]. As the drying process reduces the water content, there is an increase in the concentration of dry matter, which does not damage the physical and chemical properties of insect meal and thus increases the shelf-life [180].

Thermal processing is also a widely used technique in the food production industry that inhibits microbial reactions and extends the shelf-life of insect meals [181]. Thermal processing induces physical and chemical changes without damaging the quality of food [182]. Different types of thermal processing such as hot air drying and oven-assisted boiling show the highest result in maintaining the quality and standard of mealworm larvae [183]. Thermal processing can process the silkworm larvae into a yellowish powder by using warm water, drying, and grinding. Thermal processing can also damage some important bioactive compounds [184] and the nutritional content of insect meals [185]. Thermal processing increases the bioavailability of biomolecules by increasing the process of protein hydrolysis [186]. When *A. domesticus* and *T. molitor* are heated at 200 ◦C for 10 min, improvements in the nutritional value and AA profile were observed [187].

#### 3.4.3. Fermentation and Antibiotic Resistance

Recently, fermentation technology has been used to produce insect meal, increasing the nutritional value and quality of the meal [188]. Considerable literature is currently available on fermented insect meal, including other insect strategies such as feed additives and biofuel [189]. A particular culture of microorganisms is utilized to ferment the biomolecules in the substrate during the fermentation of the insect meal, improving its nutritional value and digestibility. Antibiotic-resistant bacteria can undoubtedly come from insects [190,191]. However, there are currently not many studies that have been published that demonstrate how frequent antibiotic resistance is in edible insects [192–195] and the antibiotic-resistant (AR) genes in different edible insects. The existence of transferable AR genes in edible insects has not yet been precisely linked by studies to rearing conditions. Additionally, no research has been conducted to ascertain the loads and dynamics of antibiotic resistance in certain microbial communities associated with edible insects. This aspect of edible insect processing needs much attention for the healthy production of food and feed.

#### **4. Potential Insects Used as Fishmeal**

Over the last two decades, numerous studies have been conducted to lower the demand for fishmeal, fish oil, and their by-products in the aquaculture industry [196], which resulted in increased incorporation of plant-derived ingredients in fish feed [197]. However, compared to fishmeal-based diets, adding these substances to aquafeeds imposes greater pressure on water and land resources [197] and generates more waste [198]. Several protein alternatives have been examined for fish feed, insect meal, and fishery by-products, showing the greatest potential to fulfill aquafeed's protein requirements in the next decades [18]. For several aquatic species, the successful incorporation of insect meal preference to fishmeal in the diet of many fresh and marine water fish has been widely reviewed [39,199,200].

#### *4.1. Black Soldier Fly*

The black soldier fly (BSF) (*Hermetia illucens*) is one of the best options currently used as an alternative source of protein in the aquaculture diet. It is one of the extensively investigated insect species due to its healthy nutritional value [199]. The insect's balanced nutritional composition makes it an ideal and possibly significant alternative to fishmeal, and the larval form is what is used to prepare BSF meals.

Bioactive compounds with nutraceutical properties, such as lauric acid, chitin, and antimicrobial peptides, are present in the BSF larvae meal. These compounds are very important for improving the growth of the fish [201]. Previous research suggests that these chemical compounds have either prebiotic or probiotic properties; they are fermented by beneficial commensal bacteria in the intestine and produce metabolites that improve the host's health [202,203]. Black soldier fly (BSF) larvae growth as fish feed is a promising approach, since it uses organic wastes while also being safe for humans and animals [201,204]. BSFLM also has an amino acid profile comparable to fishmeal [39], making it a good candidate for long-term mass production. Many experimental studies have indicated that substituting FM with BSFLM in aquaculture has no adverse impact on fish development [205–208]. One of the best features of BSFLM is that it can utilize organic wastes as the substrate, such as animal dung [209,210] and plant waste, which includes vegetables and fruit wastes [211], algae [212], and fish [163,213]. The inclusion of fish offal in the BSF diet causes black soldier fly prepupae to absorb different lipids. In the modified prepupae, omega-3 fatty acids are found in reasonable amounts, which could be used as feed for carnivorous fish and other animals. Furthermore, this insect species may offer new approaches for reducing and recycling fish offal using different processing operations.

BSFL meal was found to have a greater growth response and feed conversion ratio than FM in Nile tilapia [214]. Previous studies have shown that when BSFL meal is included in the diet of blue tilapia, *Oreochromis aureus*, it shows positive results. The replacement of FM with BSFLM for different inclusion levels greatly increases *O. niloticus* growth without causing detrimental effects. It has also been studied that BSFL meals can completely replace FM [215]. It is obvious from previous research that in yellow catfish, FM can be substituted for up to 20% of the diet [216]. There were no remarkable changes in sea bass growth, feed consumption, survival, and hematological parameters when the fishmeal was partially replaced with black soldier fly meal. Additionally, it was discovered that switching from fishmeal to BSFM at 50% for eight weeks reduced feed costs by 15.5% compared to fishmeal pricing [217]. The HI has good protein solubility, water binding capacity, and lipid binding capacity [174].

#### *4.2. Common Housefly*

Housefly (*Musca domestica*) maggot meal has a high nutritional value as an insect protein source. The housefly contains a substantial amount of proteins, lipids, and carbohydrates similar to fishmeal, improving fish growth [53]. It also comprises several biologically active compounds such as antimicrobial proteins, lectin, and chitin [218]. Maggot meal shows good potential to replace fishmeal, as the housefly has a fast reproduction rate, balanced nutrient proportion, and simple processing method [219]. Energy, protein, and micronutrients EAAs and FAs are abundant in housefly larvae. Housefly larvae are less expensive, have healthy nutritional contents, and are easier to produce than other alternative animal proteins. HFL meals, high in lysine, threonine, and methionine, can supplement protein-deficient cereals and legume-based feed for aquatic animals [220].

Houseflies can quickly digest food waste and cattle dung waste, which is organic, using nutrients from waste to reduce the volume of waste in its entirety. Maggot meal enriched with protein and oil from dried fly larvae could be used as important cattle and aquaculture fodder [150]. The fish efficiently absorb maggot meal, and its inclusion in tilapia diets appears to have no oxidative-stress-inducing effect on fish metabolism. It can be used effectively as a rich source of protein for the growth of tilapia fingerlings [151]. Wang et al. [221] observed the effects of four experimental diets supplemented with 25%, 50%, 75%, and 100% *M. domestica* (MD) larva meal on Nile tilapia. About 75% of MD meals can be included in the diet of fish without causing substantial adverse effects on the growth and development of fish. Adding maggot meal (MM) slightly enhanced fillet quality by making them tougher. It is also worth noting that several experimental diets significantly improved water quality compared to the control diet. Several investigations, including MM-based feeding experiments in different fish species, have been conducted over the last decade. Maggot meal can replace 100% fishmeal in the diet of Nile tilapia fingerlings [222] and African catfish [95] without damaging growth or nutrient utilization ability or causing oxidative stress [151,222]. Partially substituting FM diets with blowfly (*Chrysomya megacephala*) MM increased juvenile tilapia growth, feed efficiency, and survival [223].

Feeding tests were conducted on juvenile Asian bass to evaluate physiological responses to growth and fillet composition to dietary FM partly supplemented with houseflymaggot-based meal. According to the research, replacing dietary FM with up to 300 g/kg of housefly maggot meal might be achieved without harming development [224]. An eightweek rearing experiment on swamp eels (*Monopterus albus*) indicated that supplementing the food with housefly larvae had positive influences on the swamp eels' development

and immunity [225]. In conclusion, several feeding studies on various aquaculture species have shown that adding housefly maggots to fish diets may boost growth and FCR while limiting physiological stress. Feeding fish diets with housefly maggot meal is also less expensive. It is a different protein source that may be used to replace FM in aquafeeds, depending on its nutritional content, availability, growth potential, and feed efficiency. This is especially helpful in underdeveloped nations when FM imports are expensive.

#### *4.3. Mealworm*

The yellow mealworm (*Tenebrio molitor*) is a fast-growing and rapidly reproducing insect that feeds on bread and cereals. In *T. molitor* (TM), the protein content varies between 47% and 63% and lipid content between 31% and 41%. The amino acid profile is in accordance with the nutritional demand of aquatic animals [120]. Mealworms are simple to produce, have a low environmental impact, have easily manipulable nutritional content, and are highly efficient.

Recent research has shown that TM meal is an innovative protein source that can partially substitute FM in chickens [226] and aquatic species [53]. A percentage ranging from approximately 33 to 74 of FM can be replaced by TM larvae without harming the growth of gilthead seabream [227]. They also reported to have tremendous potential for replacing FM in cattle and fish [39,53,228]. It is evident from previous studies that mealworms can substitute the fishmeal of rainbow trout [229], European sea bass [230], gilthead seabream [227], and blackspot seabream [231].

An approximately 35 percent substitution of yellow mealworm may replace fishmeal in European sea bass without slowing fish growth, whereas a 70 percent of fishmeal substitution reduces fish growth [230]. According to previous studies, higher inclusion levels of mealworms in rainbow trout meal had no negative effect on weight gain but enhanced the protein content and lowered the lipid content compared to the control group [229]. A 100% replacement of fishmeal with yellow mealworm meal enhances the Pacific white shrimp lipid content but does not affect the growth rate and feed conversion ratio [232]. When a considerable amount of the fish feed is replaced with a mealworm in common catfish fingerlings and African catfish, its growth retards [233]. The HI has good protein solubility, water binding capacity, and lipid binding capacity [174]. Insects have substantially greater phosphorus levels than calcium levels. They have higher phosphorus content than mammals. Mealworms lack calcium, so primarily feeding fish with mealworms might result in calcium deficiencies and body deformities. Despite this, the mealworm insect is an excellent choice to replace fishmeal [53].

#### *4.4. Cricket*

Crickets (*Acheta domesticus*) belong to the Orthoptera order, having a good amount of crude protein ranging from 55–73% and a sufficient amount of indispensable AA except lysine and methionine, which can be supplemented in the feed [80,92]. Cricket food is high in proteins and lipids and contains vitamins and minerals [234]. Cricket meal contains a significant amount of crude protein (64.9%) and lipids (17.4%) with a good proportion of amino acids, including lysine and methionine, which are deficient in a plant-based diet [235]. Recent studies reported that 8.7% of chitin is present in cricket meals, and its supplementation in the fish diet improved the interaction of chitosan glucosamine with bacterial cell walls; in addition, the consequent alterations in the permeability of cell walls reduced the bacterial population [236,237].

Due to their excellent nutritional value, live crickets are currently sold at the commercial level in big pet stores and markets as fish bait or supplemental feed for differential ornamental fish species. Cricket meal can substitute up to 100% of fishmeal in African catfish, showing better results than the control diet [238–240]. It has the potential to partially or completely replace fishmeal in fish feed [235,241]. A recent study investigated whether substituting insects (house cricket and super worm in equal proportions) for FM in perch

feed could affect fish survival, growth parameters, and fatty acid composition [242]. Cricket meal might contain any xenobiotic ingredient, thereby altering enzyme activity.

Hanan et al. [243] described meat quality and the fatty acid profile of the insects *Zophbas morio* and *A. domestica*. These species were specifically selected for their simple breeding, standardization, and higher AA, FA, and vitamin contents. Only super worms and crickets possessed a significant concentration of vitamin A for the animals that feed on insects [82]. Fishmeal can be substituted with up to 50% cricket meal in red Nile tilapia without significantly affecting growth performance or feed consumption.

#### *4.5. Locust*

Locusts (*Locusta migratoria*) are generally produced to feed domestic and zoo animals and have also been studied for cattle feeding. They vary greatly in protein composition (29 to 70% on a dry matter basis) and lipid content (4 to 22% on a dry matter basis) [53,244]. Locusts are among the most commonly consumed insect species globally, but they have also been utilized as a supplementary protein source for chickens and fish [131]. Aquaculture development in Africa and Asia and the search for alternate protein sources led to feeding experiments with locusts and grasshoppers for catfish and tilapia [245].

A 50% inclusion level of locust meal yielded the optimum results in Nile tilapia in terms of growth. As a result, it is recommended that locust meals at a 50% inclusion rate can be added to the diet of tilapia fish without impairing fish growth. When about 20% of adult Orthoptera (grasshoppers or locusts) was added in the diet of catfish and tilapia, it did not affect catfish or tilapia digestibility or growth [246,247]. However, the use of locust meal as an alternative source of protein in aquaculture diets is still understudied, and no data on its use as feed for marine fish species are known [246,248].

#### *4.6. Silkworm*

The silkworm (*Bombyx mori*) (SW) is believed to have originated in China more than 4000 years ago [249]. Its pupae are the primary by-product of the silk industry, with about 8 kg of pupae produced for each kilogram of silk. Silkworm meal is prepared by properly grinding and drying boiled cocoons of silkworm larvae. SW meal is obtained by drying and grinding the larvae's uncoiled boiled cocoons, containing 56% protein and a substantial amount of essential amino acids [250]. Kurbanov et al. [251] investigated the effect of substitution of fishmeal with silkworm pupa protein on the growth and feed utilization ability of African catfish (*Clarias gariepinus*) fingerlings. Five isonitrogenous diets containing approximately 40% crude protein and different levels of fishmeal replacement (0–100%) were fed for 40 days at 5% live fish body weight. The growth rate and feed utilization efficiency of fingerlings fed diets with a 50:50 mixture of fishmeal and SPP were significantly higher than those fed diets containing SPP or fishmeal alone. The fishmeals of different fish species have been substituted with silkworm pupae meal and promising results were obtained, thus showing the good potential of silkworms to replace fishmeal. About 10% SWM can be successfully substituted in the diet of chum salmon and olive flounder [252].

The snakeskin gourami diet can be substituted with up to 15% SWP (silkworm pupae) without adverse effects on growth, but a 22% inclusion level of SWPM decreases growth rate and protein digestibility [253]. A 30–50% successful inclusion of defatted or nondefatted SWP can be achieved in rohu, common carp, mahseer, and rainbow trout [254,255]. A total FM replacement without adverse effects on growth has been observed in common carp and Japanese sea bass [256]. Some fish species show negative responses even when a low level of SWM is incorporated into their diet. In tilapia, a 5% inclusion level drastically decreased growth and development [257]. Jian carp also showed reduced digestive enzyme activity, heat shock protein activities, and increased oxidative stress when the diet was incorporated with about 8% of SWPM [258]. In walking catfish, the protease activity result is comparable to FM when 58% FM is substituted with SWP meal. SWP appears to be a potential source of protein for fish diets in general, except for Nile tilapia and Jian carp

gourami. SWP meal was found to be an advantageous sustainable feed element in carp diets, with benefits for boosting growth performance and specific physiological markers, according to an eleven-week feeding research on *Cyprinus carpio* [259]. In the rainbow shark (*Epalzeorhynchos frenatum*), feeding experiments revealed that SPM could substitute up to 30% of FM in the diet [260].

#### **5. Insects as Feed for Crustaceans**

Fishmeal is a commercial diet for rearing prawns and shrimp because of its high protein content, favorable amino acid and fatty acid profiles, high digestibility, and palatability [205]. Farm animals' diets have recently begun to include insect larvae meals, and previous studies showed that insect meals are a promising addition to the diets of farmed crustaceans [205,232].

#### *5.1. Shrimp*

Studies have found different results comparing the growth rate performances of commercially produced shrimp and fish fed with FM substituted with insect meal. This could result from changes in meal preparation methods or diet formulation. The nutritional value of animal protein meals is influenced by the processing technique and the quality of the raw materials [261]. For example, research on rainbow trout showed that the BSF larval diet could replace 50% of fishmeal without impairing growth performance. However, a decrease in protein utilization efficiency was reported in shrimp [262]. Without impairing shrimp growth, a full-fat BSF (*H. illucens*) meal can substitute up to 25% of fishmeal [205]. Like this study, subsequent studies on *T. molitor* full-fat meal-based diets found that when methionine was added in adequate amounts, Pacific white shrimp's nutritional value and growth performance were on par with or even superior to those of FM-based diets [167,232].

Motte et al. [263] reported that utilizing TM meal in shrimp diets significantly enhances growth when paired with FM without affecting shrimp survival or feed consumption, even when TM replaces 100% of FM. Moreover, this insect meal's very important key feature is that it improves the immune system and resistance to infection, leading to increased shrimp survival. Improving immunity is critical when shrimp production intensity is very high, causing stress and increasing sensitivity to illnesses. TM meal has good potential as an efficient feed source for commercial shrimp farming. The 50% substitution of fishmeal with mealworms for the shrimp diet is ideal for boosting shrimp growth performance, while having no negative effects [167]. TM is a suitable replacement for FM that has no negative effects on the expression of important digestive enzymes, gut microbiota, or the Pacific white shrimp's immune system [264].

#### *5.2. Prawns*

Efforts have been made to supplement the diet of prawns with insect meal. A recent study examined the effect of replacing the diet of prawns with insect meal [265]. The insect meals were prepared from the larvae of three insects: house fly, BSF, and mealworm. Incorporating the black soldier fly into the fishmeal diets of prawns resulted in considerably higher prawn growth rate and survival. The shrimp survival rate was significantly reduced when fed a diet consisting of house fly larvae, but growth performance was incomparably higher. Insect inclusion in plant-based foods had little effect on growth performance, while survival was greater in the TM and HI inclusion diets. When TM and HI were added to fishmeal and plant meal diets, they increased the protein content of the prawns' muscles. Langer et al. [266] studied the consequences of replacing fishmeal with silkworm pupae, soybean meal, and earthworm meal in the freshwater prawn *Macrobrachium dayanum* for 90 days. The diet including silkworm pupae showed the second best result, closely followed by fishmeal.

Two studies investigated the utilization of seven diets for prawns farmed at suboptimal temperatures [267,268]. All seven diets supplied to the juvenile prawns were quickly devoured. Shrimp- and silkworm-based meals provided the best growth and survivorship, while fishmeal- and black soldier fly larvae meal (BSFLM)-based diets provided the poorest growth. Survival rates were comparable for diets based on soybeans, crickets, mealworms, and fishmeal. BSFLM inclusion in the diet caused the mortality in prawns after 1 day of feeding, which might be due to some antimicrobial or toxic compounds produced by BSFL. Similarly, as the termite meal inclusion level increased in the diet, decreasing order in growth was observed in prawns. Partial replacement of FM with 35% TM in prawns showed positive results but was lower than FM. Termite meal cannot replace fishmeal as an alternate protein source for *M. rosenbergii* juveniles. However, it can be added to the diet of prawns for amino acid supplementation since its addition can improve the growth rate [268].

#### **6. Physiological Responses of Fish Using Insects as Fishmeal**

Diet formulation is the key factor for sustainable fish production, as the composition and nutrition of the diet will directly influence the fish growth performance and health status [269]. An accurate and detailed examination of the organs involved in digestion and absorption of feed, immunological response, and metabolic processes should be conducted while testing new dietary formulations [270]. It is generally known that insects contain bioactive substances such as chitin, which, at specific concentrations, can strengthen fish immune systems and promote the diversification of the gut flora of fish [271]. Recently, scientists have been working on new aquafeed formulations such as insect meal and examining their effect on the physiological response of different fish species. A review of some of these studies is presented in the subsequent sections.

Recent research has shown that rainbow trout's inflammatory response can be lowered by including HI-based meal (*H. illucens* meal) in low-FM diets [272,273]. Additionally, it has been noted that HI-based meal positively impacts the intestinal physiology of various farmed fish species [274]. According to studies by Osimani et al. [275], Zarantoniello et al. [276], and Zarantoniello et al. [165], HI-based meals contain some bioactive substances, such as chitin and medium-short FAs. These useful substances have immune-stimulating, antimicrobial, and/or anti-inflammatory properties, which have positive and beneficial effects on fish gut health [277]. It is evident from recent research that the quality of the nutrients can affect the reproductive system (with an emphasis on oocyte quality). A good aquafeed composition is essential for brood stock's proper growth and development [270]. Chemello et al. [166] observed the physiological effect of different levels of BSF prepupae meal (0, 25, 50, 75, and 100%) on fish growth performance, lipid metabolism, stress response, and reproduction. For determination of the physiological response of fish to experimental diets, a multidisciplinary approach (biometric, gas-chromatographic, histological, and molecular analyses) was used. There was not any detrimental effect on physiological responses of fish when the fishmeal was replaced with 50% BSF meal in zebrafish. However, the inclusion of a higher level of BSF meal (75% and 100%) showed a negative effect on stress response, oocyte maturation, and spawning and hatching of zebrafish.

Sudha et al. [278] revealed the inclusion of different levels of BSFM in the catfish diet and its effect on the physiological response of catfish. When the fishmeal was replaced with 20%, 40%, and 60% diets, no significant difference in growth performance was observed compared to the control group. However, the inclusion of 100% BSFM in the diet of catfish showed a reduction in growth and feed efficiency. No significant differences in catfish's amino acid profile and hematological responses were observed when fed with different BSFM. The non-significant difference in the liver and intestinal amylase and lipase activity was observed in the inclusion of BSFM. Higher levels of BSFM (80% and 100%) significantly lowered the proximate body composition of fish and increased lipase activity. Fish fed with 80% and 100% BSFM showed increased congestion in the hepatocyte. Zarantoniello et al. [276] observed a significant growth and survival reduction when fish were fed with a diet containing 50% *H. illucens*.

Additionally, a 50% HI diet substantially decreased the hepatic lipid and glycogen content but increased hepatic *hsp* 70.1 gene expression. The inclusion of 50% HI in sturgeon's

diet also badly affected gut histological morphometric parameters. Zarantoniello et al. [165] reported on the inclusion of insect meal and its effect on the physiological responses of fish. The different inclusion levels of BSF meal (0, 25, 50, 75, and 100%) in the diet of zebrafish showed a significant difference in the physiological response of fish. When the fish were fed with a high inclusion level of BSF meal (75 and 100%), severe hepatic steatosis was observed in the fish's liver. Inclusion of a high level of BSF meal resulted in a reduction in gut microbiota biodiversity, high lipid content, and significant upregulation of genes involved in immune response.

#### **7. Challenges of Using Insects as Feed in Aquaculture**

Chitin, a non-protein nitrogen molecule found in most insect cuticles, causes reduced meal digestibility and growth performance [279]. Increasing the chitin concentration in IM products to ensure a favorable response in fed species requires more investigation. Alkaline extraction may easily remove chitin [280], but it is costly and leaves chemical residues and contaminants [281].

In addition to the presence of chitin, a negative effect in the growth of aquatic animals is observed when insect meals are included in aquafeeds. This fact can be attributed to lower levels of fatty acids in the insect-based diets compared to the fishmeal control diet [13]. Many studies have reported that insects have lower levels of n-3 PUFAs [165], which results in lower n-3 PUFA levels in aquafeeds if substrates enriched with n-3 PUFAs are not used during the insect rearing. Zarantoniello et al. [276] found that a 50% substitution of fishmeal with BSFM caused a significant reduction in n-3 fatty acids, the result of which was a marked reduction in sturgeon weight and SGR observed when fed with BSF-substituted diets. In addition to fatty acids, insect meals are also deficient in some essential amino acids as compared to fishmeal, and manipulation of these components through the rearing substrate is still a big challenge [282].

Prices of insect meals are predicted to become competitive by 2023, but at present, IM prices are very high [283,284]. The main limitation of using insects is the existence of toxic compounds which can negatively affect fish physiologically, such as lowering growth and altering hematological parameters [285]. On the other hand, the substitution of FM and FO with *H. illucens* meal can reduce the content of some potentially toxic elements such as Ni, As, and Pb in fish feed, causing levels of harmful chemicals in animal feed to be below the permitted limit [286]. It is now widely acknowledged that dietary metal(loid) intake can cause chronic toxicity in aquatic species [287]. These pollutants can bioaccumulate or bio-magnify in food chains [288]. Consumption of PTE contaminates fish, thus is a major risk to human health. These PTEs should be examined in the environment and feed or food because of their hazard to feed/food safety and, eventually, human health. Non-defatted insect meals and oils show a dramatic change in FA profile, which shows huge variation in the quality of insect meal and its composition, limiting the usage of insects as feed ingredients.

Another important aspect of incorporating insect meal in aquaculture depends upon the aquaculture producer and consumer acceptance. Without consumer acceptance, adopting insects in the aquaculture sector is difficult. An accurate evaluation of factors involved in insects' production is also a big challenge, as it involves transitioning from the wild catching of potential edible insects from their habitats to their large-scale production. Intensive insect rearing requires intensive labor, as most commonly used insect-rearing methods are labor intensive. Scaling up the insect industry requires the availability of labor/staff, as only a few stages of insect rearing are automated. The safety risks (allergens, chemicals, and microbial hazards) of insect-fed animals are also increasing with the increasing demand for the inclusion of insect meals in the diet of aquatic animals [289]. The regulation of insect feed is also a critical issue for the insect-rearing industry. The regulations and guidelines regarding insect feed are different in different countries. For example, in several states of the United States, insect-based feed is allowed, while other states are waiting for FDA

permission. There is a lack of proper guidelines, regulations, and legislation regarding insect rearing and consumption [290] (Figure 3).

**Figure 3.** Challenges and solutions of insect-based meal in aquaculture industry.

#### **8. Possible Solutions to Challenges in Introducing the Insects in Aquafeed**

Chitin, which has a detrimental effect on the growth of fish, can be removed by supplementation with chitinase/chitinolytic-producing bacteria. This approach is very beneficial in inducing an immune response to fish and mitigating the environmental impact of fish waste [281]. Two major factors that can accelerate consumer acceptance and positive perception regarding the aquatic products produced through insect-based feed are the availability of information and product awareness [291,292]. To promote wider consumer acceptance, it is very important to reduce information asymmetry [292].

The environmental impact can be reduced by proper upscaling of insect production, and as a result, the insects can compete with conventional ingredients [293]. Environmental impact categories of *T. molitor* [46], *M. domestica* [294], and *H. illucens* [295] largely contribute to feeding produced from the insect-rearing industry. Therefore, providing a suitable substrate for insect rearing and increasing the efficiency of facilities will be major contributors to attaining insect meal's environmental benefits [293,296].

Another method for improving the amino acid content is defatting, but it requires intensive energy usage, increasing the environmental impact and feed cost [297]. It is better to add complementary raw material to insect meals or supplement the IM with deficient amino acids. Defatted mealworm and aquatic insect meals have higher arginine content than full-fat BSF diets [298,299]. As many AA compositions are found in insects, it is critical to analyze them before producing any insect-based feed [233,300]. More trials are needed to find a suitable feed to boost the omega-3 lipid content in insect meals, notably in HI, which tends to acclimate more easily to different diets [212,301].

Among the manufacturing techniques, extrusion can play a vital role in nutrition utilization [196]. The efficiency of extruded insect-based feed is also documented in recent studies [302,303]. Environmental impacts can be reduced by properly addressing feeding practices to minimize feed waste and FCR. Novel processing techniques are needed to address the safety concerns regarding insect feed consumption. It has been observed that various chemicals can be degraded if the insects are grown properly [289]. Preparing insect protein meal or dry pellets would save money and extend their shelf-life (relative to live food).

To optimize various stages of insect rearing, academic researchers and institutes collaborate with different EU companies to foster innovations. Studies have indicated that collaboration between academic media and entrepreneurs resulted in the development new innovative techniques. For example, a Dutch company has developed a centralized system for controlling oviposition in HI adult colonies with the help of olfactory triggers (web source). Larvae from the adult cages are transferred to a separate system by a newly developed automated device that allows counting larvae, dozing, and proper analysis. Breeding cages with pipes have also been developed to efficiently clean insect-rearing cages (web source). For promoting the insect business, there is a dire need to develop the proper legal framework to help the feed industry flourish fast (Figure 3).

#### **9. Future Perspective**

First, the insect industry must greatly increase its production capacity. Insect feed prices are currently too high. Fishmeal, high-quality SBM extracts, and soybean meal production volumes are thousands of times higher than insect meal protein and its byproducts. By expanding the production scale, insect producers can compete on price and product stability with other protein sources. Industrialization and controlled manufacturing technologies will help shareholders to scale up the industry by reducing labor-intensive insect production [304].

Further cost–benefit research will need to be conducted regularly to determine the economic effects of adding insects to animal feed to see how these alternative protein ingredients affect total production costs. Additional research is needed on insect meal to modify and improve the digestive tract of the fed animal. Multiple approaches, including histomorphology, molecular biology, and histochemistry, will be advised to assess the gastrointestinal tract health of insect-fed organisms.

It is crucial to clarify and comprehend the factors behind consumer concerns about farm animal welfare worldwide. First, one rationale is that customers give farm animal welfare a higher priority than other traits when evaluating the quality of food [305–307]. One key reason consumers buy animal-welfare-friendly items is a correlation between better human health benefits and farm animal wellbeing [308–310].

The bioconversion technique for insect production is one of the sustainable solutions to food security. In this sense, waste is a valuable resource for producing high-quality protein (insect meal) for the food system. As a result, the technology produces zero waste and lowers the need for costly protein sources such as soy meal and fishmeal in aquafeed. Utilization of organic wastes for rearing edible insects is an attractive approach that would facilitate the SDGs promoting female entrepreneurs. This approach will help build sustainable and smart areas with reduced greenhouse gas emissions, indirectly reducing the carbon footprints. The potential of the insect meal industry to meet the increasing demand of the fish feed industry is unclear and data deficient. Research on this topic is limited to tiny plots or cages with immature or juvenile fish. More research is needed to verify that insects can be produced effectively and efficiently to feed young and adult fish.

Moreover, the insect's requirement level for different fish species varies according to stages and culture systems, and more studies are needed to fill this gap. This gap needs to be filled to commercialize aquaculture insects. The long-term sustainability impact of insect rearing is unknown. The data are not available for the proper understanding of production aspects of potential insects and the requirements and risks of their accidental release, which opens a new avenue for further research. Some insects have the potential to replace fishmeal completely, such as the BSF. However, the species-specific threshold limits the complete substitution of fishmeal with insect meal. Therefore, it is very important to explore new fractionation, separation, and biorefining schemes to extract useful products from insects.

Recent studies have demonstrated that different parts of insect's meal, such as meal, oil, pulp, and paste, can be used in aquaculture. The most literature is available on insect meal and, to some extent, oil. However, the data regarding the utilization of pulp and paste are scarce, and this area of research needs to be addressed. More collaboration is needed between the feed industry, government, academia, and local farmers to explore new insect protein sources and build efficient, cost-effective, sustainable rearing systems.

#### **10. Conclusions**

The present review analyzed that insects have good potential to replace fishmeal due to their nutritional value. Among the non-conventional feed ingredients, insects have the biggest potential to replace conventional feed ingredients. Proper processing technologies can further improve the nutritional value of insects. Insect-based diets showed a positive physiological response in many fish species. Insects have great potential to use agro-industrial and plastic wastes, thus contributing to combating pollution-related environmental problems. Our review also highlighted the challenges or hurdles in using insects in aquafeed, and possible solutions to these challenges were also addressed. However, more studies need to be conducted to determine the required level for aquatic animals, which varies from species to species and with developmental stages. Most of the studies on replacing fishmeal with insect meal focus on juvenile stages, not adult ones. For this reason, this area still needs to be investigated. It is important to scale up insect farming at the industrial level with standard, cost-effective, and eco-friendly facilities and to develop suitable substrates for insects to deal with nutritional and environmental issues.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/fishes7060395/s1, Table S1: Patterns of amino acids (mg/g CP) of six commonly used

edible insect species. Sources [80,82,152–155]; Figure S1: The processing techniques of the insect'sbased feed.

**Author Contributions:** Conceptualization, W.M. and A.H; Writing-Original Draft Preparation, W.M., A.H., M.N., M.B., U.R., M.H., S.U.R. and N.R.; Writing-Review & Editing, W.M., A.H., M.N., M.B., U.R., M.H., S.U.R. and N.R.; Funding Acquisition, N.R. and W.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** All authors declare that they have no conflict of interest.

#### **References**


## *Review Lamiaceae* **as Feed Additives in Fish Aquaculture**

**Graziella Orso, Roberta Imperatore, Elena Coccia, Ghasem Ashouri and Marina Paolucci \***

Department of Science and Technologies (DST), University of Sannio, 82100 Benevento, Italy **\*** Correspondence: paolucci@unisannio.it

**Abstract:** The growing demand for high-quality food has induced a rapid expansion of the aquaculture sector. On the other hand, this sector has to overcome numerous challenges and problems triggered by the adoption of intensive farming systems, such as stress and high susceptibility to diseases. The improper use of chemicals and antibiotics has led to the development of antibiotic resistance in fish, with consequent health risks for consumers. Natural additives are increasingly used in aquaculture and, among these, medicinal plants are constantly under investigation as safe and environmentally friendly alternatives to chemicals. Great attention has been paid to *Lamiaceae* plants as feed additives capable of enhancing the growth performance, immune system, and antioxidant status of farmed fish. The aim of this review is to provide an updated picture of the employment of the *Lamiaceae* species (oregano, rosemary, sage, thyme, and mint) to enhance farmed fish health. The benefits of oregano, rosemary, sage, thyme, and mint feed supplementation on growth performance, immune system, antioxidant status, hemato-biochemical parameters, and resistance to stress, parasites, and bacteria have been described, highlighting weaknesses and drawbacks and proposing possible implementations.

**Keywords:** *Lamiaceae*; fish; health; growth performance; antioxidant; immunity; nutrition

#### **1. Introduction**

In recent decades, the aquaculture sector has shown rapid expansion in order to meet the food needs of the growing human population [1]. Aquaculture products represent an important source of high-quality animal proteins, as well as essential macro- and micronutrients. The growing demand for fish, both salty and freshwater, has prompted the aquaculture industry to adopt intensive and even ultra-intensive farming systems to increase productivity. However, intensive practices are responsible for numerous problems, such as poor water quality, overcrowding, high temperature, and poor nutrition, that contribute to lowering the growth performances of fish health and immune competence, with consequently increased stress and high susceptibility to diseases. Although aquaculture plays an important role today, it is a sector that must overcome numerous challenges that hinder its expansion, such as the spreading of infectious diseases, fish health problems, and consequent economic damage [2]. In recent years, disinfectants, chemotherapeutics and synthetic antibiotics have been used in order to prevent or mitigate the economic losses caused by diseases in farmed fish [3,4]. Unfortunately, as there are no antibiotics developed specifically for fish [4], veterinary or human antibiotics have been used, contributing to the onset of antibiotic-resistance [5]. Furthermore, the recurrent and uncontrolled use of antibiotics in farmed fish leads to the accumulation of residues of these substances in fish products, with consequent health risks for consumers [6].

Recently, researchers have paid great attention to identifying safe and environmentally friendly alternatives to antibiotics [7–9]. The use of natural additives capable of replacing pharmaceutical substances in intensive farming appears to have many potential benefits, including immunostimulation, the inhibition of pathogens in the intestinal tract, and the improvement of the absorption and utilization of nutrients [10,11]. Numerous studies have evaluated the effects of several natural feed additives, including probiotics [12],

**Citation:** Orso, G.; Imperatore, R.; Coccia, E.; Ashouri, G.; Paolucci, M. *Lamiaceae* as Feed Additives in Fish Aquaculture. *Fishes* **2022**, *7*, 349. https://doi.org/10.3390/ fishes7060349

Academic Editor: Francisco J. Moyano

Received: 27 October 2022 Accepted: 24 November 2022 Published: 26 November 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

prebiotics [13], synbiotics [14], functional amino acids [15], minerals [16], and additives of origin vegetable or phytochemicals [8,17–20].

Medicinal plants, which include herbs, spices, and their extracts, have been increasingly used in aquaculture due to their low cost and simple use. In farmed fish, medicinal plants promote a vast array of effects, including the improvement of growth [21], immunity [22], antimicrobial and anti-stress activities [23], as well as resistance against pathogens [24]. In general, the efficacy of medicinal plants and their derivatives (extracts, and essential oils) is closely related to the abundance of bioactive substances such as alkaloids, quinones, lectins, steroids, phenolic compounds, tannins, terpenoids, saponins and flavonoids [25]. Among the medicinal plants used as a feed supplement for fish, growing scientific interest is directed to aromatic plants, both as extracts and essential oils [26]. Many of these aromatic plants belong to the *Lauraceae*, *Umbelliferae*, *Myrtaceae* and *Lamiaceae* families. In particular, the plant species of the *Lamiaceae* family are among the most studied and are frequently used as feed additives in aquaculture [27]. The aim of this review is to furnish a general outlook of the main *Lamiaceae* species (oregano, rosemary, sage, thyme, and mint) on the health and pathogen resistance in farmed fish, with the aim of providing a picture as complete as possible of *Lamiaceae* employment in aquaculture, highlighting the weaknesses and drawbacks of their implementation and proposing possible alternatives.

#### **2.** *Lamiaceae* **Family**

*Lamiaceae* are distributed all over the world, although the best environmental conditions for their growth were found in the Mediterranean basin [28]. The *Lamiaceae* family includes 245 genera and approximately 8000 species [29]. Since ancient times, the dried herb, leaves, and essential oils of *Lamiaceae* plants have been used in humans to treat various respiratory diseases, rheumatoid arthritis, gastrointestinal disorders, and urinary tract infections [29]. Plants of the *Lamiaceae* family represent a natural, economical, sustainable, and safe source of feed integrators capable of enhancing the growth performance, immune system, and antioxidant status of farmed fish [30–32]. Such beneficial effects are attributable to the bioactive molecules present in *Lamiaceae* plants, such as terpenes, terpenoids, alkaloids, and flavonoids [2]. For example, the immunomodulatory properties of the *Lamiaceae* plants are mediated by the predominant terpenes, carvacrol, and thymol, which are capable of modulating inflammatory processes through the activation of ion channels, such as TRP (Transient Receptor Potential) channels, and consequently activate the NFkB pathway [26]. Moreover, carvacrol and thymol show strong antioxidant activity due to their ability to neutralize the oxygen free radicals (ROS) in tissues and cells [26].

*Lamiaceae*, as feed additives, can be administered in different forms, as a whole plant or parts (leaves or seeds), as active compounds extracted from the plant, and individually or as a combination of extracted compounds [2]. It should be emphasized that the efficacy of *Lamiaceae* plants as feed additives depends on several crucial factors such as dose, duration, time schedule of administration, and fish species [2]. In particular, the most important factor is represented by the dose which, if suitable, can induce beneficial effects, while if too low or too high, may induce either no response or even be toxic [11]. As reported in a meta-analysis study on fish diets enriched with plants, the dosages used in aquaculture vary according to the plant species used. The higher dosages are used with powdered plants (0.1–420 mg/100 g of fish × day), followed by ethanolic and aqueous extracts (0.2–160 mg/100 g of fish × day; 0.03–200 mg/100 g of fish × day, respectively), while the lower doses are used with essential oils (0.005–30 mg/100 g of fish × day) [21]. Thus, to improve the growth performances and health of a specific fish species, the challenge for researchers is to identify the optimal conditions in terms of the part of the plant to be used, doses, duration, and time schedule.

#### **3. Oregano**

Among the *Lamiaceae* family, the oregano (*Origanum vulgare* L.) is the most worldwide spread species, distributed throughout Eurasia and North Africa, and particularly abundant in the Mediterranean area [33]. The richness of the chemical composition and aromatic compounds of oregano have led to its use, since ancient times, in the pharmaceutical and cosmetic fields, as well as in the food industry as a flavoring substance [34]. The efficacy of oregano in the treatment of a wide range of human diseases has been reported in both in vitro and in vivo studies [35,36]. Furthermore, several studies have reported the growth and health-promoting role of oregano in farmed animals, primarily in terrestrial monogastric animals (poultry and pigs) [37,38] and fish (Table 1).

The biological activities attributed to oregano are related to its bioactive components, which include a wide variety of secondary metabolites, most of which are monoterpenes (carvacrol and thymol) and polyphenols (rosmarinic acid, luteolin and derivatives, chlorogenic acid, quercetin and derivatives, caffeic acid, hyperoside, rutin, *p*-coumaric, ferulic, carnosic, ursoli acids) [29,39].


**Table 1.** Studies of oregano products added to the feed of aquaculture species.


**Table 1.** *Cont*.

WG: Weight gain; FW: Final weight; FCR: Feed conversion ratio; SGR: Specific growth rate; PER: Protein efficiency ratio; BWI: Body weight index; RBC: Red blood cell count; WBC: White blood cell count; Hct: Hematocrit; Hb: Hemoglobin; LYS: Lysozyme; Ig: Immunoglobulin; IL-1β: Interleukin-1β; IL-10: Interleukin-10; TNFα: Tumor necrosis factor-α; TGFβ: Transforming growth factor-β; SOD: Superoxide dismutase; CAT: Catalase; GPx: Glutathione peroxidase; NS, non-significant effects.

#### *3.1. Oregano's Effects on Growth Performance*

Great attention has been paid by researchers to the use of oregano essential oil (OEO) in farmed fish (Table 1). OEO feed inclusion stimulates the growth performance of fish, primarily by improving the feed utilization rate and by acting on metabolic processes. Zhang et al. [43] reported that 0.15 and 0.45% of OEO supplementation, for 56 days, stimulated digestive enzymes in koi carp juveniles (*Cyprinus carpio*), increasing the activation of proteases, amylases and lipases. The same beneficial effects on intestinal enzymes have been reported for the hydroalcoholic extract of oregano (at a dose of 3% in 85 days feeding trial) in rainbow trout (*Oncorynchus mykiss*) [57]. In addition, OEO dietary supplementation may promote growth due to its beneficial effects on intestinal health. The inclusion of 1.5% of OEO in the diet for 60 days significantly improved growth performance and intestinal histomorphometry (villous height and width) in common carp fry [42]. Similarly, the addition of 0.05% of OEO to the diet of yellow-tailed (*Astyanax altiparanae)* for 90 days increased the absorption area of the intestine [40]. The study by Huley et al. [58] showed that the inclusion of different OEO concentrations (0.075, 0.15, 0.225, and 0.3%) in Nile tilapia (*Oreochromis niloticus*) juveniles for 64 days acted as a developmental stimulant of intestinal villi and, consequently, as a growth promoter.

The beneficial effects of OEO supplementation on growth performance are also, most likely, linked to the improvement of the gut microbial community [43]. Fish gut microbiota serves crucial functions in host health, growth, and development, aiding digestive functions and protecting against intestinal infections [59]. Dietary supplementation with the major monoterpenes of oregano (thymol and carvacrol) positively altered the gut microbiota of Nile tilapia [60], and resulted in improved nutrient digestibility and absorption, as well as protein conversion [50,61]. The OEO inhibited some pathogenic bacterial groups and increased commensal beneficial communities of *Corynebacterium*, *Brevinema*, and *Propionibacterium* in koi carp juveniles [43].

In contrast to the beneficial effects of OEO, Santo et al. [54] reported no significant improvement in growth performances and no significant alterations in intestinal villous height in Nile Tilapia juveniles fed with different percentages (0.025, 0.05, 0.075, 0.1, 0.125, and 0.15%) of dried oregano leaves for 30 days. Similarly, weight gain (WG) and specific

growth rate (SGR) did not significantly differ in seabream juveniles (*Sparus aurata*) fed with 0.5 and 1% oregano leaves powder for 15 or 30 days [56].

The hydroalcoholic oregano leaf extract also appeared to counteract the toxic effects of Diazinon, an organophosphate pesticide, on growth and liver metabolic enzymes (aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) in rainbow trout juveniles; in fact, doses between 0.2 and 1%, but not higher, of oregano hydroalcoholic extract dietary inclusion significantly increased the body weight index (BWI) and the SGR compared to the standard diet in a 60 day feeding trial [51].

Based on these results, the best forms of oregano feed supplement for fish to stimulate growth rate and feed conversion parameters are essential oils and hydroalcoholic extracts, while powdered oregano leaves have no beneficial effects. A possible explanation may reside in the similar percentage of bioactive constituents (carvacrol 63%; thymol 4.7%; ρ-cimene 12.8%; γ-terpinene 8.4%) in essential oils and hydroalcoholic extracts [40,54,62].

#### *3.2. Oregano's Effects on Oxidative Stress*

Oregano essential oil or hydroalcoholic extracts administered in the diet reduced the oxidative stress in different fish species, including common carp [41,43,44], rainbow trout [31,49,50,61], Nile tilapia [53,55], and catfish [63]. Oregano acted as an antioxidant activity enhancer, promoting the activities of serum and hepatic superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) enzymes accompanied by a reduction in malonaldehyde (MDA) levels [41,43].

The choice of the administered dose plays an important role in the antioxidant effect of oregano when used as a feed additive. For example, in rainbow trout juveniles, after a 60 day feeding trial with 0.6 and 1% doses of hydroalcoholic oregano leaf extract, the activity of antioxidant enzymes SOD, CAT, and glutathione peroxidase (GPX) increased, while high doses (1.4%) caused a decrease in their activities [50]. Similarly, the particular part of the plant being used appears to play an important role in determining its antioxidant effect. The use of 0.5 and 1% of oregano leaf powder for 30 days in sea bream juveniles, for example, did not cause any significant effects on liver antioxidant enzymes activities [56]; this is likely a result of the lower number of bioactive components with respect to the essential oil and the hydroalcholic extract. Further, the presence of the bioactive molecules within the vegetable matrix, as it occurs in the leaves, makes their extraction and absorption during the digestive processes difficult, with a consequent limited action. Indeed, the level of bioactive molecule uptake in the intestine represents only a limited percentage of the total quantity.

#### *3.3. Oregano's Effects on Immune Response*

The effects of dietary oregano supplementation on the immune status of farmed fish have been widely reported. The results of numerous studies carried out on rainbow trout [31,48,49], Nile tilapia [55], and koi carp [43] reported that both oregano essential oil and hydroalcoholic extract increased the non-specific immune response, mainly via improving lysozyme, protease and complement system activities. In comparison to mammals, the innate immune system represents a fundamental defense weapon in fish [64]. For example, lysozyme is capable of destroying the bacterial cell, by splitting the β-1,4 glycosidic bonds of the peptidoglycan, providing protection against fish pathogens [65]. The use of 0.02% of hydroalcoholic oregano (*O. majorana*) leaf extract for 56 days enhanced the activity of lysozyme in common carp juveniles [44]. In another study, dietary integration of 0.1% of OEO in red-bellied tilapia (*Tilapia zillii*) improved lysozyme activity levels, accompanied by the increase in proteases, antiproteases, and bactericidal activities [66].

The supplementation with immunostimulants in fish diets also beneficially improved the expression of specific immune elements, such as IgM [56] and pro-inflammatory cytokyne Interleukin-1β (IL-1β) [66]. In particular, in sea bream juveniles, supplementation with oregano leaf powder at 0.5 and 1% for 30 days improved both the innate (complement system and antibacterial activity) and adaptive (IgM) responses of skin mucus immunity

compared to the control group, while the oregano leaf powder integration did not alter the humoral immune response in the serum [56]. From this difference in the results, authors have suggested that the immune defense against pathogens resides in the antibody response of the skin mucus, which increases proportionally with the concentration of oregano leaf powder in the diet (resulting highest at the dose of 1%).

#### *3.4. Oregano's Effects on Hemato-Biochemical Parameters*

Hemato-biochemical parameters are reliable biomarkers of the health and immunity conditions of farmed fish species [67]. The incorporation of hydroalcoholic extract of the oregano aerial part into fish feed had no effect on red and white blood cell counts (RBC and WBC, respectively), leukocytes count (monocyte, lymphocyte and neutrophile), hematocrit (Hct), and hemoglobin (Hb) in rainbow trout juveniles treated with a dose of 1% [48,49] and in Nile tilapia treated with 0.2 and 0.5% [55]. On the contrary, the hematological parameters were significantly enhanced in red-bellied tilapia fed with 0.1% of OEO for 15 days [66]. Similar augmentation of RBC, WBC, thrombocytes, and hemoglobin was recorded in common carp juveniles fed a diet containing 0.02% of oregano (*O. majorana*) leaf hydroalcoholic extract for 56 days [44]. It has been suggested that the increase in the hematological parameters RBC, Hct, and Hb, may favor the tissue oxygenation and the elimination of carbon dioxide, contributing to growth [68]. Moreover, homeostasis, or the increase in such hematological parameters, indicates that the oregano supplementation had no negative effects on erythrocytes production and the destruction of mature RBC, therefore indicating that it is non-toxic [69]. Serum biochemical parameters, such as total protein, albumin, and globulin values, were enhanced by hydroalcoholic oregano leaf extract, added to the diet at the dose of 1% in rainbow trout juveniles [48,49]. Similarly, in sea bass (*Dicentrarchus labrax*) juveniles fed with 0.01% of OEO for 150 days, the improvement of total protein, glucose, triglycerides, and cholesterol occurred [47].

#### *3.5. Oregano's Effects against Pathogen Infections*

Several studies have revealed that the use of OEO, in addition to increasing growth and feed utilization, improves resistance to pathogens in common carp [41,43,44], channel catfish (*Ictalurus punctatus*) [63], zebrafish (*Danio rerio*) [45], Nile Tilapia [55], rainbow trout [31], and red-bellied tilapia [66]. Carvacrol and thymol, the most abundant phenolic components, are likely responsible for the antimicrobial activities of oregano, being able to alter the bacterial outer membrane and consequently its permeability [39]. Carvacrol, in particular, is involved in the disintegration of bacterial cells by altering the synthesis and mobility of the flagella, the fatty acid composition of the membranes, membrane proteins, and periplasmic enzymes [55,70]. The flavonoids and terpenoids contained in the oregano also contribute to the antimicrobial power, as demonstrated by the terpenoids, ρ-cymene [70].

#### *3.6. Conclusions*

Based on the literature, it appears that the best dietary supplement is represented by oregano's essential oil and hydroalcoholic leaf extract. It should be noted that fish fed the diet supplemented with oregano showed improved growth performance, immunological parameters, and antioxidant status in a dose-dependent manner and that an excessive amount of oregano could cause immunosuppression and toxicity. In this regard, on the basis of the results examined, it can be stated that it is convenient to use oregano as a feed additive for fish in the form of essential oil and hydroalcoholic extract in a concentration ranging between 0.5 and 1%, for a minimum duration of 8 weeks.

#### **4. Rosemary**

Rosemary (*Rosmarinus officinalis* L.) is a small evergreen medicinal herb, widespread in the Mediterranean region. It is widely used for farmed animals' nutrition. Both in vitro and in vivo studies have shown that rosemary-based food supplements improve oxidative stress and immune responses [71]. In particular, rosemary extract possesses antiinflammatory, anticancer, antidiabetic, hepato- and blood-protective activities [72,73]. Among *Lamiaceae* medicinal plants, rosemary presents the least chemical composition variability. Rosemary is primarily composed of terpenes β- and α-pinene, camphene, camphor, and limonene [74]. Many other compounds are also extracted from rosemary, such as polyphenols and steroids [71,75]. Among polyphenols, the most abundant are rosmarinic acid, 7-methylrosmanol naringin, and also, at lower concentrations, rutin, and ferulic acid [76]. Although there is numerous evidence to show the beneficial effects of rosemary as a feed additive in terrestrial animals, its application in aquaculture is still scarce. However, significant interest in the use of rosemary in aquatic animals has recently increased due to its efficacy as a stimulant of growth, the immune system, and health status [72,77].

#### *4.1. Rosemary's Effects on Growth Performance*

Several studies have confirmed that the oral administration of rosemary could enhance growth performances in farmed fish, such as common carp [72,77], Nile tilapia [78–80], and sea bream [81] (Table 2). Among the rosemary-based products, rosemary leaf powder is the most commonly investigated as a fish feed additive. In common carp fingerlings, different doses (1, 2, and 3%) of rosemary leaf powder positively increased, in a dose-dependent manner, the growth performances and feed conversion parameters (WG, SGR, final weight (FG), feed conversion ratio (FCR) levels) after a trial of 65 days [72].


**Table 2.** Studies of rosemary products added to the feed of aquaculture species.

WG: Weight gain; FCR: Feed conversion ratio; SGR: Specific growth rate; PER: Protein efficiency ratio; RBC: Red blood cell count; WBC: White blood cell count; Hct: Hematocrit; Hb: Hemoglobin; LYS: Lysozyme; Ig: Immunoglobulin; SOD: Superoxide dismutase; CAT: Catalase; NS, non-significant effects.

The same findings with rosemary leaf powder supplementation were also obtained in Nile tilapia fingerlings [78,80]; in particular, Naiel et al. [80] recorded better growth performance in fish fed on 0.5 and 1% of rosemary leaf powder for 60 days. Similarly, in a 65-day feeding trial, common carp juveniles fed on hydroalcoholic rosemary leaf extract (0.01, 0.25, 0.5, and 1%) showed an increase in growth performances [77]. Various studies have shown that herbal plants not only improved fish growth and nutrition, but also enhanced appetite and modified the gut microbiota composition, increasing the diversity and activity of the beneficial bacteria, while inhibiting pathogenic bacteria [2,75,82]. In agreement with these findings, rosemary leaf powder also showed a positive role in controlling nutrient uptake and enhancing the intestinal mucosal condition in rats ([83]. On the contrary, in Nile tilapia juveniles fed 90-day diets with different amounts (0.1, 0.25, and 0.5%) of commercial rosemary extract, Yilmaz et al. [79] did not report significant changes in growth performances. In addition, in gilthead seabream, growth performances and feed intake were not modified by the inclusion of different doses (0.06, 0.12, 0.18, 0.24%) of commercial rosemary extract for 84 days [81]. Such differences could be attributed to different fish species, feeding trial length, source and rosemary doses. In this regard, it is necessary to emphasize that, in the experiment conducted by Hernández et al. [81], a commercial rosemary extract powder made of a blend at the ratio 1:1 of two diterpenes (carnosic acid and carnosol) was used. Similarly, Yilmaz et al. [79] used a commercial rosemary extract composed of rosmarinic acid at 5.32%. Therefore, the lack of results may be associated with the small amount of the chemical active principles in the feed additive used. In contrast to the inclusion of powder or fresh leaf extract, it is also interesting to underline that rosemary oil did not result in an increase in growth performance, as well as growth rate (GR) and FCR in sturgeon juveniles (*Huso huso*) [84] and seabass [85].

#### *4.2. Rosemary's Effects on Oxidative Stress*

The beneficial effects of rosemary dietary-inclusion also resulted in the improvement of the antioxidant status in common carp [72] and in Nile tilapia [80]. Rosemary leaf powder supplementation at the doses of 0.5 and 1% in the diet of Nile tilapia fingerlings for 60 days significantly improved the antioxidant status via an increase in CAT activity [80]. Similarly, in a 65-day feeding trial in common carp juveniles, different doses (1, 2, and 3%) of rosemary leaf powder induced an increase, in a dose-dependent manner, of blood CAT activity, but the higher dose (3%) led to a decrease in blood SOD activity [86].

The effect of powdered rosemary leaves as antioxidant defense enhancers could be linked to its several beneficial compounds, such as rosmarinic and carnosic acids [76].

#### *4.3. Rosemary's Effects on Immune Response*

Dietary supplement with rosemary products showed an enhancement of the immune system in fish. The elevation of total immunoglobulin (Ig) levels, lysozyme and alternative complement activities of common carp juveniles fed on diets containing rosemary leaf powder in various doses (1, 2, and 3%), for 65 days, was reported [72]. The findings of Dezfoulnejad and Molayemraftar [77] confirmed the potential of oral administration of hydroalcoholic rosemary leaf extract as a stimulatory agent of the non-specific immune system in common carp juveniles. Similarly, in tilapia (*O. mossambicus*) fingerlings, the inclusion of 0.25 and 0.5% hydroalcoholic rosemary extracts for 60 days led to an improvement in the principal non-specific immunity elements (lysozyme, immunoglobulin and alternative complement) [87]. In addition, in Nile tilapia fingerlings, the oral administration of 1% of rosemary leaf powder for 60 days induced a significant increase in the expression of the immune genes related to innate and adaptive immune response, such as lysozyme, complement and immunoglobulin M (IgM) [80].

#### *4.4. Rosemary's Effects on Hemato-Biochemical Parameters*

It has been reported that rosemary bioactive compounds, such as rosmarinic acid, could positively affect thymus and spleen activities, leading to a significant increase in the WBC counts (lymphocytes T and B, monocytes and neutrophils) [88]. In fact, after 65 days of oral administration of 2 and 3% of rosemary leaf powder, WBC markedly increased in common carp juveniles [72]. Similarly, tilapia fingerlings treated with 1% of rosemary leaf powder showed a significant increase in both haematological (WBC, haematocrit and leukocrit levels) and serum biochemical (total protein, albumin and globulin levels) parameters [78]. Serum biochemical parameters are good fish health indicators [68]. Several studies have suggested the possible correlation between enhanced fish growth performance and the simultaneous increase in total protein, albumin and globulin levels due to dietary herbal inclusion [68,89]. Findings on the oral supplementation of rosemary in common carp [72,77] and in Nile tilapia [78,80] confirmed the hypothesis of the combination effects of health and growth performance in fish treated with herbal supplementation. Moreover, several vitamins (A, B, and C) and minerals (K, Ca, and Fe) present in significant quantities in rosemary could positively modulate other blood biochemical parameters due to their hypocholesterolaemic effects [90]. For example, the levels of triglycerides and LDL (low-density lipoprotein cholesterol) diminished, while HDL (high-density lipoprotein cholesterol) augmented in common carp juveniles fed on hydroalcoholic extract of rosemary at 1% in a 65-day feeding trial [77].

#### *4.5. Rosemary's Effects against Pathogen's Infections*

Some studies have evaluated the effects of rosemary as an alternative antimicrobial agent in aquaculture. The dietary application of dried rosemary leaves for 20 days improved the resistance against *Streptococcus iniae* at the 8% dose and against *Streptococcus agalactiae* at 16% dose in tilapia fingerlings [91]. Similarly, the 60-day feeding supplementation of 1% of rosemary leaf powder provided adequate protection to Nile tilapia fingerlings against the infection of *Aeromonas hydrophila* [80]. Numerous in vitro studies demonstrated that rosemary possessed antibacterial properties against Gram-positive and Gram-negative bacteria, mainly linked to its composition in phenolic compounds [92,93].

#### *4.6. Conclusions*

Based on the reported literature, both leaf powder and extract of rosemary positively affect growth performance, antioxidant status, and the general health of farmed fish. In order to improve the haemato-biochemical and non-specific immune parameters and increase the resistance against bacterial diseases, a dosage of 1% of rosemary extract or leaf powder and 60 days of administration can be recommended as useful fish feed additives.

#### **5. Sage**

Sage is the largest genus of *Lamiaceae* and includes approximately 900 species, among which *Salvia officinalis* is globally widespread and highly considered for its medical relevance [29]. In fact, *Salvia officinalis* is cultivated in numerous countries and its dried leaves are used as raw material in medicine, the food industry, and perfumery [94]. It has been found that the essential oil and leaf extract of *Salvia officinalis* have strong antimicrobial and antioxidant effects, and also exhibit immunomodulatory and anti-inflammatory activities [29]. These beneficial effects may be due to the particular chemical constituents of sage, such as tannic acid, oleic acid, carnosol and carnosic acid and some polyphenols, such as caffeic acid, p-coumaric acid, rutin, rosmarinic acid, quercetin, luteolin, and apigenin [29]. Other compounds, such as monoterpenes and terpenoids, including 1.8-cineole, α-thujone, β-thujone, β-pinene, and camphor, are present in sage [74]; α-Thujone is a neurotoxic monoterpene ketone whose amount may vary according to the harvesting time, being high after flowering and low before flowering [95]. Therefore, an important parameter that must be considered is the variation of the chemical composition of medicinal plants. This variation, in fact, could influence the biological properties of the herb.

#### *5.1. Sage's Effects on Growth Performance*

As reported for *O. vulgare* L. and *R. officinalis* L., *Salvia officinalis* has also been studied in several experiments in farmed fish [30,96,97] (Table 3). One study by Sönmez et al. [30] reported the positive effects of a 60-day dietary supplementation of sage oil (0.05, 0.1, and 0.15%) on growth performance and parameters such as SGR and FCR in rainbow

trout juveniles. The same results were shown in beluga after 42 days of dietary inclusion of sage ethanolic extract (3, 6, and 12%) [97]. This growth-promoting action could be partially attributed to the sage polyphenolic compounds, such as ursolic acid, a pentacyclic triterpenoid carboxylic acid, which induces muscular hypertrophy in rainbow trout [98]. An increase in growth performance was also reported in gilthead seabream juveniles fed for 92 days with 0.01% of a combined extract of sage and lemon verbena (*Lippia citriodora*) leaf [96].


**Table 3.** Studies of sage products added to the feed of aquaculture species.

WG: Weight gain; FW: Final weight; FCR: Feed conversion ratio; SGR: Specific growth rate; BWI: Body weight index; RBC: Red blood cell count; WBC: White blood cell count; Hct: Hematocrit; Hb: Hemoglobin; LYS: Lysozyme; Ig: Immunoglobulin; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; IL-1β: Interleukin-1β; IL-10: Interleukin-10; TNFα: Tumor necrosis factor-α; TGFβ: Transforming growth factor-β; SOD: Superoxide dismutase; CAT: Catalase; GPx: Glutathione peroxidase; G6PD: glucose-6-phosphate dehydrogenase; NS, nonsignificant effects.

#### *5.2. Sage's Effects on Oxidative Stress*

The dietary inclusion of sage protects against reactive oxygen species (ROS) by stimulating the antioxidant defenses in farmed fish [30,96]. In rainbow trout juveniles, different concentrations (0.05, 0.1, and 0.15%) of sage oil added to the diet for 30 days significantly increased liver enzyme SOD, glucose-6-phosphate dehydrogenase (G6PD) and glutathione peroxidase (GPx) activities, while an extension of the feeding trial to 60 days induced a reduction in the antioxidant enzymes activities [30]. A positive modulation on the antioxidant defense system was also reported in gilthead seabream [96]. The findings of Salomón et al. [96] have shown that a 92-day administration of 0.1% dietary additives made of sage and lemon verbena hydroethanolic leaf extract stimulated SOD and CAT gene expression in gilthead seabream fingerlings. According to the authors, the up-regulation of SOD and CAT genes could be linked to the triterpenic and polyphenolic compounds, mainly ursolic acid, present in the sage [96].

#### *5.3. Sage's Effects on Immune Response*

Great attention has been given to the utilization of the dietary inclusion of sage to fortify innate immunity in farmed fish. In beluga juveniles, the immunomodulation through the oral administration of sage ethanolic extract for 42 days (3, 6, and 12%) enhanced lysozyme and alternative complement activities, and serum immunoglobulin levels [97]. In addition, in rainbow trout juveniles, 30 days of dietary supplementation of 0.5, 1, and 1.5% of hydroethanolic extracts of sage positively affected the immune system indices (lysozyme and complement activities and total immunoglobulin levels) in a dose-dependent manner [5].

In fish, the immunomodulatory properties of the dietary supplementation of sage combined with other medicinal herbs have also been demonstrated. After a feeding trial of 28 days, the combination of sage and Spirulina platensis (*Arthrospira platensis*) increased the non-specific (lysozyme, IgM and complement) and specific (IL-1β and TNFα cytokines) immune response in Nile tilapia juveniles [99]. In sea bream fingerlings, the dietary administration of 0.1% sage and verbena hydroethanolic leaf extract stimulated the expression of lysozyme, IgM, Il-1β and TNFα, and also increased the anti-inflammatory cytokines TGF-1β and IL-10 levels [96].

#### *5.4. Sage's Effects on Hemato-Biochemical Parameters*

The dietary inclusion of sage leads to the improvement of the hemato-biochemical parameters in beluga [97] and seabream [96]. Sage ethanolic extract (3, 6, and 12%), administered for 42 days, stimulated RBC, Hct, Hb, total protein, albumin, and globulin levels in beluga juveniles [97]. Moreover, Dadras et al. [97] reported that the dietary inclusion of sage ethanolic extract decreased the serum ALT and AST levels, supporting the beneficial effect of sage on the physiological status of fish. In fact, AST and ALT enzyme activities are used as stress indicators and the increase in their blood levels indicates liver impairment and hepatocellular damage [69].

#### *5.5. Sage's Effects against Pathogen's Infections*

The positive impact of the dietary inclusion of 0.5, 1, and 1.5% hydroethanolic extracts of sage for 30 days on the non-specific and specific immune responses led to an increase in rainbow trout juveniles' resistance against infection with *S. iniae* [5]. The 28-day dietary treatment with sage leaf inclusion protected Nile tilapia juveniles against infection with *Pseudomonas aeruginosa*, causing a significant elevation of the expression of lysozyme, IgM, and pro-inflammatory cytokines (IL-1β and TNFα) [99].

#### *5.6. Conclusions*

Based on the reported literature, it could be concluded that the dietary inclusion of sage can improve immune response, antioxidant system activity and stimulate feed intake, leading to enhanced growth performance. The feed incorporation of sage extract at a dosage of between 6–12%, for 42 days, shows important immunomodulatory properties. Regarding the use of sage essential oil, the optimal dose seems to be at 0.05%, with a duration of feed supplementation between 30 and 60 days. In addition, the combination of leaf extracts from sage and other medicinal plants added at low concentrations (0.1%) in the fish diet for long periods (>90 days) could be useful for its beneficial effects in aquafeeds. However, further studies are needed to understand doses and timing of administration in order to optimize the beneficial effects of using sage as a fish feed additive.

#### **6. Thyme**

Among the *Lamiaceae* family, the use of the aromatic plant thyme (*Thymus vulgaris*) is common in traditional medicine, food, as well as the pharmaceutical and cosmetic industries [29]. Fresh or dry thyme leaves can be used, and the essential oil can be extracted from flowers. Thyme possesses antiseptic, antinflammatory, antimicrobial and antioxidative properties [100]. Thyme is characterized by well recognized and documented in vitro antibacterial potential [101,102], showing that both thyme extract and essential oil have strong activity against *Escherichia coli*, *Staphylococcus aureus*, *Citrobacter freundii*, *P. aeruginosa*, *Proteus mirabilis, Proteus vulgaris* and *Salmonella typhimurium*. Thyme is rich in monoterpenes such as ρ-cymene, γ-terpinene, carvacrol, and thymol. The concentrations of these four main compounds remain very stable in plants harvested in different seasons, suggesting

that they are the compounds that functionally and biologically support the plant [103]. Thyme is also rich in polyphenols, including p-hydroxybenzoic acid, caffeic acid, rosmarinic acid, catechin, luteolin, apigenin, and quercetin [29]. The observed activities of thyme can be ascribed, in particular, to the presence of the caffeic and rosmarinic acids, quercetin and luteolin [100]. Thyme is an immunostimulator and growth promoter in poultry and swine farming; however, knowledge concerning its efficacy in aquatic species is limited to a few studies reporting that the dietary inclusion of thyme was effective in growth stimulation, immune responses, disease resistance and antioxidant enzyme profile in different farmed fish, including Nile tilapia, rainbow trout, and common carp [78,104,105] (Table 4).


**Table 4.** Studies of thyme products added to the feed of aquaculture species.

WG: Weight gain; FW: Final weight; FCR: Feed conversion ratio; SGR: Specific growth rate; PER: Protein efficiency ratio; RBC: Red blood cell count; WBC: White blood cell count; Hct: Hematocrit; Hb: Hemoglobin; LYS: Lysozyme; Ig: Immunoglobulin; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; ALP: Alkaline phosphatase activity; IL-1β: Interleukin-1β; IL-8: Interleukin-8; TNFα: Tumor necrosis factor-α; TGFβ: Transforming growth factor-β; SOD: Superoxide dismutase; CAT: Catalase; GPx: Glutathione peroxidase; GR: Glutathione reductase; NS, non-significant effects.

#### *6.1. Thyme's Effects on Growth Performance*

Several studies have investigated the effect of dietary thyme inclusion on fish growth parameters [78,104–107]. These scientific findings have shown that thyme does not possess adverse or toxic effects and is able to maintain the physiological conditions of the alimentary tract in fish [32,108]. As for the other herbal products, the optimal concentration of thyme is a critically important factor. Rainbow trout juveniles fed on 0.05, 0.1, and 0.2% of thyme essential oil for 60 days showed the best growth performance and parameters (weight gain, SGR, and feed intake) with the dose of 0.05% [30,106]. In common carp fingerlings, the dietary administration of 1.5% of thyme leaf led to the improvement of growth performances after a 56-day feeding trial when compared to the other experimental dietary concentrations tested (0.5, 1, and 2%) [104]. In sturgeon (*Acipenser stellatus*) juveniles, 58 days of feed thyme application improved fish growth at the concentration of 2% [109] compared to the 1% inclusion dose [110].

#### *6.2. Thyme's Effects on Oxidative Stress*

The positive role of thyme in enhancing antioxidant capacity has been demonstrated in rainbow trout juveniles [30,105]. For example, 0.05 and 0.1% of thyme essential oil supplementation provided enhanced antioxidant protection, improving liver CAT, SOD, GPx, and glutathione reductase (GR) activities and decreasing MDA production after 30 days of the feeding trial [30]. Thyme essential oil or water extract could successfully mitigate oxidative stress, likely due to their high concentrations of thymol and carvacrol [32]. The antioxidant effects of thymol and its isomer carvacrol have been well documented in several in vitro and in vivo studies, including cell lines [111] and animal models, such as weaning piglets [112].

#### *6.3. Thyme's Effects on Immune Response*

Several studies have been carried out to understand the immunomodulatory effects of thyme in fish. Thyme dietary inclusion is capable of stimulating the non-specific immune response in rainbow trout, including lysozyme, alternative complement and total immunoglobulin levels [105,106]. Furthermore, dietary 1% of thyme essential oil counteracted the negative effects on immunity and intestinal inflammation induced by aflatoxin B1 in rainbow trout juveniles, significantly lowering the expression levels of TNFα, IL-8 and TGF-β [107]. The immunomodulatory effects of thyme are linked to its major bioactive components, such as carvacrol, thymol, eugenol, and cymene [106]. Thymol feed supplementation, for example, improved the immunoglobulin levels in broiler chickens [113] and in pigs' guts [114].

On the contrary, the feeding inclusion of 0.1, 0.5, and 1% of thyme essential oil for a short period (15 days) did not alter respiratory burst activity, lysozyme concentration, or alternative complement activity in Nile tilapia juveniles [108]. These results confirm the importance of the optimal choice of the duration of immunostimulant administration.

#### *6.4. Thyme's Effects on Hemato-Biochemical Parameters*

In farmed fish, the increase in blood parameters (Hb, RBC, and WBC counts) and the improvement of biochemical profile (total protein, albumin and globulin levels) suggest that the dietary inclusion of thyme products are safe feed additives able to enhance fish health and welfare. In Nile tilapia juveniles, 0.1, 0.5, and 1% of thyme essential oil for 15 days led to a significant increase in total leukocytes (monocytes, neutrophils, basophils and lymphocytes), especially at the highest dose (1%) [108]. The safety of thyme as a fish feed additive is also confirmed by the absence of negative or toxic effects on ALT and AST levels [106,108]. For example, the inclusion of up to 0.2% of thyme oils over 2 months did not alter the activity of these enzymes in rainbow trout juveniles, suggesting that thyme oils at up to 0.2% in feed can be considered as a safe additive for trout [106].

#### *6.5. Thyme's Effects against Pathogen's Infections*

Thyme also improves fish disease resistance against several bacteria and fungi, such as *Saprolegnia* spp. [104], *A. hydrophila* [106], *Yersinia ruckeri* [115], and *S. iniae* [116]. The efficacy of thyme essential oil or leaf powder could be a consequence of the increasing levels of the main immunity factors (lysozyme, alternative complement, immunoglobulin and cytokynes) and hemato-biochemical parameters. Feed supplementation of 0.05% thyme essential oil improved the resistance of rainbow trout juveniles against motile *Aeromonas* septicemia caused by *A. hydrophila* via the upregulation of the C3 and CD4 immune genes and the increase in IL-1β cytokine gene expression [106]. In fish, CD4 T helper cells provide a protective response against bacteria, fungi, and protozoa and C3 protein is crucial for the activation of both classical and alternative complement pathways [117].

#### *6.6. Conclusions*

Based on the reported literature, it can be observed that periods of 60 days of feeding supplementation with 0.5-1% of thyme essential oil can be considered a proper length of time and percentage to stimulate the cellular components of the non-specific immune response, enhance the growth performance and disease resistance against pathogens. On the contrary, short-term supplementation (such as 15 days) of 1% of thyme essential oil does not show beneficial effects. Regarding the use of thyme leaf powder, the feeding supplementation dose of 1% shows positive effects even in very long administration periods (140 days).

#### **7. Mint**

Another aromatic plant belonging to the family *Laminaceae* that captured the attention of researchers for its use in aquaculture is mint, also known as mentha or peppermint (*Mentha piperita*). Mint is a perennial herbaceous plant and is widely cultivated [118]. Peppermint is a crucial medicinal and aromatic plant, used in food since ancient times, and more recently in sanitary and cosmetic industries [119]. Several studies have confirmed its antimicrobial, antioxidant, and immunomodulatory effects [118]. The beneficial activities of mint, especially its antimicrobial effect, are due to its major compounds, such as menthol (33.8%), menthone (15.8%) and pulegone (8.3%) [119,120]. Used in perfumery and aromatherapy, pulegone and menthol are potentially toxic compounds when administered in large amount, causing liver damage in rats [121]. On the contrary, menthone has a digestive favoring effect and is non-toxic [120]. Mint also presents a high polyphenolic content (19–23%), primarily characterized by rosmarinic acid, luteolin, hesperidin and apigenin [122].

#### *7.1. Mint's Effects on Growth Performance*

The incorporation of mint into the diets of fish showed positive growth- stimulating effects, improving GR, WGR, and FCR in several fish species, such as Asian sea bass (*L. calcarifer*) [123], Nile tilapia [124], Caspian brown trout (*Salmo trutta caspius*) [125], and Caspian white fish (*Rutilus frisii kutum*) [126]. In Caspian white fish juveniles fed with 1, 2, and 3% of peppermint hydroalcoholic extracts for eight weeks, the growth parameters increased in a dose-dependent manner [126]. Mint could be considered as an appetite activator that significantly increases the daily feed intake [123]. Furthermore, the beneficial effect of mint feed inclusion on growth parameters could be attributed to its influence on intestinal enzymes (amylase and protease) and microbiota, leading to an improvement of the digestibility and availability of nutrients [126]. Interestingly, after a 60-day feeding trial, the dietary inclusion of 0.01 and 0.025% of mint essential oil enhanced intestinal health and increased the length of the intestinal villi [127]. On the contrary, the dietary supplementation, for 50 days, of mint essential oil at 0.075, 0.125, and 0.25% did not cause significant differences in growth compared to the control group in Nile tilapia fingerlings [120]. The possible explanation for this lack of beneficial effects on growth performance may be caused by small quantities employed in the study as the effect of mint is dose-dependent [126,128].

#### *7.2. Mint's Effects on Oxidative Stress*

Mint as a feed additive is effective in improving oxidative stress induced by the main environmental stressors, such as the water pollutants ammonia [129] and pesticides [124]. Nile tilapia juveniles fed on 0.25% of mint essential oil for 30 days displayed enhanced CAT, SOD, and GPx gene expression levels, allowing a reduction in the oxidative stress induced by pesticides toxicity exposure [124]. Similarly, oral administration of menthol at 0.25% improved the antioxidative status in common carp juveniles, mitigating the ammoniainduced alterations on antioxidant enzymes activities [129]. The antioxidant effects of mint are mainly attributable to the monoterpenic ketones mentone and isomentone [118].

#### *7.3. Mint's Effects on Immune Response*

Recently, great attention has been given to the immunostimulating effect of mint on different fish species, including rainbow trout [130,131], tilapia [120,127], common carp [132], sea bass [133], Caspian brown trout [125], and Caspian kutum [126] (Table 5).


**Table 5.** Studies of mint products added to the feed of aquaculture species.

WG: Weight gain; FW: Final weight; FCR: Feed conversion ratio; SGR: Specific growth rate; RBC: Red blood cell count; WBC: White blood cell count; Hct: Hematocrit; Hb: Hemoglobin; LYS: Lysozyme; Ig: Immunoglobulin; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; ALP: Alkaline phosphatase activity; SOD: Superoxide dismutase; CAT: Catalase; GPx: Glutathione peroxidase; GR: Glutathione reductase; G6PD: glucose-6 phosphate dehydrogenase; NS, non-significant effects.

A four-week period of dietary supplementation of horsemint (*Mentha longifolia*) hydroalcoholic extract at 0.1, 0.2, and 0.3% improved the non-specific immunity response (lysozyme and complement) and immune-related genes (TNFα) in rainbow trout juveniles, especially at the dose of 0.3% [131]. Similarly, in the same fish species, the improvement of lysozyme activity in a dose-dependent manner was reported after a 56-day feeding trial with 1, 2, and 3% of mint hydroalcoholic extract [130]. The enhancement of the immune system was also observed in juveniles of Caspian brown trout [125] and Caspian kutum [126]. Results of both studies showed that 56 days of dietary inclusion of 1, 2, and 3% of dried mint powder improved the immunological parameters in a dose-dependent manner. On the contrary, in Nile tilapia fingerlings, 60 days of feeding with 0.01 and 0.025% of mint

essential oil did not alter the lysozyme levels, while the activation of the complement system was significantly increased, especially at the concentration of 0.025% [127].

#### *7.4. Mint's Effects on Hemato-Biochemical Parameters*

The improvement of the haematological and biochemical parameters due to the dietary inclusion of mint dried leaf, essential oil and hydroalcoholic extract were also observed [123–126,130]. Dietary administration, for eight weeks, of mint hydroalcoholic extract at 3% improved the RBC, Hb, and WBC levels in the rainbow trout juveniles [130], Caspian brown trout [125], and Caspian white fish [126]. Mint is rich in vitamins, such as vitamins A, C, and E, and in mineral salts, such as iron, potassium, and calcium [119]. Some studies have suggested that mint used as food additive favors the intestinal absorption of iron and vitamins, increases hematopoiesis and, consequently, the hematological indices [134,135]. Moreover, mint-integrated diets enhanced serum biochemical parameters, resulting in the reduction in serum glucose, lipids, triglycerides and cholesterol levels, and in an increase in total protein, albumin and globulin levels [123,127,130].

#### *7.5. Mint's Effects against Pathogen's Infections*

Several studies have revealed the efficacy of mint dried leaf, essential oil and hydroalcoholic extract on the protection against *S. agalactiae* [120], *Vibrio harveyi* [123], and *Y. ruckeri* [130]. In rainbow trout juveniles, the oral supplementation of 1, 2, and 3% of mint hydroalcoholic extract for 56 days significantly enhanced serum bactericidal and antiprotease activity, protecting from infections and giving resistance to the pathogens [130]. Moreover, the oral supplementation of mint essential oil or hydroalcoholic extract increased fish survival in experimental challenge tests, modulated haematological RBC and WBC counts, parameters of non-specific and specific immunity (lysozyme and complement activities and Ig levels) and increased cytokines expression (TNFα, IL-1β, IL-8) [120,130,131].

#### *7.6. Conclusions*

Based on the reported literature, the fish dietary incorporation of mint is able to improve the haematological and immune response parameters and provide resistance against pathogenic infections in a dose-dependent manner. Mint hydroalcoholic extracts should be used in fish feed at a concentration range of between 2–3% for a duration of 56 days. Regarding the use of mint essential oils as a fish feed additive, low doses (0.075–0.125%) of feed supplementation shows benefits to intestinal health and immune response, while higher doses (0.25%) are necessary to stimulate growth and improve haematological parameters.

#### **8. General Conclusions and Future Perspectives**

This review summarizes the findings regarding the role of the species of *Lamiaceae* family as feed additives in aquaculture. According to studies conducted with medical herbs, oregano, rosemary, sage, thyme, and mint (whole plant, extract or essential oil) have the potential for use as safe additives in fish feed, showing benefits on growth performance, immune system, antioxidant status, hemato-biochemical parameters and resistance to stress, parasites and bacteria.

Considering the scientific literature reported in this review, it is possible to indicate that great importance must be given to the choice of suitable dosage and administration time to obtain positive effects on fish health. A specific dose may induce beneficial impacts such as immunostimulation, whereas an unfavorable dose may not cause any responses, or may even be cytotoxic. Consequently, the optimization of the dosage according to the plant and the type of material chosen is strongly recommended. Moreover, as several *Lamiaceae* plants have been shown to have a dose-dependent effect, further studies are required to understand the toxicological safety of these feed additives.

The employment of *Lamiaceae* plants is an interesting field in aquaculture; however, there are still numerous research gaps. Foremost, comparative studies concerning the part of the *Lamiaceae* plants and the type of extraction (leaves, extract, mixed, or essential oil), the optimal administration method (immersion, injection or oral administration), and the duration of administration are necessary to gather information about the best beneficial effects on fish health and the parameters of interest in aquaculture, primarily growth performance and immune response. Additionally, as the impact of a feed supplement is species-specific, further research is required on the use of the *Lamiaceae* family in order to identify the plant species and products with the best beneficial potential for each fish species of interest in aquaculture.

Moreover, in a considerable number of the reviewed studies, the chemical characterization of the fish feed supplements is absent; thus, the chemical analysis of *Lamiaceae* products used as feed additives should be encouraged, with the aim of identifying and quantifying the active molecules and establishing their proper dosages and the duration of administration. Detailed information about the chemical compositions of *Lamiaceae* species could help critically analyze their effectiveness as growth promoters, immunostimulants, and antioxidant agents. In addition, the knowledge of the chemical composition could open the way to a possible correlation between the bioactive compounds present in the fish feed supplement used and the results obtained. As has previously been reported, the chemical composition of plants is influenced by numerous factors, such as the form and type of extraction (Table 6).

**Table 6.** The major bioactive compounds identified in essential oil, hydrolcoholic extract and leaves of *Lamiaceae* plants (oregano, rosemary, sage, thyme and mint).


H-R GC: High-Resolution Gas Chromatography; GC/MS: Gas chromatography/mass spectrometry; HS-SPME-GC/MS: Headspace solid-phase microextraction- Gas chromatography/mass spectrometry.

This variation of bioactive compounds could be reflected in the biological properties of the herb used as feed additives. In our opinion, the knowledge of the chemical composition of *Lamiaceae* plants or products represents an important parameter that must be considered in order to standardize the use of medicinal herbs in fish nutrition.

In addition, the knowledge of the mechanism of action of the bioactive molecules present in medicinal plants is still scarce. The understanding of the mechanisms of action of the bioactive compounds contained in *Lamiaceae* plants used as feed additives could elucidate the cellular and molecular processes underlying their capabilities of enhancing growth performance, immune system, and antioxidant status.

In conclusion, plants of the *Lamiaceae* family represent an exciting research field in aquaculture and a natural, economical, sustainable, and safe source of feed integrators capable of enhancing the health of farmed fish. However, although these are natural products, it is necessary to take into account the criteria for the safe use of plant ingredients in diets for farmed fish according to legislation, which differs among countries. Within the European Union, the safe use of oregano, rosemary, sage, thyme, and mint as feed additives for animal nutrition is approved and governed by regulation (EC) 1831/2003 of the European Parliament and of the Council of 22 September 2003 (https://www.efsa.europa.eu/it/ applications/feedadditives/regulationsandguidance accessed on 1 November 2022).

**Author Contributions:** Conceptualization, M.P.; writing—original draft preparation, G.O. and M.P.; writing—review and editing, G.O., R.I., E.C., G.A. and M.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Review* **In Pursuit of Fish-Free Feeds: A Multi-Species Evaluation**

**Kelly B. Campbell 1, Ewen McLean 2,\* and Frederic T. Barrows <sup>3</sup>**


**Abstract:** The future growth and sustainability of fed aquaculture, and especially that for carnivorous species, will be highly dependent upon the industry stepping away from its reliance upon forage fishes as major feed ingredients. With this goal in mind, the F3 Feed Innovation Network—a consortium of researchers; businesses, including feed manufacturers and ingredient providers; NGOs; and others—energizes industry to adopt novel and promising aquafeed ingredients and formulations. All evaluated formulae are open-source and freely available on the F3 website. Moreover, the F3 diets can be readily retailored to suit user demands and/or local conditions (i.e., ingredient availability/restrictions). This presentation summarizes completed F3 trials undertaken with five species of cultured and candidate fishes. With reference to eight studies, findings are compared against conventional fishmeal (FM)/fish oil (FO)-based feeds. The described research documents the response of test animals to aquafeeds containing traditional FM/FO alternatives (e.g., soybean meal and poultry by-product meal) as well as innovative ingredients (e.g., microalgae and single-cell proteins). Depending on the species examined, account is given to the overall growth performance, health aspects, and product quality. The F3 trials demonstrate the feasibility of the complete removal of FM/FO from the diets of the tested animals.

**Keywords:** largemouth bass; pompano; amberjack; red drum; algal oil

#### **1. Introduction**

The desire to optimize aquafeeds has a long history. Like today, although not necessarily directly articulated, there was an aspiration to develop a more sustainable aquaculture from as early as the 1920s. Concerns included reducing water pollution caused by raw meats (fish, horse, seal, and sheep) and offal (liver, spleen, heart, and lungs), which were commonly used as hatchery feeds. Around the same time, and especially during war years, fish and meats employed as feeds were also rationed and or becoming more expensive [1–4]. Concurrently, culturists sought to confront problems related to the effective storage and dissemination of feed [5–8] and disease transmission from trash to cultured fish [9]. Thus, cheaper feeds based on alternative ingredients were sought. Investigations with animalprotein-free diets, however, resulted in inferior growth and feed conversion, changes in animal physiology, and increased mortalities (e.g., [10,11]). These adverse reactions were generally attributed to plant-derived toxins and nutritional inadequacies, such as vitamin deficiencies [12,13], and were so commonly described that some suggested the use of plant meals, especially in fingerling feeds, was inadvisable [14].

In the intervening years, various dietary formulations were evaluated [15,16], with pelleted feeds such as the Oregon Moist Pellet [17] and dry preparations being used in US state hatcheries and at commercial farms in the 1950s [18–21]. In the late 1950s, Edward Grassl [22–24] evaluated the use of dry diets both as feeds and medicated diets. He compared the growth of trout fed either wet chopped meats or dry pelleted animal/vegetable feeds and reported identical growth even when the pellet was fed at 50% of the amount

**Citation:** Campbell, K.B.; McLean, E.; Barrows, F.T. In Pursuit of Fish-Free Feeds: A Multi-Species Evaluation. *Fishes* **2022**, *7*, 336. https://doi.org/ 10.3390/fishes7060336

Academic Editors: Marina Paolucci and Shunsuke Koshio

Received: 6 October 2022 Accepted: 13 November 2022 Published: 17 November 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

recommended for raw feeds, provided that chopped liver was fed once a month or so. Implementation of the pelleted feeds by state hatcheries resulted in 60% improved production and a 40% reduction in food costs. Use of a vitamin mixture in the dry pellet, as recorded by Phillips et al. [25], eliminated the need for chopped liver supplements and formed the basis for trout dry pellet formulations and the development of mechanized feeders [26,27]. Across the Atlantic, similar trials were being undertaken with salmon [28]. These pioneering studies, together with the elucidation of the nutritional requirements of some species, represent foundational moments in the elaboration of the global aquafeed industry.

Between 2001 and 2010, global aquaculture production increased at an annual average of 5.8%, and between 2011 and 2018, by 4.5%. This astonishing rate of expansion, while having moderated to around 3% during 2020, continues to grow [29]. Significantly, much of the growth experienced this century has occurred in the fed aquaculture sector, which accounted for 86% and 73% of global FM and fish oil (FO) supplies, respectively, in 2020 [29]. Future projections suggest that if the use of FM/FO is to remain as-is in aquafeeds, demand will outstrip supply by 2030 [30]. This scenario has led to major reductions in the use of FM/FO over the last two decades through wiser use allocations and their replacement with alternatives, including fish processing by-products among many others [31]. Together with enhanced feed conversion efficiencies, genetic selection programs [32], and even commodity price risk hedging [33], among other strategies, feed costs have been reduced, with some of the alternative products illustrating adequate environmental performance [34–36], thereby having potential to improve industry sustainability.

The drive to replace FM/FO from aquafeeds is based not only on projected availability, which also influences raw material prices, but also on the growing concerns of active environmental and consumer lobbies. These groups point to the fact that forage fisheries influence the health and sustainability of marine and coastal ecosystems, while their prey are vital to the sustenance of marine predators including other fishes, birds, marine mammals, and humans. Seafood buyers are also becoming more knowledgeable of the range of potential contaminants that may impact food safety, including those of raw materials used during aquafeed production (review: [37]). Well-informed consumers are learned of human rights infractions that occur in some industrial fisheries and across aquaculture supply chains [38] and aware of the negative consequences of at-sea discarding [39], ghost nets [40], harmful fisheries subsidies [41], carbon emissions from fleets and feed manufacturers [42,43], and animal welfare issues [44]. These worries have resulted in the creation of a sustainability imperative driven by consumers who demand safe and ethically and environmentally responsive food production systems and base purchase decisions on these principles. The aquafeed industry is in an influential position to ensure that consumer and environmental desires are achieved. In the interim, the search for and the evaluation of suitable alternatives to FM and FO must be unwavering.

Since 2014, the F3 Feed Innovation Network (f3fin.org (accessed on 11 November 2022)) has encouraged sustainable initiatives to reduce the dependence of the aquafeed industry on forage fishes and embolden the sector to adopt novel and promising ingredients and formulations. One way in which the F3 consortium accomplishes this is through openly sharing recipes and experimental findings through its website and publications. Aquafeeds do not have a requirement for any specific ingredient but must satisfy the nutritional prerequisites of the target animal. Feeds, therefore, must provide a combination of nutrients in the correct proportions to fulfill the metabolic needs of the species in question [45]. Bearing this in mind, the F3 consortium has completed several trials in efforts to eliminate FM/FO from the feeds of a variety of widely cultivated and candidate species. Here, we recount the findings of eight of these trials. The formulations considered herein for each species studied are all open-source and freely available on the F3 website. All F3 diets may be retailored to suit the user's demands; other tested formulations are similarly deposited on the F3 website.

#### **2. Species Evaluated**

Five well-established and candidate cultured species of teleost were examined for their sensitivity to fish-free feeds (F3). They included the largemouth bass *Micropterus salmoides*, which is the preeminent farmed Perciformes, representing over 50% or around 432,000 tons of total production [46]; the Florida pompano *Trachinotus carolinus*, a strong candidate species for US aquaculture; and other members of the family Carangidae, including two species of amberjack, namely the California yellowtail *Seriola dorsalis* and kampachi *S. rivoliana*. More than 170,000 tons of pompano is cultured annually, with most production being in Asia [47], while 150,000 tons of amberjack is farmed globally, with most production being dependent on raw fish, although the availability of formulated diets has recently increased. As a major portion of compounded feeds used in Chinese mariculture is taken by farmers of pompano and red drum *Sciaenops ocellatus* [48], the latter species was also evaluated. Global production of Sciaenids exceeds 340,000 tons, with the red drum representing around 25% of the total [49]. As adults, the species evaluated herein are considered as obligate carnivores. For many marine carnivores, only a few studies have investigated the potential for concurrently replacing FM/FO in diets.

#### **3. Selection of Dietary Ingredients**

The ingredients employed in the various diets are presented in Table 1. All experimental feeds were evaluated against the response of the test species to an FM/FO-based diet. The least expensive and best performing experimental feeds from each reference were chosen for comparison for the sake of this paper; however, additional diets and corresponding performance data are available in the references listed. Dietary protein for the investigational diets was derived from both animal and plant sources and based on availability, demonstrated utility, and/or promise as a dietary component. For example, poultry by-product meal (PBM) represents a resource of considerable potential as an FM alternative. Global production of chickens is estimated to be 33 billion individuals, equivalent to 101 million tons for 2022 [50], with the largest producers being the USA, Brazil, China, and the EU Raw materials leftover from slaughterhouses and processing facilities represent about 30% of liveweight [51] or around 30 million tons. The clean unused parts of butchered poultry, including the voided intestines and culled laying hens, are ground and then rendered into meal. Corn gluten meal (CGM), a by-product of corn processing containing about 65% crude protein, and corn protein concentrate (CPC), comprising around 67% protein, have both enjoyed success as components of a variety of commercial and investigational aquafeeds, including those for amberjack [52], Florida pompano [53], red drum [54], and largemouth bass [55]. No negative effects of CGM or CPC have been reported, even when used at relatively high levels of dietary incorporation. Soybean meal (SBM; ~50% crude protein) is an excellent substitute for animal proteins in aquafeeds, even though the presence of anti-nutritional factors (ANFs) may disrupt gut function in some species [56]. Nevertheless, SBM and soy products, such as soy protein concentrate (SPC; ~63% crude protein), which has a reduced concentration of ANFs, have garnered wide use and are well represented in commercial and experimental aquafeeds across the board [57,58]. MrFeed Pro 50 (~51% crude protein), a bacterial hydrolysate made from soybean-derived cellulosic sugars, and similar products have received increased attention due to their availability, high digestibility, lower costs, safety, and sustainability [59,60]. *Spirulina* expresses 55%+ crude protein and an elevated PUFA content and, when used at high levels of supplementation, has been observed to provide a beneficial effect on animal growth, body composition, pigmentation, immunity, and reproductive performance [61]. A wide variety of FO alternatives have been assessed with a broad range of species, and flax, canola, and algal oils are not exceptional, being widely available and competitively priced. Each has been successfully used as an FO substitute with pompano, yellowtail, largemouth bass, and others [62–64].

**Table 1.** Formulations of experimental diets in which fishmeal and fish oil were replaced with a combination of different protein sources and oils. GMO = Genetically Modified Organism; ARS = Agricultural Research Service. For formulation information on FM/FO-based diets, the reader is directed to f3fin.org/resources/open-feed-formulas/ (accessed on 11 November 2022).


#### **4. Fish Holding and Husbandry**

Other than for a study with kampachi, all feeding trials were undertaken in tanks configured as recirculating systems. The study lengths, which varied from 56 to 126 days; water quality parameters, including temperature, salinity, and dissolved oxygen levels; and start weights of experimental animals are summarized in Table 2. Water quality parameters were collected using standard methods. The feeding schedule for the experimental and control diets for each species is likewise presented in Table 2. All studies were executed with appropriate regard to Institutional Animal Care and Use Committee regulations and complied with all relevant international animal welfare laws, guidelines, and policies.

**Table 2.** Experimental systems employed, stocking densities, starting weights, study lengths, water quality parameters, and feeding schedules in various studies undertaken to evaluate the impact of dietary fishmeal and fish oil replacement on the performance of established and candidate species of teleost for aquaculture.


#### **5. Data Collection**

Details relating to the precise procedures employed in data acquisition for each species may be found in the relevant publications (see Table 1 for references). Depending on the trial under consideration, the following information was compiled to assess the performance of experimental animals with each dietary treatment:

Weight gain (%) = [(Final body weight − initial body weight)/(initial body weight)] × 100;

Survival (%) = [final population/initial population] × 100;

Feed efficiency (FE) = weight gain (g)/dry feed consumed (g);

Feed conversion ratio (FCR) = weight of feed consumed (g)/weight gained by the animal (g);

Protein efficiency ratio (PER, %) = [weight gain (g, wet weight)/protein intake (g, dry weight)] × 100;

Fillet yield (%) = [fillet weight (g)/gutted weight (g)] × 100;

Hepatosomatic index (HSI, %) = [liver weight (g)/body weight (g)] × 100;

Interperitoneal fat ratio (IPF, %) = [IPF weight (g)/body weight (g)] × 100;

Fulton condition factor (K) = [fish weight (g)/(fish length, cm)3] <sup>×</sup> 100;

Viscerosomatic index (VSI, %) = [weight of viscera (g)/body weight (g)] × 100.

After gauging the above-mentioned indices, all the remains of fish samples (*n* ≥ 5) were homogenized as a composite sample and analyzed for proximate composition, when measured, using established methods: the Dumas protocol for crude protein (6.25 × N) [70], and chloroform–methanol (4:1) extraction for crude lipid [71]. A lipid droplet subsample was isolated from these ingredients and conserved in N2 at −80 ◦C for identification of their fatty acid profile by flame ionization gas chromatography. Fatty acid methyl esters (FAMEs) were prepared as described previously [72] and modified to include an additional saponification step [73]. Ash was determined after heating samples at 650 ◦C in a muffle furnace for3h[70].

Histological analyses were undertaken on the guts and livers of California yellowtail and largemouth bass. Samples were collected immediately following gross necropsies for performance characteristics (*n* ≤ 6 per treatment). Sections of liver and distal intestine (2 cm × 2 cm) were preserved in Bouin's fixative for 24 h and subsequently transferred to 70% ethanol for final fixation. Tissues were then dehydrated, embedded in paraffin, and sectioned at 5 μm before staining with H&E using standard procedures. Rankings were then performed to differentiate histopathologic changes in the liver and intestine between diets.

Criteria assessed included intestinal goblet cell density and inflammation, hepatic glycogen content, and cellular changes [66]. For largemouth bass, spleen samples were stained using Gomori's modified iron procedure for hemosiderin [74] to evaluate the staining intensity of melano-macrophage centers (MMCs), which were graded from 0 to 2 for low, medium, and high, respectively.

Taste tests of largemouth bass were informal and used 25 active consumers who were provided with blind samples and asked to prepare fish using plain methods. Each was then requested to determine whether there were differences in taste, texture, or aroma between samples. Similar studies were undertaken with kampachi. The collected data were subjected to various statistical analyses with significance set at the *p* < 0.05 level. Readers are directed to the papers noted in Table 1 for complete details.

#### **6. Observations and Discussion**

Weight gain in all marine species fed F3 diets, except pompano 2, was less than that achieved by animals fed conventional FM/FO feeds (Table 3). However, there was no impact discerned on FCR, survival, fillet yield, HSI, or *K*. In kampachi, a significantly higher VSI in the control group accounted for the increased weight gain such that once this was taken into account, no differences in weight were apparent. In largemouth bass, the only freshwater species examined, the weight gain in fish fed F3 and F2 (FO included) was equivalent to that in animals fed the conventional diet (Table 3). The FCR in F3/F2-fed largemouth bass was equivalent to that in the conventional group in two of the three trials and was elevated in one of the trials, while survival was lower in one study. Accordingly, the trials described here illustrate the potential to severely reduce, and perhaps eliminate, FM/FO from aquafeeds of facultative carnivores. Importantly, the evidence presented to support this statement originated from investigations that employed a constrained list of possible FM/FO alternatives. Additionally, the F3 recipes used were derived from a formulator's experience rather than from experiments designed to determine the optimal inclusion rates for specific ingredients. Undoubtedly, with dietary refinement, perhaps involving the inclusion of other proteins and oils or modification to their concentrations/combinations, even greater benefits than those achieved will accrue. This supposition is supported by the findings of other researchers who have successfully replaced FM/FO in diets for an ever-increasing number of species (e.g., [75–81]).

The results considered here with the F3 feeds, together with the experience of others, imply that marine species will be more demanding than freshwater fishes regarding the complete removal of dietary FM/FO. It is probable that the largemouth bass were indifferent to lipid exchange due to their essential fatty acid (EFA) requirements being met by dietary 18:3n-3 and/or 18:2n-6 PUFA [82]. Similar observations have been made with other species of freshwater fish, where a wide variety of alternative dietary lipids have been shown to facilitate growth [83–85]. These results thus provide support for the idea that FO can already be totally removed from largemouth bass diets. However, a precautionary approach should be taken since some substitute oils have been demonstrated to cause physiological disturbance [86,87]. Marine species lack the enzymatic machinery necessary to elongate or desaturate PUFAs, such that EFA requirements are met by long-chain PUFAs, *viz.* 20:5n-3 and/or 22:6n-3 [88], which, in some diets, may have been limiting. Nonetheless, the substitution of fish oil with vegetable and/or algal oil in all species examined had no significant impact on survival, suggesting that the dietary fatty acid composition, even though varying, achieved the n-3 HUFA requirements of the species examined, at least over the study length. Importantly, lipid exchange had either no impact or only a marginal impact on feed palatability, thereby underscoring the flexibility that exists for the substitution of dietary lipids. An additional advantage of using *Schizochytrium* sp.-derived algal oil, produced by controlled heterotrophic fermentation, is its contaminant-free status, which contrasts to that of some FOs [89].

**Table 3.** Response of various species to experimental diets in which fishmeal and fish oil were replaced with alternatives. FCR = feed conversion efficiency; FE = feed efficiency; HSI = hepatosomatic index; IPF = intraperitoneal fat ratio; *K* = condition factor; PER = protein efficiency ratio; VSI = viscerosomatic index. Up- and downward-pointing arrows indicate significant differences (*p* < 0.05) from fish fed a control diet.


As recorded previously for a wide variety of species [90,91], the fatty acid profiles of fillets of the assessed fish correlated well with those of their feeds (Table 4, Figure 1). One negative aspect of this trait, however, was that while n6:n3 ratios remained stable, the EPA/DHA fractions were inferior to those of control fillets. Fish oil substitution, therefore, may negatively affect the nutritional value of fillets [92,93]. Were it to be considered necessary, fillet lipids (types and levels) might be tailored to a specific use with finishing diets [94,95]. Such an eventuality might occur where significant changes in flesh quality, including firmness, juiciness, and fresh oily taste, deviate following large fluctuations in proximate composition, or, for example, when higher fillet lipid levels are required for reasons of processing, such as smoking [96]. Even given differences between the control and treatment group fillet fatty acid profiles, and subtle modifications to proximate the composition of largemouth bass, organoleptic evaluation by 25 habitual consumers resulted in 48% preferring the fishmeal–fish-oil-fed fish based on the taste, texture, and aroma, while 40% favored the F3-fed animals and 12% indicated no preference [68]. Thus, for largemouth bass, the deletion of FO from their diet had no apparent impact on consumer acceptance. Similarly, a blind taste test of kampachi resulted in 62% of participants preferring the F3-fed fish, 19% having a preference for *S. rivoliana* fed on a traditional diet, and 19% being unable to discriminate between the two dietary groups [65].



**Figure 1.** Scatter plots depicting the relationship between measured feed (y-axes) and fillet (x-axes) fatty acid content and 95% confidence intervals (dashed lines) for largemouth bass, Florida pompano, and red drum (see Table 4 for data).

Although the main goal of the F3 initiative is to eliminate the use of forage fishes in aquafeed production, an aspiration that is close to attainment for the species evaluated, some still question the practice of using animal by-products as alternative proteins. While this may be achievable with lower trophic species, a consistent observation with carnivores has been poorer overall performance when diets comprise vegetable proteins only. This is undoubtedly related to the presence of poorly digested carbohydrates and imbalances in essential amino acids (EAAs), the presence of a wide variety of anti-nutritional factors, and structural differences between plant and animal proteins [97]. These have negative impacts on growth, feed efficiency, metabolism, and health [31], and it is feasible that these effects may partly account for the reduced growth observed in the described trials herein. However, even given the presence of PBM in all F3 feeds, the marine test species failed to attain the growth recorded by control groups. Due to the variety of generally unsegregated material that is employed in PBM production, together with differences in processing and equipment, meals vary widely in their protein content and nutritional quality, lacking certain EAAs, being high in ash, and expressing variable digestibility [98,99]. Nonetheless, PBM has been successfully employed to replace relatively high levels of FM [100], although growth penalties coupled with higher FCRs and changes in body composition are known to occur in various species (e.g., [101–103]), and this may have been witnessed here.

The new and emerging technologies that modify raw materials, together with advances in process engineering, are starting to overcome many of the constraints encountered with alternative vegetable proteins, which bodes well for the future. For example, the production of plant protein concentrates and isolates removes carbohydrates, fiber, and anti-nutritional factors, resulting in products that, while more expensive, generally express an augmented EAA balance and have enhanced digestibility. However, the use of plant proteins for aquafeeds is disapproved by some who raise concerns relating to forest transitions, displacement of land use, increased use of fertilizers, eutrophication, environmental degradation, carbon footprinting, and others [104]. Given the current production strategies of established and emergent alternative proteins and their projected growth potential, it has been suggested that no single substitute protein will be able to source future demands of the animal feed industry, just as reliance on a few sources of ingredients, namely FM/FO, has created the bottlenecks we see today. Accordingly, the availability of a broad range of replacement proteins represents the soundest approach to overcome future supply constraints. Indeed, today, feed formulation scientists have a wide assortment of FM/FO alternatives [31,51,105]. Nevertheless, the aquafeed sector retains a significant dependency upon marine products [106], and it is likely that this addiction will remain for some time. Although their use will probably continue to decline in grower feeds, FM/FO will remain significant ingredients in specialty feeds, as exemplified by broodstock diets, and, perhaps, finishing feeds that may overcome fillet quality issues.

To date, most successful FM/FO replacement trials with carnivores have used diets containing blends of proteins and/or lipids that have been formulated to meet the nutrient requirements of the target species [77–79,107–110]. The broad range of potential aquafeed ingredients currently available, however, while providing strategic opportunities for formulating FM/FO-free feeds, also brings headaches for predicting optimal nutritional and economic blends, especially when mixtures might include a range of functional ingredients. Methods for overcoming some of these complexities are considered elsewhere [111–113]. One aspect of feed blending that has received limited attention is the potential to impact gut flora and fauna colonization and how this may influence nutrient absorption, etc., leading to potential for gut dysfunction. Clearly, there must be no consequences to the health of the target species when using alternative dietary ingredients. In one study with largemouth bass (Table 3), however, survival was apparently compromised by F3 feeds, although in a further two studies, no such effect was observed. Nonetheless, the detected anomaly prompted more detailed analyses of fish health. One indicator of immune function in teleosts is the status of splenic MMC [114], but evaluations thereof failed to detect differences between control and F3 treatments [67]. Moreover, the splenic index and hematocrit

levels in examined fish were similar, and histological observations of the liver and distal intestine did not reveal any microscopic changes for the F3-fed group. In California yellowtail, slight hepatic inflammation and microscopic structural changes were encountered, with F3-fed animals also expressing higher glycogen accumulation. In contrast, control fish exhibited increased hepatocellular vacuolization and eccentric nuclei, together with a higher number of goblet cells in the distal intestine [66]. The decreased presence of goblet cells in F3 fish was not associated with inflammation, which the authors suggested might have indicated a protective effect of the *Spirulina* and/or algal oils incorporated into the diets. Notable is that the inclusion of soybean meal and concentrate in great amberjack *S. lalandi* diets was also associated with increased goblet cell numbers [115].

Since it is likely that animals cultured using sustainable marine-resource-free diets, such as organically certified and other premium foods, will represent quality products [116], methods for verifying their authenticity and traceability will become an imperative [117,118]. Animal tissue δ15N is commonly employed to designate trophic position in food [119–121], and the technique has been applied to examine the relative contributions of plant and animal proteins in feeds for crustaceans [122–124] and fishes [117,125,126]. Thus, when the contribution of dietary FM declines, a corresponding decline in δ15N is encountered. This response thereby potentially provides a method for verifying the integrity of animals reared using F3 diets. To substantiate this possibility, a study was undertaken with largemouth bass [68] (Figure 2). The trial examined fish fed a commercial feed, an FM/FO-based control diet, an FM-free feed containing FO, and an F3 diet. The FM control and commercial feeds both expressed final δ15N values that were significantly higher than those for the FM-free feeds (Figure 2), no doubt reflecting the relative proportion and isotopic values of their ingredients. Substitution of the PBM from the F3 feed with another plant protein would likely shift the δ15N values lower still. The use of stable isotope ratios to discriminate between aquacultured animals fed on more sustainable feeds, therefore, is apparently operational but should probably be restricted to animals reared in contained environments.

**Figure 2.** Isotope values for largemouth bass fed one of four diets. Values are means ± 95% confidence intervals (redrawn from [68]).

Based on the findings presented using essentially carnivorous species of cultured fish, total replacement of FM/FO appears more than just a convincing and economically viable proposition. Even so, further production-length research, perhaps with adjusted dietary formulae, is warranted to ensure that such diets have no negative consequences to the overall health and welfare of farmed animals. The potential adverse outcomes that dietary

changes may have on various quality attributes, which may influence wholesale, retail, and consumer purchasing choices, also demand greater attention. Lucid though, from the considered trials, is that replacement protein/oil combinations provide products that are more secure in terms of food safety and more acceptable to discriminating consumers. The use of such nutrients will bridge gaps between the future supply and demand for FM/FO while serving global sustainability initiatives. While this might appear an over-enthusiastic conclusion, we have already demonstrated the potential for aquafeed mindset change with Pacific whiteleg shrimp *Litopenaeus vanammei* production [127,128], where F3 feeds are now firmly placed in the production sector. Similar success has been achieved with trout, largemouth bass, yellow croaker, and red seabream [129].

**Author Contributions:** Conceptualization, E.M.; draft manuscript preparation, K.B.C. and E.M.; Resources, F.T.B., K.B.C. and E.M.; Writing—review and editing, F.T.B. and K.B.C.; Project administration, K.B.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Data Availability Statement:** All author-owned experimental data are available at f3fin.org (accessed on 11 November 2022).

**Acknowledgments:** The authors express gratitude to all those who participated in the mentioned f3fin projects and especially the Anthropocene Institute for its unwavering support throughout.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Review* **Nutritional Value of Dry Fish in Bangladesh and Its Potential Contribution to Addressing Malnutrition: A Narrative Review**

**Md. Hasan Al Banna 1, Abdullah Al Zaber 2, Nahidur Rahman 3, Md Abrar Mozahid Siddique 4, Muhammad Abu Bakr Siddique 5, John Elvis Hagan, Jr. 6,7,\*, M. A. Rifat 8, Christiana Naa Atsreh Nsiah-Asamoah 9, Abdul-Aziz Seidu 10,11, Bright Opoku Ahinkorah <sup>12</sup> and Md Shafiqul Islam Khan <sup>1</sup>**


**Abstract:** Understanding the linkage between the nutrient composition of foods and individuals' recommended nutrient intake is important to address malnutrition. Despite it being a traditional and popular food item in Bangladesh, the nutrient composition of dry fish has not been reviewed yet. This study used a narrative review to assess the nutrient composition of dry fish and estimated its potential contribution to addressing some common nutritional deficiencies among children and pregnant and lactating women in Bangladesh. Records were collected from different databases, including the Web of Science, Google Scholar, PubMed, ScienceDirect, Banglajol, and ResearchGate. Data were extracted from 48 articles containing 1128 entries regarding nutrient composition. Most of the nutrient analyses estimated the proximate composition, whereas vitamin, mineral, amino acid, and fatty acid compositions were scarce in the literature. We found that dry fish has high protein and mineral content and could contribute highly to meeting the recommended nutrient intake of protein, iron, zinc, and calcium for children and pregnant and lactating women. The summarized nutrient composition data could be useful for further research to observe how dry fish could be best utilized to address malnutrition in Bangladesh. This narrative review recommends that further nutrient analysis, with emphasis on vitamin, mineral, and fatty acid compositions.

**Keywords:** Bangladesh; children; dry fish; malnutrition; minerals; nutrients; protein; vitamins; women

#### **1. Introduction**

Bangladesh is a riverine country, blessed with vast fishery resources, inland as well as marine [1]. From the viewpoint of the natural gift of aquatic resources, the aquatic food system plays a vital role in the food culture, eating habits, and lifestyle of the people. Fresh fish has versatile nourishing properties, which include highly bioavailable protein,

**Citation:** Banna, M.H.A.; Al Zaber, A.; Rahman, N.; Siddique, M.A.M.; Siddique, M.A.B.; Hagan, J.E., Jr.; Rifat, M.A.; Nsiah-Asamoah, C.N.A.; Seidu, A.-A.; Ahinkorah, B.O.; et al. Nutritional Value of Dry Fish in Bangladesh and Its Potential Contribution to Addressing Malnutrition: A Narrative Review. *Fishes* **2022**, *7*, 240. https://doi.org/ 10.3390/fishes7050240

Academic Editors: Marina Paolucci and Shunsuke Koshio

Received: 26 July 2022 Accepted: 5 September 2022 Published: 8 September 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

essential fatty acids, macro- and micro-minerals, and vitamins; therefore, it undergoes rapid microbial spoilage [2]. Thus, various preservation techniques such as drying, salting, chilling, freezing, and smoking are used to prevent microbial spoilage and keep up the nutrient quality with a view to storing throughout the year [3,4]. Among these techniques, drying is the most traditionally used method of fish processing and preservation in developing countries, including Bangladesh [5,6]. From nutritive aspects, dry fish consist of high-quality proteins, healthy fatty acids, including long-chain omega-3 fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and are a unique source of essential nutrients such as iodine, zinc, copper, selenium, and calcium [7]. Generally, dry fish is a delicious and gastronomically nourishing food that provides high protein and low calories compared to other animal protein such as beef. For example, 100 g of dry fish contains approximately 80 g of protein and 300 calories of energy, whereas animal meat contains almost two times more calories but low protein compared to dry fish [7]. Moreover, dry fish is considered a healthy food item for individuals and an entirely natural product that contains almost equivalent omega-3 fatty acid content and antioxidant properties to fresh fish [7]. Dry fish contributes a big share of micronutrients to the diet of the low socioeconomic population groups in South and Southeast Asia [8].

Dry fish (locally known as Shutki) is a popular and traditional food item among Bangladeshi people because of its high nutritional value, good taste, aroma, and distinctive flavor [9]. Moreover, people prefer some fish species, for instance, Bombay duck and ribbon fish, as dried rather than fresh for consumption. Evidence shows that people often find it reasonable to include dry fish in their diet to avoid heart diseases, diabetes, and obesity [7]. Dry fish is of low cost, affordable to low socioeconomic groups, and usually consumed with vegetables, oil, and spices in mixed dishes along with the major staple rice; thus, it helps improve individuals' dietary diversity and nutrition security [1,7].

Nowadays, there are different areas of Bangladesh, including Charfashion in Bhola, Dublar Chor in Khulna, Kutubdia, Khuruskul, Moheskhali, Sonadia, and St. Martin Island in Cox's Bazar, and Alipur, Mohipur, Rangabali, and Kuakata in Patuakhali, where dry fish is commercially produced [1,9]. Here, mainly three categories of fish, including large fish, elongated fish, and small fish, are used for dry fish production [10]. About 20% of the artisanal catch is processed for dry fish production by the sun-drying method [11]. In addition to fresh fish and seafood products, dry fish has created potential market demand in Bangladesh and abroad [12]. In 2018–2019, Bangladesh exported a total of 2339.36 metric tons of dry fish to different countries, including India, Singapore, Hong Kong, Malaysia, the UK, the USA, and the United Arab Emirates, and earned approximately USD 4 million [13].

In Bangladesh, dry fish has market demand all around the year although their availability is somewhat more seasonal, mostly during the winter. Market demand for dry fish is high during spring and early monsoon when the supply of fresh fish from capture fisheries and aquaculture is the lowest [9]. There are regional and cultural differences in dry fish consumption; for instance, the highest consumption occurs in the Chattogram and Sylhet divisions, followed by moderate in the Dhaka division, low in the Barishal and Rajshahi divisions, and rare in the Khulna division [9]. The choice of type of dry fish consumption also varies geographically. For example, people from the Chattogram region consume mainly dried fish of marine origin, while people from Sylhet and Dhaka consume dry fish of both freshwater and marine origin.

In recent decades, Bangladesh has stepped forward to achieve self-sufficiency in food production; however, food and nutrition insecurity and malnutrition remain major public health issues [14]. Moreover, a recent study reported a moderate level of nutrition literacy among Bangladeshi adults [15]. According to Bangladesh Demographic and Health Survey 2014, the prevalence of childhood malnutrition is high (for instance, stunting: 36.1%, wasting: 14.3%, and underweight: 32.6%) [16,17]. Micronutrient deficiencies (such as iron deficiency anemia and zinc and calcium deficiencies) are still highly prevalent among children and women of reproductive age [17]. Therefore, the choice of a diet with high nutritional value is important to prevent the malnutrition burden. In this aspect, the

inclusion of dry fish in the diet could be a consideration for improving dietary diversity and nutrient supply to the body.

Although drying causes some changes in fish flesh, it is still an excellent source of essential nutrients [7]. However, the use of spoiled raw fish, insecticides, and pesticides, poor hygiene and sanitation during preparation, the traditional way of processing, and longterm traditional storage conditions may lead to the nutrient loss and quality deterioration of dry fish [18–20]. A previous investigation conducted in the Chattogram region of Bangladesh identified that the nutritional value of dry fish undergoes deterioration with high storage time [18]. Another study in Bangladesh reported that deteriorative changes in dried fishes may result in browning reactions and develop rancidity when the moisture content is comparatively high [21]. Therefore, sufficient precautionary measures such as using fresh raw fish, proper drying, maintaining personal hygiene and sanitation, and proper storage and packaging must be taken into consideration during dry fish production.

There is a lack of scientific documentation and quantitative information on the nutrient composition of dry fish prepared from fish species captured on the coast of Bangladesh. The "Food Composition Table for Bangladesh" prepared by the Institute of Nutrition and Food Science (INFS), University of Dhaka, Bangladesh, contains 381 food items but only four dry fish items [22]. Exploration of food composition data is essential for providing basic information on various aspects of nutrition, understanding dietary choices, and developing basic tools to improve food and nutrition security [23]. Therefore, the purpose of our study was to perform a narrative review on the nutrient composition of dry fish in Bangladesh, summarize quantitative data on the nutrient composition of dry fish, and observe their potential contribution to meeting the recommended nutrient intake (RNI) of some nutrients with high public health importance for children and pregnant and lactating women in Bangladesh.

#### **2. Materials and Methods**

#### *2.1. Data Sources and Search Strategy*

To obtain the nutrient composition of dry fish prepared from fish species captured in Bangladesh, we conducted a narrative review with a literature search in the following databases: Web of Science, PubMed, Google Scholar, ScienceDirect, Banglajol (Bangladeshbased database), and ResearchGate. The following search strategy was used to collect records from the Web of Science and PubMed:

("Dried fish" OR "dry fish" OR fish) AND (Nutrient OR Composition OR "Nutrient composition" OR Vitamin OR Mineral OR Quality OR "Proximate composition" OR "Nutrient analysis") AND (Bangladesh)

Keywords, including "dry fish", "dried fish", nutrient composition, and Bangladesh, were used to conduct a manual record search in the databases of Google Scholar, ScienceDirect, Banglajol, and ResearchGate. No filter was applied while searching the Web of Science and PubMed. Database searching was conducted from June 2021 to December 2021. All citations were imported into Mendeley software and checked for duplication. Then, screening was conducted to identify eligible records.

#### *2.2. Inclusion and Exclusion Criteria*

Initially, titles and abstracts were screened using a checklist. The checklist consisted of three questions: (i) Is the article original research? (ii) Does the article contain the nutrient composition of dry fish? (iii) Does the article consider dry fish produced from fishes available in Bangladesh? Articles that qualified for initial screening were screened for full text. The full-text screening was conducted considering several criteria, including the number of fish species analyzed, name of the species, place of sample collection, number and types of nutrients considered for analysis, methods for nutrient analysis, and statistical representation of the data. In addition, the following inclusion criteria were followed: (1) publication date: no time restriction was applied, (2) language: no language restriction was applied, (3) laboratory methods: no article was excluded due to the study design or the laboratory methods used for nutrient analysis, and (4) sample type: only articles with

dry fish nutrient composition data were considered. We excluded records if irrelevant to the research question, and not peer reviewed. Finally, articles that met the inclusion criteria were considered for data extraction and analysis.

#### *2.3. Selection Process*

Four researchers independently conducted the literature search and screened titles and abstracts. They also conducted full-text screening according to the screening strategy. Another reviewer assessed the variance of the number of records at each stage of the selection process. To minimize the bias, any discord in the selection process, such as database searching and inclusion and exclusion of the articles, was settled through a discussion among all the researchers. At first, the literature search was performed in different databases and sites. Then, all the records were imported into Mendeley software for duplication checking. After excluding the duplicates, an initial screening was conducted using the checklist. Articles that qualified for initial screening were then screened for full text. Finally, articles that qualified for the screening process and inclusion criteria were considered for data extraction.

#### *2.4. Data Extraction*

Four reviewers independently conducted data extraction from the selected articles. Data were extracted in a Microsoft Excel spreadsheet and included the name of the species (local, common, and scientific names) and nutrient composition per 100 g of edible dry fish. Some articles presented nutrient composition per 1 kg of weight or in other units, such as ppm or percentage. In these cases, we converted the unit into per 100 g of edible dry fish. In the original articles, nutrient compositions were often represented in values with standard deviation, as several samples were analyzed. However, we did not consider standard deviation during data extraction and synthesis. Some common and commercially important dry fishes were analyzed by different researchers, but we considered all the findings and included them in the data extraction spreadsheet. This means that several entries were considered for a single dry fish if reported in different articles. Fatty acid and lipid profiles showed a wide range of variation in results: individual fatty acid content, total saturated fatty acid (SFA), monounsaturated fatty acid (MUFA), polyunsaturated fatty acid (PUFA), EPA, DHA, total cholesterol, etc. We considered total SFA, MUFA, PUFA, EPA, DHA, and cholesterol. In the data extraction spreadsheet, we kept the cells blank for the following: missing value, no result, trace, not detected, and unreported information (such as English name and local name of the species). The data extraction spreadsheet is provided as Supplementary Material.

#### *2.5. Assessment of Potential Contribution of Dry Fish to Addressing Malnutrition*

We focused on four nutrients based on the availability of nutrient composition data and the nutritional importance of nutrients in the Bangladesh context, including protein, zinc, iron, and calcium. Protein was considered because of low protein consumption among children in Bangladesh. Micronutrients such as zinc, iron, and calcium were considered based on data showing both national and global deficiencies [24,25]. For each of the four nutrients, we presented a calculation where we compared the nutrient content of a given uncooked dry fish item to the daily RNI for women and children at different life stages. These calculations highlighted the relative variation in the nutrient composition and density (nutrient content per unit of dry fish) among the dry fish items. For every calculation, we considered five dry fish species or items that have the highest nutrient content according to the data extraction and synthesis. We also used two reference fish for comparison: Tilapia (*Oreochromis niloticus*) and Thai pangas (*Pangasianodon hypophthalmus*). Tilapia and Thai pangas were selected because they are the most commonly consumed fish, with the highest market accessibility in Bangladesh. Nutritional values fluctuate during processing and cooking, and other dietary factors influence the absorption of particular nutrients. As a result, these calculations are not meant to provide any individual dietary advice. Rather, they help provide an estimate of how certain dry fish contributes nutrients to the diet. The percentage of what a serving of fish covers for the RNI was calculated for pregnant women, lactating women, infants 6–12 months old, and children 1–2 years old [26].

While calculating how one serving of dry fish could meet the RNI of certain nutrients for pregnant and lactating women and children, we considered a daily serving of 50 g for pregnant and lactating women and 25 g for children based on a previously used method [27,28]. We assumed 10% bioavailability for iron [26]. The RNI for iron for pregnant women was estimated based on the FAO/WHO (2004) [26] value for women aged 19–50 years old, as no specific value for pregnant women is given. The daily value of 29.4 mg closely aligns with the Institute of Medicine's recommendation of 27 mg and the Indian Council of Medical Research's (ICMR) recommendation of 35 mg for pregnant women [29,30]. The RNI for protein for children 12–23 months old and pregnant and lactating mothers was directly received from the ICMR (2011) [29]; however, the ICMR does not directly mention the RNI for protein for infants 6–12 months old. Because of this, we considered the median body weight of boys and girls at 9 months of age, which is the average and median value between 6 and 12 months, and then calculated the average standard body weight [31]. The standard body weight was then multiplied by 1.69 to obtain the recommended daily protein intake [29]. For zinc, moderate bioavailability was assumed [26]. We calculated the daily zinc requirement by averaging the requirement across the three trimesters of pregnancy and the first 12 months of lactation, using a value of 7.5 mg for pregnancy and 8.5 mg for lactation. For the calcium requirements of the target populations, the FAO/WHO (2004) recommendation was followed [26]. The calculation showing how one serving of dry fish could meet the RNI of the target group is provided as Supplementary Material.

#### **3. Results**

Our search yielded 2139 articles. After screening titles and abstracts, 1939 records were excluded. After a full-text screening of the 198 records, we finally included 48 articles that had a nutrient composition of 1128 entries on dry fish from Bangladesh (Figure 1). Characteristics of the included studies [18,21,22,32–76] such as the number of samples analyzed, sample collection location, etc., are summarized in Table S1 (Supplementary Material).

**Figure 1.** Selection process of the records.

#### *3.1. Proximate Composition of Dry Fish Prepared from Fish Species Captured in Bangladesh*

Of the total 1128 entries, 702 (62.23%) had the proximate composition of dry fish. Total protein content was determined in 14.54% (*n* = 164) of entries, and it was found that the average protein content was 56.63 g per 100 g of dry fish with the highest (77.68 g) in Churi (*Trichiurus savala*) and the lowest (26.73 g) in Ilish (*Tenualosa ilisha*). Total fat and ash content were analyzed in 15.70% (*n* = 177) and 15.25% (*n* = 172) of entries, respectively, and showed that Rupchanda (*Pampus chinensis*) and Tengra (*Mystus tengra*) contained the highest amount of fat and ash, respectively (Table 1).

Protein deficiency malnutrition is still prevalent among nutritionally vulnerable population groups such as children and pregnant and lactating women in Bangladesh. Being a good source of high-quality animal protein, dry fish can contribute to meeting individuals' daily protein requirements. For example, a serving of Ribbon fish (Churi) could fulfill 49.79% and 54.70% of daily recommended protein intake for pregnant and lactating mothers, respectively, and provides 100% of the recommended protein intake for children up to two years old. The potential contribution of dry fish to individuals' recommended protein intake is higher than that of fresh Thai pangas and Tilapia in Bangladesh (Figure 2).

**Table 1.** Analyzed nutrients, average nutrient content, and name of dry fish species with the highest and the lowest nutrient content.



**Table 1.** *Cont.*

\* Duplication (same species analyzed by several researchers) was considered. \*\* Values such as range, zero, not detected, missing values, and trace were not considered while calculating the average.

**Figure 2.** Potential contribution (%) of dry fish to the recommended nutrient intake (RNI) of protein for children and pregnant and lactating women in Bangladesh. Local names: Churi (*Trichiurus savala*), Bele (*Awaous grammepomus*), Taki (*Channa punctata*), Bele (*Glossogobius giuris*), Punti (*Puntius puntio*), Thai Pangas (*Pangasianodon hypophthalmus*), and Tilapia (*Oreocbromis mossambicus*).

#### *3.2. Minerals*

The content of minerals such as iron, zinc, calcium, phosphorus, magnesium, sodium, potassium, manganese, and copper was assessed in 23.76% (*n* = 268) of entries. The average mineral content with the highest and the lowest values is presented in Table 1.

Iron (Fe) was estimated in 4.43% (*n* = 50) of entries. The average Fe content was 13.15 mg per 100 g of dry fish which ranged from 45.10 mg (*Coilia neglecta*) to 2.80 mg (*Channa striata*) (Table 1). The bioavailability of iron is the extent to which dietary iron is absorbed by the body; therefore, highly bioavailable iron is good for health. Iron from dry fish (i.e, haem iron) has more bioavailability than non-haem iron and can meet iron demands at critical stages of the life cycle. Our analysis shows that a daily serving of Olua, Bata, or Loitta could fulfill 100% of the recommended intake of iron for children up to two years and lactating mothers (Figure 3). For a pregnant woman, a daily serving of Olua, Bata, and Churi meets 76.70%, 68.03%, and 52.72% of her daily iron needs, respectively (Figure 3). According to our data, the potential contribution of dry fish to individuals' recommended iron intake is higher than that of fresh Thai pangas and Tilapia in Bangladesh (Figure 3).

Zinc (Zn) was analyzed in 3.37% (*n* = 38) of entries, and we found that the average zinc content of dry fish was 4.31 mg (per 100 g of dry fish). The zinc content ranged from 0.23 mg to 19.30 mg per 100 g, and the highest amount was identified in dried Tengra (*Mystus tengra*). In many low- and middle-income countries including Bangladesh, zinc is deficient in diets. Thus, zinc-rich dry fish can contribute to reducing the gap. As demonstrated in Figure 4, a daily serving of dried Tengra provides 100% of the recommended intake of zinc for children aged up to two years and pregnant and lactating mothers. Like other nutrients, dry fish contributes much more to individuals' recommended zinc intake compared to reference fresh fish (Figure 4).

**Figure 3.** Potential contribution (%) of dry fish to the RNI of iron for children and pregnant and lactating women in Bangladesh. Local names: Olua (*Coilia neglecta*), Bata (*Cirrhina reba*), Loitta (*Harpadon nehereus*), Kachki (*Corica soborna*), Churi (*Trichiurus lepturus*), Thai Pangas (*Pangasianodon hypophthalmus*), and Tilapia (*Oreocbromis mossambicus*).

**Figure 4.** Potential contribution (%) of dry fish to the RNI of zinc for children and pregnant and lactating women in Bangladesh. Local names: Tengra (*Mystus tengra*), Kata mach (*Osteogeniosus militaris*), Lakhua (*Polynemus indicus*), Kachki (*Amblypharringodon microlepin*), Rita (*Rit rita*), Thai Pangas (*Pangasianodon hypophthalmus*), and Tilapia (*Oreocbromis mossambicus*).

In 3.90% (*n* = 44) of the entries, calcium (Ca) content was reported. According to the data analysis, the calcium content of dry fish varied from 3590 mg (dry Chela) to 33.70 mg (dry Rita) per 100 g of dry fish with an average of 954.61 mg. (Table 1). Figure 5 represents how one serving of dry fish could cover the recommended calcium intake of the target population. The calculation shows that one serving of dry fish could significantly meet the calcium requirement for women and children. We found one serving of the respective dry fishes (i.e., Chela, Olua, Bata, Punti, and Khailsa) could meet 100% of the daily dietary calcium requirement of the women and children. Similarly, dry fish are ahead in the case of potential contribution to the recommended intake of calcium compared to the referenced raw fish (Figure 5).

#### *3.3. Vitamins*

Only 0.71% (*n* = 8) of entries were found to report the vitamin content of the dry fish. Data show that the average folate, thiamine, riboflavin, and vitamin B6 content of the dry fish was 22 μg, 0.16 mg, 0.68 mg, and 0.43 mg per 100 g of dry fish, respectively. Based on the data, vitamin content was found to be higher in dried Fesha (*Engraulis tellara*) compared to other dried fishes. Moreover, a study reported that the vitamin D content of dried Giant sea perch (local name: Vetki) was 4.7 μg [22].

#### *3.4. Fatty Acids and Amino Acids*

Major fatty acid and amino acid contents of the dry fish were found in 13.30% (*n* = 150) of entries. The average fatty acid and amino acid content per 100 g of dry fish with the highest and lowest values is presented in Table 1. According to the data, the three most common fatty acids were palmitic acid, palmitoleic acid, and leic acid with an amount of 47.15 g, 11.53 g, and 11.35 g per 100 g of dry fish, respectively. The highest amount of palmitic acid and palmitoleic acid was found in the Koladia (*Otolithoides pama*) and Bol koral (*Lates calcarifer*) species, respectively (Table 1). Moreover, the PUFA content of dried Kauwa fish (*Megalaspis cordyla)* varied from 26.74% (traditional drying) to 30.45% (solar drying) in which docosahexaenoic acid and eicosapentaenoic acid were prominent [72]. The amino acid content of the dry fish shows that they contain all the essential amino acids, including sulfur-containing amino acids (e.g., methionine) which are lacking in plant protein and lysine which is absent in terrestrial meat proteins. According to the data, the highest amount of essential amino acids was found among three species, namely, Churi (*Lepturacanthus savala*), Coral (*Lates Calcarifer*), and Rupchanda (*Pampus chinensis*) (Table 1).

#### **4. Discussion**

The analysis of fish consumption data presented by the International Food Policy Research Institute (2006/7) shows that dried fish is one of the most frequently consumed categories of fish in Bangladesh [9]. Although dry fish plays a significant role in the diet and nutrition of the people in Bangladesh, there is a dearth of information about evidencebased documentation of the nutritive aspects of dry fish and its potential contribution to the recommended nutrient intake of vulnerable groups such as children and pregnant and lactating women. This study reviewed and accumulated the available nutrient composition data on dry fish produced in Bangladesh and represents its potential contribution to meeting the nutrient requirement of children and pregnant and lactating women. We found that most of the studies analyzed dry fish for proximate composition (i.e., moisture, protein, fat, and ash) rather than minerals, vitamins, and fatty acids. Our review suggests that future research should focus on the analysis of nutrients including vitamins, minerals, fatty acids, and amino acids. By documenting the available nutrient composition data on dry fish, our review provides a baseline resource for fisheries, nutrition researchers, and policymakers to better understand the need to include dry fish in food-based interventions to reduce malnutrition in Bangladesh.

Our analysis showed that different kinds of dry fish were able to meet the daily recommended intake of protein, iron, zinc, and calcium for children up to 2 years old and pregnant and lactating mothers. Additionally, dry fish contain more nutrients than the considered reference fresh fish (Thai pangas and Tilapia). A recent review, focusing on the global context, on fish nutrient composition conducted by Byrd et al. (2021) [28] also considered the RNI in the study. Generally, the RNI is a more conservative estimate than the estimated average requirement (EAR), which provides a concrete scientific basis for meeting the requirements of nearly all individuals in a group and the adequacy of diets [26].

The nutrient content of a particular fish species could vary from one habitat to another and season to season due to the variation in the amount and quality of food consumed by the fish and also their movement [77–79]. In our review, we found that the nutrient content of dried Loitta was analyzed in twelve studies; however, the crude protein content varied from 32.21 g to 67.21 g (per 100 g of dry fish). This inconsistency in the nutrient content of the same fish species might be due to the drying methods of raw fish (whether sun, solar, or mechanically dried), fish capture season, source of raw fish collection, and quality of the raw fish. Therefore, future comparative studies evaluating the effect of different drying techniques on nutrient composition are recommended to better understand the quantitative differences in the nutrient content of dry fish samples from the same species.

Evidence shows that the nutrient content of the dry fishes mainly varies due to the nutrient composition of their respective raw fishes and geographical differences [80]. According to the extracted data, dry fishes prepared from marine sources were more nutritious considering their proximate composition and fatty acid and amino acid contents. For instance, Churi (*Trichiurus savala*) had comparatively higher protein content, Rupchanda (*Pampus chinensis*) had higher fat content, Tuna (*Tunnus albacores*) had comparatively higher lauric acid content, and Churi (*Lepturacanthus savala*) had comparatively higher essential amino acid content than the other dry fishes. Marine fish species are rich in nutrients such as polyunsaturated fatty acids [81]; therefore, even after drying, they could retain a substantial amount of nutritive properties. A previous study reported no significant effect of drying on the fatty acid profile and composition of the dried cod heads [82]. Bangladeshi dry fish from freshwater sources contains higher protein than the respective freshwater dry fish in Northeast India as investigated by Ullah et al. (2016) [83]. Based on the extracted data, we found that mineral content was higher among small dry fish species, including Olua (*Coilia neglecta*), Tengra (*Mystus tengra*), Chela (*Salmostoma acinaces*), and Khailsa (*Colisa fasciata*), than the large dry fish. Several studies also reported that small fish and their dried products are good sources of minerals [28,84,85].

Sun drying is the most used fish drying technique in Bangladesh. It is a traditional preservation method of fish that is carried out in the open air using sunlight to evaporate the water and the airflow to carry away the vapor [86]. Though sun drying has several advantages, including a simple operation technique and economical convenience, it has some demerits too. The major constraints of traditional fish drying include dependency on weather, a long drying period (2–3 days), and the required hygienic handling of raw materials [86].

#### *4.1. Hazards Associated with Dry Fish Production in Bangladesh and Some Recommendations*

Dry fish is proven to have a higher nutritional profile that is important for public health. However, the quality of dried fish can be degraded due to various hazards in the production chain. Rasul and his colleagues (2020) [20] summarized the chemical and microbiological hazards of dried fish in Bangladesh. They reported that several dried fishes were contaminated with a high content of heavy metals (for example, Pb, Cd, and Cr) and pesticide residue (dichlorodiphenyltrichloroethane, heptachlor, endrin, aldrin, and dieldrin), and highly pathogenic *E. coli*, *Salmonella* sp., and *Vibrio* sp. were found in a few dried fish samples that may cause serious health hazards after consumption [20]. They also reported that lipid oxidation occurred in some dried fishes from Bangladesh which are responsible for the unpleasant flavors and odors. Sun-based drying affects polyunsaturated lipids and can promote lipid oxidation, which can reduce the nutritive value and functional quality and raise consumer health risks [7]. Two recent Bangladeshi studies conducted by Hoque et al. (2021) [87] and Rakib et al. (2021) [88] reported that heavy metals pose moderate-to-high health risks to the dry fish consumer. There are several factors associated with these health hazards which include traditional drying techniques, the use of harmful pesticides, anthropogenic contamination, atmospheric deposition, the lack of maintaining proper hygiene and sanitation, improper packaging and storage, and water pollution [20,88]. To minimize and prevent these hazards of dry fish production in Bangladesh, some recommendations include: (i) developing improved and cost-effective methods of fish drying [89], (ii) designing effective packaging and storage facilities, (iii) ensuring heavy metal decontamination strategies, (iv) organizing public health awareness programs for dry fish producers regarding basic hygiene and sanitation practices, the adverse effect of chemical contaminants in dry fish, and the importance of the quality of the raw materials used, and (v) providing training on safe dry fish production and waste management to dry fish producers.

The use of harmful chemicals in dry fish production is a special concern. In Bangladesh, dry fish producers usually use harmful chemicals (such as a mixture of organochlorine) to protect dry fish from insect infestation and to increase shelf life [19] without considering their deleterious impacts on human health. A study found the presence of harmful chemicals such as DDT and heptachlor in some dry fish samples in Bangladesh [90]. These pesticides are used to protect fish from insect infestation; however, they are associated with serious health problems, including cancer and non-allergic reactions, and environmental hazards [71]. Considering the situation, the following recommendations can be highlighted to prevent the use of harmful chemicals or insecticides during dry fish production: (i) Ensuring strict implementation of laws and policies related to harmful pesticide use. In many countries, including Sweden, Japan, and the USA, organochlorine chemicals have been banned due to their potential harm to human health. Therefore, the government of Bangladesh may strictly implement the updated pesticide legislation and policies for fishery products. (ii) Developing and implementing safe insect control strategies instead of using harmful insecticides. For instance, red pepper and turmeric (separately or mixed) have insect- and bacteria-repealing characteristics and thus can be used in fish drying. Evidence shows that the pretreatment of fish with 10–12% salt for 10–12 h can reduce infestation [91]. (iii) Sensitizing dry fish producers to the harmful impacts of pesticides on human health and the food system. (iv) Taking initiatives to prevent environmental pollution so that heavy metals and harmful chemicals cannot enter the aquatic ecosystem.

#### *4.2. Implications for Practice*

The findings of our study (i.e., up-to-date data on the nutrient composition of dried fish) can be useful to policymakers, public health practitioners, and nutrition experts for developing nutrition-based programs and interventions to improve the country's food and nutrition security. Such programs and interventions could encompass formulating dietary guidelines, updating the food composition table, promoting dry fish production and consumption, and formulating nutrition-education-related materials.

Again, an area that requires further exploration is assessing the shelf lives of dried fish during storage over a period of time to explore possible nutritional changes that may occur when dried fish is under storage. Assessing the impact of consuming dried fishes on growth and nutritional status among vulnerable populations is another key research area that has to be further explored.

Inclusion of nutritious food items in the diet is important for health and survival. Globally, the recent coronavirus (COVID-19) pandemic has imposed a new set of challenges on humans to maintain a diversified and healthy diet [92–94]. Energy, protein, and micronutrient (especially vitamin A, B complex, C, and D, zinc, iron, and selenium) deficiencies are associated with impaired immune function and an increased risk of infection and mortality among vulnerable populations as well as COVID-19 patients [94,95]. Evidence shows that zinc, magnesium, and vitamin C have a potential role in reducing the severity of the infection and inflammatory response associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), while folate and vitamin D may have a role in antagonizing the entry of virus in host cells [96,97]. Zinc has the potential to reduce viral replication and increase immune responses as well as act as a prophylactic which might provide an additional shield against the initiation and progression of COVID-19 [98]. Since dry fish contain high nutritional properties, including protein and minerals (Fe, Zn, and Ca), the inclusion of dry fish in the diet could be a consideration especially for at-risk groups during this COVID-19 pandemic [99]. For instance, in Myanmar, the use of dry small fish powder provides an opportunity for accessible and acceptable forms of micronutrients required to improve the health status of young children during this pandemic [100]. The example from the Myanmar study [100] suggests the potential fortification of staple cereal and grain-based complementary foods with dried fish powder that can improve the nutrient quality of foods that are used to feed young children in most developing countries. A similar concept could also be replicated for Bangladesh. Dry fish of high nutritional quality could be considered for powder preparation and be used to improve the nutritional quality of complementary food for young children. Bangladesh is a disaster-prone country. As dry fish could be stored for a longer period of time, it could be included in the ration after any disaster, including floods and cyclones.

#### **5. Conclusions**

According to available information, dry fish possess high amounts of nutritional properties, especially protein, zinc, iron, and calcium. In particular, dry fish from marine sources and small fish species are high in protein, fatty acids, amino acids, and minerals. Dry fish significantly contributed to the recommended intake of protein, iron, zinc, and calcium for children up to two years and pregnant and lactating mothers. It is imperative that policymakers along with food and nutrition experts focus on promoting the nutritional value of dried fish and encouraging particularly vulnerable populations (children and pregnant and lactating women) to include dry fish in their diet. Further research in this area may emphasize analyzing vitamin and mineral composition rather than proximate analysis. Further research may highlight and analyze the essential fatty acid composition (omega-3, -6, and -9) of different species of fishes and its impact on improving the cognitive development and functioning/performance of children and the elderly, respectively.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/fishes7050240/s1, Table S1: Characteristics of included studies (N = 48), data extraction file, and calculation of RNI.

**Author Contributions:** M.H.A.B.: conceptualization, study design, literature search and screening of articles, writing—original draft (Abstract, Introduction, Results interpretation, Discussion, and Conclusion) and editing. A.A.Z.: literature search and screening of articles, data management and analysis. N.R.: literature search and screening of articles, data curation. M.A.M.S.: literature search and screening of articles. M.A.B.S., J.E.H.J., C.N.A.N.-A., A.-A.S., B.O.A. and M.S.I.K.: visualization, validation, writing—reviewing and editing. M.A.R.: conceptualization and supervision, study design, visualization, validation, writing—original draft (Materials and Methods), critical review for intellectual content, editing. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding. The article processing charge (APC) was funded by Bielefeld University, Germany.

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** Data are contained within the article or Supplementary Material.

**Acknowledgments:** The authors would like to express their humble gratitude to Bielefeld University, Germany, for providing financial support through the Institutional Open Access Publication Fund for the APC.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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