4.

EFFECT OF DIETARY SPIRULINA ON GROWTH,

DIGESTIBILITY, COLORATION AND LEUCOCYTE
COUNT IN XIPHOPHORUS HELLERI

4.1. INTRODUCTION

With the increasing demand as a possible source of protein to be used

directly or indirectly for human consumption, Spirulina had gained popularity and

importance throughout the world. The potential of Spirulina as a protein source was

evident as early as 1940 when it was used as part of the diet of the village people in

Africa and Mexico (Baldia et al., 1991). More recently, Spirulina had found

application in various fields like agriculture, waste-water treatment (Borowitzka and

Borowitzka, 1988), aquaculture, nutrient recycling and production of valuable

chemicals (-carotene and phycocyanin). Spirulina cultivation is widespread for

aquaculture application particularly to the use of the pigments as feed for tropical fish

(Vonshak and Richmond, 1988). Spirulina is used in aquaculture as a liquid feed to

young fishes and in dehydrated form to enrich foods of ornamental fish, shell fish and

bivalves.

Spirulina is one of the most concentrated natural source of nutrition for

all animals. Spirulina has 60-70% protein by weight and is the richest source of

vitamin B12, -carotene, essential fatty acids and minerals. Besides, there are eight

amino acids such as isoleucine, leucine, lysine, methionine, phenylalanine, threonine,

tryptophane and valine in Spirulina. More recently, there has been new interest

concerning the therapeutic effects of Spirulina as growth promotor and probiotic or

booster for the immune system in animals and fish. In short, Spirulina is a powerful

tonic for the immune system. Many authors have studied the effect of Spirulina diet

37
or extract on consumption, growth, nucleic acids, digestive enzymes, and immune

responses in various animals (Hayashi et al., 1994; Qureshi et al., 1995; Qureshi and

Ali, 1996; Portoni et al., 1996; Nandeesha et al., 1998). However, there is no such

study has been carried on the effects of Spirulina diet on feed intake, digestability,

growth, coloration and leucocyte count in ornamental fishes. Hence, the present

investigation has been carried out to analyse the impact of different levels of Spirulina

diet on growth, digestability, coloration and leucocyte count in red swordtail,

Xiphophorus helleri.

4.2. MATERIALS AND METHODS

For the experiment, 45 days old 300 juveniles of X. helleri were

collected from the laboratory bred brooders. Healthy and active juveniles were

divided into five groups and offered with different levels of Spirulina diet (0, 5, 10, 15

and 20% diet).

Each group consisting of 20 individuals was reared in circular cement

tank containing 100 l of water (width: 58.5 cm; height: 40 cm; 120 l capacity).

Triplicates were maintained for each Spirulina diet. The tanks were filled with

dechlorinated well water (Temperature : 28.210C; pH : 7.50.05; salinity: 0.570.01

ppt; water hardness : 322 mg CaCO 3 l 1 ; DO: 4.370.18 ml l-1).

Feed formulation was done according to Hardy (1980). The dried and

powdered ingredients of diets were blended at first to make a homogenous mixture.

Subsequently mixed with suitable level of Spirulina powder with an aliquot of boiled

water and stream cooked for 15-20 minutes. After moderate cooling, pellets

(2 mm size) were prepared with a hand operated pelletizer and dried in sunlight. After

drying, diets were separately stored in refrigerator for experimental use. The

38
composition of control diet and different levels of Spirulina incorporated diets were

given in Table 2.1.

Protein and lipid contents of experimental diets were determined in

spectrophotometer following Lowry et al. (1951) and Bragdon (1951) respectively.

Moisture content was analysed by drying in an electric hot air oven at 1000C. Mineral

content of the test diets was estimated following the method of Paine (1964). Nitrogen

free extract (NFE) was calculated by subtracting the total of the protein, lipid and

mineral contents from the dry weight of the feed samples.

A known quantum of different levels of Spirulina diets were kept in

petri dish in the corresponding tanks for about 1 hr. Growth was measured at an

interval of 25 days by weighing all the individuals in the tanks. For other relevant

details regarding this, please refer chapter 2. The experiment was conducted for a

period of 175 days to complete two breeding in test animals.

Nutrient digestability

Digestability was determined following the chromic oxide method

(Furukawa and Tusukahara, 1966). The experimental X. helleri were fed with test

diets containing 1% chromic oxide as indicator. Faeces were collected after 4 hr of

feeding over the period of 7 days at each sampling. Analysis of feed, faeces and

digestability of protein were calculated. The apparent digestability of protein was

determined at regular intervals of 25 days.

Faecal collection

Although the chromic oxide method eliminates the need for total

collection of faeces, satisfactory faecal sampling is essential to ensure accurate

measurement of the ratio of undigested nutrient to indicator in the faeces. In each

39
case, the faeces were collected from the water over a 7-day period and pooled for the

fish fed each diet. The faecal material was freeze-dried and used for further analysis.

Chromic oxide analysis by wet acid digestion (Furukawa and Tusukahara, 1966)

Samples of dried feed or faeces containing 1% Cr2O3 were wrapped in

a small piece of filter paper and transferred to a digestion flask (Micro Kjeldahl).

Following the addition of 5 ml of concentrated nitric acid and a 5 minutes waiting

period, the samples was heated for 20 minutes or until a white precipitate formed.

After cooling, 3 ml of 70% perchloric acid was added and the green coloured mixture

reheated for 10 additional minutes after changing color to yellow, orange or red. After

cooling to room temperature, add 50 ml of distilled water and transferred to 100 ml

volumetric flask. The yellow solution was mixed well, allowed to stand for 5 minutes.

Subsamples transferred to colorimetric tubes and read at 350 nm in a

spectrophotometer. Per cent chromic oxide was calculated from a prepared standard

curve where Y = the optical density at 350 nm and X = the Cr2O3 content of the

sample (mg / 100 ml).

Calculation of digestability by this method was based on the

assumption that the amount of chromic oxide in the feed (1%) and the amount voided

in the faeces was the same over equal periods of time. Therefore digestion coefficients

can be calculated for crude protein and nitrogen free extract following appropriate

chemical analysis for the nutrients and chromic oxide in the consumed feed and the

faeces. This method eliminates the necessity of a quantitative collection of all faeces

required by direct method (Maynard and Loosli, 1969).

% nutrient in faeces
Apparant digestability (%) = 100  100 u u
% nutrient in feed
% Cr2 O 3 in feed
% Cr2 O 3 in faeces

40
 W1  W0
Protein efficiency ratio =
Dp

Where W1 = final wet weight (g), W0 = Initial wet weight (g) and Dp = dry protein

intake (g).

4.3. RESULTS

An increase in Spirulina levels with the basal diet (45% protein), the

protein and mineral contents were increased while nitrogen free extract showed the

opposite trend (Table 2.1). With the increasing level of Spirulina in the diet, fat

percentage declined because of the low fat content of Spirulina.

The mean body length and weight of X. helleri increased with increase

in Spirulina levels upto a level (15%) and declined thereafter. The mean body weight

of test fish fed Spirulina at the 0, 5, 10, 15 and 20% diets was 0.70, 0.85, 0.99, 1.38

and 1.35 g wet weight respectively on day 100 (Table 4.1; Fig. 4.1). Similar trend was

also observed in mean body length (Fig 4.1). The slope values (b) obtained for mean

body length (0.28) and weight (0.018) in fish fed with 15% Spirulina diet was higher

than those fed with other Spirulina diets. However, slope of the length and weight

gain of fish fed with 15% Spirulina diet did not differ significantly (P>0.05) as

compared to fish fed with 20% Spirulina diet; but it significantly (P<0.05) differed

with fish fed with 10% Spirulina diet. But, there were significant (P<0.05) differences

in slopes of weight gain between 15% Spirulina diet and other Spirulina diets. Two-

way ANOVA showed that the different levels of Spirulina and rearing period hold

highly significant effect (P<0.01) on the mean body length and weight in X. helleri

(Table 4.2).

Fish fed with 15% Spirulina diet exhibited the maximum feeding (feed

consumption and feeding rate) and growth (weight gain and specific growth rate

41
(SGR) parameters and low feed conversion ratio (FCR) value than those fed with

other levels (Tables 4.3 and 4.4). Feeding rate, SGR and protein efficiency ratio

(Table 4.5) were decreased with increase in rearing period from day 0 to till the

commencement of breeding in all treatments (Fig 4.2 and 4.3); however, feed

conversion ratio (FCR) and apparent digestability coefficient (ADC) were showed the

opposite trend (See Tables 4.4 and 4.5). Duncans multiple range test showed that

PER and ADC between Spirulina treatments differed significantly (P < 0.05) with

better values in fish fed with 15% Spirulina diet. The mean specific growth rate of

X. helleri at pre-spawning period was high as compared to mean SGR at post-

spawning (Fig 4.4), while feeding rate and FCR elicited the opposite trend (Fig. 4.4).

The differences in the feeding and growth parameters of X. helleri were highly

significant (Table 4.6; ANOVA: P<0.01) in relation to Spirulina levels.

Total carotenoid content in fins, skin and muscle of X. helleri was

increased with increase in rearing period and Spirulina levels upto a middle level and

declined thereafter. Maximum carotenoid content was observed in fish consuming

15% Spirulina diet in all three tissues while control group elicited the low carotenoid

content. Duncans multiple range test showed that carotenoid content between

Spirulina treatments differed significantly (P<0.05) with better values in fish fed 15%

Spirulina diet. Fins exhibited the maximum coloration followed by skin and muscle in

all the treatments (Table 4.7; Fig. 4.5).

The amylase activity was higher in the foregut than midgut and

hindgut. The secretion of protease was high in midgut followed by hindgut and

foregut. Lipase activity was highest in the hindgut of X. helleri followed by midgut

and foregut. The foregut secreted meagre amount of lipase as compared to hindgut

42
(Table 4.8; Fig. 4.6). Fish fed with 15% Spirulina diet elicited higher activity of

digestive enzymes than other treatments.

The number of lymphocytes, neutrophils and monocytes were

increased with an increasing of Spirulina levels upto 15% level and thereafter they

declined (except lymphocytes) (Table 4.9; Fig. 4.7). However, thrombhocytes and

basophils showed the opposite trend.

4.4. DISCUSSION

The present investigation reveals that, fish fed with 15% Spirulina diet

elicited the maximum feeding and growth parameters (feed intake, mean body length

and weight, specific growth rate and protein efficiency ratio); it may be due to the

high amount of protein (50%) and growth stimulatory effect of Spirulina in the diet.

Spirulina has been identified as a potential protein source for animal feed (Braun,

1988). It contains high protein and many essential amino acids, gamma linolenic acid,

beta carotene and phycocyanin pigments, vitamins and minerals in large quantities.

James and Sampath (2004c) found that 45% animal protein or plant protein

significantly enhanced the feed consumption and growth rate in X. helleri, which

supports the observations made in the present study. Nandeesha et al. (1998) found

that growth rate and protein efficiency ratio improved in carp fed with Spirulina

incorporated diet which also supports the findings made in the present study. Scaria

et al. (2000) found that ornamental fishes guppy (Poecilia reticulata) and platy

(Xiphophorus maculatus) consumed maximum amount of Spirulina substituted feed

than those fed with mushroom and azolla. Sayed (1994) observed that high feed

intake, body weight gain and specific growth rate in fish silver seabream

(Rhabdosargus sarba) which consumed Spirulina substituted diet than those

43
consuming soybean meal and chicken offal meal. Maximum growth rate was found in

fishes fed with Spirulina diet than non-Spirulina diets (Daniel and Kumuthakalavalli,

1991; Okada et al., 1991). Nakazoe et al. (1986) reported that, 5% supplementation of

Spirulina resulted the higher body weight gain in nibbler, Girella punctatus.

Keshavanth et al. (1986) recorded good growth of Deccan masheer Tor Khudree

when fed with diets containing S. plantensis in place of fish meal. Mustafa et al.

(1994a) found that 3-5% incorporation of Spirulina meal produced a significant

enhancement of feed utilization and growth in one year-old red sea bream, Pagrus

major, supports the present investigation. Aravindan et al. (2001) reported that dietary

E carotene contents (10-30 mg 100g-1) increased the specific growth rate (in terms of

mean body length and weight) as compared to non-E-carotene diet in gold fish

Carassius auratus. The relatively high feeding rate and FCR values at post-spawning

were probably due to allocation of a larger proportion of the feed to maintenance

(Brett and Groves, 1979) and spawning (James and Sampath, 2003a).

The protein digestability data support the growth trend. The protein

digestability of fish fed on higher levels of Spirulina incorporated diets (15 and 20%)

did not differ significantly (P>0.05) and it was significantly (P<0.05) better than the

control and lower levels of Spirulina diet (Table 4.5). Digestability evaluation studies

with common carp using S. platensis have shown that maximum protein digestability

can be obtained at an incorporation level of 50% in a 28% protein diet (Umesh et al.,

1994). Previous studies have also revealed that, an improvement in the protein

digestability of cultivable fishes fed diets with Spirulina (Atack and Matty, 1979;

Atack et al., 1979). Spirulina has no cellulosic cell wall which helps in better

digestion and absorption (Becker and Venkataraman, 1984). The better digestability

44
of protein observed in the Spirulina incorporated diet must be a result of better

absorption which enhanced the growth in X. helleri.

Eventhough the red swordtail fish is brightly colored, dietary

substitution of Spirulina significantly further enhanced the coloration in the fins and

skin. The increase in carotenoid content in skin, fins and muscle of X. helleri in

relation to dietary carotenoid content of Spirulina diets demonstrates that, the fish has

capacity to utilize it efficiently. Similar observations in the muscle of trout and

salmon have been made by a few authors earlier (Storebakken et al., 1987; Bjerkeng

et al., 1990). A dose-dependent carotenoid content has been reported in the muscle of

Arctic char and salmon (Bjerkeng et al., 1990; Ando et al., 1994; Halten et al., 1997;

Wathne et al., 1998). Paripatananont et al. (1991) found that 36-37 mg astaxanthin

kg-1 diet produced maximum coloration in goldfish, C. auratus and the coloration was

stable even after 2 months. They also reported that feeding astaxanthin could be a

suitable way for gold fish producers to stimulate color among fish grown in an algae

free environment. In ornamental fishes, (unlike salmon and Arctic char) the

pigmentation was highly found only in the skin and fins and this might be due to

acquiring, digesting, utilizing dietary carotenoids and transporting more directly to the

skin and fins rather than storing in muscle (Aravindan et al., 2001). The low

carotenoid content in the muscle of X. helleri, indicates that the assimilated carotene

is directly transported to the skin and fins to provide necessary pigmentation.

According to Schiedt et al. (1985) this was achieved by establishing reductive

metabolic pathways from muscle to the skin and fins. In salmon, Arctic char and

trout, the pigmentation of integument and fins occurs only during sexual maturation

and a reduction in the muscle carotenoid is an indication that the carotenoids are

mobilized directly to the integument and fins from the muscle during that season.

45
High density lipoproteins have been demonstrated to be responsible for the carotenoid

transport from muscle to integument in salmon (Ando and Hatano, 1988). Moreover,

several abiotic and biotic factors are also expected to influence the ingestion,

mobilization and metabolism of carotenoids like other feed constituents (Halten et al.,

1996). It is likely that, a similar mechanism operates in X. helleri also.

The present study also revealed that supplementation of 15% Spirulina

diet increased the chosen gut enzyme activities (amylase, protease and lipase) while

10 and 20% Spirulina diets reduced the enzyme activities in X. helleri.

Nandeesha et al. (1998) found that higher levels of Spirulina (60-100%)

supplementation reduced the intestinal protease and lipase in Cyprinus carpio and it

supports the observations made in the present study. Field beans and groundnut leaf

meals increased the amylase activity in the foregut and midgut; prawn head meal and

chicken intestine diets showed an elevated amylase secretion in the foregut but

decreased gradually in the mid and hind guts of Labeo rohita (Sethuramalingam and

Haniffa, 2002). They also reported that prawn head and chicken intestine meals

produced the higher secretion of protease in the midgut of L. rohita. Red swordtail

fish exhibited the maximum lipase activity in hindgut of all the treatments. On the

contrary, lipase activity was high in midgut followed by hindgut in cultivable fishes

(Sastry, 1974; Sethuramalingam and Haniffa, 2002).

Lymphocytes, neutrophils and monocytes were the leucocytes types

with positive response to the addition of Spirulina in the diet. Feeding processed form

of Spirulina platensis enhanced specific and non-specific immunity against

Edwardsiella ictaluri infection in channel catfish, Ictaluras punctatus (Duncan and

Kiesins, 1996). James et al. (2006) found that, Xiphophorus helleri fed with 8%

Spirulina enhanced the lymphocyte and monocyte populations among leucocytes

46
which inturn increased the resistance. The powder form and cell extracts of Spirulina

were found to enhance immunity in Ictaluras punctatus by increasing phagocytic

activity (Portoni et al., 1996). and chitosan were used as immunomodulatory

feed additive in carp nutrition (Victor et al., 2004) which supports the findings of

present study.

Based on the result of the present investigation, it was observed that,

15% Spirulina diet produced more growth, coloration and leucocytes count in red

swordtail fish, X. helleri than other treatments. However, 20% Spirulina diet produced

more coloration in swordtail fish than 15% Spirulina diet; but it did not show

significant (Duncans multiple range test: P<0.05) difference between them. Hence

15% Spirulina diet is considered as optimum level to maximise the feed intake,

growth, coloration and leucocytes count in X. helleri. Spirulina could be used as

nutrition / immunodulatory feed additive in red swordtail fish nutrition.

47
Table 4.1. Effect of different levels of Spirulina on mean body weight (g wet

 
weight) and length (mm) in red swordtail, Xiphophorus helleri as
a function of time. Each value is the mean X r SD of three
observations.

Levels of Spirulina (%)

Rearing
period Mean body weight Mean body length
(days)
0 5 10 15 20 0 5 10 15 20
0.132 0.132 0.132 0.132 0.132 22.58 22.58 22.58 22.58 22.58
0
0.02 0.02 0.02 0.02 0.02 1.26 1.26 1.26 1.26 1.26
0.316 0.332 0.341 0.353 0.363 25.80 27.55 29.10 30.60 31.80
25
0.03 0.02 0.01 0.02 0.02 2.56 2.87 2.21 1.62 1.08
0.431 0.466 0.511 0.606 0.685 26.30 27.80 29.40 31.00 32.50
50
0.05 0.02 0.01 0.02 0.01 1.27 1.94 2.15 1.41 1.02
0.545 0.588 0.681 0.947 0.947 31.80 36.80 36.90 42.00 42.27
75
0.05 0.01 0.01 0.01 0.01 2.71 4.51 2.37 2.76 4.41
0.702 0.852 0.988 1.379 1.345 35.60 36.80 42.30 45.40 45.00
100*
0.01 0.01 0.01 0.01 0.04 2.29 2.32 1.19 1.88 1.18
0.904 0.969 1.207 2.090 2.085 40.25 41.75 44.12 52.63 52.38
125
0.01 0.03 0.01 0.08 0.08 2.17 1.09 2.89 1.80 2.12
1.145 1.244 1.507 2.683 2.645 43.00 46.63 50.00 63.50 61.50
150
0.06 0.02 0.01 0.01 0.04 1.22 0.99 2.24 4.72 1.87
1.672 1.721 2.251 3.280 3.250 48.25 51.75 57.38 73.00 71.63
175
0.03 0.02 0.05 0.01 0.40 2.50 1.48 3.16 2.78 3.46

* Breeding commenced
Table 4.2. Two-way ANOVA for mean body weight and length in
Xiphophorus helleri as a function of Spirulina levels and rearing
period.

Source of variance SS df MS F-value P-value

Mean body weight

Between Spirulina levels 65.04 7 9.29 2.87 P < 0.01

Between rearing period 11.34 4 2.84 3.56 P<0.01

Interaction 9.00 28 0.32 1.97

Residual 0.06 80 0.01

Total 85.43 119

Mean body length

Between Spirulina levels 27005.90 7 3857.99 2.75 P < 0.01

Between rearing period 3811.67 4 952.92 3.44 P < 0.01

Interaction 2064.65 28 73.74 1.85

Residual 1228.80 160 7.68

Total 34111.02 199

Table 4.3. Effect of different levels of Spirulina on feed consumption (g dry
matter) and feeding rate (mg g-1 live fish day-1) in red swordtail,

X r SD of three observations.
Xiphophorus helleri as a function of time. Each value is the mean

Rearing Levels of Spirulina (%)

period (days) 0 5 10 15 20
Feed consumption
25 6.63 0.52 7.24 0.43 7.30 0.03 8.21 0.02 8.51 0.17
50 6.22 0.47 6.44 0.08 7.48 0.23 11.12 0.25 14.66 0.24
75 4.15 0.38 4.46 0.07 6.67 0.03 10.56 0.03 12.70 0.15
100* 5.48 0.28 6.29 0.11 9.26 0.15 13.48 0.03 13.58 0.36
125 4.53 0.07 4.41 0.08 6.98 0.12 11.81 0.06 11.53 0.18
150 9.15 0.05 12.80 0.05 12.47 0.48 14.60 0.50 15.02 0.03
175 4.33 0.30 4.33 0.36 7.46 0.46 10.59 0.32 10.30 0.48
Feeding rate
25 79.88 0.85 84.19 4.42 85.01 0.48 94.77 0.26 99.82 4.17
50 39.35 0.51 38.81 1.13 43.48 1.81 63.16 3.13 80.95 4.00
75 20.86 0.51 21.12 0.77 28.02 0.23 37.95 0.97 40.07 0.90
100* 25.84 1.29 27.19 0.49 34.46 0.11 37.01 0.39 35.78 1.52
Mean feeding
rate at 41.49 42.89 47.74 58.25 64.16
pre-spawning
125 65.66 1.49 58.99 2.43 74.21 1.86 93.92 3.79 87.02 2.74
150 101.237.28 102.364.70 103.404.97 77.49 5.91 72.12 2.53
175 38.04 4.64 34.54 3.06 49.48 2.77 40.71 1.58 38.95 1.27
Mean feeding
rate at 68.31 75.30 75.70 70.71 66.03
post-spawning

*Breeding commenced
Table 4.4. Effect of different levels of Spirulina on weight gain (g wet

 
weight), specific growth rate (% day-1) and feed conversion ratio

X r SD of three observations.
in red swordtail, Xiphophorus helleri. Each value is the mean

Rearing Levels of Spirulina (%)

period (days) 0 5 10 15 20
Weight gain
25 3.02 0.34 3.20 0.09 3.39 0.02 3.60 0.28 3.84 0.27
50 2.28 0.36 2.67 0.06 3.39 0.15 4.89 0.03 6.42 0.75
75 1.28 0.07 1.55 0.18 2.04 0.03 4.36 0.51 3.42 0.05
100* 1.30 0.67 2.69 0.11 3.07 0.14 4.83 0.08 4.32 0.04
125 0.86 0.01 0.88 0.05 1.05 0.11 3.03 0.06 2.52 0.09
150 0.96 0.22 1.10 0.20 1.20 0.07 2.85 0.23 2.24 0.46
175 1.59 0.15 1.87 0.05 2.97 0.03 3.25 0.60 2.31 0.04
Specific growth rate
25 2.58 0.09 2.63 0.04 2.75 0.02 2.84 0.11 3.01 0.02
50 1.22 0.07 1.35 0.04 1.61 0.06 2.11 0.09 2.54 0.34
75 0.61 0.09 0.67 0.09 0.78 0.01 1.32 0.16 0.96 0.01
100* 0.59 0.03 1.02 0.04 1.00 0.05 1.15 0.03 1.00 0.02
Mean SGR at
1.25 0.81 1.42 0.74 1.54 0.06 1.86 0.07 1.87 0.04
pre-spawning
125 1.08 0.01 1.03 0.11 0.99 0.13 1.89 0.10 1.56 0.11
150 0.94 0.19 1.00 0.19 0.89 0.06 1.28 0.13 0.95 0.20
175 1.51 0.28 1.27 0.02 1.60 0.02 0.94 0.02 0.79 0.02
Mean SGR at
1.18 0.24 1.10 0.12 1.16 0.01 1.37 0.09 1.10 0.03
post-spawning
Feed conversion ratio
25 2.20 0.08 2.26 0.07 2.15 0.01 2.29 0.12 2.23 0.11
50 2.77 0.23 2.42 0.02 2.21 0.03 2.28 0.01 2.32 0.30
75 3.27 0.46 2.91 0.30 3.27 0.03 2.46 0.30 3.71 0.09
100* 5.89 0.25 2.34 0.05 3.03 0.19 2.30 0.22 2.93 0.10
Mean FCR at
3.53 0.41 2.48 0.25 2.67 0.49 2.33 0.07 2.80 0.59
pre-spawning
125 5.30 0.11 5.07 0.41 6.69 0.82 3.59 0.09 4.80 0.20
150 10.05 2.38 11.99 2.10 10.51 0.35 4.89 0.04 6.99 0.43
175 2.11 0.22 2.32 0.25 2.51 0.18 4.41 0.04 4.28 0.46
Mean FCR at
5.82 0.26 6.46 0.06 6.57 0.26 4.30 0.54 5.36 0.47
pos- spawning
* Breeding commenced
Students t test
Mean SGR at pre-spawning Mean SGR at post-spawning
15 Vs 10% Spirulina : t = 10.58; P < 0.01 15 Vs 10 Spirulina : t = 26.65; P < 0.01
15 Vs 20% Spirulina : t = 4.99; P < 0.01 15 Vs 20 Spirulina : t = 5.99; P< 0.01
Table 4.5. Effect of different levels of Spirulina on protein efficiency ratio

 
(%) and apparent digestability co-efficient (%) in red swordtail,
Xiphophorus helleri. Each value is the mean X r SD of three
observations.
Rearing Levels of Spirulina (%)
period
(days) 0 5 10 15 20

Protein efficiency ratio

25 0.91 0.05a 0.92 0.05 a 0.97 0.03 b 1.02 0.04 c 1.00 0.01 c
50 0.54 0.03 a 0.67 0.05 b 0.98 0.04 c 1.09 0.04 d 1.08 0.04 d
75 0.55 0.02 a 0.66 0.01 b 0.77 0.10 c 0.86 0.10 d 0.76 0.08 c

100* 0.55 0.30 a 0.65 0.01 b 0.72 0.04 c 0.84 0.03 d 0.75 0.03 c
Mean PER
at pre- 0.64 0.73 0.86 0.95 0.90
spawning
125 0.33 0.04 a 0.42 0.01 b 0.44 0.04 b 0.53 0.01 c 0.45 0.02 b
150 0.19 0.03 a 0.21 0.01 b 0.24 0.06 c 0.41 0.02 e 0.31 0.06 d
175 0.46 0.03 a 0.64 0.14 b 0.83 0.13 c 0.96 0.11 e 0.86 0.06 d

Mean PER
at post- 0.33 0.44 0.50 0.63 0.54
spawning

Apparent digestability co-efficient

25 55.25 0.49a 57.08 0.62 a 60.40 0.45 a 66.95 0.54 b 66.83 0.62 b
50 67.90 1.54 a 68.80 0.75 a 71.63 0.74 b 81.75 1.12 c 81.05 0.74 c
75 68.13 1.53 a 68.55 0.58 a 78.63 0.72 b 87.30 0.50 c 83.70 0.77 c
100* 58.75 0.91 a 61.33 0.68 a 69.15 0.54 b 88.43 0.87 c 84.40 0.71 c
125 63.63 1.34 a 66.53 0.63 a 80.13 0.49 b 90.23 0.63 c 88.23 0.82 bc
150 61.90 0.29 a 74.45 0.66 b 87.58 0.49 c 96.88 0.44 d 93.73 0.57 d
175 62.35 0.42 a 76.03 0.51 b 88.03 0.49 c 97.55 0.70 d 96.23 0.53 d

* Breeding commenced

Values (mean r SD) with different superscripts in the same row are significantly

different (P<0.05).
Table 4.6. Two-way ANOVA for selected food utilization parameters in
Xiphophorus helleri as a function of Spirulina levels and rearing
period.

Source of variance SS df MS F-value P-value

Feeding rate
Between Spirulina levels 69153.15 6 11525.53 3.07 P<0.01
Between rearing period 2026.13 4 506.53 3.60 P<0.01
Interaction 14013.57 24 583.90 2.07
Residual 482.84 70 6.90
Total 85675.69 104
Weight gain
Between Spirulina levels 68.28 6 11.38 3.07 P<0.01
Between rearing period 79.86 4 19.96 3.60 P<0.01
Interaction 27.76 24 1.16 2.07
Residual 5.08 70 0.07
Total 180.98 104
Specific growth rate
Between Spirulina levels 39.96 6 6.66 3.07 P<0.01
Between rearing period 2.70 4 0.68 3.60 P<0.01
Interaction 6.48 24 0.27 2.07
Residual 1.16 70 0.02
Total 50.29 104
Feed conversion ratio
Between Spirulina levels 486.37 6 81.06 3.07 P<0.01
Between rearing period 22.64 4 5.66 3.60 P<0.01
Interaction 108.99 24 4.54 2.07
Residual 52.73 70 0.75
Total 670.72 104
Table 4.7. Effect of feeding different levels of Spirulina on carotenoid

 
contents (g 100 1 mg wet tissue) in fins, skin and muscle of red
swordtail, Xiphophorus helleri. Each value is the mean X r SD
of three observations.

Rearing Levels of Spirulina (%)

period (days) 0 5 10 15 20
Fins
0.025 0.060 0.072 0.093 0.107
50
0.003a 0.004b 0.006c 0.005d 0.005e
0.037 0.060 0.110 0.140 0.140
75
0.002a 0.002b 0.012c 0.004d 0.003d
0.109 0.182 0.320 0.463 0.384
100*
0.002a 0.004b 0.006c 0.017e 0.006d
Skin
0.014 0.023 0.030 0.072 0.084
50
0.001a 0.001b 0.001c 0.005d 0.004e
0.030 0.052 0.103 0.128 0.127
75
0.001a 0.002b 0.010c 0.002d 0.001d
0.082 0.133 0.278 0.377 0.291
100*
0.002a 0.004b 0.008c 0.004e 0.005d
Muscle
0.003 0.010 0.015 0.029 0.025
50
0.001a 0.002b 0.003c 0.003e 0.004d
0.016 0.022 0.026 0.040 0.028
75
0.001a 0.002a 0.002a 0.001b 0.002a
0.046 0.094 0.142 0.176 0.169
100*
0.002a 0.001a 0.003ab 0.001b 0.003ab

* Breeding commenced

Values (mean SD) with different superscripts in the same row are significantly
different (P < 0.05).
Table 4.8. Impact of feeding various levels of Spirulina on specific activity

 
(g mg-1 of body protein hr-1) of digestive enzymes in the digestive
tract of Xiphophorus helleri. Each value is the mean X r SD of
three estimations.

Amylase Protease Lipase

(g maltose mg-1 of (g tyrosine mg-1 of (g lipase mg-1 of

Spirulin
protein hr-1) protein hr-1) protein hr-1)
a levels
(%)
Foreg Midg Hindg Foreg Midg Hindg Foreg Midg Hindg
ut ut ut ut ut ut ut ut ut

121.06 263.17
226.72 191.34 77.88 192.11 40.34 176.25 190.79
0
5.09 5.47 4.78 19.16 1.05 9.31 10.57
17.46 17.46

192.11 318.33
238.72 227.71 168.22 260.54 72.47 195.00 244.74
5
8.35 9.49 9.40 19.87 1.86 1.97 2.91
21.22 10.88

265.47 347.46
289.87 203.60 236.85 271.06 99.91 248.59 314.62
10
4.67 12.84 9.12 10.85 2.47 8.11 6.18
13.12 12.05

411.76 328.14 323.70 415.80 275.22

243.52 336.85 125.83 341.71
15
10.03 21.70 3.41 1.39
14.98 17.29 15.12 14.89 24.59

382.31 318.43 373.70

300.06 220.01 323.44 126.25 292.23 339.13
20
5.72 10.22 26.89 2.26 3.65 0.91
11.59 24.83 29.77

Students t test

Amylase in foregut
15 Vs 10% Spirulina : t = 4.39; P < 0.05
15 Vs 20% Spirulina : t = 4.51; P < 0.05

Protease in midgut
15 Vs 10% Spirulina : t = 12.49; P < 0.01
15 Vs 20% Spirulina : t = 2.53; P < 0.05

Lipase in hindgut
15 Vs 10% Spirulina : t = 9.79; P < 0.01
15 Vs 20% Spirulina : t = 4.15; P < 0.05
Table 4.9. Effect of different levels of Spirulina on leucocyte counts (%) in

X r SD of three observations.
red swordtail, Xiphophorus helleri. Each value is the mean

Levels of
Spirulina Lymphocytes Thromphocytes Monocytes Neutrophils Basophils
(%)

15.50 26.50
0 21.50 1.80 20.00 2.50 16.50 1.50
1.20 2.29

18.50 24.00
5 24.75 1.09 14.75 2.95 18.00 1.22
2.18 1.12

21.50 10.00
10 35.50 1.12 12.25 3.42 20.75 2.17
1.50 1.18

22.00 6.00
15 40.50 2.87 8.00 0.83 23.50 1.66
1.50 0.12

20.00 6.50
20 46.00 3.22 8.25 1.78 19.25 2.09
1.14 0.66
 4 (a)
0 Spirulina Y = 0.04 + 0.008x; r = 0.930
5% " Y = 0.06 + 0.008x; r = 0.956
10% " Y = - 0.009 + 0.011x; r = 0.938

Mean body weight (g wet weight)

15% " Y = - 0.17 + 0.018x; r = 0.964
3 Y = - 0.14 + 0.018x; r = 0.964
20% "

(b)
80 0 Spirulina Y = 21.23 + 0.148x; r = 0.983
5% " Y = 22.23 + 0.163x; r = 0.974
Y = 22.40 + 0.189x; r = 0.980
10% "
Y = 20.68 + 0.279x; r = 0.973
15% "
Mean body length (mm)

60 Y = 21.87 + 0.264x; r = 0.974

20% "

40

20

0
0 25 50 75 100 125 150 175 200

Rearing period (days)

Fig. 4.1. Effect of different levels of Spirulina on mean body

weight (a) and length (b) in red swordtail, Xiphophorus
helleri reared for 175 days.
 140 0 Spirulina
5% "
10% "
120
15% "
20% "
Feeding rate (mg g -1 live fish day -1)

100

80

60

40

20

0
25 50 75 100 125 150 175

Rearing period (days)

Fig. 4.2. Effect of different levels of Spirulina on feeding rate

in red swordtail, Xiphophorus helleri as a function of
time.
 7
(a)
0 Spirulina
5% "
6
10% "
15% "

Weight gain (g wet weight)

5 20% "

4 (b)
Specific growth rate (% day-1)

0
25 50 75 100 125 150 175

Rearing period (days)

Fig. 4.3. Effect of different levels of Spirulina on weight gain

(a) and specific growth rate (b) in red swordtail,
Xiphophorus helleri as a function of time.
 80 Pre-spawning
(a)
Post-spawning

(mg g-1 live fish day -1)

60

Feeding rate
40

20

2 (b)
Specific growth rate

1.5
-1
(% day )

0.5

8 (c)
Feed conversion ratio

0
0 5 10 15 20
Levels of Spirulina (%)

Fig. 4.4. Effect of different levels of Spirulina on feeding

rate (a), specific growth rate (b) and feed conversion
ratio (c) in red swordtail, Xiphophorus helleri as a
function of time.
 0.5 Fins 0 Spirulina
5% "

0.4 10% "

15% "
20% "
0.3

0.2

0.1

0
Carotenoid concentration (mg 100-1 mg wet tissue)

0.5
Skin

0.4

0.3

0.2
(b)
0.1

0.2 Muscle

0.15

0.1

0.05

0
50 75 100
Rearing period (days)

Fig. 4.5. Effect of feeding different levels of Spirulina on

carotenoid contents in fin, skin and muscle of red
swordtail, Xiphophorus helleri as a function of time.
 0 Spirulina
500

(g maltose mg-1 of protein hr -1)

5% "
10% "
400 15% "
20% "

Amylase
300

-1 200

100

500
-1
(g tyrosine mg of protein hr )

400
Protease

300
-1

200
-1

100

400
-1
(g lipase mg of protein hr )

300
Lipase

200
-1

100
-1

0
Foregut Midgut Hindgut

Fig. 4.6. Impact of feeding various levels of Spirulina on

specific activity of digestive enzymes in the digestive
tract of Xiphophorus helleri.
 60 Lymphocytes
Thromphocytes
Monocytes
Neutrophils
50
Basophils

40
Leucocyte counts (%)

30

20

10

0
0 5 10 15 20
Spirulina levels (%)

Fig. 4.7. Effect of different levels of Spirulina on leucocyte

counts in red swordtail, Xiphophorus helleri.