Evaluation of the nutritional value of Artemia nauplii for European seabass(Dicentrarchus labrax L.) larvae

Al A.El-Dhhr ,Rshwn S.Rshwn ,Smy Y.EL-Zeem ,Shim A.Shhin ,Mon M.Mourd ,Mohmmed F.El Bsuini

a Animal and Fish Production Department,Faculty of Agriculture- Saba Basha,Alexandria University,Alexandria,21526,Egypt

b National Institute of Oceanography and Fisheries (NIOF), Cairo,11562,Egypt

c Department of Animal Production,Faculty of Agriculture,Tanta University,Tanta,31527,Egypt

d Faculty of Desert Agriculture,King Salman International University,South Sinai,46618,Egypt

Keywords: European seabass Gastrointestinal histology Antioxidants Vitamin C Vitamin E DHA

ABSTRACT Adapted European seabass (Dicentrarchus labrax L.) larvae at 25 days post-hatching (dph) with a primary weight and length of 23 mg and 9.92 mm,start the weaning up to 46 dph using the weaning micro-diet (54% crude protein from fish meal,powder milk,and poultry egg) concurrently with Artemia enriched with fortification emulsions (0.6 g/L) of DHA selco® as a control group (DHAS) or Fish oil +20% Vitamin C (FOVC),or Fish oil +20% Vitamin E (FOVE),or Fish oil +10% Vit.C +10% Vit.E (FOCE),or Fish oil only (FO).At 46 dph,groups of larvae fed enriched Artemia with DHAS and FOVE exhibited the highest final body weight,weight gain,average daily gain,specific growth rate,feed intake,protein efficiency ratio,and survival% as well as the lowest feed conversion ratio.Meanwhile,larvae in FOVC displayed the lowest final body weight,total length,weight gain,average daily gain,specific growth rate,feed intake,protein efficiency ratio,and survival% as well as the highest feed conversion ratio.Gastrointestinal histological assessment exhibited no pathological alteration as well as an improvement in the structure with DHAS,FOVE,FOVC,and FOCE co-additives compared to FO.Glutathione peroxidase enzyme (GPx) recorded the highest rates (P < 0.05) in groups fed Artemia supplemented with DHAS and FOVE followed by FOCE and FOVC.While the lowest record for GPx activity was noted in the FO group.In conclusion,Using augmented Artemia nauplii with fish oil +vitamin E in single or in companion with vitamin C as cheap antioxidants support D.labrax larval growth,survival,and antioxidant efficacy during the critical weaning period.

1.Introduction

Aquaculture is seen as a feasible solution in providing a large durable part of high-quality protein,restocking,and conserving overfished stocks and endangered aquatic species (Cámara-Ruiz et al.,2019;Mzengereza et al.,2021;Zaki et al.,2020).Nevertheless,aquaculture faces numerous barriers (Bentzon-Tilia et al.,2016;Hannon et al.,2013;Naylor et al.,2021).As extensive aquaculture expands,adverse impacts and stressful circumstances for aquatic animals and systems,such as larval mortality,pathogen outbreaks,and water pollution,become more prevalent (Mohapatra et al.,2013;Wang et al.,2008).The efficacy of aquaculture relies on several interrelated aspects,such as the broodstock,which starts with larvae,and the aquatic environment.

Acquiring high-quality eggs and larvae is the opening step towards sustainable aquaculture (Bobe,2015;Lieke et al.,2021;Olafsen,2001).Fish larvae production is often hindered by high mortality rates,which can be caused by pathogens or environmental factors (Lieke et al.,2021;Teiba et al.,2020).Aquatic species have a natural survival rate of less than 1% to sexual maturity,with significant mortality in the early stages(Vadstein et al.,2013).The need for growth and immune stimulants is vital,especially in the initial stages,and antibiotics were the widely employed approach before their use was restricted as a result of the harmful consequences of unbalanced usage (Schar et al.,2021).Antibiotics and other synthetic materials are progressively being switched with environmentally adequate substitutes (Dossou et al.,2021;El Basuini et al.,2022;Zaineldin et al.,2021).

Modulating feed is a key verified procedure to alter the growth and immune system of aquatic species (Magnadottir,2010;Martins et al.,2019;Murray et al.,2010;Tarnecki et al.,2019).Larval nutrition is one of the biggest challenges in aquaculture,and improving nutritional protocols at this stage,especially during weaning and conversion from live feed to commercial feed,is the bottleneck (Lipscomb et al.,2020).The early co-feeding period has shown that the stomach is adapted to commercial feeds,allowing early weaning with a preferable developmental rate when weaning begins toward the termination of the larval stage (Hamre et al.,2013).In the fish hatchery,the importance of live feed as initial feeding for fish larvae is well recognized.Rotifers andArtemiaform the mainstay of any marine hatchery due to numerous characteristics.Artemiahas substantial advantages,most notably the speed and ease of hatching,the reduction in the cost of culturing,and the availability of nauplii hatched from commercially available constant cysts (Manfra et al.,2012).Unfortunately,there are many limitations againstArtemiaincluding its extreme sensitivity (Libralato,2014).

Enrichment of live prey with essential fatty acids (EFAs) is important to achieve better and significant larval survival and development through metamorphosis (Copeman et al.,2002;Smith et al.,2002).Fresh microalgae or commercial augmentation products are popular fortified techniques,but due to the difficulty of microalgae production,commercial enrichment products are more generally employed in the enrichment procedures (Fu et al.,2021).To ensure optimal health and growth of aquatic animals,it is vital to provide an adequate level of highly unsaturated fatty acids (HUFAs) in the diet because of lacking in the desaturation and elongation routes required for the biogenesis of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (Mourente et al.,2000).Fish oil (FO) comprises a high content of n-3 polyunsaturated fatty acids (PUFA) e.g.,DHA and EPA.Thus it is commonly employed as a lipid source for aquatic animals (Ruxton et al.,2005;Tur et al.,2012).Polyunsaturated fatty acids are more susceptible to oxidative stress,necessitating the deployment of a powerful antioxidant system.The activity of glutathione peroxidase is a potent mechanism,which is elevated throughout larval development to preserve the cells by catalyzing the breakdown of H2O2to H2O (Fernández-Díaz et al.,2006;Wang et al.,2006).Vitamins are necessary for normal functions in aquatic organisms at the cellular level,as well as growth and development,emphasizing that vitamin deficiencies lead to the emergence of clear disease symptoms (Dawood et al.,2018).Vitamins C and E are key antioxidant supplements applied in food manufacturing and have been demonstrated to minimize oxidative stress in animals (Gao et al.,2013;Jiménez-Fernández et al.,2015).

In this sense,the present trial was conducted on the European sea bass,Dicentrarchus labrax,which is one of the most valuable extensively farmed marine species in the world (Bjørndal et al.,2019;Grigorakis,2007;Mladineo et al.,2020).The study objects to assess the replacement of the high-priced commercial product DHA selco® with fish oil augmented with vitamin C,vitamin E or a mixture thereof (as antioxidants) or with fish oil without any additive in enriching the nutritional value of live prey (Artemia nauplii) as a co-feed during weaning of European sea bass larvae.

2.Materials and methods

2.1.Experimental animals and rearing conditions

The broodstock of European seabass (D.labrax) from the Marine Hatchery of National Institute of Oceanography and Fisheries (NIOF),Alexandria,Egypt with weights of 1.3—1.7 kg (males) and 1.5—2 kg(females) at a 2 ♂:1♀ ratio was stocked in 2 m3tanks (3 fish/tank).The spawning induction was performed by infusing fish with HCG at concentrations of 1000 IU/kg for females and 10—50 IU/fish for males.After spawning (24—36 h),eggs were assembled from the broodstock tanks and allocated into 2 m3tanks with constant aeration to secure proper eggs flip.Following hatching,larval tanks were enriched with microalgae (Nanochloropsis oculate) at an initial density of 500 ×103cells/ml,which continually reduces until final cessation with larval development up to 20 days post-hatching (dph).Rotifer was provided to larval tanks at a level of 5 individual/ml from 3 dph to 6 dph and gradually extended to a final density of 10—15 ind./mL with larval development up to 20 dph.The live feed (Artemia nauplii) was primarily introduced to the larvae (9 dph) at a density of 0.01 ind./mL with a gradual increase to become the main prey (4 ind./mL).

European seabass larvae (20 dph) were re-allocated into 15 experimental tanks (30 L) for adapting to trial conditions in terms of temperature (21.6◦C),pH (6.91),dissolved oxygen (>4.5 ppm),ammonia(<0.02 ppm),salinity (33),photoperiod (12 h light: 12 h dark),and a stocking density of 15 larvae per litter (450 larvae/tank).Adapted European seabass larvae at 25 dph with an initial weight and length of 23 mg and 9.92 mm,start the weaning up to 46 dph using the weaning micro-diet (54% crude protein — Table 1) concurrently with the live food(enrichedArtemia).

Table 1 European Sea bass (Dicentrarchus labrax L.) weaning diet components and chemical analysis.

2.2.Artemia enrichment and feeding regime

Five enrichment emulsions (0.6 g/L) were prepared as follows:

-The control (DHAS): 0.6g DHA selco® emulsified in 1 litter filtered water (35—40◦C) for 5 min and then cooled to room temperature.

A standard solution (solution A) was prepared as a base for other treatments emulsions: 0.1 g Lecithin +50 g fish oil emulsified in 50 g filtered water (35—40◦C) for 5 min and then cooled to room temperature.

-FOVC: 25g solution A+2.5g vitamin C well mixed for 5 min,then 1.3 ml from resulting mixture emulsified in 1 litter filtered water(35—40◦C) for 5 min and then cooled to room temperature.

-FOVE: 25g solution A+2.5g vitamin E well mixed for 5 min,then 1.3 ml from resulting mixture emulsified in 1 litter filtered water(35—40◦C) for 5 min and then cooled to room temperature.

-FOCE: 25g solution A +1.25g vitamin C+1.25g vitamin E well mixed for 5 min,then 1.3 ml from resulting mixture emulsified in 1 litter filtered water (35—40◦C) for 5 min and then cooled to room temperature.

-FO: 1.2 ml from solution A emulsified in 1 litter filtered water(35—40◦C) for 5 min and then cooled to room temperature.

Artemia was developed in the enrichment emulsions for 24 h and gathered using a plankton net and rinsed three times in room temperature seawater according to Adloo et al.(2012).The micro-diet (pellet size 100—179 μm) and live food were manually offered 4 times a dayad libtium.

2.3.Growth variables,feed utilization,and survival rate

Initial body weight (W0),final body weight (Wt),total length (TL),feed intake (FI),and numbers of larvae were recorded during the trial period (t) to compute the following indices (Shadrack et al.,2022;Zaineldin et al.,2021):

2.4.Glutathione peroxidase (GPx) activity

Glutathione peroxidase (GPx) in the whole larvae body was assessed using Nicotinamide Adenine Dinucleotide Phosphate Hydrogen(NADPH) peroxidase coupled response (Flohé &Günzler,1984).Briefly,whole larval tissues were homogenized (Hettich model EBA 12R,Germany) in cold buffer (50 mM phosphate buffer,pH 7.0,5 mM EDTA) at a ratio of 1w: 4—8v(sample weight: buffer volume) and centrifuged for 10—20 min at 4000 rpm and 2—8◦C to obtain a supernatant fluid which examined spectrophotometrically at 340 nm to detect GPx activity according to the following equation:

where F is a constant that is used to convert absorbance per minute (A/min) into enzymatic units (U);RV=Reaction volume;SV=Sample volume;BV=Buffer volume (mL) used to dilute 1 g of tissue during the enzyme extraction;6.22=NADPH molar extinction coefficient (Mm./cm).

2.5.Histological investigations

For a histological examination,three post-larvae were gathered haphazardly from each tank and primary preserved for 48 h at room temperature with 4% formaldehyde fixative followed by ethanolic dehydration in 70%,80%,90%,and 100% concentrations and then embedded in paraffin for sectioning at a thickness of 7 μm (Rotary Microtome 2145,Leica Microsystems).Sections were placed on clean glass slides and stained with hematoxylin and eosin (H &E) and screened with light microscopy (LEICA,Leica Microsystems AG,Wetzlar,Germany) at magnifications of ×10 and ×40.The histo-assessment was completed for five villi using the ImageJ program software.

2.6.Statistical analysis

Experiment data were subjected to variance normality and homogeneity check using Shapiro-Wilk and Levene tests.The results of the One-way ANOVA and LSD’s post hoc test were provided as a mean of three replicates with standard errors (SE) using SPSS (SPSS Inc.,Ver.20,Richmond,VA,USA).

3.Results

3.1.Larval growth indices,nutrient efficacy,and survival rate

Growth performance,feed consumption,and survival rate of European bass (Dicentrarchus labraxL.) larvae at 46 days post-hatch (dph) are presented in Table 2.Groups of larvae fed enrichedArtemiawith DHAS and FOVE exhibited the highest final body weight,weight gain,average daily gain,specific growth rate,feed intake,protein efficiency ratio,and survival% as well as the lowest feed conversion ratio.Meanwhile,Larvae in FOVC displayed the lowest final body weight,total length,weight gain,average daily gain,specific growth rate,feed intake,protein efficiency ratio,and survival% as well as the highest feed conversion ratio.In addition,no remarkable alteration (P>0.05) among FOVC,FOCE,and FO in terms of final body weight,average daily gain,specific growth rate,and feed intake.The highest final total length (FTL) values were recorded with larval groups fedArtemiasupplemented with vitamin E(FOVE) in single or in companion with vitamin C (FOCE).The condition factor (K) of larvae fedArtemiawith DHAS or FO displayed higher values and the lowest K value was in the FOCE group.

Table 2 Growth performance,feed consumption,and survival rate of European bass(Dicentrarchus labrax L.) larvae at 46 days post-hatch (dph) fed enriched Artemia nauplii with mean ±SE.

3.2.Histological characteristics

Fig.1 and Table 3 show the gastrointestinal photomicrograph and morphometric analyses of the esophagus,stomach,and intestine of European bass (Dicentrarchus labraxL.) larvae (46 dph).

Fig.1.Photomicrograph of a transverse section (TS) in the esophagus,stomach,and intestine of European bass (Dicentrarchus labrax L.) larvae (46 dph).DHAS: DHA selco® as a control group;FOVC: Fish oil +20% Vit.C;FOVE: Fish oil +20% Vit.E;FOCE: FOVE: Fish oil +(10% Vit.C +10% Vit.E);FO: Fish oil.Where,arrows point to mucosa (mu),submucosa (Smu),muscular (mus),goblet cells (Go),gastric epithelial gland (Gg),and lumen (＊).

Table 3 Gastrointestinal morphometric analyses of esophagus,stomach,and intestine of European bass (Dicentrarchus labrax L.) larvae (46 dph).

3.2.1.Esophagus

Groups fedArtemiaenhanced with DHAS,FOVE,FOVC,and FOCE co-additives had the highest values of the esophagus folds count,and the lowest count was in the FO group.Goblet cells recorded the highest counts in DHAS,FOVC,and FOCE groups followed by the FO group and the lowest count was in the FOVE group.The highest mean length of the esophagus villi was noticed in FOVE and FOCE larvae compared to other groups (DHAS,FOFC,and FO).

3.2.2.Stomach

Groups fedArtemiaaugmented with FOVC,FOVE,and FO cosupplements had the lowest values of stomach folds,and the highest fold was in the DHAS group that was co-significant with the FOCE group.Stomach fold length in the ofD.labraxat 46 dph was improved (P<0.05) in DHAS,FOVE,and FOCE followed by FOVC while the lowest fold was in the FO group.

3.2.3.Intestine

Larvae fed enrichedArtemiawith FOVE exhibited higher intestinal villi count followed by DHAS and FOCE while the lowest counts were in FO and FOVC.Also,larvae in groups FOVE and DHAS displayed the highest goblet cell counts (P<0.05) followed by FOVC and FOCE groups while the minimal count was recorded in the FO group.The largest length of villi in the intestine was observed in FOVC,DHAS,and FOCE groups,while the least was noted in the FO group.

3.3.Glutathione peroxidase activity (GPx)

Fig.2 presents the activities of the glutathione peroxidase enzyme(GPx) in experimental groups.GPx recorded highest rates (P<0.05) in groups fedArtemiasupplemented with DHAS (118.67 ± 40.11,U/mg)and FOVE (102.71 ±28.69,U/mg) followed by FOCE (77.55 ±7.18,U/mg) and FOVC (39.4 ± 10.3,U/mg).While the lowest record for GPx activity was noted in the FO group (5.97 ±0.94,U/mg).

Fig.2.Glutathione peroxidase activity of European bass (Dicentrarchus labrax L.) larvae (46 dph).DHAS: DHA selco® as a control group;FOVC: Fish oil+20% Vit.C;FOVE: Fish oil +20% Vit.E;FOCE: FOVE: Fish oil+(10% Vit.C+10% Vit.E);FO: Fish oil.

4.Discussion

Commercially,Artemiaand rotifers are commonly employed as live meals for aquatic animals’ larvae and fry (Dhont et al.,2013).Many types of aquatic organisms have benefited from the enrichment of live food,which has been proven to boost their survival and growth performance (Kandathil Radhakrishnan et al.,2020;Rasdi &Qin,2016).It is critical to augment live prey with essential fatty acids (EFAs) to enhance larval survival and growth (Copeman et al.,2002;Smith et al.,2002).Because aquatic animals lack the desaturation and elongation paths required for the manufacture of EPA and DHA,it is vital to provide a suitable content of highly unsaturated fatty acids (HUFAs) in their diet to guarantee ideal health and performance (Oboh et al.,2017).Fish oil(FO) is frequently employed as DHA and EPA source for aquatic species(Ruxton et al.,2005;Tur et al.,2012).Commercial DHA (DHA selco®)contains both vitamin C and vitamin E,as well as most of the elements that larvae require at this age,making it highly-priced.This study is attempting to purpose a low-cost alternative that performs a similar antioxidant impact.

Results of larvae fed enrichedArtemiawith DHAS and FOVE exhibited the highest final body weight,weight gain,average daily gain,specific growth rate,feed intake,protein efficiency ratio,and survival%as well as the lowest feed conversion ratio.In addition,the highest final total length (FTL) values were recorded with larval groups fedArtemiasupplemented with vitamin E in single or in companion with vitamin C.The beneficial impacts of Vitamin E in single or in companion with vitamin C may be linked to their antioxidant amplitude,which benefits in various ways starting with fish oil protection against peroxidation.

The highly unsaturated fatty acids (HUFAs) in fish oil are exceedingly susceptible to lipid peroxidation under natural conditions,particularly during storage and preparation of feed (Hasanpour et al.,2017;J.;Wang et al.,2016).Several studies have shown that oxidized fish oil can cause oxidative stress in aquatic animals and harm their development,metabolism,antioxidant,and hepatic mitochondrial role(Chen et al.,2019;Jian Gao et al.,2012;Song et al.,2019;Yin et al.,2019).Vitamins are required for basic cellular activities as well as growth and development in aquatic animals (Dawood et al.,2018).Mainly vitamin E and vitamin C act as potent antioxidants (Gao et al.,2013;Jiménez-Fernández et al.,2015),in addition to roles of Vitamin C as the electron donor,co-factor in the synthesis of hormones and collagen (El Basuini et al.,2017),and regenerate and/or afford vitamin E from α-tocopheryl (Jiménez-Fernández et al.,2015).

Results of larvae fed enrichedArtemiawith a mixture of Vitamin E and Vitamin C (FOCE) exhibited better performance and survival rate compared to those fed on solo Vitamin C (FOVC).The existence of an interaction mechanism of vitamin C/E has been proposed as a justification for the wide range of superior performance and vitamin E deficiency insensitivity stated in farmed aquatic species (Dawood et al.,2018).Because Vitamin E is the most basic hydrophobic antioxidant,elevating dietary PUFA would speed up the auto peroxidation cycle of Vitamin E,hence expanding the need for this nutrient (Izquierdo et al.,2001,2019).In this context,Atalah et al.(2012) declared that increasing vitamin E levels from medium to high improved larval performance in terms of total length at medium dietary HUFA levels.Furthermore,a rise in vitamin E increased the concentration of HUFAs in the larval polar lipids,indicating that vitamin E has an antioxidant impact.Moreover,Lee et al.(2015) stated that adding Vitamin E to the diet promotes larval development and reduces lipid peroxidation in tissues in both seabass and seabream.Also,Tocher and Glencross (2015)stated that expanding Vitamin E levels improve larval development.In addition,the affirmative outcomes were recognized with enriching live feed on HUFA and Vitamin C as results of shielding HUFAs from oxidation,maintaining Vitamin E level,developing larval tissue,and supporting larval development in milkfish,Chanos chanos(Gapasin et al.,1998),cuttlefish,Sepia officinalisL.(Koueta et al.,2002),Acipenser persicusandHuso huso(Noori et al.,2011),Senegalese sole,S.senegalensis(Jiménez-Fernández et al.,2018),Patagonian red octopus,Enteroctopus megalocyathus(Hernández et al.,2019),Anabas testudineus,Bloch,1792 (Singh et al.,2019).

The digestive efficiency of larvae is expected to be lower than that of adults due to their simpler digestive system.Most fish species,on the other hand,have digestive enzymes from the start of external feeding(Martínez-Lagos et al.,2014).Moreover,the diet has long been known to have an impact on the gut and its biological function (Buddington et al.,1997).The function of villi is to absorb nutrients,whilst the blind end provides for a longer retention time for nutritional content within the digestive tract while mucus-secreting goblet cells contain antimicrobial features,making transit through the intestinal epithelium easier,and keeping the gut epithelium intact (Firdaus-Nawi et al.,2013).The results of the histological study show that a functioning digestive tract with no pathological alterations occurred in any of the experimental groups,with a considerable improvement in histological structure,such as fold length,villi length,and goblet cell count,notably in the DHAS and Vitamin E in a single form or companion with vitamin C.The improvement in the tissue structure is due to the integrity of the tissues from oxidative damage during the stages of development.Furthermore,the beneficial effects observed in the current study for intestinal histometric parameters can be linked to the modulation of the gut microbiota that is relevant to the prevention of intestinal disorders and imbalances.Unsaturated lipids and antioxidant vitamins have a substantial impact on the development of gut structure and functions (El Kertaoui et al.,2019;Turchini et al.,2022).Affirmative effects of implementing Vitamin E ±Vitamin C on the digestive tract are previously reported in channel catfish,Ictalurus punctatus,Rafinesque 1818 (He et al.,2017),Solea senegalensis(Jiménez-Fernández et al.,2018),pikeperch,Sander lucioperca(El Kertaoui et al.,2019),Rainbow Trout,Oncorhynchus mykiss(Rahimnejad et al.,2021).

Organisms have developed effective systems to keep a balance between lipid peroxides and antioxidant system components (e.g.glutathione peroxidase) to combat oxidative stress induced by lipid peroxidation (Olsen et al.,2013;Song et al.,2018).During larval development,glutathione peroxidase activity is heightened to defend cells by speeding the breakdown of H2O2 to H2O (Fernández-Díaz et al.,2006;Wang et al.,2006).Also,GPx is well interlinked with the immune system (Hasanpour et al.,2017).Results of feeding on enrichedArtemiawith DHA selco®,Vitamin E,and a mixture of Vitamin E and Vitamin C displayed higher GPx activity compared to other groups,which highlight the antioxidant potential of Vitamin E and C.Several studies have shown that vitamins E and C,either alone or in combination,have a favorable outcome on GPx (Dawood et al.,2018).In this context,Alkaladi (2019) stated that the oxidative stressors caused by zinc oxide nanoparticles on the liver and gills of Nile Tilapia are alleviated by vitamins E and C with a healthier antioxidant system (GPx).According to Dawood et al.(2020 a,b),Selenium Nanoparticles and/or vitamin (E or C) have a favorable impact on Nile Tilapia’s antioxidant status (GPx).

In the future of aquatic larval production,it will be critical to track biological reactions to external feeds at a molecular level,as well as focus on developing an exact nutritional formula capable of completely replacing rotifers andArtemiain larval feeding without compromising performance or survival rate.

5.Conclusion

Eventually,using enrichedArtemia naupliiwith DHA selco® (DHAS)or fish oil +vitamin E as a single or combined antioxidant with vitamin C is recommended during the critical weaning phase to supportD.labraxlarval growth,survival,and antioxidant efficacy.

CRediT authorship contribution statement

Alaa A.El-Dahhar:Conceptualization,Validation,Resources,Data curation,Visualization,Supervision,Project administration,Funding acquisition.Rashwan S.Rashwan:Conceptualization,Methodology,Software,Validation,Formal analysis,Investigation,Resources,Data curation,Visualization,Project administration,Funding acquisition.Samy Y.EL-Zaeem:Conceptualization,Validation,Resources,Data curation,Visualization,Supervision,Project administration,Funding acquisition.Shaimaa A.Shahin:Conceptualization,Methodology,Formal analysis,Investigation,Resources,Data curation,Visualization,Supervision,Project administration,Funding acquisition.Mona M.Mourad:Conceptualization,Methodology,Software,Validation,Formal analysis,Investigation,Resources,Data curation,Visualization,Supervision,Project administration,Funding acquisition.Mohammed F.El Basuini:Conceptualization,Methodology,Software,Validation,Formal analysis,Investigation,Resources,Data curation,Writing —original draft,Writing — review &editing,Visualization,Project administration,Funding acquisition.

Declaration of competing interest

The authors declare no conflict of interest.