Carbohydrate fractionation and nitrogen compounds, mineral status, and gas production in arboreal cotton and cactus silages

[Fracionamento de carboidratos e compostos nitrogenados, status mineral e produção de gases em silagens de algodão arbóreo e palma forrageira]

ABSTRACT

The aim of this study was to evaluate the fractionation of carbohydrates and nitrogen compounds, mineral status, and in vitro gas production of arboreal cotton silages combined with different cactus levels (0, 150, 300, 450 and 600g.kg^-1 on dry matter basis), distributed in a completely randomized design with 5 treatments and 4 replicates. For carbohydrate fractionation, there was an increase in fraction B2, and a decrease in fraction C (P<0.05). In nitrogen compounds, there was a decrease in B3 and C fractions (P<0.05). A quadratic effect was observed on mineral matter, crude protein, nitrogen, potassium and latency phase (P<0.05). Under the experimental conditions, the use of cactus in arboreal cotton silages increases the levels of minerals, carbohydrates and gas production in the silage, however, it reduces the protein content in the silages when cactus levels above 150 g.kg are inserted in arboreal cotton silages.

Keywords:
forage conservation; Gossypium hirsutum L; Opuntia stricta [Haw.] Haw; tropical forages

RESUMO

Objetivou-se avaliar o fracionamento de carboidratos e compostos nitrogenados, o perfil mineral e a produção de gases in vitro de silagens de algodão arbóreo combinadas com níveis de palma forrageira (0, 150, 300, 450 e 600 g.kg^-1 na matéria seca), distribuídos em delineamento inteiramente ao acaso, com cinco tratamentos e quatro repetições. Para o fracionamento de carboidratos, houve aumento na fração B2 e diminuição na fração C (P<0,05). Nos compostos nitrogenados, houve decréscimo para as frações B3 e C (P<0,05). Observou-se efeito quadrático sobre matéria mineral, proteína bruta, nitrogênio, potássio e fase de latência (P<0,05). Nas condições experimentais, o uso da palma forrageira em silagens de algodão arbóreo aumenta os teores de minerais, carboidratos e produção de gases na silagem, porém reduz o teor de proteínas nas silagens quando são inseridos níveis de palma forrageira acima de 150g.kg^-1 em silagens de algodão arbóreo.

Palavras-chave:
conservação de forragem; Gossypium hirsutum L; Opuntia stricta [Haw.] Haw; forrageiras tropicais

INTRODUCTION

Alternatives have been used as a resource for animal feed, capable of preserving the nutritional characteristic of the feed and meeting the nutritional requirements of animals (Shurson, 2020). Among the methods used, silage is characterized by a food preservation technique that allows its use for supply in times of feed shortage in dryland regions, reducing the production cost (Mehmood et al., 2020). However, the quality of the silage depends on factors intrinsic to the ensiled material such as adequate levels of dry matter and soluble carbohydrates, low buffering capacity and particle size necessary to avoid losses due to unwanted fermentations (Borreani et al., 2018).

Cactus pear is a Cactacea adapted to dryland regions used as a source of feed and water for ruminants. Because of specific characteristics, such as high biomass production, high levels of water-soluble carbohydrates and energy and low content of dry matter, neutral detergent fiber and crude protein, its use alone is not recommended. Thus, the use of cactus pear in silages should be associated with other bulky feed sources, ensuring adequate levels of fiber and protein to have a feed that meets the needs of the animals, maintaining normal conditions in the rumen and allowing synchronization between the supply of energy and nitrogen for ruminal microorganisms (Matias et al., 2020).

Arboreal cotton (Gossypium hirsutum L. var. marie-galante Hutch) is a forage species well adapted to semi-arid conditions. Due to characteristics such as neutral detergent fiber (565.1g.kg^-1 dry matter), dry matter (217.7g.kg^-1 natural matter) and crude protein (245.1g.kg^-1 dry matter) (Figueira et al., 2020), the use of arboreal cotton can nutritionally complement the cactus pear when used in combination in silage, aiming to correct the low levels of dry matter, physically effective fiber and crude protein that cactus has (Silva et al., 2022). Furthermore, cactus can reduce the buffering capacity (47.5 mEq × 10⁻³) that tree cotton, being a forage legume, has (Kazemi and Tohidi 2023). Figueira et al. (2020), when evaluating exclusive arboreal cotton silages, they found that they have low levels of non-fibrous carbohydrates (245.3 g.kg^-1 dry matter) and high levels of fraction C (indigestible fraction) for the fractionation of carbohydrates (146.4g.kg^-1 total carbohydrates) and nitrogenous compounds (224.4g.kg^-1 crude protein), in relation to arboreal cotton silages containing natural additives.

The ensiling process may present factors that promote inefficient fermentation, requiring the use of additives that improve the fermentation process in ensiling (Yitbarek and Tamir, 2014). Therefore, the association of cactus with forage plants that have considerable levels of physically effective fiber, such as arboreal cotton, is essential to maintain normal rumen functions and salivary secretion (Banakar et al., 2018). Furthermore, the content of water-soluble carbohydrates (150.6g.kg^-1 dry matter) present in cactus (Brito et al., 2020) can benefit the fermentation process of cotton silages through the growth of the lactic acid bacteria population (Zhang et al., 2023).

The determination of minerals, carbohydrate fractions, nitrogenous compounds, and nutrient digestion rates are extremely important for the animal nutritionist. This information can be used in feed formulations, contributing to minimizing energy and nitrogen losses and promoting greater efficiency of microbial synthesis (Magalhães et al., 2019) and improving the bioavailability of minerals for animals (Kazemi and Tohidi 2023), considering the nutritional requirements of ruminants. Thus, the association of cactus with arboreal cotton in the silages composition that can be used in diets offered to ruminants, can contribute to the supply of nutrients to ruminal microorganisms and benefit the animals' productive performance.

To the best of our knowledge, there are no studies evaluating the nutritional quality of the association of arboreal cotton with cactus in the composition of silages, so we hypothesize that the association of 60% cactus in arboreal cotton in the composition of mixed silages increases the concentration of carbohydrate fractions and nitrogen compounds that are quickly degraded, improves the mineral composition of silages, and increase the gas production.

The aim of this study was to evaluate the mineral composition, carbohydrate fractionation, nitrogen compounds and gas production kinetics of arboreal cotton silages combined with different levels of cactus pear.

MATERIALS AND METHODS

The present study was appreciated and approved by the Ethics Committee on the Use of Animals (CEUA) of the Brazilian Agricultural Research Corporation (Embrapa Semiárido; Opinion no. 0004/2016).

The study was carried out at the Federal University of Agreste de Pernambuco, in Garanhuns, state of Pernambuco, Brazil, located in the mesoregion of Agreste Meridional de Pernambuco, at 8º53'25'' South latitude and 36º29'34'' West longitude, at 896 m above sea level. The region’s climate is classified as Aw' tropical, with an average annual temperature of 21.2 ºC and an average annual rainfall of 897 mm and characterized by hot, dry summers and mild and humid winters, with relative humidity ranging from 75 to 83 %.

Inclusion levels of cactus pear (0, 150, 300, 450 and 600 g.kg^-1 on dry matter basis) were evaluated in arboreal cotton silages, in a completely randomized design, with 5 treatments and 4 replicates, totaling 20 experimental silos. Cotton [Gossypium hirsutum L. var. marie-galante (Hutch)] and the cactus pear Mexican Elephant Ear variety, IPA-200016 (Opuntia stricta Haw) used for making silages came from an area established on the experimental farm of the Federal University of Agreste de Pernambuco. Arboreal cotton was harvested manually 24 months after planting, collecting the upper third of the plants. For cactus pear, first-order cladodes were collected manually from a plantation established 12 months ago in the field.

The harvested material was processed in a stationary forage harvester (PP-35, Pinheiro Máquinas, Itapira, São Paulo, Brazil), to particles of average size of 2.0 cm. The material was homogenized manually. During homogenization, the treatments were formulated, adding the proportions of cactus to the arboreal cotton, according to the experimental levels. After homogenization, ensiling was carried out in experimental silos (10 cm in diameter, 55 cm in height), equipped with a Bunsen valve to allow fermentation gases to escape. At the bottom of the experimental silos, 2 kg dry sand were deposited, protected by a cotton fabric, preventing the ensiled material from coming into contact with the sand, allowing the effluent to drain in. Samples of the non-ensiled material (original material) were collected for further laboratory analysis (Table 1). After being sealed, silos were kept for 90 days in a covered shed. Samples of the ensiled material were collected for further laboratory analysis (Table 2).

Samples of green material and silages were pre-dried in a forced ventilation oven at 55 °C for 72-h and ground to 1- and 2-mm particles in a knife mill (Wiley, Marconi, MA 580, Piracicaba, Brazil). The contents of dry matter (DM, method 967.03), mineral matter (MM, method 942.05), crude protein (CP, method 981.10) and ether extract (EE, method 920.29), and acid detergent fiber (ADF, method 973.18) were determined following the methodological procedures of AOAC (Official…, 2016). Neutral detergent fiber (NDF) was quantified according to Van Soest et al. (1991).

The content of neutral detergent fiber corrected for ash and protein (NDFap; using thermostable α-amylase with sodium sulfide) in silages was determined according to Licitra et al. (1996) and Mertens (2002).

Table 1
Chemical composition of cactus pear and arboreal cotton before silage

Variables (g.kg-1 dry matter)	Cactus pear	Arboreal cotton
Dry matter1	74.80	308.19
Mineral matter	152.74	59.05
Organic matter	847.26	940.95
Ether extract	19.65	23.57
Crude protein	74.29	167.21
Neutral detergent fiber	280.71	443.07
Acid detergent fiber	177.73	361.45
Total carbohydrates	795.24	750.17
Non-fibrous carbohydrates	545.49	307.10

• ¹
  Expressed in g.kg^-1 of natural matter

Table 2
Chemical composition of silages

Variables (g.kg-1 dry matter)	Cactus pear levels (%)
	0	15	30	45	60
Dry matter1	314.8	281.2	273.5	238.5	197.8
Mineral matter	59.2	60.8	60.2	63.1	71.7
Organic matter	940.7	939.2	934.1	936.9	928.3
Ether extract	20.4	15.5	15.2	14.4	7.4
Neutral detergent fiber	448.6	456.6	455.2	487.8	451.6
NDFap	371.2	377.0	379.2	379.1	379.2
Acid detergent fiber	418.9	338.6	360.1	356.9	346.6
Non-fibrous carbohydrates	341.4	319.4	345.5	309.6	352.8

• ¹
  Expressed in g.kg^-1 of natural matter; NDFap= Neutral detergent fiber corrected for ash and protein

Fermentative losses, buffering capacity and aerobic stability of silages are available in Melo et al. (2022). For carbohydrate fractionation, total carbohydrates (TC) were calculated from the equation proposed by Sniffen et al. (1992), where:

$TC=1000-\left(CP+EE+MM\right)$ (Equation 1)

were divided into fractions A+B1, B2 and C. Non-fiber carbohydrates (NFC), corresponding to fractions A+B1 were estimated by the equation (Hall, 2003):

$NFC=TC–NDFap$ (Equation 2)

The indigestible NDF, corresponding to fraction C, was obtained after 288 hours of in situ incubation in rumen fistulated goats (Valente et al., 2011). Fraction B2, corresponding to the digestible fiber, was obtained by the difference between NDFap and fraction C.

Protein was analyzed and calculated for the five fractions, A, B1, B2, B3 and C by the CNCPS system (Sniffen et al., 1992). The content of non-protein nitrogen (NPN), neutral (NDIN) and acid (ADIN) detergent insoluble nitrogen were determined according to Licitra et al. (1996). Fraction A (non-protein nitrogen - NPN) was determined by the equation:

$A=Nt-N1/Ntx1000$ (Equation 3)

where Nt = total nitrogen in the sample, and N1 = trichloroacetic acid insoluble nitrogen content. Fraction B1 refers to the soluble protein, rapidly degraded in the rumen, obtained by the formula:

$B1=N1-N2/Ntx100$ (Equation 4)

where: N2 = borate phosphate buffer insoluble nitrogen.

Fractions B2 and B3 consist of insoluble protein with an intermediate-slow degradation rate in the rumen. Values of fractions B2, B3 and B1 + B2 were estimated by the equations:

$B2=N2-NIDN/Ntx100$ (Equation 5)

$B3=NDIN-ADIN/Ntx100$ (Equation 6)

$B1+B2=100-\left(A+B3+C\right)$ (Equation 7)

Fraction C, formed by indigestible insoluble protein in the rumen and intestine, was determined by the residual nitrogen content of the sample after treatment with acid detergent (ADIN).

Mineral analyzes were carried out in the soil laboratory of the Brazilian Agricultural Research Company (Embrapa Semiárido). The concentrations of nitrogen (N), potassium (K), phosphorus (P), copper (Cu), iron (Fe), manganese (Mn), zinc (Zn), calcium (Ca), magnesium (Mg), sulfur (S) and chloride content were determined according to the methodologies described by Nogueira and Souza (2005).

The in vitro semi-automatic gas production technique proposed by Theodorou et al. (1994). One gram of sample was placed in glass vials (160mL), which were added with 90mL nutrient medium. Subsequently, 10mL ruminal fluid (from three rumen fistulated goats) was added to each flask, which was kept under CO₂ flow, sealed and placed in a greenhouse at a constant temperature of 39ºC during the incubation period. The pressure (P; in psi) originated by gases accumulated in the upper part of the vials was measured by a portable pressure transducer (Datalogger Universal Logger AG100) connected at its end to a needle (0.6 mm). Pressure was read more frequently during the initial fermentation period and subsequently reduced (2, 4, 6, 8, 10, 12, 15, 18, 21, 24, 30, 36, 48 and 72-h of incubation). Pressure data were converted into gas volume (1 psi = 4.859 mL gas) using the equation:

$V=5.1612P-0.3017;R^2=0.9873$ (Equation 8)

Generated at the Gas Production Laboratory (GPL), from the Federal University of Agreste Pernambucano (UFAPE), based on 937 observations. From each pressure reading, the total produced by the vials without substrate (blank) was subtracted for each sample. The cumulative gas production data were analyzed using the Gompertz two-compartmental model, cited by Schofield et al. (1994):

$V\left(t\right)=Vf1/\left[1+e^{(2–4Kd1(L-T))}\right]+Vf2/\left[1+e^{(2–4Kd2(L-T))}\right]$ (Equation 9)

where: V(t) = maximum total volume of gas produced; Vf1 = maximum volume of gas for the fast digestion fraction (non-fiber carbohydrates; NFC); Kd1 = specific growth rate for the rapid degradation fraction; Vf2 = maximum volume of gas for the slow digestion fraction (fibrous carbohydrates; FC); Kd2 = specific growth rate for the slow degradation fraction; L = duration of initial digestion events (latency phase), common to both phases, and; T = fermentation time.

Data were analyzed using the PROC REG of the Software Statistical Analysis System University (SAS University) through analysis of variance and regression at 5% probability. As a criterion for choosing the regression models, the significance of the parameters estimated by the models and values of the coefficients of determination were adopted. The following statistical model was used:

$Y=\mu +Tj+eij$ (Equation 10)

where: μ = overall mean; Tj = effect of the level of inclusion of cactus pear; eij = residual error.

RESULTS AND DISCUSSION

The contents of TC (P=0.003) increased linearly following the increase in cactus pear levels in the composition of cotton silages (Table 3). This may be due to the higher levels of cactus that were inserted during ensiling, since cactus has higher TC content in its composition compared to cotton (Table 1). This result is beneficial for providing greater availability of substrates for fermentation, in addition to being characterized as a good energetic quality for ruminal microorganisms (Araújo et al., 2023).

Table 3
Fractionation of carbohydrates and nitrogen compounds in arboreal cotton silages associated with different levels of cactus pear

Variables	Cactus pear levels (g.kg-1 DM)					SEM	P value
	0	150	300	450	600		L	Q
	Fractions of carbohydrate compounds (g.kg-1TC)
TC (g.kg-1 DM)1	780.8	781.3	781.6	797.4	801.4	4.45	0.003	0.419
A+B1	432.1	411.6	431.5	388.2	438.5	10.19	0.744	0.058
B22	147.6	173.5	147.5	251.4	267.0	28.65	0.003	0.318
C3	420.2	414.9	420.9	360.3	294.4	29.49	0.005	0.109
	Fractions of nitrogen compounds (g.kg-1CP)
CP (g/kg DM)4	130.4	147.7	128.2	125.1	116.5	4.17	0.002	0.039
A5	21.9	24.9	21.4	20.9	19.4	0.02	<0.001	<0.001
B1+B26	793.6	777.9	791.7	813.3	814.6	8.58	0.012	0.212
B37	170.0	181.6	170.2	152.9	155.2	7.99	0.036	0.422
C8	14.4	15.5	16.6	12.9	10.7	1.45	0.046	0.052

• DM= dry matter; CP= crude protein; TC= total carbohydrates; Fractions of carbohydrate compounds: A+B1 = soluble fraction; B2 = potentially degradable fiber; C = indigestible fiber; Fractions of nitrogen compounds: A - non-protein nitrogen; B1+B2 - high and average rumen nitrogen degradation fraction; B3 - slow nitrogen degradation fraction; C - unavailable nitrogen fraction; SEM = standard error of mean; L= Linear; Q= Quadratic; Significant at the 5% probability level. Equations: ¹ŷ= 783.68 + 0.33x, R²= 0.49; ²ŷ= 134.05 + 2.11x, R²= 0.75; ³ŷ= 443.41 - 2.04x, R² = 0.76; ⁴ŷ= 134.59 + 0.34x - 0.011x², R²= 0.65; ⁵ŷ = 22.70 + 0.050x - 0.0018x², R²= 0.65; ⁶ŷ= 782.76 + 0.516x, R²= 0.61; ⁷ŷ= 177.69 - 0.39x, R²= 0.60; ⁸ŷ= 16.02 - 0.07x, R²= 0.46.

There was no effect of the levels of cactus pear on fractions A + B1 in the tested cotton silages (P>0.05; Table 3). Godói et al. (2024), evaluating cactus silages associated with different forages adapted to the semiarid region, mention that the fraction A + B1 can provide an increase in pH, due to the higher content of soluble sugars and pectin, which provides cellulolytic microorganisms with more suitable conditions in the rumen. Thus, the similarity between the silages for fraction A + B1 infers that most carbohydrates are available for use by ruminal microorganisms.

The inclusion of cactus pear in cotton silages promoted a linear increase in fraction B2 (P=0.003) (Table 3). Cotton silages containing 450 and 600 g.kg^-1 cactus pear in their composition showed higher proportions of fraction B2. This fact is possibly related to the higher NDF content in tree cotton compared to cactus. For mixed silage, adequate proportions of components in the silage can help in the balanced supply of energy during the degradation of nitrogenous compounds slowly degraded in the rumen, since the B2 fraction corresponds to carbohydrates with slower ruminal degradation. Therefore, forages with a higher content of the B2 fraction may present a reduction in food intake and longer chewing and rumination time (Oliveira et al., 2020a), being, on the other hand, a mechanistic measure used to evaluate the effect of NDF and their fractions in digesta retention in the rumen. According to Oliveira et al. (2016), the B2 fraction contributes to rumen pH balance, rumination and saliva production. However, it causes a low rate of passage through the rumen reticulum canal, reducing total dry matter intake, affecting the animal's performance. Therefore, it is necessary and pertinent to observe the nutritional composition of silages before offering them to ruminants.

The inclusion of cactus pear in cotton silages promoted a linear reduction for fraction C (P=0.005) (Table 3). Fraction C of carbohydrates corresponds to indigestible fibers, and they influence rumen filling, which promotes lower intake per unit of time, due to their indigestibility. Therefore, its reduction is positive, since lower concentrations of this fraction imply greater digestibility of fibrous carbohydrates (Godói et al., 2024).

Fractionation of nitrogen compounds is important in the development of more efficient diets, since crude protein is a nutrient that can increase the costs of the diet supplied to ruminants, thus, knowledge of these fractions allows the choice of high-quality protein foods to compose the animals’ diets (Pas et al., 2021). In this study a quadratic effect was found for CP (P=0.039), with higher CP content in arboreal cotton silages containing 150g.kg^-1 DM of cactus (147.7g.kg^-1 DM; Table 3). Forages with high values of protein and/or fiber have few soluble carbohydrates, therefore, silage of arboreal cotton together with cactus pear can balance the contents of dry matter, crude protein and fiber, making them more suitable for preservation and ruminant feeding (Villalba et al., 2021). The protein content of silages was favored by the crude protein content of the arboreal cotton (167.21g.kg^-1 DM; Table 1), providing the silages with crude protein values above the minimum required (70g.kg^-1 CP; Van Soest, 1994) to ensure proper functioning of the rumen microbiota.

The inclusion of cactus pear in arboreal cotton silages resulted in a quadratic effect was verified for fraction A (P<0.001), with a higher content of this fraction in arboreal cotton silages containing 150g.kg^-1 DM of cactus (24.9 g.kg^-1 CP; Table 3). Higher CP content of cotton resulted in a greater conversion of the silage protein into non-protein nitrogen (Fraction A) because it is highly susceptible to proteolysis (Van Soest, 1994). However, high proportions of NPN can result in greater nitrogen losses due to the lack of the carbon skeleton readily available for microbial protein synthesis to occur.

The inclusion of cactus pear in arboreal cotton silages resulted in an increase in the levels of fraction B1 + B2 (P=0.012) and a linear reduction in fractions B3 (P=0.036) and C (P=0.046) (Table 2). Among the protein fractions of the studied silages, the largest proportion consisted of the fraction B1 + B2. As it presents a rapid ruminal degradation rate in relation to fraction B3, fraction B1 + B2 tends to be extensively degraded in the rumen, contributing to meet the nitrogen requirements of the ruminal microorganisms; however, the rapid proteolysis of these fractions in the rumen can lead to the accumulation of peptides and allow their escape into the intestine, since the use of peptides is considered to limit protein degradation (Moreira et al., 2013).

Higher contents of cactus pear in arboreal cotton silages reduced the B3 fraction of the protein, which may improve rumen degradation by reducing the content of degraded protein in the rumen, but which may have high intestinal digestibility (Titze et al., 2019). Thus, in relation to ruminant nutrition, it is necessary to include sources of carbohydrates with rapid ruminal degradation, such as cactus pear, to favor synchronization between energy and protein, maximizing the synthesis of microbial protein (Oliveira et al., 2020b). The increase in cactus pear reduced the content of fraction C in cotton silages, enabling a better use of nitrogen compounds.

There was a reduction in nitrogen in silages (P=0.002; Table 4), in agreement with the reduction in CP and fraction A in silages as the levels of cactus inclusion increased, as seen in Table 3. Therefore, this reduction in N is possibly associated with the lower content of this macromineral in cactus, compared to cotton. Fermentation in silage can modify the amount of nitrogen and nitrogen compounds due to the action of proteolytic enzymes and other microorganisms such as Clostridium and Enterobacter, which act in the deamination and decarboxylation of proteins (Kung Jr. et al., 2018; Campos et al., 2023). However, more studies are necessary, with the determination of the macromineral contents of the ingredients used in the composition of the silages tested here, so that inferences can be made on their mineral composition.

Table 4
Macromineral (g.kg^-1) and microminerals (mg.kg^-1) composition in arboreal cotton silages associated with different levels of cactus pear.

Variables	Cactus pear levels (g.kg-1)					SEM	P value
	0	150	300	450	600		L	Q
	Macrominerals (g.kg-1DM)
N1	2.1	2.4	2.0	2.0	1.9	0.06	0.002	0.036
P	3.9	4.9	3.7	4.2	3.8	0.26	0.241	0.381
K2	30.9	30.6	29.0	28.4	31.8	0.71	0.870	0.008
Ca3	9.7	10.5	10.4	11.5	14.6	0.39	<0.001	0.001
Mg4	3.6	3.9	3.9	4.4	5.6	0.15	<0.001	0.001
Na5	747.2	735.5	668.0	612.5	464.7	13.81	<0.001	<0.001
	Microminerals (mg.kg-1DM)
S	1.1	0.8	1.1	0.9	1.0	0.10	0.809	0.413
B	34.8	32.3	37.8	36.9	20.3	4.46	0.103	0.057
Cu6	14.1	16.0	17.5	18.3	20.2	0.62	<0.001	0.725
Fe	129.8	141.5	117.3	129.7	135.2	6.80	0.970	0.353
Mn7	45.2	42.5	38.3	38.5	36.2	1.31	<0.001	0.285
Zn	16.5	16.1	15.1	16.3	15.3	0.83	0.494	0.773

• N= nitrogen; P= phosphorus; K= potassium; Ca= calcium; Mg= magnesium; Na= sodium; S= sulfur; B= boron; Cu= cooper; Fe= iron; Mn= manganese; Zn= zinc; DM= dry matter; SEM = standard error of mean; L = Linear; Q = Quadratic; Significant at the 5 % probability level; Equations: ¹ŷ= 2.32 - 0.08x, R²= 0.84; ²ŷ= 31.40 - 0.16x + 0.0026x², R²= 0.62; ³ŷ= 9.16 + 0.072x, R²= 0.77; ⁴ŷ= 3.34 + 0.031x, R²= 0.81; ⁵ŷ= 746.06 + 0.37x - 0.082x², R²= 0.98; ⁶ŷ= 14.34 + 0.096x, R²= 0.98; ⁷ŷ= 44.55 - 0.15x, R²= 0.91.

A quadratic effect was observed for K (P= 0.008) with a higher concentration of this macromineral (31.8g.kg^-1 DM) in silages containing 600g.kg^-1 DM of cactus in its composition (Table 4). Cactus pear with high levels of K, the highest content of this nutrient was verified with the presence of 150 and 600g.kg^-1 DM of cactus in cotton silage. This effect has a direct relationship with the osmolarity of the medium, in this sense, silages of 150 and 600g.kg^-1 of cactus in cotton silage possibly had higher osmotic pressure. According to Wiendl (2013) potassium acts to increase protein nitrogen and decrease the content of non-protein nitrogen, producing more digestible dry matter and protein in the silage, which corroborates the findings in Table 3.

Levels of cactus pear in cotton silages resulted in a linear increase in the levels of Ca (P<0.001), Mg (P<0.001) (Table 4). Cactus pear has high levels of Ca and Mg in its composition, so the increase in the level of cactus pear in arboreal cotton silages has increased the content of these minerals in silages, mainly due to the hydrocolloid properties and mucilage production of cactus, which provides greater water and mineral retention capacity due to its hydrophilic properties (Araújo et al., 2023). Another factor that may have caused the increase and preservation of Ca is its immobility in the plant tissue (Campos et al., 2021), making its losses during fermentation difficult. Magnesium, in turn, which is lost during the ensiling process is related to its soluble fraction, and the less mobile fraction that is bound to the fiber and chlorophyll (Campos et al., 2021), effects that justify the preservation of Mg. There was no effect of the levels of cactus pear on the P (P> 0.05; Table 4).

A quadratic effect was observed for Na (P<0.001; Table 4), with exclusive arboreal cotton silages (0% cactus) presenting the highest concentration of this mineral (747.2 kg^-1 DM) in relation to the other silages evaluated. For cacti with Crassulacean Acid Metabolism (CAM), Na is considered an essential mineral element, as it is essential for the regeneration of phosphoenolpyruvate, the substrate of the first carboxylation in this pathway, its deficiency in CAM plants induces chlorosis and necrosis in addition to failures in flower development (Bhatla and Lal, 2018). The decrease in Na is linked to its ease of mobilization and regulation of osmolarity promoting the movement from a hypertonic to a hypotonic medium, an effect that promoted the dissociation of Na ions with increasing moisture (inclusion of cactus pear), favoring its dilution and losses during the formation of effluents (Tavakkoli et al., 2011).

There was no effect of the levels of cactus on the macromineral P (P> 0.05; Table 4) in the tested cotton silages. There was also no effect of cactus inclusion levels on the microminerals S, B, Fe and Zn (P> 0.05; Table 4) in the silages.

Among the micronutrients, the increase in the proportion of cactus pear in cotton silages increased the concentration of Cu (P<0.001) and reduced the concentration of Mn (P<0.001) (Table 4). The increase in Cu in silage may be associated with its affinity for nitrogen compounds of the amino group; soluble nitrogen, such as amino acids. This affinity is observed by the increase in fraction B1 + B2 of nitrogen compounds that refer to true proteins. The reduction in Mn occurs due to its free form or low molecular weight complexes causing losses, in addition to the decrease in these minerals directly affecting the number of lactic acid bacteria that accumulate Mn to avoid toxicity by oxygen peroxide (Pahlow et al., 2003).

For the in vitro gas production, the increase in the levels of cactus pear in arboreal cotton silages promoted a quadratic effect for the variables V(t) (P=0.024) (Table 5). The inclusion of 60 % cactus pear in arboreal cotton silages resulted in a greater estimated volume of gas (209.4mL.g^-1 DM). This result was expected because, according to Guimarães Jr et al. (2008), more fermentable forages, rich in non-fibrous carbohydrates, such as cactus, present higher maximum potential values together with high gas production rates, leading to greater fermentation of the material in a shorter incubation time. A similar result was observed by Cunha et al. (2022) when including forage cactus in elephant grass silages. The authors observed that the inclusion of up to 30% of cactus in silages increased V(t) to 255.8 mL.g^-1 DM and attributed this result to the higher content of non-fibrous carbohydrates that cactus has, in relation to elephant grass.

The increase in the levels of cactus pear in arboreal cotton silages promoted a quadratic effect for Latency phase (P<0.001) (Table 5). The latency phase represents the time between the beginning of the incubation and the microbial activity on the tested substrate (Arreola et al., 2019). Thus, it was observed that the presence of cactus pear in the composition of arboreal cotton silages provided a reduction in the latency phase in relation to the control treatment, with a shorter latency period with the inclusion of 30% of cactus in arboreal cotton silages (4.26 h), this period also corresponding to the lowest V(t) (180.5 mL.g^-1 DM). This characteristic is related to the presence of readily fermentable substrates and to the physical and chemical characteristics of the samples, which can favor microbial fermentation (Bertrand, 2019). There was no effect of the levels of cactus pear on Vf1, Kd1, Vf2, Kd2 (P>0.05; Table 5).

Table 5
In vitro gas production in arboreal cotton silages associated with different levels of cactus pear

Variables	Cactus pear levels g.kg-1)					SEM	P value
	0	150	300	450	600		L	Q
Vf1 (mL.g-1 DM)	47.5	41.8	47.3	47.0	76.9	10.09	0.063	0.104
Kd1 (mL.h-1)	0.13	0.12	0.13	0.14	0.08	0.02	0.262	0.374
Vf2 (mL.g-1 DM)	142.6	144.2	134.1	142.6	133.7	9.12	0.517	0.944
Kd2 (mL.h-1)	0.03	0.03	0.03	0.03	0.02	0.01	0.950	0.214
V(t) (mL.g-1 DM)1	188.9	185.4	180.5	189.1	209.4	6.50	0.046	0.024
Latency phase (h)2	5.71	4.53	4.26	4.36	4.79	0.196	0.006	<0.001

• Vf1 - maximum volume of gas for the fast digesting fraction (non-fiber carbohydrates; NFC), Kd1 - specific growth rate for the rapid degradation fraction, Vf2 - maximum volume of gas for the slow digesting fraction (fibrous carbohydrates; FC), Kd2 - specific growth rate for the slow degradation fraction, Latency phase - duration of initial digestion events (latency time), common to both phases, V(t) - maximum total volume of gas produced by adjusting the two-compartment model; SEM= Standard Error of the Mean; L= Linear; Q = Quadratic; Significance at 5% probability; Equations: ¹ŷ= 190.42 - 0.86x + 0.019x², R²= 0.95; ²ŷ= 5.65 - 0.082x + 0.0011x², R²= 0.97.

CONCLUSIONS

Under the experimental conditions, the use of cactus in arboreal cotton silages increases the levels of minerals, carbohydrates and gas production in the silage, however, it reduces the protein content in the silages when cactus levels above 150 g.kg^-1 DM are inserted in arboreal cotton silages. Therefore, more studies are necessary and pertinent to observe the effect of silage as an exclusive feed or in the composition of diets for animals.

ACKNOWLEDGEMENTS

To the Coordination for the Improvement of Higher Education Personnel - CAPES for granting post-doctoral scholarships (Process 8882.316819/2019-01).

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Publication Dates

• Publication in this collection
  21 Feb 2025
• Date of issue
  Mar-Apr 2025

History

• Received
  20 Mar 2024
• Accepted
  11 Sept 2024