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Silage is a method for preserving forage nutrients from their lactic acid fermentation, carried out by epiphytic bacteria under anaerobic conditions (Liu et al. 2016). It is used to segregate the excess forage that occurs during rainy season and to have this food to cover part of the deficit of pastures in dry season. Silages produced in tropical areas are generally composed of forage grasses with low nutritional value but high biomass production, good leaf proportion, rusticity and adaptation to a great diversity of soils and adverse climatic conditions, highlighting the species of Cenchrus genus (García et al. 2014).
For several decades, the Institute of Animal Science (ICA) has been accumulating experience in methods to improve the fermentation process and nutritional value of tropical forage silages, including varieties and hybrids of Cenchrus. In this sense, the effect of pre-drying has been evaluated (Michelena and Molina 1990), the inclusion of protein scrubs such as Moringa oleifera and Tithonia diversifolia to increase protein level and improve mixed silage quality (Gutiérrez et al. 2014 and Morales et al. 2016), the use of energy additives such as molasses (Domínguez et al. 1982) and sweet potato tubers (Ipomoea batatas) (Rodríguez et al. 2019 and Rodríguez et al. 2020) to improve availability of easily fermented carbohydrates, and the use of biotechnological products such as VITAFERT that provides lactic bacteria, organic acids and microbial growth factors that favor the fermentation process (Gutiérrez et al. 2014, Morales et al. 2016 and Rodríguez et al. 2019). Recently, it was described the effect of including higher levels of sweet potato tubers (up to 50%, humid base) in association with the use of pure Lactobacillus pentosus LB-31 strain (Rodríguez et al. 2020) on the nutritional value of mixed silages.
Microbial additives obtained from pure strains of lactic acid bacteria are safe inoculants, easy to use, not corrosive to agricultural machinery, and do not pollute the environment (Ozduven et al. 2017). The most widely used to stimulate lactic fermentation and improve the quality of silages have been homofermentative bacteria (McDonald et al. 1991), including several species of Lactobacillus, Enterococcus and Pediococcus (Muck et al. 2018) genera, that have demonstrated potential to improve fermentation processes and guarantee good quality of the obtained product (Porto et al. 2017).
Therefore, the objective of the present study was to evaluate the effect of the inclusion of sweet potato tubers and P. pentosaceus LB-25 on the nutritional value of mixed silages of C. purpureus x C. glaucum (OM-22 hybrid) and M. oleifera, by analyzing its chemical composition and indicators of its in vitro fermentation.
Materials and Methods
Silage obtaining
Forages used for silage production. Plants were collected in experimental areas of the Institute of Animal Science (ICA) of Cuba. Plants of C. purpureus x C. glaucum (OM-22 hybrid) and M. oleifera (moringa) of 90 and 60 days of age, respectively, were collected by manual cut. They were established in typical red ferralitic soil, fast drying and uniform profile (Hernández et al. 2015). Both forages were passed through a forage mill until they reached a particle size of 20-30 mm and ground material was spread for pre-drying under the sun on a flat surface with drainage until reaching a DM superior to 30%.
Additives used for silage production. Sweet potato tubers were used as energy additives to increase the content of easily fermented carbohydrates. Small and medium-sized sweet potatoes were used, without washing, but without adhering soil. Tubers were cut lengthwise into pieces, and, then, they were reduced in size by thinly laminating them. This process was carried out at the time silages were prepared to avoid starch oxidation as much as possible.
The pure culture of the strain of lactic acid bacteria P. pentosaceus LB-25 was used as microbial additive, obtained from the ICA Bank of Microorganisms. This strain was identified by sequencing the ribosomal RNA 16S gene and its sequence is deposited at GenBank (access number: FR717465). The strain was grown in Rogosa broth (Oxoid, UK), at 37 °C, for 18-24 h. After incubation period, its purity was verified and concentrations of 109 cells·mL-1 were guaranteed, determined by counting in a Neubauer chamber.
Microsilos preparation. Microsilos preparation was carried out according to the methodology proposed by Rodríguez et al. (2016) in hermetically sealed glass tubes (120 mm height x 70 mm diameter). First, a mixture was prepared with the forages to be ensiled (OM-22 and moringa, 50:50 in humid base), which constituted the fiber and protein core of silages to be evaluated. Later, this core was mixed with the three levels of sweet potato tubers (0, 25 and 50%) and then, all treatments were uniformLy sprayed with urea (1%) and ammonium sulfate (0.2%), diluted in water at a proportion of 1: 2, also on a humid base. After mixing with urea and ammonium sulfate, it was left to rest for 30 minutes before adding the microbial inoculum to treatments. For this inoculation, 75 mL of medium with the corresponding inoculum for each 1.5 kg of the material to be ensiled were sprayed for each combination of forage and sweet potato (5% v/w). Treatments without microbial inoculum were sprayed with the same amount of distilled water to ensure that all treatments were homogeneously moistened.
After a last mixing, treatments were introduced into glass containers, in layers that were compacted with a tamper tool. At the end, containers were hermetically sealed. Microsilos, with approximately 300 g of fresh matter, were kept under anaerobic conditions for a period of 64 days. The evaluated treatments were:
Forage of OM-22: moringa (F)
Forage of OM-22: moringa+25% of sweet potato tubers (F+25% B)
Forage of OM-22: moringa+50% of sweet potato tubers (F+50% B)
Forage of OM-22: moringa+P. pentosaceus LB-25 (F+LB-25)
Forage of OM-22: moringa+25% of sweet potato tubers+P. pentosaceus LB-25 (F+25% B+LB-25)
Forage of OM-22: moringa+50% of sweet potato tubers+P. pentosaceus LB-25 (F+50% B+LB-25)
At the end of silage process, microsilos were opened and a sample of 10 g was taken from each, 90 mL of distilled water was added and it was mixed in orbital shaker at 250 rpm for 15 minutes, at 20ºC. Then, the mixture was filtered through gauze and pH was measured to the filtrate (Everich pH meter, PHSJ-3F, China). Additionally, approximately 200 g were taken from each microsilo, they were dried to constant weight in a forced air oven, with regulated temperature (60ºC) and then they were ground in a hammer mill until reaching a particle size of 1 mm. Subsequently, half of the dry material from each microsilo was individually stored in sealed nylon bags for chemical composition analysis. The rest of dry material was homogeneously mixed by treatment and the obtained pool was also stored in sealed nylon bags until it was used for in vitro evaluations.
In vitro evaluation of the fermentative capacity of the obtained silages. Two in vitro tests were performed to evaluate the nutritional value of the obtained mixed silages, in which gas production technique described by Theodorou et al. (1994) was used. An amount of 1.0 g of each treatment was incubated in 100 mL glass bottles, with culture medium (Menke and Steingass 1988) and an inoculum of ruminal microorganisms in a proportion of 0.20 mL of the total incubation volume (80 mL).
Ruminal content of two stabulated adult Siboney cows, fed ad libitum with grass forage and free access to water and mineral salts, was used as inoculum. Rumen content was collected through the rumen cannula, before offering food in the morning, and it was kept in independent closed thermoses until reaching the laboratory, where they were filtered through gauze and mixed in equal proportions. During the process, inocula temperature was maintained at 39 ± 1ºC, and anaerobic conditions by continuous flow of CO2. Bottles were sealed and incubated in a bath, at controlled temperature (39ºC). This moment was taken as zero hour of incubation. In both tests, bottles without substrate were incubated as control.
In the first test, to determine in vitro fermentation kinetics of obtained silages, gas production was measured up to 144 h, using an HD8804 manometer coupled to a TP804 pressure calibrator (DELTA OHM, Italy). After each measurement, gas was released to equalize the external and internal pressures of bottles. Gas volume was estimated from pressure data, using a pre-established linear regression equation (Rodríguez et al. 2013). Gas volume was expressed per gram of incubated organic matter (incOM). To estimate gas production kinetics, the exponential model described by Krisnamoorthy et al. (1991) was used:
Where Y is accumulated gas production (mL g-1 incOM) at an incubation time t (h), D is fermentation potential of the substrate under incubation conditions (asymptote of the curve, mL g-1 incOM), c is fermentation fractional rate of (h-1) and L is the Lag phase of fermentation (h).
In the second test, gas production was measured only up to 24 hours of incubation (3, 6, 9, 12, 18, and 24 h). At the end of incubation, bottles were opened and their contents were filtered to nylon bags (50 μm of porosity and 28 cm2 of surface area), previously tared in an analytical balance (Sartorius BL 1,500 ± 0.0001). Bags with fermentation residues were dried up through constant weight in a forced air oven with regulated temperature at 60ºC, for 72 hours. By this means, apparent in vitro degradability of DM (DAIVMS) of evaluated silages was determined by gravimetry, as the difference between incubated substrate and fermentation residue, expressed as a proportion of incubated substrate and multiplied by 100 (%).
In addition, 5 mL of sample were taken from the filtrate to determine the NH3 content, which were preserved until the time of their analysis.
Chemical analysis. To determine the chemical composition of silages, DM, OM and crude protein (CP) were determined (AOAC 2016). Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were quantified by the procedure described by Van Soest et al. (1991). To obtain ammonia concentration in the in vitro test, the colorimetric technique proposed by Chaney and Marbach (1962) was used.
Statistical analysis. For the analysis of chemical composition of evaluated silages, a completely randomized experimental design, with a factorial arrangement (3x2), was used, in which the main factors were the 3 levels of sweet potato inclusion (0, 25 and 50%) and the inclusion of microbial additive (without additive and with P. pentosaceus LB-25). Microsilos were considered as an experimental unit (5).
In the 24 h in vitro trial, a randomized block design with a factorial arrangement (3x2) was used, where analyzed factors were the same as in the previous case and incubations were considered as a block (4). In this experiment, bottles were considered as an experimental unit (3 bottles per treatment).
In both cases, variables were analyzed by ANOVA. In case differences were found (P <0.05), means of treatments were compared by Duncan (1955) test. InfoStat statistical package (Di Rienzo et al. 2012) was used for these analyzes.
In the case of the variable in vitro accumulated gas production, the fermentation was divided a priori into two phases. The first phase included three sampling times between the start and the 9 hours of incubation (3, 6 and 9 h) and the second phase was considered from that moment to the end of incubation (12, 16 and 24 h). Since in vitro accumulated gas production is measured over time on the same experimental unit, the methodology proposed by Gómez et al. (2019) was applied. For the analysis of these data, SAS (2013) statistical package, version 9.3, was used.
Results and Discussion
The chemical composition of forages and sweet potato tuber used in silage production was previously reported (Rodríguez et al. 2020). In all cases, forage DM values were higher than 30% as suggested by Michelena and Molina (1990).
When evaluating the effect of additives on DM yield, estimated as the proportion between obtained silage DM and the DM of materials to be conserved and expressed as percent, there was no interaction among factors nor effect of microbial additive LB-25 (P> 0.05). However, sweet potato inclusion level affected this indicator (P <0.05) by decreasing with 50% inclusion of the tuber (figure 1). In all the treatments, DM yield showed values higher than 96.6%, which could be related to the high DM levels of the preserved forage mix and to the type of microsilo used in the study, which does not have losses due to lixiviation.
Figure 1 Effect of inclusion level of the energy additive on DM yield (%) of evaluated silages (SE = ± 3.12)
Regarding pH, there was no interaction between the effects of the two evaluated additives nor the effect of LB-25 microbial additive (P> 0.05). However, the inclusion of sweet potato influenced on this indicator of fermentation (P = 0.003) although no differences were observed between 25 and 50% of tubers (figure 2).
High pH values observed in the treatments without sweet potato did not affect the organoleptic characteristics of microsilos nor were there signs of putrefaction or the presence of filamentous fungi. The maximum pH value for obtaining quality silage is between 3.8 and 4.2 (McDonald et al. 1991). However, other research has also found that, at high DM content of forage to be ensiled, good products preserved at higher pH can be obtained (Castle and Watson 1984 and Dumont 1994). In fact, it is argued that it is common to find silages with higher pH values when they have been produced with pre-dried forages (dos Santos et al. 2013).
There was also no interaction of evaluated factors on indicators of chemical composition (P>0.05). After analyzing individual factors, it was observed that sweet potato level affected DM, OM, NDF and ADF (P <0.0001), while the inclusion of LB-25 strain increased NDF (P=0.048). None of the additives had an effect on CP content of evaluated silages (P> 0.05) and the values obtained for this indicator were superior to 10.0% for all treatments.
Table 1 shows the effect of sweet potato inclusion level and the inclusion of LB-25 on indicators of chemical composition of the evaluated silages. DM and NDF contents decreased with the increase of tuber level because the pre-dried forage core was replaced by a fresh material with a lower content of DM and fiber (P <0.0001). However, OM content increased and ADF content decreased with the inclusion of 25% of the tuber (P <0.0001), but, in both cases, there was no difference between silages with 25 and 50% of this additive.
Table 1 Effects of sweet potato and LB-25 strain inclusion on the chemical composition of the evaluated silages
| Sweet potato level | |||||
|---|---|---|---|---|---|
| Tuber (%) | DM (%) | OM (%) | NDF (%) | ADF (%) | CP (%) |
| 0 | 45.4c | 89.0a | 70.2c | 51.0b | 10.6 |
| 25 | 38.7b | 89.8b | 62.3b | 44.3a | 10.9 |
| 50 | 32.2a | 90.0b | 55.8a | 41.5a | 11.4 |
| SE | 0.52 | 0.12 | 1.14 | 1.13 | 0.30 |
| P | <0.0001 | <0.0001 | <0.0001 | <0.0001 | 0.2404 |
| Inclusion of LB-25 | |||||
| LB-25 | DM (%) | OM (%) | NDF (%) | ADF (%) | CP (%) |
| Without LB-25 | 39.0 | 89.5 | 61.5 | 44.7 | 11.0 |
| With LB-25 | 39.0 | 89.7 | 64.6 | 46.8 | 10.9 |
| SE ± | 0.41 | 0.09 | 0.90 | 0.90 | 0.24 |
| P | 0.3996 | 0.2898 | 0.0480 | 0.1695 | 0.7224 |
AbcValues with different letters per row differ at P<0.05 (Duncan 1955)
Even though differences in DM content were observed, all evaluated treatments showed DM superior to 30%, which is the minimum recommended value to obtain quality silages (Michelena and Molina 1990). The high levels obtained of NDF could be associated to cutting age of forages and the disappearance of soluble material during anaerobic fermentation (Rodríguez et al. 2016 and Rodríguez et al. 2019). On the other hand, increase of OM can be related to the higher OM content of sweet potato.
Due to the low protein content of sweet potato tubers, a decrease of CP content was expected with the increase of this additive in silage, as found by Neiva et al. (2001) by increasing sugarcane levels as a source of soluble carbohydrates. However, the fact that there were no differences could be related to the availability of nonstructural carbohydrates provided by the tuber, that could favor nitrogen retention processes through microbial protein synthesis. In any case, CP values found were higher than the 6-8% PB suggested by Mertens (1994), as a minimum content so that this nutrient does not limit the fermentation of carbohydrates by the rumen microorganisms.
Regarding the effect of the inclusion of LB-25 microbial additive on NDF content, similar results were found by Junges et al. (2013) when evaluating a mixture of L. plantarum, L. brevis and E. faecium as a microbial additive. These authors related this increase of NDF content by including microbial additives, with the fermentation of part of the soluble components of forage to be preserved by the inoculated bacteria, which leads to the proportional increase of insoluble components. However, Ozduven et al. (2017), when inoculating with L. buchneri and P. acidilactici observed that these microorganisms improved the fermentation characteristics and decreased NDF and ADF content of silages of sunflower plants.
In vitro fermentation of silages.Table 2 shows the parameters of equations of the best fit to the experimental data obtained according to the exponential model of Krishnamoorthy et al. (1991), which was able to explain a high percent of the variability of data of accumulative in vitro gas production for all treatments (R2> 0.9783).
Table 2 Kinetic parameters of the curve of accumulated production of in vitro gas when fermenting evaluated silages, according to the exponential model of Krishnamoorthy et al. (1991)
| Treatments | Parameter D*(mL g-1 MOinc) | SE± | Parameter c (h-1) | SE± | Parameter L (h) | SE± | SE of the curve ± | R2 |
|---|---|---|---|---|---|---|---|---|
| F | 433.97 | 8.590 | 0.04 | 0.003 | 7.25 | 0.397 | 20.109 | 0.9835 |
| F + 25% B | 543.10 | 10.177 | 0.05 | 0.003 | 6.88 | 0.381 | 26.528 | 0.9815 |
| F + 50% B | 619.63 | 11.919 | 0.05 | 0.003 | 6.87 | 0.384 | 32.264 | 0.9790 |
| F+ LB-25 | 468.57 | 10.682 | 0.04 | 0.003 | 7.34 | 0.459 | 23.265 | 0.9810 |
| F + 25% B+ LB-25 | 538.24 | 11.189 | 0.05 | 0.003 | 6.83 | 0.430 | 28.362 | 0.9783 |
| F + 50% B+ LB-25 | 662.69 | 11.502 | 0.05 | 0.003 | 6.82 | 0.351 | 30.795 | 0.9831 |
*Parameters were significant in all cases (P< 0.0001)
It was observed that gas production potential (parameter D) tended to increase with sweet potato inclusion level. The same trend was show by the microbial additive when used with forages alone and 50% of sweet potato. Regarding the fractional rate or parameter c, there was also a tendency to increase when including sweet potato, but, at the same inclusion level of the tuber, there were no differences in the magnitudes of this parameter when including the microbial additive or not. On the other hand, the Lag phase (parameter L) showed an inverse trend because with the inclusion of sweet potato, the time required to start fermentation tended to decrease, but, like for fractional rate, this indicator was not affected by the inclusion of Pediococcus strain.
The increase of potential and fractional rate of gas production when sweet potato was included could be due to the greater amount of soluble carbohydrates available for fermentation, which corroborates the increase of nutritional value of silages by including this energy additive. Similar responses were observed when replacing Cenchrus forage with better quality T. diversifolia forage (Morales et al. 2016) or including up to 15% of sweet potato in mixed silages of Cenchrus and M. oleifera (Rodríguez et al. 2019).
For the variable accumulative production of in vitro gas, there was no interaction between sweet potato level, the inclusion or not of LB-25 and sampling times, in the two phases of fermentation in which the sampling times were distributed a priori (P> 0.05).
Table 3 shows the individual effects of factors analyzed in the accumulated production of in vitro gas. It was appreciated that the effect of energy additive on accumulated gas production had the following performance in both fermentation phases: level 50%> level 25%> level 0% (P<0.0001). Similarly, it was observed that the microbial additive improved gas production in the second phase of fermentation (P=0.0470). Regarding the factor sampling time, in both phases, gas production increased over time (P <0.0001).
Table 3 Effect of sweet potato level, LB-25 inclusion and sampling time on accumulated production of in vitro gas (mL g-1 incOM) during the two phases of fermentation
| Sweet potato level (%) | |||
|---|---|---|---|
| Tuber (%) | Gas production 1st phase | Gas production 2nd phase | |
| 0 | 32.1a | 137.2a | |
| 25 | 55.3b | 187.3b | |
| 50 | 60.7b | 225.7c | |
| SE | 2.12 | 3.78 | |
| P | <0.0001 | <0.0001 | |
| Inclusion of LB-25 | |||
| LB-25 | Gas production 1st phase | Gas production 2nd phase | |
| WithoutLB-25 | 48.2 | 179.0 | |
| With LB-25 | 50.5 | 187.8 | |
| SE | 1.73 | 3.09 | |
| P | 0.3340 | 0.0470 | |
| Sampling times within the same phase (h) | |||
| Time (h) | Gas production 1st phase | Time (h) | Gas production 2nd phase |
| 3 | 13.3a | 12 | 128.4a |
| 6 | 47.0b | 18 | 186.9b |
| 9 | 87.8c | 24 | 234.9c |
| SE | 2.12 | EE | 3.78 |
| P | <0.0001 | P | <0.0001 |
AbcValues with different letters per row differ at P<0.05 (Duncan 1955)k 3u
Indicators of in vitro fermentation of silages. At 24 hours, there was no interaction between tuber level and microbial additive inclusion, nor the effect of LB-25 inclusion in DAIVMS (P>0.05). However, sweet potato level influenced on this gravimetric indicator (figure 3). DAIVMS increased with 50% of tuber inclusion, but no differences were observed between the other two inclusion levels (P=0.0207).
Figure 3 Effect of sweet potato tuber inclusion level on DAIVMS (%) after 24 h of fermentation of the evaluated silages (SE = ±4.42)
The observed DAIVMS was higher than that reported by Rodríguez et al. (2019) for silages of C. purpureus (cv. Cuba CT-169) and M. oleifera, with the same cutting age and lower sweet potato inclusion levels (5, 10 and 15%). This confirms the importance of increasing inclusion levels of energy additives in mixed silages of tropical forages, to improve fermentation conditions for their conservation and their nutritional quality as feed for ruminants (Mühlbach 2001).
For N-NH3, there was also no interaction between sweet potato level and LB-25 inclusion, nor microbial additive effect (P> 0.05). However, sweet potato level influenced on this indicator (P<0.05), when an increase was observed after including 25 and 50% of tuber regarding silage with only forage (figure 4).
Figure 4 Effect of inclusion level of the energy additive on N-NH3 (mg 100 mL-1) after 24 hours of in vitro fermentation of the evaluated silages (SE = ± 3.94)
Results demonstrated higher ruminal concentrations of N-NH3 than those reported by Rodríguez et al. (2019). These differences could be caused by the higher levels of moringa used and urea inclusion in the mixtures to be ensiled. In addition, ammonia N concentrations above 20 mg 100 mL were observed in all treatments, superior to ruminal values considered optimal to maximize microbial protein synthesis (2-13 mg 100 mL-1) and ruminal fermentation rate of food (3-25 mg 100 mL-1) (Boucher et al. 2007).
The lack of effects of microbial additive, except in the NDF content of silage and in the accumulated production of in vitro gas from 12 to 24 h, could be related to the preserved forage species. A meta-analysis of 130 articles showed that the effect of microbial additives varies according to the forage species (Oliveira et al. 2017). That study showed that the use of microbial additives, regardless of the conserved forage species, improved the production of lactic acid and reduced the concentrations of butyric acid and ammoniacal nitrogen in silage. However, inocula reduced pH in many temperate and tropical forages, including legumes, but it did not influence on this indicator when corn, sorghum and sugar cane were preserved. Similarly, they observed that it did not influence on yield of corn and sorghum silos, and this indicator worsened in sugar cane silages.
Conclusions
The inclusion of sweet potato tubers in mixed silages of C. purpureus x C. glaucum (OM-22 hybrid) and M. oleifera improved the nutritional value of conserved materials, with the best results with the inclusion of 50%. On the other hand, the use of P. pentosaceus LB-25 strain, at 5% (v/w), did not influence on nutritional quality of the evaluated silages, except NDF increased its content.
