4.1. Dry Matter Intake
Cattle systems in the tropics are characterized by meat and milk production under the use of monoculture pastures of low nutritional value, affecting diet consumption parameters and efficiency in the use of the nutrients consumed. In the present study, the inclusion of species such as
T. diversifolia and L. leucocephala in diets based on improved pastures contributed to higher food intake and greater N retention. The steers increased their dry matter intake when the diets offered included species with a high protein value and low NDF content, thus achieving an average intakes of 3% and 3.2% of live weight in the
B. decumbens + T. diversifolia and in the Cayman +
L. leucocephala diets, respectively. These results are in line with previous information reported by other authors [
41], who observed higher intake levels as the CP level increased in the diet and aligned with other studies that reported increased intake in diets with added
L. leucocephala [
42,
43].
The inclusion level reached in the
T. diversifolia diet was 32.7%, while the
L. leucocephala reached an 18.4% inclusion level. These results are consistent with other studies that have reported levels of inclusion of
T. diversifolia between 15% and 35% in grazing of lactating cows, without altering the animals’ voluntary intake [
10,
13]. The NDF and ADF values reported in this study for
T. diversifolia are below those reported in other studies [
44] (NDF: 53%; ADF: 48.18%) and above those reported by other investigations [
45] (NDF: 33.35% and ADF: 12.21%) for similar regrowth ages (56 and 60 days), and establishment of the shrubby cuttings. Both investigations were carried out in different environmental conditions than those of this study (2456 m.a.s.l and 2905 m.a.s.l, respectively). Chemical composition of this shrub fluctuates in response to altitude and, according to other authors [
46], the composition of
T. diversifolia is mainly related to factors such as soil fertility, environmental temperature, and the level of solar radiation, which can influence the plant maturation process and structural carbohydrates. This composition of the
T. diversifolia did not represent a limiting factor to forage intake with respect to the
B. decumbens and Cayman diets.
The
L. leucocephala intake values reported in our study are consistent with those reported [
47]. A diversified supply of fodder stimulates voluntary intake in ruminants during grazing, since they can select and consume plant materials of the best nutritional quality [
15]. It has been reported that increases in voluntary intake by providing
L. leucocephala in the diet is associated with a higher DM rate degradation and a higher passage rate [
47]. In our study, the forages offered in the Cayman grassland, by including
L. leucocephala in rows, was 1.7 times greater (2591 kg DM ha
−1) than in the Cayman grassland and
B. decumbens in monoculture (1513 kg DM ha
−1; 1481 kg DM ha
−1, respectively), as well as 1.3 times greater than in
B. decumbens +
T. diversifolia grassland (1965 kg DM ha
−1). Likewise, the DM and CP
L. leucocephala content values are consistent with the quality parameters referenced for this type of legume [
48] and with those reported by other studies under similar conditions of the tropics [
49,
50]. However, the NDF and ADF values found in our study for both
L. leucocephala and
T. diversifolia are above those reported in other studies at similar sampling ages [
43,
45,
49,
50]. According to several studies [
51,
52] high levels of fiber constitute physical factors that limit the DM intake and degradation rate. However, similar values of NDF and ADF in
L. leucocephala to those found in our study have been reported [
47], and when included at 20% in the diet did not generate any effect on the DM intake. However, the same level generated a reduction in NDF digestibility. In our experiment, the greater protein contribution in the diet by the
L. leucocephala inclusion favored the protein degradation, in part due to supply of adequate energy levels at the rumen, not limiting total DM degradation and DM intake. However, individually, both
L. leucocephala and Cayman grass presented acceptable in vitro DM and NDF digestibility values.
The inclusion of
L. leucocephala in silvopastoral systems contributes to improving soil fertility and, therefore, pasture productivity through biological N
2 fixation [
53]. At the same time, other studies [
12,
54] indicate that the arrangement in rows can generate a better distribution of the N
2 fixed by
L. leucocephala, as well as provide better microclimatic conditions where the grass is less exposed to direct solar radiation and wind effects. The above contributes to the increase in the nutritional quality of the grass in terms of CP and gross fiber [
49]. In our study, no differences were found in the nutritional quality of Cayman associated with
L. leucocephala, compared to the Cayman in monoculture. This may be due to the
L. leucocephala low planting density (1428 seedlings ha
−1), and to the early age of the crop (one year), due to a not yet significant N contribution by the legume to the grassland, as well as a less homogeneous N distribution in the pasture. The N levels are significantly higher below the rows of
L. leucocephala (0.9 t N ha
−1 at 0.2 m deep), being 27% more than in grassland [
55].
On the other hand, in our study the soil was characterized as a compact soil of low aeration. Additionally, although the soils in this study corresponded to neutral soils [
56], and phosphorus fertilization was performed, the P levels in the soil were low compared to other studies in plots established in the same research center [
57], and which indicate a moderate P deficiency (5–15 mg kg
−1) [
56]. P deficiency in the soil can directly affect
L. leucocephala growth, limit nodular development, and therefore have an indirect effect on the symbiotic N
2 fixation [
58]. Maybe, in our study, these physical and chemical characteristics would influence the root development of the legume and a higher planting density will be needed to generate a greater impact on the physical soil structure and the nutritional grass quality associated.
The DM and CP levels found in our study for both plots of
B. decumbens are consistent with those reported by other authors at similar ages and under tropical conditions [
41]. However, CP levels are low, especially in consideration of the high NDF content found. Therefore, energy may have been a limiting factor to ensure an intake level for these pastures. In general, the growth of grasses in all plots was slow and this led to long resting periods (50 days), which could explain the high NDF values reported in this study. Inclusion of both protein sources in this study generated a greater volume of urine, which is consistent with reports by other authors, who attributed this effect to the greater N ingestion [
59], given the increased water intake that is required to digest the proteins and for N metabolism. Furthermore, the contribution of N supplied with the diet of the silvopastoral system, in turn, increased the concentration of N-NH
3 in the rumen and at the same time increased the amount of urine N compared to the other treatments. However, the daily contribution of CP for treatments that included species with high protein value was 84 and 147 g kg DM
−1. These CP values do not exceed the range of requirements for ruminants (100 to 170 g kg DM
−1; [
8]). In this sense, the total amounts of N excreted per N consumed is low. Therefore, the inclusion of
T. diversifolia and
L. leucocephala in the diet leads to better dietary N use and, in turn, can improve the use of the low-quality forage and lead to better performance in terms of live weight gain [
13,
16,
60,
61]. Additionally, other studies have reported that the exponential increase in N excretion through urine is generated with N intakes above 400 g d
−1 [
62,
63]. In our study, the highest N consumed corresponded to 228 g d
−1, and the N excreted/N consumed ratio was 61% and 42% for the
T. diversifolia and the silvopastoral system, respectively. Therefore, our results suggested that the mixture of Cayman
+ L. leucocephala has the potential to favor N retention and thus favor DM degradation under tropical conditions. Finally, this can contribute to improving N use efficiency at the ruminal level [
64].
Use of the silvopastoral system and the inclusion of
T. diversifolia in the
B. decumbens diet generated a lower N excretion in dung compared to the monoculture diets. This is consistent with several studies [
62,
65] where no direct relationship was found between N ingestion and N excretion in dung. These results suggest that protein forages could have contributed to better amino acid digestibility at the intestinal level since the fecal N corresponds largely to the undigested N of the food [
66].
The
B. decumbens diet in monoculture had the lowest N retention, which is characteristic of the low-quality diets used in the tropics [
67]. The N excretion in dung and urine as a proportion of the ingested N decreased with
T. diversifolia inclusion, generating a better N balance (
Table 3). These results are consistent with data reported by other authors [
68], who found a higher percentage of N retention (35.5%) resulting from the inclusion of 35%
T. diversifolia with respect to the 100% grass-based diet. Likewise, other studies found that N excretion in dung, as a proportion of the N ingested, showed linear and quadratic reductions [
69].
4.2. Ruminal Parameters
In our study, rumen fluid pH values remained within an optimal range for the degradation of dry matter, ranging between 6.3 and 6.7, without being affected by the different treatments. These results are consistent with other studies where animal diets are based on forages [
70]. Likewise, in another study where the inclusion of different CP levels were evaluated through the supply of
L. leucocephala flour, pH levels were not affected [
71]. On the other hand, the ruminal N-NH
3 concentration was affected by the different diets; where the inclusion of protein forages increased the amount of N-NH
3 in the rumen over the days, the silvopastoral system diet presented the larger increase on the 15th day of measurement. The minimum required N-NH
3 concentration for maximum microbial growth in the rumen is 50 mg L
−1 [
72]. Likewise, another study reported that levels of 50–80 mg L
−1 appear to be sufficient to favor fiber digestion, because when they obtained higher N-NH
3 levels they did not significantly increase the degradation characteristics of food in the rumen [
73]. According to this information, the N-NH
3 levels reported in our study under
L. Leucaena in the Cayman diet would be meeting the minimum requirement at the ruminal N level, which could be used in the synthesis of microbial proteins at the level ruminal [
74]. In the same way, the N-NH
3 concentration in the rumen is a gross predictor of the efficiency of the dietary N conversion into microbial N [
73]. Although this same diet has generated a higher concentration of urine N, it also generated a greater N retention. Despite this, it is not possible to infer that under the
L. Leucocephala inclusion in the diet it has contributed to efficient microbial protein synthesis, since predictive factors such as the DPR and UDPR level, as well as the passage rate, were not quantified in this study.
The production of VFA in the rumen is influenced by dietary carbohydrate fraction and degradability. In our study, the molar acetate concentration was higher with the
L. leucocephala inclusion, which could relate to a greater CP availability in the diet, since the CP promotes the growth of ruminal bacteria and with it greater acetate production arising from cellulose degradation [
75]. Similar results were reported in the other studies, where an increase in the protein level supplied to lambs increased acetic acid concentration and the acetic:propionic ratio, as well as higher N-NH
3 concentration [
76]. In this sense, in our study, with the inclusion
T. diversifolia in the diet of
B. decumbens, a greater total bacteria production was found at the 15th day of measurement. This was followed by the diet including
L. Leucocephala, both of which were the diets with greater CP contribution supplied in this trial. These results are consistent with those reported in other studies [
77,
78], where a greater total bacteria production in the rumen was evidenced by increasing CP in the diet. On the other hand,
L. leucocephala has a non-protein amino acid called mimosine (α-amino, 3-hydroxy-4-pyridinepropanoic acid). This component fluctuates between 40 to 120 g kg DM
−1 according to its physiological state and plant fraction [
11] and, during ruminal fermentation, can contribute to the increase in acetate production as well as to an increase in the N-NH
3 content and organic matter degradation [
79]. On the other hand, if the higher N-NH3 level found in this study under the silvopastoral system would influence the higher growth of ruminal microorganisms, would be promoting cellulose degradation [
80], which would contribute to acetic acid synthesis. However, the acetic:propionic ratio in the ruminal fluid was higher in
B. decumbens grass in monoculture, but this diet presented quantitatively lower production of propionate.
Other populations quantified in our study, such as
F. succinogenes,
S. ruminantium, and Protozoa, were not significantly affected by the different diets (
p > 0.05), although the Protozoa populations showed a significant increase during 15th day of measurement under the
T. diversifolia inclusion in the
B. decumbens-based diet (
p < 0.001). This result could trigger higher emissions of enteric CH
4, since, methanogens associated with ciliated protozoa are responsible for 9% to 37% of methane production in the rumen [
79]. Therefore, by increasing the population of protozoa, the activity of the methanogenic bacteria associated with them could be increased. These results are contrary to those reported by various studies where
T. diversifolia was included and reduced the protozoa population significantly in in vitro conditions [
81,
82], which is mainly attributed to the content of secondary metabolites such as condensed tannins (CT) and total phenols [
83,
84,
85]. However, in another study that evaluated
T. diversifolia between 30 and 60 days of regrowth, the presence of CT and total phenols was low, both in leaves and in the mixture of leaves and stems, mainly finding alkaloid type metabolites [
86].
On the other hand, the presence of CT in the plant negatively affects its palatability, thus reducing DM intake [
87]. However, in our study, the results indicate an increase in the DM intake with respect to the diet offered in
B. decumbens and the Cayman in monoculture, despite there being high levels of the shrub in the diet (35%). Therefore, according to the previous studies, it may be that the concentrations of CT and phenols in the ration were not high enough to generate an effect in reducing the population of protozoa. In contrast, the diet offered has high levels of fiber and did not show variation in pH, which could be a reason to stimulate the population of protozoa in the rumen [
88,
89].
Better physical and chemical characteristics of the soil than those observed in this study are necessary to improve the performance and the nutritional quality of
B. decumbens grass, and thereby lead to better results on ruminal parameters when this grass is associated with protein forages such as
T. diversifolia. Likewise, the concentration of tannins and phenols in the plant depends on edaphoclimatic factors [
83]. Therefore, further research is necessary to evaluate the tannin concentration in
T. diversifolia and its relationship with the population of protozoa and methanogenic bacteria, to identify the role of this shrub more accurately on the population of protozoa and methanogenic bacteria in the rumen.
Prevotella ruminicola 23 is one of the most abundant and important microorganisms for ruminal fermentation; 50% of the population grows well under cultivation in the lab and has a high frequency within the 16S gene [
37,
38]. Its population was affected by the inclusion of protein forages in the diet, showing an increase on the 15th day of measurement, being slightly higher with the
T. diversifolia inclusion for the same period. This result suggests that the diet is providing enough energy for multiplication and microbial growth [
34]; this benefit is only evidenced up to 15 days after consuming protein forages (
T. diversifolia and
L. leucocephala). This result is consistent with those reported by another study [
76], which found greater abundance of
Prevotella when lactating cows were fed legume hay (
Medicago sativa). Likewise, other authors found that the total count of viable bacteria, cellulolytic bacteria, and proteolytic bacteria counts increased when
L. leucocephala leaf meal was supplied without thermal processing [
71].
The higher acetate production on the 15th day of measurement found that
L. leucocephala may be related to the greater abundance of
Prevotella, since acetate corresponds to one of its main fermentation products [
90]. However, it is known that
L. leucocephala has secondary compounds such as condensed tannins whose total concentration can fluctuate between 1.8–4% [
91] and can affect the DM intake [
50], as well as causing rumen defaunation [
92]. In our study, the tannin content in
L. leucocephala was not quantified; therefore, the level of this secondary metabolite ingested by the animals during this test is not known. However, under the inclusion
L. leucocephala level (18%) obtained, neither the intake parameters nor the total protozoa population were affected. Likewise, in a study where the CT effect extracted from
L. leucocephala (CIAT 734) and
Desmodium ovalifolium on the parameters of fermentation and cellulolytic bacteria was evaluated,
L. leucocephala had a lower effect on digestibility, fibrolytic population bacteria, and total AVF production [
93].