Custom services
text in 
English (pdf)
Article in xml format
Article references
Send this article by e-mail
Cited by SciELO 
Similars in
SciELO 
Permalink
Grasses and forages are the main and the most economic source of nutrients for ruminants in the tropics (Ramos-Trejo et al. 2013). However, the production of these plants varies through the year, and in the dry season the availability of biomass is not enough to satisfy the animal requirements, but in the rainy season forage biomass surpluses are produced, which may be lost if no conservation procedures are implemented. Hay and silage production are the only option available for farmers that conserve forage at a large scale for its use in dry season. Silages have advantages respect to hay production because a higher amount of forage can be conserved in less time, are less influenced by climate, are easier to manage and reduce substantially deterioration due to rainfall and losses in the field although a decrease of nutritive value is observed (CharmLey 2001).
In the tropic, silages are generally elaborated using grasses with low content of protein and high fibre and this result in a material of low quality. C. purpureus is a forage species highly utilized in Cuba due to higher biomass production, good leaves production, rusticity and adaptation to a great diversity of soils and adverse climate conditions (García et al. 2014). Among the clones obtained and utilized in Cuba as forage, the cultivar CT-169 is one of the highest height, fastest growth and highest dry matter yield (Martínez et al. 2010).
Forage tree legumes, other legumes and plants can be used in tropical silages to increase protein content. Moringa oleifera is a shrub with high nutritional quality and good green biomass production (Nouman et al.2014). The high protein content is utilized to improved nitrogen balance in mixed silage elaborated with the excess of forage biomass obtained in the rainy season (Roa and Galeano 2015).
The objective of the present experiment was to evaluate the effect of inclusions of M. oleifera forage in the nutritive valued of silage of C. purpureus cv. Cuba CT-169, considering the chemical composition and gas production.
Material and Methods
Obtained the evaluated silage. Treatments consist in the evaluation of five silages obtained by the mixture of five proportion of C. purpureus and M. oleifera (100:0, 80:20, 60:40, 40:60, 20:80 on a humid base respectively). Forage of C. purpureus cv. Cuba T-169 and M. oleifera were of 60 and 50 days of age respectively and collected in areas of the Instituto de Ciencia Animal (ICA) in Cuba previously established in red ferralitic soil (Hernández et al. 1999) without fertilization or irrigation. When both forages were harvested, they were milled and dried in the sun to increase dry matter content (DM) at 30 % (Reyes et al. 2008).
Different proportions of forage were mixed and compacted in microsilos elaborated in PVC tubes (24 cm x 10cm) with a capacity of 450 g of fresh forage (Gutiérrez et al. 2015). Finally, they were hermetically closed and allocated in a protected and dry placed for 62 days. Ten microsilos per treatment were prepared.
At the end of the ensilage process they were opened and a sample of nearly 100 g was taken from each treatment and mixed until homogeneously. A pool of ensilage fresh material per treatment was stored in bags hermetically sealed and put in refrigeration (-4 ºC) for the in vitro evaluation.
In vitro experimental procedure. The in vitro technique of gas production in glass bottles, described by Theodorou et al. (1994), was used. An amount of 1.0 g of fresh matter of each treatment was incubated in bottles of 100 mL in a culture medium (Menke and Steingass 1988) and an inoculum of ruminant microorganism in the proportion of 0.20 of the total inoculation volume (80 mL).
The inoculum of the ruminal content of three Nubia adult goats (Capra hircus) was utilized. They were fed ad libitum with grass forage and free access to water and mineral salts. The ruminal content of each animal was orally collected before feed offering in the morning and then conserved in independent closed thermos until the arrival to the laboratory when were filtered through several layers of gauze and the three inoculum were mixed in equal proportion.
During the process inoculum were maintained at 39 ± 1 ºC, and the anaerobiosis conditions using a continue flow of CO2. Bottles were sealed and incubated in bath at controlled temperature of incubation (39 ºC). This moment was considered as cero time of incubation.
Gas production was measured at 2, 4, 6, 8, 12, 21, 24, 30, 48, 72, 96 y 120 h by mean of a manometer HD 8804, coupled to a pressure calibration TP804 (DELTA OHM, Italy). After each measurement gas was liberated until equalize external and internal pressure in the bottle. The volume of gas was estimated from data pressure by mean of a linear regression equation pre-established (Rodríguez et al.2013).
Gas volume was expressed in gram of organic matter (OM) incubated (OM inc). For estimating gas production kinetics, the monophasic model of Gompertz was applied:
Where:
Y- gas production at t time (mL g-1 OM inc)
A- potential of gas production (asymptote) when t=
C- is a constant factor of the microbial efficiency (h-1)
t- incubation time
It was estimated the incubation time needed to reach the maximum velocity (TVmax) of gas production starting from the second derivative of the Gompertz model evaluated in zero (inflexion point in the sigmoidal model).In addition, the maximum velocity of gas production (Vmax; mL g-1 ON inc h-1) was studied substituting TVmax in the first derivate of the model (Rodríguez et al. 2013).
Estimation of microbial biomass synthesis. At the end of incubation, bottles were opened and the content filtered through nylon bags, previously weighed, with a porosity of 50 µm and a surface of 28 cm2. The bags with the fermentation residue were dried in a forced air oven with a temperature regulated at 60 °C during 72 h.Microbial biomass synthesis (MBS, mg) was estimated by gravimetry, as the difference between DM and the content of neutral detergent fiber (NDF) in the solid residue of the fermentation (Blümmel et al. 1997).
On the other hand, efficiency of microbial biomass synthesis (EMBS, g g-1fermented DM) was estimated as the rate between MBS and fermented DM. Fermented DM was estimated as the differences between incubated DM and the content of NDF from the solid residue of fermentation. As another alternative for estimating microbial efficiency, the partition factor (PF) was also determined as the relation between fermented DM and gas volume produced in the in vitro system (Blümmel et al. 1997).
Chemical analysis. In the samples of the different ensilage treatment and solid residue from the fermentation, DM, OM and crude protein was determined (AOAC 2016). NDF was obtained from the procedure described by Van Soest et al. (1991).
Statistical analysis. A completely randomized experimental design was employed considering the silage as treatment (5) and each bottle as an experimental unit (5).
The results of gas production were repeated measures in the same experimental unit and, therefore, analyzed according to MANOVA to know if an interaction between treatments and sampling hours was present. To relate the number of times in which gas production was measured, the analysis of the fermentation was divided a priori, in the three phases and four sampling times according to the concepts of MANOVA. The initial phase was defined since the beginning of fermentation until 8 hours, the intermediate from that moment up to 30h and the final phase from that instant up to 120 h. When the interaction was evident between treatments and times, an analysis of variance of split plot was carried out for interaction means. On the other hand, estimates of synthesis and efficiency were analyzed by ANOVA.
In both cases, when differences were evident (P˂0.05), means of treatments were compared using the Duncan (1955) test. The response of indicators, at the level of inclusion of M. oleifera, was studied by means of regressions and the use of Pearson coefficient to determine the degree of correlation between the two variables of efficiency of microbial synthesis (MBS and PF) that was studied in the experiment. In all cases, the use of InfoStat was applied (Di Rienzo et al. 2010).
Results and Discussion
Table 1 shows the chemical composition of the ensiled products that were evaluated in this study. M. oleifera decreased DM content regarding silage with 100 % of C. purpureus (P˂0.001). Cardenas et al. (2013) appreciated that the inclusion level of protein shrubs did not affect DM content of mixed silages with C. purpureus (variety Taiwan 144 A), but in these experiment the effect of pre-dried process, applied in both plant materials, could affect the results. On the other hand, the content of organic matter increased when the inclusion level of protein shrub was increased (P˂0.001). However, the treatment with 40 % of M. oleifera showed an unexpected performance because the OM content was lower than the other treatments.
Table 1 Chemical composition of the evaluated mixed silage.
abcde Values with different letter per column differ at P˂0.05 (Duncan 1955)
When the content of M. oleifera augmented, an increase of CP level in the silage was produced (P˂0.001). Other authors also reported linear increments in the CP content in mixed silage when inclusion level of protein shrubs is increased (Cárdenas et al. 2013). CP content of studied silages was superior considering the range of values reported for this indicator (9-14 % of CP) and by McDonald et al. (1987), and also higher to the protein content considered when ensiling maize with 4 different shrub legumes (50:50 humid base) (Phiri et al. 2007).
The inclusion of M. oleifera also linearly diminished NDF content (P˂0.001) although the treatment with 40 % of M. oleifera showed the same unexpected performance obtained for OM at this inclusion level. The increase appreciated in CP content and decrease of crude fiber content of silages as a response to the inclusion of Moringa in ensiled mixtures was in correspondence with the chemical composition of used forage, because this protein shrub showed a protein content 2.5 times higher than that of the grass but its NDF content was 15 % lower (Gutiérrez et al. 2015). Similar results were found with the inclusion of growing levels of Tithonia diversifolia in mixed silages based on C. purpureus (Morales et al. 2016).
Table 2, shows the nonlinear regression equations obtained when evaluating the effect of inclusion levels of M. oleifera on the studied indicators of chemical composition. All the equation point out that the inclusion level of M. oleifera had a quadratic effect on the performance of all analyzed variables, with an elevated coefficient of determination (R2 >0.8860), superior to that obtained when equations of linear regressions were used. The above discussion indicates that the addition of growing levels of M. oleifera improve the quality of silages obtained since there is a tendency of CP content increase and fiber decrease.
Castaño and Villa (2015) concluded that varieties of C. purpureus like Cuba OM-22 can be easily conserved. Nevertheless, from a practical point of view mixed silage production, including protein shrubs, allow to improve the nutritional value of silage because tropical grasses contain a low nutritional quality, even though they do not undergo an anaerobic fermentation process for their conservation.
However, the inclusion of high levels of CP could have a negative influence (in practical terms) on the quality of the obtained silage because it is known that with a higher CP content in fresh forage, its buffer capacity or resistance to pH change will be higher during the fermentation process (Otero and Esperance 1994). Singh et al. (1996) indicated that the highest values of ammonia N and pH are associated to high values of populations of proteolytic anaerobic bacteria. Likewise, Bolsen (1999) stated that this buffer effect, due to high levels of nitrogen, influence on fermentation process because it requires the production of extra quantities of lactic acid to lower the pH value, needed for a good preservation of silage, which increase DM losses. Therefore, probably under production conditions, it is recommended to include only between 20 and 40 % of the shrub because the experience show that, practically, it is easier to guarantee availability of grass forage compared to M. oleifera.
Table 2 Non linear regression between level of used M. oleifera (independent variable) and indicators of chemical composition in the evaluated mixed silages
The analysis of MANOVA of in vitro gas production of (mL g-1 OM inc) illustrates that there was an interaction between treatments and times in which the gas was measured (P˂0.05) for the different fermentation phases (initial, intermediate and final) (table 3).
Table 3 Effect of the level of inclusion of M. oleifera in the profile of in vitro gas production in the mixed silage evaluated (mL g-1 OMinc).
In the initial phase of fermentation, at 2 h of incubation, all inclusion levels of M. oleifera increased gas production in comparison with silage without moringa which was the treatment with the lowest gas production up to 8 h of incubation (P˂0.001), although, during this time, this treatment showed no differences regarding silage with 60 % of the protein shrub. The treatment with 80 % of M. oleifera shows the highest gas productions up to 8h, but not differing from the treatment with 40 % at 4 h, or the treatment with 20 % at 6 h or in the treatment with 40 % at 8 h.
In the intermediate phase of fermentation, it could be also observed that the lowest gas production in all times corresponded to the silage with 100 % of C. purpureus (P˂ 0.001) except at 30 h, which did not differ from the treatment with 20 % of M. oleifera. This treatment was followed by the treatment with 20 % of the shrub plant while no high differences were estimated among the silages with M. oleifera.
The highest gas production was found in the treatment with M. oleifera regarding silage with 00 % of C. purpureus in the initial and intermediate fermentation phases. This could be attributed to the chemical composition in both forages. It is known that M. oleifera has high values of soluble and easily fermentable carbohydrates (Rodríguez et al. 2017).
However, in the final fermentation phase, it was appreciated how gas production of treatment with 100 % of C. purpureus increased regarding the other treatments and was higher at 96 and at 120 h. This could be explained because the most fermentable materials of this shrub (the remains from the silage process) were degraded in the first phases and the fact that treatments with different protein contents are being compared, because it is known that gas contribution of carbohydrates is superior to those of proteins (Makkar 2002).
Kinetic parameters of in vitro fermentation of evaluated silages are shown in table 4. It is important to point out that the applied model showed a high coefficient of determination for all treatments (R2>0.9077). This consideration allows stating that it was able to explain a high percentage of variability of the obtained experimental data.
Table 4 Kinetic parameters according to Gompertz model of the in vitro fermentation in the evaluated mixed silages.
SE of the parameters were significant different at P˂ 0.0001
1 SE of the curve
The treatment with 100 % of C. purpureus tended to show the highest potential of gas production. The maximum velocity was obtained in the mixed silage with 60% of M. oleifera, while the inflexion point of the curve (TVmax), corresponding to the moment in which the maximum velocity is reached, was achieved, first, in the treatment with 40 % of M. oleifera and later in the ensilage with 100 % of C. purpureus. The lower TVmax when using 40 % of M. oleifera could be due to a higher microbial efficiency, as it is noticed in the value reached in the parameters C, which tended to be the highest of the rest of silages. These results indicated that, as the extension of gas production is higher when grass is ensiled alone, this result could be related to the conditions mentioned above, regarding the highest contribution of gas from the fermentation of carbohydrates, in contrast to that of protein and it should not be assumed as an indicator of better quality, because when 40 % of M. oleifera is included, better indicators are appreciated related to the microbial actions as TVmax and parameters C.
On the other hand, no differences were observed between treatments in MBS (P=0.5383). However, differences were achieved regarding the EMBS (figure 1)
Mixed silage with 40 % of M. oleifera showed a better efficiency of synthesis compared to the other studied treatments, except in the silage with 20 % of the shrub (P ˂ 0.05). The rest of the treatments did not differ among them. The EMBS showed a quadratic performance and the equation is given below.
In figure 2, the effect of inclusion level of M. oleifera on PF increased when the inclusion level of the shrub was augmented, also in a quadratic direction (y = 0.001 x2 + 0.051 x + 3.375; R2 = 0.9196; SE =0.2138), reaching a maximum with the inclusion of 40 % (P˂0.001), although this treatment did not differ from the silage with 20 % of M. oleifera.
PF is an indicator representing the fermented substratum distribution between the microbial biomass and the final products of the fermentation. Variation of this relation demonstrates changes in the microbial yield per unit of produced volatile fatty acids. A high PF indicates that a high amount of the fermented substratum was transformed into microbial cells and viceversa. However, it should be point out that the value of PF obtained for mixed silages with 40 % of M. oleifera surpasses lightly the theoretical parameter defined by Blümmel et al. (1997). In this respect, there are evidences indicating that the presence of anti- nutritional substances may provoke values above the theoretical parameter (Baba et al. 2002). It is also considered that determination at 120 h of incubation influence on the results due to possible processes of microbial recycling in the in vitro system. Nevertheless, it should be stressed that the coefficient of Pearson between EMBS and PF was high (0.9740; P˂0.01) allowing to consider that increases in the efficiency of microbial synthesis are reached when between 20 % and 40 % of M. oleifera is included on mixed silages.
Figure 1 Effect of the inclusion level of M. oleifera on the EMBS (mg g-1 fermented DM) in the evaluated mixed silages
Figure 2 Effect of the inclusion level of M. oleifera on PF (mg mL-1) of the mixture of evaluated silages.
It is concluded that the inclusion of M. oleifera on mixed silages improved the nutritional quality of the products obtained when levels of protein are increased and NDF are reduced regarding silage of 100% of grasses.
Silage with 40 % of inclusion of M. oleifera was the best combination of the studied levels due to adequate levels of CP, lower NDF values and the fermentation showed high Vmax, low TVmax and the highest efficiency of microbial synthesis.
