The Effect of Ensiling on the Starch Digestibility Rate of Rehydrated Grain Silages in Pigs Depends on the Hardness of the Maize Hybrid

Abstract

The aim of the present study was to determine the in vitro starch digestibility kinetics of rehydrated maize grain silages in pigs and to investigate the relationship between the in vitro starch digestibility rate and the physical properties of the mature grain. Grains of seven commercial maize hybrids were harvested at physiological maturity, rehydrated, and ensiled with a commercial inoculant during different ensiling periods (0, 21, and 95 days) in five replicates using a completely randomized design. The starch digestibility rate was determined using first-order kinetics following an in vitro digestibility procedure mimicking the stomach and small intestine of pigs. The tested hybrids differed in their physical properties (test weight, kernel size, and density and hardness), digestion coefficients, and starch digestibility rate (p < 0.05). The starch digestibility rate increased with an increasing ensiling period, with average values of 0.588, 1.013, and 1.179 1/h for 0, 21, and 95 days of ensiling period, respectively. However, the effect of ensiling was more pronounced in hybrids with higher grain hardness, reaching a rate of 1.272 1/h in hybrids with higher grain hardness compared to 1.110 1/h in hybrids with lower grain hardness. In conclusion, ensiling results in higher availability of starch to digestive enzymes, and the duration of ensiling and hardness of the maize hybrid should be considered when formulating the pig diet.

1. Introduction

Worldwide, maize is the main feed in pig nutrition due to its high production quantities, high available energy content, and relatively long storage [1,2]. Maize grains with a low buffering capacity [3] and sufficient simple sugars allow easy fermentation or ensiling [2]. Rehydrated or high-moisture grain silage is an excellent component in the liquid feeding of pigs [4]; it does not require drying, which reduces costs, and it does not produce greenhouse gasses [5]. Rehydrated or high-moisture maize grain silage can completely replace dry grain in the diets of pigs [6] and piglets [7], and it can replace up to 40% in broiler diets [8]. Pigs fed rehydrated maize grain silage (RMGS) achieve similar or lower daily gains and feed intakes compared to dry maize grain but a better feed conversion ratio [6,9] due to higher energy digestibility and higher metabolizable energy content [6]. The starch in RMGS is exposed to a breakdown in the protein matrix through the ensiling process, which increases starch digestibility [9] and significantly increases the metabolizable energy content (4231 kcal/kg compared to 3635 kcal/kg in dry grain) [10].

Starch utilization capacity is more associated with the characteristics of the source than with the monogastric species to which it is fed [11]. Maize hybrids commonly used in animal nutrition differ in their properties. Although their chemical composition is considered quite similar, there are major differences in their endosperm structure [12,13,14]. Maize endosperm contains vitreous and floury regions, which differ in the arrangement of the protein matrix surrounding the starch granules. In vitreous endosperm, the granules are tightly packed into the protein matrix, while the floury endosperm is loosely packed with empty spaces filled with air [15]. In addition, the prolamin zein proteins are the primary proteins in the starch–protein matrix [16], and cross-linking between the zein proteins encapsulates the starch granules in the starch–protein matrix, with their extensive cross-linking resulting in a vitreous endosperm [17]. Therefore, these two types of endosperm differ in their texture. They also differ in their chemical composition; Zurak et al. [18] showed that vitreous endosperm has a higher content of amylose, zein, and starch lipids, while floury endosperm has a higher content of non-starch lipids. Maize hybrids vary in the proportion of vitreous and floury endosperms, resulting in differences in grain vitreousness [12]. Grain physical properties are related to vitreousness, with maize hybrids with higher grain vitreousness having a higher density, test weight, and hardness, as well as a lower flotation index compared to hybrids with lower grain vitreousness [19,20].

Differences in grain vitreousness and physical properties affect the nutritional value of the maize grains [21]. Previous studies have shown that maize hybrids with higher grain vitreousness have lower starch digestibility rates in both monogastric and ruminant animals [22,23], suggesting that properties related to the endosperm structure affect the availability of starch to digestive enzymes. In general, intrinsic factors such as granule size, the presence of pores in the granule surface, the ratio of amylose to amylopectin, the degree of crystallinity, and the chain-length distribution of amylopectin affect the digestibility of starch from different sources [24]. Enzymatic hydrolysis occurs faster in starches that have a higher proportion of amylopectin compared to amylose, a higher proportion of short compared to long amylopectin side chains, a lower degree of crystallinity, smaller granules, and granules with pores and channels [25,26,27]. All of these effects can also be observed in maize grains, with the genotype determining these characteristics in maize hybrids [28]. In addition to the intrinsic starch properties, starch contains additional components such as proteins and lipids that may be located on the surface or embedded within the native starch granules [25]. These minor components could reduce enzyme binding by blocking the adsorption sites, thus reducing starch digestibility [28]. All of the above-mentioned properties that influence starch digestibility are related to the proportion of different starch digestion fractions, with a higher content of resistant starch in sources with a higher amylose content, a higher proportion of small, spherical B-type granules, and a higher proportion of amylose–lipid complexes, among other properties [29]. As mentioned for maize, the starch granules in cereals are embedded in a protein matrix containing prolamin, which acts as a barrier for the starch against digestive enzymes [12,18,28,30,31]. As a result, the starch digestibility rate is lower in cereal grains with a higher prolamin content [28,30,31].

As previously mentioned, the ensiling of rehydrated maize grain leads to the degradation of the starch–protein matrix via proteolysis caused by epiphytic and inoculated bacteria, thereby exposing the starch granules to digestive enzymes [32]. The extent of this effect depends on the maize genotype, i.e., grain vitreousness, and the duration of silage storage [33,34]. These studies showed that the content of total zeins decreased in the grains of all tested maize genotypes and with an increasing storage time of both high-moisture and whole-plant maize silage, resulting in increased starch digestibility. However, the extent to which ensiling affects starch digestibility in different maize hybrids or how grain physical properties could be related to starch digestibility in RMGS has not been investigated so far, especially in pigs. The hypothesis of the present study is that different maize hybrids will respond differently in their starch digestibility rate during the ensiling of rehydrated maize grain and that these differences will depend on physical properties. The aim of the present study was, therefore, to determine the starch digestibility kinetics in the RMGS of seven commercial maize hybrids that differ in their physical properties and to investigate the relationship between the starch digestibility rate during different ensiling periods and the physical properties of the mature grain.

2. Materials and Methods

2.1. Rehydration and Ensiling of Maize Grain

Seven commercially available maize hybrids provided by the Bc Institute (Zagreb, Croatia) were used for the study (

Table 1. Maize hybrids tested in the present study.

Abbreviation	Hybrid	Type	FAO Maturity Group
H1	Bc 344	hard dent	300
H2	Bc 418b	hard dent	460
H3	Bc 424	dent	460
H4	Bc 525	dent	510
H5	Bc 572	hard dent	500
H6	Kekec	semi-flint	330
H7	Pajdaš	hard dent	490

). These hybrids were selected because they are the most commonly used hybrids in Croatia, produced by a domestic breeding company. In addition, hybrids belonging to different FAO maturity groups were selected; FAO maturity groups from 300 to 500 are the most commonly used maize hybrids in Croatia and the region.

The tested maize hybrids were grown in the growing season of 2018 in a test field of Rugvica Station in central Croatia, near Zagreb (45°45′12″ N, 16°15′02″ E). All hybrids were grown under the same environmental conditions using an intensive production system [35]. The soil of the test field is classified as Gleysols with a heavy clay texture and with 47.5% of silt, 51.4% of clay, and 1.0% of sand in a depth of 0 to 30 cm. The soil contained 2.87% of organic matter, and the pH was 7.45. Each hybrid was planted on a 6 m-wide (8 rows) and 50 m-long plot, following the recommendations of the Bc Institute (Zagreb, Croatia) for optimum planting density. The daily average minimum and maximum temperatures (°C) and precipitation (mm/day) for the vegetation season were the following: 8.95, 22.47, and 0.84 for April, 13.07, 25.04, and 1.54 for May, 15.83, 26.6, and 3.72 for June, 16.85, 27.87, and 2.30 for July, 16.85, 29.82, and 1.47 for August, 11.79, 24.92, and 2.11 for September, and 6.07, 18.97, and 2.54 for October, respectively [36].

Once the hybrids had reached physiological maturity, the ears were harvested by hand at five locations (five replicates) within each plot. The total weight of the ears in each replicate was 20 kg. The ears were shelled, and the moisture content of the grains in each replicate of each hybrid was determined via drying at 103 ± 2 °C for 24 h [37]. A representative sample of 1 kg of grains was taken from each replicate to analyze the physical properties using the quartering method [38]. The samples were dried at 40 °C to 12% moisture and stored at 4 °C until analysis. The main nutrient composition of the tested hybrids is shown in Supplementary Table S1.

The grains of the tested maize hybrids were rehydrated and ensiled immediately after harvesting at the Department of Animal Nutrition of the University of Zagreb Faculty of Agriculture, where all analyses were also carried out. The grains of each replicate were rehydrated by adding water to achieve a moisture content of 32% (Supplementary Table S2). First, the grains were placed in plastic bags, and the required amount of water was added. The plastic bags were sealed and placed in a room with an average daily temperature of 20 ± 2 °C. The bags were turned every 8 h to ensure complete rehydration after 2 days. The rehydrated grains of each replicate were then ground with a hammer mill (B2m, Ino Brežice, Brežice, Slovenia) through a 5 mm sieve. The ground grains were thoroughly mixed, and 3 kg samples were taken from each replicate using the quartering method [38]. The samples were then sprayed with an inoculant solution (BIO-SIL^®, Dr. Pieper Technologie und Produktentwicklung GmbH, Wuthenow, Germany) containing lyophilized cultures of Lactiplantibacillus plantarum 8862 and 8866. According to the manufacturer’s instructions, the inoculant was used at a concentration of 300,000 CFU/g. The entire batch for each replicate was divided into three parts, each containing 1 kg of sprayed ground grains and representing a time point during ensiling (0, 21, and 95 days). The subsample representing 0 days of ensiling was immediately stored at −20 °C for further analysis, while the other two subsamples were immediately packed in 280 × 360 mm airtight nylon bags (Status, d.o.o., Metlika, Slovenia) and vacuum-sealed with a vacuum sealer (SmartVac, Status d.o.o., Metlika, Slovenia). These two bags of each replicate were ensiled at 20–25 °C for 21 and 95 days and stored at −20 °C for further analysis.

2.2. Analyses of Physical Properties of Maize Grain

In the analysis of physical properties, each replicate of the tested hybrids was analyzed in at least triplicate. The weight and volume of 200 kernels per replicate were recorded and multiplied by 5 to obtain 1000 kernel weight and volume. To obtain the test weight, the weight of 1000 kernels was divided by the volume of 1000 kernels. The kernel dimensions (height, length, and width) were measured with a digital caliper, and the data were used to calculate the kernel sphericity [39]. Grain hardness was determined using the Stenvert hardness test according to the method described by Pomeranz et al. [40]. The parameters of Stenvert kernel hardness were the time required to grind 17 mL of grits, the height of the grits in the grinding column, and the ratio of coarse (>0.7 mm) to fine particles (<0.5 mm; C/F) in the grits. According to the method, harder kernels have a longer grinding time, a lower height of grits, and a higher C/F. The density was determined based on the kernel weight and the volume occupied by it using a pycnometer with ethanol as a solvent. The flotation index was determined by stirring the maize grains in a sodium nitrate solution with a relative density of 1.25 [21]. The breakage susceptibility was determined using the HT-I drop-test device, which was constructed according to the scheme described by Kim et al. [41].

2.3. Analysis of Chemical Properties of Ensiled Maize Grain

To calculate the kinetics of starch digestibility in vitro, the contents of total starch and total sugar were determined in all frozen samples of ensiled maize grain (including 0 days of ensiling; Supplementary Table S1). The silages were thawed, dried at 40 °C for 48 h, and milled with a laboratory mill (Cyclotec 1093, Foss Tocator, Hoganas, Sweden) through a 1 mm sieve. The moisture content of all samples was determined by drying at 102 °C for 4 h [38].

The total starch content was determined using an enzymatic method (Total Starch Assay Procedure, amyloglucosidase/α-amylase method, Megazyme, Wicklow, Ireland) according to the method AOAC 996.11 [42]. The total sugar content was determined using a modification of Luff–Schoorl and Nelson–Somogyi methods [43,44]. The sample (4 g) was mixed with 125 mL of water in a shaker for 30 min. Carrez I and Carrez II solutions were added, followed by water up to 250 mL. After filtration, 25 mL of the solution was mixed with 15 mL of a 7.2% HCl solution and incubated in a water bath at 65–70 °C for 5 min. After cooling, the mixture was neutralized with a 28% NaOH solution and filled up to 25 mL with water. An aliquot (0.5 mL) was mixed with copper reagent and incubated in a boiling water bath for 10 min. After the aliquot had cooled, 1 mL of arsenic color reagent was added, and the mixture was allowed to stand for 5 min. Then, the mixtures were diluted with 15 mL of water and thoroughly mixed, and the absorbance was measured at 450 nm. Free glucose was quantified using a glucose standard curve (Kemika, Zagreb, Croatia).

2.4. In Vitro Starch Digestion of Ensiled Maize Grain

In vitro starch digestion was determined using a method that mimics the digestion process in the stomach and small intestine of the porcine digestive system [30] using the dried and milled samples of ensiled maize grain. Samples were incubated with 5 mL of 0.05 mol/L HCl solution containing 5 mg/mL pepsin (P7000; Sigma-Aldrich, Schnelldorf, Germany) for 30 min at 37 °C with horizontal shaking. After the first incubation, which mimics digestion in the stomach, the pH was adjusted to 5.2 by adding 20 mL of 0.1 mol/L sodium acetate buffer. Subsequently, 5 mL of the enzyme mixture containing pancreatin (amylase activity 48318 FIP-U/g; AppliChem, Darmstadt, Germany), amyloglucosidase (A7095; Sigma-Aldrich, Schnelldorf, Germany) and invertase (I4504; Sigma-Aldrich, Schnelldorf, Germany) were added. The mixtures were incubated for 5 h, and aliquots were taken at 0, 0.25, 0.5, 0.75, 1, 2, 3, 4, and 5 h after the addition of the enzyme mixture. The amount of released glucose was determined calorimetrically using a glucose oxidase-based method (D-Glucose Assay Kit, GOPOD format, Megazyme, Wicklow, Ireland). All samples were analyzed in duplicate on two consecutive days, and the average values were calculated for each incubation time point.

2.5. In Vitro Starch Digestion Calculations

The proportion of digested starch, i.e., the digestion coefficient (C_t), for each time point was calculated using the following equation:

$${\mathrm{C}}_{\mathrm{t}}={\displaystyle \frac{\mathrm{a}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{g}\mathrm{l}\mathrm{u}\mathrm{c}\mathrm{o}\mathrm{s}\mathrm{e} \mathrm{p}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{e}\mathrm{n}\mathrm{t} \mathrm{a}\mathrm{t} \mathrm{t}\mathrm{i}\mathrm{m}\mathrm{e} \mathrm{t}\times 0.9}{\mathrm{a}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{s}\mathrm{t}\mathrm{a}\mathrm{r}\mathrm{c}\mathrm{h} \mathrm{i}\mathrm{n} \mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e} \mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}}}\times 100$$

(1)

Starch digestibility follows first-order kinetics, described by the following equation [30]:

$${\mathrm{C}}_{\mathrm{t}}={\mathrm{C}}_0+{\mathrm{C}}_{\mathrm{\infty }}\times \left(1-{\mathrm{e}}^{-{\mathrm{k}}_{\mathrm{d}}\mathrm{t}}\right)$$

(2)

where C_t is the starch digested at time t (% of total starch), C₀ is the starch digested at 0 min (% of total starch) and represents soluble starch fraction, C_∞ is the potential digestibility of starch (% of total starch) and represents potentially digestible starch fraction, k_d is the starch digestibility rate (1/h), and t is the incubation time (min). The nonlinear, iterative least-squares Marquard method, implemented in the NLIN procedure within the SAS software (version 9.4; SAS Institute Inc., Cary, NC, USA), was used to model the starch disappearance curve and obtain C₀, C_∞, and k_d.

2.6. Statistical Analysis

The results were analyzed using SAS statistical software (version 9.4; SAS Institute Inc., Cary, NC, USA). The experiment was conducted in a completely randomized design with five replicates. Using the MIXED procedure, differences between hybrids in the analyzed physical properties were determined using an analysis of variance with the hybrid as a fixed effect with a one-way model as follows:

$$Y_{ij}=\mu +{\alpha }_i+e_{ij}$$

(3)

where µ is the population mean, α_i is the fixed treatment effect of hybrid i, and e_ij is the random error with mean 0 and variance σ². Using the MIXED procedure, differences in in vitro starch digestibility kinetics were determined using an analysis of variance with the hybrid, the ensiling period, and their interaction as fixed effects with a one-way model as follows:

$$Y_{ijk}=\mu +{\alpha }_i+{\beta }_j+{\left(AB\right)}_{ij}+e_{ijk}$$

(4)

where µ is the population mean, α_i is the fixed treatment effect of hybrid i, βj is the fixed treatment effect of ensiling period j, (AB)_ij is the effect of the interaction of the hybrid with the ensiling period, and e_ijk is the random error with mean 0 and variance σ². Mean values were defined using the least-squares means statement and compared using the PDIFF option; the letter groups were determined using the PDMIX macro procedure. The relationship between the physical properties and the in vitro starch digestibility rate was analyzed using the CORR procedure. The threshold for statistical significance was defined as p < 0.05.

3. Results and Discussion

3.1. Physical Properties of Tested Maize Hybrids

Grain physical properties are easy to determine with simple and rapid methods and have, therefore, often been used to assess quality differences between maize hybrids. In addition, some physical properties, such as hardness, are important indicators of the nutritional value of maize grains [45,46]. This suggests that physical properties could also be related to the kinetics of starch digestibility, as the intrinsic properties of the maize grains endosperm influence both. The maize hybrids tested in the present study differed in their physical properties (

Table 2. 1000 kernel weight and volume, test weight, and kernel dimensions of seven tested maize hybrids (n = 5).

Hybrid	1000 Kernel Weight	1000 Kernel Volume	Test Weight	Kernel
				Height	Length	Width	Sphericity
	g	mL	kg/hL	mm
H1	321.5 d	434.4 b	74.01 c	11.30 c	8.51 ab	4.52 bc	0.654 bc
H2	305.8 e	422.8 b	72.34 d	11.37 c	8.58 ab	4.38 c	0.662 b
H3	327.4 cd	456.2 a	71.77 d	11.77 b	8.61 ab	4.83 a	0.669 ab
H4	342.7 ab	465.8 a	73.57 c	12.25 a	8.48 b	4.37 c	0.629 c
H5	333.4 bc	428.4 b	77.83 a	11.21 c	8.39 b	4.69 ab	0.700 a
H6	263.7 f	340.2 c	77.49 a	11.33 c	7.76 c	4.51 c	0.665 b
H7	346.7 a	452.6 a	76.61 b	11.75 b	8.73 a	4.69 ab	0.667 b
SEM	3.9	4.9	0.30	0.12	0.08	0.06	0.011
p	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	0.0102

a–f Different letters indicate a statistically significant difference in the presented physical properties between maize hybrids at p < 0.05. SEM—standard error of the mean.

and

Table 3. Density, flotation index, Stenvert hardness parameters, and breakage susceptibility of grains from seven tested maize hybrids (n = 5).

Hybrid	Density	Flotation Index	Stenvert			Breakage Susceptibility
			Time	Height	C/F
	g/mL	%	mm			%
H1	1.249 bc	40.90 c	4.84 c	8.15 c	0.664 bc	36.62 b
H2	1.230 d	75.90 a	4.45 d	8.55 b	0.620 d	29.69 d
H3	1.232 cd	64.00 b	4.43 d	8.64 b	0.634 cd	25.57 e
H4	1.223 d	74.20 a	3.42 e	9.19 a	0.462 e	33.99 bc
H5	1.276 a	6.60 d	5.23 b	7.97 d	0.752 a	37.27 ab
H6	1.261 ab	9.06 d	5.60 a	8.10 cd	0.726 a	40.76 a
H7	1.253 b	11.10 d	5.00 bc	8.08 cd	0.672 b	30.73 cd
SEM	0.006	2.03	0.08	0.06	0.013	1.39
p	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001

a–e Different letters indicate a statistically significant difference in the presented physical properties between maize hybrids at p < 0.05. Time—time required to grind 17 mL of grits; Height—the height of the grits in the grinding column; C/F—the ratio of coarse (>0.7 mm) to fine (<0.5 mm) particles in grits from 20 g of maize grain; SEM—standard error of the mean.

).

The results obtained for physical properties are comparable to those obtained in previous studies [20,46,47,48], although the hybrids in the present study have a narrower range of values. The 1000 kernels of the maize hybrids tested averaged 320.2 g and 428.6 mL, giving an average test weight of 74.80 kg/hL. The 1000 kernel weight is associated with grain size and hardness, while the test weight is positively associated with grain vitreousness and could give an indication of the nutritional value of the grains [47,49]. Furthermore, grains of hybrids with higher 1000 kernel weight also had larger kernels (higher height, length, and width), while kernel height was negatively correlated with the Stenvert grinding time and C/F value. Of the hybrids tested, hybrid H6 had the lowest values for 1000 kernel weight and volume, with approximately 20% lower values compared to the other hybrids, in agreement with the lower values for 1000 kernel weight and test weight in flint compared to dent hybrids [50]. This hybrid also had among the smallest grains, reflected in the kernel height, length, and width of the tested hybrid (Table 2). On average, the tested hybrids had kernels 11.60 mm high, 8.44 mm long, and 4.57 mm wide. Kljak et al. [47] have shown that kernels with lower height and width in 80 commercial maize hybrids have a higher grain test weight and hardness.

The grain density and the flotation index are two properties that can be used as a direct measure of maize hybrid hardness [20], with higher density and a lower flotation index indicating maize hybrids with higher grain hardness [47]. The hybrids tested in the present study had an average kernel density of 1.256 g/mL and a flotation index of 40.25%. The latter property showed great variability in the tested hybrids. The values ranged from 6.60 to 75.90%, indicating a variable proportion of less dense grains. The tested hybrids were categorized into two groups based on this property: H1–H4 had a higher flotation index of 63.75% on average, while H5–H7 had a lower flotation index of 8.92% on average. This differentiation of the hybrids into two groups based on the flotation index was also reflected in the estimation of grain hardness using the Stenvert hardness test. According to this test, harder grains require more time to grind and yield coarser grits, which is reflected in a lower height in the collection tube and a higher proportion of coarse particles [47]. For hybrids H1–H4, it took 4.29 s to grind 20 g of grains and obtain 8.63 cm high grits in the collection tube, with an average C/F of 0.596. For hybrids H5–H7, it took 5.28 s to grind 20 g of grain and obtain 8.05 cm grits in the collection tube, with an average C/F of 0.717. Consequently, the first group consisted of hybrids with a lower grain hardness and the second group of those with a higher grain hardness.

3.2. Starch Digestibility Kinetics of Tested Maize Hybrids

Digestion coefficients during in vitro starch digestibility procedure were determined after an incubation time of 0.25, 0.5, 0.75, 1, 2, 3, 4, and 5 h in maize grains before ensiling and RMGS after 21 and 95 days of ensiling. A significant effect of the hybrid and the ensiling period and their interaction was observed for all incubation time points except the last one (

Table 4. Digestion coefficients (C_t) of seven tested maize hybrids after 0, 21, or 95 days of the ensiling period at different time points during in vitro starch digestibility incubation (n = 5).

Hybrid	Ensiling Period	Time of Incubation/h
		0	0.25	0.50	0.75	1	2	3	4	5
		Ct/%
H1	0	7.50 dC	21.36 cB	31.49 dC	39.15 eC	48.45 dC	74.35 cB	94.44 aB	100.00 a	99.97
	21	10.82 cdA	26.07 cdA	40.43 dB	53.68 cB	63.91 cB	96.65 aA	100.00 A	100.00	100.00
	95	9.52 eB	26.03 eA	43.99 dA	57.42 dA	72.33 cA	97.12 bA	99.17A	100.00	100.00
H2	0	7.44 dB	21.60 cC	31.70 dC	42.71 bcC	52.24 cC	80.17 aC	85.98 dB	96.83 cB	99.55
	21	10.31 dA	26.91 bcB	44.09 bB	58.69 bB	73.25 aB	94.70 aB	99.94 A	100.00 A	99.46
	95	10.56 dA	29.43 bcA	48.73 bA	64.18 bcA	77.80 bA	100.00 aA	100.00 A	100.00 A	99.56
H3	0	10.29 bcB	22.40 bcC	31.89 dC	40.73 deC	47.24 dC	73.49 cC	86.33 dB	99.97 a	98.41
	21	11.22 bcA	25.24 dB	40.91dB	55.08 cB	64.87 cB	92.44 bB	100.00 A	99.79	99.74
	95	11.99 bcA	27.21 dA	47.05 c A	65.53 bA	72.49 cA	100.00 aA	100.00 A	100.00	100.00
H4	0	12.02 aA	23.87 aB	38.83 aA	44.79 aC	56.82 aB	77.46 bC	94.35 aB	100.00 a	99.99
	21	11.18 bcB	26.55 cA	43.69 bB	57.61 bA	68.28 bA	96.56 aA	99.82 A	100.00	98.66
	95	10.38 dB	24.72 fB	40.09 eA	55.15 eB	69.02 dA	92.24 cB	99.24 A	100.00	100.00
H5	0	10.19 cA	23.52 abC	34.10 cC	41.68 cdC	54.15 bcC	73.50 cC	92.20 bB	98.46 bB	100.00
	21	11.39 bcB	27.77 abB	46.78 aB	60.85 aB	74.17 aB	95.04 aB	100.00 A	100.00 A	100.00
	95	12.65 aC	33.23 aA	52.62 aA	69.31 aA	80.80 aA	98.27 abA	100.00 A	100.00 A	100.00
H6	0	9.64 cB	23.66 aC	35.67 bB	45.09 aC	55.53 abC	79.29 abC	90.29 cB	99.38 a	100.00
	21	11.78 bA	27.07 bcB	44.19 bA	58.37 cB	68.41 bB	92.07 bB	99.71 A	100.00	100.00
	95	11.71 cA	28.62 cA	45.35 dA	64.05 bcA	80.03 abA	97.15 bA	98.68A	100.00	99.45
H7	0	11.05 bB	23.56 aC	35.01 bcC	43.83 abC	55.51 abC	78.70 abC	91.17 bcB	99.53 a	100.00
	21	12.82 aA	28.87 aB	45.71 aB	60.61 aB	71.88 aB	95.46 aB	99.55 A	100.00	100.00
	95	12.52 bcA	30.49 bA	50.06 bA	62.86 cA	80.43 aA	99.06 abA	100.00 A	100.00	99.70
SEM		0.29	0.40	0.53	0.64	0.87	0.71	0.59	0.27	0.33
p	Hybrid (H)	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	0.119
	Period (P)	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	0.740
	H × P	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	0.017

a–e Different lowercase letters indicate a significant difference in the digestion coefficients between tested maize hybrids within the same ensiling period at p < 0.05. A–C Different uppercase letters indicate a significant difference in the digestion coefficients between different ensiling periods within the same maize hybrid at p < 0.05. SEM—standard error of the mean.

). The significant effect of the hybrid was expected, as found in previous studies in maize grains and RMGS [18,22,33,34].

With a range of 7.44–12.82%, the digestion coefficients in all maize samples were determined at the beginning of the intestinal phase of the in vitro digestion procedure (Table 4, Figure 1). This result indicates that the soluble starch fraction was present in all maize samples, most likely due to the acidic conditions during the gastric phase of the in vitro digestion procedure [51]. After the start of the intestinal phase, starch digestibility increased rapidly, reaching digestion coefficients of 47.24 to 80.80% after 1 h of incubation, depending on the hybrid and ensiling period. In the RMGS samples, maximum starch digestibility was reached after 2 h of incubation, while, in samples before ensiling, digestion coefficients gradually increased up to 4 h of incubation (99.17%), reaching maximum values after 5 h of incubation (99.70%).

When considering the effect of the ensiling period on the digestion coefficients, there is a clear difference between RMGS and grains before ensiling (Figure 2). This result is consistent with the solubilizing effect of the acidic conditions in silages on the protein matrix, which leads to a higher availability of starch for the digestive enzymes [33,34]. In addition, the difference in digestion coefficients between RMGS ensiled for 21 and 95 days was determined at incubation time points from 0.25 to 2 h. For example, the digestion coefficients for grains and RMGS after 21 and 95 days of ensiling were 42.57, 57.84, and 62.64% after 45 min of incubation and 52.85, 69.25, and 76.13% after 1 h of incubation, respectively. These results are consistent with previous findings showing that the prolonged exposure of the starch–protein matrix of maize grains leads to a more extensive solubilization of the protein matrix [33,34,52], which explains why RMGS after 95 days of ensiling reached higher digestion coefficient values earlier than RMGS after 21 days of ensiling. More specifically, Duvnjak et al. [53] have shown that a more intensive degradation of 27 kDa γ-zein leads to a higher starch availability and that the reduction in this zein protein in maize grain silages is primarily caused by enzymatic proteolysis.

The significant interaction between the hybrids and the ensiling period indicates that some hybrids respond differently, depending on the ensiling period. Digestion coefficients increased more for some hybrids in RMGS compared to rehydrated grains than for others. This higher increase was mainly observed for hybrids H5–H7 (Table 4), especially at time points up to 1 h of incubation. For example, the digestion coefficients after 30 min of incubation for grains and RMGS after 21 and 95 days of ensiling of these hybrids were 34.93, 45.56, and 49.34%, respectively. In contrast, the average values for the same digestion coefficients for hybrids H1–H4 were 33.48, 42.28, and 44.97%, respectively. Interestingly, the hybrids that showed a higher extent of ensiling on starch digestibility coefficients were also hybrids with higher grain hardness. Although this result was unexpected, ensiling appeared to have a more intensive solubilizing effect on the starch–protein matrix of harder maize grains. Possibly, the reason for this result is an intensive degradation of 27 kDa γ-zein during ensiling [53]. As Caballero-Rothar et al. [54] have shown, this zein protein is associated with harder, more vitreous endosperm. In addition, Kljak et al. [12] have shown that maize hybrids with higher vitreousness have smaller starch granules. Since higher vitreousness is associated with higher grain hardness [19], the exposure of starch granules after the degradation of the starch–protein matrix during ensiling may increase the extent of starch digestibility due to the larger surface area per molecule in hybrids with higher grain hardness [26,27].

Based on the starch digestibility coefficients determined during the 5-h incubation in the in vitro digestibility procedure, the parameters of starch digestibility kinetics were estimated (

Table 5. Kinetics of in vitro starch digestibility [soluble (C₀) and potentially digestible fraction (C_∞) and starch digestibility rate (k_d)] of seven tested maize hybrids after 0, 21, or 95 days of the ensiling period (n = 5).

Hybrid	Ensiling Period	C0	C∞	kd
		%	%	1/h
H1	0	6.97 dAB	103.22 aA	0.537 bC
	21	7.63 cdA	96.67 aB	0.920 dB
	95	6.28 eB	96.54 aB	1.075 dA
H2	0	6.82 d	96.70 cdA	0.637 aC
	21	7.31 cd	95.27 bB	1.076 bB
	95	7.48 d	95.02 bcB	1.243 bA
H3	0	10.27 bA	101.31 bA	0.579 bC
	21	8.23 bcB	95.55 bB	0.910 dB
	95	8.19 cB	94.61 cB	1.161 cA
H4	0	11.32 aA	94.88 e	0.630 aC
	21	8.00 bcdB	95.25 b	1.016 cA
	95	7.01 deC	96.12 ab	0.959 eB
H5	0	10.15 bcA	97.40 cA	0.564 bC
	21	8.46 bB	93.82 cB	1.117 aB
	95	10.17 aA	91.46 eC	1.356 aA
H6	0	9.34 cA	95.48 deA	0.649 aC
	21	9.34 aA	93.37 cdB	0.989 cB
	95	7.84 cdB	94.12 cdB	1.219 bA
H7	0	10.07 bc	95.97 deA	0.619 aC
	21	10.02 a	92.50 dB	1.064 bB
	95	9.31 b	93.03 dB	1.242 bA
SEM		0.29	0.45	0.012
p	Hybrid (H)	<0.001	<0.001	<0.001
	Period (P)	<0.001	<0.001	<0.001
	H × P	<0.001	<0.001	<0.001

a–e Different lower-case letters indicate a significant difference in the starch digestibility kinetics parameters between tested maize hybrids within the same ensiling period at p < 0.05. A–C Different upper-case letters indicate a significant difference in the starch digestibility kinetics parameters between different ensiling periods within the same maize hybrid at p < 0.05. SEM—standard error of the mean.

). However, it should be noted that the statistical iteration for the best fit to the nonlinear curve is not ideal for describing biological processes, as the sum of the contents of soluble and potentially digestible starch fractions was higher than 100%. Regardless, the hybrid, the ensiling period, and their interaction significantly affected the contents of the soluble and potentially digestible starch fraction, as well as the starch digestibility rate. On average, over the ensiling periods, H5 and H7 had the highest content of soluble starch fractions and, for hybrid H6, the lowest content of the potentially degradable starch fraction. The contents of the soluble starch fraction determined in the present study were higher than the 0.7% in maize grain and 3.4% in steam-flaked maize grain reported by Giuberti et al. [30]. The differences in the results may be due to the maize hybrid or the enzymes used in the in vitro digestibility procedures. However, the present study used rehydrated maize grain for the in vitro starch digestibility analysis. Therefore, it is possible that the processing of the maize grain before the in vitro digestibility procedure caused variations. On the other hand, Zurak et al. [18] reported a range of 1.11 to 6.90% for the soluble starch fraction content in 30 maize hybrids, while Brambillasca et al. [55] reported values closer to 10% for dry, soaked, and ensiled rehydrated sorghum grains.

Giuberti et al. [30] reported the content of the potentially digestible starch fraction of 95%, consistent with the range in the present study. The ensiling duration reduced the estimated content of the soluble and potentially digestible starch fraction. On average for the tested hybrids, the content of the soluble starch fraction decreased by in order of 9.28, 8.43, and 8.04%, while the content of the potentially digestible fraction decreased by an order of 97.85, 94.63, and 94.41% for grain and RMGS after 21 and 95 days of ensiling, respectively. The small but significant decrease in the contents of these fractions indicates that the silages were not completely inactive after 21 days of ensiling and that the processes continued during the stable phase [56].

The average starch digestibility rate was 0.588 1/h for rehydrated maize grain, 1.013 1/h for RMGS after 21 days, and 1.179 1/h for RMGS after 95 days of ensiling. The obtained results are difficult to compare with those of previous studies because different kinetic models or statistical approaches are used to calculate starch digestibility kinetics. Furthermore, studies involving in vitro starch digestibility of RMGS for monogastric animals are lacking. Those that could be compared refer to dry grain; the values obtained in the present study are similar to the lower values of the range reported by Zurak et al. [18] (0.73–1.63 1/h). Higher values were expected when the effects of processing the grains before ensiling and a high proportion of soluble starch were considered.

The almost twofold increase in the starch digestibility rate in RMGS compared to grains before ensiling confirms the extent of the effect of ensiling on the starch–protein matrix in maize grains. In addition, the further increase with a longer ensiling period confirms that silages are not completely inactive after the completion of the fermentation phase. Although this increase implies a higher proportion of digested starch in the intestine, it must be considered that rapidly digestible starch stimulates insulin secretion, which could consequently inhibit feed intake and increase adiposity [57]. In addition, it is important to ensure an optimal proportion of resistant starch, as its fermentation in the gut leads to the production of short-chain fatty acids that prevent the excessive growth of pathogenic bacteria [58]. Ultimately, the difference between digestible and resistant starch plays an important role in the microbial composition in the hindgut [59], the pig body composition, and subsequent growth [57]. It also plays an important role in the carbon footprint of feed production, as drying consumes more energy than ensiling [60].

In the hybrids tested, the differences in the starch digestibility rate increased with an increasing ensiling period. For rehydrated grains, the range was between 0.537 and 0.649 1/h, while, for RMGS after 21 days of ensiling, it was between 0.910 and 1.117 1/h. After 95 days of ensiling, the tested hybrids were divided into two groups based on grain hardness; hybrids with a higher hardness had a higher starch digestibility rate compared to hybrids with a lower grain hardness (1.272 vs. 1.110 1/h). This observation is consistent with higher digestion coefficients in the group of hybrids with a higher grain hardness.

3.3. Relationship Between Physical Grain Properties and Starch Digestibility Rate

The relationship between the physical grain properties and the starch digestibility rate of grains before ensiling and RMGS was evaluated, and the results for tested hybrids are shown in Figure 3. In addition to the rates for each ensiling period, the ratio between the rates at different ensiling periods was included to evaluate the extent of the ensiling of rehydrated maize grain on starch digestibility rate in terms of physical properties.

The starch digestibility rate of the rehydrated grains showed only a few correlations with physical properties; significant correlations were found for the 1000 kernel weight and volume (r = −0.36 and −0.34, respectively) and kernel width (r = −0.40). These results indicate that the hybrids with the higher starch digestibility rate were hybrids with lighter and smaller but thicker kernels. These results are in agreement with the expected higher starch digestibility rate in hybrids with grains of lower hardness [41], as the mentioned physical properties are also related to grain hardness [19,20]. However, it should be noted that processing before ensiling may affect starch and that more correlations would be found if the grains had not been processed [61].

Only one significant correlation was found between the starch digestibility rate of the RMGS after 21 days of ensiling and the physical properties of tested hybrids. Hybrids with a higher test weight had a higher starch digestibility rate (r = 0.47). Since this property is an indirect measure of grain hardness [19,20], the positive correlation implies that ensiling led to a higher increase in starch digestibility in hybrids with a higher grain hardness. In addition, the higher increase in hybrids with a higher grain hardness was supported by significant correlations with the 1000 kernel weight (r = 0.44), Stenvert height (r = −0.38), flotation index (r = −0.36), kernel density (r = 0.42), kernel height (r = 0.34), and kernel sphericity (0.38), as determined by the ratio between the starch digestibility rate of the rehydrated grains before ensiling and after 21 days of ensiling (k_d21/k_d0). Hybrids with higher grain hardness also had a higher 1000 kernel weight, density, width, and sphericity [19,20,47].

The relationship between the starch digestibility rate and physical properties in RMGS of the tested hybrids, which was only hinted at with one significant correlation in RMGS after 21 days of ensiling, was confirmed with the number of significant correlations in RMGS after 95 days of ensiling. The starch digestibility rate of RMGS after 95 days of ensiling correlated with the test weight (r = 0.49), Stenvert time (r = 0.69), Stenvert height (r = −0.69), C/F (r = 0.78), flotation index (r = −0.58), kernel density (r = 0.55), kernel height (r = −0.69), kernel width (r = 0.35), and kernel sphericity (r = 0.62). These correlations confirm the shift in the starch digestibility rate as a function of grain hardness. In addition, the increase in the number of significant correlations indicates that the effect of ensiling on starch digestibility of rehydrated maize grain was greater when the ensiling period was longer, so it could be detected with several physical properties indicating grain hardness. The effect of a longer ensiling period on starch digestibility agrees with previous findings [33,34,52], indicating a greater degradation of the starch–protein matrix [53] and possibly the effect of starch granules’ size on starch digestibility when the starch–protein matrix was degraded [26,27]. The extent of the ensiling period effect was also confirmed by the ratio between the starch digestibility rate in rehydrated grain before and after 95 days of ensiling (k_d95/k_d0) and the ratio between the starch digestibility rate after 21 and 95 days of ensiling (k_d95/k_d21).

4. Conclusions

The starch digestibility rate of rehydrated maize grain silage increased after ensiling, and the values increased with the ensiling duration. The effect on in vitro starch digestibility was observed in all tested hybrids. However, the extent to which the starch digestibility rate increased depended on the grain hardness of the tested hybrids; hybrids with higher grain hardness showed higher rates than hybrids with lower grain hardness. Moreover, the effect was more pronounced during the longer ensiling period. These results suggest that ensiling increases the availability of starch to digestive enzymes and that the magnitude of this effect increases with time. As the use of RMGS depends on the silo size and the rate at which it is used, farmers should consider the time from the start of ensiling to optimize the composition of pig diets and provide a sufficient amount of other nutrients to utilize the readily available starch. In addition, farmers should also consider grain hardness to foresee the effects of hardness on starch digestibility during ensiling and potentially faster starch digestibility. Furthermore, future studies should focus on incorporating the kinetics of starch digestibility into the evaluation of the energy value of ensiled grains, its effects on the microbiota, and the ecological footprint of different maize hybrids.