1. Introduction
Larvae of many aquatic species either have complete dependence on zooplankton live food as a basal diet, or they have significantly better performance when started on live food [
1]. Live food is commonly regarded as “living capsules of nutrition”, rich in proteins, vitamins, carbohydrates, minerals, amino acids, and fatty acids [
2]. As a superior nutritional prey, some zooplankton contain high levels of digestive enzymes [
3] and are capable of producing appetite-stimulating effects on larvae [
4]. Live food organisms are able to swim freely in the water column, thereby being constantly accessible to finfish and crustacean larvae [
5,
6]. Their jerking movements are likely to stimulate larval feeding responses [
7]. On the contrary, formulated feeds often accumulate on the water surface or some slowly sink to the bottom, whereby becoming less accessible to larvae [
5]. Zooplankton such as rotifers and
Artemia are by far the most commonly utilized live food in the cultivation of finfish and crustaceans [
8].
Substitution of live food by formulated diets has been emphasized [
9]. However, the sole application of a formulated diet may seem like a far-fetched idea due to low its digestibility and the deterioration of water quality [
6,
7]. Even though the use of live food in larval rearing has been reported to improve larval growth performance, survival, and disease resistance [
1,
10,
11], the cultivation and management of live food for aquatic production is costly and unpredictable [
12]. Multiple studies have demonstrated the success of total live food replacement or reduction in aquaculture [
9,
13]. It is important to understand the nutritional requirements of fish larvae in order to facilitate the optimization of diets and feeding protocols, which may subsequently enhance larval quality [
7,
14]. Consequently, several studies have emphasized developing practical methods to improve the nutritional status of live food with essential nutrients [
15,
16,
17,
18,
19,
20].
By taking advantage of primitive feeding characteristics, the manipulation of the nutritional status of zooplankton is achievable by pre-feeding them through the so-called “bioencapsulation” or “enrichment” protocols. Through enrichment techniques, essential nutrients lacking in zooplankton, prophylactics, and therapeutics can be delivered to fish larvae via zooplankton live food. The application of enriched live food is reflected in enhanced growth, survival, stress tolerance, and microbial diversity for a variety of aquatic species [
19,
21,
22,
23,
24]. A very important aspect of live food enrichment is its reproducibility and predictability, which are crucial in commercial hatcheries. Hence, it is necessary to constantly produce high-quality live food on a large scale [
15]. However, producing enriched live food with consistent levels of the important nutrients can be complex. This review aimed to emphasize the significance of live food and the implementation of different enrichment techniques to incorporate nutrients such as minerals, vitamins, microalgae, lipids, and probiotics to enhance the nutritional status of the live food and to subsequently boost the health of the aquatic animals.
2. Enrichment with Fatty Acids
Highly unsaturated fatty acids (HUFA) with 20 or more carbon atoms are one of the major sources of metabolic energy during the embryonic and pre-feeding larval stages in fish. However, these energy sources rapidly declined during the endogenous feeding stage [
25]. The n-3 series HUFA docosahexaenoic acid (DHA, 22:6n-3) and eicosapentaenoic acid (EPA, 20:5n-3), and the n-6 series HUFA arachidonic acid (ARA, 20:4n-6), play significant roles in fish larval development; thus, the deficiency of HUFA may impair fish growth, reproduction, and survival, causing pale or swollen liver, myocarditis, intestinal steatosis, lordosis, fin erosion, and shock syndrome [
26]. HUFA are synthesized in very small concentrations from their precursors alpha-linolenic acid (ALA, 18:3n-3) and linoleic acid (LA, 18:2n-6) [
27] due to the lack of delta-5 and delta-6 desaturases and elongases in marine fish larvae [
28]. Therefore, HUFA must be incorporated through live foods such as copepods, rotifers and
Artemia to meet the requirements for larval growth [
27]. The requirements of HUFA in fish and crustaceans have been widely studied. The effects of dietary HUFA in the juveniles of golden pompano (
Trachinotus ovatus) [
29], yellowtail (
Seriola dumerili) [
30], Asian seabass (
Lates calcarifer) [
31], and Pacific white shrimp (
Litopenaeus vannamei) [
32] are among the most recently published studies.
Enrichment of live food with commercial oil emulsion (Super Selco, DHA Selco, Selco S.presso) is a common practice [
33,
34,
35,
36,
37,
38,
39,
40]. Several studies have assessed the dietary fatty acid profiles of copepods and enriched
Artemia [
39,
41,
42]. The predominant fatty acids in copepods are DHA, EPA, and palmitic acid, while DHA, EPA, and oleic acid are the predominant fatty acids in
Artemia enriched with Super Selco and DHA Selco [
41]. Apart from the absolute amount of HUFA, the dietary DHA/EPA ratio is suggested to impact the normal growth and development of certain fish species [
43,
44]. The average DHA/EPA ratio for copepods ranged between 1.83 and 5.5 whereas the DHA/EPA ratio for
Artemia enriched with DHA Selco ranged from 1.4 to 2.2 [
41,
42]. The DHA/EPA ratio of
Artemia enriched with Super Selco at 600 mg/L for 16 h was reported at 0.2 [
39], whilst enrichment at 200 and 300 mg/L for 24 and 20 h, respectively, brought about 0.68 and 0.3 DHA/EPA ratio, respectively [
38,
41]. Altogether,
Artemia enriched with DHA Selco recorded a higher DHA/EPA ratio than that of Super Selco. The instability of HUFA and the catabolism of these compounds by
Artemia in addition to low DHA retention efficiency in
Artemia during the first 24 h post enrichment might be the contributing factors to this variation [
38,
41,
45]. Commercial emulsions are more stable and effective as the primary emulsions are mainly made from HUFA-rich fish oils and emulsified with egg yolk and seawater. However, these forms of enrichment formula are low in efficiency but are cheap alternatives in developing countries [
7]. Higher DHA and EPA contents and DHA/EPA ratio were recorded in the freshwater cladoceran
Moina micrura enriched with commercial emulsion (Maxepa MERCK, Delhi, India) in addition to gelatine, egg yolk, and Celin [
19]. Modifications of dietary fatty acid compositions of rotifers and
Artemia should be made in line with those of copepods.
Boosting of the nutritional status of rotifers [
46,
47],
Artemia [
48,
49], copepods [
50,
51,
52,
53,
54,
55,
56,
57], and
Moina [
58] through algal enrichment techniques is a common practice to boost the quality of the otherwise nutrient-deficient feed. Microalgae is a rich source of HUFA and polyunsaturated fatty acids (PUFA) [
43,
59,
60]. It is easier to control the essential fatty acid (EFA) composition of enrichment emulsions when microalgae-derived oil is used in comparison with purified fish oils [
61]. Due to the high cost and difficulty in producing, concentrating, and storing live microalgae, the development of different forms of microalgae as a replacement to live microalgae has become a major focus of research [
62]. A cheaper microalga paste has been used in aquaculture practice as an alternative to live microalgae [
63]. Rotifers fed on microalgal pastes (
Nannochloropsis oculata and
Chlorella vulgaris) at equal quantities were rich in palmitic acid, linoleic acid, and EPA after 48 h of exposure to the microalgal diet. However, the DHA content was only recorded at 6 mg/g dry weight (DW). Nevertheless, the DHA content was enough to improve the growth, development, and stress resistance of fish larvae [
64]. This study underlined the importance of enriching rotifers fed to larvae with multiple microalgal species over monospecific diets.
A previous study investigated the fatty acid composition of rotifers enriched with a mixture of DHA-enriched
C. vulgaris (Super fresh Chlorella V12, SV, Chlorella Industry, Tokyo, Japan) and DHA emulsion (Bio Chromis, Chlorella Industry, Tokyo, Japan) for 12 h [
43]. The DHA content in enriched rotifers increased from 0.1 to 15.4 % and the DHA/EPA ratio was highest in the treatment. DHA was found to be dominant in rotifers enriched with DHA- and arachidonic acid (AA)-rich oils extracted from the dinoflagellate
Crythecodinium sp. and the fungus
Mortierella alpina, respectively, in addition to EPA-rich marine oil [
65]. Rotifers have a better retention rate of EPA compared to DHA, regardless of the ratio in their enrichment [
44]. Enrichment of rotifers can be achieved either through short-term enrichment (alteration of the lipid content of the rotifers just before larval feeding) and long-term enrichment (feeding of rotifers on a complete diet) [
66,
67]. Enriched DHA was stable in rotifers at 10 °C for at least 24 h post-enrichment under starving conditions, whereas a higher temperature of 20 °C significantly decreased the DHA level during starvation [
68]. Rotifers emptied their gut at a reduced rate as culture temperature decreased from 26 °C to 4 °C [
69]. Moreover, microalgae are often added to the enrichment formula to promote “green water” to maintain the nutritional quality of zooplankton [
64,
70,
71]. The larvae of rainbow trout (
Oncorhynchus mykiss) [
72], Russian sturgeon (
Acipenser gueldenstaedtii) [
73], Atlantic sturgeon (
Acipenser oxyrinchus) [
74], caspian kutum (
Rutilus frisii kutum) [
75], yellowtail flounder (
Limanda ferruginea) [
65], gilthead seabream (
Sparus aurata) [
28], and greater amberjack (
S. dumerili) [
24], whitefish (
R. kutum) fry [
76], and juvenile milkfish (
Chanos chanos) [
77] have been reared with live food enriched with essential fatty acids.
The high contents of EPA, DHA, and some digestive enzymes in copepods are among the important properties that make them a superior live food to
Artemia and rotifers [
6]. Therefore, it is recommended to enrich zooplankton in order to meet copepod HUFA levels. The enrichment emulsions are commonly prepared using commercial emulsions such as DHA Selco and Super Selco. To meet the copepod DHA/EPA ratio, it is recommended that
Artemia and rotifers be enriched with DHA Selco. Even though studies on HUFA enrichment in
Moina are fairly limited, a study has successfully enriched
Moina with Maxepa. Additionally, HUFA enrichment can be performed using microalgae, either live or pastes. It is recommended that microalgae pastes be used as a cheaper alternative to live microalgae, and the application of multiple microalgal species over monospecific diets would be very beneficial. Moreover, a combination of commercial emulsions and microalgae in an enrichment mixture would be advantageous in terms of enhancing the DHA/EPA ratio.