Journal of Applied Phycology

https://doi.org/10.1007/s10811-019-01977-5

Enriched Ulva pertusa as partial replacement of the combined fish

and soybean meals in juvenile abalone Haliotis asinina (Linnaeus)
diet
Rena Santizo-Taan 1 & Myrna Bautista-Teruel 1 & Jean Rose H. Maquirang 1

Received: 27 August 2019 / Revised and accepted: 24 October 2019

# Springer Nature B.V. 2019

Abstract
The potential of enriched Ulva pertusa meal as feed ingredient in abalone juveniles, Haliotis asinina was evaluated. Four
isonitrogenous and isolipidic diets were formulated which contain 27% protein and 5% lipid. Enriched U. pertusa meal replaced
0% (control), 10%, 20%, and 30% of protein from fish and soybean meals in the formulated diets. Thirty randomly selected
abalone juveniles with average weight and shell length of 0.45 g ± 0.01 and 12.71 mm ± 0.01, respectively, were placed in each of
the twelve 60-L oval fiberglass tanks equipped with a flow-through seawater system. Abalone were given diets at 3–5% body
weight daily for 120 days in three replicate samples. Results showed no significant differences (p > 0.05) in percent weight gain,
shell length, specific growth rate, feed conversion ratio, and protein efficiency ratio even up to 30% replacement level. Apparent
dry matter digestibility of U. pertusa meal in abalone diet was 92%. Crude protein content of the abalone fed diets 1 (10%
enriched U. pertusa meal) and 3 (30% enriched U. pertusa meal) was significantly (p < 0.05) higher than those fed on basal diet.
Enriched U. pertusa meal can partially replace fish and soybean meals as protein source in formulated diets for abalone, Haliotis
asinina juveniles.

Keywords Abalone . Green algae . Haliotis asinina . Nitrogen enrichment . Ulva pertusa

Introduction sustainability of the aquaculture industry, research is continu-

Globally, fish meal has been used as major protein source in
aquafeed even in abalone culture industry (Guzman and Viana
1998; Jung et al. 2016; Myung et al. 2016). Balanced amino
acid composition, high protein content, better food palatabil-
ity, digestion, and absorption made fish meal a primary ingre-
dient in the aquafeed industry (Miles and Chapman 2006;
Gatlin III et al. 2007). In the last decade, more than 68% of
global fish meal production has been used for aquatic feeds
(Tacon and Metian 2008). However, the cost of fish meal has
increased over the years due to aquaculture expansion, in-
creasing demand for protein sources, and the effects of
weather-related events (Hardy 2010; Shamshak and
Anderson 2010; Myung et al. 2016). To support the
* Rena Santizo-Taan
santizorena@gmail.com
1
Aquaculture Department, Southeast Asian Fisheries Development
Center, Tigbauan, 5021 Iloilo, Philippines
ously conducted to find alternative protein sources for fish
meals that have good nutritional value, are cost effective,
and are readily available (Watanabe 2002; Gatlin III et al.
2007; Tacon and Metian 2008). The use of soybean meal as
substitute for fish meal has been successfully studied in finfish
diets (Kikuchi 1999; El-Saidy and Gaber 2002; Riche and
Williams 2011; Zhang et al. 2016) and abalone (Guzman
and Viana 1998) without affecting animal performance.
Though the outcome of replacing fish meal with soybean meal
is promising, its utilization will only be limited to a few spe-
cies such as salmonids (Hardy 2010) since prolonged use of
feeds containing more than 30% soybean meal would have
adverse effects on the digestive system of the animal, like
intestinal enteritis (Gatlin III et al. 2007). Furthermore, soy-
bean meal contains anti-nutritional factors including trypsin
inhibitor (Arndt et al. 1999; Francis et al. 2001) and non-starch
polysaccharides which may negatively affect nutrient utiliza-
tion and reduced feed efficiency, thus limiting its use in some
aquatic feeds (Gatlin III et al. 2007). Even though most ter-
restrial plant ingredients are readily available at a lesser cost
than fish meal, their relatively low protein content, unbalanced

essential amino acid profile, high levels of fiber, starch as well
as the presence of one or more anti-nutritional factors, make
their usage in aquafeeds to be limited (Krogdahl et al. 2003;
Hardy 2010).
Marine macroalgae have been cultivated for centuries and
utilized for human and animal food. They are rich in soluble
dietary fibers, proteins, minerals, vitamins, antioxidants, phy-
tochemicals, and polyunsaturated fatty acids containing low
caloric value (Fleurence 1999; Mohamed et al. 2012).
However, protein of non-enriched macroalgae is generally
low (11–19%), but it can be increased up to more than 30%
if cultured in nutrient-enriched medium (Viera et al. 2011).
Currently, attention was given on seaweeds with elevated pro-
tein content as potential ingredients in fish and crustacean
diets (Valente et al. 2006; Marinho-Soriano et al. 2007;
Azaza et al. 2008; Silva et al. 2015; Zhu et al. 2016).
Mustafa and Nakagawa (1995) reviewed the benefits of using
algae as feed ingredient. Various seaweed species, such as red
algae Porphyra spp. (Davies et al. 1997; Soler-Vila et al.
2009; Silva et al. 2015), Gracilaria spp. (Valente et al. 2006;
Marinho-Soriano et al. 2007; Pereira et al. 2012), brown algae
Saccharina (Laminaria) japonica (Xia et al. 2012) Undaria
pinnatifida (Ansary et al. 2019) and Sargassum muticum
(Pereira et al. 2012); green algae Ulva sp. (Waseff et al.
2001), Ulva lactuca (Xia et al. 2012; Zhu et al. 2016), and
Ulva rigida (Valente et al. 2006) have been evaluated for
incorporation into aquafeed. Marine algae have been widely
considered as potential alternative to fish meal not just be-
cause they can be cultured under enrichment condition to en-
hance their nutritional profile but due to their potential in
imparting additional health benefits to the animal
(O’Mahoney et al. 2014). Moreover, marine algae can also
be an important lipid source in fish diets (Nakagawa et al.
1987) as well as feed binder (Hashim and Saat 1992). The
nutrient content of algae however differs according to species
and seasonal conditions (Fleurence 1999). Ulva is one good
candidate for aquafeed since it has strong affinity for growth in
high nitrogen concentrations and can thrive unattached in
sheltered marine waters and estuaries. Nonetheless, Ulva is
not grown commercially outside Asia because there is no
market for it (Bolton et al. 2009). In the Philippines, they are
commonly found in the intertidal zones and labeled as nui-
sance species as they could over-proliferate producing blooms
or ‘green tide’ (Largo et al. 2004).
In world aquaculture production, abalone only contributes
relatively small portion but this commodity is considered as
one of the most highly prized seafood delicacies; therefore, the
value of its production is very important to many countries
(Cook 2014). The growth of global abalone aquaculture has
been brought about by the decline of wild stocks as the de-
mand continue to increase primarily from Asian countries
(Cook and Gordon 2010). In the Philippines, farming of
Haliotis asinina is an emerging aquaculture industry that
J Appl Phycol
interests the public because of the increase in demand in the
global market. In 2013–2014 the abalone farm production of
the Philippines was eight tonnes (Cook 2014) which is four
tonnes higher than in 2010. Abalone are known to be herbiv-
orous and in their natural environment, they feed on
macroalgae (Viana et al. 1993). In abalone culture total depen-
dence on macroalgae as food resulted in variable growth rates
of the animal due to differences in nutritive content of sea-
weed (Bautista-Teruel and Millamena 1999). Thus, utilization
of formulated diets in abalone culture is recommended.
FitzGerald (2008) identified some positive reasons for the
use of artificial diets which include enhanced growth rates of
the cultured animal, biosecurity, sea, and seasonal indepen-
dence. Moreover, numerous studies have reported that en-
hanced growth rate was observed in abalone fed with artificial
diet than those fed with fresh seaweeds alone (Viana et al.
1993; Fleming et al. 1996; Naidoo et al. 2006; Bansemer
et al. 2016; Bautista-Teruel et al. 2016). Enriched Ulva sp.
has been tested to improve the growth rate of Japanese abalo-
ne, Haliotis discus hannai and European abalone, Haliotis
tuberculata (Shpigel et al. 1999).
This study aimed to determine the effects of enriching
macroalgae U. pertusa and utilize it as partial replacement to
fish and soybean meals as protein source in the formulated
diets of abalone H. asinina juveniles.
Materials and methods
Ulva pertusa enrichment
Ulva pertusa were collected from the intertidal zone of Brgy.
Buyuan Tigbauan, Iloilo, Philippines. Algae were washed
with sand-filtered sea water; epiphytes were removed and in-
cubated for 24 h at ambient temperature in 60-L fiberglass
oval tanks.
Initial experiment was conducted on the incubation of
U. pertusa (5 g fresh weight) in 1-L beakers containing
ozonated sea water having 0, 30, 60, 90, and 120 ppm of
ammonium chloride (NH4Cl) done in three replicates. The
seaweed was taken after 0, 3, 6, 12, and 24 h, rinsed with
ozonated sea water, then fresh water. Collected samples were
then oven dried in a mechanical convection oven at 60 °C for
8 h. The dried algae were powdered through a grinder (Krups,
F203), sieved (150 μm) and stored in the cold room (16 °C)
until used. Dried enriched U. pertusa was analyzed for its
crude protein content through Kjeldahl method. U. pertusa
enriched with 30 ppm NH4Cl at 6 h incubation time had sig-
nificantly higher (p < 0.05) crude protein content (27%) thus
was chosen for enrichment trial (Table 1). Enrichment was
conducted in 30 days to collect 1100 g (dry weight) of
enriched U. pertusa needed for diet preparation and proximate
analysis.
J Appl Phycol

Table 1 Crude protein content (%) of U. pertusa after enrichment trial Feeding experiment
Ulva pertusa NH4Cl Incubation time Crude protein
treatment (ppm) (h) content (%) Twelve rectangular (28 cm × 22.5 cm × 7.5 cm) trays with
floaters were each suspended into 60-L fiberglass tanks con-
1 0 0 11.81 taining individual aeration, supplied with filtered sea water in
(non-enriched)
2 30 3 25.92
a flow-through system at a flow rate of 500 mLmin−1. Seven
6 27.00
hundred abalone H. asinina juveniles were sourced from the
Abalone Hatchery of the Aquaculture Department, Southeast
9 26.24
Asian Fisheries Development Center (SEAFDEC/AQD). The
12 20.42
experiment was conducted in the ground floor of the Nutrition
24 18.46
Building of SEAFDEC/AQD with natural photoperiod of 12 h
3 60 3 14.11
light and 12 h dark. After 14 days of acclimation to the culture
6 16.12
environment and basal diet, 30 abalone juveniles with an av-
9 13.54
erage weight and shell length of 0.45 g ± 0.01 and 12.71 mm ±
12 13.36
0.01 (mean ± SEM), respectively, were randomly stocked into
24 13.48
each of the trays in four treatment groups with three replicates.
4 90 3 13.21
Abalone were fed with the respective experimental diets ac-
6 15.72
cording to treatments at 3–5% average body weight once ev-
9 16.17
ery 1600 h. Every morning, the uneaten feeds and fecal mat-
12 16.75
ters were siphoned out from the bottom of the experimental
24 15.17
tanks. Water quality parameters were monitored daily in the
5 120 3 16.54
whole duration of the experiment. The water temperature,
6 16.24 salinity, pH, dissolved oxygen, and ammonia were maintained
9 16.17 at 27–29 °C, 30–32 ppt, 8–8.2, 5–5.7 mg L−1, and 0–0.26,
12 15.17 respectively. Sampling was done every 30 days by measuring
24 14.71 individually the shell length of abalone using plastic vernier
caliper and its weight through analytical balance. The feeding
experiment lasted for 120 days. During stocking, 100 juve-
niles of abalone were sacrificed for initial carcass composi-
Experimental diets tion. Abalone samples were pooled per treatment and
sacrificed for proximate analysis at the end of the experiment.
Four isonitrogenous (27%) and isolipidic (5%) experimental
diets were formulated (Bautista-Teruel and Millamena 1999)
with varying amount of enriched U. pertusa to partially re- Growth parameters
place the protein from fish and soybean meals at 0% (basal
diet), 10% (diet 1), 20% (diet 2), and 30% (diet 3). Enriched The dietary performance was evaluated by determining the
U. pertusa contained 27% crude protein and 0.36% crude fat. percent weight gain (%WG), specific growth rate (SGR), shell
Danish fish meal and defatted soybean meal were the major length (SL) increase, survival rate, and feed conversion ratio
protein sources. A mixture of cod liver oil and soybean oil (FCR) computed as follows:
(1:1) were used as lipid sources. Wheat flour and sodium %WG = [(final weight−initial weight)/initial weight] × 100
alginate were the carbohydrate and binder, respectively. All SGR = [(ln ave. final weight−ln ave. initial weight) no. of
dry ingredients were sieved through a 710 μm mesh prior to days] × 100
mixing for 10 min in a feed mixer (Artisan, 5KSM150). SL = [(final SL−initial SL)/initial SL] × 100
Mixture of oils were added to the mixed dry ingredients and Survival (%) = (final no. of abalone/initial no. of abalone) ×
mixing was done for 10 min. The pre-cooked (90 °C) wheat 100
flour (1:3 w/v) was added last, mixed for 10 min and the FCR = total weight of dry feed given/wet weight gain
produced dough was allowed to pass through the pelletizer
(Hobart Canada, N50) with 2 mm die hole diameter. The pel-
letized diets were steamed for 5 min, cooled, and processed In vivo dry matter digestibility
through the pelletizer again. The strands were oven-dried in a
dry air mechanical convection oven (Memmert Wisconsin Digestibility of enriched U. pertusa was tested as an in-
Oven, 600) at 60 °C for 6 h, grounded and stored in the cold gredient in abalone diet. The indirect method by Cho
room (16 °C) prior to use. et al. (1982) was adapted to determine the dry matter
 J Appl Phycol

digestibility of enriched U. pertusa. There were two diets
formulated, a reference diet and a test diet (Table 4). The
formulation of the reference diet was the same as the basal
diet used in feeding experiment. On the other hand, the
test diet contained 70% reference diet and 30% enriched
U. pertusa. Both diets were provided with 1% chromic
oxide (Cr2O3) as inert indicator. The digestibility chamber
described by Montaño-Vargas et al. (2002) was modified
and used in this study. One-and-a-half-liter plastic funnel-
shaped chamber with a 50 mL collecting bottle (Falcon
tube) attached at the bottom served as the digestibility
chamber. On both sides of the lateral funnel walls, two
circular openings at 3 cm diameter were made and cov-
ered with black net to allow constant flow of water
through the chambers. Moreover, the top of the chambers
was covered with black net to prevent abalone from es-
caping. Four abalone with an average shell length of 38 to
40 mm and body weight of 26 to 30 g were placed inside
each of the 20 chambers. Ten chambers were suspended
inside each of the two 250-L fiberglass conical tank sup-
plied with flow-through aerated sea water having an av-
erage flow rate of 300 mL min−1. Abalone were acclimat-
ed to feed on the basal diet (without Cr2O3) for 7 days
and starved for 1 day to empty their gut. Thereafter, aba-
lone were fed with reference and test diets once daily at
0800 h at 3% body weight. Fecal collection started after 3
days of feeding and lasted until day 30. Abalone were
allowed to feed for 4 h and at 1200 h uneaten feeds were
siphoned to avoid contamination of feces and the
collecting tube were carefully attached to the chamber to
facilitate fecal collection. Fecal matters were collected ev-
ery 1400 h and 1600 h, rinsed carefully with distilled
water, pooled according to treatment, weighed, and stored
in −80 °C until chromic oxide analysis.
The chromic oxide content of both feces and feeds were
analyzed using the method described by Montano-Vargas
et al. (Montaño-Vargas et al. 2002) observed in triplicate.
Samples weighing 110 mg were ashed overnight at 450 °C,

Table 2 Formulation (%) and proximate composition (dry weight basis, %) of the experimental diets

Ingredients Experimental diets

(g (100 g)−1 diet)
basal 1 2 3
(10% replacement) (20% replacement) (30% replacement)

Danish fish meal 17.0 15.0 13.5 12.0

Defatted soybean meal 17.0 15.0 13.5 12.0
Spirulina 7.5 7.5 7.5 7.5
Sodium alginate 7.5 7.5 7.5 7.5
Wheat flour 18.0 18.0 18.0 18.0
Rice bran 19.95 14.45 7.95 1.45
Cod liver oil 1.5 1.5 1.5 1.5
Soybean oil 1.5 1.5 1.5 1.5
a
Vitamin mix 3.0 3.0 3.0 3.0
b
Mineral mix 4.0 4.0 4.0 4.0
Dicalcium phosphate 3.0 3.0 3.0 3.0
Butylated hydroxytoluene 0.05 0.05 0.05 0.05
Enriched Ulva pertusa 0.00 9.5 19.0 28.5
Proximate composition (%)
Protein 27.72 28.07 27.42 28.06
Fat 5.31 5.11 5.23 5.29
Carbohydrate 46.55 45.11 44.25 43.82
Ash 11.46 13.99 16.92 18.93
C
Estimated energy (kcal kg−1) 3449 3387 3338 3351
a
Vitamin mix (g (100 g)−1 diet): thiamine HCl 0.012 g; riboflavin 0.01 g; folic acid 0.003 g; paraminobenzoic acid (PABA) 0.04 g; pyridoxine HCl
0.004; niacin 0.08 g; Ca pantothenate 0.02 g; inositol 0.4 g; ascorbic acid 0.4 g; biotin 0.0012 g; α-tocopherol 0.045 g; menadione 0.008 g; vitamin B12
0.00018 g; vitamin A 10,000 IU; cholecalciferol 200 IU; and ethoxyquin 0.04 g
b
Mineral mix (g (100 g)−1 diet): NaCl 0.04 g; MgSO4 . 7H2O 0.6 g; NaH2PO4 . 2H2O 1.0 g; KH2PO4 1.28 g; Ca(H2PO4)2. H2O 0.8 g; Fe-lactate 0.1 g;
Ca-lactate 0.14 g; ZnSO4 . 7H2O 0.0141 g; MnSO4 . 4H2O 0.0648 g; CuSO4 . 5H2O 0.00124 g; CoCl2 . 6H2O 0.0004 g; and KIO3 0.00012
c
Computed based on standard physiological fuel values of 9 kcal g−1 lipid and 4 kcal g−1 protein and carbohydrate (Brett and Groves 1979).
J Appl Phycol

Table 3 Formulation (%) and proximate composition (dry weight basis, matter digestibility coefficient of the diets (ADC) and test
%) of reference and test diets for digestibility study
ingredient were calculated using the following equation
Ingredients (g (100 g)−1 diet) Reference diet Test diet (Maynard and Loosli 1969):
ADC of dry matter of diet (%) = [% marker in diet/% marker
Danish fish meal 17.0 11.90 in feces)] × 100
Defatted soybean meal 17.0 11.90 ADC of dry matter of test ingredient (%)
Spirulina 7.5 5.25 = [ADC of test diet−(proportion of reference diet in test diet ×
Sodium alginate 7.5 5.25 ADC of reference diet)]/proportion of test ingredient in test
Wheat flour 18.0 12.6 diet
Rice bran 18.95 12.965
Cod liver oil 1.5 1.05
Soybean oil 1.5 1.05
a
Vitamin mix 3.0 2.1 Chemical analysis
b
Mineral mix 4.0 2.8
Dicalcium phosphate 3.0 2.1 Proximate analysis of enriched and non-enriched U. pertusa,
Butylated hydroxytoluene 0.05 0.035 experimental diets, digestibility diets, and abalone carcasses
Enriched Ulva pertusa 0.00 30 taken before and after feeding trial were determined following
Chromic oxide 1.00 1.00 the standard methods (AOAC 2000). Moisture was deter-
Proximate Analysis mined using a moisture analyzer (Mettler Toledo, MJ33),
Crude protein 29.38 ± 0.01 27.32 ± 0.36 crude protein (Tecator Kjeltec Systems 2300 FOSS) based
Crude fat 6.79 ± 0.04 5.91 ± 0.13 on a semi-micro Kjeldahl method with 6.25 as factor, crude
Fiber 2.93 ± 0.10 3.15 ± 0.23 fat (FOSS, Soxtec 2055) was extracted through Soxhlet ex-
Moisture 0.16 ± 0.15 0.51 ± 0.03 traction method, and crude fiber (Fibertec 1020, FOSS) from a
Ash 12.80 ± 0.00 20.65 ± 0.01 fat-free material sample by dilute acid and alkali treatment.
NFE 48.09 ± 0.06 42.96 ± 0.45 Ash was measured by heating the samples in a muffle furnace
(62700 FOSS) at 550 °C.

cooled and thereafter 15 mL of digestion solution was added.

The digestion solution was prepared by mixing 2% sodium
molybdate (Na2MoO4), prepared in 150 mL of distilled water,
150 mL sulfuric acid (H2SO4), and 200 mL perchloric acid
(HClO4). The samples were then heated until they turned to
yellow or reddish in color and were then maintained at that
temperature for additional 15 min then allowed to cool. The
samples were then transferred to 200 mL volumetric flasks
and diluted to mark with distilled water. The absorbance was
read in spectrophotometer (UV-VIS Shimadzu, UV mini
1240) at 440 nm and the concentration of chromic oxide
was calculated using a standard curve. The apparent dry
Statistical analysis
Results were expressed as mean ± SEM and were analyzed for
normal distribution and homogeneity of variances. Data for
enrichment trial were analyzed using factorial analysis.
Results of the growth trial and body composition were sub-
jected to one-way analysis of variance (ANOVA) and
Duncan’s Multiple Range Test (DMRT) as post hoc analysis
to determine significant differences among treatment means
using SPSS Package version 16. Differences were considered
significant at p < 0.05.

Table 4 Growth of abalone juveniles fed with increasing replacement levels of enriched U. pertusa

Dietary treatments Parameters

Initial SL (mm) d Initial ABW (g) d Final SL (mm) Final ABW (g) Weight gain (%) SGR (% day −1)

Basal 12.82 ± 0.15 0.46 ± 0.02 19.42 ± 0.16 1.37 ± 0.04 197.82 ± 12.08 0.90 ± 0.04
Diet 1 (10%) 12.65 ± 0.19 0.44 ± 0.02 19.04 ± 0.09 1.27 ± 0.03 188.78 ± 18.21 0.90 ± 0.06
Diet 2 (20%) 12.69 ± 0.02 0.44 ± 0.03 19.40 ± 0.14 1.33 ± 0.05 201.06 ± 25.51 0.94 ± 0.07
Diet 3 (30%) 12.70 ± 0.08 0.43 ± 0.03 19.33 ± 0.31 1.31 ± 0.06 205.66 ± 24.68 0.96 ± 0.07
d
Not included in statistical analysis
SL, shell length; ABW, average body weight; and SGR, specific growth rate
 J Appl Phycol

Table 5 Survival and feed efficiency of abalone juveniles fed with Body Composition
increasing replacement levels of enriched U. pertusa

Dietary treatments Parameters The crude protein, crude lipid, and ash composition of initial and
final groups of abalone fed on different dietary treatments are
FCR PER Survival (%) given in Table 6. Result showed that the crude protein content
Basal 1.87 ± 0.07 2.14 ± 0.08 86.67 ± 1.92
of the abalone fed diet 1 (68.21 ± 0.12) and diet 3 (68.74 ± 0.88)
were significantly (p < 0.05) higher than that of abalone fed on the
Diet 1 (10%) 1.83 ± 0.11 2.20 ± 0.13 88.89 ± 1.11
basal diet. Meanwhile, no significant differences were observed
Diet 2 (20%) 1.71 ± 0.13 2.36 ± 0.18 84.44 ± 2.94
(p > 0.05) in the crude protein content of abalone fed diets 1, 2,
Diet 3 (30%) 1.69 ± 0.11 2.38 ± 0.15 84.44 ± 4.84
and 3. The highest value (6.28 % ± 0.07) for crude fat content was
FCR, feed conversion ratio and PER, protein efficiency ratio exhibited by abalone fed on the basal diet which was significantly
different (p < 0.05) from the rest of the treatments. The ash con-
tent of abalone fed diet 3 was lowest at 8.85 ± 0.01 and this was
significantly different (p < 0.05) from all other treatments.
Results

Ulva pertusa enrichment

The crude protein content (dry weight) of U. pertusa increased
from 11.81 to 27% through enrichment with 30 ppm ammo-
nium chloride for 6 h (Table 1).
Feeding and digestibility
Formulation and proximate composition of the four experi-
mental and digestibility diets are shown in Table 2 and
Table 3, respectively. Abalone readily accepted the formulated
diets and no diseases or abnormalities were observed in the
whole duration of the experiment. Results for growth perfor-
mance are indicated in Table 4 and feed efficiency and surviv-
al rate in Table 5. No significant differences (p > 0.05) among
all treatments were observed in terms of shell length, average
body weight, weight gain (WG), and specific growth rate
(SGR). Survival rates of abalone ranged between 84 and
89% and were not significantly (p > 0.05) affected by the
dietary substitution of fish and soybean meals with enriched
U. pertusa. FCR and PER were also not significantly (p >
0.05) affected with the dietary treatments. Results from digest-
ibility study indicated that the dry matter digestibility of ref-
erence diet and test diet were 78% and 84%, respectively.
Moreover, the test ingredient (enriched U. pertusa) was 92%
digestible.
Discussion
There are no negative effects on growth, survival, feed efficiency,
and body composition observed in abalone fed diets with
enriched U. pertusa even up to the highest replacement of 30%
in the abalone diet. Macroalgae are rich in bioactive compounds
that have potential biological effects on the animal including anti-
bacterial and anti-viral activity, anti-oxidant and anti-
inflammatory properties (O’Sullivan et al. 2010). In
H. laevigata fed Ulva lactuca and Spyridia filamentosa, en-
hanced anti-viral activity was observed in the digestive gland
(Dang et al. 2011). With these added health benefits, abalone
fed diets containing macroalgae may develop resilience to stress
thus improving the scope for growth (Bansemer et al. 2014;
Kemp et al. 2015).
The use of ammonium chloride as an agent to enrich the
protein content of U. pertusa was again proven effective with
protein increase from 11.81 to 27% which is comparable to
previous studies that used enriched macroalgae as feed for
abalone (Shpigel et al. 1999; Viera et al. 2011). Bansemer
et al. (2014) suggests that feeding abalone with macroalgae
cultured in a nitrogen/protein enriching media resulted to im-
proved growth rates compared with corresponding non-
enriched algae treatments. Result supports the fact that Ulva
spp. is capable of assimilating exogenous inorganic nitrogen
for amino acid and protein synthesis (Hernández et al. 2002).

Table 6 Crude protein, fat, and ash (%) of abalone juveniles fed with experimental diets for 120 days

Proximate composition Initial d Basal Diet 1 Diet 2 Diet 3

Crude protein 62.75 ± 0.16 64.97 ± 0.21b 68.21 ± 0.12a 66.89 ± 0.20 ab 68.74 ± 0.88 a
Crude fat 2.17 ± 0.02 6.28 ± 0.07 a 4.87 ± 0.02 c 4.88 ± 0.04 c 5.28 ± 0.04 b
Ash 16.55 ± 0.16 11.91 ± 0.30 a 10.21 ± 0.03 b 9.92 ± 0.03 b 8.85 ± 0.01 c
a,b,c
Values in the same row with different superscript letters are significantly different (p<0.05)
d
Not included in statistical analysis
J Appl Phycol
Throughout the experiment, diets were readily ingested in
abalone indicating that U. pertusa incorporated in the diets
could be accepted by the experimental animal. The protein
content of all dietary treatments met the required protein level
of 27% for H. asinina juveniles (Bautista-Teruel and
Millamena 1999) to support normal growth and metabolic
activities. Apparent digestibility coefficients (ADC) of dry
matter and test ingredient (enriched U. pertusa) at 84 and
92% were higher than other species fed with macroalgae
(Laurencia botryoides) (Fleming et al. 1996). High
macroalgal digestibility could be attributed to the natural di-
gestive system of the abalone which is anatomically and bio-
chemically adapted to macroalgal-based food (Erasmus et al.
1997; Harris et al. 1998).
In the present experiment, replacing fish and soybean meals
with enriched U. pertusa meal up to 30% did not have a negative
impact on weight gain (%), final shell length, final average body
weight, SGR, survival, FCR, and PER of abalone juveniles. The
study of Viera et al. (2011) displayed the same result wherein,
abalone fed enriched macroalgae diets showed higher perfor-
mance in terms of specific growth rate, weight gain, increase in
shell length, and FCR values than those fed on non-enriched
diets. This result could be related to the comparable protein con-
tent of enriched U. pertusa meal with fish and soybean meals.
Fleming (1995) suggests that nitrogen is a limiting factor for
growth in Haliotis sp. and the maximum growth of abalone
can only be achieved when sufficient protein in the correct pro-
portion of amino acids is supplied in the feed (Shpigel et al. 1999;
Naidoo et al. 2006). The protein quality of the diet measured
through protein efficiency ratio (PER) suggests that protein from
enriched U. pertusa meal is comparable to that of fish and soy-
bean meals as demonstrated by the no significant difference in
PER values among all treatments.
Similarly, proximate composition of the abalone showed that
the biochemical composition of the abalone meat was affected by
the diets (Mai et al. 1995; Thongrod et al. 2003; Myung et al.
2016). Crude protein content in abalone fed diets containing
enriched U. pertusa were generally higher than those fed on basal
diet indicating an improved protein deposition in abalone meat.
Overall, this study demonstrated that enriched U. pertusa
meal can be an alternative ingredient in abalone diet as partial
substitute to fish and soybean meals. Inclusion of enriched
U. pertusa meal at 30% of the diet did not manifest any neg-
ative effects on growth, feed efficiency, protein efficiency ra-
tio, and body composition of abalone juveniles.
Acknowledgments The author would like to acknowledge the chemists
of the Laboratory Facilities for Advanced Aquaculture Technology
(LFAAT) for doing the proximate analysis, Mr. Richard Tantiado for his
assistance, and Juan Miguel Taan for data analysis.
Funding information The author would like to acknowledge the
Aquaculture Department, Southeast Asian Fisheries Development
Center (study code: FD-01-M2015T) for providing funds for this study.
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