ABSTRACT

Several studies on the kinetics of sugarcane’s fiber digestion have been published, but, to date, no study has evaluated the influence of sugarcane rind on the digestion of fresh sugarcane by ruminants. This study aimed to evaluate the effects of sugarcane components (rind and pith) on chemical composition, in vitro digestibility, metabolizable energy, and sugarcane quality. A randomized block design was used in a split-plot scheme with five sugarcane genotypes [plot] (RB068027, RB058046, RB987917, RB867515, and RB855536) and three sugarcane components [sub-plot] (rind, pith, and whole cane), Each treatment consisted of four replicates. The chemical composition, in vitro gas production, in vitro digestibility, metabolizable energy, and sugarcane quality were evaluated. No interaction between components and genotypes was observed for the variables analyzed herein. Although the rind had a higher crude protein content, it showed a large amount of insoluble crude protein. The rind had higher fibrous fractions, comprising 87.33 % of the indigestible fraction of the neutral detergent fiber (NDF). The sugarcane rind showed ~ 71.20 % more lignin than the pith tissue. Further, the rind decreased by 6.5 % in vitro dry matter digestibility compared to the whole sugarcane. The in vitro NDF digestibility of the rind was 18.38 % lower than the whole sugarcane. The RB068027 genotype showed the lowest sugarcane quality. Despite the higher content of potentially digestible neutral detergent fiber (pdNDF) in the rind, its high lignin content influences the quality of the final fibrous fractions of sugarcane and negatively impacts the nutritional value. The genotypes do not differ nutritionally, but RB855536 presented higher biomass and energy yields.

Saccharum officinarum L.; digestion; fibrous fractions; forage

Introduction

Sugarcane is grown extensively in America, Africa, Asia, and Oceania on account of its ease of cultivation and outstanding production of green mass, which facilitate its use in ruminant feeding during the dry season (characterized by low rainfall and high temperatures), which results in a shortage of forage (Bento et al., 2018Bento CB, Filoso S, Pitombo LM, Cantarella H, Rossetto R, Martinelli LA, et al. 2018. Impacts of sugarcane agriculture expansion over low-intensity cattle ranch pasture in Brazil on greenhouse gases. Journal of Environmental Management 206: 980-988. https://doi.org/10.1016/j.jenvman.2017.11.085
https://doi.org/10.1016/j.jenvman.2017.1... ). Thus, sugarcane is an excellent option for farmers as it has advantages such as great nutritional value and forage production per area, concurring with the period of forage shortage compared to tropical forages (Gomes et al., 2016Gomes RS, Oliveira TS, Pereira JC, Vieira RAM, Silva CJ, Leonel FP, et al. 2016. Kinetics of digestion of low-quality forage grazed by beef cattle fed supplements containing increasing levels of rumen undegradable protein. Revista Brasileira de Zootecnia 45: 563-571. https://doi.org/10.1590/S1806-92902016000900009
https://doi.org/10.1590/S1806-9290201600... ). However, sugarcane production in Brazil focuses on the sugar-energy industry rather than on animal nutrition, making selecting varieties with better nutritional value for animal feeding necessary (Carvalho et al., 2022Carvalho CAB, Zuccari AM, Pereira W, Moura AM, Schultz N, Paiva AJ, et al. 2022. Evaluation of sugarcane for animal feed in the Baixada Fluminense - RJ. Revista Ciência Agronômica 53: e20218230. https://doi.org/10.5935/1806-6690.20220055
https://doi.org/10.5935/1806-6690.202200... ).

Sugarcane deserves some attention due to its nutritional limitations, such as low protein and mineral levels and low-quality fibrous fractions. Among the nutritional limitations, the fibrous fractions significantly impact feed digestion, and protein and minerals can be corrected by supplements (Gomes et al., 2016Gomes RS, Oliveira TS, Pereira JC, Vieira RAM, Silva CJ, Leonel FP, et al. 2016. Kinetics of digestion of low-quality forage grazed by beef cattle fed supplements containing increasing levels of rumen undegradable protein. Revista Brasileira de Zootecnia 45: 563-571. https://doi.org/10.1590/S1806-92902016000900009
https://doi.org/10.1590/S1806-9290201600... ). Sugarcane constituents with high lignification are in the strongly recalcitrant rind (Maziero et al., 2013 Maziero P , Jong J , Mendes FM , Gonçalves AR , Eder M , Driemeier C . 2013. Tissue-specific cell wall hydration in sugarcane stalks. Journal of Agricultural Food Chemical 61: 5841-5847. https://doi.org/10.1021/jf401243c
https://doi.org/10.1021/jf401243c... ). The tissue architecture of lignin and the suberin lamellae’s aromatic fraction may be a significant physicochemical factor limiting rumen microorganisms’ degradation of sugarcane (Maziero et al., 2013 Maziero P , Jong J , Mendes FM , Gonçalves AR , Eder M , Driemeier C . 2013. Tissue-specific cell wall hydration in sugarcane stalks. Journal of Agricultural Food Chemical 61: 5841-5847. https://doi.org/10.1021/jf401243c
https://doi.org/10.1021/jf401243c... ).

Moreover, the existence of a genetic variability effect between sugarcane genotypes on fibrous fraction is a possibility. Fiber-related traits were neglected by selection, resulting in cultivars having more genetic variability available for fiber-related than for sugar-related traits (Cursi et al., 2021). There are many studies on sugarcane’s chemical composition and fiber digestion kinetics, but to date, no study has evaluated the influence of the rind on the digestion of fresh sugarcane. In addition to mechanical protection and water retention, the rind can drain the stored carbon into the stalk, which accumulates sucrose (Wang et al., 2013 Wang J , Nayak S , Koch K , Ming R . 2013. Carbon partitioning in sugarcane (Saccharum species). Frontiers in Plant Science 4: 201. https://doi.org/10.3389/fpls.2013.00201
https://doi.org/10.3389/fpls.2013.00201... ). Based on the above, it was hypothesized that: 1) the fibrous fractions of the sugarcane pith would have as much impact on the nutritional value as the fibrous fractions of the rind; and 2) there would be a nutritional difference between genotypes. Thus, the present study aimed to evaluate the effect of components (rind and pith) of five sugarcane genotypes on chemical composition, in vitro digestibility, metabolizable energy, and sugarcane quality.

Materials and Methods

Location

The experiment was conducted in Bom Jesus do Itabapoana, Rio de Janeiro, Brazil (21°08’13” S, 41°39’30” W, 85 m altitude). The climate of the northern state of Rio de Janeiro is partial Aw, i.e., humid tropical with rainy summers and dry winters, with an average annual temperature of 23 °C and rainfall of 1,200 mm according to the Köppen-Geiger classification (Alvares et al., 2013Alvares CA, Stape JL, Sentelhas PC, Gonçalves JLM, Sparovek G. 2013. Köppen's climate classification map for Brazil. Meteorologische Zeitschrift 22: 711-728. https://doi.org/10.1127/0941-2948/2013/0507
https://doi.org/10.1127/0941-2948/2013/0... ).

Area and experimental design

The soil was prepared with plowing, harrowing, and furrowing, according to Portz et al. (2013)Portz A, Resende AS, Teixeira AJ, Abboud ACS, Martins CAC, Carvalho CAB, et al. 2013. Recommendation of fertilizers, correctives and organic matter management for the main crops in the State of Rio de Janeiro = Recomendação de adubos, corretivos e de manejo de matéria orgânica para as principais culturas do Estado do Rio de Janeiro. p. 370-376. In: Freire LR, Balieiro FC, Zonta E, Anjos LHC, Pereira MG, Lima E, et al. eds. Liming and fertilization manual of the State of Rio de Janeiro = Manual de calagem e adubação do Estado do Rio de Janeiro. UFRRJ, Rio de Janeiro, RJ, Brazil (in Portuguese).. Before planting, some soil samples were sent for analysis at the Analysis Center of the Universidade Federal Rural do Rio de Janeiro, Campos dos Goytacazes, Rio de Janeiro. The soil presented the following chemical composition: pH in H₂O = 5.8 P (mehlich) = 8 mg dm³; K = 21.5 mg dm³; Na = 0.0 mg dm³; Ca = 1.5 cmol dm³ ; Mg = 0.7 cmol dm³; Al = 0.0 cmol dm³; H + Al = 2.7 cmol dm³; CEC (t) = 1.1 cmol dm³; CEC (T) = 2.2 cmol dm³; SB = 2.2 cmol dm³; BS = 45.6 %; OM = 1.7 %; Fe = 59.5 mg dm³; Cu = 0.3 mg dm³; Zn = 300.6 mg dm³; and Mn = 18.6 mg dm³. The area was fertilized following the recommendations of the Liming and Fertilization Manual of the State of Rio de Janeiro for sugarcane crops (Portz et al., 2013Portz A, Resende AS, Teixeira AJ, Abboud ACS, Martins CAC, Carvalho CAB, et al. 2013. Recommendation of fertilizers, correctives and organic matter management for the main crops in the State of Rio de Janeiro = Recomendação de adubos, corretivos e de manejo de matéria orgânica para as principais culturas do Estado do Rio de Janeiro. p. 370-376. In: Freire LR, Balieiro FC, Zonta E, Anjos LHC, Pereira MG, Lima E, et al. eds. Liming and fertilization manual of the State of Rio de Janeiro = Manual de calagem e adubação do Estado do Rio de Janeiro. UFRRJ, Rio de Janeiro, RJ, Brazil (in Portuguese).). Four hundred kg ha¹ of formulated NPK 08-28-16 was applied. In order to reduce area heterogeneity [soil fertility]) the randomized block used a split-plot design. The factors were arranged per the experimental design as main factor plot genotypes and sub-plot factor components of sugarcane. There were five sugarcane genotypes from the Interuniversity Network for Development of the Sugar-Energy Sector (INDSES), with four replicates for each genotype. The genotypes were RB867515 [G1], RB855536 [G2], RB068027 [G3], RB058046 [G4], and RB987917 [G5]. Three components of sugarcane (rind, pith, and whole cane) were evaluated. Each replicate had four lines, 4 m long, and a spacing of 1.20 m, totaling 19.2 m² of useful area per replicate.

The harvest was carried out in Aug 2020. Ten whole sugarcanes were harvested from the third row of each plot, and their weight recorded. Next, five stalks were taken for sugarcane quality testing. Five canes were rinded with a spoon so that all the pith in the rind was removed, and the other five canes had the aerial part (whole cane) stripped off.

Chemical composition

The sugarcane samples were taken to the Animal Nutrition Laboratory of the Universidade Estadual do Norte Fluminense (UENF) and separated into rind, pith, and whole sugarcane. They were dried at 55 °C for 72 h, ground in a Wiley mill (R-TE-648, Tecnal) with a 1-mm-sieve, and stored in airtight plastic containers. All samples were analyzed for total dry matter (DM, method 967.03; AOAC, 2019), crude fat (CF, AOAC Method 2003.06; Thiex et al., 2003Thiex NJ, Anderson S, Gildemeister B. 2003. Crude fat, hexanes extraction, in feed, cereal grain, and forage (Randall/soxtec/submersion method): collaborative study. Journal of AOAC International 86: 899-908. https://doi.org/10.1093/jaoac/86.5.899
https://doi.org/10.1093/jaoac/86.5.899... ), ash (method 942.05; AOAC, 2019), and crude protein ([N × 6.25] CP, AOAC Method 984.13 and AOAC Method 2001.11; AOAC, 2019; Thiex et al., 2002Thiex NJ, Manson H, Anderson S, Persson J. 2002. Determination of crude protein in animal feed, forage, grain, and oilseeds by using block digestion with a copper catalyst and steam distillation into boric acid: collaborative study. Journal of AOAC International 85: 309-317.). Neutral detergent insoluble fiber (aNDF) was determined using the fiber analyzer (Tecnal TE-149). Sodium sulfite and two standardized heat-stable α-Amylase solution additions were used according to the INCT-CA method F-001/1, as described by Detmann et al. (2012)Detmann E, Souza MA, Valadares Filho SC, Queiroz AC, Berchielli TT, Saliba EOE, et al. 2012. Methods for Food Analysis = Métodos para Análise de Alimentos. INCT - Ciência animal 1ed. Suprema, Visconde Rio Branco, Brazil (in Portuguese).. The acid detergent fiber (ADF) was analyzed also according to the INCT-CA-F-003/1 method described by Detmann et al. (2012)Detmann E, Souza MA, Valadares Filho SC, Queiroz AC, Berchielli TT, Saliba EOE, et al. 2012. Methods for Food Analysis = Métodos para Análise de Alimentos. INCT - Ciência animal 1ed. Suprema, Visconde Rio Branco, Brazil (in Portuguese). and the lignin (sa) content by Möller (2009) Möller J . 2009. Gravimetric determination of acid detergent fiber and lignin in feed: interlaboratory study. Journal of AOAC International 92: 74-90. https://doi.org/10.1093/jaoac/92.1.74
https://doi.org/10.1093/jaoac/92.1.74... . Non-fibrous carbohydrate (NFC) was estimated as NFC (g kg¹) = 1000 – CP – CF – Ash – aNDF. The content of neutral detergent soluble (NDS) was obtained by subtracting NDS = 1000 – aNDF. Neutral detergent insoluble crude protein (NDICP) was determined by analyzing the aNDF residues for Kjeldahl nitrogen (Licitra et al., 1996Licitra G, Hernandez TM, Van Soest PJ. 1996. Standardization procedures for nitrogen fractionation of ruminant feeds. Animal Feed Science and Technology 57: 347-358. https://doi.org/10.1016/0377-8401 (95)00837-3
https://doi.org/10.1016/0377-8401 (95)00... ).

For the analysis of indigestible neutral detergent fiber (iNDF), the rind, pith, and whole sugarcane were processed in a Wiley mill with a 4-mm-sieve and stored in 13 × 7 cm nylon bags, 50 µm of pore diameter, a ratio of 25 mg of DM cm² of the bags’ surface. The bags with samples were tied on a steel chain with a 250 g anchor and introduced into the rumen of four cannulated sheep for 240 h. Next, the material was taken from the rumen and washed under running water until there were no traces of ruminal residue. Subsequently, the samples were dried in a forced-air oven at ± 55 °C for 72 h, and the weight was determined on an analytical scale for further aNDF analysis according to the INCT-CA F-001 method /1, as described by Detmann et al. (2012)Detmann E, Souza MA, Valadares Filho SC, Queiroz AC, Berchielli TT, Saliba EOE, et al. 2012. Methods for Food Analysis = Métodos para Análise de Alimentos. INCT - Ciência animal 1ed. Suprema, Visconde Rio Branco, Brazil (in Portuguese)..

Gas production kinetics, in vitro digestibility, and metabolizable and net energy

Four cannulated sheep were used in this study. The Ethics Committee approved all experimental procedures on the Use of Experimental Animals, protocol 419/2017. The animals weighed 50 kg (standard deviation = 4.1 kg) and were used as donors of ruminal fluid. They were kept in collective stalls with troughs and drinkers. Before ruminal fluid collections, the sheep were adapted to a diet of Tifton 85 hay and concentrate feed (roughage:concentrate ratio [80:20]) with 100 g d¹ of sugar for 14 days. After this period, the ruminal fluid collections were initiated moments before daytime feeding, as Yáñez-Ruiz et al. (2016)Yáñez-Ruiz DR, Bannink A, Dijkstra J, Kebreab E, Morgavi, DP, O'Kiely P, et al. 2016. Design, implementation and interpretation of in vitro batch culture experiments to assess enteric methane mitigation in ruminants - a review. Animal Feed Science and Technology 216: 1-18. https://doi.org/10.1016/j.anifeedsci.2016.03.016
https://doi.org/10.1016/j.anifeedsci.201... recommended.

The ruminal fluid (liquid and solid) was collected at several points on the liquid-solid interface of the ruminal environment via cannula using a collecting cup. A buffer solution described by McDougall (1948)McDougall EI. 1948. The composition and output of sheep's saliva. Biochemical Journal 43: 99-109. was added. Two hundred mg (standard deviation = 10 mg) of rind, pith, and whole sugarcane samples from the five sugarcane genotypes were added in amber penicillin flasks (100 mL) with 20 mL of the previously prepared inoculum [ratio 1:4; ruminal fluid and buffer solution, respectively, according to Goering and Van Soest (1970)Goering HK, Van Soest PJ. 1970. Forage Fiber Analysis. USDA-ARS, Washington, USA.]. The free space in the flasks was immediately saturated with CO₂. Next, the flasks were sealed and taken to a water bath at 39 °C, where they were shaken during incubation to homogenize the inner content. In vitro incubations were conducted in two consecutive runs, each with the sample in triplicates.

Time profiles of accumulated gas production were obtained using a non-automated device. A 0 to 8 psi manometer (0.05 increments) was attached to a three-way plastic valve. One of the ways was connected to a silicone tube (i.d. 5 mm; 1.5 m in length) with a 20 gauge needle attached to the loose extremity of the tube. The second way was attached to the manometer by a small piece of the silicone tube (i.d. 5 mm; 0.3 m in length) and plastic clamps. The third way was connected by another silicone tube (i.d. 5 mm; 1.3 m in length) to the top of a graduated 25 mL pipette (0.1 mL increments), which had its conical end connected to the stem of a separating funnel (1,000 mL) by the same type of silicone tube (i.d. 5 mm; 0.4 m in length). The funnel and pipette were attached to a metal support stand in a vertical and static position. The connecting system was filled with resazurin solution (0.1 g L¹) to the zero mark of the pipette, i.e., it allowed for atmospheric pressure equilibration. The system was cautiously filled to avoid the formation of air bubbles. Pressure and volume were taken at: 0, 1, 2, 3, 4, 6, 8, 10, 12, 16, 20, 24, 30, 36, and 48 h after the ruminal inoculum was added. The pressure and cumulative volume of the fermentation gases were obtained by summing the readings corrected to the mark after zero.

The model used to estimate the cumulative gas production was proposed by Groot et al. (1996)Groot JCJ, Cone JW, Williams BA, Debersaques FMA, Lantinga EA. 1996. Multiphasic analysis of gas production kinetics for in vitro fermentation of ruminant feeds. Animal Feed Science and Technology 64: 77-89. https://doi.org/10.1016/S0377-8401 (96)01012-7
https://doi.org/10.1016/S0377-8401 (96)0... :

where: G is the amount of gas produced per unit of dry matter (DM) at time t after the incubation started, A, the asymptotic gas production (mg g¹ DM), B, the time (h) after incubation in which half of the asymptotic gas was formed representing the speed of gas production, C, a constant that determines the sharpness of the curve change; and R_M the maximum gas production rate when the microbial population does not limit the fermentation and digestion is not reduced by chemical or structural barriers of the potentially digestible material.

The determination of in vitro digestibility was focused on a single digestion step of the ruminal fluid, omitting the step with pepsin recommended by Tilley and Terry (1963)Tilley JMA, Terry RA. 1963. A two-stage technique for the in vitro digestion of forage crops. Grass and Forage Science 18: 104-111. https://doi.org/10.1111/j.1365-2494.1963.tb00335.x
https://doi.org/10.1111/j.1365-2494.1963... . The buffer solution was the same as mentioned above. For each sample (rind, pith, and whole cane), triplicates of approximately 200 mg of air-dried samples were weighed and placed in 100 mL amber penicillin flasks with 20 mL of buffer solution and inoculum. The free space in the flasks was immediately saturated with CO₂, sealed, and taken to a water bath at 39 °C.

After 48 h of incubation, the flasks were withdrawn from the water bath, washed with hot distilled water (above 90 °C), and the incubated material filtered through quantitative filter paper (55 L s¹ m² air permeability). The resulting material was dried (55 °C 24 h¹ followed by 105 °C 16 h¹) and weighed to obtain the apparently undigested residue of dry matter (DM). Next, that material was analyzed for in vitro digestibility of NDF implementing the methodology described by Detmann et al. (2012)Detmann E, Souza MA, Valadares Filho SC, Queiroz AC, Berchielli TT, Saliba EOE, et al. 2012. Methods for Food Analysis = Métodos para Análise de Alimentos. INCT - Ciência animal 1ed. Suprema, Visconde Rio Branco, Brazil (in Portuguese).. The potentially digestible fraction was determined by subtracting NDF from iNDF.

The digestibility (D) of DM and NDF was calculated according to the Eq. (3):

where: M = mass of DM or NDF incubated (g); R = DM or NDF residue from incubation (g); B = DM or NDF residue of the blanks (g).

Metabolizable energy (ME) and net energy (NE) of the rind, pith, and whole sugarcane of the five genotypes were estimated using the equations by Menke and Steingass (1988)Menke KH, Steingass H. 1988. Estimation of the energetic feed value obtained from chemical analysis and in vitro gas production using rumen fluid. Animal Research and Development 28: 7-55.:

where: GP is the net gas production over 24 h (mL mg¹ DM).

Sugarcane quality

Five culms from each experimental plot were taken to the Coagro (Cooperativa Agroindustrial do Estado do Rio de Janeiro Ltda.) to conduct the technological analyses according to the methodologies suggested by CONSECANA (2006). Technological analyses were performed only for the whole sugarcane.

The automatic hydraulic press method performed the brix and polarization of sugarcane (POL) analyses from the juice (Codistil). Brix (%) was analyzed using a digital refractometer (Acatec RDA8600) with automatic reading and corrected temperature. An automatic saccharimeter was used to determine the POL (Acatec DAS2500). It was calibrated at 20 °C with a wavelength between 587 and 589.4 nm and fitted with a continuous flow polarimetric tube. The percentage of POL was obtained by the following Eq. (6):

The apparent purity of the juice was obtained by the ratio between POL and Brix, according to Eq. (7):

Total recoverable sugars (TRS) were determined based on Eq. (8):

where S is sucrose (%), and P represents the purity calculated by the ratio between % POL and % Brix.

The reducing sugars were calculated according to Eq. (9):

Equations (6), (7), (8), and (9) were proposed by CONSECANA (2006).

At the end of the cycle (360 days), the sugarcane was harvested and weighed by cutting two linear meters of the second line of each experimental replicate. Next, the weights of total biomass (stalks, leaves, and straw) were recorded and used to estimate the tons of stems per hectare (TSH) and corrected for DM content, expressed in t ha¹of DM.

Statistical analysis

The chemical composition, cumulative gas production, metabolizable and net energy estimates, and in vitro digestibility were compared by Tukey test at 0.05 significance using the SAS MIXED package using REPEATED statement and option = SUBJECT = Block × genotypes for analyzing split-plot design (SAS OnDemand Academics, SAS Institute Inc.). No interaction was observed between genotypes and components in the analyzed variables. Thus, the genotype was tested with residue (a [numerator degree of freedom = 4 and denominator degree of freedom = 42]) and the components with residue (b [numerator degree of freedom = 2 and denominator degree of freedom = 42]).

The following statistical model was used:

where Y_ijk is the value observed for the variable under study referring to the k-th replicate of the i-th sugarcane genotype in the j-th component (whole sugarcane without aerial part; sugarcane without rind, and only the rind); μ, the mean of all experimental units for the variable under study; α_i the effect of the sugarcane genotype with i = 1,2,3,4,5; b_k the random effect of the k-th block on the observation, αb_ik the residue (a) associated with the plot; β_j the effect of the component with i = 1,2,3; αβ_ij, the interaction between sugarcane genotypes and components, and e_ijk, the residue (b) associated with the split-plots.

Sugarcane biomass and quality were compared by Tukey test at 0.05 significance using the SAS GLM package (SAS OnDemand Academics, SAS Institute Inc.). The following statistical model was used:

where Y_ij is the value observed for the variable under study referring to the k-th replicate in the i-th sugarcane genotype, µ, the mean of all experimental units for the variable under study, α_i, the effect of the sugarcane genotype with i = 1,2,3,4, b_j, the random effect of the j-th block on the observation, and e_ij, the error associated with observation Y_ij.

Results

Chemical composition

There was no interactive effect (p ≥ 0.05) between components and genotypes on chemical composition (Tables 1 and 2). In the genotypes, G4 presented lower contents for CP (p = 0.042) and CF (p = 0.035) than G2, but it did not differ from the others (Tables 1 and 2). As for the components, although the rind had a higher CP content (p < 0.001), it had a large amount of NDICP (p < 0.001), approximately 34.55 % (10.19/15.57) more than the whole sugarcane and 55.10 % more than the pith. The CF content was also higher for the rind than the whole sugarcane (p < 0.001), approximately 33.88 % more and 72.54 % more than the pith (Tables 1 and 2). However, the rind had lower DM (p < 0.001) and NFC (p < 0.001) content compared to the pith. Ash contents for the rind were higher (p = 0.003) than the pith, but they did not differ from the whole sugarcane (Tables 1 and 2).

Fibrous fractions

There was no interaction effect (p ≥ 0.05) between components and genotypes on fibrous fractions (Tables 1 and 3). The genotypes did not affect (p ≥ 0.05) the fibrous fractions of sugarcane (Tables 1 and 3). However, regarding the components, the rind impacted these fractions, presenting 34.52 % more NDF than whole sugarcane, of which 87.33 % corresponds to the indigestible fraction of NDF (Tables 1 and 3). Furthermore, the potentially digestible fraction of the rind was higher by 52.71 % than whole sugarcane and by 52.68 % more than pith. On the other hand, the NDS contents of the rind were higher (p < 0.001) by 42.21 % than the pith (Tables 1 and 3). Contents of ADF (p < 0.001) and indigestible dry matter (iDM) (p < 0.001) had similar behavior to NDF for the rind, pith, and whole sugarcane. As regards lignin contents, the rind was 71.20 % higher than the pith (p = 0.002) (Tables 1 and 3). The average values of the components for neutral detergent fiber content, indigestible neutral detergent fiber content, potentially digestible neutral detergent fiber content, and in vitro neutral detergent fiber digestibility are presented in Figures 1A-D.

Gas production kinetics, in vitro digestibility, and metabolizable and net energy

There was no effect of interaction (p ≥ 0.05) between components and genotypes on gas production, in vitro digestibility, and energy (Tables 1 and 4). The genotypes did not affect (p ≥ 0.05) the gas production, in vitro dry matter digestibility (IVDMD), in vitro neutral detergent fiber digestibility (IVNDFD), nor the metabolizable and net energy of the sugarcane (Tables 1 and 4). When comparing the components, gas production for the rind was lower (p = 0.001) than that of the pith (Tables 1 and 4) within 24 h of in vitro incubation. However, there was no difference in components regarding sugarcane’s metabolizable (p = 0.085) and net (p = 0.091) energy. Metabolizable and net energy for the rind were lower than the pith, approximately 9.61 and 12.38 % (Table 3), respectively. The rind presented IVDMD 6.5 % lower (p < 0.001) than the whole sugarcane. Additionally, IVNDFD was 18.38 % lower (365.59/447.91) (p < 0.001) than the whole sugarcane (Tables 1 and 4). There was no run effect (p = 0.526).

Sugarcane quality and biomass

Genotype did not affect on TSH (p = 0.173) or biomass (p = 0.771) (Table 5). However, G3 presented lower (p < 0.001) Brix than G1, G2, and G4. POL contents of the G3 genotype differed (p = 0.002) only in G1 and G5. Apparent purity was affected by genotypes (p < 0.001). G5 had 6.34 % more purity than G4. As regards TRS (p < 0.001) and RS (p < 0.001) sugars, only G2 did not differ from G3 (Table 5).

Discussion

Nutritional quality is essential in choosing a sugarcane variety for ruminant nutrition and productivity. However, one of the limitations of sugarcane in ruminant feeding is the low protein content and fiber digestibility. When the industry selects varieties, little attention is paid to the variables of the plant that affect its nutritional value. This study observed a difference between genotypes for the CP content. G2 was higher (19.8 g kg¹) than G4 (14.0 g kg¹) (Tables 1 and 2). However, 67.82 % of the CP is in the rind, from which 81.34 % is in the form of NDICP (Tables 1 and 2). Neutral detergent insoluble crude protein represents the B3 fraction of protein fractioning, i.e., the fraction slowly degraded in the rumen because it adheres to the cell wall and is highly escapable from rumen degradation (Sniffen et al., 1992Sniffen CJ, O'Conor JD, Van Soest PJ, Fox DG, Russell JB. 1992. A net carbohydrate and protein system for evaluating cattle diets: II. Carbohydrate and protein availability. Journal of Animal Science 70: 3562-3577. https://doi.org/10.2527/1992.70113562x
https://doi.org/10.2527/1992.70113562x... ; Lanzas et al., 2008Lanzas C, Broderick GA, Fox DG. 2008. Improved feed protein fractionation schemes for formulating rations with the Cornell Net Carbohydrate and Protein System. Journal of Dairy Science 91: 4881-4891. https://doi.org/10.3168/jds.2008-1440
https://doi.org/10.3168/jds.2008-1440... ). The CF content was lower for G4 than for the other genotypes (Tables 1 and 2). However, the rind has higher CF content than the pith, and the sugarcane rind’s wax, cutin, and suberin can explain this difference. They are polymerized fatty substances in the cell wall and reduce water loss from the plant (Nawrath, 2002Nawrath C. 2002. The biopolymers cutin and suberin. The Arabidopsis Book 1: e0021. https://doi.org/10.1199/tab.0021
https://doi.org/10.1199/tab.0021... ). The wax of the rind has always been attractive for industrial applications, mainly in the cosmetic and pharmaceutical industries (Nuissier et al., 2002Nuissier G, Bourgeois P, Grignon-Dubois M, Pardon P, Lescure MH. 2002. Composition of sugarcane waxes in rum factory wastes. Phytochemical 61: 721-726. https://doi.org/10.1016/S0031-9422 (02)00356-4
https://doi.org/10.1016/S0031-9422 (02)0... ). The pith has lower DM than the rind (Tables 1 and 2). This fact is due to the thickness and impermeability of the fatty substances of the rind, thereby preventing water loss. Furthermore, the rind is structurally divided into the outer and inner rind. The outer rind comprises dead cells, providing structural support and protection against mechanical damage and pathogens. The inner rind comprises living tissue, including the phloem, responsible for storing and transporting water and solutes throughout the plant (Rosell et al., 2017Rosell JA, Olson ME, Anfodillo T, Martínez-Méndez N. 2017. Exploring the bark thickness-stem diameter relationship: clues from lianas, successive cambia, monocots and gymnosperms. New Phytologist 215: 569-581. https://doi.org/10.1111/nph.14628
https://doi.org/10.1111/nph.14628... ). The higher NCF for the pith than the rind is due to the higher sucrose content in the pith. Ash had a higher content for the rind than the pith (Tables 1 and 2).

As regards the fibrous fractions, the rind showed higher levels of fiber than the pith. This result is due to the hemicellulose (NDF minus ADF), cellulose (ADF minus lignin), and lignin that grant greater rigidity, impermeability, and resistance to microbiological and mechanical attacks on plant tissues (Liu et al., 2018 Liu Q , Luo L , Zheng L . 2018. Lignins: biosynthesis and biological functions in plants. International Journal of Molecular Sciences 19: 1 - 16 . https://doi.org/10.3390/ijms19020335
https://doi.org/10.3390/ijms19020335... ). It was observed that the indigestible fraction (iNDF) accounted for most NDF (Figures 1A and B). The indigestibility is probably related to the lignin of the rind and the compact organization of cellulose microfibrils in the hemicellulosic polysaccharide matrix covalently linked to a complex lignin structure (Vega-Sánchez and Ronald, 2010Vega-Sánchez ME, Ronald PC. 2010. Genetic and biotechnological approaches for biofuel crop improvement. Current Opinion in Biotechnology 21: 218-224. https://doi.org/10.1016/j.copbio.2010.02.002
https://doi.org/10.1016/j.copbio.2010.02... ). Even so, lignin is the main component of the plant cell wall and is responsible for resistance to degradation (Bottcher et al., 2013Bottcher A, Cesariano I, Santos AB, Vicentini R, Mayer JLS, Vanholme R, et al. 2013. Lignification in sugarcane: biochemical characterization, gene discovery, and expression analysis in two genotypes contrasting for lignin content. Plant Physiology 163: 1539-1557. https://doi.org/10.1104/pp.113.225250
https://doi.org/10.1104/pp.113.225250... ). Thus, pdNDF presented low values, thereby reducing the fibrous fractions’ availability to the ruminal microorganisms (Figure 1C). The iDM showed the same behavior as the iNDF (Tables 1 and 3). The lignin of the rind was 71.21 % higher than the pith. The low lignin content in the pith is due to the negative correlation between lignin and sucrose, which caused a dilution effect. Lignin drastically reduces the efficiency of saccharification in the pith since tissues rich in syringyl (S) are more susceptible to enzymatic hydrolysis than those rich in guaiacyl (G) (Bottcher et al., 2013Bottcher A, Cesariano I, Santos AB, Vicentini R, Mayer JLS, Vanholme R, et al. 2013. Lignification in sugarcane: biochemical characterization, gene discovery, and expression analysis in two genotypes contrasting for lignin content. Plant Physiology 163: 1539-1557. https://doi.org/10.1104/pp.113.225250
https://doi.org/10.1104/pp.113.225250... ). The most common monolignols for lignin polymer formation are p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) residues. They are secreted in the apoplast and deposited in the cell wall by extracellular peroxidases and laccases (Bottcher et al., 2013Bottcher A, Cesariano I, Santos AB, Vicentini R, Mayer JLS, Vanholme R, et al. 2013. Lignification in sugarcane: biochemical characterization, gene discovery, and expression analysis in two genotypes contrasting for lignin content. Plant Physiology 163: 1539-1557. https://doi.org/10.1104/pp.113.225250
https://doi.org/10.1104/pp.113.225250... ; Dixon and Barros, 2019 Dixon RA , Barros J . 2019. Lignin biosynthesis: old roads revisited and new roads explored. Open Biology 9: 190215. https://doi.org/10.1098/rsob.190215
https://doi.org/10.1098/rsob.190215... ; Llerena et al., 2019Llerena JPP, Figueiredo R, Brito MS, Kiyota E, Mayer JLS, Araujo P, et al. 2019. Deposition of lignin in four species of Saccharum. Scientific Reports 9: 1-19. https://doi.org/10.1038/s41598-019-42350-3
https://doi.org/10.1038/s41598-019-42350... ).

Cell wall digestibility is complex and can be influenced by several factors such as porosity, surface area, ratio of lignin monomers, cellulose crystallinity, degree of polymerization that limits the access of cellulolytic enzymes to cell wall polysaccharides, lignin, suberin, and cross-links with hemicellulose (Pu et al., 2013Pu Y, Hu F, Huang F, Davison BH, Ragauskas AJ. 2013. Assessing the molecular structure basis for biomass recalcitrance during dilute acid and hydrothermal pretreatments. Biotechnology for Biofuels and Bioproducts 6: 1-13. https://doi.org/10.1186/1754-6834-6-15
https://doi.org/10.1186/1754-6834-6-15... ). In the present study, the rind presented lower IVNDFD content (Tables 1 and 4) than the pith, probably due to the higher contents of CF and lignin in the rind, thereby corroborating Pu et al. (2013)Pu Y, Hu F, Huang F, Davison BH, Ragauskas AJ. 2013. Assessing the molecular structure basis for biomass recalcitrance during dilute acid and hydrothermal pretreatments. Biotechnology for Biofuels and Bioproducts 6: 1-13. https://doi.org/10.1186/1754-6834-6-15
https://doi.org/10.1186/1754-6834-6-15... . For Wilson and Mertens (1995)Wilson JR, Mertens DR. 1995. Cell wall accessibility and cell structure limitations to microbial digestion of forage. Crop Science 35: 251-259. https://doi.org/10.2135/cropsci1995.0011183X003500010046x
https://doi.org/10.2135/cropsci1995.0011... , the rumen microorganisms cannot digest lignin and suberin (plant cell wall biopolymers). It prevents access to the polysaccharide matrix in the cell wall and affects digestibility. On the other hand, the pith showed higher IVNDFD content, which was caused by the higher saccharification, i.e., the number of sugar monomers released through enzymatic hydrolysis of cell wall polysaccharides (Ding et al., 2012Ding S, Liu Y, Zeng Y, Himmel ME, Baker JO, Bayer EA. 2012. How does plant cell wall nanoscale architecture correlate with enzymatic digestibility? Science 338: 1055-1060. https://doi.org/10.1126/science.1227491
https://doi.org/10.1126/science.1227491... ). Unlike most grasses, the overall digestibility of sugarcane does not decrease with maturity. Instead, there is a slight increase since the accumulation of soluble cell contents (sugars) offsets the decline in cell wall digestibility. The ability to maintain high digestibility with increasing maturity gives an important advantage to sugarcane as a feed crop, especially in the critical dry season when all other grasses and forages decline in quality and availability (Preston, 1977Preston TR. 1977. Nutritive value of sugar cane for ruminants. Tropical Animal Production 2: 125-142.). Furthermore, the pith also presented higher gas production (24 h) and IVDMD than the rind (Tables 1 and 4; Figure 1D). Another interesting report in this study was the similarity between the rind and pith regarding metabolizable (ME) and net energy (NE). This result is because of the contents of CP and CF in the rind. Even though the rind has a high content of NDICP, the equations for estimating ME and NE do not consider this fraction, only the CP content. Moreover, the equation does not consider the pdNDF levels. The rind, for example, showed a higher content of pdNDF than the pith (Figure 1C). All these factors may have influenced the ME and NE values.

In addition to nutritional characteristics, sugarcane’s production potential and quality are essential for the sugarcane industry and animal nutrition. However, the fibrous fraction (indigestible) affects the best use of sugarcane by animals. Five different genotypes were evaluated in this study, and no differences in productivity (TSH) were observed, although G1 produced 15.36 % less than G2 (Table 5). However, the Brix and POL contents varied between genotypes (Table 5). For Barnes (1974)Barnes AC. 1974. The Sugarcane. Leonard Hill Books, London, UK., the higher the Brix degree, the better the nutritional value of sugarcane since approximately 90 % of sugarcane’s dry matter consists of (soluble) carbohydrates. These carbohydrates are divided into fibrous (NDF, mainly) and non-fibrous, represented mainly by sucrose, although this also contains starch and reducing sugars (glucose and fructose). Sucrose is the primary pathway through which the phloem transmits carbohydrates from leaves to the rest of the plant to provide carbon and energy for the growth and accumulation of reserve products (Felix et al., 2009 Felix JM , Papini-Terzi FS , Rocha FR , Vêncio RZN , Vicentini R , Nishiyama Jr MY , et al . 2009. Expression profile of signal transduction components in a sugarcane population segregating for sugar content. Tropical Plant Biology 2: 98-109. https://doi.org/10.1007/s12042-009-9031-8
https://doi.org/10.1007/s12042-009-9031-... ). In sugarcane, ripening is a physiological process that involves the synthesis of sugars in the leaves, translocation of products, and sucrose storage in the stalk (Patrick et al., 2013Patrick JW, Botha FC, Birch RG. 2013. Metabolic engineering of sugars and simple sugar derivatives in plants. Plant Biotechnology Journal 11: 142-156. https://doi.org/10.1111/pbi.12002
https://doi.org/10.1111/pbi.12002... ). Polarization (POL) is an indicator of cane ripeness. The unripe cane has a high content of reducing sugars and color precursor compounds, resulting in low POL values with a dark-colored juice (Pereira et al., 2017Pereira LFM, Ferreira VM, Oliveira NG, Sarmento PLVS, Endres L, Teodoro I. 2017. Sugars levels of four sugarcane genotypes in different stem portions during the maturation phase. Anais da Academia Brasileira de Ciências 89: 1231-1242. http://dx.doi.org/ 10.1590/0001-3765201720160594
http://dx.doi.org/ 10.1590/0001-37652017... ). For Rhein et al. (2016)Rhein AFL, Pincelli RP, Arantes MT, Dellabiglia WJ, Kölln OT, Silva MA. 2016. Technological quality and yield of sugarcane grown under nitrogen doses via subsurface drip fertigation. Revista Brasileira Engenharia Agrícola Ambiental 20: 209-214. https://doi.org/10.1590/1807-1929/agriambi.v20n3p209-214
https://doi.org/10.1590/1807-1929/agriam... , POL is one of the main characteristics of sugarcane quality, along with purity and TRS. In the case of purity, G4 and G5 genotypes showed 80.93 % and 86.47 %, respectively. Purity indicates the sucrose content and is related to the sugarcane’s ripeness. The higher the purity, the greater the sucrose accumulation. As the sugarcane ages, purity tends to decrease and sugar’s color may change, reducing its nutritional value. The goal is to obtain purity greater than 80 % (CONSECANA, 2006). However, the purity of the sugarcane juice can be influenced by mineral and vegetable impurities added to the sugarcane at harvest (Oliveira et al., 2012Oliveira FM, Aspiazú I, Kondo MK, Borges ID, Pegoraro RF, Vianna EJ. 2012.Technology assessment of sugarcane varieties influenced by different fertilization and water stress. Revista Ceres 59: 832-840. https://doi.org/10.1590/S0034-737X2012000600014 (in Portuguese, with abstract in English).
https://doi.org/10.1590/S0034-737X201200... ). Although the genotypes did not affect energy concentration (Tables 1 and 4), the gas production (24 h), ME, and NE showed high mean values in G2 for pith and low values in G4. It was also observed that the POL and purity values did not differ between G2 and G4. However, G2 presented 10.82 % more biomass and 10.15 % more TSH than G4 (Table 4), thereby demonstrating the potential for ruminant nutrition. As regards the reducing sugars (RS), G3 showed a lower value (16.36 %) than other genotypes (but not statistically different from G2), which means this genotype will convert less sucrose into glucose and fructose. The SR tends to follow the POL, increasing with ripening (Durán-Soria et al., 2020Durán-Soria S, Pott DM, Osorio S, Vallarino JG. 2020. Sugar signaling during fruit ripening. Frontiers in Plant Science 11: 564917. https://doi.org/10.3389/fpls.2020.564917
https://doi.org/10.3389/fpls.2020.564917... ). In the present study the SR presented the same behavior as POL (Table 4). The TRS values showed the same behavior as RS (Table 5). The TRS indicates the total sugars in sugarcane, mainly sucrose and reducing sugars, and it is the most critical parameter for the industry and farmers (Costa et al., 2011Costa CTS, Ferreira VM, Endres L, Ferreira DTRG, Gonçalves ER. 2011. Growth and yield of four varieties of sugarcane (Saccharum sp.), in the third ratoon. Revista da Caatinga 24: 56-63 (in Portuguese, with abstract in English).).

Forage quality is an essential factor for adjusting intake, improving the efficiency of nutrient utilization, and reducing concentrate feedstuffs in the diet of ruminants (Tafaj et al., 2005Tafaj M, Kolaneci V, Junck B, Maulbetsch A, Steingass H, Drochner W. 2005. Influence of fiber content and concentrate level on chewing activity, ruminal digestion, digesta passage rate and nutrient digestibility in dairy cows in late lactation. Asian-Australasian Journal of Animal Science 18: 1116-1124. https://doi.org/10.5713/ajas.2005.1116
https://doi.org/10.5713/ajas.2005.1116... ). Low fiber digestibility is the main limiting factor for high-performance beef or dairy cattle-fed sugarcane-based diets (Corrêa et al., 2003Corrêa CES, Pereira MN, Oliveira SG, Ramos MH. 2003. Performance of Holstein cows fed sugarcane or corn silages of different grain textures. Scientia Agricola 60: 621-629. https://doi.org/10.1590/S0103-90162003000400003
https://doi.org/10.1590/S0103-9016200300... ). However, the digestibility of sugarcane does not decrease with maturity because the accumulation of soluble cell contents (sugars) offsets the decrease in cell wall digestibility (Preston, 1977Preston TR. 1977. Nutritive value of sugar cane for ruminants. Tropical Animal Production 2: 125-142.).

Although the rind has a higher content of pdNDF than the pith, the high lignin content in the rind influences the quality of the final fibrous fractions of sugarcane and directly impacts the nutritional value. The genotypes do not differ nutritionally. However, the G2 presents higher biomass and energy yields than the others, making it more attractive in ruminant nutrition.

Acknowledgments

This research was supported by Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), process numbers E-26/010.100974/2018, E-26/010.002458/2019, and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

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