1. Introduction
The importance of animal ownership for indigenous communities throughout the inter-Andean region has been well- documented (Instituto Nacional de Estadísticas y Censos [INEC], 2022; Simbaña Pulupa y Tayupanta Escobar, 2014). Cattle and sheep farming is essential for the economy and family labor force, as well as for communities 'worldviews’ (Mugnier et al., 2021). Animals have frequently been observed to demonstrate a greater degree of resilience in comparison to crops, thus serving as a sustainable solution to economic challenges, in addition to their role in providing manure, animal traction, and animal protein for the daily diet of peasant families (Palate Moreta, 2022; Pulido, 2018).
Nevertheless, the transition from traditional systems of land use known as "chacra" as a food provider for local families, to pastures for cattle and sheep grazing, has been identified as a recent socio-environmental phenomenon. The decline in agricultural productivity, caused by the erosion and degradation of hillsides, along with the limited availability of cultivable land due to a constant process of land division, have disrupted the balance of productive practices; consequently, families have been compelled to adopt livestock husbandry, cows and sheep specifically, as a means of subsistence (Grijalva et al., 2013; Yánez-Yánez et al., 2017).
Environmental problems have been observed in indigenous communities along the extensive Andean region which have been attributed to intensive agriculture on hillside terrains, the indiscriminate use of agrochemicals that contribute to soil and water contamination, and soil loss. In addition, traditional livestock management practices affect natural vegetation, contributing to soil erosion, factors that ultimately lead to low productivity and increased rural poverty (Bustamante, 2017; Espinosa et al., 2022). The “paramo” ecosystem's grasslands are frequently used for sheep grazing in a continuous grazing system of native or naturalized plant species with low productivity and nutritional value, which negatively impacts animal performance. Statistics show that sheep mortality can exceed 30%, primarily due to infectious diseases and fetal death resulting from metabolic disorders, as well as the consequences of inadequate grass and grazing management (Grijalva et al., 2016; León et al., 2018; Pulido, 2018).
It has been demonstrated by several authors that pastures can exhibit deficiencies and imbalances in one or more minerals (Palate Moreta, 2022; Suttle, 2016). The deficit and/or excess of minerals in soils of volcanic origin has been identified as a direct cause of low production and reproductive performance of ruminants under grazing (Grijalva et al., 2016). The solution to this problem appears to lie in a transition from traditional forages to the utilization of foreign pastures, a strategy that has been demonstrated to ensure high pasture persistence and generate greater economic returns for Indigenous families within the study area.
Considering the above, the purpose of this study is to evaluate two distinct scenarios for the intensification of grasslands based on perennial ryegrass (Lolium perenne) and white clover (Trifolium repens). These scenarios will be implemented with rest intervals of 45 and 60 days in locations situated between 3.000-3.400 m a.s.l. used for grazing sheep and cows.
2. Materials and Methods
The research was conducted in the Chimborazo River micro-watershed, situated in the San Juan parish of the Riobamba canton, Chimborazo province, with geographical coordinates of 9825450 N and 746718 E. The mean temperature is 14.2 °C with a maximum of 21.3 °C and a minimum of 8.3 °C (Instituto Nacional de Meteorología e Hidrología [INAMHI], 2016).
For the soil component, soil samples were collected at altitudes of 3.500-3.700 and 3.000-3.400 m a.s.l in the micro-watershed, at depths of 0-10 and 11-20 cm, to evaluate bulk density (Da, g cm-3) at the beginning of January 2016 and December 2023 using the known volume cylinder method with the equation [1].
Soil samples were obtained to determine pH levels using a potentiometer in a 1:2.5 ratio of aqueous solution. Total nitrogen was determined by calculating the organic matter obtained through the wet combustion method. Assimilable phosphorus was determined by colorimetry using the modified OLSEN method, while potassium [K], calcium [Ca], and magnesium [Mg] were determined by the Atomic Absorption Spectrophotometry [AAS] technique.
For the pasture component, three plots for sheep and cow grazing were selected in six communities of the micro-watershed, which were identified as intensification scenarios: a) a natural grassland scenario composed of high Andean grasses such as Stipa ichu, Holcus lanatus, Rumex acetocella, and Paspalum sp.; here, grazing was conducted at intervals of 60-75 days; b) the mixed pasture scenario composed of perennial ryegrass (Lolium perenne) and white clover (Trifolium repens) which was grazed at intervals of 45 days; and c) mixed pasture scenario similar to scenario b, but grazed at 60-day intervals.
In each intensification scenario, "semi-permanent exclusion cages" were installed, where grass samples were taken to evaluate the effect of each scenario on chemical composition and nutritional value. The proximate components were determined in the laboratory using the official methods proposed by (Latimer, 2019). The minerals present in pastures were analyzed using the Atomic absorption spectrometry [AAS] method, and the colorimetry method was used for phosphorus. The results obtained were expressed as a percentage or ppm (dry basis).
The equation [2], proposed by Cañas (1998), was used to estimate Total Digestible Nutrients [TDN] in percentages, based on the proximate values mentioned above:
Where:
Y = Total Digestible Nutrients.
CF = Crude fiber.
EE = Ether extract.
NFE =Nitrogen free extract.
CP = Crude protein.
To calculate energy partition, the equations [3], [4] and [5], described by the National Research Council (2001), were used:
Where:
DE = Digestibility energy (Mcal kg-1).
TDN = Total digestibility of nutrients (%).
ME = Metabolizable energy (Mcal kg-1).
NEL = Net energy lactation (Mcal kg-1).
The stocking rate and carrying capacity were calculated based on the animal inventory, grazing area, and dry biomass yield (per ha-1year-1). These variables were expressed in Animal Units [AU], with a reference value of 1.0 AU for a "dry" cow weighing 400 kg (Bonilla Cárdenas & Lemus Flores, 2012). Daily measurements were taken of milk production for lactating crossbred cows in each grazing scenario, starting from the calving date and continuing for nine months of lactation.
To calculate emissions (equations [6] and [7]), the production of CH4 per unit of animal product was considered as an appropriate index for comparing Greenhouse Gas [GHG] emissions from livestock under different feeding conditions (Bonilla Cárdenas & Lemus Flores, 2012). Consequently, the conversion of CH4 to a percentage of gross energy intake [GEI] was done following recommendations provided by The Intergovernmental Panel on Climate Change [IPCC] (2006), which suggest the utilization of a gross energy value for feed equivalent to 18.45 MJ kg-1 of dry matter, and an energy value for CH4 of 55.65 MJ kg-1 of dry matter.
Where:
CH4 = Methane emissions (g kg -1 of dry matter).
DMI = Dry Matter Intake (g day-1).
GEI = Gross Energy Intake (MJ day-1).
Blood samples were obtained by puncturing the jugular vein of sheep in a "fasting catabolism" condition, for which 5.0 ml of blood per animal was collected. The enzymatic activity of aspartate aminotransferase [AST] and alanine aminotransferase [ALT], collectively known as GOT (glutamic-oxaloacetic transaminase), and alkaline phosphatase [AP] was measured using the method of the International Federation of Clinical Chemistry [IFCC]. For the ALT enzyme, the UV kinetic method was used, which is based on the kinetic determination of ALT activity, expressed in international units [IU] per liter.
The research data were analyzed using the analysis of variance [ANOVA] method, with Tukey's test at the 5% level used to compare the means of the various treatments. This analysis was conducted using Infostat software version 8 (Di Rienzo et al., 2020).
3. Results
Samples from grazing sites at lower altitudes (3.000 and 3.400 m a.s.l.) showed significantly higher values for most physicochemical properties of soil (Table 1), suggesting that soils in these regions might be more conducive to livestock farming. However, the analysis of soil bulk density [BD] did not reflect significant differences. Regarding soil minerals, statistical differences were reflected between the sites, with levels of Ca, P, and Mg being significantly higher at the lowest altitudinal level.

Table 1 Physicochemical properties of soils used for raising bovine and ovine livestock in high Andean micro-watersheds.**
The variables DM, CP, TDN, DE, ME, and NEL differ significantly within the pasture use scenarios and between these options and the natural grassland scenario (Table 2). The response in CP is inversely correlated with NDF and ADF (p < 0.0001). These results can be explained by the fact that the latter form of use is normally grazed at bi-monthly or even quarterly frequencies by the surrounding communities, which would result in a greater accumulation of dry matter. Statistical differences in Ca and P were observed between pastures and natural grassland, as well as between scenarios with pastures and different grazing intervals. The higher level of intensification or shorter grazing interval explains a higher mineral content in pastures.

Table 2 Chemical composition and nutritional value of pastures in various scenarios of pasture intensification in high Andean micro-watersheds.**
As demonstrated in Table 3, high Andean ecosystems can only support a maximum sustainable animal load of 0.1 AU ha-1 year-1, which could range between 0.05-1.0 AU ha-1 year-1 due to their fragile environmental conditions associated with their biophysical characteristics, particularly low temperatures, periods of frost and seasonal rains, which hinders the intensification of natural pasture management. The analysis of CH4 from carbohydrate fermentation suggests an increase in emissions as structural components, specifically FDN and FDA, increase.

Table 3 Animal stocking rate and carrying capacity, milk production, and estimated CH4 emissions in three scenarios of grazing intensification in high Andean micro-watersheds.
The results of the blood analysis in sheep grazing on high Andean micro-watersheds meadows and pastures are shown in Table 4. The mean mineral levels in their blood are within a normal range. However, a high degree of variation was observed in the ALT and AF enzymes, relative to established reference ranges. On the other hand, the AST enzyme demonstrated a profile that falls within the considered normal range.

Table 4 Mean levels of calcium, phosphorus, potassium, magnesium, and activity of various enzymes in the blood serum of sheep grazing on natural grasslands and mixed pastures in high Andean micro-watersheds.*
4. Discussion
The findings of this research, under the high grazing scenario, demonstrate a prevailing tendency toward soil acidification, a phenomenon consistent with observations reported in the literature by several researchers. An acidic soil is characterized by high concentrations of Al+3 and H+ ions as the pH decreases, which impacts the chemical, physical, and biological characteristics of the soil, reducing plant growth and nutrient availability such as Ca, Mg, P, K, and other micro- elements (Espinosa et al., 2022).
Several authors have reported that soils of volcanic origin tend to retain large amounts of water and organic matter, (Espinosa et al., 2022; Yánez-Yánez et al., 2017), which coupled with the slow decomposition and mineralization of organic matter, could potentially limit the availability of nutrients to plants.
In lower areas that are actively used for livestock activity, the total nitrogen content of the soil is higher due, in part to, the contribution of nutrients from animal excreta. Soils with levels below 0.10-0.25% of N are classified as deficient. However, it is important to note that total N is not always a useful measure in soils of volcanic origin, since this component is very dynamic and is normally associated with the mineralization of organic matter, losses due to nitrification and denitrification, leaching, immobilization, and volatilization (Espinosa et al., 2022; Maresma Galindo, 2020).
On the other hand, the status of P indicates a more favorable situation in low-elevation sites, suggesting a significant contribution of animal excreta from grazing in these pastures. Levels lower than 10 ppm of P are often considered deficient, and this is precisely what occurs in the highest and most delicate sites associated with the “paramo” ecosystem. However, the absorption of this element by grass root systems may be limited by pH and allophane clay (Espinosa et al., 2022). Except for K, which demonstrates deficiency at the highest altitude, the other two elements (Ca and Mg) exhibit significantly higher values at the lower sites. Conversely, erosive processes contribute to the leaching of nutrients in the study areas located at higher altitudes above sea level.
The analysis of soil BD does not reveal significant differences. In this regard, Chinchilla et al. (2011) states that the density of andosol soils is generally less than 0.90 g cm-3. These results are consistent with the findings of González Ponce (2009), who reported variations from 0.77 to 0.95 g cm-3 over a seven-year grazing period. It is important to note that all values reported by these authors are lower than the reference value of 1.3 g cm-3, which is associated with soil compaction problems.
Several authors agree that the content and yield of DM in pastures generally increase with an increase in the frequency of rest or grazing. However, there is a simultaneous decrease in the nutritional value and animal consumption due to an increase in plant maturity (Merlo-Maydana et al., 2017; León et al., 2018; Pulido, 2018).
Our results demonstrate a negative correlation between crude protein [CP] and both NDF and ADF, indicating that an increase in NDF with advancing maturity increases is typically related to a decline in CP content, as suggested by several authors (Apráez et al., 2014; Posada Ochoa et al., 2013; León et al., 2018). In this regard, García-Bonilla et al. (2014) state that FDA is related to digestibility, with lower FDA values indicating higher Digestibility. Lignin did not show differences between alternatives. These results differ from other studies that reported lignin values of 18.2% in natural grasslands (Albarracín et al., 2015).
In relation to Ca and P, the higher level of intensification or shorter grazing interval caused a higher content of macro-mineral elements. In comparison to alternative intensification strategies, natural grassland exhibited significantly lower values, indicating a substantially restrictive environment concerning the provision of nutrients necessary for optimal animal productivity (Grijalva et al., 2016; Suttle, 2016; López-Vigoa et al., 2019; Palate Moreta, 2022).
The findings of this research indicate a correlation between elevated methane emissions and a high concentration of crude fiber, characterized by low protein content and digestibility, particularly in the high scenario. This observation aligns with the conclusions reported by several authors, suggesting consistency in the scientific community's findings indicating that GHG emissions or ruminal methane production constitute an energy loss that significantly contributes to GHG emissions into the environment. The magnitude of this phenomenon appears to be influenced by the consumption, chemical composition, and digestibility of pastures (Apráez et al., 2014; Bonilla Cárdenas & Lemus Flores, 2012; Garnsworthy, 2018; IPCC, 2006; Lovett et al., 2005; Ribeiro Pereira et al., 2015).
The mean concentrations of Ca, P, K, Mg and the activity of various enzymes in the blood serum of sheep grazing on natural grasslands and mixed pastures in high Andean micro-basins demonstrate trends similar to those reported by several authors, which may be indicative of a physiological response in grazing sheep according to Gioffredo (2011). However, considerable inter-individual variation was observed, with values ranging from below the minimum or normal range to well above the average.
Furthermore, the ALT and AF enzymes also show considerable variation in the established reference ranges (Avellanet et al., 2007). Conversely, the AST enzyme fell within a considered normal range, which agrees with the results reported by other authors (Djokovic et al., 2017). It has been established that the activity of the AF enzyme is associated with Ca content in the diet. An increase in AF levels has been observed during periods of dietary calcium deficiency, but not necessarily phosphorus deficiency (Braun et al., 2010; de Oliveira et al., 2014). This could potentially alter the calcium-to-phosphorus ratio and cause problems in animals exposed to changes in the quality of pastures or natural grasslands.
5. Conclusions
The natural grassland, which is based on a "pajonal" plant community, is frequently used for the grazing of sheep and cattle in Indigenous communities. This is done in a continuous grazing system of low productivity and quality, regardless of the stocking rate, which has a detrimental effect on the productive performance of the animals. The use of foreign pastures, such as ryegrass and clovers, at early utilization frequencies, appears to contribute to the intensification of livestock as a low-emission grazing proposal in the Andean ecoregion.
Funding
Universidad Central del Ecuador, Dirección de Investigación´, project SENIOR DI-CONV-2022-006.
Contributor roles
Jorge Eduardo Grijalva-Olmedo: conceptualization, methodology, project administration, writing – original draft.
Paola Mercedes Palate-Moreta: investigation, resources
Roy Roger Vera-Vélez: methodology, writing – review & editing.
Raúl Armando Ramos-Veintimilla: conceptualization, methodology.
Jean-François Tourrand: validation, writing – review & editing.
Arnulfo Portilla-Narváez: investigation, formal análisis.