Conservation and chemical composition of Leucaena leucocephala plus fresh or wilted Pennisetum purpureum mixed silages
Conservación y composición de ensilajes mixtos de Leucaena leucocephala con Pennisetum purpureum fresco o presecado
Ángel Santana P,1* Ph.D, Mario Cisneros L,2 Ph.D, Yordan Martínez A,1 Ph.D, Yoandris Pascual S,1 M.Sc.
1Universidad de Granma, Facultad de Medicina Veterinaria, Centro de Estudios de Producción Animal, Cuba.
2Instituto de Investigaciones Agropecuarias Jorge Dimitrov, Granma, Cuba.
Received: July 2014; Acepted: February 2015.
Objective. Quantify the effects of mixing Leucaena (L) with King grass forage, fresh (K) or wilted (Kp), on the fermentation process and chemical composition of mixed silages. Materials and methods. Silos were produced mixing several proportions (kg:kg) K:L and Kp:L (100:0; 75:25; 60:40; 50:50; 40:60 and 0:100) of both types of plants, under a completely randomized design of four replications. The quantity of organic acids (butyric, acetic, lactic), pH, ammonia nitrogen percent and some of the typical bromatologic nutrients of the forage before and after ensiling were measured. The treatment effects were evaluated through variance and regression analysis. Results. The results clearly proved the differences (p<0.05) between King grass and Leucaena which promote its mixing and wilting: better legume contents of crude protein (24 vs. 7%), dry matter (33.77 vs. 22.05%) and crude fiber (26.53 vs. 32.5%). Clear benefits on the conservation process of mixed silages were also measured: higher lactic productions and less butyric, acceptable pH (4.02-4.8) and protein degradation (<8%). In addition, a positive effect on the chemical composition of the aforementioned silages was quantified (crude protein, dry matter and crude fiber progressive improvement). Conclusions. Mixed K+L silages are better than pure K if L is included below 25% in KL and up to 40% when K has been wilted. Higher inclusions of L will worsen the conservation process and will limit its elaboration.
Key words: Ammonia, fermentation, lactic acid, nutrients, ph (Source: DeCS).
Objetivo. Cuantificar los efectos de la adición de forraje de Leucaena leucocephala (L) en el proceso fermentativo y la composición química de ensilajes mixtos con King grass fresco (K) o presecado (Kp). Materiales y métodos. Se ensilaron en condiciones de laboratorio diferentes proporciones (kg:kg) K:L y Kp:L (100:0; 75:25; 60:40; 50:50; 40:60 y 0:100), bajo un diseño totalmente al azar de cuatro repeticiones por cada una. Se midió la cantidad de ácidos orgánicos (butírico, acético, láctico), pH, porcentaje de nitrógeno amoniacal y los nutrientes típicos de la bromatología en el forraje antes de ensilar y en los ensilados resultantes. Los efectos de los tratamientos se midieron mediante análisis de varianza y regresión. Resultados. Los resultados demostraron las diferencias (p<0.05) de la composición química entre el King grass y la Leucaena que animan a su deshidratación previa y mezclado: mejores tenores de proteína cruda (24 vs. 7%), de materia seca (33.77 vs. 22.05%) y fibra cruda (26.53 vs. 32.5%) en la leguminosa; así como los beneficios en la preservación del ensilaje mixto (mejores producciones de ácido láctico y menores del butírico, aceptable pH (4.02-4.8) y degradación de la proteína(<8%) y en la bromatología de estos (avance progresivo en la concentración de proteína, materia seca y fibra). Conclusiones. Los ensilajes mixtos K+L son mejores que los de K siempre que L se adicione hasta 25% con K y hasta 40% con K presecado. Con inclusiones mayores de Leucaena se empeora la conservación, lo que limita su elaboración.
Palabras clave: Ácido láctico, amoníaco, fermentación, nutrientes, ph (Fuente: DeCS).
It has been shown that for typical small farmers in the tropics, conservation of surplus forage as silage can be important because this way they can thus avoid the influence of climatic conditions that are adverse to developing hay and because various technological variants can be applied according to changing economic conditions, infrastructure and levels of knowledge (1).
Historically, producers have different species and varieties of grasses to feed their animals which, at the same time, are the basic materials to be preserved for scarcity periods. However, today it is more common to use other forage families to combine with the classic grass-based diets (2).
On the other hand, silage fermentation as warranty of conservation, is a complex process involving relationship between characteristics of the original feed, applied technology, epiphytic microflora of the environment and prevailing weather conditions. (3)
It is known that the grasses are better preserved than legumes due to a higher content of soluble carbohydrates and because they are lower in protein and minerals; but they have lower nutritional value and animal productions are consequently lower. In contrast, leguminous plants produce less desirable fermentations, as they have less fermentable carbohydrates and because they have greater protein and mineral content; however, its use stimulates consumption of silage, digestibility of nutrients, and production aspects such as weight gain and milk yield (2,4,5).
Contrary to this, some research has not reported significant improvements, but rather reductions in some percentage parameters, such as nutrient digestibility coefficients that are included in the nutritional evaluation of silage. Using silage that has the progressive addition of legumes, however, close dependencies with mixed forage species (1) are indicated.
The objective of this research was to quantify the effects of adding Leucaena leucocephala forage in the fermentation process and chemical composition of silage mixed with fresh or wilted King grass (Pennisetum purpureum cv. King grass).
MATERIALS AND METHODS
Site and type of experiment. The experiment was carried at laboratory level in the research facilities of the Centro de Estudios de Producción Animal, Faculty of Veterinary Medicine, Universidad de Granma; eastern region of Cuba (Coordinates: 20°, 16’,52.596” North latitude, 76°, 43’, 36.192” East longitude, and altitude of 112.5 m asl), tropical brown soil free of carbonates, 3.3% of organic matter, and pH from neutral to slightly acid (6).
Procedure. Several Cullison type of microsilos (1.2 L of capacity), with Bunsen copper valves to avoid air entering, were used to ensile different mixtures of fresh King grass (K) or sun pre-dehydrated (Kp) during 4 hours having 80 days of regrowth (pre-bloom stage) plus Leucaena (L). Both type of forages were chopped (1–1.5 cm long) and mixed in proportions (kg:kg) of 100:20; 75:25; 60:40; 50:50; 40:60 and 0:100 of King grass:Leucaena.
Forage was established in dry areas for a period of 14 months and Leucaena foliage consisted of leaves and tender stems considered to be edible.
Laboratory analysis. 75 days after ensiling the silos were opened to measure pH using a potentiometer, organic acids (lactic, acetic and butyric acids) by volume (Lepper-Flieg method), NH3 to N Total by micro diffusion and bromatologic components using AOAC methods (7): dry matter (MS), crude protein (PB), crude fiber (FB), nitrogen free extract (ELN), ether extract (EE), organic matter (MO) and total ash (CZ).
The samples were dried and ground before doing bromatological analysis and fresh silages were diluted in deionized water for organic acids quantification.
Experiment design and statistical analysis. The research was conducted under a 2x6 factorial design (factor 1, level of dryness of King grass: fresh K or pre-dried for 4 hours Kp; factor 2, six ratios described) with four replications per treatment (N=48). One-Way analysis of variance for bromatology was conducted on mixed fodder (K, Kp and L) as sources of variation and Two-Ways analysis of variance for pH and NH3 (the two factors described and their interaction as fixed effects). Correlation and regression analysis were done too. In al cases values of p<0.05 were considered significant.
Also, variation adjustment (Y) in bromatology and synthesis of organic acids was tried with three types of regression equations (linear, quadratic and cubic), where the independent variable (X) was the percentage of Leucaena in silage.
The best equation was selected according to the highest value of R2 (for more accuracy), as long as it was higher by at least 0.05 units compared to less complex ones (for ease of calculations). The standard error of Y estimates was included and it was also considered that residuals were normally distributed.
The values of “X” where “Y” has its maximum and minimum points in quadratic equations were determined as equaling zero with the first derivatives. All statistical analysis were performed using the Statistica de StatSoft program (8).
Table 1 shows some important differences between the three forages used before ensiling. It should be noted that MS significantly increased in dehydrated King grass forage in comparison with fresh grass, and it was also higher than the quantified value in Leucaena. PB contents found in this legume are greater than the grass in both forms.
Similarly, Leucaena is also favored in its FB and EE content, which may improve the chemical composition of silage when mixed with grass.
On the other hand, the fractions MO and ash showed no important differences between the two forages studied.
The variation in synthesis of lactic, acetic and butyric acids with the progressive addition of Leucaena to ensiled mixture is shown in figure 1. Importantly, butyric acid production, both KL and KPL, increased from 50% inclusion of the legume in comparison with the control (0% of Leucaena), with greater emphasis on KL treatment. In this sense, when making calculations with equations, it was found that KPL has a minimum production of this acid with inclusions of up to 27% of the legume and equal to 100 King grass (0 Leucaena), with a rate of 47K:53L.
For lactic acid, the plotted variation indicated that this is synthesized in the same, or slightly higher, quantities than grass alone when small amounts of Leucaena are included. The estimates indicate that adding up to 7% of Leucaena in the mix with KL and up 11% KPL improves lactate synthesis.
It is also seen in figure 1 that with KL it was possible to produce lactic acid in a similar concentration with that of control KL with 0% Leucaena, in a mixture with 69.5:30.5 (Kp: L) according to the equation for this acid.
In the case of acetic acid in KL, there was no trend that would fit the evaluated equations. However, in KPL a gradual increase was observed, but with less variation, as more Leucaena was added to the mixture, which also showed the beneficial effect of wilting the grass.
Table 2 shows the pH of the mixtures and the percentage of ammonium nitrogen relative to the quantified total that is classically related to the level of protein deterioration. Comparing the mean pH values between treatments is less important that observing its variation as the percentage of Leucaena increases in the mixture, in order to assess the practical effects of the latter. An increase for both types of silage is observed, although in KL inclusion at 25% Leucaena caused a significant rise in pH from 4.02 to a value close to the ideal 4.
With the previously wilted grass in the control treatment (100% Kp), the pH begins to progressively increase starting at 4.19, which is statistically equal to its similar percentage of Leucaena in Kl, which is the initial cause so that the recorded measurements with successive additions of Leucaena exceed 5 in 50:50 proportion.
In KL the pH increased highly adjusted (R2=0.78) to the equation pH=4.09+0.01 (% L), the same as in the KPL mixtures was adjusted (R2=0.87) pH=4.12+0.02 (% L), which indicates an intercept, or initial pH with pure grass, higher in this latter mixture and also a greater slope that signals the greater effect that Leucaena has when King grass was dehydrated beforehand.
The effects caused by the progressive addition of Leucaena to the bromatology of the mixed silage is shown in figure 2. The contribution of each forage silage mix is perceived. Thus, it was found that the concentrations of PB increased almost linearly with the proportion of Leucaena.
The MS content is also influenced by the original forages, considering that fresh King grass forage contains about 22% and Leucaena nearly 34, so different KL mixtures were in that range. The same effect can be seen for KPL and the FB of KPL; however, the observed variation in the FB of KL did not conform to the models tested.
Significant differences between PB contents of Leucaena and grass, in its two forms, is one of the most important encouraging elements in mixing these two, with the aim of improving the nutritional quality of the resulting mixed silage. Other research (9,10) has confirmed the differences of these concentrations between grasses and legumes. This principle is also reflected in the composition of the mixtures used, as evidenced by higher values than single grasses that are reported in combinations with several tropical legume species (11,12) which are logically determined by the proportions of the two types of plants found in the mixture (13,14).
From the point of view of fermentation, if the silage does not have more than 0.5% of butyric acid in the MS, the nutritional quality and fermentation (15) is not affected, but above this is proof that Clostridia has dominated the conservation and therefore the response in animals is not positive.
The results show that the percentage of butyric acid reaches about 0.5% only when L represents more than 60% of the KL mixture and more than 80 in KPL.
All this happens in grasses-legumes mixtures where a new substrate is created and there are, in addition to the sugars needed for fermentation, greater sources of nitrogen and vitamins than when pure grasses are ensiled. However, the “best” would be to leave it to the point where the sugars do not limit and/or the buffering effects do not impose (1.3).
It has been known for a long time that wilting tropical forages is one of the best ways to lead fermentation to a lactic pattern (16), which is the most desirable in fermentation because it is stronger than others, causes greater decreases in pH, reduces MS loss and stimulates silage consumption by ruminants. It is advisable to avoid Pennisetum silage when it has not been dried previously or any kind of additive is used (17).
Certainly in this experiment this was demonstrated, since the lactic acid synthesis initially increases more in KPL than in KL. In Rye grass+Clover silage, a lactic acid synthesis above 5% and butyric acid below 0.04 in the MS was obtained, and the pH of the mass was 4.5. However, it was considered acceptable because the dry matter exceeded 43%, and it also coincided with investigations with triticale+vetch mixtures that possess pH values above 4.5 and dry matter greater than 40 (17,18).
The theory of the various origins of acetic acid in silage explains the irregularities seen in this and similar experiments, including studies using corn to try to reach a better fermentation (19-20). When this acid is produced in concentrations higher than 3% based on dry matter, it can reduce consumption of silage, although such a statement is still inconsistent. In a study of corn silage mixed with a legume (50:50), the pH was 4.66, even though it has good fermentation, nutritional quality and NH3-N accounted for only 6% (14).
In Sorghum almum mixtures with Arachys hipogea and Arachys hipogea ensiled without additives, pH values above 5 (21) were recorded, and the main cause is attributed to the lack of soluble carbohydrates. The opposite effect was found when mixtures of corn stubble with legume Lupinus exaltatus and L. rotundiflorus, adding molasses as a carbohydrate source, and it was possible to lower the pH to values close to 4, although there is a tendency to be higher with greater proportions of the legumes (20).
The strength of synthesized acids in silage can be summarized with pH measurement and the values of this variable decrease to intervals of 3.6-4.5 ideally, so that the values obtained in the present investigation are considered acceptable for preserving mixtures with less than 50% Leucaena, whether or not the King grass was pre-dried, even though pre-drying and the presence of legumes are valid reasons so that the pH reaches above normal values; for this reason, two samples of silage can have the same pH, but different concentrations of acid and vice versa.
Moreover, the values of the interaction between the form of King grass forage (fresh or pre-dried) vs. the percentage of Leucaena in the mixture denote that it is not possible to isolate the influence of both factors in the pH. This means that, regarding the acidity of the silage, it is necessary to consider each one as separate material when the grass is combined independently (fresh or pre-dried) and percent of legume; while regarding the NH3 it is feasible to say that a lower relative protein degradation is obtained when the King grass is wilted before silage, since the value of the interaction is not significant.
Overall, the results in fermentation of different mixtures are closely linked to specific factors depending on the original forages. But those values also show that the local environmental could influence the spontaneous development of various microorganisms, since adding bacterial cultures to induce fermentation showed specific and precise effects, regardless of the forage characteristics, which can be pure legumes and other types of forage (21.22).
The influence of Leucaena has been well established, since this forage has MS high tenors (about 30%) and experiments done on their ensilability conclude that it is good for mixed silage, but does not cause significant decreases in the pH of the preserved mass (3).
The proteolytic potential of the silage depends on the protein content and the affinity of the proteinase in the forage; additionally, the solubilization process of the proteins depends on a decrease in the pH and the content of MS in the material as an indicator of the osmotic pressure in the environment, although the proteolysis is inhibited more by the rapid drop in pH that by osmolarity due to limitations it imposes on the proteolytic bacteria and Clostridia (23). In addition, other factors of the composition of the original feed, such as the presence of polyphenols, also affect the preservation of legume proteins (24).
This interdependence is valid for all kinds of ensiled materials. In research with silages made from orange peels it was considered very good at 8.3% NH3, which shows a low process of deamination and protein degradation, even when the pH dropped to 3.7 and produced acidity that ensured the stability of the preserved mass (25).
Furthermore, deterioration of the protein did not go above 11 and 15% of the total N (Table 2), which are accepted as limitations since at proportions higher than NH3 it is released very rapidly in the rumen of animals and causes significant urinary losses of this fraction (26). It is important to highlight that when fermentation is done by adding organic acids, and not naturally as in this investigation, lower percentages of NH3-N are obtained (26,27).
N ammonia ratios found are in accordance with these principles, although this increases when a greater proportion of Leucaena is in the mixture. Regardless of this, the increased PB achieved is one of the main motivations for using mixed silage of grasses with legumes or species of other families that are richer in this nutrient (9,10,13,20).
The results that were quantified in the MS, PB and FB show the expected benefit of mixing, especially with respect to PB, and confirm that if the chemical composition of forages is known, it could certify a MS content in silage that also ensures good preservation and high nutritional value. Under this principle, a very wet forage mixed with another drier one will improve the parameters of conservation and nutritional quality, whenever the used proportions are controlled. Hence the recommendation to combine cereal high in MS with wet forage to solve the disadvantages of both extreme situations (16).
In a similar investigation with a legume and cereal (berseem+triticale), it was found that the chemical composition in the mix is better than with just cereal, but not when compared with the legume, while fermentation favors the mixture in silage in relation to the legume alone (28), so it was preferred over the two pure silages.
From the results in fermentation measurements, bromatology, pH and N ammonia, it can be concluded that the mixed silage of fresh King grass with Leucaena is well preserved when up to 25% of the latter is included, while the previously dehydrated King grass (KPL) has best results when 45% legume was added. With this option the nutritional quality of silage is improved, there are benefits in conserving the ensiled mass, chemical composition and the protein content, whose solubility is not affected. Increasing proportions of Leucaena above the previous limits leads to undesirable fermentation that do not guarantee the preservation of the silage mass.
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