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Article

Rehydrated Corn Grain Silage and Exogenous Protease: Effects on Dairy Cow Performance, Metabolism, and Starch Digestibility

by
Jefferson R. Gandra
1,2,3,4,*,
Rafael M. Mattos
2,
Thais M. D. M. Soares
2,
Cibeli A. Pedrini
3,
Antônio C. Martinez
2,
Euclides R. Oliveira
3,
Erika R. S. Gandra
1,
Wallison R. F. Vasconcelos
4 and
André C. Andrade
1
1
Veterinary Medine Faculty, Universidade Federal do Sul e Sudeste do Pará, FAMEV|IETU, Campus de Xinguara, Xinguara 68557, PA, Brazil
2
Veterinary Medine Faculty, Universidade Estadual de Maringá, Programa de Pós-Graduação em Saúde e Produção Animal Sustentável, Campus de Umuarama, Umuarama 87500, PR, Brazil
3
Animal Science Faculty, Universidade Federal da Grande Dourados, Programa de Pós-Graduação em Zootecnia, Dourados 79825, MS, Brazil
4
Animal Science Faculty, Universidade Federal Rural do Amazonas, Programa de Pós-Graduação Integrado em Zootecnia nos Trópicos, Campus de Parauapebas, Parauapebas 68515, PA, Brazil
*
Author to whom correspondence should be addressed.
Dairy 2025, 6(1), 1; https://doi.org/10.3390/dairy6010001
Submission received: 3 December 2024 / Revised: 19 December 2024 / Accepted: 22 December 2024 / Published: 26 December 2024
(This article belongs to the Section Dairy Animal Nutrition and Welfare)
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Abstract

:
Twenty-four Girolando cows (107 ± 56 days in milk, milk yield 22.0 ± 10.25 kg/d, and 529 ± 103 kg body weight) were used in a completely randomized design. The cows were divided according to the following experimental diets: 1—CON (basal diet with ground dry corn) 2—RCS (diet with total replacement of ground corn by rehydrated corn grain silage) 3—RCSP (diet with total replacement of ground corn by rehydrated corn grain silage + 500 g ton−1 of CINBENZA DP100®, NOVUS International, Inc (Chesterfield, Missouri, U.S.A.). enzymatic extract of Bacillus licheniformis, protease activity 600 IU g−1). Cows fed RCS + RCSP showed higher DMI, milk yield, and better efficiency (MY/DMI) compared to animals fed CON diet. Cows fed the CON diet had higher fecal concentration, and lower total starch digestibility and milk protein content compared to animals supplemented with RCS + RCSP. Cows fed RCSP showed a 2.96% superiority in total starch digestibility compared to animals fed RCS. Animals supplemented with RCSP presented higher milk yield (17.45%) and milk fat content (13.76%) than cows fed RCS. The inclusion of protease in rehydrated corn silage improved dairy cow performance, enhancing dry matter intake, milk yield, and productive efficiency. It also increased starch digestibility and milk protein and fat content, optimizing nutrient utilization.

1. Introduction

Rehydrated corn grain silage is an alternative feed option for ruminants that enhances the nutritional value of corn by improving its digestibility and stability. This improvement occurs through mechanisms such as increased starch gelatinization during the reconstitution process, which disrupts the starch structure and enhances enzymatic access, and the ensiling process, which promotes fermentation and reduces anti-nutritional factors. Additionally, the microbial inoculant used during ensiling supports a more favorable fermentation profile, enhancing lactic acid production and stabilizing the silage while potentially improving ruminal microbial activity and nutrient utilization. Through rehydration and ensiling, the grains undergo fermentation, resulting in a feed with increased energy availability and extended storage life compared to dried grains [1]. This process preserves high moisture corn, making it more palatable and easier to digest for livestock, while also reducing potential losses during storage. Given the growing interest in sustainable and efficient livestock production, rehydrated corn grain silage offers a practical solution for producers aiming to optimize feed resources, reduce waste, and maintain consistent nutritional quality [2].
The starch digestibility of rehydrated corn grain silage is a key factor influencing energy intake and milk production in dairy cows. During the rehydration and ensiling processes, structural changes in the starch granules increase their susceptibility to enzymatic breakdown in the rumen, leading to higher starch availability and absorption in the small intestine [3]. This improved starch digestibility not only provides a more consistent energy supply for lactating cows but also enhances feed efficiency and milk yield. Furthermore, optimizing starch digestibility in dairy cow diets through the use of rehydrated corn grain silage aligns with sustainable feeding strategies by improving nutrient utilization efficiency. Enhanced starch digestibility allows for better absorption of energy from the diet, potentially reducing the need for supplemental energy sources and minimizing feed waste. This leads to a more efficient use of resources, lower environmental impact from feed production, and reduced dependency on energy-intensive grain treatments such as dry grinding or chemical processing. By maintaining high nutritional standards, this approach contributes to both economic and environmental sustainability in animal production [4].
The addition of exogenous proteases to rehydrated corn grain silage presents notable advantages in enhancing starch digestibility for dairy cows by targeting corn prolamins, which encapsulate starch granules and restrict enzymatic access. Degradation of these prolamins directly influences ruminal metabolism by increasing the availability of starch for microbial fermentation, leading to more efficient utilization of dietary energy. This improved digestibility enhances feed efficiency, as more energy is absorbed from the diet, reducing the need for additional energy supplementation and improving overall cow performance [5]. Proteases break down these prolamins, releasing the starch and improving its accessibility to digestive enzymes in the rumen and small intestine. This action supports more efficient starch utilization, leading to enhanced energy availability and better feed efficiency. By integrating exogenous proteases into rehydrated corn grain silage, dairy producers can achieve improved milk yield and performance, as the enzymes help unlock the feed’s full nutritional potential, making this a valuable tool in sustainable dairy nutrition [6].
Based on the above, we hypothesize that the total replacement of ground corn grain by rehydrated corn silage with addition of exogenous protease improves the productive performance and metabolism of dairy cows. The objective of this study was to evaluate the replacement of total ground corn grain by rehydrated corn silage with addition of exogenous protease on dry matter intake, total starch digestibility, milk yield and composition, plasma biochemical profile, microbial protein synthesis, and fatty acid profile of milk from dairy cows.

2. Materials and Methods

2.1. Animals and Experimental Design

This trial was carried out on a commercial farm in the Umuarama region, PR between January and May 2023. Twenty-four Girolanda (3/4 to 7/8) cows (107 ± 56 days in milk, milk yield 22.0 ± 10.25 kg/d, and 529 ± 103 kg body weight) were used in a completely randomized design with repeated measures over time. The trial lasted 56 days. Sampling was performed on days 0, 7, 14, 28, 42, and 56.
The cows were housed individually in a free stall (25 m2) with free access to water. Cows were fed twice daily (700 and 1400 h) at TMR with 60:40 forage to concentrate ratio (Table 1) formulated to achieve or surpass the nutrient requirement estimates of NASEM [7].
The cows were divided according to the following experimental diets: 1—CON (basal diet with ground dry corn) 2—RCS (diet with total replacement of ground corn by rehydrated corn grain silage) 3—RCSP (diet with total replacement of ground corn by rehydrated corn grain silage + 500 g ton−1 of CINBENZA DP100®, NOVUS Internacial, Inc. enzymatic extract of Bacillus licheniformis, protease activity 600 IU g−1). The exogenous enzyme complex CINBENZA DP100®, NOVUS Internacial, Inc. enzymatic extract of Bacillus licheniformis, protease activity 600 IU g−1 was added at a dosage of 500 g/ton at the time of ensiling the rehydrated corn.
The reconstitution process for the corn grain was carried out by adding water to the dry ground corn to achieve a target moisture content of 35%. The rehydrated corn was allowed to rest for 24 h to ensure uniform moisture distribution before being ensiled. The ensiling process was conducted in trench silos covered with plastic sheeting to maintain anaerobic conditions. A microbial inoculant containing (Lentilactobacillus buchneri (CCT 3746, 5.0 × 1010 CFU/g), and Lentilactobacillus hilgardii (CCT 5840, 5.0 × 1010 CFU/g)) was applied at an inclusion rate of 8 g/ton of fresh matter during ensiling. The silos were sealed and opened after 45 days of fermentation. The particle size of the dry ground corn prior to reconstitution was approximately 2 mm, while the reconstituted and subsequently ground corn had an average particle size of 1.5 mm, measured using a particle size analyzer with a standardized sieve set. These parameters were carefully controlled to ensure consistency and reliability of the experimental treatments.

2.2. Sampling and Chemical Analysis

According to AOAC International [8], samples of corn silage, rehydrated corn grain silage, and concentrate components were examined for the presence of ether extract (method 920.39), crude protein (N × 6.25, Kjedahl method 984.13), and DM (method 930.15). According to Van Soest et al. [9], samples were also examined for the presence of lignin, neutral detergent fiber (aNDF), and acid detergent fiber (aADF). The detergent solutions included alpha amylase and sodium sulfite. As explained by Hendrix [10], the starch content of the samples was determined using an enzymatic degradation technique and the glucose absorbance was evaluated using a spectrophotometer. The amount of fodder and concentrate given to the cows, together with their daily orts, were measured in order to assess their dry matter intake. Throughout the sampling period, ort samples were taken every day and frozen for further analysis of the DM content.
Fecal samples (500 g) were collected directly from the rectal ampulla of each animal before each milking to measure fecal starch content according to Hendrix [10]. The calculation of total starch digestibility was performed according to Fredin et al. [11].
Cows were milked twice daily (600 and 1600 h) with milk yield recorded daily. Milk samples were collected on days 0, 7, 14, 28, 42, and 56 to determine concentrations of fat, protein, lactose, defatted dry extract, total dry extract, and milk urea nitrogen by mid-infrared method, while the flow cytometry technique was used to determine the variable Somatic Cell Count (SCC).
The body condition score (BCS) was assessed using the methodology established by Wildman et al. [12] and later refined by Edmonson et al. [13]. This assessment involves visual and tactile evaluations of body fat reserves at specific anatomical points on the cow’s body. The scoring system operates on a biological scale ranging from 1 to 5, with increments of 0.25 points. A score of 1 indicates a very thin cow, while a score of 5 reflects a very obese cow. This scoring method is independent of the cow’s body weight or size parameters, such as height, thoracic circumference, and length [12,13]. Additionally, the cows’ body weights were measured using a digital scale, with all weighings conducted prior to milking each morning.
Blood samples were collected into plasma and serum tubes on day 15 of each experimental period by puncture of coccygeal vessels, after the morning milking and before feeding. No blood samples were collected after the afternoon milking shift. Blood samples were centrifuged (2000× g for 15 min) and the supernatant (plasma and serum) was stored frozen for further analyses.
Concentrations of glucose, total cholesterol, triglycerides, total protein, albumin, urea, and urea nitrogen were analyzed using commercial kits (Bioclin, Belo Horizonte, Brazil) by enzymatic colorimetric methods with absorbances analyzed on a spectrophotometer (SBA-200 CELM, São Paulo, Brazil). Whole blood samples were collected into tri-potassium EDTA tubes for complete blood cell counts using an automatic analyzer (BC-2800 Vet; Mindray Animal Medical, Mahwah, NJ, USA).
Urine samples were collected 4 h after the morning feeding. Urine samples were filtered and frozen for further determination of N content according to AOAC [8]. Daily urinary volume was estimated based on the ratio between total creatinine excretion and creatinine concentration presented in urine spot samples [14]. The daily creatinine excretion (CE) was estimated as follows: CE (mg/kg LW) = 32.27 − 0.01093 × LW (kg). Urine creatinine concentration was determined by an enzymatic colorimetric method using commercial kits (Laborlab™, Osasco, Brazil) and absorbance was measured in a biochemistry analyzer (SBA-200, CELM™, Sao Caetano do Sul, Brazil).
Microbial protein synthesis was estimated based on the urinary excretion of purine derivatives (PD; allantoin and uric acid) according to Chen and Gomes [15]. Aliquots (10 mL) of urine were diluted in sulfuric acid (40 mL, 0.036 N) to avoid purine derivative destruction and uric acid precipitation. Allantoin and uric acid concentrations in urine were determined by a colorimetric method according to Fujihara et al. [16]. Excretion of PD was calculated as the sum of allatoin and uric acid excreted in urine (mmol/d). Absorbed microbial purines (Pabs, mmol/d) were calculated based on the equation Pabs = (PD − 0.512 × LW0.75) ÷ 0.70, wherein 0.70 is the recovery of absorbed purines and 0.512 × LW0.75 is the endogenous excretion of PD [17]. Ruminal synthesis of microbial N (Nmic, g/d) was calculated according to Chen and Gomes [15], as follows: Nmic = (70 × Pabs) ÷ (0.83 × 0.134 × 1000), in which 70 is the N content in purines (mg N/mol), 0.134 is the ratio of purine N to bacterial N [18], and 0.83 is the intestinal digestibility of microbial purines.
Samples for the fatty acid profile in milk were collected. One sample (about 200 mL) was taken from the morning milking and one from the afternoon milking, according to Feng et al. [19]. The samples were centrifuged for the extraction procedure at 17,800× g for 30 min at 4 °C and almost 19,300× g for 20 min at 4 °C. The separated fat (300–400 mg) was methylated, and methyl esters were formed according to Kramer et al. [20]. Two internal standards, C18:0 and C19:0, were used to correct losses during the methylation process. Extraction of fat from foods was performed according to the method by Folch et al. [21] and methylation was performed according to Kramer et al. [20]. Lipids were extracted by homogenizing the sample with a 2:1 chloroform and methanol solution. Then the lipids were isolated after adding a 1.5% NaCl solution. Fatty acids were quantified by gas chromatography (GC Shimatzu 2010, Kyoto, Japan, with automatic injection), using a capillary column SP-2560 (100 m × 0.25 mm diameter and 0.02 mm thickness, Supelco, Bellefonte, PA, USA). The initial temperature was 70 °C for 4 min (13 °C/min) until reaching 175 °C, maintained for 27 min., then a new increase of 4 °C/min was started up to 215 °C, and maintained for 31 min. Hydrogen (H2) was used as carrier gas with a flow rate of 40 cm/s. During the identification process, four standards were used: standard C4-C24 fatty acids (Supelco® TM 37, EUA, San Luis, AZ, USA), vaccenic acid C18:1 trans-11 (V038-1 g, Sigma®, EUA, San Luis, AZ, USA), C18:2 CLA trans-10, cis −12 (UC-61M 100 mg), and C18:2 cis-9, trans-11 (UC-60M 100 mg), (NU-CHEK-PREP USA®, EUA, San Luis, AZ, USA) for identification of the fatty acids that are formed during the biohydrogenation of unsaturated fatty acids.

2.3. Statistical Analyses

The data obtained were submitted to SAS [22], checking the normality of the residues and the homogeneity of variances by PROC UNIVARIATE. The data were analyzed by PROC MIXED according to the following model:
Yijk = µ + Ai + Dj + Tk + Dj × Tk + eijk
where Yijk = dependent variable, µ = general mean, Ai = animal effect (i = 1 to 24), Dj = diet effect (j = 1 to 3), Tk = time effect (k = 1 to 5), Dj × Tk = diet × time interaction effect, and eijk = error. The random effect of the model (random) was characterized by Ai. The degrees of freedom were corrected by DDFM = kr. The data obtained were subjected to analysis of variance and analyzed by repeated measures in time by the PROC MIXED command (SAS, 2004). Differences among treatments were evaluated by orthogonal contrasts to test the effect of diets: C1 (Control vs. RCS + RCSP); C2 (RCS vs. RCSP). Significance was declared when p ≤ 0.05.

3. Results

Dry matter intake (kg/d and %BW) was influenced (p ≤ 0.005) by the experimental diets. Cows fed RCS + RCSP showed higher DMI (p = 0.011) compared to animals fed the CON diet (15.13 vs. 15.98 kg/d)(2.83 vs. 2.98%BW) (Table 2). Additionally, animals supplemented with RCSP presented higher DMI (p = 0.035) (kg/d and %BW) compared to cows fed RCS.
Throughout the experimental period, cows fed RCSP showed higher DMI (p = 0.003) than the other experimental diets. Animals supplemented with RCS showed intermediate DMI and cows on the CON diet showed lower intake than the other animals (Figure 1).
Cows fed RCS + RCSP showed better efficiency (p = 0.042) (MY/DMI) compared to animals fed the CON diet (1.09 vs. 1.16). Additionally, animals supplemented with RCSP presented better efficiency (p = 0.046) (MY/DMI) compared to cows fed RCS.
The experimental diets strongly influenced the excretion (%) and total starch digestibility (%) (p < 0.0001). Cows fed the CON diet had higher fecal concentration (7.45 vs. 6.10%) and lower total starch digestibility (88.45 vs. 93.90%) (p < 0.0001) compared to animals supplemented with RCS + RCSP. Additionally, cows fed RCSP showed a 2.96% superiority (p = 0.015) in total starch digestibility compared to animals fed RCS.
Cows fed CON showed higher fecal starch excretion throughout the experimental period. However, cows supplemented with RCSP reduced fecal starch excretion from the 28th day of evaluation (Figure 2). Animals that received the RCS diet showed intermediate levels between the 14th and 42nd days of evaluation, showing no difference to the CON cows on the 56th experimental day.
Milk yield was influenced (p ≤ 0.005) by the experimental diets. Cows fed RCS + RCSP showed higher milk yield (16.80 vs. 18.74 kg/d), FCM (18.56 vs. 20.11 kg/d), and ECM (19.03 vs. 20.45 kg/d) compared to animals fed the CON diet (Table 3). Animals supplemented with RCSP presented higher (p ≤ 0.011) milk yield (17.45%), FCM (22.50%), and ECM (22.04%) than cows fed RCS.
Cows fed RCSP showed higher milk yield throughout the experimental period compared to the other animals evaluated (Figure 3). However, cows supplemented with RCS showed an intermediate level of milk yield, being superior to CON animals only on the 14th and 42nd days of the experimental period.
Cows fed RCS + RCSP showed higher (p ≤ 0.008) milk protein (kg/d and %) compared to animals fed the CON diet. On the other hand, cows supplemented with RCSP showed higher (p ≤ 0.038) milk fat (kg/d and %), milk lactose (kg/d), and total solids (kg/d) compared to animals supplemented with RCS.
Cows fed RCS + RCSP showed lower (p = 0.005) milk urea nitrogen concentration (mg/dL) compared to animals supplemented with the CON diet (12.78 vs. 10.61 mg/dL). Animals supplemented with RCSP showed lower (p = 0.032) somatic cell counts compared to cows fed RCS.
Cows fed RCS + RCSP showed higher (p ≤ 0.041) concentrations of milk fatty acids: C6:0; C15:1; C16:1, and C20:0 compared to cows supplemented with the CON diet (Table 4). However, cows fed RCSP showed higher (p ≤ 0.034) concentrations of milk fatty acids: C15:1 and C16:1 compared to animals supplemented with RCS.
Cows fed RCS + RCSP had higher (p = 0.024) plasma glucose (60.98 vs. 75.98 mg/dL) and lower (p = 0.033) total cholesterol (155.83 vs. 130.60 mg/dL), urea (28.42 vs. 22.57 mg/dL), and blood urea nitrogen (12.33 vs. 9.80 mg/dL) concentrations compared to animals supplemented with the CON diet (Table 5).
Cows fed RCS + RCSP had higher (p = 0.012) microbial synthesis compared to animals supplemented with the CON diet (Table 6). Animals fed RCSP showed greater (p ≤ 0.015) excretion of total purines (mmol/d), absorbable purines (mmol/d), as well as greater (p = 0.036) synthesis of nitrogen and microbial protein compared to cows supplemented with RCS.

4. Discussion

The addition of protease derived from Bacillus licheniformis during the ensiling process of rehydrated corn grains is hypothesized to enhance the productive performance of lactating cows. This improvement is attributed to the degradation of the protein matrix composed of zein, predominantly found in the endosperm of “Flint”-type corn grains, facilitating greater starch availability. While this process likely contributes to improved starch degradability by the ruminal microbiota, the potential effects of protease cannot be entirely isolated from the broader benefits associated with the rehydration and ensiling processes. Together, these factors may act synergistically to improve nutrient utilization and performance outcomes.
As starch reaches the rumen, its fermentation process begins alongside other dietary carbohydrates. The microorganisms involved are primarily bacteria and protozoa, with bacteria being the main agents responsible for starch digestion. Starch hydrolysis starts with the adhesion of ruminal bacteria to the starch granules, where interactions occur involving van der Waals forces with the substrate surface. This process begins with a hydrophobic ionic interaction that neutralizes the charges of both the bacterial cell membrane and the substrate, particularly calcium (Ca) and magnesium (Mg), as both exhibit negatively charged exteriors [9].
In whole corn grains, as well as grains with vitreous endosperm, bacterial adhesion is hindered by the pericarp and the protein matrix surrounding the starch, which are more resistant to digestion as they impede enzymatic activity [23]. This constitutes the focal point for the difference in starch digestion between diets using rehydrated corn grain silage with added protease. During the ensiling process, the pericarp is broken during grain grinding, hydration accelerates bacterial adhesion, and protease action breaks down the protein matrix surrounding the starch granules. Consequently, starch from rehydrated corn grain silage treated with protease becomes readily available, expediting bacterial adhesion to starch granules and, subsequently, the starch degradation process.
With the increased rate of attack on starch granules, a greater proportion of starch is degraded. The products of this degradation—oligosaccharides, disaccharides, or monosaccharides—are more readily absorbed by bacteria and utilized for microbial protein synthesis or volatile fatty acid (VFA) production. VFAs represent the primary energy source for ruminants [24].
In this study, cows fed the diet containing rehydrated corn grain silage with protease (RCSP) exhibited higher intake compared to those fed rehydrated corn grain silage without protease (RCS) and the control diet (CON) throughout the experimental period (Table 2; Figure 1). The rehydration of high-moisture corn improves the hydration of the endosperm, making starch more accessible to microbial and enzymatic action. The addition of exogenous protease, in turn, degrades the protein matrix that encapsulates starch granules, enhancing their availability for ruminal and intestinal digestion. This leads to greater starch utilization efficiency and increased metabolizable energy production, thereby promoting dry matter intake (DMI). Furthermore, the high starch digestibility reduces residues in the lower gastrointestinal tract, optimizing the animal’s energy balance, which further stimulates voluntary DMI and improves productivity [25].
In an evaluation involving cows receiving rehydrated corn grain silage as a replacement for ground dry corn in diets using sugarcane as the forage source, dry matter intake was higher with rehydrated corn grain silage [26].
Similarly, a study assessing diets with rehydrated corn grain silage replacing ground dry corn reported an effect on dry matter intake compared to other treatments [27]. Sheep fed rehydrated corn grain silage supplemented with amylolytic enzymes demonstrated higher dry matter intake than the control group without the addition of amylolytic enzymes [28].
In the evaluation of feed efficiency, expressed as milk yield per kilogram of dry matter intake (MY/DMI), animals fed RCSP achieved superior results compared to the other treatments (Table 2). Despite the higher dry matter intake (DMI) observed in these diets, the increased efficiency is attributed to improved starch availability and utilization as an energy source. This enhanced digestibility leads to greater production of volatile fatty acids (VFAs) in the rumen and increased glucose absorption (Table 6) in the small intestine, thereby boosting the efficiency of nutrient conversion into metabolizable energy [29]. As a result, the cow can meet its maintenance and production requirements with reduced energy waste and improved feed efficiency, ensuring higher milk yield (Table 3) relative to DMI, even under conditions of increased intake.
Animals fed rehydrated corn grain silage exhibited greater feed efficiency than those fed ground dry corn [30]. This finding aligns with a study that observed a positive interaction when evaluating the effects of rehydration and ensiling of corn grain on ruminal degradability [31].
One method for evaluating starch digestibility in the diet of lactating cows is through the analysis of fecal starch [11]. Fecal starch concentration has proven to be an accurate predictor of starch degradation and digestibility in dairy cows [32]. The starch content found in feces was lower in cows fed rehydrated corn grain silage (5.51%), and the addition of protease further reduced fecal starch content (4.69%), while the control group exhibited the highest value (7.45%) (Table 2; Figure 2), supporting the hypothesis of this study. Fecal starch levels above 5% indicate inadequate processing of starch-rich grains in the diet [33]. This finding can be attributed to the processing of corn grains during silage production and the protease action derived from Bacillus licheniformis, which contributes to the weakening of the protein matrix formed by zein, a protein highly concentrated in the endosperm of vitreous-type corn grains.
Milk yield was 17% higher (2.96 kg/day) for cows fed RSCP compared to those fed RCS and 20% higher (3.42 kg/day) than cows fed the control diet (Table 3; Figure 3). This increase can be attributed to the enhanced starch availability from the degradation of the protein matrix surrounding the starch granules by the exogenous protease, which improves fermentation by rumen microorganisms. This process results in greater production of volatile fatty acids (VFAs), particularly propionate, and promotes the synthesis of high-quality microbial protein, which supports milk synthesis. These VFAs, once absorbed, are metabolized in the liver, with propionate being converted into glucose, which is essential for lactose synthesis in the mammary gland, a key determinant of milk volume [27].
However, it is important to acknowledge that other studies [34,35] have reported no significant differences in milk yield or composition when using protease-treated silages. These discrepancies could be attributed to variations in experimental conditions, such as the type of forage used, the duration of silage fermentation, and differences in starch content and structure between diets. In this study, the silage duration may have influenced the extent of protease activity and starch availability, and the specific forage composition could have affected the balance of energy and protein available for milk production.
Future research should focus on isolating the effects of exogenous protease from those of other factors, such as forage type, silage fermentation dynamics, and basal diet composition, to better understand its potential and limitations in dairy cow diets. While the results of this study are promising, a more nuanced analysis, including acknowledgment of the study’s limitations, provides a balanced perspective on the implications of these findings.
The difference in milk production observed with the ensiling process and protease addition in this study contrasts with results from a study where replacing wet corn grain silage with ground dry corn showed no differences in milk yield or composition, reporting that the post-ruminal starch digestion of dry ground corn compensated for the high ruminal starch digestibility in cows consuming wet corn grain silage [36].
In a complementary study to this one by our research group, Durães et al. [34] reported that addition of protease to rehydrated corn silage (SGR) resulted in significant improvements in milk yield and milk yield corrected for 3.5% fat compared to the control diet. Specifically, cows fed the SGR diet exhibited a 9.64% higher milk yield (average of 17.93 kg/day) and a 10.8% increase in milk yield corrected for 3.5% fat. Production efficiency was also enhanced, with SGR-fed cows achieving 7.58% greater efficiency (1.34 kg of milk per unit of dry matter intake). These findings align with the hypothesis that protease enhances starch availability by degrading the protein matrix surrounding starch granules, thus improving fermentation by ruminal microorganisms and increasing the production of volatile fatty acids, particularly propionate.
However, it is important to contextualize these results in the broader literature, which includes studies reporting divergent outcomes [36,37,38]. For instance, previous research has shown no significant differences in milk yield or composition when wet corn grain silage was replaced with dry ground corn. This discrepancy was attributed to the compensatory effects of post-ruminal starch digestion in dry ground corn, which offset the benefits of high ruminal starch digestibility observed in wet corn grain silage diets. These contrasting findings highlight the multifactorial nature of dietary effects on milk production, which are influenced by the type of forage, starch source, and ensiling duration.
Our results emphasize the potential of protease-treated rehydrated corn silage to enhance productive performance in lactating cows. Nevertheless, they also underline the need for further studies to isolate the effects of protease from other dietary factors and to explore its performance under varying feeding conditions, forage types, and starch sources.
The site of starch digestion may shift from the rumen to the intestine, leading to greater starch digestion in the large intestine for dry ground corn treatments compared to wet corn [29]. Another factor to consider is the ensiling duration, as the corn grain protein matrix is degraded during the ensiling process, facilitating ruminal microbial access to starch granules [35]. The progressive degradation of the corn protein matrix by ruminal microbial activity and fermentation end products likely explains the increase in starch degradability over the storage period. While this does not directly affect feed intake, it may positively influence the digestibility of dry matter and nutrients, ultimately contributing to increased milk production.
The quantities, as well as the fat and protein contents of the milk yield, were higher in cows fed RCSP. This can be attributed to increased volatile fatty acid production and microbial protein synthesis by the ruminal microbiota, due to the greater availability of starch ingested by the cows. The impact of grain processing on milk fat content is inconsistent in the literature. However, the greater energy supply provided by the efficient digestibility of starch supports the mobilization of acetate and butyrate for milk fat synthesis. Thus, the interaction between starch digestibility, hepatic metabolism, and mammary biosynthesis explains the increased fat content in the milk of cows fed these diets [35].
In a recent published work, Durães et al. [34] reported a strong correlation between short-chain fatty acids and milk composition. The results indicate a clear correlation between ruminal fermentation parameters and milk production and composition. The inclusion of protease in the SGR diet led to significant increases in ruminal acetate, propionate, and total volatile fatty acid (VFA) concentrations, as well as quadratic behavior for butyrate synthesis, which aligns with enhanced milk production outcomes. Specifically, cows fed the SGR diet exhibited a 9.64% higher milk yield and a 10.8% higher milk yield corrected for 3.5% fat compared to the control diet. The linear increase in VFAs, particularly propionate, is a key driver of gluconeogenesis in dairy cows, which supports the observed increase in production efficiency and milk yield. Although milk fat, crude protein, and lactose contents remained unchanged, the quadratic behavior observed in milk fat content may reflect the nuanced effects of altered butyrate concentrations on milk fat synthesis. These findings underscore the role of optimized ruminal fermentation in enhancing both the quantity and efficiency of milk production [34].
The significant increase in milk production observed in the RCSP (Rehydrated Corn Silage with Protease) group on day 56 may impact the final statistical results. A potential physiological explanation for this increase could be related to the adaptation of the rumen microbiota to the diet, particularly with the inclusion of protease. Protease supplementation may enhance the digestibility of both protein and starch, leading to more efficient fermentation processes in the rumen. This, in turn, could result in greater production and availability of volatile fatty acids (VFAs), which are crucial precursors for milk synthesis. Furthermore, the protease could improve the overall nutrient utilization, particularly protein, thereby contributing to the observed increase in milk yield. This effect may become more pronounced after several weeks of dietary adaptation, leading to the significant increase in milk production observed on day 56 (Figure 3).
Higher milk fat content was reported in cows fed corn silage with processed grains [39]. Conversely, higher milk fat content was observed in cows fed whole-plant corn silage with unprocessed grains compared to processed grain silages [40].
The increased supply of amino acids resulting from more efficient protein synthesis (Table 5), supported by the effective digestibility of starch and the action of exogenous protease, contributes to the optimization of hepatic and mammary metabolism, leading to an increase in milk protein concentration. Microbial protein provides a balanced composition of essential amino acids, which are transported to the mammary gland for the synthesis of milk proteins [41].
Milk urea nitrogen (MUN) levels were lower in cows fed diets containing rehydrated corn grain silage (Table 3). Increased starch fermentation in the rumen resulted in higher microbial protein synthesis (Table 5), which was associated with greater protein synthesis in the mammary gland [42]. Among the diets, cows fed the CON diet exhibited a higher MUN value (12.78 mg/dL) compared to the average MUN levels of diets containing rehydrated corn grain silage (10.63 mg/dL). This difference can be explained by the lower energy availability in the rumen, which limits the utilization of nitrogen produced during ruminal fermentation, leading to increased ammonia absorption [43].
The lower MUN levels observed for the RCSP and RCS treatments, along with the reduced plasma urea nitrogen values for these diets (Table 5), indicate that the ensiling process may be creating a more favorable fermentation pattern for nitrogen utilization. This result also supports the hypothesis of this study.
The body weight and body condition score (BCS) of the cows were not influenced by the diets. However, it was observed that the animals maintained an adequate body condition for optimal milk yield performance. It is suggested that cows at the beginning of lactation should ideally have a body condition score of 3.00 ± 0.25 points, cows in mid-lactation 3.25 ± 0.25, and cows at the end of lactation 3.50 ± 0.25 [44]. Body weight and BCS were not affected by the processing of corn grains [45]. Cattle fed diets based on processed corn grains produced similar amounts of milk while maintaining their body weight and condition scores [46].
Milk fatty acids with carbon chains C15:1, C16:0, and C20:0, classified as medium- and long-chain fatty acids, showed an increase in cows fed diets containing rehydrated corn grain silage compared to the control (Table 4). Saturated milk fatty acids with chains of 18 carbons or more are primarily derived from the diet, with stearic acid being the most abundant fatty acid in milk [47].
The increased excretion of odd-chain fatty acids, such as pentadecenoic acid (C15:1), in the milk of dairy cows is directly associated with enhanced microbial protein synthesis in diets with higher total starch digestibility. During ruminal fermentation, microorganisms synthesize lipids containing odd- and branched-chain fatty acids, derived from the metabolism of precursors such as propionate and valeric and isovaleric acids. The high starch digestibility in these diets promotes a more efficient ruminal environment, increasing microbial protein production and, consequently, the release of microbial lipids into the gastrointestinal tract [48]. After digestion and absorption, odd-chain fatty acids from microorganisms are incorporated into the animal’s tissues and into milk lipid synthesis in the mammary gland, resulting in higher concentrations of C15:1 in the milk. Thus, the interaction between ruminal fermentation, microbial metabolism, and mammary synthesis explains the correlation between higher starch digestibility and increased excretion of odd-chain fatty acids in milk [49].
A positive relationship has been observed between the concentration of non-esterified fatty acids in the blood and 18-carbon fatty acids in bovine milk, indicating that cows with higher lipomobilization exhibit a greater proportion of 18-carbon fatty acids in their milk [50]. Long-chain fatty acids, ranging from 18 to 20 carbons, reach the mammary gland via the plasma and originate from the diet or fatty acid synthesis in bovine adipose tissue. These long-chain fatty acids can represent up to 55% of the fatty acids present in milk [51].
The increased supply of non-structural carbohydrates, with high starch content, can lower ruminal pH and microbial synthesis efficiency, while the lack of carbohydrates with a faster fermentation rate reduces the energy available for bacterial growth [52] (Table 5). A reduction of 1.2 mg dL−1 was observed for silages with processed grains [53]. The increase in starch digestibility in the rumen can affect pH and volatile fatty acid production in the rumen [40]. Higher ruminal propionate concentrations, which affected the acetate/propionate ratio, have been reported for lactating cows fed corn silages with processed grains [39,53].
Several factors influence starch digestion in ruminants, altering the dynamics and the amount of starch that will be fermented in the rumen or reach the intestine. Factors such as harvest maturity, moisture content, endosperm type, particle size, storage method, and fermentation duration of the silo affect starch digestibility [54,55]. The total starch digestibility in lactating cows can range from 70 to 100% [27,54]. Total starch digestibility values close to 95% are considered adequate, with values above 98% being ideal [11]. The values found for total starch digestibility for RCSP (95.30%) were higher than RCS (94.48%), which was also higher than CON (92.54%).
The increase in microbial protein synthesis should be considered alongside the amount of Rumen Undegradable Protein (RUP) flowing into the hindgut, as both factors contribute to the overall supply of amino acids for milk protein synthesis. While microbial protein synthesis provides a significant portion of the amino acids available for milk production, the RUP fraction, which bypasses ruminal fermentation, also plays a crucial role in supporting milk protein synthesis by providing essential amino acids directly to the small intestine. Therefore, a comprehensive discussion of milk protein synthesis must account for both the contributions of microbial protein and RUP to the amino acid pool, highlighting the balance between ruminal fermentation and post-ruminal protein supply [39,56].
Plasma glucose was higher for animals that received diets with rehydrated corn grain silage (Table 6), which helps validate the hypothesis of this study. The concentration of glucose present in the plasma is influenced by the production of glucose precursors, such as ruminal propionate [49], as well as by the duodenal absorption of glucose [57]. The stimulation of lactose synthesis and the subsequent increase in milk production are linked to the increase in plasma glucose concentration [58,59,60].
The increase in productivity of animals fed with rehydrated corn grain silage and the addition of protease can be justified by the improved efficiency of their digestive tract’s digestibility, with the lower fecal starch concentration serving as evidence, along with the reduction in milk urea nitrogen (MUN), of the increase in plasma glucose concentration, and the increase in long-chain fatty acids in milk.

5. Conclusions

The supplementation of dairy cows with rehydrated corn silage combined with the addition of protease yielded promising results, showing significant improvements in productive performance and milk quality. The increase in dry matter intake, milk yield, and productive efficiency reflects a positive effect of protease on digestion and nutrient utilization, enhancing cow performance.
Furthermore, the improvement in starch digestibility and the increase in milk protein and fat content indicate enhanced utilization of both energy and protein sources in the diet. These results suggest that the inclusion of protease in the diet of dairy cows can be an effective strategy to optimize milk production and quality, contributing to the sustainability and profitability of dairy farming.

Author Contributions

Conceptualization, J.R.G. and R.M.M.; methodology, J.R.G. and E.R.O.; software, C.A.P. and J.R.G.; validation, E.R.S.G., T.M.D.M.S. and R.M.M.; formal analysis, J.R.G. and C.A.P.; investigation, R.M.M. and T.M.D.M.S.; resources, A.C.M., E.R.O. and E.R.S.G.; data curation, W.R.F.V. and A.C.A.; writing—original draft preparation, J.R.G. and E.R.S.G.; writing—review and editing, C.A.P. and A.C.M.; visualization, W.R.F.V. and A.C.A.; supervision, J.R.G.; project administration, J.R.G.; funding acquisition, J.R.G., A.C.M. and E.R.O. All authors have read and agreed to the published version of the manuscript.

Funding

This review received no external funding.

Institutional Review Board Statement

The animal study protocol was approved by the Animal Ethics Committee of the State University of Maringá (CEUA/UEM, No. 2106091020, approved on 16 November 2020), following the precepts of Law 11,794 of 8 October 2008, Decree 6899 of 15 July 2009, as well as the standards issued by the National Council for the Control of Animal Experimentation (CONCEA).

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors upon request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Dairy 06 00001 g001
Figure 1. Dry matter intake throughout the experimental period according to the experimental diets. Letters a, b, and c indicate statistical differences among treatments (CON, RCS, and RCSP) at each time point (p < 0.05).
Figure 1. Dry matter intake throughout the experimental period according to the experimental diets. Letters a, b, and c indicate statistical differences among treatments (CON, RCS, and RCSP) at each time point (p < 0.05).
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Figure 2. Fecal starch excretion throughout the experimental period according to the experimental diets. Letters a, b, and c indicate statistical differences among treatments (CON, RCS, and RCSP) at each time point (p < 0.05).
Figure 2. Fecal starch excretion throughout the experimental period according to the experimental diets. Letters a, b, and c indicate statistical differences among treatments (CON, RCS, and RCSP) at each time point (p < 0.05).
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Figure 3. Milk yield throughout the experimental period according to the experimental diets. Letters a, b, and c indicate statistical differences among treatments (CON, RCS, and RCSP) at each time point (p < 0.05).
Figure 3. Milk yield throughout the experimental period according to the experimental diets. Letters a, b, and c indicate statistical differences among treatments (CON, RCS, and RCSP) at each time point (p < 0.05).
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Table 1. Ingredients and chemical composition of experimental diets.
Table 1. Ingredients and chemical composition of experimental diets.
IngredientsExperimental Diets 1
CONRCSRCSP
Corn silage60.0060.0060.00
Corn meal26.00--
Rehydrated corn grain silage 2-26.0026.00
Rehydrated corn grain silage + protease--26.00
Soybean meal11.0011.0011.00
Urea1.001.001.00
Mineral mix 32.002.002.00
Chemical composition
Dry matter51.7049.0949.13
Organic matter (%DM)93.8495.4795.46
Crude protein (%DM)15.1015.2415.52
Fat (%DM)3.813.173.31
Non-fiber carbohydrate (%DM)39.2639.1239.24
Starch (%DM)23.4523.2523.32
Neutral detergent fiber (%DM)35.6330.9531.23
Neutral detergent fiber (%DM), physically effective24.1523.5623.67
Acid detergent fiber (%DM)19.9017.8217.96
Lignin (%DM)4.094.454.59
Ash (%DM)6.164.534.54
Total nutrient digestible (%DM) 464.5966.3866.46
Net energy lactation 4 (Mcal/kg DM)1.441.491.49
1 CON (control diet); RCS (diet with total replacement of ground corn by rehydrated corn grain silage); RCSP (diet with total replacement of ground corn by rehydrated corn grain silage + 500 g/ton of CINBENZA DP100® enzymatic extract of Bacillus licheniformis, protease activity 600 IU/g). 2 RCS nutritional values (DM = 57.60; CP = 9.34 (%DM); starch = 61.51 (%DM); pH = 3.91; ammonia = 13.48 (%TN); lactate = 4.89 (%DM)); RCSP nutritional values (DM = 57.74; CP = 8.93 (%DM; starch = 67.43 (%DM); pH = 4.05; ammonia = 25.73 (%TN); lactate = 7.74 (%DM)). 3 Mineral mix (Ca 110 g/kg; P 42 g/kg; S 18 g/kg; Mg 20 g/kg; Na 123 g/kg; Co 14 mg/kg; Cu 600 mg/kg; Cr 20 mg/kg; Fe 1050 mg/kg; I 28 mg/kg; Mn 2000 mg/kg; Se 18 mg/kg; Zn 2800 mg/kg; biotin 80 mg/kg; vitamin A 240,000 UI/kg; vitamin D 100,000 UI/kg vitamin E 100,000 UI/kg. 4 Calculated according to NASEM, 2021.
Table 2. Dry matter intake, fed efficiency, and starch digestibility according to experimental diets.
Table 2. Dry matter intake, fed efficiency, and starch digestibility according to experimental diets.
ItemExperimental Diets 1SEM 2p-Value 3
CONRCSRCSP DietTimeIntC1C2
Dry matter intake, kg/d15.1315.5716.390.2070.0050.0030.0120.0110.035
Dry matter intake, %BW2.832.923.030.0290.0180.0750.0100.0300.106
Efficiency
MY/DMI 41.091.101.220.0250.0480.0330.0960.0420.046
FCM/DMI 51.311.331.350.0280.2310.5150.3350.5650.423
ECM/DMI 61.321.341.360.0260.8310.2310.4370.1180.528
Fecal starch, (%)11.457.514.690.012<0.00010.0880.730<0.00010.015
Total starch digestibility, (%)88.5592.4995.310.254<0.00010.0880.730<0.00010.015
1 CON (control diet); RCS (diet with total replacement of ground corn by rehydrated corn grain silage); RCSP (diet with total replacement of ground corn by rehydrated corn grain silage + 500 g/ton of CINBENZA DP100® enzymatic extract of Bacillus licheniformis, protease activity 600 IU/g). 2 SEM (standard error of the mean). 3 Int (interaction effect diet × time); C1 (control vs. RCS + RCSP); C2 (RCS vs. RCSP). 4 Milk yield/dry matter intake. 5 Fat correct milk, 3.5/dry matter intake. 6 Energy correct milk/dry matter intake.
Table 3. Milk yield and composition according to experimental diets.
Table 3. Milk yield and composition according to experimental diets.
ItemExperimental Diets 1SEM 2p-Value 3
CONRCSRCSP DietTimeIntC1C2
kg/d
Milk yield16.8017.2620.220.5180.0050.4630.0300.0410.013
Fat correct milk 3.5%18.5617.5622.660.5740.0010.5640.0550.0210.002
Energy correct (Mcal/d)19.0317.8922.950.5520.0020.5020.4610.0320.011
Fat0.6770.6140.8190.0220.0120.5470.2140.1240.002
Protein0.5570.6050.7210.0150.0050.3210.1540.0020.231
Lactose0.8090.7470.9960.0250.0020.1500.0210.2780.001
Total solids2.412.252.610.0640.1110.1920.0650.9120.038
Percentage (%)
Fat4.033.564.050.0830.0010.8150.4350.7750.003
Protein3.323.513.570.0400.0310.3710.0370.0080.318
Lactose4.374.424.550.0250.0520.0130.3900.0740.098
Total solids13.2713.1513.090.1090.3380.6980.1460.1320.124
Milk urea nitrogen (mg/dL)12.7810.7810.450.5330.0020.0050.0780.0050.962
Somatic cell count (Log10)2.622.832.300.0570.0230.2090.0690.7700.032
Body weight, kg5455345417.8000.125<0.00010.4590.1370.160
Body weight movement, kg7.254.705.172.1680.5620.0010.4470.2910.850
Body condition score3.123.042.980.0350.1660.0070.1360.1060.356
Body condition score movement0.0310.0680.0370.0240.4850.3120.1120.4500.350
1 CON (control diet); RCS (diet with total replacement of ground corn by rehydrated corn grain silage); RCSP (diet with total replacement of ground corn by rehydrated corn grain silage + 500 g/ton of CINBENZA DP100® enzymatic extract of Bacillus licheniformis, protease activity 600 IU/g). 2 SEM (standard error of the mean). 3 Int (interaction effect diet*time); C1 (control vs. RCS + RCSP); C2 (RCS vs. RCSP).
Table 4. Milk fatty acids composition according to experimental diets.
Table 4. Milk fatty acids composition according to experimental diets.
Fatty Acids (g/100 g)Experimental Diets 1SEM 2p-Value 3
CONRCSRCSP DietTimeIntC1C2
C4:01.6151.6291.6030.0080.312<0.00010.0560.9250.129
C6:01.6141.5831.5840.0070.0360.0230.0750.0100.939
C8:02.9292.9002.9260.0080.1010.2950.2590.2060.088
C10:06.8026.7546.7330.0230.3820.0360.3410.1850.672
C12:04.1984.2064.2130.0040.3890.0040.4510.2220.520
C14:011.3111.2911.280.0180.509<0.00010.0070.2850.626
C14:10.0550.0530.0540.0010.1510.0010.0040.0530.895
C15:01.4611.4621.4630.0040.9570.0760.2250.8610.810
C15:10.1950.2010.2100.0020.003<0.00010.1860.0240.006
C16:027.4127.4727.680.0460.041<0.00010.0540.1140.034
C16:10.9760.9610.9560.0040.0430.7900.6660.0290.601
C17:00.1750.1680.1670.0020.214<0.00010.8970.2230.334
C17:10.4140.4230.4120.0040.354<0.00010.7020.6660.173
C18:014.3014.2914.160.0290.1130.1150.0210.2180.088
cis 11,C18:17.187.157.190.0130.5680.4390.0300.5450.387
cis 9,C18:113.5113.6013.560.0310.5440.5980.0880.3290.619
trans-10,cis-12 C18:21.5461.5531.5320.0070.404<0.00010.4490.7760.187
cis-9,cis-12,cis-15 C18:31.5531.5611.5360.0020.1770.0080.3340.7010.085
C20:00.8470.8520.8570.0060.0280.8970.9590.0410.243
C22:00.8510.8550.8500.0020.5570.2240.5540.5570.225
C24:00.1600.1610.1630.0020.4470.2250.5210.4120.842
Summary
Ʃ 4-a 14-C 428.5328.4228.400.0330.1460.0080.1610.1120.724
Ʃ above de 16-C 569.8069.9169.920.0330.1800.0050.1580.0660.822
Ʃ SFA 673.6973.6373.700.0340.7400.7520.2610.7160.504
Ʃ UFA 725.3225.4025.340.0340.6860.7070.2210.5590.527
Ʃ MUFA 822.3422.3922.370.0330.8600.8080.1470.6230.870
Ʃ PUFA 93.953.963.920.0100.4520.0020.5640.0870.124
Ʃ OCFA 102.2512.2532.2520.0050.1240.2250.8740.5560.789
Sat/insat 112.912.892.900.0060.2240.0870.6650.3350.442
1 CON (control diet); RCS (diet with total replacement of ground corn by rehydrated corn grain silage); RCSP (diet with total replacement of ground corn by rehydrated corn grain silage + 500 g/ton of CINBENZA DP100® enzymatic extract of Bacillus licheniformis, protease activity 600 IU/g). 2 SEM (standard error of the mean). 3 Int (interaction effect diet × time); C1 (control vs. RCS + RCSP); C2 (RCS vs. RCSP). 4 Fatty acids with 4 to 14 carbons. 5 Fatty acids with more than 16 carbons. 6 Saturated fatty acids. 7 Unsaturated fatty acids. 8 Monounsaturated fatty acids. 9 Polyunsaturated fatty acids. 10 Odd-chain fatty acids. 11 Total saturated/unsaturated fatty acid ratio.
Table 5. Plasmatic metabolites according to experimental diets.
Table 5. Plasmatic metabolites according to experimental diets.
ItemExperimental Diets 1SEM 2p-Value 3
CONRCSRCSP DietTimeIntC1C2
Glucose (mg/dL)60.9875.5676.394.094<0.00010.0780.8100.0240.912
Total cholesterol (mg/dL)155.83134.00127.207.5660.042<0.00010.3190.0330.616
Triglycerides (mg/dL)217.98204.36209.443.7720.3200.0030.6470.1610.575
Total protein (g/L)8.669.108.460.1560.149<0.00010.0470.6720.057
Albumin (g/L)3.363.283.640.0960.1700.0160.7500.5820.072
Urea (mg/dL)28.4221.2423.900.752<0.00010.0720.1050.0330.396
Blood urea nitrogen (mg/dL)12.339.2210.370.326<0.00010.0720.1050.0330.396
1 CON (control diet); RCS (diet with total replacement of ground corn by rehydrated corn grain silage); RCSP (diet with total replacement of ground corn by rehydrated corn grain silage + 500 g/ton of CINBENZA DP100® enzymatic extract of Bacillus licheniformis, protease activity 600 IU/g). 2 SEM (standard error of the mean). 3 Int (interaction effect diet*time); C1 (control vs. RCS + RCSP); C2 (RCS vs. RCSP).
Table 6. Microbial protein synthesis according to experimental diets.
Table 6. Microbial protein synthesis according to experimental diets.
ItemExperimental Diets 1SEM 2p-Value 3
CONRCSRCSP DietTimeIntC1C2
Uric acid (mmol/L)1.351.381.740.0570.1680.0290.9590.2960.115
Urine allantoin (mmol/L)40.9839.3742.211.4750.7460.4290.0250.9540.447
Allantoin milk (mmol/L)0.9010.8660.9280.0320.6580.4510.0650.9840.445
Total purines (mmol/L)43.2341.6244.881.5100.6940.377<0.00010.9950.394
Uric acid (mmol/d)20.7921.1827.040.9620.1440.0030.5060.2780.099
Urine allantoin (mmol/d)656.28592.96659.2026.0850.3810.239<0.00010.2870.064
Allantoin milk (mmol/d)15.6713.0419.930.8240.3870.2350.0020.2880.075
Total purines (mmol/d)692.75627.19706.1826.6650.0210.2260.0230.0850.001
Absorbable purines (mmol/d)674.71607.68686.5226.6700.0180.3350.0030.0950.015
Microbial synthesis (g/d)
Nitrogen490.54441.81499.1319.3900.0020.3380.0210.0120.036
Crude protein306527613119121.190.0020.3380.0210.0120.036
1 CON (control diet); RCS (diet with total replacement of ground corn by rehydrated corn grain silage); RCSP (diet with total replacement of ground corn by rehydrated corn grain silage + 500 g/ton of CINBENZA DP100® enzymatic extract of Bacillus licheniformis, protease activity 600 IU/g). 2 SEM (standard error of the mean). 3 Int (interaction effect diet*time); C1 (control vs. RCS + RCSP); C2 (RCS vs. RCSP).
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MDPI and ACS Style

Gandra, J.R.; Mattos, R.M.; Soares, T.M.D.M.; Pedrini, C.A.; Martinez, A.C.; Oliveira, E.R.; Gandra, E.R.S.; Vasconcelos, W.R.F.; Andrade, A.C. Rehydrated Corn Grain Silage and Exogenous Protease: Effects on Dairy Cow Performance, Metabolism, and Starch Digestibility. Dairy 2025, 6, 1. https://doi.org/10.3390/dairy6010001

AMA Style

Gandra JR, Mattos RM, Soares TMDM, Pedrini CA, Martinez AC, Oliveira ER, Gandra ERS, Vasconcelos WRF, Andrade AC. Rehydrated Corn Grain Silage and Exogenous Protease: Effects on Dairy Cow Performance, Metabolism, and Starch Digestibility. Dairy. 2025; 6(1):1. https://doi.org/10.3390/dairy6010001

Chicago/Turabian Style

Gandra, Jefferson R., Rafael M. Mattos, Thais M. D. M. Soares, Cibeli A. Pedrini, Antônio C. Martinez, Euclides R. Oliveira, Erika R. S. Gandra, Wallison R. F. Vasconcelos, and André C. Andrade. 2025. "Rehydrated Corn Grain Silage and Exogenous Protease: Effects on Dairy Cow Performance, Metabolism, and Starch Digestibility" Dairy 6, no. 1: 1. https://doi.org/10.3390/dairy6010001

APA Style

Gandra, J. R., Mattos, R. M., Soares, T. M. D. M., Pedrini, C. A., Martinez, A. C., Oliveira, E. R., Gandra, E. R. S., Vasconcelos, W. R. F., & Andrade, A. C. (2025). Rehydrated Corn Grain Silage and Exogenous Protease: Effects on Dairy Cow Performance, Metabolism, and Starch Digestibility. Dairy, 6(1), 1. https://doi.org/10.3390/dairy6010001

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