Effect of fresh cassava leaves and water spinach as protein sources in diets on N and amino acid digestibility in growing crossbred pigs 

Chhay Ty, Le Duc Ngoan^*, Le Dinh Phung^*

Center for Livestock and Agriculture Development (CelAgrid),
Pras Teat village, Rolous commune, Kandal Stung district, Kandal province. PO Box 2423 Phnom Penh 3, Cambodia
chhay@celagrid.org
Hue University of Agriculture and Forestry, Vietnam, 102 Phung Hung Street. Hue city, Vietnam 

Abstract

The objective of this experiment was to study effect of fresh cassava leaves and water spinach as protein sources in diets on N and amino acid digestibility in growing crossbred pigs. A balance experiment was arranged as a Latin-square design and included 4 male castrates of 50.3 (1.44) kg live weight. Four treatments included diets based on rice bran, broken rice and fishmeal (as the control diet – CTRL), and with fresh cassava leaves (FCL) or fresh water spinach (WS) or mixture of WS and FCL with ratio 1:1 (WSFCL) as the main protein source. Feed intake and nutrient digestibility were measured.

The control diet had higher feed intake than diets with fresh cassava leave, water spinach and mixture of fresh cassava leave and water spinach as the main protein source. Fresh water spinach was more palatable than the cassava leaves as reflected in higher total dry matter intake but not differences in proportion of the diet (26.7% for fresh cassava leaves and 26% for fresh water spinach and 26.3% for fresh cassava leave mix with water spinach). Digestibility of DM, OM, N, CF, NDF and essential amino acid in WS diet was higher than those in other treatments. There appeared to be some synergism effects between fresh cassava leaves and the fresh water spinach as the N retention of pigs fed the mixture fresh water spinach and fresh cassava leave diet (21.0g/day) was almost as high as on the fresh water spinach diet (24.6g/day) and higher than cassava leave diet  (16.7 g/day). In conclusion, fresh water spinach and mixing water spinach with cassava leaves could be a good protein sources for pigs. 

Key words:   Digestibility, growing pigs, fresh cassava leaves, water spinach

Introduction

In Cambodia, pig production is important for poor farmers in terms of main family income sources, money savings and festivals. Normally, farmers keep around 1-3 heads/ household. Most still practice the traditional scavenging system or a form of semi confinement using poor quality rice by-products without protein/amino acid supplementation and local and/or crossbred between local and exotic breeds. For these reasons pigs grow slowly and take a long time to reach slaughter weight.  

Recently, many researchers have developed systems to use protein-rich forages for pigs to replace expensive and imported fish meal and soybean meal. Several studies have been done on amino acid profiles of forages rich in protein and found that Lysine, Isoleucine and Leucine are  limiting amino acids for pigs (Le Duc Ngoan and Lindberg, 2001; Le Van An et al., 2003). Apparent ileal digestibility of some local protein sources have been studied in growing pigs (Bui Huy Nhu Phu et al. 2000; Le Duc Ngoan and Lindberg, 2001; Le Van An et al. 2003). Positive effects of supplementation of Methionine and Lysine in diets based on cassava foliages were reported by Nguyen Thi Loc et al., (2003) and Du Thanh Hang et al., (2009b). Mixing cassava leaves with water spinach in pig diets let to improve intake and animal growth rates (experiment 1 and 2).  However, it is not known if this is due to improving digestibility of the protein in water spinach and cassava leaves or an improvement in the amino acid profile balance in the diets.

Objective

The aim of the present experiment was therefore, to study the total tract digestibility of DM, N, OM, CF and NDF on crossbred pigs fed diets contained the fresh cassava leaves, fresh water spinach and a mixture of fresh water spinach and cassava leaves as protein sources with a basal diet of rice bran, broken rice and fish meal in Cambodia.

Materials and Methods

Experimental animal and treatments

Four crossbred (Hainam breed x Large White) castrate male pigs with a body weight of 50.3kg (SEM±1.44) were used in this study. There were four dietary treatments, including Control diet (CTRL), Fresh cassava leaves (FCL), Fresh water spinach (WS), and Mixture of fresh water spinach and fresh cassava leaves (WSFCL) (50:50 DM basis). The control diet was composed of fish meal (FM), broken rice (BR), rice bran (RB) and premix, while other foliages treatment were composted of the same ingredients in CTRL treatment but low level of fish meal in the diets, however, all diets were balance crude protein and energy. The treatments were:

• CTRL: Control diet

• FCL: Fresh cassava leaves

• WS: Water spinach

• WSFCL: Mixture of fresh water spinach and fresh cassava leaves (50:50 DM basis)

Experimental feeds and feeding

The cassava variety used in this experiment was known locally breed as called Damlong Kor (sweet variety) and was harvested everyday for pigs after about 3-4 months planting. Stems and petioles were removed from the leaves and then the leaves were chopped into small pieces (about 2-3cm) and offered to pigs immediately. Water spinach was chopped into small pieces and offered immediately at the same time as the fresh cassava leaves (FCL).  

The daily feed offer in dry matter was calculated based on feed consume daily and varied from 2.5-4.5% of the body weight. Pigs were fed thrice daily with equal rations at 8:00, 12:00 and 17:00h and given as a wet mash. The amount of feed offer was reduced if refusals were observed. Fish meal (FM), broken rice (BR), rice bran (RB) and premix were thoroughly mixed together and then fed to pig first prior foliage (FCL, WS and mixture WSFCL). The mixture of FCL and WS were in the ratio 50:50 in DM basis (the proportion used in this experiment was different in experiments 1 and 2 which used 40:60 and 60:40 in DM basis for water spinach and cassava leaves respectively and resulted showed that both proportion were not different on daily weight gain and by the ways, in order to make easy in practical work, new proportion was assigned with 50:50 in DM basis in this study). Water was permanently supplied through low pressure nipples. The animals were weighed at the beginning of the trial and every 12 days. Basal diet was very homogenized, through this method, intake of feed ingredients were calculated, if feed refusals were observed. 

Experimental design

The experiment was conducted in a 4*4 Latin Square (Figure 1) with the experimental period of 12 days comprising 7 days of adaptation to each diet followed by 5 days to collect feces, feed refusal and urine. All pigs were housed in metabolism cages during the whole trial (48days). 

Sample collection

Samples of feeds offered, refusals (in case), urine and faeces were collected daily and weighed, and 10% of the collected amount as faecal sample were stored at -18⁰C until the end of each collection period. A representative sample was obtained from every treatment, mixed thoroughly by hand and then homogenized in a coffee grinder and dried at 60⁰C prior to analyses of DM, OM, HCN, N CF, NDF and amino acids. Urine was collected in a plastic bucket to which sulphuric acid was added to maintain the pH below 4.0 (20ml of concentrated H₂SO₄). The volume of urine was measured every day and 10% of the total volume was stored at -18⁰C until the end of each period, when a sample was taken for N analysis.

Chemical analyses

The chemical composition of the diets, faeces and urine was determined using the Association of Official Analytical Chemists (AOAC) methods (1990). Dry matter (DM) was measured by drying fresh samples at 60^oC for 24 hours. Total nitrogen (N) was determined on fresh samples by the Kjeldahl method and CP was calculated from total nitrogen (N*6.25). Crude fibre (CF) and Neutral detergent fiber (NDF) was determined according to Van Soest et al (1991). All samples were analyzed in duplicates. Amino acids of feed ingredient and faeces were analyzed according to Spackman et al. (1958) on an ion exchange column using HPLC. Samples were weighed as mg and then adding 100 ml HCl 6N to hydrolyze at 110°C for 22 hours, then checking pH with V ml Na2CO3 and take 25 ml of mix add 100 ml reagent l to load of sorbet tip and elute sample by adding 50ml iso-octane and 100ml chloroform to emulsify sample, transfer part of the organic layer in vial to evaporate, after that re-dissolve in 1 ml HPLC mobile to LC/MS analysis. (Standard 20 nmol/ ml)

%AAs: (S_sample x C_standard x V_first x V_final x M)/ (S_standard x V_taked mix x W (g)).

Statistical analyses

Analysis of variance was performed according to a 4*4 Latin-square design using the general linear models of Minitab statistic software (Minitab 2007 version 15.12). Sources of variation were: column, row, treatments and random error. When the F-test was significant at P<0.05, pair wise comparisons were performed using Turkey’s procedure (Minitab Statistical Software). The model used was:

Y_ijk = μ + T_i + P_j +A_k + e_ijk

In where, Yijk = Dependent variables

μ = overall mean

T_i = treatment effect (i=1-4)

P_j = column effect (j=1-4)

A_k = row effect (k=1-4)

e_ijk = random error 

Results and discussion

Chemical composition of feeds ingredients

Chemical compositions of feed ingredients were determined during the trial and indicated some remarkable differences between fresh cassava leaves and fresh water spinach. The content of DM, OM and NDF in fresh cassava leaves was significantly higher than in water spinach but it was in the other way around for CP and CF. DM contents of basal diet ingredients were almost similar, around 90%. The highest content of CP was found in fish meal with 59.9%. Crude fiber was not found in broken rice, while it was 25% in rice bran. Organic matter and CP of water spinach in our study were in agreement with Bui Huy Nhu Phuc (2000) with 88.8 and 26.4%, respectively, but NDF content was higher, 38.7% compared with 22.9%. HCN content in this study was 357.7 mg/kg DM that was in agreement with experiment 2 and 3 (351 and 378mg/kg DM, respectively) and higher than the study of Nguyen Duy Quynh Tram and Preston (2004) with 269mg/kg DM.  

 The essential amino acids in cassava leaves have similar value with water spinach. Both forages (cassava leaves and water spinach) have closed value of amino acid with fish meal except Iso, Leu, Lys, Phe, Thr and Val, which were higher in fish meal. Amino acid profile in broken rice were similar value with rice bran but in rice bran was high on Arg, His, Leu, Phe and Val. However, the amino acid content in water spinach and cassava leaves in this study were lower than other studies of Bui Huy Nhu Phuc et al., (2000); Le Thi Men et al., (2005) on water spinach and Bui Huy Nhu Phuc et al., (2000), Woolfe. (1992) cited by Le Van An. (2003), Bui Huy Nhu Phuc and Lindberg (2001) on cassava leaves. The total essential amino acids of  water spinach and cassava leaves in this study were 22% and 28%, respectively which were lower than above studies nearly twice times (over 45%).  

Amino acid content in fish meal in this study was lower than fish meal that has protein content 64.5, 56.8, 65.0 and 62.2% which studied by Famino et al., (2004), La Van Kinh et al., (no year), NRC (1994), respectively. The low content of fish meal in present study might be due to fish meal sellers who put some material such as sand or soil or other heavy materials in order to get high profit, even though, CP content is high (59.9%) but this value can be also effect by adding some materials such as urea, animal hair or skin or nail. 

Feed intake

Total intake of DM, CP, CF, NDF and intake (g/kg live weight) were highest in control diet (CTRL) and then water spinach (WS) and mixture of water spinach and fresh cassava leaves (WSFCL), and lowest in fresh cassava leaves (FCL) treatment. The DM intake as percentage of live weight (LW) on WS was 3.15% that is in agreement with Prak Kea et al., (2003), who used the same diet but lower than that reported by Chhay Ty et al., (2005a) on both WS and WSFCL. This is due to pigs used in previous studies fed broken rice at 2% of live weight (LW) and WS fed ad libitum while in the present study WS and other ingredients are offered based on formula which was planned during the experiment. However, feed intake as percentage of LW was improved when pigs fed mixture WSFCL as agreed with Chhay Ty et al., (2005a) and Bounhong et al., (2004). DM intake as percentage of LW in FCL (2.60%) in this study was ranged to that reported by Du Thanh Hang (2005) (2.7 to 3.3%), for crossbred pigs fed a 2:1 mixture of ensiled cassava root and rice bran and fresh cassava leaves that had been washed prior to feeding and similar values reported by Nguyen Duy Quynh Tram and Preston (2004) and Bounhong et al., (2004) (2.6 and 3.11%,  respectively), who used similar diets as in the present study but no fish meal. However, a much higher intake (4.4%) was recorded for pigs fed broken rice and cassava leaves that had been ensiled before feeding (Chhay Ty et al. 2003b). It would appear that ensiling the leaves increases their palatability and the low voluntary intake could have been due to characteristics of fiber of ingredients and the presence of cyanogenic glycosides and tannins in fresh cassava leaves. According to Ravindran et al., (1987), the bitter taste of the cassava leaves could negatively influence pig intake. 

The HCN intake in FCL was nearly twice higher than mixture WSFCL. This result is similar with the study of Chhay Ty et al., (2005a), who reported that got up to 125mg/day on FCL alone compared with 83.9mg/day for mixture WSFCL. The intake of HCN (mg/kg live weight) (2.52) in this study was higher than that reported by Getter and Baine (1938) with 1.4 mg/kg BW but lower than Butler, (1973) with 4.4 mg/kg LW; Tewe, (1992) with 3.5 mg/kg LW; Chhay Ty et al., (2005a) with 5.1 and 7.4 mg/kg LW for WSFCL and FCL, respectively but supplementing with 0.3% Met in diets. Du Thanh Hang, (2005) recorded that even higher intakes (from 6.0 to 15 mg/kg live weight) when feeding fresh cassava leaves with ensiled cassava root and rice bran. Gómez, (1991) suggested that 100 mg HCN/kg feed could be the permissible maximum level for animal. However, many studies mentioned above did not report any health problem or death of animals caused by HCN intoxication. 

Faecal characteristics in pigs fed fresh cassava leaves, fresh water spinach

Faeces of pigs fed CTRL diet had higher DM, CF and NDF contents than those of FCL, WS and WSFCL. However, faeces of pigs fed CTRL diet had lower contents of OM and N than other diets. A total fresh faeces excretion was not significant difference among treatments (P>0.05) but faeces excretion in dry matter was lowest in WS (P<0.001). The DM content of faeces, excretion in dry matter and water in this study were higher than previous studies reported by Chhay Ty et al., (2005a). This might be due to the LW of pigs used in this study (50.3±1.44kg) were heavier than study of Chhay Ty et al., (2005a) with 15.1kg. In addition, in that experiment pigs were fed broken rice at 2% of body weight and WS fed ad libitum while in the present study WS and other ingredients were offered based on formula which was planed during the experiment. 

Nutrient digestibility

Digestibility of DM, OM, N, CF and NDF in WS treatment was higher than other treatments of CTRL, WSFCL and the lowest was FCL. The low digestibility of FCL could be affected by anti nutrition compounds in this plant such as HCN and tannin because HCN is colorless, volatile and extremely poisonous when hydrocyanic acid is generated (Van Soest, 1994). However, Stosic and Kaykay (1981) noted that small quantities of HCN ingest on a regular basis, though not large enough to cause mortality, might be sufficient to affect the general health and productivity of the animal. According to Humphreys (1988), intake of feeds containing over 20mg HCN/100g are potentially dangerous to livestock. Tannins have been considered as anti-nutrients due to a range of adverse effects including reduced feed intake and digestibility (Chung et al. 1998 and Onwuka, 1992). Tannin content in cassava leaves is reported to increase with maturity and varied between cultivars, from 30-50g/kg DM (Ravindran, 1993).

However, the digestibility coefficient of DM for FCL in this study (77.1%) was similar to the value of 77% as reported by Chhay Ty et al., (2003b) when the cassava leaves was ensiled but lower than that reported by Nguyen Duy Quynh Tram and Preston (2004) with 89% and Bounhong et al., (2004) with 84%. The digestibility of N in FCL was 67.3%, higher than that reported by Chhay Ty et al., (2005a) with 60% and Bounhong et al., (2004) with 65% but lower than that reported by Nguyen Duy Quynh Tram and Preston (2004) with 74%. This might be due to low intakes of FCL and high intake of broken rice in their study, which would have the effect on increasing digestibility. In term of digestibility for DM and N in WS treatment was 83.3 and 80.6%, respectively, and ranged values with reported by Chhay Ty et al., (2005a) with 89 and 82% for DM and N, respectively for pig fed WS 47% in the diet. Prak Kea et al., (2003) reported that DM and N digestibility of diet with 35% water spinach as the only supplement to broken rice (in DM) were 89 and 77%, respectively. 

The digestibility of CF and NDF was observed high for WS treatment than other treatments especially CTRL treatment, this could be cause of high feed intake and fiber intake in CTRL diet. Ogle (2006) mention that high CF content in the diets reduces the nutrient digestibility, especially of CP, fibrous feeds in the diet also lead to an increased rate of passage of digesta through the gut and reduced ileal and total tract digestibility and affect on gut size and development, particularly the large intestine (An et al. 2004 and Jorgensen et al. 1996). Nearly all CF digestion takes place in the caecum and colon, where bacteria break down fermentable carbohydrates that have escaped digestion in the stomach and small intestine. However, levels of more than 7-10% of CF in the diet will generally result in decreased growth rates (Kass et al. 1980). Ravindra et al., (1987) observe in pigs a depression in digestibility of DM, EE, cell wall components and hemicelluloses in diets in which CLM is replaced by coconut oil meal. Sarwat et al., (1988) also observe lower digestibility of DM and OM when CLM is included in a sorghum base diets of growing pigs. Bui Huy Nhu Phuc et al., (1996) report a significant reduction in apparent digestibility of DM, OM, CF and EE as the level of inclusion of CLM meal increase from zero to 216g/kg diet.  

Digestibility of essential amino acids of CTRL and WS diets were higher than those of FCL diet. Combining WS and FCL increased AA digestibility compared to FCL. The digestibility of essential amino acids in FCL treatment ranged with study of Bui Huy Nhu Phuc et al., (2000), who showed digestibility of Arg 80, 82; His 77,79; Iso 70,69; Leu 74,76; Lys 79,79; Met 70, 71; Phe 76,78; Thr 66,68 and Val 67, 66% for dry and ensiled cassava leaves which used at level of 15% in the diet and basal diet from cassava root meal and soybean meal, respectively. 

Nitrogen balance

Daily N intake was highest for CTRL diet followed by WS and WSFCL and lowest in FCL (P<0.001). This means total N excretion was highest for CTRL but not differences in N excretion among FCL, WS and WSFCL. Daily N retention is superior for CTRL (28.1g/d) and WS (24.6g/d) but low in FCL (16.7g/d). There appeared to be some synergism and interaction between FCL and WS as the N retention on WSFCL (21.0g/day) was almost as high as on the WS (24.6g/day). In previous experiment on growth performance with the same diets, we indicated that the mixture of cassava leaves and water spinach may even be superior to the water spinach alone, thus providing support for the hypothesis that mixtures of fresh cassava leaves and water spinach may have a complementary action in providing the required nutrients for animals. The N retention in FCL (16.7g) in this study was higher than that of Chhay Ty et al., (2003b, 2005a), Nguyen Duy Quynh Tram and Preston (2004) and Bounhong et al., (2004). In the present study, fish meal is added at level of 6% in the diet that may improve amino acid in the diets. Eggum (1970) reported that imbalance in the diets containing cassava leaves may be a factor affecting nutrient digestibility of pigs as well as the study of Bui Huy Nhu Phuc et al., (2001), who reported that Met as well as Lys was the major limiting amino acids for N retention, as well as biological value. Chhay Ty et al., (2005a) reported that supplementing with 0.3% of Met in fresh cassava leaves improved significantly digestibility of DM and OM. Furthermore, Adegbola (1977) concluded that when Met was added 0.2% in diet with 20% of dry cassava leaves, it would be improved amino acid as well protein quality. 

 The relationship between N intake and N retention was plotted. It can be seen from figure 10 that, there was linearly increased (R² = 0.88) nitrogen retention as nitrogen intake increased. The result in this study was similar with study of Nguyen Duy Quynh Tram and Preston (2004). The authors reported that relationship between N intake and N retention was very high (R² = 0.86).

 Conclusions

• Control diet had higher feed intake than diets with fresh cassava leave, water spinach and mixture of fresh cassava leave and water spinach as the main protein source. Fresh water spinach was more palatable than the cassava leaves as reflected in higher total dry matter intake but not differences in proportion of the diet (26.7% for fresh cassava leaves and 26% for fresh water spinach and 26.3% for fresh cassava leave mix with water spinach).

• Digestibility of DM, OM, N, CF, NDF and essential amino acid in WS diet was higher than those in other treatments.

• There appeared to be some synergism effects between fresh cassava leaves and the fresh water spinach as the N retention of pigs fed the mixture fresh water spinach and fresh cassava leave diet (21.0g/day) was almost as high as on the fresh water spinach diet (24.6g/day) and higher than cassava leave diet  (16.7 g/day).

Acknowledgements 

The authors would like to express their gratitude to the MEKARN project financed by the SIDA-SAREC Agency and to the Center for Livestock and Agriculture Development (CelAgrid), for providing resources for conducting this experiment.

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