Introduction
Maize and sorghum are feedstuffs commonly used for feedlot diets, and dry grain processing (i.e. milling and rolling) is the primary technique utilized (Samuelson et al., Reference Samuelson, Hubbert, Galyean and Löest2016; Pinto and Millen, Reference Pinto and Millen2018). It is well known that grain processing may improve grain digestibility, compared to its natural form (Owens et al., Reference Owens, Zinn and Kim1986). However, even when milled, cereal grain starch (STA) granules may still exhibit some resistance to microbial degradation in the rumen due to the protein matrix surrounding STA granules (McAllister et al., Reference McAllister, Rode, Major, Cheng and Buchanan-Smith1990; Hoffman et al., Reference Hoffman, Esser, Shaver, Coblentz, Scott, Bodnar, Schmidt and Charley2011). Among the available alternative grain processes, processing based on ensiling high moisture grains has been shown to increase the ruminal fermentation of STA over that of dry forms (Oba and Allen, Reference Oba and Allen2003). However, the use of high moisture grain silage has been questioned due to some operational problems. According to Paulino (Reference Paulino2018), there are no machines in Brazil with high mill capacity of high moisture grains. Thus, grain harvesting is usually faster than the milling process. This lack of synchronization between harvesting and milling processes may constrain the use of high moisture grains in larger operations. An alternative would be to use reconstituted grain silage (Paulino, Reference Paulino2018).
Digestibility is recognized as the main source of variation in the energetic value of feedstuffs for ruminants. The in vivo estimation of digestibility is considered to be the most accurate method available (Dijkstra et al., Reference Dijkstra, Forbes and France2005). However, increasing concern for animal welfare is increasing the community pressure on ethics committees for animal use in experiments. As a result, ethics committees have recommended stricter protocols and the reduction or, if possible, the replacement of animals with alternative laboratory methods (Hartung, Reference Hartung2008; Pinto et al., Reference Pinto, Fonseca, Brandao, Gern, Guimarães, Carvalho, Brito, Viccini and Martins2017). In addition, experiment length and the costs to the acquisition of animals, feeding and labour are important limitations of in vivo procedures (Doke and Dhawale, Reference Doke and Dhawale2015).
Alternative procedures, such as in situ and in vitro procedures, are viable options to reduce the use of animals, study duration and the amount of feedstuffs used in digestibility studies. In addition, a greater number of different feedstuffs or diets can be evaluated simultaneously using both in vitro and in situ procedures. Several studies have evaluated degradability estimations from in situ and in vitro procedures (Krizsan et al., Reference Krizsan, Nyholm, Nousiainen, Südekum and Huhtanen2012; Ali et al., Reference Ali, Van Duinkerken, Cone, Klop, Blok, Spek, Bruinenberg and Hendriks2014; Krieg et al., Reference Krieg, Seifried, Steingass and Rodehutscord2017). However, only a few studies have correlated the digestibility results from in situ or in vitro procedures with the values obtained by in vivo procedures (National Research Council, 2001; Ghoorchi et al., Reference Ghoorchi, Lund, Larsen, Hvelplund, Hansen-Møller and Weisbjerg2013; Lopes et al., Reference Lopes, Ruh and Combs2015). Furthermore, most in vitro and in situ studies have evaluated individual ingredients rather than complete diets (Holt et al., Reference Holt, Yang, Creech, Eun and Young2016; Zanetti et al., Reference Zanetti, Menezes, Silva, Silva, Rotta, Detmann, Engle and Valadares Filho2017; Fernandes et al., Reference Fernandes, Ávila, Pereira and Ferraretto2018).
Therefore, we hypothesized that (1) ensiling ground maize or sorghum grains after reconstitution reduce potentially degradable fraction in the rumen (b), increase the readily soluble fraction (a) and rate constant for degradation of faction b (c) of dry matter (DM), organic matter (OM) and STA; and (2) the in vivo digestibility of diets can be estimated by different incubation times when in situ and in vitro procedures are used. The aims of this study were (1) to evaluate the effects of ensiling ground maize or sorghum grains after reconstitution on fraction a, fraction b and c of DM, OM and STA; and (2) to estimate an appropriate incubation time when in situ or in vitro procedures can be used to estimate in vivo digestibility.
Materials and methods
Ensilage process
Approximately 60 days before the beginning of the experiment, the ensilage process of flint maize with 74.51% of vitreous endosperm (Dombrink-Kurtzman and Bietz, Reference Dombrink-Kurtzman and Bietz1993) and sorghum grains was carried out. For this, approximately 6000 kg of each grain were ground in a hammer mill (DMP-2, Nogueiras, São João da Boa Vista, São Paulo, Brazil) with a 3-mm sieve, and the DM content was measured (method 934.01; AOAC, 2012). Subsequently, each ground grain was reconstituted to reach a moisture content of 35%. The hydrated grains were ensiled in laboratory silos with a mean density of 1000 kg/m3. An additional 6000 kg of each grain was ground and stored dry. Therefore, two grains (maize and sorghum) with two processing methods (dry ground and reconstituted) were used in this study.
Animals, facilities and experimental design
The experiment was conducted in the Experimental Feedlot of the Animal Science Department at the Universidade Federal de Viçosa, Viçosa, MG, Brazil. Four rumen-cannulated Nellore young bulls (age = 8 ± 1.0 months; initial body weight = 262 ± 19.6 kg) were used in this experiment. Bulls were assigned into a 4 × 4 Latin square with four treatments and four replicates per treatment. Bulls were kept in a tie stall barn with concrete floor and equipped with water and feed troughs. The experimental periods, lasting 19 days each, encompassed 14 days for adaptation (Machado et al., Reference Machado, Detmann, Mantovani, Valadares Filho, Bento, Marcondes and Assunção2016) and 5 days for the in vivo digestibility and in situ and in vitro degradability procedures, as described below.
Evaluation of in vivo digestibility
Four experimental diets, consisting of 0.28 maize silage and 0.72 of concentrate (DM basis), were evaluated. The diets differed in the main grain type of the concentrate, as follows: dry ground maize (DGM), dry ground sorghum (DGS), reconstituted maize grain silage (RMS) or reconstituted sorghum grain silage (RSS). The diet was formulated according to the BR-CORTE recommendations (Valadares Filho et al., Reference Valadares Filho, Marcondes, Chizzotti and Paulino2010) to provide 130 g of crude protein/kg DM on a total DM basis and to support an average daily gain of 1.2 kg/day. The chemical composition of the feedstuffs used in the experimental diets is presented in Table 1, and the proportions of feedstuffs and the chemical composition of the diets are presented in Table 2.
Chemical composition of the feedstuffs used in the experimental diets
DM, dry matter; OM, organic matter; CP, crude protein; EE, ether extract; apNDF, neutral detergent fibre corrected for residual ash and residual nitrogenous compounds.
a Non-fibre carbohydrates = 1000 − [(crude protein + NDF corrected for residual ash and residual nitrogenous compounds + EE + ash], calculated according to Detmann and Valadares Filho (Reference Detmann and Valadares Filho2010).
b Urea plus ammonium sulphate in a 9 : 1 ratio.
Feeds and chemical composition of experimental diets used to estimate in vivo digestibility and in situ and in vitro ruminal degradation parameter of DM, OM and STA
a The diets differed in the main grain type of the concentrate, as follows: DGM, DGS, RMS or RSS.
b Premix guarantees (per kg of DM): 200–220 g of Ca, 10 mg of Co (Min), 500 mg of Cu (Min), 22 g of S (Min), 333 mg of Fe (Min), 178.41 mg of F (Max), 10 g of P (Min), 25 mg of I (Min), 17 g of Mg (Min), 1500 mg of Mn (Min), 1100 mg of monensin, 100 × 109 CFU of Saccharomyces cerevisiae (Min), 6.6 mg of Se (Min), 50 g of Na (Min), 100 000 IU of vitamin A (Min), 13 000 IU of vitamin D3 (Min), 150 IU of vitamin E (Min) and 2000 mg of Zn (Min).
c Urea + ammonium sulphate in a 9 : 1 ratio.
d NDF corrected for residual ash and residual nitrogenous compounds.
e Non-fibre carbohydrates = 1000 − [(crude protein − crude protein from urea + urea) + NDF corrected for residual ash and residual nitrogenous compounds + EE + ash], calculated according to Detmann and Valadares Filho (Reference Detmann and Valadares Filho2010).
All ingredients of the diets were weighed separately, then mixed at the time of feeding, such that a total mixed ration (TMR) was provided twice per day (8.00 h and 16.00 h). Diets were adjusted daily to allow for approximately 5% refusals of the offered total on an as-fed basis, in order to provide ad libitum treatments.
Intake was recorded during the data collection period (from day 15 to 19). Also, diet ingredients and refusals were sampled daily and stored at −20°C until further analysis. Each diet ingredient and refusals sample were combined per animal for each period (percent as-fed basis), dried in a forced-air oven (55°C) for 72 h, and ground in a knife mill (Tecnal, Piracicaba, São Paulo, Brazil) with a 1-mm sieve. These ingredients were analysed individually and used to calculate the dietary composition.
To estimate diet digestibility, total faeces collection was performed for five consecutive days (from day 15 to 19) of each experimental period. Faeces were collected from droppings on the concrete floor and placed in 30 litres buckets. At the end of each collection day (24 h), the buckets containing the samples were weighed, homogenized and a subsample per day was dried in a forced-air oven at 55°C for 72 h, and ground in a knife mill (Tecnal, Piracicaba, São Paulo, Brazil) with a 1-mm sieve. Furthermore, a composite faecal sample from the 5 days of collection was made for each animal per period, based on the DM content of the faeces collected on the individual days.
In situ degradation procedures
From day 15 to 19 of each experimental period, the in situ degradability of the DGM, DGS, RMS, RSS grains and each TMR was evaluated. All ingrediets of the diets were collected daily and stored at −20°C between day 4 and 10 of each experimental period, dried in a forced-air oven at 55°C for 72 h, and ground in a knife mill (Tecnal, Piracicaba, São Paulo, Brazil) with a 2-mm sieve. To compose the diet, maize silage and concentrate were weighed separately, maintaining the same roughage : concentrate ratio on a DM basis.
Approximately 5 g of dried sample was individually weighed into nylon bags (Sefar Nitex, Switzerland; 50-μm porosity, 400 cm2 surface area) and incubated in each animal. The incubation for each grain was carried out in the same animal that was receiving the corresponding treatment in the in vivo procedure. The sample mass to bag surface area was 12.5 mg/cm2. Incubation was performed to allow the following ruminal degradation times: 0, 1, 2, 3, 4, 6, 12, 24, 48, 72 and 96 h. The number of bags varied as a function of the incubation time to guarantee enough residual samples after incubation (i.e. more bags per sample were incubated for the longer incubation times relative to the shorter incubation times).
Samples were incubated in the rumen by attaching the bags to a steel chain with a weight at the end, to allow for continual immersion within ruminal contents. Bags were placed into the rumen in the reverse order of incubation hours such that all bags were removed at the same time for washing. After the incubation period, the bags were washed in running water followed by washing with cold tap water by hand by the same person. The endpoint for washing was the high clarity of rinse water (Zanetti et al., Reference Zanetti, Menezes, Silva, Silva, Rotta, Detmann, Engle and Valadares Filho2017). The 0 h bags were not incubated in the rumen, but as with the incubated bags, they were rinsed in running water. Samples were oven-dried at 55°C for 72 h. In sequence, bags were placed in an oven at 105°C for 2 h and weighed. The residues of each diet were removed from the nylon bags, ground in a knife mill (Tecnal, Piracicaba, São Paulo, Brazil) with a 1-mm sieve, and placed in a labelled plastic bag to obtain a sample of each diet per animal/incubation time. Residual samples in the bags of different time points were used to estimate the parameters of ruminal degradation.
In vitro degradation procedures
An in vitro degradability evaluation of the experimental TMR was performed for each experimental period. A system of four 4 litre digestion jars, equipped with slow rotation and a temperature controller, was used (TE-150; Tecnal, Piracicaba, SP, Brazil). The procedures for sample collection and the processing of maize silage and the concentrates of the experimental diets were similar to those performed for the in situ procedure. Ingredients were ground in a knife mill (Tecnal, Piracicaba, São Paulo, Brazil) with a 1-mm sieve. For the in vitro procedure, TMR samples were weighed (approximately 0.5 g/bag) into filter bags (F57, Ankom Technology, Macedon, NY, USA). Maize silage and concentrate were weighed separately, to reach the same TMR roughage : concentrate ratio as the in vivo trial. The bags were then heat-sealed and incubated in ruminal fluid for 0, 2, 4, 6, 12, 24, 48, 72 and 96 h. For each incubation, each jar received three bags of the diet/time point plus one bag with no samples (blanks) as a correction factor.
Two hours before the start of the in vitro procedure, two buffer solutions were made: (1) buffer A (10 g/l KH2PO4, 0.5 g/l MgSO4⋅7H2O, 0.5 g/l NaCl, 0.1 g/l CaCl2⋅2H2O and 0.5 g/l urea) and (2) buffer B (15 g/l Na2CO3, 1 g/l Na2S⋅9H2O). The buffers were stored separately in a heated room (39°C) for the temperature stabilization. Then, in a 10 litre container, approximately 1064 ml of solution B were added to 5320 ml of solution A. The ratio (1 : 5) was adjusted until the pH was 6.8 at 39°C (Holden, Reference Holden1999). After the pH adjustment, 1600 ml of the combined buffer solution were added to each of the digestion jar with the samples. The jars were purged with CO2 gas for 30 s before the lid was closed.
For the ruminal digesta collection, eight 1 lltre preheated to 39°C thermos bottles were used. Approximately 1 litre of rumen fluid from each bull was collected 2 h post-feeding, and put into four thermos bottles. Also, wet particulate matter of about 30 g was collected in eight different points of the rumen of each bull, and placed in four other thermos bottles. Rumen fluids and contents were transported back to the heated room (39°C) immediately after collection so that rumen digesta was collected within 15 min of its use. For the inoculum preparation of each animal, approximately 200 g of solid rumen contents were weighed and added to approximately 1 litre ruminal fluid in a blender. The blender was purged with CO2 gas and blended for 2 min (adapted from method 3 DAISYII Incubator, Ankom Technology and Benedeti et al., Reference Benedeti, Fonseca, Shenkoru, Marcondes, Paula, Silva and Faciola2018). The blended digesta was filtered through four layers of cheese cloth into a preheated (39°C) 2 litre flask (Holden, Reference Holden1999; Benedeti et al., Reference Benedeti, Fonseca, Shenkoru, Marcondes, Paula, Silva and Faciola2018). The flask was continuously purged with CO2 gas. Then, 400 ml of this inoculum were added to each digestion jar that already contained the 1600 ml of the combined buffer solution and the samples. It is noteworthy that each digestion jar received the inoculum and bags of the respective treatment. The jar was purged with CO2 gas for 30 s before the lid was closed.
Similar to the in situ procedure, bags were placed into each jar in the reverse order of the incubation hours (starting at 96 h) such that all bags were removed at the same time for washing. In order to maintain the anaerobic conditions during each incubation, the jars were continuously purged with CO2 gas immediately after the lid was opened, during the incubation time (approximately 2 s) and 30 s before the lid was closed.
After the incubation period, the bags were rinsed with tap water (similar to that performed for the in situ procedure) and oven-dried at 55°C for 72 h. Residual samples in the bags of the different time points were used to estimate the parameters of the ruminal TMR degradation of DM and OM. In this procedure, grain degradability was not evaluated alone, due to the in vitro incubator capacity limitation.
Chemical analyses
All collected ingredients, refusals and faeces were ground through a 1-mm screen for laboratorial analysis. These samples were analysed for DM, OM, nitrogen (N) and ether extract (EE), according to the AOAC (2012) methods 934.01, 930.05 and 981.10 and AOAC (2006) method 945.16, respectively. Neutral detergent fibre (NDF) analysis was performed according to the techniques described by Mertens (Reference Mertens2002), without the addition of sodium sulphite, but with the addition of thermostable alpha-amylase to the detergent. The NDF content was corrected for residual ash and protein (apNDF). Estimations of neutral detergent insoluble nitrogen followed the technique described by Licitra et al. (Reference Licitra, Hernandez and Van Soest1996). Non-fibre carbohydrates were calculated according to Detmann and Valadares Filho (Reference Detmann and Valadares Filho2010) by difference between the total feed (1000 g/kg of DM) and the contents of ash, crude protein, apNDF and EE. STA analysis was performed following the recommendations of Zinn (Reference Zinn1990). In situ residues were analysed for DM, OM and STA, and in vitro residues were analysed for DM and OM, according to previously described methods.
Degradation models and statistical analysis
For the in situ and in vitro evaluations, the DM, OM and STA degradation profiles were estimated using the Ørskov and McDonald (Reference Ørskov and McDonald1979) asymptotic function:
where Yt = degraded fraction of DM, OM or STA in time ‘t’, g/kg; a = readily soluble fraction, g/kg; b = potentially degradable fraction in the rumen, g/kg; c = rate constant for degradation of b, per h and t = time, h.
Estimated times for the in situ and in vitro incubations to assess the in vivo digestibility of DM, OM and STA were defined as the time in which in situ or in vitro degradation equalled in vivo digestibility. This can be obtained using the following equation:
where T = estimated time; a = readily soluble fraction, g/kg; b = potentially degradable fraction in the rumen, g/kg and c = rate constant for degradation of b, per h.
The parameters a, b and c of the described models were evaluated using the NLIN procedure of SAS (version 9.4, SAS Institute Inc., Cary, NC), from the Marquardt algorithm, to obtain the parameters of the non-linear regression for each animal and period.
A confidence interval was adopted to in vivo digestibility instead of a single average value of in vivo digestibility. The asymptotic confidence intervals for the in vivo digestibility of DM, OM and STA (1 − α = 0.95) and the analysis of the degradation parameters were performed using the PROC MIXED procedure of SAS (version 9.4, SAS Institute Inc., Cary, NC) following the model:
where Yijkm = response variable; μ = general constant; Gi = fixed effect of grain i; Pj = fixed effect of processing method j; (GP)ij = fixed effect of the interaction of grain i and processing method j; ak = random effect of animal k; pl = random effect of experimental period l and eijkm = residual random error.
In order to verify the biologically relevant issues of the fixed effect, three orthogonal contrasts tested the effects of grain type, processing method and their interaction, considering 0.05 as the critical level of the probability for type-I error.
Results
In situ degradation parameters of maize and sorghum with different processing methods
The ruminal degradation parameters of DM, OM and STA of reconstituted or DGM and sorghum grains estimated from in situ incubations are presented in Table 3. There was an interaction (P < 0.01) between the type of grain and processing method for the fraction a, fraction b and c estimates for DM, OM and STA. Regarding DM and OM degradation parameters, maize grains presented greater (P < 0.01) fraction a, lower (P < 0.01) fraction b and greater (P < 0.01) c, compared to sorghum grains. Similarly, reconstituted ground grain silages presented a greater (P < 0.01) fraction a, lower (P < 0.01) fraction b and greater (P < 0.01) c, compared to dry grains.
Ruminal degradation parameters of DM, OM and STA of reconstituted or DGM and sorghum grains estimated from in situ incubations
DGM, dry ground maize; DGS, dry ground sorghum; RMS, reconstituted maize grain silage; RSS, reconstituted sorghum grain silage; a, readily soluble fraction (g/kg); b, potentially degradable fraction in the rumen (g/kg); c, rate constant for degradation of b (per h).
a,b,cWithin row, means without a common superscript significantly differ (P < 0.05).
In relation to the STA degradation parameters, the interaction showed that the RMS and RSS had a greater (P < 0.01) fraction a, lower (P < 0.01) fraction b and greater (P < 0.01) c, compared to DGM and DGS, respectively. In addition, RMS presented a greater (P < 0.01) fraction a, lower (P < 0.01) fraction b and greater (P < 0.01) c, compared to RSS. However, no difference was observed for fraction a (P = 0.80) or fraction b (P = 0.89) between DGM and DGS. We observed that c was greater (P < 0.01) for RMS and DGM than for RSS and DGS, respectively.
In situ degradation parameters of total mixed rations
In situ ruminal degradation parameters of DM, OM and STA of diets based on reconstituted or DGM and sorghum grains are presented in Table 4. There was an interaction (P < 0.01) between the grain type and processing method for all in situ degradation parameters evaluated. Regarding DM, OM and STA degradation parameters, diets based on reconstituted grain silage presented a greater (P < 0.01) fraction a and lower (P < 0.01) fraction b in relation to diets based on dry ground grains. Furthermore, the RMS-based diet presented a greater (P < 0.01) fraction a and lower (P < 0.01) fraction b, compared to the RSS-based diet. However, there was no difference (P > 0.05) for fraction a and fraction b of the DM, OM, and STA between the DGM- and DGS-based diets.
Ruminal degradation parameters of DM, OM and STA of diets based on reconstituted or DGM and sorghum estimated from in situ incubations
a, readily soluble fraction (g/kg); b, potentially degradable fraction in the rumen (g/kg); c, rate constant for degradation of b (per h).
a,b,c Within row, means without a common superscript significantly differ (P < 0.05).
1 Diets were composed of 0.28 maize silage and 0.72 of concentrate (DM basis). Diets differed in the main grain type of the concentrate, as follows: DGM, DGS, RMS or RSS.
There was no difference (P > 0.05) in the c of DM and OM between the RSS- and DGS-based diets. On the other hand, both the RMS- and RSS-based diets presented a greater (P < 0.01) c in relation to the DGM- and DGS-based diets, respectively. With regards to the c of STA, there was no difference (P = 0.67) between the RSS-based diet and the DGS-based diet. However, the RMS-based diet presented a greater (P < 0.01) c in relation to the DGM-based diet. Similarly, there was no difference (P = 0.07) in the c of STA between the DGM- and DGS-based diets. However, the RMS-based diet presented a greater (P < 0.01) c of STA in relation to the RSS-based diet.
In vitro degradation parameter evaluation of total mixed rations
In vitro ruminal degradation parameters of DM and OM of diets based on reconstituted or DGM and sorghum grains are presented in Table 5. There was effect of the grain type and processing method for fraction a and fraction b of the DM and OM. Maize-based diets and reconstituted grain silage-based diets presented a greater (P < 0.01) fraction a and lower (P < 0.01) fraction b, in relation to the diets containing sorghum and dry grain-based diets, respectively. There was interaction (P < 0.05) between the grain type and processing method for c of the DM and OM. There was no difference (P > 0.05) in the c for DM and OM for diets based on RSS and DGM compared to DGS-based diets. On the other hand, diets based on RMS presented a greater (P < 0.01) c for DM and OM than diets based on DGM and RSS.
Ruminal degradation parameters of DM and OM of diets based on reconstituted or DGM and sorghum estimated from in vitro incubations
a, readily soluble fraction (g/kg); b, potentially degradable fraction in the rumen (g/kg); c, rate constant for degradation of b (per h).
a,bWithin row, means without a common superscript significantly differ (P < 0.05).
1 Diets were composed of 0.28 maize silage and 0.72 of concentrate (DM basis). Diets differed in the main grain type of the concentrate, as follows: DGM, DGS, RMS or RSS.
Estimation of in vivo digestibility through in situ and in vitro procedures
The in situ incubation time to access in vivo degradability of DM, OM and STA ranged between 19 and 24 h (Table 6, Fig. 1). On the other hand, in vivo degradability estimates of DM and OM were obtained after 25–37 h of in vitro incubation.
Suggested incubation times for in situ (A) and in vitro (B) procedures and incubation time intervals in which the in situ and in vitro degradability predicts the in vivo digestibility of DM, OM and STA for different diets.
Means and confidence intervals (1 − α = 0.95) of the in vivo digestibility coefficients, in situ degradability coefficients with 24 h and in vitro degradability coefficients with 36 h of incubation for the DM, OM and STA of diets based on reconstituted or DGM and sorghum
a Diets were composed of 0.28 maize silage and 0.72 of concentrate (DM basis). Diets differed in the main grain type of the concentrate, as follows: DGM, DGS, RMS or RSS.
Discussion
Degradation parameters of the grain and total mixed rations
STA is the main component in maize and sorghum grains, representing approximately 74% of these cereals. Consequently, variations in STA degradation parameters had a direct effect on the degradability of DM and OM, which explains the similar behaviour among these three components in the evaluated grains and diets.
We hypothesize that reconstituted grain silage would promote changes in the degradation parameters of DM, OM and STA. Corroborating with our hypotheses the reconstituted grain silages presented a greater fraction a and c and lower fraction b of DM, OM and STA for the in situ procedure compared to the dry grains for all evaluated components. However, the increase in the fraction a and c and reduction in fraction b of DM, OM and STA when the grains were reconstituted, in relation to dry grains, was more prominent for maize than for sorghum, characterizing the interaction effect. In addition, diets based on reconstituted grain silage and maize grain presented a greater fraction a and lower fraction b of DM and OM for in vitro procedure, compared to those based on dry grain and sorghum grain, respectively. Also, diets based on RMS presented greater c of DM and OM than diet based on RSS. The prolamins present in maize and sorghum and how the processing methods adopted act on these proteins maybe can explain the results obtained in the in situ and in vitro procedures.
The prolamins of sorghum (kafirins) and maize (zeins) are similar (Duodu et al., Reference Duodu, Taylor, Belton and Hamaker2003) and deserve special attention because they are water insoluble and resistant to ruminal degradation (Sniffen, Reference Sniffen1974; Gibbon and Larkins, Reference Gibbon and Larkins2005). These proteins are responsible for the formation of protein bodies that constitute the matrix that surrounds the STA granules in the cereal endosperm. Despite the similarity between the prolamins of maize and sorghum grains, kafirins are considered more hydrophobic than zeins (Duodu et al., Reference Duodu, Taylor, Belton and Hamaker2003). Although the same amount of water was used in the grain reconstitution process, the greater hydrophobicity by kafirins has been responsible for a lower water uptake capacity by the sorghum particles. Consequently, the lower water uptake capacity of the particle in sorghum may have contributed to the reduction of the amount of solubilized material and the rate of this material's colonization during the ensilage or in the rumen, which would explain the obtained results. Moreover, maize and sorghum prolamins presented differences in their organization within the grain and their interaction with non-protein components, such as STA (Duodu et al., Reference Duodu, Taylor, Belton and Hamaker2003). Kafirins contain a higher proportion of cross-linked fractions and a greater propensity to form intermolecular disulphide-cross linkages that could facilitate the formation of more covalent bonds compared to zeins. Therefore, enzymatic digestion by microorganisms or animals and STA solubility can be lower in sorghum grains compared to maize grains (Rooney and Pflugfelder, Reference Rooney and Pflugfelder1986; Duodu et al., Reference Duodu, Taylor, Belton and Hamaker2003; Belton et al., Reference Belton, Delgadillo, Halford and Shewry2006).
The physical, chemical and biological processes resulting from the reconstitution and ensilage of the grains may cause the prolamins to break down or even break from the structure of the STA granules (Hoffman et al., Reference Hoffman, Esser, Shaver, Coblentz, Scott, Bodnar, Schmidt and Charley2011; Junges et al., Reference Junges, Morais, Spoto, Santos, Adesogan, Nussio and Daniel2017; Valadares Filho et al., Reference Valadares Filho, Silva, Pacheco, Menezes, Godoi, Alhadas, Silva, Paulino and Rennó2018), increasing the area exposed to the enzymatic action of ruminal microorganisms (Hoffman et al., Reference Hoffman, Esser, Shaver, Coblentz, Scott, Bodnar, Schmidt and Charley2011). This set of factors may lead to an increase in the amount of soluble material and in the c. Similar results were verified by other studies (Pereira, Reference Pereira2012; Silva et al., Reference Silva, Borges, Lopes, Silva, Vieira, Duque, Borges, Rodrigues and Gonçalves2014; Arcari et al., Reference Arcari, Martins, Tomazi and Dos Santos2016) in which the reconstitution and ensilage of maize and sorghum promoted an increase in fraction a and reduction in fraction b of DM and STA.
According to Valadares Filho et al. (Reference Valadares Filho, Silva, Pacheco, Menezes, Godoi, Alhadas, Silva, Paulino and Rennó2018), the breakdown of prolamins in the reconstituted maize and sorghum silages is corroborated by a reduction of the insoluble nitrogen and an increase of non-protein nitrogen in reconstituted grain silage, compared to dry grains. Although the ensiling process changes the protein fractions for both grains, the reduction of insoluble nitrogen and increase of non-protein nitrogen over the ensiling time is greater for maize grains compared to sorghum grains (Valadares Filho et al., Reference Valadares Filho, Silva, Pacheco, Menezes, Godoi, Alhadas, Silva, Paulino and Rennó2018), demonstrating the greater resistance of sorghum prolamins to proteolysis during the ensiling process.
Although there was no difference between the fractions a and b of DM, OM and STA when comparing dry maize and dry sorghum diets in the in situ procedure, the c for the dry maize was greater, which can also be justified by the greater hydrophobicity of sorghum prolamins compared to maize (Duodu et al., Reference Duodu, Taylor, Belton and Hamaker2003); this would hamper the ruminal colonization of the STA particles, as previously discussed.
In vivo digestibility estimation
Alternative procedures for estimating in vivo digestibility have been studied for several years. The first studies establishing the relationship between in vivo digestibility and laboratory procedures were reported by Walker (Reference Walker1959) and Tilley and Terry (Reference Tilley and Terry1963). More recently, other studies (Krizsan et al., Reference Krizsan, Nyholm, Nousiainen, Südekum and Huhtanen2012; Ghoorchi et al., Reference Ghoorchi, Lund, Larsen, Hvelplund, Hansen-Møller and Weisbjerg2013; Lopes et al., Reference Lopes, Ruh and Combs2015) have been dedicated to the improvement of methods for predicting in vivo digestibility. However, the use of varied parameters and models (Tilley and Terry, Reference Tilley and Terry1963; Stern et al., Reference Stern, Bach and Calsamiglia1997; Krizsan et al., Reference Krizsan, Nyholm, Nousiainen, Südekum and Huhtanen2012) makes it difficult to compare results between procedures. In addition, most studies do not conduct the in situ and in vitro studies together with in vivo digestibility, and these studies are realized with feedstuff evaluations, mostly forage, rather than complete diets (Tilley and Terry, Reference Tilley and Terry1963; Ferraretto, et al. Reference Ferraretto, Fredin and Shaver2015; Holt et al., Reference Holt, Yang, Creech, Eun and Young2016). Therefore, the use of a standardized ruminal environment and the evaluation of complete diets may favour more accurate results.
Regardless of the evaluated diet, the time intervals of 19–24 h of in situ incubation and 25–37 h of in vitro incubation allow for the adequate estimation of in vivo digestibility for the evaluated components. Although the diets presented more than one incubation time, whose degradabilities were within the in vivo digestibility confidence interval, we decided to adopt common times between the diets that facilitated the incubation process. Therefore, 24 h of in situ incubation is recommended to estimate the in vivo digestibility of DM, OM and STA and 36 h for the in vitro digestibility of DM and OM. Some reasons may account for differences in the in situ and in vitro incubation times for in vivo digestibility estimations, such as the relatively slow fermentation of the fibre and the longer time required for the development and colonization of microorganisms in the in vitro procedures (Dewhurst et al., Reference Dewhurst, Hepper and Webster1995).
It is also worth noting that this is a preliminary study and further studies are needed to evaluate diets composed of different feedstuffs, processing methods and roughage : concentrate ratios, in order to verify the influence of these factors on the incubation times required for the estimation of in vivo digestibility.
In conclusion, reconstituted grain silage and maize grains present greater fraction a and c and lower faction b of DM and OM than dry ground grains and sorghum grains, respectively. Furthermore, the reconstitution process promotes grains protein matrix breakdown increasing STA availability. The incubation times required for in vivo digestibility estimations of DM, OM and STA are 24 h for in situ and 36 h for in vitro procedures.
Financial support
This work was supported by the National Council of Scientific and Technological Development (CNPq); the National Institute of Science and Technology in Animal Science (INCT – CA); the Coordination of Improvement of Personal Higher Education (CAPES); the Foundation for Research Support of the State of Minas Gerais (FAPEMIG) and Cargill.
Conflict of interest
The authors declare that they have no competing interests.
Ethical standards
The experiment was conducted following the approval of the animal handling and procedures described herein by the Ethics Committee for Animal Use (protocol CEUAP/DZO/UFV 42/2016).
