Physiological and Chemical Response of Urochloa brizantha to Edaphic and Microclimatic Variations Along an Altitudinal Gradient in the Amazon

Abstract

Urochloa brizantha (Brizantha) is cultivated under varying altitudinal and management conditions. Twelve full-sun (monoculture) plots and twelve shaded (silvopastoral) plots were established, proportionally distributed at 170, 503, 661, and 1110 masl. Evaluations were conducted 15, 30, 45, 60, and 75 days after establishment. The conservation and integration of trees in silvopastoral systems reflected a clear anthropogenic influence, evidenced by the preference for species of the Fabaceae family, likely due to their multipurpose nature. Although the altitudinal gradient did not show direct effects on soil properties, intermediate altitudes revealed a significant role of CaCO₃ in enhancing soil fertility. These edaphic conditions at mid-altitudes favored the leaf area development of Brizantha, particularly during the early growth stages, as indicated by significantly larger values (p < 0.05). However, at the harvest stage, no significant differences were observed in physiological or productive traits, nor in foliar chemical components, underscoring the species’ high hardiness and broad adaptation to both soil and altitude conditions. In Brizantha, a significant reduction (p < 0.05) in stomatal size and density was observed under shade in silvopastoral areas, where solar radiation and air temperature decreased, while relative humidity increased. Nonetheless, these microclimatic variations did not lead to significant changes in foliar chemistry, growth variables, or biomass production, suggesting a high degree of adaptive plasticity to microclimatic fluctuations. Foliar ash content exhibited an increasing trend with altitude, indicating greater efficiency of Brizantha in absorbing calcium, phosphorus, and potassium at higher altitudes, possibly linked to more favorable edaphoclimatic conditions for nutrient uptake. Finally, forage quality declined with plant age, as evidenced by reductions in protein, ash, and In Vitro Dry Matter Digestibility (IVDMD), alongside increases in fiber, Neutral Detergent Fiber (NDF), and Acid Detergent Fiber (ADF). These findings support the recommendation of cutting intervals between 30 and 45 days, during which Brizantha displays a more favorable nutritional profile, higher digestibility, and consequently, greater value for animal feeding.

1. Introduction

Urochloa brizantha (Brizantha), also known as Brachiaria brizantha, is a highly versatile forage grass, adapted to diverse ecological zones, valued for its productivity [1,2]. In the Peruvian tropics, Brizantha is the most cultivated pasture, thanks to its low incidence of phytosanitary problems and its rapid regrowth after grazing. Studies have provided evidence that Brizantha presents a remarkable adaptation to acid soils, tolerates drought conditions, and contributes significantly to the improvement of soil fertility [3,4,5]; in turn, this increases the availability of nutrients such as calcium, magnesium, and potassium [6]. These benefits, related to soil conservation, position Brizantha as a promising alternative for livestock systems in the Peruvian tropics [7,8], with a remarkable potential to promote the sustainability of agricultural systems [9].

Brizantha has demonstrated a biomass yield comparable to other species [10]; it has a good performance in terms of dry matter digestibility and high nutritional value [11,12] and accumulates approximately 50% of its dry matter in the leaves, which maintain adequate levels of essential nutrients for ruminal fermentation [13]. Likewise, Brizantha is characterized by a higher proportion of neutral detergent fiber in the stems and high crude protein content in the leaves, which contributes to better forage digestibility [14]. In terms of soil fertility, nitrogen availability significantly increases crude protein content and in vitro dry matter digestibility while reducing neutral detergent fiber and acid detergent fiber levels. However, parameters such as cutting age and climatic conditions also significantly influence the chemical composition of this forage [15,16].

In the Peruvian tropics, Brizantha is grown along altitudinal gradients ranging from 50 to more than 1200 m in altitude. This geographical factor generates significant variations in soil and climatic conditions that can influence the physiological and chemical properties of Brizantha. The temperature decreases by between 0.4 and 0.6 °C for every 100 m increase in altitude [17]. Conversely, soil properties such as pH, organic matter content, and moisture tend to increase, reaching maximum values at altitudes near 3000 m [18]. Consequently, the combined effects of edaphoclimatic and management variations in pasture physiology are not fully understood [19].

Peruvian small-scale farmers manage Brizantha in mixed production systems that include both silvopastoral and monoculture areas within the same farm. Research comparing Brizantha production in these systems has reported varying results. Some studies found no significant differences in forage accumulation between silvopastoral and monocultural systems [20], while others have evidenced higher forage and biomass production in silvopastoral systems [21]. The presence of trees in silvopastoral systems modifies the morpho-physiological characteristics of pastures [22,23], mainly due to the shading generated by tree canopies, which creates different microclimates compared to monocultural systems. This shading reduces the proportion of lignified tissues in Brizantha, which can improve its digestibility [24]. In addition, the greater availability of daily sunlight hours in monocultural systems, compared to silvopastoral systems, may influence the structure and stomatal density of Brizantha leaves. Under shaded conditions, stomatal density tends to decrease on both adaxial and abaxial leaf surfaces [25,26,27].

Understanding the morpho-physiological responses of Brizantha to altitudinal gradient and to shading and full-sun conditions typical of silvopastoral and monocultural areas is essential for optimizing the productive management of forage grass. However, studies on Brizantha in aspects such as foliar chemical properties and their relationship with soil properties and stomatal morphometry along an altitudinal gradient, as well as under different productive management conditions, are still limited. In this study, it is hypothesized that altitudinal gradient, full-sun and partial-shade tree conditions, and soil properties affect chemical properties and stomatal morphometry of Brizantha. Therefore, the objective of this study was to determine the influence of altitudinal gradient and production management on the chemical and physiological properties of Brizantha.

2. Materials and Methods

2.1. Study Area Location

The study was conducted between March 2023 and June 2024 in the Peruvian tropics, encompassing an altitudinal gradient. The lower zone of the gradient included the district of Yurimaguas, at 170 masl, in the Loreto region. The middle zone comprised the districts of Cuñumbuqui, at 503 masl, and Zapatero, at 661 masl, both in the San Martín region. The upper zone included the district of Calzada, at 1110 masl, also in the San Martín region. A total of 24 Brizantha plots were established, evenly distributed across the four districts. Six plots were installed in each district: three under monoculture management (full sunlight) and three under silvopastoral management (partial tree shade). All plots were georeferenced using a Garmin 65S GPS device (Table 1).

In each district, two automatic weather stations (HUNAN Rika, RK900-05, Changsha, China) were installed—one in full sun within monoculture areas and the other under partial shade in silvopastoral areas—resulting in a total of eight stations for the entire experiment, as reported by Murga-Orrillo et al. [28]. Daily microclimatic data from these stations were used to determine average temperature, relative humidity, and solar radiation during the study period (Table 1).

Table 1

Latitude (ϕ), longitude (λ), altitude (φ, masl), average temperature (°C), relative humidity (RH, %), and solar radiation (W/m²) during the study period in the experimental Brizantha plots located at 170, 503, 661, and 1110 m above sea level.

masl	Condition	ϕ	λ	φ	°C	HR (%)	W/m2
170	Shade	5°33′36″	76°47′60″	155
		5°58′48″	76°11′24″	178
		5°54′0″	76°10′12″	170	26.2 ± 1.2	93.5 ± 3.9	37.5 ± 25.8
	Sun	5°53′24″	76°12′0″	192
		5°58′12″	76°10′48″	157	26.5 ± 1.3	89.9 ± 4.2	144.4 ± 56.3
		5°53′24″	76°11′24″	169
503	Shade	6°31′12″	76°31′12″	571	25.7 ± 1.5	83.7 ± 7.8	108.8 ± 47.7
		6°31′48″	76°31′12″	373
		6°32′24″	76°28′48″	554
	Sun	6°30′36″	76°30′36″	572	26.5 ± 1.7	80.4 ± 7.9	146.0 ± 49.5
		6°31′12″	76°30′0″	466
		6°32′60″	76°29′24″	483
661	Shade	6°36′36″	76°29′24″	648	25.2 ± 1.6	81.0 ± 9.1	61.9 ± 29.2
		6°32′60″	76°31′12″	724
		6°35′60″	76°29′24″	652
	Sun	6°32′60″	76°30′36″	651
		6°30′36″	76°32′24″	637	25.6 ± 1.7	78.8 ± 8.1	163.6 ± 57.8
		6°20′60″	76°17′60″	652
1110	Shade	6°2′24″	77°2′60″	1112	23.1 ± 1.1	86.1 ± 5.0	52.6 ± 32.0
		6°2′24″	77°4′48″	1035
		6°2′60″	77°3′36″	1260
	Sun	6°2′24″	77°2′24″	1009	24.2 ± 2.0	80.5 ± 7.0	161.3 ± 52.6
		6°2′24″	77°0′36″	1138
		6°4′48″	77°0′36″	1104

Adapted from Murga-Orrillo et al. [28,29].

2.2. Trees in Silvopastoral Areas

The silvopastoral areas of the estate were evaluated in 12 livestock systems (3 estates at 4 different altitudes). Each area had a minimum of 5 trees per hectare, with a canopy diameter exceeding 20 m² and a trunk diameter greater than 10 cm, providing partial shade to Brizantha. Additionally, the total tree height was measured using a clinometer (Suunto, PM-5/1520, Vantaa, Finland), following the methodology of Korning and Balslev [30]. The canopy area was calculated by multiplying the diagonals of its projection.

2.3. Soil Sampling and Analysis

Soil samples were collected from the 24 experimental plots, with approximately 0.5 kg per sample. A 2.5 cm diameter auger was used to extract samples to a depth of 20 cm (Riverside Brand, Arequipa, Peru). In the laboratory, samples were air-dried, ground, and sieved through a 2 mm mesh. Organic matter was determined using the Walkley and Black method [31], and available P was analyzed following the Olsen et al. method [32]. Exchangeable cations (Ca²⁺, Mg²⁺, K⁺, and Na⁺) were extracted with a 1 N potassium chloride (KCl) solution and quantified by emission spectrometry, based on Hunter [33]. Soil texture was assessed using the Bouyoucos hydrometer method [34]. Soil pH was determined using a potentiometric method with a 1:2.5 soil-to-water ratio. Electrical conductivity (EC) was measured using a conductivity meter with a 1:1.5 soil-to-water aqueous extract. CaCO₃ content was quantified using the volumetric gas evolution method.

2.4. Sample Collection and Biomass

The Brizantha plots were measured 5 m × 5 m (25 m²), with 12 plots exposed to full sunlight and 12 under partial tree shade. Each experimental plot underwent a uniformity cut at 5 cm above the soil surface. Samples were collected from a 1 m² area within each plot at 15, 30, 45, 60, and 75 days after uniformity cutting, resulting in a total of 120 samples (5 intervals × 24 plots). Sampling areas were marked using a PVC frame measuring 1 m per side. Fresh biomass was weighed in situ using a balance with 0.1 g precision (T-Scale S29B, Taippei, Taiwan). In addition, plant height, leaf area, and stem diameter were measured on 600 plants with 15 to 20 tillers each (5 plants × 24 plots × 5 evaluations). Leaf area was calculated by multiplying the width and length of leaves from the middle third of the tiller, applying a correction factor of 0.7468 for Brizantha, following the methodology described by Bianco et al. [35]. Stem diameter was measured using a digital caliper (Truper, Tlalnepantla de Baz, México). Samples were dried in Kraft paper bags at ambient temperature for two days, followed by drying in a hot-air oven (ODHG-9070A, Dongguan, China) at 60 °C for three days until a constant weight was achieved, yielding dry biomass. Leaves and stems were separated, and leaf samples were ground for subsequent chemical or bromatological analysis.

2.5. Chemical or Bromatological Analysis

Ground leaf samples were analyzed in the Animal Nutrition and Food Bromatology Laboratory (LABNUT/UNTRM). Ash content was determined gravimetrically by incineration in a muffle furnace [36]. Fat content was evaluated using high-temperature solvent extraction [37,38]. Protein content was measured via the Kjeldahl method [36]. Non-nitrogen extract content was calculated by the difference [36]. Fiber, neutral detergent fiber, and acid detergent fiber were assessed using ANKOM methodologies [35], and in vitro dry matter digestibility was measured using the DAISYII incubator [39]. Gross energy was determined with an adiabatic bomb calorimeter [36].

2.6. Stomatal Length and Density

To determine stomatal density and length under a full sun in monoculture areas and partial shade in silvopastoral areas, Brizantha samples were collected from the eight experimental plots equipped with weather stations (Table 1). From each plot, three plants were selected, each with five fully developed leaves. Stomatal samples were taken from the middle region (midsection) of the fourth leaf blade, on both the adaxial and abaxial surfaces. These samples were treated with a brilliant green solution for preparation. Stomatal images were obtained using an Optika microscope (OPTIKA Srl, Ponteranica, Italy) with 200× magnification and a computer image capture system (OPTIKA PROVIEW, version x64, 4.11.20805.20220506). Measurements included stomatal length in micrometers (μm) and density per square millimeter (mm²).

2.7. Data Analysis

Principal Component Analysis (PCA) was performed to identify discriminant factors related to altitude, full-sun and partial-shade management, and evaluation ages, for physiological variables and leaf chemical properties of Brizantha. In the PCA, discriminants that showed clustering were subsequently subjected to Tukey’s mean comparison tests (p < 0.05). Also, tree dasometry between altitudes, and soil properties between altitudes and management, were subjected to Tukey’s mean comparison tests (p < 0.05). The association between soil properties and chemical properties of Brizantha was evaluated by Pearson correlation analysis (p < 0.05). The correlation coefficient (r) was classified according to the following criteria: perfect (|r| = 1), very strong (0.8 ≤ |r| < 1), strong (0.6 ≤ |r| < 0.8), moderate (0.4 ≤ |r| < 0.6), weak (0.2 ≤ |r| < 0.4), and insignificant (|r| < 0.2). For stomata length and density, which did not present normal distribution of data, the Kruskal–Wallis test (p < 0.05) was applied to compare altitudes, and the Wilcoxon test (p < 0.05) was applied to compare management in full sun and partial shade. Statistical analyses were conducted using various R environment packages [40], including readr, FactoMineR, factoextra, ggplot2, tidyverse, agricolae, ggpubr, car, grid, patchwork, gridExtra, RColorBrewer, ExpDes.pt, ggthemes, corrplot, and seriation.

3. Results

3.1. Silvopastoral Tree Families

Throughout the altitudinal gradient, in the silvopastoral areas of the cattle farms, different tree families were identified according to altitude. At 170 m, 13 families were recorded, with 70% of the trees belonging to Simaroubaceae, Asteraceae, and Fabaceae. The remaining 30% consisted of Rubiaceae, Sapindaceae, Meliaceae, Rhamnaceae, Rutaceae, Euphorbiaceae, Bixaceae, Bignoniaceae, Myristicaceae, and Apocynaceae. At an altitude of 503 m, five families were identified, where 75% of the trees belonged to Malvaceae and Fabaceae, while the remaining 25% belonged to Rutaceae, Vochysiaceae, and Sapindaceae. At 661 m, 10 tree families were recorded: Anacardiaceae, Proteaceae, Rubiaceae, Fabaceae, Malvaceae, Vochysiaceae, Rutaceae, Euphorbiaceae, Annonaceae, and Sapotaceae, with no evidence of dominance among them. Similarly, at 1110 m, nine families were identified: Apocynaceae, Fabaceae, Urticaceae, Annonaceae, Meliaceae, Sapindaceae, Malvaceae, and Melastomataceae, also without dominance of any particular family. In Figure 1, the mean comparison tests of tree dendrometry at 170, 503, 661, and 1110 m in altitude did not show a clear altitudinal influence pattern. This is because the trunk diameter showed significant differences (p < 0.05), with the highest mean observed at 661 m compared to the other altitudes (Figure 1A). Similarly, the crown area showed significant differences (p < 0.05), with the highest mean at 661 m, followed by 1110 m, 503 m, and finally 170 m, respectively (Figure 1B). In turn, the tree height showed significant differences (p < 0.05), with higher means at 170, 661, and 1110 m when compared to 503 m (Figure 1C).

The presence of trees in silvopastoral areas at altitudes of 170, 503, 661, and 1110 m led to a reduction in temperature by 0.3 °C, 0.8 °C, 0.4 °C, and 1.1 °C, respectively, compared to monoculture areas (Table 1). Similarly, relative humidity increased by 3.6%, 3.3%, 2.2%, and 5.6%, while solar radiation decreased by 106.9 W/m², 37.2 W/m², 101.7 W/m², and 108.4 W/m², respectively. Regarding the altitudinal gradient, temperature decreased by 0.33 °C for every 100 m increase in altitude in silvopastoral areas, and by 0.27 °C in monoculture areas. However, neither relative humidity nor solar radiation exhibited a defined pattern in response to increasing altitudes (Table 1).

3.2. Soil Properties Along the Altitudinal Gradient

The soil properties (Table 2) showed significant differences (p < 0.05) across the altitudes of 170, 503, 661, and 1110 m. pH, electrical conductivity, CaCO₃ content, phosphorus, potassium, clay proportion, and cation exchange capacity had higher means at 503 and 661 m compared to the altitudes of 170 and 1110 m. Organic matter also showed significant differences (p < 0.05), with the highest mean at 661 m, intermediate values at 503 and 1110 m, and the lowest at 170 m. Regarding soil texture, sand content showed significant differences (p < 0.05), with the highest mean at 170 m, intermediate at 1110 m, and lower values at 503 and 661 m. Silt content also showed significant differences (p < 0.05), with the highest mean at 1110 m, intermediate at 503 and 661 m, and the lowest at 170 m. Furthermore, using the USDA texture triangle with the texture data from Table 2, the soil has a sandy loam texture at 170 m, clay at 503 m, clay loam at 661 m, and loam at 1110 m. When comparing the mean values of soil properties between full-sun and partial-shade areas, no significant differences were observed (p < 0.05).

3.3. PCA of Brizantha Variables

The PCA, which explained 75.2% of the total variability for the variables fresh and dry biomass, plant height, stem diameter, and leaf area of Brizantha, shows that the altitude of 503 m is separated along Dim2 (21.7%) from the altitudes of 170, 661, and 1110 m (Figure 2A). However, this PCA does not show separation between full-sun and partial-shade management (Figure 2B). Regarding the days after cutting, the PCA differentiates along Dim1 (53.5%), with plant height, leaf area, and fresh and dry biomass as the main contributing variables (Figure 2C). Similarly, the PCA explaining 63.4% of the total variability in the foliar chemical properties of Brizantha shows that the altitudes differ from each other along Dim2 (24.8%) (Figure 2D). Nevertheless, this PCA also does not show separation between full-sun and shade management (Figure 2E). On the other hand, the PCA does differentiate between days after cutting along Dim1 (38.6%), mainly for variables related to fiber, protein, acid detergent fiber, neutral detergent fiber, and in vitro dry matter digestibility (Figure 2F).

3.4. Growth Variables

A more detailed analysis of the physiological variables in relation to altitude and Brizantha growth stages is presented in Table 3. At the altitudes of 170, 503, 661, and 1110 m, significant differences (p < 0.05) were observed in fresh and dry biomass only on day 30 of growth, with the highest means at 170 m, followed by 503 and 661 m, and the lowest at 1110 m. Plant height at 15, 45, and 75 days after cutting showed significant differences (p < 0.05), with the highest mean at 503 m, followed by 170 and 661 m, and the lowest at 1110 m. Stem diameter showed significant differences (p < 0.05) at 60 days, with higher means at 170 and 503 m compared to 661 and 1110 m. Leaf area at 15 and 45 days also showed significant differences (p < 0.05), with the highest mean at 503 m and lower means at 170, 661, and 1110 m.

Regarding plant age (Table 3), fresh and dry biomass, as well as plant height, showed significant differences (p < 0.05), with the highest means at 75 days, followed by progressively lower means at 60, 45, 30, and 15 days. Leaf area also showed significant differences (p < 0.05), with the highest means recorded at 30 and 75 days, intermediate values at 45 and 60 days, and the lowest at 15 days. On the other hand, stem diameter did not show significant differences (p < 0.05) at any of the evaluated ages.

3.5. Foliar Chemical Properties

In the chemical properties of Brizantha (Table 4), ash content showed significant differences (p < 0.05), with higher means at 661 and 1110 m compared to 170 and 503 m across all evaluations. Regarding fat content, significant differences (p < 0.05) were observed only at 75 days, with the highest mean at 661 m, intermediate values at 503 and 1110 m, and the lowest at 170 m. Crude protein content showed significant variation (p < 0.05) at 45, 60, and 75 days: at 45 days, higher means were observed at 170, 503, and 1110 m compared to 661 m; at 60 days, higher means were found at 170 and 1110 m, followed by 503 m, with 661 m showing the lowest mean; and at 75 days, the highest mean was recorded at 1110 m, intermediate values at 170 and 503 m, and the lowest at 661 m. Neutral detergent fiber content showed significant differences (p < 0.05) only at 45 days, with the highest mean at 503 m compared to 170, 661, and 1110 m. Similarly, acid detergent fiber showed significant differences (p < 0.05) only at 15 days, with higher means at 170 m than at the other altitudes. In vitro dry matter digestibility showed significant differences (p < 0.05) at 15 and 45 days. At 15 days, the highest mean was observed at 503 m, followed by 661 and 1110 m, with the lowest mean at 170 m. At 45 days, the highest mean was found at 170 m, followed by 1110 m, while 503 and 661 m had lower means. Energy content showed significant differences at 15 and 30 days, with the highest mean at 1110 m compared to 170, 503, and 661 m.

Considering the age of Brizantha in relation to its chemical properties (Table 4), ash content showed significant differences (p < 0.05), with higher means at 15 days, intermediate values at 30 and 45 days, and lower means at 60 and 75 days. Regarding protein content, significant differences (p < 0.05) were observed, with higher means at 15 and 30 days, intermediate at 45 and 60 days, and lower at 75 days. Fiber content also showed significant differences (p < 0.05), with higher means at 60 and 75 days, and lower means at 15, 30, and 45 days. For neutral detergent fiber, significant differences (p < 0.05) were recorded, with the highest mean at 75 days, intermediate values at 60, 45, and 30 days, and the lowest at 15 days. Similarly, acid detergent fiber showed significant differences (p < 0.05), with the highest mean at 75 days, intermediate at 60 days, and lower means at 45, 30, and 15 days. In contrast, in vitro dry matter digestibility showed significant differences (p < 0.05) at all evaluated ages, with the highest mean at 15 days, followed by progressively lower means at 30, 45, 60, and 75 days. Lastly, fat, non-nitrogen extract, and energy contents did not show significant differences (p < 0.05) at any of the evaluated ages.

3.6. Association Between Foliar Chemical Properties and Soil

Soil properties and pasture chemical properties were correlated (Figure 3). Ash content showed a moderate positive correlation (p < 0.05) with soil pH, electrical conductivity, organic matter, potassium, clay, and cation exchange capacity, but a moderate negative correlation (p < 0.05) with sand. On the other hand, protein content exhibited a strong negative correlation (p < 0.05) with pH, electrical conductivity, clay, and cation exchange capacity, but a moderate negative correlation (p < 0.05) with potassium. Fiber showed a moderate positive correlation with potassium (p < 0.05). Non-nitrogen extract presented a moderate negative correlation (p < 0.05) with pH, electrical conductivity, and cation exchange capacity. Meanwhile, neutral detergent fiber showed a moderate negative correlation (p < 0.05) with organic matter. Energy exhibited a strong negative correlation (p < 0.05) with pH and a moderate negative correlation (p < 0.05) with electrical conductivity, clay, and cation exchange capacity.

3.7. Morphometry of Brizantha Stomata

In the stomata (Figure 4), stomatal length was significantly greater (p < 0.00023) at 170 m compared to 1110 m; similarly, at 661 m, it was significantly greater (p < 0.00023) compared to 503 and 1110 m (Figure 4A). The number of stomata per mm² showed significantly higher medians (p < 0.014) at 170 and 503 m compared to 661 m (Figure 4B). On the other hand, both stomatal length and number showed significant differences (p < 0.0044), with higher medians in sun-exposed plants compared to those under partial shade (Figure 4C,D), where the presence of trees reduced temperature and solar radiation, and increased relative humidity (Table 1). According to their location on the leaf, stomatal length showed significant differences (p < 0.000065), with higher medians on the abaxial surface than on the adaxial surface (Figure 4E); however, stomatal number did not show significant differences based on leaf surface location (Figure 4F).

4. Discussion

4.1. Tree Family in Altitudinal Gradient

The distribution of tree families and their dendrometric characteristics (Figure 1) was not influenced by the altitudinal gradient or soil fertility (Table 2), but rather by anthropogenic factors. During the land-use change from natural forest to silvopastoral areas, the trees that were retained and/or integrated were generally found along the borders of the pastures and served multiple purposes, including shade, live fences, fruit production, timber, or soil conservation. Other studies have reported that tree families such as Fabaceae, Melastomataceae, and Rubiaceae are found across all altitudinal levels of tropical forests [41,42]. However, in this study, only the Fabaceae family was distributed across all altitudes within the evaluated silvopastoral gradient, likely favored for its role in soil conservation, biological nitrogen fixation, and/or pod fruit production. The integration of Fabaceae trees into agroforestry systems can rehabilitate degraded soils, enhance biodiversity, and support wildlife habitats [43]. Fabaceae trees are economically important as they provide protein-rich food while offering multiple ecosystem services [44]. These Fabaceae and other multipurpose tree families can improve the livelihoods of smallholder farmers in the Peruvian Amazon while maintaining ecosystem services in this increasingly vulnerable region. Anthropogenic influence was also evident in tree dendrometry, as significant differences (p < 0.05) with higher mean trunk diameters were observed at 661 m altitude (Figure 1A). Although previous studies have associated larger trunk diameters with higher soil CaCO₃ content [17,45], in this study, CaCO₃ levels in the soil were significantly higher (p < 0.05) at 503 and 661 m altitudes (Table 2).

4.2. Soil Properties

The altitudinal gradient did not influence soil properties, as CaCO₃ was present only at mid-altitudes (503 and 661 m), where the soils showed significant differences (p < 0.05), with higher mean values of pH, electrical conductivity, phosphorus, potassium, cation exchange capacity, organic matter, and clay content compared to soils at lower and higher altitudes (170 and 1110 m) (Table 2). Moreover, these properties were positively correlated with CaCO₃ (p < 0.05, 0.4 ≤ |r| < 0.8), suggesting that CaCO₃ had a favorable effect on soil characteristics (Figure 3). Other studies support the benefits of CaCO₃ in soil, stating that it plays a key role in clay distribution and influences electrical conductivity and cation exchange capacity [46]. Additionally, CaCO₃ increases soil pH, extractable calcium, and organic carbon [47].

4.3. Physiological Variables and Chemical Properties of Brizantha

The physiological variables and chemical properties of Brizantha did not show differentiation between full-sun and partial-shade management in monoculture and silvopastoral areas, respectively, according to the PCA (Figure 2B,E). This indicates the hardiness of this forage grass to microclimatic variations caused by tree canopy shading in silvopastoral areas, where temperature decreased by 1.1% to 4.5%, relative humidity increased by 2.8% to 7.0%, and solar radiation was reduced by 25.5% to 74.0% across the altitudinal gradient from 170 m to 1110 m (Table 1). Other studies have found that moderate partial shade does not significantly reduce biomass production compared to full-sun exposure [48,49].

Physiological variables of Brizantha at 503 m altitude were distinguishable from those at 170, 661, and 1110 m in the PCA (Figure 2A). Similarly, the Tukey test showed significant differences (p < 0.05) at 503 m, with higher means in plant height at 15, 45, and 75 days, stem diameter at 60 days, and leaf area at 15 and 45 days (Table 3). These differences could be attributed to soil fertility, as the 503 m altitude showed significantly higher mean values (p < 0.05) of CaCO₃, potassium, and phosphorus compared to the other altitudes (Table 2). Apollon et al. [50] found that CaCO₃ application increases the height of Brizantha, while potassium and phosphorus interact to improve leaf area and dry matter of the shoots [51].

Foliar ash contains calcium, phosphorus, potassium, and micronutrients [52]. In the chemical properties of Brizantha, ash content increased with altitude, showing significant differences (p < 0.05), with lower means at lower altitudes (170 and 503 m) and higher means at higher altitudes (661 and 1110 m) (Table 4). These results suggest that Brizantha is more efficient at absorbing calcium, phosphorus, and potassium as altitude increases, independent of soil fertility, since soils had higher availability of CaCO₃, phosphorus, and potassium at mid-altitudes (503 and 661 m) (Table 2). Altitude and harvest timing significantly affect the nutritional value and productivity of Brizantha [53,54].

With increasing cutting age of Brizantha, ash content, crude protein, and IVDMD significantly decrease (p < 0.05), while fiber, NDF, and ADF increase (Table 4). These findings suggest that harvesting Brizantha at intervals between 30 and 45 days yields higher nutrient content (ash), higher protein levels, and greater digestibility. After 45 days, fiber content increases, reducing digestibility. Other studies recommend cutting intervals between 28 and 42 days for optimal regrowth and productivity [55]. Longer cutting intervals tend to increase dry matter content and fiber fractions while reducing crude protein levels [56]. Quintino et al. [57] observed a decline in vitro dry matter digestibility with advancing plant age, while Costa et al. [56] reported that longer intervals between harvests boost dry matter production but lower protein levels.

4.4. Stomatal Morphometry

Stomatal length and number showed significant differences (p < 0.05), but no clear pattern was observed in relation to the altitudinal gradient (Figure 4A,B). However, both traits exhibited significant differences (p < 0.05), with higher medians in full-sun areas compared to shaded areas (Figure 4C,D). In other words, the reduction in solar radiation-ranging from 25.5% (108.8 ± 47.7 W/m²) to 74.0% (37.5 ± 25.8 W/m²) due to tree shading-affected stomatal length and number compared to full-sun areas, where solar radiation ranged from 144.4 ± 56.3 W/m² to 163.6 ± 57.8 W/m² (Table 1). Shading generally reduces stomatal density in most plant species [58]. However, stomatal size in other species often increases under shaded conditions [27].

5. Conclusions

The conservation and integration of trees in silvopastoral systems reflected a clear anthropogenic influence, evidenced by the preference for species of the Fabaceae family, likely due to their multipurpose nature. This selection highlights not only deliberate management decisions but also contributes to the functional biodiversity of silvopastoral systems.

Although the altitudinal gradient did not show direct effects on soil properties, intermediate altitudes (503 and 661 m) revealed a significant role of CaCO₃ in enhancing soil fertility, with significantly higher values (p < 0.05) of pH, EC, P, K, and CEC. These edaphic conditions at mid-altitudes favored the leaf area development of Brizantha, particularly during the early growth stages, as indicated by significantly larger values (p < 0.05). However, at the harvest stage, no significant differences were observed in physiological or productive traits, nor in foliar chemical components, underscoring the species’ high hardiness and broad adaptation to both soil and altitude conditions.

In Brizantha, a significant reduction (p < 0.05) in stomatal size and density was observed under shade in silvopastoral areas, where solar radiation and air temperature decreased while relative humidity increased. Nonetheless, these microclimatic variations did not lead to significant changes in foliar chemistry, growth variables, or biomass production, suggesting a high degree of adaptive plasticity to microclimatic fluctuations.

Foliar ash content exhibited an increasing trend with altitude, indicating greater efficiency of Brizantha in absorbing Ca, P, and K at higher altitudes, possibly linked to more favorable edaphoclimatic conditions for nutrient uptake.

Finally, forage quality declined with plant age, as evidenced by reductions in protein, ash, and In Vitro Dry Matter Digestibility (IVDMD), alongside increases in fiber, Neutral Detergent Fiber (NDF), and Acid Detergent Fiber (ADF). These findings support the recommendation of cutting intervals between 30 and 45 days, during which Brizantha displays a more favorable nutritional profile, higher digestibility, and consequently, greater value for animal feeding.

Author Contributions

H.M.-O.—data curation, formal analysis, funding acquisition, investigation, methodology, project administration, validation, visualization; writing—original draft; writing—review and editing. L.A.A.L.—conceptualization, data curation, investigation, methodology. M.A.M.-F.—data curation, formal analysis, funding acquisition, investigation, validation. J.C.C.—data curation, investigation, resources, supervision, visualization. J.S.-R.—investigation, methodology, software. A.C.A.-C.—data curation, formal analysis, software. C.I.P.S.—investigation, methodology, software. M.R.G.—data curation, investigation, supervision. A.A.G.-T.—conceptualization, data curation, methodology. N.L.M.V.—data curation, investigation, validation. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

Data is contained within the article. The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author(s).

Acknowledgments

We would like to thank the livestock farmers from Yurimaguas (Loreto region), Cuñumbuque, Zapatero, and Calzada (San Martín region) for making their cattle available for this research. We also thank the field technicians and the technical and financial managers of the project.

Conflicts of Interest

The authors declare no conflicts of interest.

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Figures and Tables

Figure 1 Trunk diameter (A), crown areas (B) and plant height (C) of trees from silvopastoral areas at 170, 503, 661 and 1110 m altitude. The same lowercase letters above the means do not show significant differences between them, by Tukey’s test (5%).

Figure 2 Principal Component Analysis (PCA) for Brizantha variables: Fresh weight (FW), dry weight (DW), plant height (PH), stem diameter (SD), and leaf area (LA) evaluated across (A) altitudes (170, 503, 661, and 1110 m), (B) Environmental management (full-sun vs. partial-shade), (C) Days after cutting (15, 30, 45, 60, and 75 days). PCA for chemical properties of Brizantha leaves: Ash, protein, Non-Nitrogen Extract (NNE), Neutral Detergent Fiber (NDF), Acid Detergent Fiber (ADF), In Vitro Dry Matter Digestibility (IVDMD), and Energy evaluated across (D) altitudes, (E) Environmental management, (F) Days after cutting.

Figure 3 Pearson correlation matrix (p < 0.05) between physical and chemical properties of the soil, and chemical properties of Brizantha. EC—electrical conductivity; CaCO₃—calcium carbonate; OM—organic matter; P—phosphorus; K—potassium; CEC—cation exchange capacity; NNE—Non-Nitrogen Extract; NDF—Neutral Detergent Fiber; ADF—Acid Detergent Fiber; IVDMD—In Vitro Dry Matter Digestibility.

Figure 4 Length and number of stomata by altitudinal origin (A,B), by environmental condition of full sun and partial shade (C,D) and location on the leaf (E,F). ** Significant difference (p < 0.05), *** very significant difference (p < 0.01), **** highly significant difference (p < 0.0001).

Table 2

Physical and chemical soil properties at altitudes of 170, 503, 661, and 1110 m.

mals	pH	EC	CaCO3	OM	P	K	Sand	Silt	Clay	CEC
		dS/m	%							cmolc/kg
170	4.2 b	0.1 b	0.0 b	2.7 b	2.3 bc	62.0 c	56.0 a	24.0 b	20.0 b	9.2 b
503	7.4 a	0.8 a	2.3 a	5.1 ab	4.2 a	385.8 a	29.3 b	25.3 ab	45.3 a	35.8 a
661	7.1 a	0.7 a	1.6 ab	6.0 a	3.8 ab	342.0 ab	35.3 b	25.7 ab	39.0 a	31.3 a
1110	4.4 b	0.1 b	0.0 b	3.8 ab	2.2 c	138.0 bc	40.3 ab	35.3 a	24.3 b	12.3 b
DMS	0.4	0.2	2.3	2.5	1.6	221.3	17.0	11.3	10.2	8.6
Enviroment
Shade	5.9 a	0.4 a	1.4 a	5.1 a	3.1 a	280.8 a	36.2 a	30.2 a	33.7 a	22.8 a
Sun	5.6 a	0.4 a	0.6 a	3.7 a	3.1 a	183.1 a	44.3 a	25.0 a	30.7 a	21.4 a
DMS	1.3	0.3	1.4	1.5	1.1	157.2	11.6	6.5	10.4	11.0

MSD—Minimum Significant Difference; EC—electrical conductivity; CaCO₃—calcium carbonate content; OM—Organic matter; P—phosphorus; K—potassium; CEC—cation exchange capacity. Means followed by the same letter in the columns did not differ statistically from each other by Tukey’s mean comparison test (p < 0.05). Adapted from Murga-Orrillo et al. [29].

Table 3

Fresh weight (FW) and dry weight (DW), plant height (PH), stem diameter (SD), and leaf area (LA) of Brizantha at 15, 30, 45, 60, and 75 days after cutting at 170, 503, 661, and 1110 m altitude.

Day	m	FW	DW	PH	SD	LA
		kg/ha		cm		cm2
15 (n = 120)	170	3783.3 a	1755.5 a	30.1 ab	0.27 a	5.9 c
	503	5158.3 a	494.8 a	35.8 a	0.39 a	30.8 a
	661	2898.3 a	906.8 a	24.5 b	0.40 a	18.6 b
	1110	4491.7 a	1325.9 a	25.9 ab	0.33 a	5.7 c
	MSD	3628.3	924.9	11.0	0.2	7.7
30 (n = 120)	170	7366.7 a	2785.0 a	36.6 a	0.43 a	17.2 a
	503	5225.0 ab	1838.6 ab	38.3 a	0.35 a	32.0 a
	661	5916.7 ab	173.9 ab	42.5 a	0.29 a	33.7 a
	1110	4108.3 b	1353.4 b	31.8 a	0.34 a	34.8 a
	MSD	2701.4	1097.7	15.0	0.2	22.4
45 (n = 120)	170	8033.3 a	2912.0 a	41.0 ab	0.25 a	17.8 ab
	503	7808.3 a	2581.9 a	55.2 a	0.40 a	32.3 a
	661	7073.3 a	1982.9 a	39.7 ab	0.24 a	11.0 b
	1110	7666.7 a	2453.6 a	35.5 b	0.33 a	28.4 ab
	MSD	6135.4	1860.4	19.2	0.2	17.8
60 (n = 120)	170	11,183.3 a	3667.6 a	52.4 a	0.36 a	22.4 a
	503	11,925.0 a	4058.6 a	61.8 a	0.35 a	31.9 a
	661	10,000.0 a	3423.8 a	46.0 a	0.17 b	19.1 a
	1110	10,491.7 a	3403.4 a	46.8 a	0.31 ab	18.9 a
	MSD	7508.8	2466.2	24.9	0.2	18.8
75 (n = 120)	170	14,566.7 a	5061.8 a	71.0 ab	0.32 a	39.3 a
	503	14,425.0 a	4715.8 a	72.9 a	0.33 a	37.2 a
	661	12,683.3 a	4331.8 a	60.8 ab	0.25 a	38.7 a
	1110	13,100.0 a	4722.4 a	53.8 b	0.31 a	11.4 a
	MSD	7581.4	2732.2	18.9	0.1	46.4
Gradient (n = 600)	Day
	15	4082.9 d	1370.7 d	29.1 d	0.35 a	15.2 b
	30	5654.2 cd	2037.7 cd	37.3 cd	0.35 a	29.4 a
	45	7645.4 c	2482.6 c	42.8 bc	0.31 a	22.4 ab
	60	10,900.0 b	3638.3 b	51.8 b	0.30 a	23.1 ab
	75	13,693.8 a	4707.9 a	64.6 a	0.30 a	31.7 a
	MSD	2790.7	946.0	9.9	0.1	14.0

MSD—Minimum Significant Difference. Means followed by the same letter in the columns did not differ statistically from each other by Tukey’s mean comparison test (p < 0.05).

Table 4

Chemical properties of Brizantha evaluated at 15, 30, 45, 60, 75 days after cutting, at 170, 503, 661 and 1110 m altitude.

Day	masl	Ash	Fat	Protein	Fiber	NNE	NDF	ADF	IVDMD	Energy
		%								kcal/kg
15 (n = 24)	170	8.3 b	1.7 a	8.3 a	28.2 a	43.5 a	68.0 a	39.4 a	43.9 b	3635.5 ab
	503	10.9 a	1.3 a	6.5 a	28.7 a	42.1 a	66.3 a	36.1 b	48.9 a	3458.2 b
	661	12.3 a	1.7 a	7.4 a	26.7 a	42.7 a	65.5 a	35.4 b	47.9 ab	3480.3 b
	1110	11.5 a	1.8 a	7.6 a	29.3 a	47.4 a	66.5 a	36.3 b	46.0 ab	3813.8 a
	MSD	2.5	0.9	2.1	3.1	7.9 a	3.5	3.0	4.8	278.1
30 (n = 24)	170	8.4 b	1.6 a	7.5 a	28.5 a	43.9 a	68.3 a	37.3 a	43.9 a	3614.1 ab
	503	9.4 ab	1.7 a	6.7 a	29.0 a	44.6 a	68.5 a	35.9 a	46.3 a	3653.4 ab
	661	11.4 a	1.3 a	6.8 a	27.1 a	43.7 a	67.0 a	35.0 a	45.5 a	3507.9 b
	1110	11.2 a	1.9 a	8.1 a	28.4 a	47.9 a	68.5 a	36.8 a	45.9 a	3837.2 a
	MSD	2.1	0.8	2.3	3.1	7.1	3.9	3.4	5.9	313.8
45 (n = 24)	170	7.9 b	2.0 a	8.3 a	27.6 b	43.9 a	68.7 ab	36.4 a	46.5 a	3654.2 a
	503	9.5 ab	1.4 a	6.6 a	29.6 ab	43.8 a	70.7 a	36.2 a	42.8 bc	3582.4 a
	661	9.8 ab	1.8 a	4.1 b	31.5 a	44.2 a	67.2 b	37.8 a	40.8 c	3580.3 a
	1110	10.4 a	1.9 a	8.1 a	27.1 b	48.5 a	66.9 b	36.1 a	45.4 ab	3804.1 a
	MSD	2.3	1.0	2.3	2.5	7.3	3.3	2.6	3.4	257.0
60 (n = 24)	170	7.5 b	1.7 a	7.9 a	30.3 a	43.3 a	69.0 a	37.6 a	43.5 a	3691.4 a
	503	8.8 b	1.4 a	6.1 b	29.4 a	43.9 a	69.9 a	38.4 a	40.7 a	3650.6 a
	661	10.4 a	2.0 a	4.1 c	32.7 a	42.2 a	67.5 a	37.9 a	41.0 a	3565.8 a
	1110	8.9 ab	2.1 a	8.8 a	30.8 a	45.3 a	67.4 a	36.2 a	44.3 a	3910.6 a
	MSD	1.5	0.8	1.5	3.6	6.5	4.0	3.7	3.7	364.2
75 (n = 24)	170	7.2 b	1.4 b	6.3 ab	31.5 a	45.8 a	71.1 a	40.0 a	39.7 a	3733.3 a
	503	8.2 b	1.9 ab	5.8 ab	32.3 a	41.3 a	68.4 a	37.7 a	41.78 a	3591.4 a
	661	10.5 a	2.2 a	4.4 b	32.1 a	42.3 a	66.6 a	38.0 a	40.9 a	3579.4 a
	1110	10.2 a	1.6 ab	6.5 a	32.0 a	48.3 a	71.1 a	38.6 a	40.7 a	3892.0 a
	MSD	1.8	0.6	2.0	3.1	7.4	5.6	3.0	3.3	335.3
Gradient (n = 120)	Day
	15	10.7 a	1.6 a	7.4 a	28.2 b	43.9 a	66.6 b	36.8 b	46.7 a	3596.9 a
	30	10.1 ab	1.6 a	7.2 a	28.5 b	44.8 a	67.9 ab	36.4 b	45.3 ab	3654.2 a
	45	9.4 ab	1.8 a	6.8 ab	28.7 b	45.3 a	68.5 ab	36.5 b	43.9 bc	3654.2 a
	60	8.9 b	1.8 a	6.7 ab	30.8 a	43.7 a	68.4 ab	37.5 ab	42.3 cd	3704.6 a
	75	9.0 b	1.8 a	5.8 b	31.9 a	44.4 a	69.3 a	38.6 a	40.8 d	3699.0 a
	MSD	1.4	0.4	1.3	1.7	3.7	2.1	1.6	2.3	175.4

MSD—Minimum Significant Difference. NNE—Non-Nitrogen Extract; NDF—Neutral Detergent Fiber; ADF—Acid Detergent Fiber; IVDMD—In Vitro Dry Matter Digestibility. Means followed by the same letter in the columns did not differ statistically from each other by Tukey’s mean comparison test (p < 0.05).

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