Nutrient Digestibility in Labeo rohita Fingerlings Fed Citric Acid Acidified Phytase Sprayed Diet

Syed Zakir Hussain Shah1*, Muhammad Afzal2, Mahroze Fatima3,

Syed Makhdoom Hussain4 and Tanveer Ahmed5

1Department of Zoology, University of Gujrat, Gujrat, Pakistan

2Fish Nutrition Laboratory, Department of Zoology, Wildlife and Fisheries, University of Agriculture, Faisalabad

3Department of Fisheries and Aquaculture, University of Veterinary and Animal Sciences, Lahore

4Department of Zoology, Government College University, Faisalabad

5Aquaculture Biotechnology Laboratory, Department of Zoology, Wildlife and Fisheries, University of Agriculture, Faisalabad

ABSTRACT

A factorial experiment under completely randomized design was conducted by using citric acid (CA) supplementation (3 levels) and phytase (PHY) supplementation (3 levels) as the main effects. CA was supplemented at 0, 1.5 and 3% and PHY was added at 0, 750 and 1000 FTU/kg diet in a 3 × 3 factorial arrangement. All the nine test diets were contained 37.6% crude protein and 4.72 kcal/g gross energy. Results showed improved (p<0.05) dry matter, crude protein, crude fat and gross energy digestibility by fingerlings when fed PHY sprayed diet. Similarly, CA addition in the diet resulted in increased (p<0.05) digestibility of these nutrients. Also, the minerals digestibility was significantly (p<0.05) affected by top spraying of phytase on the diet pellets. Fish having CA acidified diet also showed improved (p<0.05) minerals digestibility performance. Interaction plots showed synergistic effect of both the supplements for crude fat and gross energy digestibility. Again, interaction data showed synergistic effect on minerals digestibility except Fe. Inclusion of both the supplements independently and in combination in the diet resulted in less nutrient contents in fish feces. In conclusion, addition of CA and PHY in the Labeo rohita diet may improve the nutrient digestibility which may lead to improved growth performance and less pollution in the water bodies.

Article Information

Received 23 September 2015

Revised 02 May 2019

Accepted 12 June 2019

Available online 11 January 2021

Authors’ Contribution

SZHS and MF conducted the experiment and wrote the manuscript. MA supervised the experiment manuscript write up. SMH and TA helped in the data collection and analysis.

Key words

Citric acid, Dietary acidification, Nutrient digestibility, Phytate, Rohu

DOI: https://dx.doi.org/10.17582/journal.pjz/20150923011725

* Corresponding author: zakiruaf@gmail.com

0030-9923/2021/0002-0459 $ 9.00/0

Copyright 2021 Zoological Society of Pakistan

INTRODUCTION

Fishmeal is being used as a major protein source in aquafeeds (Hardy, 1995) as it has high protein content, balanced amino acid profile, utmost digestibility and presence of relatively less anti-nutritional factors. However, due to increasing demand, unstable supply and high price of fishmeal, other alternative protein sources have long been of interest (Lunger et al., 2007). Attempts are being made to use plant proteins in place of fishmeal in aquafeeds (Cain and Garling, 1995). However, these low cost plant proteins have many problems including the presence of anti-nutritional factors like phytate (myo-inositol-1, 2, 3, 4, 5, 6-hexaphosphates). Up to 80% of total phosphorus contents in plant ingredients are present in phytate form which are unavailable to agastric and mono-gastric fish species (NRC, 1993). Therefore, undigested phosphorus is excreted into water which results in eutrophication (Liebert and Portz, 2005). Besides, phytate makes insoluble chelate complexes with indispensable minerals (Papatryphon et al., 1999). Also, it reduces the availability of protein, fat and vitamins by making complexes with them (Liu et al., 1998; Sugiura et al., 2001).

Agastric fish species including L. rohita lack enzyme system to break the phytate. Supplementation of exogenous phytase (PHY) in plant based fish feed has resulted in increased bioavailability of nutrients resulting in improved growth performance. Phytase is an enzyme chemically known as myo-inositol-hexaphosphate phosphohydrolase which catalyzes the hydrolysis of phytate into inositol and orthophosphoric acid (Lei et al., 2013) resulting in release of bound nutrients. Previous studies showed improved nutrients digestibility in L. rohita fingerlings fed on phytase supplemented diets (Hussain et al., 2011a, b, c; Baruah et al., 2007b).

Another approach being applied in animal nutrition to break the phytate is the use of organic acids. Among these organic acids, citric acid (CA) is considered as the most promising acidifier. Zyla et al. (1995) reported that CA dephosphorylate the phytate in vitro. Beside this, these organic acids also help to reduce the gastric pH, increase the activity of digestive enzymes, improve the nutrient digestibility and reduce the bacterial load (Hossain et al., 2007). Previous studies indicated that CA (3%) improve the P utilization in L. rohita (Baruah et al., 2005) while enhanced apparent absorption of P and Mg in Oncorhynchus mykiss was also observed in CA (5%) supplemented group (Sugiura et al., 2001).

An important aspect of PHY is that it is a pH dependent enzyme and performs better at lower pH (2.5 and 5.0-5.5) (Simons et al., 1990). However, L. rohita is an agastric fish species having poor acid secretion in its gut, resulting in higher pH (above 6). Therefore, phytase performance can be enhanced by adding organic acids in the diet which may lower the dietary pH leading to decreased pH in fish gut. Present research was conducted to investigate the influence of CA acidification in PHY treated diet in improving the nutrient digestibility performance by L. rohita fingerlings. Information based on this research may prove useful to develop less expensive and less polluting feed for Labeo rohita.

 

MATERIALS AND METHODS

The experiment was carried out in Fish Nutrition Laboratory, Department of Zoology and Fisheries, University of Agriculture, Faisalabad, Pakistan. The experiment lasted for 60 days.

Experimental diets

Basal diet was formulated having 37.6% crude protein and 4.72 kcal/g gross energy. Fish meal and soybean meal was added as protein source in basal diet. Fish oil was used as lipids source. Composition of basal diet is shown in Table I. All ingredients were ground, sieved (0.5 mm) and were thoroughly mixed in electric mixer for 10-20 minutes with gradual addition of fish oil. In dry mixed ingredients citric acid was added at three concentrations 0%, 1.5% and 3% and partitioned into three test diets. Inert marker (1% Chromic oxide) was added for digestibility analysis. Dough for extrusion was made with addition of 10-15% of water. Pellets (2 mm) were made by using had pelletizer. These pellets were sprayed by following Jackson et al. (1996) with three concentrations of phytase; 0, 750 and 1000 FTU/kg diet. Hence, supplementation of citric acid and phytase resulted in nine test diets, ranging from T1 having 0% CA and 0 FTU/kg PHY to T9 containing 3% CA and 1000 FTU/kg PHY. Diets with 0 FTU/kg phytase concentration was sprayed with equal amount of water to make moisture contents constant. Pellets were dried up till 8-10% moisture contents remained and stored at 4oC until initiation of feeding trial.

Feeding and experimental units

Fingerlings of Labeo rohita (average weight 9 g) were taken from Government Fish Seed Hatchery, Faisalabad, Pakistan. Before feeding trial, fingerlings were treated with 5 g NaCl/L to make them ectoparasites free and acclimatized in V shaped tanks while feeding on basal diet (Allan and Rowland, 1992) with constant supply of oxygen. A group of 9 fish was stocked in each experimental V shaped tank of 70 L capacity (UA system), peculiarly designed for fecal collection from aquatic media. Triplicate tanks were allocated to each test diet. Water quality parameters (DO 5.8-7.3 mgL-1, pH 7.4-8.6 and temp 24.9-28.7oC) were monitored constantly among all tanks. Fish were fed 2% of live wet body weight twice a day and feeding was adjusted according to weekly weight measure of fish. After three hours of feeding all uneaten diet was removed with washing of tanks. After two hours of washing fecal material was collected via feaces collection tube of V shaped tanks equipped with two valves. Fecal material was collected by subsequent opening of valve I and II and was oven dried at 60oC and stored for further analysis.

Chemical analysis

At the end of feeding trial samples of feed ingredients, test diets and fecal material were homogenized with the help of pestle and mortar and analyzed following AOAC (1995). Dry matter contents were determined by drying in oven at 105oC until constant weight. Crude protein (CP) was calculated by N×6.25 while N was determined by micro Kjeldahl apparatus while crude fat by ether extraction method through Soxtec HT2 1045 system. Gross energy was measured with the help of adiabatic oxygen bomb calorimeter (Parr Instrument Co., Moline, USA). For estimation of minerals, test diets and feces samples were digested in boiling nitric acid and perchloric acid mixture (2:1). After recommended dilutions, minerals were estimated on atomic absorption spectrophotometer (Hitachi Polarized Zeeman AAS, Z-8200, Japan) except P, Na and K. Na and K were estimated on flame photometer (Jenway PFP-7, UK) while P was estimated calorimetrically (UV/VIS spectrophotometer Hitachi U-2001) at 720 nm absorbance. Chromic oxide contents were measured by using UV-VIS 2001 spectrophotometer at 350 nm absorbance after oxidation with molybdate reagent (Divakaran et al., 2002).

 

Table I. Composition and proximate analysis of test diets.

CA level (%) PHY level (FTU/kg) Test diets	0			1.5			3
	0	750	1000	0	750	1000	0	750	1000
	T1 (Control)	T2	T3	T4	T5	T6	T7	T8	T9
Fish meal (%)	36	36	36	36	36	36	36	36	36
Soybean meal (%)	30	30	30	30	30	30	30	30	30
Rice polish (%)	14	14	14	14	14	14	14	14	14
Wheat flour (%)	10	10	10	8.5	8.5	8.5	7	7	7
Fish oil (%)	6	6	6	6	6	6	6	6	6
Vitamin premix (%)	1	1	1	1	1	1	1	1	1
Minerals mixture (%)	1	1	1	1	1	1	1	1	1
Ascorbic acid (%)	1	1	1	1	1	1	1	1	1
Chromic oxide (%)	1	1	1	1	1	1	1	1	1
Total	100	100	100	100	100	100	100	100	100
Proximate analysis
Dry matter (%)	97.43	97.5	97.67	97.4	97.43	97.4	97.53	97.47	97.53
Crude protein (%)	37.5	37.8	37.7	37.63	37.53	37.93	37.73	37.43	37.5
Crude fat (%)	8.7	8.5	8.87	8.9	8.87	8.67	8.7	8.9	8.63
Gross energy kcal/g	4.72	4.66	4.74	4.72	4.73	4.72	4.69	4.68	4.68

 

Calculations and statistical analysis

Apparent nutrient digestibility coefficients (ADC%) of test and reference diets were calculated following NRC (1993).

Resulting data was subjected to two way analysis of variance (Steel et al., 1996). Significant differences among means were calculated by Tukey`s Honestly Significant Difference test at 5% significance level (Snedecor and Conhran, 1991). The CoStat computer package (Version 6.303, PMB 320, Monterey, CA, 93940 USA) was used for statistical analysis while interaction plots were made by using Minitab15.

 

RESULTS

Effect of CA and PHY supplementations on nutrients content in feces is shown in Table II. Both the supplements result in less excretion of dry matter, crude protein, crude fat and gross energy in feces in comparison with the control group (T1), however, minimum excretion was observed in T9 diet accompanied with 3% CA and 1000 FTU/kg phytase level. Similarly, addition of both the additives (CA and PHY) resulted in reduced amount of minerals in fish feces (Table II). Among all the tried levels of phytase, minimum excretion of minerals was recorded at 1000 FTU/kg level. Similarly, CA supplementation also resulted in less discharge of minerals in feces. Also, both the additives act synergistically (p<0.05) to decrease the mineral excretion in fish feces. However, their synergistic effect was not significant for Fe excretion in feces.

Nutrients digestibility results are presented in Table III. Top spraying of phytase on extruded pellets showed improved absorption (digestibility) of dry matter, nutrients (crude protein and crude fat) and gross energy as compare to control group. However, different phytase supplementation levels showed statistically non-significant differences (p<0.05) among themselves for dry matter, crude protein and gross energy digestibility while crude fat was maximum digested by L. rohita at phytase level of 1000 FTU/kg. Similarly, dry matter, nutrients and gross energy digestibility was also enhanced by CA addition in the diet.

Main effect data of PHY addition indicated that top spraying of phytase to plant based diet improved the minerals (P, Ca, Mg, Na, K, Fe, Cu and Zn) digestibility significantly (p<0.05) except Mn. However, both the phytase levels showed same effect (p>0.05) for most of the minerals except Fe and Mn which showed highest digestibility at 750 FTU/kg and 1000 FTU/kg phytase levels respectively.

Dietary addition of CA also improved (p<0.05) mineral absorption as compare to control group. Ca, Na, Mn and Cu showed higher digestibility at 1.5% CA level while other minerals (Mg, K and Fe) showed utmost digestibility at 3% CA level.

 

Table II. Concentration of nutrients in feces of fish fed CA and PHY containing diets.

CA level (%) PHY level (FTU /kg) Test diets

0

1.5

3

Analysis of variance

0

750

1000

0

750

1000

0

750

1000

T1 (Con-trol)

T2

T3

T4

T5

T6

T7

T8

T9

PSE*

CA

PHY

CA× PHY

Dry matter (%)

77.06a

66.20b

65.00b

65.26b

56.96d

55.83d

62.33c

53.60e

50.03e

0.911

S

S

NS

Crude protein (%)

26.90a

21.53c

21.90bc

22.16bc

18.90d

18.63d

23.03b

18.63d

17.96d

0.693

S

S

NS

Crude fat (%)

6.43a

5.73b

5.20c

5.03c

4.53e

4.76d

5.13c

4.40e

3.93f

0.093

S

S

S

Gross energy (%)

3.65a

3.21c

3.29b

3.24b

2.63e

2.49f

3.32b

2.75d

2.52f

0.035

S

S

S

P (%)

0.521a

0.452b

0.449c

0.434d

0.357f

0.336g

0.417e

0.330g

0.275h

0.007

S

S

S

Ca (%)

0.547a

0.455b

0.450b

0.446c

0.367d

0.362d

0.451b

0.337e

0.332e

0.004

S

S

S

Mg (%)

0.423a

0.345c

0.344c

0.351b

0.227f

0.225f

0.348c

0.247d

0.233e

0.004

S

S

S

Na (mg/g)

0.558a

0.449c

0.452c

0.450c

0.355d

0.351d

0.476b

0.343e

0.354d

0.004

S

S

S

K (%)

0.954a

0.757c

0.774b

0.678d

0.461h

0.481f

0.662e

0.470g

0.457i

0.004

S

S

S

Mn (μg/g)

77.66b

83.66a

75.66bc

69.33cd

67.66d

64.66d

75.66bc

64.00d

72.66c

0.000

S

NS

S

Fe (μg/g)

0.314a

0.2163b

0.2153b

0.2163b

0.1163cd

0.1173c

0.2146b

0.116cd

0.1146d

0.001

S

S

NS

Cu (μg/g)

41.33a

38.00bc

36.00c

38.14bc

26.01de

27.33d

39.03b

25.66de

24.33e

0.000

S

S

S

 

Data are means of three replicates. Means within rows having different superscripts are significantly different at P < 0.05. *PSE, pooled; SE, √MSE/n (where MSE, mean-squared error).

 

Table III. Effect of CA and PHY supplementation on apparent digestibility coefficient (ADC %) of nutrients in L. rohita fingerlings.

CA level (%) PHY level (FTU/kg) Test diets

0

1.5

3

Analysis of variance

0

750

1000

0

750

1000

0

750

1000

T1 (Control)

T2

T3

T4

T5

T6

T7

T8

T9

PSE*

CA

PHY

CA×PHY

Dry matter

30.76f

40.45e

41.68e

40.94e

48.67c

49.74c

43.942e

51.75b

54.94a

1.079

S

S

NS

Crude protein

36.85d

50.03b

49.08b

48.08b

55.79a

56.93a

46.45c

56.33a

57.90a

1.921

S

S

NS

Crude fat

36.12f

40.36e

48.55d

50.10c

55.06b

51.79c

48.26d

56.59b

59.96a

1.378

S

S

S

Gross energy

31.75f

39.47d

39.05d

39.52d

51.10b

53.77a

37.80e

48.48c

52.05b

0.922

S

S

S

P

36.87g

44.42f

44.66f

46.61e

56.10c

58.82b

48.80d

59.37b

66.12a

0.923

S

S

S

Ca

44.54g

53.74f

54.29ef

54.49e

62.60d

63.21c

54.46e

65.21b

66.09a

0.349

S

S

S

Mg

36.32f

47.14d

47.32d

46.13e

65.43a

65.53a

46.67de

62.39c

64.32b

0.579

S

S

S

Na

33.15e

47.07c

46.81c

46.92c

58.11b

58.67ab

44.04d

59.50a

58.24b

0.561

S

S

S

K

33.11h

47.02f

45.85g

52.39e

67.73a

66.36c

53.83d

67.13b

67.99a

0.307

S

S

S

Mn

45.51d

40.68e

46.93c

52.01b

52.91ab

54.76a

46.18c

55.17a

48.55c

1.461

S

NS

S

Fe

33.53d

54.13c

54.46c

54.32c

75.43ab

75.23ab

54.48c

75.15b

75.69a

0.233

S

S

NS

Cu

33.97d

39.67cd

42.21c

39.61cd

59.48ab

55.46b

37.78d

59.30ab

61.75a

2.427

S

S

S

 

Data are means of three replicates. Means within rows having different superscripts are significantly different at P < 0.05. *PSE, pooled; SE, √MSE/n (where MSE, mean-squared error).

 

DISCUSSION

Effects of different phytase inclusion methods in the fish diet have been studied including pre and post-treatments. Top spraying (post-treatment) of phytase on feed pellets can retain the phytase activity in the diet (Liu et al., 2013). Improved nutrients digestibility has been reported in carnivorous fish species by top spraying the phytase on dried pellets (Vielma et al., 2004; Wang et al., 2009; Tudkaew et al., 2008). However, there is scarcity of literature concerning the effect of phytase applications through top spraying on nutrients digestibility in the diets of non-carnivorous; agastric fish species.

Present study demonstrated that phytase top spraying to plant based L. rohita diet significantly enhanced the apparent digestibility of dry matter, crude protein, crude fat, gross energy and minerals. Likewise, improved apparent nutrients and minerals digestibility was also observed in our previous study (Hussain et al., 2011a, b, c) in L. rohita fingerlings fed on plant based diets sprayed with graded levels of phytase. Baruah et al. (2007a, b) also observed improved nutrients and minerals absorption in response to phytase supplementation (through spraying) to L. rohita diet. Recently, Liu et al. (2013), while working on another agastric carp species, grass carp (Ctenopharyngodon idellus), reported enhanced ADC% of crude protein, crude lipid, dry matter and minerals in phytase sprayed group in comparison of fish fed on control diet. This increased nutrient digestibility will ultimately lead to improved growth performance of fish (Hussain et al., 2011b).

Inclusion of organic acids in the diet of agastric fish species is an increasing practice now days, as such fishes have no acid secretion mechanism in the gut. Results of present study showed that citric acid supplementation to L. rohita feed enhanced the crude protein, crude fat, gross energy and minerals digestibility. Increased nutrient absorption will not only improved the nutrient profile of fish, but would be helpful in reduction of nutrient loss in feces, which will decrease the need of nutrient supplementation in feed and aquatic pollution caused by nutrient loss in feces. The positive effects of diet acidification by CA supplementation on nutrients absorption were also reported by Baruah et al. (2007a, b) in L. rohita fingerlings fed on plant based diet. Rainbow trout also showed enhanced minerals absorption in response to CA addition to plant based diet (Sugiura et al., 2001). Citric acid increases the bioavailability of nutrients to fish in several different ways. It physically affects the chemical bonds between phytate and different nutrients resulting in release of these bounded nutrients (Atapattu and Nelligaswatta, 2005). Also, being a strong chelator of Ca and P, it removes these minerals from the phytate making it less stable and more susceptible to endogenous phytases (Khajepour and Hosseini, 2010). Furthermore, it helps in increasing the pepsin activity and in decreasing the intestinal microbial load by lowering the gastric pH (De Wet, 2005).

In the present study, CA and PHY synergistically enhanced the crude fat and gross energy digestibility. However, both the additives showed statistically non-significant interaction for the digestibility of dry matter and crude protein digestibility. So far, few studies have been conducted to investigate the nutrient digestibility in response to combined effect of CA and PHY. Similar to our study, Baruah et al. (2007b) also reported a non-significant interaction between CA and PHY on dry matter and crude protein digestibility in L. rohita fingerlings. In this study, a positive interaction between CA and PHY was observed on minerals absorption. CA may have provided optimum conditions for PHY to work by lowering the intestinal pH which lead to their synergitic effect on minerals digestibility. Furthermore, diet acidification lowers the rate of gastric emptying (Mayer, 1994) which also provide more time to the phytases for action. In agreement to our findings, improved minerals absorption was also reported by Baruah et al. (2007a) and Sugiura et al. (2001) in L. rohita and rainbow trout respectively fed on CA acidified phytase treated plant based diets.

 

CONCLUSION

Both the supplements (CA and PHY) act synergistically to improve the nutrient digestibility in L. rohita fingerlings which may lead to improved growth performance. Furthermore, more nutrient digestibility mean less excretion of these nutrients in the water bodies which may result in less water pollution. The information gathered in this study would contribute to develop the more nutritionally digestible feed for fish, however, more investigations on how increased nutrient digestibility improve the growth and nutrient retention and excretion in fish are needed. In addition, the effect of CA and PHY on digestive enzyme activities also require further evaluation

 

Statement of conflict of interest

The authors have declared no conflict of interest.

 

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