Nutritional Quality of Opuntia ssp. at Different Phenological Stages: Implications for Forage Purposes

The study aimed to evaluat the chemical composition of forage cactus cladodes of the genus Opuntia spp. at different stages of phenological development. The experiment was conducted in a completely randomized design, with a 4×3 factorial scheme, with four species of cactus ( Opuntia stricta Haw, Opuntia undulata Griffiths, Opuntia atropes Rose and Opuntia larreri FAC Weber) and three stages of cladode development (young, intermediate, and mature). The chemical composition of Opuntia cladodes varied according to species and phenological stage. Dry matter (DM), neutral detergent fiber (NDF) and acid detergent fiber (ADF) concentrations increased, and crude protein (CP) decreased with cladode maturation. The species O. atropes had the lowest levels of organic matter (OM), CP, total carbohydrates, and non-fibrous carbohydrates, as well as the highest values of mineral matter, NDF and ADF. Calcium and phosphorus contents increased with cladodium maturation. The concentrations of boron, copper and manganese were higher in the young and intermediate stages. The cladodes of O. stricta , O. undulata and O. larreri showed better nutritional quality for feeding domestic ruminants. Young and intermediate cladodes are more proteinaceous and less fibrous. These results can be useful in selecting the forage cactus species and in optimizing the use of cladodes for feeding different categories of ruminants in semiarid regions.


Introduction
The lack of forage for animal feeding during the dry season is one of the main difficulties faced by domestic ruminant breeders in the Brazilian semiarid region. However, this barrier has been circumvented by the use of succulent stems (cladodes) of Cactaceae of the genera Opuntia and Nopalea, commonly called "forage cactus", as a source of water and energy to supplement herds in the dry season (Oliveira et al., 2021).
The genus Opuntia has about 300 species, being one of the most used in Northeast Brazil for foraging purposes (Lucena et al., 2013). Its CAM photosynthetic metabolism (Crassulaceous Acid Metabolism) favors the efficient use of water (Ferraz et al., 2017), enabling cultivation in arid and semi-arid regions (Edvan et al., 2020).
Forage cactus are an important resource for semiarid regions and has been widely studied from a nutritional point of view (Cordova-Torres et al., 2015;Vazquez-Mendoza et al., 2017;Pessoa et al., 2020). However, some aspects need a better understanding, such as nutritional quality, especially mineral composition of cladodes as a function of species/genotype and plant development stage. Thus, this study aimed to evaluate the chemical-bromatological and mineral composition of Opuntia species at different stages of phenological development, analyzing its implications for forage purposes.

Obtaining Samples
Cladode samples of four species of Opuntia (Table 1) were collected in May 2018 at the experimental station of the Instituto Agronômico de Pernambuco (IPA) in Arcoverde (-8.43° S, -37.06° W; altitude 683.9m), state of Pernambuco, Brazil. Planting was established in December 2014 (spacing 1.0 x 0.5 m) and handled in an upland system with classic cultural treatments, including periodic weeding and organic fertilization (20 t.ha -1 of cattle manure each two years). The local climate based on the Köppen-Geiger classification is BSh (hot and dry) with averages of annual temperature of 24.9 ± 1.97 °C, reactive humidity of 78.19 ± 8.24% and accumulated precipitation of 798.1 mm per year (INMET, 2019). ISSN 2166-0379 2022  The soil physicochemical characteristics in the cultivation area (0 to 20 cm deep) were analyzed according to the methodology proposed by Teixeira et al. (2017) ( Table 2). The local climate data during the trial (2014 to 2018) were provided by the automatic meteorological station of the National Institute of Meteorology (INMET) (Figure 1).  Cladodes were collected from five plants of each genotype (n=5) at three phenological stages: young (expanding cladodes, with fragile structure and light green color, located at the distal or lateral ends of the plant), intermediate (cladodes with rigid structure and dark green color, located in the middle part of the plant) and mature (fully expanded cladodes, with rigid structure and whitish-green color, located in primary order, just above the base). After collection, the samples were cleaned, chopped, and dehydrated in a forced air oven at a temperature of 55°C for 72 hours. Then, crushed in a knife mill with a 1 mm sieve, identified, packed in plastic containers with airtight closure and stored at 25 °C.
Samples were mineralized in sulfuric acid in order to evaluate nitrogen (N); in a mixture of nitric-perchloric acid, in order to determine phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), sulfur (S), copper (Cu), manganese (Mn), iron (Fe ) and zinc (Zn); and incinerated to determine boron (B). N concentration was determined by the Kjeldahl method; P by molybdenum blue colorimetry; K by emission flame photometry; Ca, Mg, Cu, Mn, Fe and Zn through atomic absorption spectrophotometry; and S by barium sulfate turbidimetry, according to Malavolta et al. (1997). The concentration of B was determined by the azomethine-H colorimetry method (Silva, 2009).

Statistical Analysis
The experimental design used was completely randomized in a 4 x 3 factorial scheme ISSN 2166-0379 2022 (genotypes of four species and three phenological stages of the cladode) and five replications. The data met the assumptions for parametric analysis and were subjected to analysis of variance at the 5% probability level (P <0.05). Treatment means were compared by the Tukey test using the Statistical Analysis System (SAS ® ) software (Cody, 2015). The statistical model adopted was: Yijk = μ + Gi + Pj + GPij + Eijk Where: Yijk is the observed value for the analyzed variable, μ overall mean, Gi is the effect of the genotype of species i, Pj is the effect of the phenological stage j, GPij is the effect of the interaction between Gi x Pj, and Eijk the experimental error associated with Yijk.

Journal of Agricultural Studies
Data were also submitted to multivariate analysis for exploratory purposes. In the cluster analysis, the Gower distance, and the hierarchical method UPGMA (Unweighted Peer Group with Arithmetic Mean) were used. In the Principal Component Analysis (PCA) treatments were plotted in relation to the first two components (PC1 and PC2). These evaluations were performed with the prcomp function in the statistical program R (R Core Team, 2018).

Chemical Composition of Cladodes
There was interaction (P <0.05) between the genotypes and phenological stages of the cladode in EE concentration, but for DM, OM, CP, and MM isolated effects of the factors were verified (P <0.05) ( Table 3). The EE content in EPP was higher in young cladodes and APP in the intermediate stage (22.68 g.kg -1 DM) compared to the young (19.56 g.kg -1 DM), but it presents similar content to that of mature cladodes (20.36 g). g.kg -1 DM). Concentrations in intermediate and mature cladodes were higher in APP and V-19 (Table 3).
NDF and ADF contents differed (P <0.05) between cladode species and developmental stages ( TC and NFC concentrations were not influenced (P >0.05) by the interaction between the factors and there was no effect (P >0.05) of developmental stages on these variables either (Table 4). Regarding the genotypes, it was found that the concentrations of TC (738.9 g.kg -1 DM) and NFC (433.12 g.kg -1 DM) in F-08 were lower (P <0.05) than the others.  EPP (Opuntia stricta); APP (Opuntia undulata); F-08 (Opuntia atropes); V-19 (Opuntia larreri). SEM, standard error of mean; G, genotypes; P, phenological stages; G x P, interaction of genotypes and phenological stages. Means followed by lowercase letters differ in the rows and capital letters in columns (P <0.05).
The P concentration in EPP was higher (P <0.05) in mature cladodes, similar to V-19, but V-19 exhibited a similar value in the intermediate stage (Figure 2b). In the APP genotype, the lowest concentration occurred in young cladodes. The P content in young and intermediate cladodes was higher in APP and F-08, but the highest concentration in mature cladodes was found in APP.

Multivariate Data Analysis
In the exploratory data analysis using PCA (Figure 4), the first two components (PC1 and PC2) explained 56.5% of the total data variation using all variables (Figure 4a), but by selecting the characteristics with the greatest potential for discrimination (11 variables) the contribution of these same components increased to 81.5% (Figure 4f).
When all variables were used, the factor loadings CP, NFC, ADF, Fe, NDF and Ca showed a greater contribution for PC1 (Figure 4b The arrows represent the direction of the feature, and the color gradient represents each one's contribution to the components. From blue to red, from low to high contributions. E J distribution of treatments as a function of the first two components for all characteristics (E) and selected characteristics (J). The color gradient indicates the contribution of the first two components to explain the variation of treatments, also represented by the size of the circles that varies from 0.25 to 0.75 (E) and 0.20 to 0.80 (J). EPP (Opuntia stricta), APP (Opuntia undulata), F-08 (Opuntia atropes), V-19 (Opuntia larreri).

Discussion
Opuntia ssp. are succulent plants and have a low DM content compared to other traditional forage plants. The values found (58.6 to 70.86 g.kg -1 DM) were lower compared to other studies with forage cactus (90.7 to 112.0 g.kg -1 NM) performed by Iná cio et al. (2020) and Barros et al. (2018). This may be related to the higher cladodes water content in this study since they were collected in the rainy season. The lower concentration of DM in mature cladodes is probably due to increase in the proportion of supporting tissues that make possible supporting the expansion of new structures, in addition to the thickening and lignification of the cell wall (Chapman et al., 2014).
Due to the low content of DM in cladodes, the nutritional requirements of ruminants, fed exclusively on palm, may not be achieved, and it is necessary to complement diets based on Opuntia with other sources of roughage to optimize consumption. On the other hand, its inclusion in the diet reduces voluntary water intake by animals (Costa et al., 2012;Gouveia et al., 2019) since it supplies part of their water needs.
The CP contents observed in this research (56.06 to 73.92 g.kg -1 DM) were higher than those reported by Siqueira et al. (2018)  The difference in CP content between mature and young cladodes can be explained by the cuticle thickening and the nitrogen components translocation to the developing structures. Mature cladodes have thicker cuticles due to expansion of the water storage parenchyma (very slowly degradable) at the expense of cell content (Astello-Garcí a et al., 2015). In this study, the CP in APP cladodes (71.08 g.kg -1 DM) and V-19 (71.24 g.kg -1 DM) are above the minimum necessary (70.0 g.kg -1 DM) for ruminal microorganisms adequate growth and development, responsible for the degradation of nutrients in the fibrous fraction (Silva et al., 2011).
Opuntia cladodes showed high concentrations of MM, similar to those described by Donato et al. (2014) (141.0 g.kg -1 DM) and Souza et al. (2010) (152.0 g.kg -1 DM). However, the F-08 species stood out in relation to the others, indicating that the accumulation of minerals differs within the genus. The increase in EE concentrations with cladode maturation, evidenced mainly in APP, acts as a plant energy reserve for carrying out biochemical and physiological processes under water stress conditions (Alves et al. 2016).
The increase in NDF and ADF contents with cladodium development (Table 4) was similar to what occurs in other forage plants. This fact was previously verified by Pinos-Rodrí guez et al. (2010), by demonstrating that there is a linear relationship between the maturation of Opuntia and the increase in fibrous fractions. This happens because as the physiological age of the plant advances, the cell wall becomes thicker with an increase in the concentrations of cellulose, hemicellulose, and lignin, implying an increase in fibrous proportions. However, it seems to have a negative impact on the content of nitrogenous compounds, since with the maturation of the cladodes the CP is lower (64.88 g.kg -1 DM).
The higher ADF concentration in mature F-08 cladodes may indicate that the DM digestibility in this species tends to be lower compared to the others, as these characteristics are inversely proportional. On the other hand, the NDF of the studied species (254.8 to 357.6 g.kg -1 DM) are below that recommended in the ruminant diet (350.0 to 450.0 g.kg -1 DM) and can reduce the total chewing time and saliva secretion, essential to keep the rumen environment stable and maximize production (Vilela et al., 2010). Therefore, Opuntia cladodes must be provided in association with a physically effective fiber source, such as silage, hay, crop residues and sugarcane bagasse, in order to avoid possible metabolic disturbances.
TC were the most abundant DM components, and the genotypes have a high concentration of NFC, which was not altered by cladode maturation. NFC contents are higher than most bulky feeds commonly used in rations, suggesting that Opuntia cladodes have the potential to replace other sources of energy concentrate in the diet (Costa et al., 2012;Felix et al., 2016). Furthermore, the rapid fermentation of these carbohydrates favors ruminal microorganisms and contributes to microbial protein synthesis (McDonald et al., 2011).
As minerals are directly related to nutrient absorption, physiological status, DM intake and performance of ruminants, the nutritional requirements of each animal category must be considered when confronting with the amount of minerals present in Opuntia cladodes. Mayer and Cushman (2019) found higher concentrations of K (47.8 to 73.6 g.kg -1 DM) and Mg (14.2 to 21.2 g.kg -1 DM), and when compared with the maximum values allowed for beef cattle during the pregnancy and lactation, found that it was above tolerable levels. The mineral concentrations in the studied cactus forage species meet the nutritional requirements of cattle in these categories, but the tolerance of Ca is exceeded in all genotypes and K tolerance is exceed in F-08 and V-19.
The P concentration in mature cladodes (Figure 2b) is certainly related to the participation of this mineral in the photosynthesis and respiration processes of plants with CAM metabolism, since more developed organs produce a greater amount of photosynthates (Queiroz et al., 2015). In animal nutrition, cladodes with higher P contents may contribute to supplement part of the deficiency often found in goats raised extensively in semiarid regions (Barbosa et al., 2018).
Ca was the most abundant macromineral in this study (41.72 to 81.8 g.kg -1 DM) and EPP and F-08 contents increased with cladode maturation. According to Hernandez-Urbiola et al. (2011), the concentration of this mineral in O. ficus increased with the plant aging, corroborating the results of this research. Ca accumulation in cladodes may be related to heat stress, where it is able to mitigate the effects by improving stomatal function and other cellular processes (Contreras-Padilla et al., 2011).
Higher Ca than P contents, result in a high Ca:P ratio. The unbalance of these minerals in the forage can cause the appearance of kidney stones and affect the performance ). However, the main form of Ca in cladodes is calcium oxalate (McConn & Nakata 2004;Tovar-Puente et al., 2007), which makes this mineral unavailable and inhibits poisoning in animals. From a nutritional point of view, both minerals are interesting for livestock feed, essential for bone formation, ionic balance, and signal transmission within cells (Agabriel 2010).
The concentration of B, Cu and Mn in young and intermediate cladodes suggests that they are required in greater amounts by developing tissues. This nutrient redistribution process is essential to the plant life cycle, as some minerals increase in the area part, others decrease, resulting in translocation (Lima et al., 2011). Thus, the cladodes supply in young and intermediate stages, located in the extremities or median portion of the plant, can be aimed at animal categories with greater demand for these minerals.
Among the microminerals, Mn was the most abundant in Opuntia species (41.6 to 317.7 mg.kg -1 DM), showing typical behavior of a nutrient synthesized in larger amounts in young structures (Figure 3c). Alves et al. (2017) observed differences in Mn concentration between secondary and tertiary order cladodes, despite not defining a pattern of accumulation in the plant, ranging from 228.5 to 512.5 mg.kg -1 DM in O. stricta, from 102.5 to 139.47 mg. kg -1 DM for O. atropes, and from 323.5 to 523.9 mg.kg -1 DM for O. larreri.
In the exploratory analysis using PCA, the variables EE, P, Mg, S, B, Cu, Zn and Mn had a minor contribution to the divergence between treatments (Figure 4b; 4c). When disregarding them, the accumulated value of the first principal components was greater than 80%, which according to Cruz et al. (2012) is sufficient to explain the variability of the data. In the formation of groups, F-08 presented lower nutritional quality compared to the other species, using all characteristics and selected characters (Figure 4e; 4j), explained by the strong contribution of the variables ADF and MM in mature cladodes, while Ca, K, and MM explain in the young and intermediate stages of development.

Conclusions
The cladodes of the species O. stricta, O. undulata and O. larreri showed a better nutritional quality for ruminant feeding. Young and intermediate cladodes are more proteinaceous and less fibrous. These results can be useful in selecting the forage cactus species with superior nutritional quality and in optimizing the use of cladodes for feeding ruminants of different categories in semiarid regions.