Ruminal metabolism, blood parameters and animal behavior of bulls submitted to sub-acute ruminal acidosis (SARA) receiving different buffers in high-concentrate dietsABSTRACT.
This study aimed to evaluate the supplementation of four different buffers into a high-grain diet on ruminal fermentation, blood metabolites, and feeding behavior of feedlot cattle. Five rumen-cannulated bulls (492 ± 24 kg) were distributed in a 5 x 5 Latin square design, through the treatments: CONT (no buffer), RUMO, RUMF, BICA and ALGA. The buffers did not alter (p > 0.05) the apparent digestibility of nutrients, ruminal fluid pH, volatile fatty acid profile, and acetic/propionic ratio compared to the CONT. Except for propionic and isovaleric acids, which were different (p < 0.05) among treatments at 4 and zero hours after feeding, respectively. Ruminal lactic acid accumulation was greater (p < 0.05) in BICA, while ammoniacal nitrogen concentrations were highest (p < 0.05) in CONT and lowest in RUMF. Blood glucose and creatinine were unaffected (p > 0.05), whereas urea and lactate concentrations were reduced (p < 0.05) in RUMO. Among the enzymes, only gamma-glutamyl transferase and creatine kinase showed treatment effects (p < 0.05). Furthermore, feeding and drinking times were unaffected by treatments; however, rumination increased (p < 0.05) in BICA, and idleness was higher in CONT. Overall, buffer inclusion modified ruminal and metabolic responses, indicating a possible modulation of ruminal acidosis.
Keywords:
animal production; Lithothamniun calcareun; nutritional disorder; rumen modulation; sub-acute acidosis
Introduction
To support their high genetic potential for meat and milk production, beef cattle and dairy cows need to receive energy-rich diets with high-carbohydrate and low fiber levels (Ornaghi et al., 2017; Rivaroli et al., 2020; Torrecilhas et al., 2021; Matos et al. 2023). These diets alter the modulation of microbiota in the rumen by reducing the pH and changing the volatile fatty acid composition towards increasing propionate, butyrate and lactate, and reducing the acetate to propionate ratio (Carvalho et al., 2021; Khalouei et al., 2021). The decrease in the rumen’s pH may lead to sub-acute ruminal acidosis (Plaizier et al., 2017). This disorder affects the production and health of beef cattle and dairy cows by decreasing production, nutrient utilization, rumen epithelium functionality and feed intake, as well as by causing inflammation, laminitis, and diarrhea (Callaway & Martin 1997; Plaizier et al., 2018).
SARA is considered a nutritional disorder, which can occur through the intake of fermentable carbohydrates in sufficient amounts to cause non-physiological accumulation of acids in the rumen, and there is considerable reduction in pH than more three hours (Nagaraja & Titgemeyer, 2007; Han et al., 2021). Ruminal pH is a critical factor in normal rumen function due to its effect on microbial populations, fermentation products, and physiological functions of the rumen, particularly motility and absorption. Therefore, the non-physiological accumulation of organic acids and the consequent reduction of pH below normal (less than 5.6) have a significant impact on microbial activity, rumen function, productivity and animal health. In beef cattle fed with high-grain diets, ruminal pH can range from 5.6 to 6.5, with an average pH typically around 5.8 to 6.2, and may drop below 5.6 during the feeding cycle (Nagaraja & Titgemeyer, 2007). Thus, the acid-base balance in the rumen requires time synchronization between acid production, its neutralization by the saliva and absorption via diffusion by the ruminal epithelium (Jaramillo-López et al., 2017). To prevent this process, there is systemic buffering with basically three systems: the body's buffer system, renal regulation and pulmonary regulation. In addition to these buffers, ruminants have buffering through saliva. A review by Herod et al. (1978) analyzed in vitro 35 different compounds with rumen fluid from cattle with high-grain diets regarding the buffering power of these compounds. The authors found that the addition of betonite and calcium bicarbonate (NaHCO3) had no effect on changing the pH, but the inclusion of calcium hydroxide (Ca(OH)), magnesium oxide (MgO), potassium bicarbonate (K2CO3), sodium bicarbonate (Na2CO3), potassium disodium (K3PO4) and sodium hydroxide (NaOH) showed a good capacity to neutralize acidity and significantly increased the ruminal pH. Furthermore, the compounds Al(OH)3, dolomite, MgSO4, NaH2PO4 and ZnSO4 had no effect on rumen pH.
The supplementation of buffer substances can be a way to limit the adverse effects of rumen acidosis. These substances have a direct effect on the pH of rumen fluids, such as neutralizing acidity through the sequestration of hydrogen (H+) and increasing the buffering capacity of the rumen fluid (Hernández et al., 2014) and can be added to complete rations at a dosage of 0.5 to 2.5% based on dry matter (Gastaldello Júnior et al., 2013). Sodium bicarbonate, disodium carbonate, magnesium oxide, potassium carbonate and anhydrous limestone are the compounds most commonly used as buffers (Valente et al., 2017). However, studies have been carried out with the objective of testing the possible effects of using a combination of these compounds and seaweed in the diet of cattle with nutritional disorders (Almeida et al., 2012; Carvalho et al., 2016; Neville et al., 2019).
Lithothamniun calcareun is a marine coralline alga belonging to the calcareous algae group, characterized by the deposition of calcium and magnesium carbonate in its cell walls in the form of calcite crystals (Boeckaert et al., 2008), which can present positive effects on the control of ruminal pH in animals receiving high-grain diets (Cruywagen et al., 2015).
This study was carried out to evaluate the effects of four different buffers in feedlot cattle fed with a high-concentrate diet, evaluating ruminal modulation, blood and behavioral ingestive parameters.
Material and methods
The development of this study was approved by the State University of Maringá (UEM) ethics committee for animal use with protocol CEUA Nº: 1583251120.
Location, facilities and animals
This study was carried out at the Rosa and Prado feedlot facility, located at the UEM Iguatemi Experimental Farm.
Five bulls with an average body weight of 492 ± 24 Kg and average age of 30 ± 6 months, adapted with rumen cannula were utilized, randomized across individual pens with a concrete floor measuring 10 m2, partially covered, with automatic drinkers and concrete feeders (2.0 m x 0.4 m x 0.5 m).
Experimental design and treatments
The study was carried out in a 5 x 5 Latin square, where four different buffers and a control treatment were tested in five animals and five data collection periods. The animals were randomized across treatments. The experimental period had 14 days of rest adaptation for the bulls (non-sub-acute ruminal acidosis) when they received a high-forage (corn silage) diet, and 3 days of SARA induction for data collection. During the three days of sub-acute acidosis induction, the animals received a high-concentrate diet in the proportion of 20% corn silage and 80% concentrate (finely ground corn, ground black oat, soybean meal and mineral salt). The experimental periods followed the chronology as shown in Table 1.
The bulls were fed once a day (at 8:00 a.m.) throughout the study. The amount of feed was adjusted to 2.0% of the animal’s body weight and both the amount of feed provided and leftovers were recorded. The leftovers were collected only with three days of sub-acute acidosis induction and 12 hours after the morning feeding.
The animals were randomly assigned to five treatments: CONT (diet without the addition of buffering compounds), RUMO (diet containing 10 g Kg-1 of dry matter with the inclusion of RUMOX®, based on sodium bicarbonate, calcium carbonate, magnesium oxide, magnesium, sodium carbonate, calcareous seaweed, honey aroma, neotame, sodium saccharin, maltodextrin and silicon dioxide), RUMF (diet containing 10 g Kg-1 of dry matter with the inclusion of RUMOXF®, based on sodium bicarbonate, calcium carbonate, magnesium oxide, calcareous seaweed, honey aroma, neotame, sodium saccharin, maltodextrin and silicon dioxide); BICA (diet containing 10 g Kg-1 of dry matter with the inclusion of sodium bicarbonate) and ALGA (diet containing 10 g Kg-1 of dry matter with the inclusion of seaweed - Lithothamnium calcareum) (Table 2).
Data collection and sampling
The feed and leftover samples were collected per animal in each experimental period and, the fecal grab samples were collected on the second day of sub-acute acidosis induction at zero, 2, 4, 8 and 12 hours after the morning feeding. The samples were dried in an oven with forced air circulation at 55ºC for 72h and were processed in a Wiley mill using 1 mm sieves for the chemical composition assay and 2 mm sieves for apparent digestibility.
The rumen fluid was collected from the ventral sac. Ruminal pH was measured at the time of collection with the aid of a portable pH meter (AK103 - Akso; Porto Alegre, Rio Grande do Sul, Brazil) previously calibrated with pH 4.0 and 7.0 standards, as recommended by the manufacturer, and the samples were collected on days zero, 1 and 2 of each experimental period at zero, 2, 4, 6, 8, 10 and 12 hours after the morning feeding. For the analysis of lactic acid, ammoniacal nitrogen and volatile fatty acids, samples were collected on the second day of sub-acute acidosis induction of each experimental period at zero, 2, 4, 8 and 12 hours after the morning feeding, using 50 mL falcon tubes, and the samples were abruptly frozen with the aid of liquid nitrogen and stored at -20° C for further analysis.
Blood from each head of cattle was collected on the second day of sub-acute acidosis induction at two hours after feeding the animal of each experimental period via jugular vein puncture, in vacuum tubes (Vacutainer®) with EDTA K2 to obtain plasma and with a clot activator to obtain serum. In sequence, the samples were centrifuged at 2,500 rpm for 15 min and the supernatant was collected, stored in an Eppendorf tube and frozen in a freezer at -20°C for further analysis.
Animal feeding behavior data was obtained on the third day of acidosis induction of each experimental period. Records of the time spent in different activities were obtained by visually observing the animals every five minutes, performed by a trained team for 12 uninterrupted hours. Data were collected to estimate the duration of periods spent feeding, drinking, ruminating and idling (Bürger et al., 2000) with modifications (Silva et al., 2006). The total time spent on each activity was determined by summing the repetitions. Moreover, the number of visits to the water drinker and feeder were recorded.
Laboratorial analysis
Feed, leftovers and feces samples were analyzed for dry matter (DM - method 930.15), crude protein (CP - method 984.13), ash (method 924.05) and ether extract (EE - method 920.29) according to the (Association of Officiating Analytical Chemists, 2005). Organic matter (OM) was calculated as 1000 - ash. The cell wall components such as neutral detergent fibers (NDF) were assayed using filter bags (F57 - Ankom) and a neutral detergent solution including thermostable amylase and sodium sulphite. In sequence, acid detergent fibers (ADF) were determined by the Mertens (2002), as shown in Table 2. Indigestible neutral detergent fibers (iNDF) were determined by 288 h of in situ ruminal incubation (Huhtanen et al., 1994).
Lactic acid concentrations present in the rumen fluid were determined using the method described by Pryce (1969) with adaptations, where lactic acid was converted to acetaldehyde by heating with sulfuric acid, then acetaldehyde was reacted with p-hydroxybiphenyl to produce a coloured complex. The ammoniacal nitrogen concentrations in the rumen fluid were determined by the colorimetric method (Chaney & Marbach, 1962). The readings were performed by spectrophotometry using the VersaMax™ ELISA Microplate Reader equipment with the SoftMax® Pro software.
The concentrations of acetic, propanoic, butyric, valeric and isovaleric acids in the ruminal fluid samples were determined by gas chromatography using a Shimadzu© GC-2010 Plus chromatograph equipped with an AOC-20i automatic injector, Stabilwax-DA™ capillary column (30 m, 0.25 mm ID, 0.25 µm df, Restek®) and flame ionization detector (FID), after acidification with 1 M o-phosphoric acid p.a. (Ref. 100573, Merck©) and fortification with a mixture of free volatile acids (Ref.
46975, Supelco®). An aliquot of 1 µL of each sample was injected with a split ratio of 40:1, using helium as a carrier gas with a linear velocity of 42 cm s-1, obtaining the separation of the analytes in a chromatographic run of 11.5 minutes. The inlet and detector temperatures were, respectively, 250 and 300°C, with initial column temperature of 40°C. The column temperature ramp started with a gradient from 40 to 120°C at a rate of 40°C min.-1, followed by a gradient from 120 to 180°C at a rate of 10°C min.-1, and from 180 to 240°C at a rate of 120°C min.-1, keeping the temperature at 240°C for another 3 minutes at the end. For the quantification of analytes, a calibration of the method was performed with dilutions of WSFA-2 standard (Ref. 47056, Supelco®) and glacial acetic acid (Ref. 33209, Sigma-Aldrich®) analyzed under the conditions described above. Peak determination and integration were performed using GC solution v. 2.42.00 (Shimadzu®), according to the methodology described by Del Valle et al. (2018).
In the blood plasma, the glucose, urea, lactate and creatinine concentrations were determined. In the blood serum, total proteins and fractions, gamma-glutamyl transferase (GGT), aspartate aminotransferase (AST) and creatine kinase (CK) concentrations were measured. The analyses were performed using commercial kits (GoldAnalisa®, Belo Horizonte, Minas Gerais, Brazil) according to the manufacturer's instructions and the readings were performed in a spectrophotometer (Bioplus, 2000®, São Paulo, SP).
Statistical analysis
All the data were evaluated for normality of residuals by Shapiro-Wilk test, and homogeneity of variances by Bartlett test. In the sequence, data were analyzed using the Mixed procedure of SAS (v. 9.4, SAS Inst. Inc., Cary, NC) with the following model:
Yijkl = μ + Si + C(S)ij + Pk + El + eijkl
Where:
Yijkl = dependent variable;
μ = overall mean;
Si = fixed effect of square;
C(S)ij = random effect of bull nested within square;
Pk = fixed effect of period;
El = fixed effect of treatments;
eijkl = residual error.
For the analyses of pH, lactic acid, ammoniacal nitrogen and volatile fatty acids, data were considering the following model:
Yijklm = μ + Si + C(S)ij + Pk + El + Tm+ E x Tlm + eijklm
Where:
Yijkl = dependent variable;
μ = overall mean;
Si = fixed effect of square;
C(S)ij = random effect of bull nested within square;
Pk = fixed effect of period;
El = fixed effect of treatments;
Tm = repeated measures in time;
E x Tlm = interaction between treatment and time;
eijklm = residual error.
Finally, significance was declared at p ≤ 0.05 and trends at 0.05 < p ≤ 0.10. Tukey test was used to compare means when differences were observed.
Results
Apparent digestibility
The inclusion of different single compounds (sodium bicarbonate [BICA] or seaweed [ALGA]) or various combinations of other compounds (sodium bicarbonate, calcium carbonate, magnesium oxide, magnesium carbonate, sodium carbonate, calcareous seaweed, honey aroma, neotame, sodium saccharin, maltodextrin and silicon dioxide [RUMO] and [RUMF]) did not change (p > 0.05) the apparent digestibility of dry matter, organic matter, crude protein, ether extract, neutral detergent fiber and non-fibrous carbohydrates of SARA-induced bulls (Table 3).
Rumen pH
The ruminal pH results are presented in graphical form in Figure 1. The ruminal pH of bulls in non-sub-acute acidosis had no difference (p > 0.05), where the means varied from 6.64 to 6.74 (Figure 1a). This was similar to the ruminal pH of animals on sub-acute acidosis induction, where the means varied between 5.95 for BICA, the lowest treatment value, and 6.09 for RUMO, the highest treatment value. However, when observing the pH values according to the hours after feeding (Figure 1b) and considering that animals with a pH lower than 5.80 are in sub-acute acidosis, it can be observed that animals that received the RUMO treatment showed a pH mean greater than 5.80 over the 12 hours of observation, while the animals that received CONT, RUMF and ALGA treatments showed pH means close to 5.70 over the 12 hours of observation, and the animals that received the BICA treatment showed an average pH of 5.60 over the 12 hours.
The prevalence time of pH lower than 5.80 and lower than or equal to 5.6 (Figure 1c) showed no difference (p > 0.05) across treatments, but RUMO had the lowest prevalence time at 240 minutes with pH lower than 5.80, and 48 minutes with pH lower or equal to 5.60. ALGA and BICA were the treatments that showed greater prevalence time with pH lower than 5.80 (348 and 336 minutes, respectively), followed by RUMF and CONT (264 and 252 minutes, respectively). For prevalence time with pH lower or equal to 5.60, BICA showed the greatest time (216 min.), followed by RUMF, ALGA and CONT, at 180, 156 and 144 minutes, respectively. When the maximum and minimum pH is analyzed (Figure 1d) no differences were observed (p > 0.05) across treatments. However, the CONT treatments showed the lowest pH, followed by BICA, RUMF, ALGA and RUMO (4.81, 4.85, 4.97, 5.23 and 5.53, respectively).
Ruminal pH. a. Ruminal pH of animal in non-sub-acute ruminal acidosis. b. Ruminal pH of animal in sub-acute ruminal acidosis induction. c. Prevalence time of ruminal pH below 5.8 and below or equal to 5.6 of animal in sub-acute ruminal acidosis induction. d. Minimum and maximum pH of animal in sub-acute ruminal acidosis induction. CONT (without addition of buffers); RUMO (sodium bicarbonate, calcium carbonate, magnesium oxide, magnesium carbonate, sodium carbonate, calcareous seaweed, honey aroma, neotame, sodium saccharin, maltodextrin and silicon dioxide); RUMF (sodium bicarbonate, calcium carbonate, magnesium oxide, calcareous seaweed, honey aroma, neotame, sodium saccharin, maltodextrin and silicon dioxide); BICA (calcium bicarbonate); ALGA (marine algae - Lithothamnium calcareum).
Volatile fatty acid
Diet effect
The treatments had no effect (p > 0.05) on total concentration of volatile fatty acids (VFA) (Table 4). Thus, the various buffers used, as well as their combinations, did not alter (p > 0.05) the ruminal metabolism of total VFA.
Time effect
For treatments CONT, RUMO and RUMF, the collection times of rumen fluid (before and 12 hours after feeding) had no effect (p > 0.05) in the concentration of total volatile fatty acids (Table 4). However, before the morning feeding, the concentration of total volatile fatty acids was lower (p < 0.05) in the BICA and ALGA treatments. Nevertheless, two hours after the morning feeding, the concentration of VFA increased and remained close to 105.3 mmL-1 (Table 4).
Acetic acid concentrations were similar and unaffected (p > 0.05) by the CONT, RUMO and RUMF treatments. On the other hand, before the morning feeding, acetic acid concentrations were low, then increased (p < 0.05) two hours after feeding and remained high until the end of the collection period (12 hours).
The time of collection had no effect on propionic acid concentrations in the CONT and RUMO treatments. Concentrations were low before the morning feeding, then increased in the first two hours and remained high until the last collection time (12 hours) in the RUMF, BICA and ALGA treatments (Table 4).
Before the morning feeding, the acetic/propionic ratio was at 2.43, then was reduced (p < 0.05) to 2.05 two hours after feeding and remained at these levels until the end of the day (Table 4).
For the other fatty acids (butyric, isobutyric, valeric, isovaleric and hexanoic), the collection time of the ruminal fluid was not important, with the exception of butyric and hexanoic fatty acids, with an increase in concentration two hours after the morning feeding (Table 4).
Ruminal lactic acid
Before the morning feeding, lactic acid concentrations in the ruminal fluid were similar (p > 0.05) in bulls of all treatments (Table 5). Lactic acid concentrations in the ruminal fluid varied from 1.43 mML-1 in bulls fed with the BICA diet, to 1.83 mML-1 in bulls fed with the RUMO diet. However, from the second to the twelfth postprandial hours, lactic acid concentrations were higher (p < 0.01) in the ruminal fluid of bulls fed with BICA, compared to the concentrations observed in the ruminal fluids of bulls fed with other diets (Table 5). Ruminal fluid lactic acid concentrations in bulls fed with the CONT, RUMO, RUMF and ALGA diets were similar (p > 0.05) at all times studied. For these four diets, lactic acid concentrations ranged from 1.43 to 3.02 mMol L-1, while the concentrations observed in bulls fed with the BICA diet were above 5.0 mMol L-1 two hours after SARA induction, up until 12 hours from collection time.
Collection times, before and after the morning feeding had no effect (p > 0.05) on lactic acid concentrations in the ruminal fluid of bulls fed with the CONT, RUMO, RUMF and ALGA diets, with the exception of an increase observed at 12 hours in bulls fed with ALGA (Table 5). However, in BICA bulls, there was a linear increase (p < 0.05) in lactic acid concentrations as a function of collection time, from 1.66 before the morning feeding to 8.83 mMol L-1 12 hours after feeding (Table 5).
Ruminal N-NH3
Before the morning feeding (0 hour) and two hours after, ammoniacal nitrogen (N-NH3) concentrations were higher (p < 0.05) in the ruminal fluid of bulls fed with the CONT (14.6 and 16.7 mg dL-1) and RUMO (14.2 and 15.4 mg dL-1) diets, lower in bulls fed with the RUMF diet (8.11 and 8.77 mg dL-1) and intermediate in bulls fed with the BICA (10.2 and 12.86 mg dL-1) and ALGA (11.7 and 15.9 mg dL-1) diets (Table 6). Four hours post-feeding, N-NH3 concentrations were higher (p < 0.05) in the ruminal fluid of bulls fed with CONT (12.5 mg dL-1), lower for those fed with RUMO (7.04 mg dL-1) and RUMF (4.79 mg dL-1) and intermediate for those fed with BICA (8.57 mg dL-1) and ALGA (8.21 mg dL-1) (Table 6). Eight hours post-feeding, N-NH3 concentrations in the ruminal fluid of bulls fed with the CONT diet (9.43 mg/dL) were higher for cattle fed with the RUMF diet (5.20 mg dL-1). The concentration of N-NH3 was 7.79, 7.42 and 7.44 mg dL-1 for bulls fed with RUMO, BICA and ALGA, respectively (Table 6). In the last collection of the day (12 hours), the concentrations of N-NH3 in the ruminal fluid of bulls were higher (p < 0.05) for CONT (11.8 mg dL-1), lower for RUMO (6.29 mg dL-1) and ALGA (7.02 mg dL-1), and intermediate for RUMF (9.96 mg dL-1) and BICA (8.95 mg dL-1) (Table 6). In general, regardless of the ruminal fluid collection time, the highest N-NH3 concentrations were observed in the ruminal fluid of bulls fed with CONT (13.0 mg dL-1), lowest for RUMF (7.3 mg dL-1) and intermediate for RUMO (10.2 mg dL-1), BICA (9.6 mg dL-1) and ALGA (10.0 mg dL-1) (Table 6).
Parameters in blood plasma
Parameters were measured in blood plasma before the morning feeding and two hours after, on the second day of ruminal acidosis induction (Table 7). Thus, these parameters could be altered due to the induction of acidosis on the previous day.
Before the morning feeding and two hours after, glucose concentrations were similar (p < 0.05) in the blood plasma of bulls from all diets (Table 7). Similarly, collection times had no effect (p > 0.05) on blood plasma glucose concentrations (Table 7).
Before the morning feeding, blood plasma urea concentrations were higher (p < 0.05) in bulls fed with CONT (16.5 mg dL-1) and BICA (15.2 mg dL-1), lower for bulls fed with RUMO (12.1 mg dL-1) and RUMF (11.6 mg dL-1), and intermediate for the animals fed with ALGA (13.1 mg dL-1). However, after 2 postprandial hours, blood plasma urea concentrations of bulls showed no differences (p < 0.05) across diets. Likewise, observed concentrations before and 2 hours later were similar (p > 0.05).
Lactate concentrations were higher (p < 0.05) in the blood plasma of bulls fed with CONT (18.5 mg dL-1), RUMF (20.1 mg dL-1) and ALGA (17.2 mg dL-1), and lower for RUMO (10.0 mg dL-1) and BICA (12.7 mg dL-1) (Table 7).
High blood plasma lactate values are characteristic of animals suffering from ruminal acidosis. In this way, cattle fed with the RUMO and BICA diets had better comfort in relation to ruminal acidosis related to blood lactate concentrations.
As observed for glucose concentrations, diets and blood collection times had no effect (p > 0.05) in blood plasma creatinine concentrations (Table 7). Creatinine values ranged from 14.9 to 20.1 mg dL-1 across diets and collection times. This variation is within the values observed for blood creatine concentrations in ruminants (Jain & Jain, 1993; Kramer, 2000).
Blood serum parameters
Diets and blood collection times (before feeding and 2 hours after) had no effect (p < 0.05) on total protein, albumin and globulin concentrations in the blood serum of bulls subjected to SARA induction (Table 8). The variations in the blood serum observed across diets and collection times are considered normal, according to reference data (Jain & Jain, 1993; Kramer, 2000).
Gamma-glutamyl transferase (GGT) concentrations were lower (p < 0.05) in the blood serum of cattle fed with RUMO (9.60 u L-1) compared to the other diets (Table 6), where it had similar concentrations (p > 0.05). Although kidney tissue has the highest levels of GGT, this enzyme in the blood appears to originate from the hepatobiliary system. Its activity is increased in all forms of liver disease. GGT can be elevated in even small subclinical levels of liver dysfunction. Thus, in this study, the RUMO diet showed greater protection of the hepatic system in cattle. On the other hand, observed concentrations before feeding and 2 hours after were similar (p > 0.05). Therefore, the short time between blood collections (2 hours) did not influence the concentrations of this enzyme.
The concentrations of the enzyme aspartate aminotransferase (AST) were lower (p < 0.05) in the blood plasma of cattle fed with BICA (44.0 IU L-1) in relation to the concentrations observed in the blood serum of animals on the other diets, and there was no difference (p < 0.05) between the concentrations of the animals fed with CONT, RUMO, RUMF or ALGA (Table 8). On the other hand, collection time (before and 2 hours after feeding) had no effect (p > 0.05) on this enzyme.
Creatinine kinase enzyme concentrations were similar (p > 0.05) in the blood serum of cattle fed with CONT and RUMF both before and 2 hours after feeding, but were higher in comparison to RUMO, BICA and ALGA (Table 8). On the other hand, creatine kinase enzyme concentrations were higher two hours after feeding, compared to concentrations observed before feeding (Table 8).
Animal behaviour
Animal feeding behaviour (Table 9) showed no difference (p > 0.05) in feeding and drinking time (minutes) across diets. When analyzing rumination time, a difference (p < 0.0176) was observed. The BICA treatment had the longest rumination time (109.00 minutes) followed by ALGA (81.00 minutes), RUMF (80.00 minutes), CONT (59.00 minutes) and RUMO (55.20 minutes), respectively. For the idleness time parameter, a difference was observed (p < 0.0023) among diets, as the animals that received the CONT and RUMOX diets showed an increase in their idle time. However, the RUMOX diet did not differ from other diets. In regards to the number of visits to the feeder, CONT was different from BICA (p < 0.03). Finally, no difference was observed (p > 0.05) in the number of visits to the water drinker across all diets.
Discussion
Apparent digestibility
Apparent digestibility was not altered by the inclusion of different buffers in the diets. However, the apparent digestibility of all nutrients was high (greater than 70% for dry matter and organic matter, greater than 80% for crude protein and non-fiber carbohydrates). The high apparent digestibility for all treatments, including CONT, could be explained in part by the diet’s composition (high content of grains rapidly degraded in the rumen) and the feeding system (restricted intake, 2% of body weight). In general, cattle finished in feedlot and finished with high grain diets have high apparent digestibility (Cruz et al., 2014; Ornaghi et al., 2017; Souza et al., 2019; Mottin et al., 2020). Sharma et al. (2018) carried out a long review of studies carried out using various buffers in diets for beef cattle, dairy cows and sheep, and concluded that these compounds have little significant effect on feed intake and apparent digestibility of dry matter, organic matter, crude protein, ether extract, neutral detergent fiber and carbohydrates, as observed in this study. Thus, the use of buffers would have little effect on the utilization of nutrients in the diets.
Ruminal pH
Ruminal pH is a critical factor in the normal and stable function of the rumen, because it has a profound effect on microbial populations, fermentation products, and physiological functions, mainly motility and absorptive function (Nagaraja & Titgemeyer, 2007; Abdela, 2016). Thus, low ruminal pH for prolonged periods can negatively affect dry matter intake and fiber degradation, resulting in reduced productive performance and, subsequently, large financial losses (Vyas et al., 2014). Furthermore, there may be a reduction in fiber digestibility at low ruminal pH due to the reduction of the fibrolytic population, given the reduced ability of fibrolytic bacteria to bind to feed particles (Calsamiglia et al., 2002).
In this study, no significant differences were observed in ruminal pH across diets (Table 4 and Figure 1). In animals fed with high-grain diets, according to Nagaraja & Titgemeyer (2007), ruminal pH can range from 6.50 to 5.60, with an average pH around 5.80 to 6.20, and it can drop below 5.60 for a period during the feeding cycle. Moreover, in this study, we considered that animals showing ruminal pH below 5.80 were in sub-acute acidosis (Beauchemin et al., 2003). Thus, observing the development of the ruminal pH over 12 hours, it is considered that all treatments had sub-acute acidosis except for RUMO. Furthermore, Gozho et al. (2007) suggest that a drop in ruminal pH below 5.60 for more than 180 minutes was used as the threshold to characterize sub-acute acidosis. In this way, we observed that the use of 10 g Kg-1 of dry matter with the inclusion of sodium bicarbonate was not effective in reducing sub-acute acidosis.
Volatile fatty acids
The mean overall concentration of volatile fatty acids (all treatments) was 102.6 mML-1, with no differences among treatments. Thus, the average concentration of fatty acids is close to the concentrations observed by several authors with the use of different buffers (Erdman, 1988; Iwaniuk & Erdman, 2015; Watanabe et al., 2019; Khalouei et al., 2021; Minami et al., 2021). Likewise, the concentrations of acetic, propionic, butyric, isobutyric, valeric, isovaleric and hexanoic acids, and the ratio between acetic and propionic acids were also not altered by the buffers, remaining close to the concentrations observed in other studies (Erdman, 1988; Iwaniuk & Erdman, 2015; Watanabe et al., 2019; Khalouei et al., 2021; Minami et al., 2021).
Lactic acid
Only the BICA diet (calcium bicarbonate) was not efficient in reducing lactic acid production. According to Nagaraja & Titgemeyer, 2007), the reason the pH drops below 5.60 is the accumulation of volatile fatty acids, which is a combination of overproduction (increased substrate), possibly decreased absorption, and the fact that, when pH is near 5.0 or below for a sustained period, the growth of lactate-fermenting bacteria is inhibited. Hence, lactate begins to accumulate. Ruminal lactate acid concentrations in SARA and acute acidosis mostly range from 0.17 to 0.74 and 50 to 120 mM, respectively (Arik et al., 2019). In this study, lactate acid concentrations between 1.81 (RUMO) and 6.48 mM L-1 (BICA) were observed, showing accumulation of lactate acid. When pH is near 5.0 or less for a sustained period, the growth of lactate-fermenting bacteria is inhibited, and lactate begins to accumulate in the ruminal environment, which is the result of increased production and decreased acid fermentation. The increase in lactic acid production is essentially due to the establishment of an amino acid-tolerant lactobacillus population, which accumulates in two isomers: D-lactate (produced in lesser quantity) and L-lactate (Jaramillo-López et al., 2017). Thus, decreased fermentation occurs because the pH is below the range required for lactate-fermenting bacteria to be active (Nagaraja & Titgemeyer, 2007) Total VFA concentration generally increases at the onset of acidosis but, with its progression, VFA concentrations decline drastically due to the destruction of normal bacterial flora, and ruminal dilution of fluid influx, to compensate for increased osmolality (Nagaraja & Titgemeyer, 2007).
There is a change in the ruminal microbiota, and bacteria that degrade fibers, such as Butyrivibrio fibrisolvens, Bacteroides succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens cease growth with a pH lower than 6.00. Starch-degrading species such as Selemonas ruminatiun and Streptococcus bovis continue to grow at a pH closer to 5.00 (Kozloski, 2011). Furthermore, one population of protozoa did not survive prolonged exposure to pH below 5.50 (Quinn et al., 1962). The less species of bacteria and protozoa present in the rumen, the less stable the microflora, and the harder to maintain the pH close to normal during periods of sudden dietary changes (Soto et al., 2013).
Ruminal N-NH3
The evolution of the kinetics of N-NH3 concentrations in the ruminal fluid of cattle showed a similar dynamic for the CONT, RUMO, RUMF, and BICA treatments, with significant reduction 4 hours after the morning feeding (Table 6), with the exception of BICA. In this way, all buffers used in the diets were Maeda et al. (2007) able to reduce the production of N-NH3 in the rumen, with emphasis on the RUMF treatment. Still, the others (RUMO, BICA, and ALGA) showed a better ability to buffer the rumen when compared to CONT. In general, N-NH3 concentrations in the rumen are strongly elevated before and in the first two hours after feeding, and decrease after 4 hours (Fregadolli et al., 2001; Maeda et al., 2007; Prado et al., 2010).
Blood plasma parameters
Blood parameters were not influenced by the inclusion of various buffers. Glucose and urea levels ranged from 62.3 to 70.0 mg dL-1 and from 13.5 to 18.4 mg dL-1, respectively. In general, the blood levels of glucose and urea in ruminants vary from 60 to 80 mg dL-1 for glucose and from 15 to 30 mg dL-1 for urea. Thus, the levels found in this study are within the normal range for ruminants. However, Xu et al. (2016) observed a significant reduction in dairy cows with sub-acute acidosis.
Glucose concentrations in the blood plasma range from 62.5 to 71.4 mg dL-1. These values are in agreement with the values considered normal (Jain & Jain, 1993; Kramer, 2000). In ruminants, diets and postprandial time have little effect on blood glucose concentrations, since glucose is metabolized very rapidly in the rumen and transformed into VFAs.
Before the morning feeding, blood plasma urea concentrations were higher (p < 0.05) in bulls fed with CONT (16.5 mg dL-1) and BICA (15.2 mg dL-1), lower for bulls fed with RUMO (12.1 mg dL-1) and RUMF (11.6 mg dL-1), and intermediate for the animals fed with the ALGA diet (13.1 mg dL-1). However, after 2 postprandial hours, blood plasma urea concentrations of bulls showed no differences (p < 0.05) across diets. Likewise, observed concentrations before and 2 hours later were similar (p > 0.05).
Urea concentrations observed before and after feeding in blood plasma are within ruminant standards (5 - 20 mg dL-1) (Jain & Jain, 1993; Kramer, 2000). Changes in the concentrations of urea in the blood of the animals are due to the degradation of the protein in the rumen. A higher rate of degradation means higher concentrations of urea in the blood. Thus, the CONT and BICA diets had lower buffering power in the rumen after the induction of ruminal acidosis. On the other hand, the RUMO and RUMF diets were more efficient in reducing protein degradation in the rumen; ALGA was neutral.
The levels of lactate in the blood ranged from 12.1 to 18.9 mg dL-1; these values being normal for cattle in normal health conditions. Although without significant differences, lactate levels were 30% higher in the CONT treatment animals at 17.9 mg dL-1, versus the 14.6 mg dL-1 mean of the other treatments. However, Xu et al. (2016) observed higher levels of lactate in the blood of dairy cows with sub-acute acidosis.
Blood serum parameters
The levels of total proteins, albumins, and globulins were not altered by the treatments. Levels of these components were normal for animals in good health. However, Xu et al. (2016) observed lower levels of total proteins, albumins, and globulins in the blood of dairy cows with sub-acute acidosis. When the liver is injured or diseases like diarrhea cause dehydration, the levels of total proteins, albumins, and globulins will be increased.
Gamma-glutamyl transferase, aspartate aminotransferase, creatinine, and creatinine kinase are increased when the animal’s muscle or liver is damaged. In our study, treatments did not change creatinine, gamma-glutamyl transferase, and aspartate aminotransferase levels. Although without significant numerical differences, in the CONT treatment animals, creatine kinase levels were 70% higher. In dairy cows, levels of gamma-glutamyl transferase, aspartate aminotransferase, creatinine, and creatinine kinase are increased when animals are in sub-acute acidosis (Xu et al., 2016).
AST is an enzyme that catalyzes the conversion of the nitrogenous portion of an amino acid to an amino acid residue. This enzyme is essential for energy production in the Krebs cycle and is found in the cytoplasm and mitochondria of many cells, primarily the liver, heart, skeletal muscle, kidney, pancreas, and red blood cells. This enzyme is released into the blood in large amounts when there is damage to the hepatocyte membrane. Thus, the absolute elevation of aspartate aminotransferases has great diagnostic significance in acute liver diseases. Therefore, the addition of sodium bicarbonate to the diet enabled better protection of the hepatic system, while the other diets were neutral.
Animal behavior
When the animals exhibit sub-acute acidosis, feed intake and performance might be reduced, even though the animal does not appear sick (Owens et al., 1998). Because of this, this study analyzed the animals’ intake behaviour, and significant differences were observed across treatments in rumination time, idleness time, and number of visits to the feeder. For rumination time, BICA showed the greatest time (109.00 minutes), which may be correlated with the amount of acid in the ruminal environment, and one possibility to control the acidosis. This is because, according to Bailey (1961) saliva secretion during rumination is similar to that while eating (25- and 20-mL min.-1, respectively) and about 2 times greater than during resting in cattle (10 mL min.-1). Moreover, saliva production is very important for rumen function, because it has high buffering capacity as a result of high concentrations of bicarbonate (125 mEq mL-1) and phosphate (20 mEq mL-1). Consequently, idleness time in the BICA treatment was reduced.
Furthermore, the BICA treatment showed the highest number of visits to the feeder, and CONT showed the lowest number (17.40 and 11.80 visits). Greater meal frequency and wider distribution of daily intake throughout the day lead to better synchronization in time between bouts of acid and saliva production, and absorption and passage of organic acids from the rumen. The time periods between feeding events allow timely metabolization of acids and elimination through urine of protons absorbed from the rumen (González et al., 2012). However, some factors can influence the animal’s ingestive behavior, such as the grain utilized, particle size, carbohydrate availability, low ruminal pH, and the feed intake control mechanisms. In addition, the use of sodium bicarbonate can show alterations in animal behaviour (González et al., 2012). Besides, short-term feed intake behaviour can be summarized by meal size and frequency, eating rate, and distribution of intake throughout the day. Moreover, it is important to note that the method used to calculate these short-term feeding behaviour measurements may have a great influence on the outcomes (Nielsen, 1999; González et al., 2012).
Conclusion
The present results showed all treatments were submitted in sub-acute ruminal acidosis. However, only the RUMO treatment showed less impact on the ruminal pH and ruminal lactate acid, being a mixture of sodium bicarbonate, calcium carbonate, magnesium oxide, magnesium, sodium carbonate, calcareous seaweed, honey aroma, neotame, sodium saccharin, maltodextrin, and silicon dioxide. In contrast, the animals that received the BICA treatment were more susceptible to ruminal acidosis than those with other treatments, which had lower ruminal pH and greater time prevalence with pH < 5.80 or ≤ 5.60. However, further studies must be carried out to define the best dose for animal supplementation and possible financial impact on cattle production.
Data availability
Not applicable.
Acknowledgements
This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES (Brasília, Brazil), Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq (Brasília, Brazil) and the company Safeeds Nutrição Animal (Paraná, Brazil). The authors gratefully acknowledge the company for financing and providing the products used in this research, which made it possible to develop this work. The mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendations or endorsements by the Department of Animal Sciences of the State University of Maringá, Paraná, Brazil.
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Edited by
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Associate Editor in charge:
Leandro Dalcin Castilha
