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Introduction
In developing countries, there is a large dependence on grains and oilseeds as the main source of protein for livestock. The high demand for these energy and protein sources causes a constant increment of grain prices. This situation has led to the search for the use of under-utilized tropical legume seeds in animal feeding. The ecological conditions prevailing in the tropical regions allow the growth of a large variety of species with nutritional potential. Legumes are a good source of crude protein (CP, Siddhuraju and Becker 2005), amino acids (Mugendi et al. 2010), and minerals (Arrivalagan et al. 2014). Mucuna pruriens (L) DC. Var (Wight) Burck called Velvet bean is originally from tropical Asia (Huisden et al. 2014) and has high CP content, carbohydrates and acceptable levels of fiber for animal feeding (Safwat et al. 2015). This legume can be an alternative to animal nutrition. However, it contains a variety of toxic secondary metabolites such as phytates, trypsin inhibitors, L-3,4-Dihydroxyphenylalanine (L-Dopa), cyanogenic glycosides, tannins, oxalates, saponins and lectins have been identified in M. pruriens (Iyayi et al. 2008), which reduce digestibility of the diet (Bhat and Karin 2009).
The heat labile characteristic of some antinutritional factors reported for M. pruriens, such as cyanogenic glycosides (Acamovic and Brooker 2005), trypsin inhibitors, tannins (Siddhuraju and Becker 2001) and phenols (Preet and Punia 2000), allows their elimination with thermic treatments (Del Carmen et al. 1999, Iyayi et al. 2008). L-Dopa in M. pruriens is not heat labile and solubilization is one way to eliminate it (Josephineand and Janardhanan 1992). Thus, soaking of this seed with calcium hydroxide (Safwat et al. 2015), water (Tuleun and Igba 2008) sodium bicarbonate, citric acid and tamarind extract reduces this metabolite (Siddhuraju and Becker 2001). Inclusion of 40% of MP in the starter phase and 60% in finishing phase have been reported as an adequate percentage for poultry diets (Vadivel et al. 2011). In the same way, the inclusion of boiled MP is viable up to 60% resulting in an improvement in productive performance (Tuleun et al. 2009), however when raw MP is toxid to birds (Tuleun and Igba 2008). Thus, although MP contains several secondary metabolites, some methods have been reported to be effective to eliminate most L-Dopa and other toxic secondary metabolites from the seeds, which allows its utilization in broiler diets without compromising their productive performance. Therefore, the objective of this study was to evaluate the effect of soaked Mucuna pruriens in tap water, acid or alkaline solution + boiling on its L-Dopa content and on the growth performance of broilers and carcass yield.
Materials and methods
Study area
The study was carried out at the poultry production facility of the Faculty of Veterinary Medicine and Animal Science, University of Yucatan, Merida, Yucatan, Mexico. Animals were kept in a poultry house with an ambient temperature between 22 - 24 °C (CONAGUA 2016).
Processing methods and experimental diets
The treatments used to eliminate L-Dopa from MP seeds were: a) soaked seeds in water; b) soaked seeds in acetic acid solution (200 ml L-1; pH = 3.0) and c) soaked seeds in calcium hydroxide solution (40 g L-1, pH = 12.3). Each batch was soaked during 24 h in plastic pots, after the seeds were rinsed with tap water until the solution residues were eliminated. The soaked seeds were boiled for 1 h in a metal buckets in a gas stove, and were rinsed again with water, finally the boiled seeds were dried in an electric oven for 48 h. Then, seeds were ground in a hammer mill (Azteca©) (particle size between one to two mm) and stored at room temperature (22 - 25 °C) in a plastic container until diets elaboration. Four diets were formulated: A) Control without MP (C); B) 25% inclusion of soaked MP in tap water + boiling (WMP); C) 25% inclusion of soaked MP in acetic acid solution + boiling (AAMP); D) 25% inclusion of soaked MP in calcium hydroxide solution + boiling (CHMP).
Mucuna pruriens and diets composition
Chemical composition of treated MP seeds and experimental diets were determined according to AOAC (2000). The composition of treated MP seeds are shown in Table 1, and the diets used during starter and finishing phases are shown in Table 2 and 3. To determine L-Dopa content the treated MP seeds were extracted with a solvent mixture made up with acetonitrile, water and formic acid (50:50:1, respectively), the extracts obtained were analyzed using a Smartline UV Detector 2500 Knauer HPLC with a Hypersil Gold HILIC column according to Takashi et al (2011).
Table1: Chemicalcomposition (%) of treated Mucuna pruriens seeds.
| Treatment | Dry matter | Crude protein | Ether extract | Crude fiber | Ash |
| Soacked in Water | 92.01 | 21.54 | 2.75 | 7.99 | 2.15 |
| Soacked in Acid | 91.76 | 22.80 | 2.90 | 7.91 | 2.19 |
| Soacked in Alkali | 92.99 | 22.48 | 1.88 | 7.92 | 5.38 |
Table 2: Composition (%) and proximal analyses of experimental diets containing treated Mucuna pruriens seeds during the starter phase.
| Ingredient | Control | WMP | AAMP | CHMP |
| Maize | 61.0 | 46.5 | 47.7 | 47.4 |
| Mucuna pruriens seeds | - | 25.0 | 25.0 | 25.0 |
| Soybean meal | 34.6 | 23.4 | 22.3 | 22.6 |
| Calcium carbonate | 1.5 | 1.6 | 1.6 | 1.6 |
| Calcium orthophosphate | 1.1 | 1.3 | 1.3 | 1.3 |
| Vegetable oil | 1.2 | 1.3 | 1.2 | 1.2 |
| Methionine 99% | 0.1 | 0.3 | 0.3 | 0.3 |
| Lysine 99% | - | 0.2 | 0.3 | 0.3 |
| NaCl | 0.3 | 0.3 | 0.3 | 0.3 |
| Minerals premix1 | 0.1 | 0.1 | 0.1 | 0.1 |
| Vitamins premix2 | 0.1 | 0.1 | 0.1 | 0.1 |
| Total | 100.0 | 100.0 | 100.0 | 100.00 |
| Determined analysis | ||||
| Dry matter | 85.3 | 89.4 | 89.0 | 89.0 |
| Crude protein | 21.4 | 21.4 | 21.4 | 21.4 |
| Crude fiber | 0.9 | 2.1 | 2.7 | 2.4 |
| Ether extract | 1.6 | 2.4 | 3.3 | 3.0 |
| Ash | 6.1 | 4.7 | 3.5 | 4.4 |
| Calculated analysis | ||||
| Metabolizable energy | 3.0 | 3.0 | 3.0 | 3.0 |
| Calcium | 0.8 | 0.8 | 0.8 | 0.8 |
| Available phosphorus | 0.4 | 0.4 | 0.4 | 0.4 |
| Sodium | 0.1 | 0.1 | 0.1 | 0.1 |
| Arginine | 0.9 | 1.0 | 0.9 | 0.9 |
| Lysine | 1.0 | 1.0 | 1.0 | 1.0 |
| Methionine | 0.5 | 0.5 | 0.5 | 0.5 |
| Meth+Cys | 0.8 | 0.8 | 0.8 | 0.8 |
| Threonine | 0.6 | 0.6 | 0.6 | 0.6 |
| Tryptophan | 0.2 | 0.2 | 0.2 | 0.2 |
1Minerals premix: manganese, 65 mg; iodine, 1 mg; iron, 55 mg, copper, 6 mg; zinc, 55 mg; selenium, 0.3 mg.2Vitamins premix: vitamin A, 8,000 UI; vitamin D, 2,500 UI; vitamin E, 8 UI; vitamin K, 2 mg; vitamin B12, 0.002 mg; riboflavin, 5.5 mg; pantothenate of calcium, 13 mg; niacin, 36 mg; choline, 500 mg; folic acid, 0.5 mg; thiamine, 1 mg; pyridoxine, 2.2 mg; biotin, 0.05 mg. WMP: seeds soaked I water + boiling, AAMP: seeds soaked in acid solution + boiling and CHMP: seeds soaked in alkaline solition + boiling.
Table 3: Composition (%) and proximal analyses of experimental diets containing treated Mucuna pruriens seeds during the finishing phase.
| Ingredient | Control | Water | Acid | Alkaline |
| Corn grain | 69.0 | 54.6 | 55.8 | 55.5 |
| Mucuna pruriens seeds | - | 25.0 | 25.0 | 25.0 |
| Soybean meal | 26.9 | 15.5 | 14.4 | 14.7 |
| Calcium carbonate | 1.4 | 1.5 | 1.5 | 1.5 |
| Calcium orthophosphate | 1.1 | 1.2 | 1.2 | 1.2 |
| Vegetable oil | 1.0 | 1.1 | 0.9 | 1.0 |
| Methionine 99% | 0.2 | 0.3 | 0.3 | 0.3 |
| Lysine 99% | 0.1 | 0.4 | 0.4 | 0.4 |
| Minerals premix1 | 0.1 | 0.1 | 0.1 | 0.1 |
| Vitamins premix2 | 0.1 | 0.1 | 0.1 | 0.1 |
| NaCl | 0.3 | 0.3 | 0.3 | 0.3 |
| Total | 100.0 | 100.0 | 100.0 | 100.0 |
| Determined analysis | ||||
| Dry matter | 87.8 | 89.0 | 89.1 | 89.0 |
| Crude protein | 19.6 | 19.6 | 19.6 | 19.6 |
| Crude fiber | 1.2 | 2.6 | 2.8 | 2.5 |
| Ether extract | 2.2 | 2.8 | 2.8 | 2.4 |
| Ash | 4.9 | 4.7 | 4.2 | 5.2 |
| Calculated analysis | ||||
| Metabolizable energy | 3.1 | 3.1 | 3.1 | 3.1 |
| Calcium | 0.8 | 0.8 | 0.8 | 0.8 |
| Available phosphorus | 0.4 | 0.4 | 0.4 | 0.4 |
| Sodium | 0.1 | 0.1 | 0.1 | 0.1 |
| Arginine | 0.7 | 0.7 | 0.7 | 0.7 |
| Lysine | 1.0 | 1.0 | 1.0 | 1.0 |
| Methionine | 0.5 | 0.5 | 0.5 | 0.5 |
| Meth+Cys | 0.7 | 0.7 | 0.7 | 0.7 |
| Threonine | 0.5 | 0.5 | 0.5 | 0.5 |
| Tryptophan | 0.2 | 0.2 | 0.2 | 0.2 |
1Minerals premix: manganese, 65 mg; iodine, 1 mg; iron, 55 mg, copper, 6 mg; zinc, 55 mg; selenium, 0.3 mg.2Vitamins premix: vitamin A, 8,000 UI; vitamin D, 2,500 UI; vitamin E, 8 UI; vitamin K, 2 mg; vitamin B12, 0.002 mg; riboflavin, 5.5 mg; pantothenate of calcium, 13 mg; niacin, 36 mg; choline, 500 mg; folic acid, 0.5 mg; thiamine, 1 mg; pyridoxine, 2.2 mg; biotin, 0.05 mg
Experimental animals
During the experiment, 96 sexed 21 days-old Hubbard chicks were used. The birds were divided into two blocks with 48 birds each (first block between August - September and the second block between September - October). There were 24 replicates per treatment. Similar number of males and females were assigned to each treatment. The animals were handled individually in metal cages (40 x 40 x 40 cm), when starting the trial the broiler chickens were identified and weighed. The starter phase was between week four and five of age and the finishing phase comprised week six to seven. The animals had free access to water and were feed ad libitum.
Growth performance
Live weight gain (LWG) and feed intake (FI) were registered weekly. The FI was determined by difference between the offered and the consumed food, the feed conversion ratio (FCR) was calculated as the ratio between feed intake and live weight gain. At the end of the rearing periods, all birds were slaughtered to determine carcass yield (CY) (carcass without drumsticks and head), in addition the liver weight (LW) was recorded.
Statistical analysis
The collected data were analyzed using the General Linear Model (GLM of SAS) using a randomized complete block design (SAS 2010). The sex of the brolilers chickens was included as fixe deffect in the statistical model. Least-Squares Means were compared using the PDIFF option of SAS when significant effects (p < 0.05) between treatments were detected.
Results and discussion
In this study it was found that raw seeds contained 2.0% of L-Dopa (Table 4). This value was lower than that reported by others studies (3.5 - 5.7%) (Da-houda etal. 2009, Cassani et al. 2016). The L-Dopa content in MP has been associated to seed colors, black varieties have a higher amount of this secondary compound, whereas white varieties contain a lower percentage and mottled varieties have intermediate values (Gurumoorthi et al. 2003). Mixed seeds were used in the current study, with predominance of white seeds. Probably, for this reason L Dopa content was lower in the current study compared to earlier studies. CHMP treatment reduced 71.0% of L-Dopa content (Table 4). This result is similar to previous reports where soaked MP in sodium bicarbonate (pH = 8.5) reduced L-Dopa 68.0% (Gurumoorthi et al. 2003). Similarly, Ukachukwu and Szabo (2003) when soaked MP in calcium hydroxide achieved 63.0% elimination of L-Dopa relative to raw seeds.
Table 4: Quantification of L-Dopa in treated Mucuna pruriens seeds.
| Sample | Content (g kg-1) |
| Raw seeds | 20.0 |
| Seeds soaked in water + boiling | 11.8 |
| Seeds soaked in acid solution + boiling | 9.3 |
| Seeds soaked in alkaline solution + boiling | 5.8 |
The reduction of L-Dopa content was lower in AAMP and WMP treatment (Table 5). These values are comparable to earlier reports. Siddhuraju and Becker (2001) found a reduction of 54.0% with tamarind extract and 40.0% when citric acid was used, while in other reports a reduction of only 27.0% was reached when MP was soaked in water (Diallo and Berhe 2003). The chemical structure of L-Dopa allows its elimination easier in an alkaline medium (Siddhuraju and Becker 2001). In the current study, CHMP treatment (pH = 12.3) eliminated L-Dopa in a greater proportion than the other treatments and would be the recommended treatment for MP for inclusion in broiler diets.
Table 5: Productive performance of broiler chicks fed diets containing Mucuna pruriens seeds treated with water, an acid solution and an alkaline solution during the starter phase. Values are least square means values ± standard deviation.
| Treatments | Initial live weight (kg) | Feed intake (kg) | Live weight gain (kg) | Feed conversion ratio |
| Control | 0.71 ± 0.080a | 1.82 ± 0.215a | 1.03 ± 0.129a | 1.78 ± 0.143b |
| WMP | 0.73 ± 0.088a | 1.76 ± 0.170a | 0.95 ± 0.095ab | 1.86 ± 0.148ab |
| AAMP | 0.70 ± 0.089a | 1.76 ± 0.249a | 0.94 ± 0.149b | 1.90 ± 0.289a |
| CHMP | 0.75 ± 0.060ª | 1.80 ± 0.222a | 0.98 ± 0.132ab | 1.83 ± 0.101ab |
Means within the same column with different superscript differ (p < 0.05). WMP: seeds soaked I water + boiling, AAMP: seeds soaked in acid solution + boiling and CHMP: seeds soaked in alkaline solition + boiling.
The inclusion of 25.0% of treated MP in diets for broiler chickens did not affect FI (p > 0.05). This inclusion level is higher than those recommended in other studies, where MP was processed with different treatments. Tuleun and Igba (2008) recommend an inclusion of up to 20.0% of soaked seeds + boiling. Iyayi et al. (2008) suggest 15.0% with MP soaked in water and Akinmutimi and Okwu (2006) suggested up to 10.0% inclusion of heated MP. Young birds are more susceptible to L-Dopa because the ingestion of this compound causes a reduction of animal growth (Del carmen et al. 1999, Gurumoorthi et al. 2008). In the current study, broilers in the AAMP group had the lowest LWG and higher FCR compared to C (p < 0.05). Nevertheless, although the broilers from AAMP treatment had the lowest daily live weight gain, this was superior to the 41.8 g day-1 reported by Tuleun et al. (2011).
When the three groups offered MP are compared, no significant differences were found for LWG and FCR (p > 0.05), however broilers growth in the CHMP treatment was similar to C (p > 0.05). These results suggest that CHMP treatment improved MP nutritive value. According to Tuleun and Patrick (2007), soaking and boiling MP seeds increases the digestibility of protein and carbohydrates compared to raw seeds and when soaking is realized in an alkaline solution it is further improves due to higher elimination of L-Dopa, thus reducing its negative effect on birds (Siddhuraju and Becker 2001).
During the finishing phase, there were not statistical differences (p > 0.05) in FI, LWG and FCR between treatments (Table 6). These results indicate that up to 25.0% of treated MP could be used in broiler diets without affecting the performance. This level of inclusion is higher to that recommended by Vadivel and Pugalenthi (2010) with soaked MP in a sodium bicarbonate solution + autoclaving (11%). Others have suggested up to 10.0% of heated MP without negative effects (Del Carmen et al. 1999) or up to 6.0% of roasted MP (Emenalom and Udedibie 1998). Other authors have suggested levels not higher than 20.0% when using soaked MP in water + boiling, or fermented (Tuleun et al. 2009, Tuleun et al. 2011). The results obtained during this growing phase suggest that WMP, AAMP or CHMP treatments were sufficient to reduce L-Dopa present in MP, and the broiler performance was not affected.
Table 6: Productive performance during the finishing phase and carcass yield of broiler chicks fed diets containing Mucuna pruriens seeds treated with water, an acid solution and an alkaline solution. (Least square means values ± standard deviation).
| Treatments | Feed intake (kg) | Liveweight gain (kg) | Feed conversion Ratio | Carcass yield (%) | Liver weight (g) | Liver (g)/ carcass (kg) |
| Control | 2.3 ± 0.3a | 1.0 ± 0.1a | 2.3± 0.2a | 77.1 ± 3.5a | 47.7 ± 7.2a | 22.4 ± 3.4b |
| WMP | 2.4 ± 0.4ª | 0.9 ± 0.2a | 2.4 ± 0.2a | 76.3 ± 2.9a | 49.3 ± 10.1a | 24.6 ± 4.5a |
| AAMP | 2.4 ± 0.3a | 1.0 ± 0.2a | 2.4 ± 0.3a | 75.6 ± 3.3a | 50.1 ± 10.5a | 25.1 ± 3.9a |
| CHMP | 2.4 ± 0.3a | 1.0 ± 0.2a | 2.4 ± 0.2a | 76.4 ± 3.2a | 50.9 ± 6.4a | 24.4 ± 3.2ab |
Means within the same column with different superscripts differ (p < 0.05)
Table 6 shows the data regarding the carcass yield. Broilers had an average carcass yield of 76.0 % without statistical differences between treatments (p > 0.05) However, birds fed with WMP or AAMP had heavier livers than the C group (p < 0.05). There were not differences in liver weight between broilers fed CHMP in comparison to those in the C group (p > 0.05). According to Emenalom and Udedibie (1998) and Emenalom and Nwachukwu (2006) toxic compounds in the diet causes an increase in liver size. However, no effect on growth performance was observed in broilers fed MP in the present experiment. Due to the higher growth rates of broiler in a short period of time the MP content after treatment possibly did not affect the broiler performance in the finishing phase, but a long term effect might be observed if broilers are fed MP for longer periods of time. These aspects warrant further research to assess the effect of feeding MP in laying hens which are fed during longer periods.
The alkaline solution was the most effective treatment to reduce L-Dopa from M. pruriens seeds. Broilers fed MP soaked seeds in acetic acid solution had lower growth rate during the starter phase. Nevertheless, the presence of L-Dopa in the diet did not affect negatively the performance of broilers during the finishing phase; therefore 25.0% of M. pruriens seeds soaked in alkaline solution and boiled could be included in broiler diets without unfavorable effects on feed intake, live weight gain and feed conversion ratio.
