Deutscher Tropentag 2005
Stuttgart-Hohenheim, October 11-13, 2005
Conference on International Agricultural Research for Development
Effects of Different Processing Methods of Pigeon Pea (Cajanus cajan) on the Haematology
of African Catfish (Clarias gariepinus) Larvae.
Ogunji, Johnny O.1, 3; Uwadiegwu, Nicholas1; Osuigwe Donald I.2; Wirth, Manfred3
1Ebonyi State University Abakaliki, Nigeria. Department of Animal Production and Fisheries Management.
2Michael Okpara University of Agriculture Umudike, Nigeria. Department of Fisheries
3Institute of Freshwater Ecology and Inland Fisheries. Müggleseedamm 310 D- 12587 Berlin, Germany.
The need to substitute fishmeal in animal feed has necessitated the use of plant derived feedstuffs.
However, problems of anti nutritional factors in tropical legumes have limited their widespread
usage and direct incorporation into animal feeds. Different processing methods have been
deviced to remove or reduce the concentration of these factors. In this study Cajanus cajan was
subjected to four different processing methods. These included milling raw, toasting, boiling and
soaking. Effect of the different processing methods on the haematology of Clarias gariepinus
larvae was evaluated. This was aimed at determining the best processing method(s) for optimum
utilisation of pigeon pea meal for fish production. Twenty-one test diets were formulated to
contain about 39% crude protein. Results obtained showed that Haematocrit (PCV),
Haemoglobin concentration, RBC and WBC counts decreased significantly (P<0.05) with
increasing dietary levels of raw pigeon pea. Fish groups fed diets from other processing methods
showed lower values than the control when compared with the initial status. Soaking for 16 hours
enhanced the best fish weight gain and haematological values and seems to be the best processing
method for Cajanus cajan.
The need to substitute fishmeal in animal feed has necessitated the use of plant derived feedstuffs.
Legume seeds have been highly favourable because of their rich protein composition, energy and,
mineral content and widespread distribution in the tropics. However, only few of these plant
proteins have been utilized and investigated (Tacon and Jackson 1985; Webster et al., 1992;,
Ogunji and Wirth 2001). The presence of anti nutritional factors in these legumes have limited
their widespread usage and direct incorporation into animal feeds.
Pigeon pea (Cajanus cajan) seed is one of the tropical legume seeds that has been scarcely used
in fish feed production in spite of its crude protein and energy profile. Like other legume seeds,
its nutritive value is masked by the occurrence of anti nutritional factors, example trypsin
inhibitors haemaglutinin and saponin (Grimaud, 1988; Francis et al., 2001).
Some anti-nutritional factors are known to have negative effects on haematological parameters.
Concanavalin A (Con-A) causes agglutination of red blood cells (RBC) in monogastrics (Liener,
1989), while saponins are known to cause erythrocyte haemolysis and reduction of blood (Checke
1971). Anaemia was also reported to be associated with nutritional toxicity (Dick et al. 1976).
Gossypol severely reduces blood packed cell volume (PCV) and haemoglobin (Hb) concentration
in rainbow trout (Herman 1970).
Haematology has been developed and well utilised in assessing the health of man and livestock.
Svobodova et al., (1991) opined that ichthyo-haematology would be useful in the assessment of
suitability of feeds and feed mixture, evaluation of fish conditions, determination of toxic effect
of substances as well as diagnosis of disease.
The removal of anti-nutritional factors in pigeon pea using different processing methods is
important to make it safe for use in fish feed production. Different processing methods have been
devised to remove or reduce the concentration of these factors. In this study C. cajan was
subjected to four different processing methods. These are raw, toasting, boiling and soaking. The
effect of these processing methods on the haematology of Clarias gariepinus larvae was
Differently processed pigeon pea (Cajanus cajan) seed meal (PPSM) were obtained by (a)
toasting the seed by frying at a temperature of 120OC, b) subjecting the seeds to atmospheric
boiling for 1hr and thereafter sun dried, c) soaking the seeds in water for 16 hours and
thereafter sun dried and d) raw seeds. These were then milled using a hammer mill. The
differently processed PPSM were used to formulate 20 different isonitrogenous (Crude protein
39% ) diets at different dietary levels such that diets A1 to A 5, B1 to B5, C1 to C5 and D1 to D5
had raw -, toasted -, boiled and soaked – PPSM respectively at different dietary levels. Diet A0
had no PPSM but of the same nutritional regime as the other and served as control. Table 1 shows
a typical dietary inclusion rate used for the formulation of the experimental diets.
Table 1: Typical dietary inclusion rate used for the formulation of the experimental diets
Experimental Diets A – D
55 41 39
45 50 55 60 65
Palm Kernel Cake
Vit./Min. Premix **
1 1 1
2 2 2
100 100 100
**Vitamin and trace minerals supplied per Kg of final feed:
Vitamin A 24000 IU, Vitamin D3 2000 IU, Vitamin E 200 mg, Vitamin K3 8 mg, VitaminB1 20 mg, Vitamin B2
30 mg, Vitamin B6 12 mg, Panthonic acid 50 mg, Biotin 0.8mg, Niacin 150 mg, Vitamin B12 0.05mg, Folic acid
4mg, Vitamin C 500mg, Choline chloride 600mg, Inositol 200mg, Batanine 200mg, Cobalt 2mg, Iron 40mg, Iodine
5mg, Manganese 30mg, Copper 4mg, Zinc 40mg, Selenium 0.20mg, Lysine 100mg, Methionine 100mg, Antioxidant
After 5 days of acclimation and feeding with the control diet each test diet was randomly
assigned using CRD (Completely Randomized design) to triplicate lots of 10 liter capacity
aquaria each containing seven C. gariepinus larvae of average weight 0.46g. The fish were fed
5% of their body weight per day in two portions for 56 days in static water. The aquaria were
cleaned and water completely replaced by siphoning every other day to avoid fouling. Water
temperature, dissolved oxygen (DO) and PH were monitored and remained relatively stable.
Temperature was maintained at 26 ± 0.2oC, dissolved oxygen between 6.5 and 8.0 mg/L and pH
between 6 and 8. No critical values were detected in any of the tanks.
Fish were tranquilized with 150mg/L of tricane methane sulponate (ms222) (Wagner et al., 1997)
for blood collection. Blood samples were collected from ten fish from the pool at the
commencement of the feeding trial. At the end of the experiment 10 fish per feeding group and
two fish from control were sampled. Blood was taken from the caudal artery using 2ml plastic
syringe and needle treated with anti – coagulant and put in sample bottles. Haematocrit (PCV)
was determined with microhaematocrit using heparinized capillary tubes (25mm). Red blood cell
(RBC) and white blood cell (WBC) counts were determined as described by Blaxhall and Diasley
(1973). Haemoglobin (Hb) concentration was determined as described by Wedemeyer and
Results and Discussion
The haemoglobin concentration, PCV and RBC decreased significantly with increased dietary
PPSM (Table 2). This may have been due to the increasing presence of anti-nutritional factors
arising from increasing dietary PPSM in the diets. Pigeon pea seed contains protein inhibitors
(Typsin and Chymotrypsin) and amylase inhibitors which affect the activity of digestive enzymes
thereby causing digestive losses (Faris and Singh, 1990). These may have contributed to the
decreasing haematological parameters observed in this work. Ant-nutritional factors have been
reported to have deleterious effects and can interfere with food utilization, health and production
of animals (Makkar et al., 1993). Liener (1994a,b) recorded that protease inhibitors including
trypsin are potential anti-nutrients and are known to decrease growth performance. Tacon (1992)
reported that nutritionally deficient diets decrease haemoglobin concentration; reduce haematocrit
and red blood cell volume. Dick et al., (1976) found that nutritional toxicity is associated with
anaemia in fish. Osuigwe et al., (2003) corroborated these observations by attributing the low
values RBC, WBC, PCV count and Hb concentration of Clarias gariepinus fed raw Jack bean
seed meal to the anti-nutritional factors inherent in the plant ingredients.
Fish fed group D diets recorded Hb concentration, PCV and RBC counts similar to those fed
the control diet but significantly different from those fed group A, B and C diets. That is to
say that fish fed diets containing soaked PPSM conferred better hematological profile on C.
gariepinus than those fed diets containing other types of PPSM. This observation agrees with the
report that overheating reduces the biological value of legumes (McDonald et al., 1987). Heating
solubilizes and reduces nitrogenous compounds in legume seeds (Udedibie and Nwaiwu, 1987;
Osuigwe et al., 2003). This seems to be responsible for the inferior performance of fish fed diets
containing toasted and boiled PPSM. However, the WBC counts for fish fed heated PPSM
generally were similar to those fed the control diet than those fed diet containing soaked PPSM.
This may be attributed to wider fluctuations in values usually associated with WBC counts.
It is therefore concluded from this study that soaking for 16 hours is the best processing method
for PPSM used in C. gariepinus diets.
Table 2: Haematological parameters (mean) and weight of Clarias gariepinus larvae fed
four differently processed diets of pigeon Pea.
Haemoglobin PCV (%)
33.3n 70.00i 8.20g 22.00i
1.30bcd 33.1 n 69.50hi 7.37 d 22.05i
A1 0.98abc 25.15 e 51.50e 5.83 c 18.00 d
A2 0.93abc 23.25 d 49.50 d 4.75 b 16.50 c
A3 0.70ab 20.65 c 47.75 c 4.40ab 15.50b
A4 0.65 a 18.20 b 47.00b 4.23 a 15.00ab
A5 0.62 a 17.58 a 45.50a 7.60 def 14.50a
B1 1.12abce 31.00 hi 69.00gh 7.90efg 20.50ef
B2 1.03abcd 30.50gh 69.50 hi 7.85defg 21.25ghi
B3 1.03abcd 29.50 f 68.00f 8.00efg 20.67fg
B4 1.3bcde 31.00 hi 70.00i 7.75defg 21.00fgh
B5 1.58 def 30.00fg 69.50 hi 7.54de 20.00e
C1 1.4cde 31.17i 68.50fg 7.73defg 21.00fgh
C2 1.54cde 32.00jk 69.00gh 7.90efg 20.50ef
C3 1.24bcde 31.37i 68.50fg 7.85defg 20.50ef
C4 1.35cde 32.00jk 69.50hi 7.90efg 20.50ef
C5 1.44cde 31.62l j 68.50fg 8.05efg 20.00e
D1 1.51cde 32.12jkl 69.50hi 8.20g 21.00fgh
D2 2.39f 32.67lmn 69.17gh 8.07efg 21.75ij
D3 2.13fgh 32.78mn 70.00i 8.07efg 21.25ghi
D4 2.13fgh 33.23 n 68.50fg 8.10fg 20.00e
D5 2.21gh 32.37klm 69.50 hi 8.12fg 21.50hij
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