1. Introduction
Optimizing animal feed is crucial in modern agriculture, not only due to its significant impact on production costs but also because of its direct influence on productivity and animal welfare. With over 50% of production costs linked to feed [
1], it is essential to develop strategies that ensure efficient resource use while promoting sustainability in agricultural production [
2].
In the Azores, agricultural production is dominated by the livestock sector, particularly the dairy industry. Cows graze on pastures year-round, with minimal stabling structures. To maintain pasture productivity for animal feed, farmers often apply synthetic nitrogen fertilizers to the soil to stimulate rapid plant growth, as nitrogen is a critical component of essential molecules. However, nitrogen fertilizers are also associated with environmental issues, such as ammonia and nitrogen oxide emissions and nitrate leaching into watercourses.
Research has shown that nitrogen deficiency is a significant limiting factor for many plant species in organic systems, underscoring the importance of nitrogen fertilization for high-demand crops [
3]. Thus, incorporating nitrogen-fixing crops like legumes as commercial crops, cover crops, temporary green manures, or in intercropping systems, including agroforestry integration, is beneficial.
Legumes used as green manure can be incorporated into the soil, increasing its organic matter, improving water retention, and enhancing soil aeration [
4]. Green manures improve soil’s physical, chemical, and biological properties. They also help control weeds, prevent erosion, and enrich the soil with nitrogen, reducing the need for synthetic fertilizers [
5].
Green manuring offers an economical and sustainable alternative to chemical fertilizers, which are often costly and environmentally harmful. The choice of green manure plants depends on local conditions, such as soil type, climate, subsequent crops, and soil management goals [
6].
In the Azores, feeding cattle with “green manures” locally known as “outonos” was common for many years but has become rare today. “Outonos” typically consist of forage crops mixed with legumes and grasses, traditionally used in rotation with corn [
7]. In the Azores, an intercrops “outonal” crop includes lupins,
Vicia faba oats, and more recently, vetch.
Lupinus luteus is a resilient and nutritious plant known for thriving in acidic soils with low organic matter, contributing to soil fertility through nitrogen fixation [
8].
Vicia faba minor varieties are recognized for their high productivity and protein content, adapting well to loamy soils unsuitable for many legumes, and can be grown as forage for green manure [
9]. Sativa vetch (
Vicia sativa L) is an annual legume planted in the fall, providing quality fodder and widely used in intercropping with cereals like triticale and oats. It adapts to various soils and climates and shows significant growth potential in spring [
10]. Oats (
Avena sativa L) are primarily grown for their grains and as animal fodder.
This study aims to evaluate both chemically and biologically the agricultural practice of “outonos”, specifically the intercropping of Lupinus luteus, Vicia faba minor oats, and vetch, in their fresh form. Additionally, it investigates how wilting and ensiling affect the chemical composition of the silage. This research contributes to a deeper understanding of sustainable agricultural practices, aiming to optimize forage production for animal feed.
4. Discussion
The silage process involves the fermentation of stored crops, such as corn, sorghum, or grasses, which are cut and compacted in silos to create an oxygen-free environment. Silage production is essential to meet the need for a constant and nutritious feed supply for livestock, ensuring continued milk and meat productivity throughout the year, regardless of climatic and seasonal variations. Additionally, silage is an efficient solution for utilizing forage surpluses, reducing waste, and contributing to the sustainability of agricultural activities [
22,
23].
To maintain pasture productivity, farmers often rely on synthetic nitrogen fertilizers, which are essential for rapid plant growth but are associated with environmental issues such as ammonia emissions and nitrate leaching [
3]. On the other hand, nitrogen-fixing crops like lupine and vetch have proven effective when grown in combination, adapting well to various soil and climate conditions [
24]. These crops (like lupine and vetch) can be used as green manure to improve soil quality, thereby reducing the need for chemical fertilizers [
5,
25].
The average productivity obtained for these crops was 1948 kg of dry matter per hectare, based on multiple samples collected at a time of year when the photoperiod is short and temperatures are lower, factors that generally reduce plant growth. Soil saturation due to excess rainfall, together with a decrease in temperature and photoperiod, significantly limits the production potential of pastures. However, the yields found in this study are higher than those reported by [
26], who, in a perennial ryegrass pasture under temperate oceanic climate conditions, achieved an average yield of 65.4 kg of dry matter per hectare in one of the spring months. This contrast highlights the effectiveness of these crops, even in adverse environmental conditions.
Despite the high productivity potential, in the Azores, the practice of using mixed legume and grass forages, known as “outonos”, has declined. This decline is clear even though it is effective when rotated with crops such as corn [
7]. Traditionally, these mixtures include lupins,
Vicia faba, oats, and vetch, plants recognized for their adaptability and nitrogen-fixing capability [
8,
9,
10,
27]. Therefore, promoting the cultivation of “outonos” could enhance forage productivity and sustainability in the region.
Although these crops are highly perishable due to their high moisture content, confirmed in this study by the dry matter (DM) value of fresh samples being only 10% (
Table 1), effective preservation strategies such as ensiling are essential to maintain the nutritional quality of the forage through controlled fermentation [
28]. Ensiling legumes like alfalfa (
Medicago sativa L.) or red clover (
Trifolium pratense L.) requires special care due to their high protein content and low soluble carbohydrate content, which favors fermentation by clostridia [
29]. Wilting can contribute to better fermentation of these legume silages [
30]. The DM content is a critical parameter in silage production, directly influencing the fermentation process and the stability of the final product [
31]. In this study, wilting significantly (<0.001) increased the dry matter (DM) content of the forage, especially after 96 h (S4) of wilting (26.47%). The increase in DM is beneficial because forage with higher DM content tends to ferment better, producing less effluent and increasing storage efficiency [
32]. This increase is crucial because the reduction in moisture decreases the activity of undesirable microorganisms, such as clostridia, which can compromise the quality of the silage [
33].
The pH of silage is a critical indicator of the efficiency of the fermentation process. Lower pH values are desirable because they indicate predominant lactic acid fermentation, contributing to the preservation of the forage [
34]. In this study, a progressive decrease in pH was observed with increasing wilting time. A pH below 4.5, as observed in the conditions of 48 (S3) and 96 h (S4) of wilting, is ideal for inhibiting the growth of undesirable microorganisms [
34]. These values are consistent with previous studies on ryegrass silage, where similar results were observed [
35,
36,
37]. However, it is important to note that lower pH values alone do not necessarily reflect the dominance of lactic acid fermentation. Future studies should include the analysis of organic acids and alcohols to provide a more comprehensive understanding of the fermentation process.
The proportion of ammonia nitrogen reflects the proteolysis within the silo. Elevated values of ammonia nitrogen indicate the development of undesirable bacteria and can suggest that soluble nitrogen is not effectively assimilated by rumen bacteria, leading to protein value losses [
38]. However, nitrogen can be utilized by rumen microorganisms if there are sufficient readily available carbohydrates in the diet.
For the samples considered in this study, the average %N-NH
3/N total values are within the expected range for grass silages, which can be considered of intermediate or good quality [
39]. The reduction in ammonia nitrogen content with increasing wilting time suggests that wilting limits protein degradation, resulting in greater protein retention in the silage. This effect is particularly important for maintaining the nutritional value of the silage.
Wilting, by reducing the initial moisture content of the forage, contributes to achieving stability more quickly [
40,
41].
The data show that wilting increases dry matter (DM), thereby potentially reducing effluent production, which is beneficial for environmental conservation and waste management. However, this advantage comes with a trade-off: reduced feed value and lower energy concentration of the forage (
Table 4). The gross energy (GE) decreased from 13.94 MJ/KgDM in fresh forage to 13.43 MJ/KgDM in silage S4 (
p = 0.03), indicating a loss of soluble energy components during wilting. Digestible energy (DE) also showed a significant decrease, from 10.78 MJ/KgDM in fresh forage to 8.38 MJ/KgDM in silage S4 (
p < 0.001), likely due to an increase in less digestible fibrous components. Similarly, metabolizable energy (ME) decreased from 8.84 MJ/KgDM in fresh forage to 6.87 MJ/KgDM in silage S4 (
p < 0.001), reflecting a lower efficiency in converting digestible energy into metabolizable energy. Net energy for lactation (NEL) also showed a notable decrease, ranging from 6.18 MJ/KgDM in fresh forage to 5.51 MJ/KgDM in silage S4 (
p = 0.02).The difference of 0.7 MJ NEL/KgDM between fresh forage and silage S4 resulted in a significant energy loss, considering a yield of almost 2 t of DM/ha, leading to a reduction of 1400 MJ NEL/ha in energy yield. While wilting can reduce effluent production, the associated energy losses due to reduced forage nutritional quality must be considered. Thus, the benefits of wilting in terms of reducing effluent and quickly stabilizing silage must be balanced against the disadvantages of reduced feed value and energy concentration.
In the samples from this study, it was observed that fresh forage had the highest crude protein content (22.51% DM), which decreased with increasing wilting time, reaching 19.67% in S4 (
Table 1). These values are in line than those found by Hartinger et al., [
42], who reported average CP contents for lucerne silages of between 4.41 and 24.88%DM The variation in CP content was minimal among the different treatments, suggesting that wilting did not have a large impact on protein concentration.
NDF, ADF, and ADL are the chemical components most commonly used to predict forage digestibility [
43]. Regarding preservation, in the case of grass silage, the values obtained indicate the amount of substrate available for fermentation. High values suggest overly mature grasses with fewer available free sugars, which may not ferment sufficiently to lower the pH to the ideal range for good preservation. In the case of corn silage, fiber values are not good indicators of the amount of available substrate, as corn silage generally has enough sugars to complete fermentation regardless of fiber content [
44].
Significant changes (
p < 0.001) in the fibrous components of the forage (
Table 2) with increasing wilting time were evident, notably in NDF, ADF, and ADL. This indicates a higher total fiber content and less digestible components, suggesting reduced digestibility and nutritional value, as confirmed by decreases in dry matter digestibility (DMD) and organic matter digestibility (OMD) (
Table 1). DMD and OMD values dropped significantly after 96 h of wilting, likely due to increased lignin content, which is highly resistant to digestion. The initial reduction in hemicellulose, followed by a slight increase, suggests an initial loss of fermentable carbohydrates, crucial for efficient fermentation. The increase in lignin content may further compromise the quality of the silage [
45].
The reduction in forage digestibility and nutritional value due to increased wilting time is further supported by gas production data (
Table 5). The amount of gas produced in in vitro fermentation reflects the extent of fermentation and the digestibility of forage [
46], being directly proportional to the rate at which the substrate is degraded [
47]. According to Chesson and Forsberg [
48], during the initial incubation period, the soluble and rapidly fermentable fraction of the substrate (soluble carbohydrates) is fermented and microbial protein is synthesized. Once this phase is completed, the fermentation of insoluble but potentially degradable components, such as the NDF fraction, begins.
The fermentation kinetics, described using McDonald’s methodology [
19], suggest that longer wilting times reduce gas production from the immediately soluble fraction (a). Fresh forage consistently showed higher gas production across all incubation times, indicating better fermentability and higher digestibility. In contrast, silage samples, particularly those with longer wilting times, exhibited lower gas production values. For instance, at 96 h of incubation, fresh forage produced 54.14 mL of gas per 200 mg DM, whereas silage with 96 h of wilting (S4) produced only 38.82 mL. This trend of decreasing gas production with increasing wilting time reflects the increase in fiber content, especially cellulose and lignin, which are less fermentable, thus reducing the amount of carbohydrates available for fermentation.