In vitro Ruminal Acid Load and Methane Emission Responses to Supplemented Lactating Dairy Cow Diets with Inorganic Compounds Varying in Buffering Capacities

Document Type: Research Articles

Authors

1 Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

2 PDepartment of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

Abstract

Using 24 hours in vitro cultures of rumen microorganisms, this study investigates the effect of buffering capacity of 2 inorganic compounds (M1=119.43 and M2=116.50 meq/L) on the in vitro rumen acidogenic value (AV), medium pH, dry matter disappearance (INVDMD) and methane emission of lactating dairy cow diets containing various forage to concentrate ratios as 40:60 (FC40:60) and 30:70 (FC30:70) in a completely randomized design. Inorganic compounds were included in the experimental diets at the rate of 0.0, 10 or 20 g/kg DM. Diet with higher amount of concentrate caused a decline in medium pH, an increase in both AV and IVDMD. The acidogenic value of FC40:60 containing M2 at 20, M1 at 10 and 20 g/kg DM and FC 30:70 plus M1 and M2 at 20 g/kg DM was the lowest. The lowest level of CH4 emission (mL/0.20 g DM) was observed in FC30:70 plus M1 at the rate of 10 g/kg DM, while the highest level belongs to FC40:60 plus M1 at 10 g/kg DM and FC30:70 containing M1 at 20 and M2 at 10 g/kg DM. It has been concluded that the higher buffering capacity of a lactating diet might reduce the rumen acid load and increased IVDMD, while a diet with higher amount of concentrate causes to decline rumen methane emission.

Keywords


INTRODUCTION

The problem primarily met in dairy cow feeding is to provide an energetically high-density ration without jeopardizing ruminal ecosystem, animal welfare and production performances (Zebeli et al. 2008). Enhancing energy supply through increased use of concentrates or rapidly fermentable fiber can swallow the rumen into acidosis. Subacute ruminal acidosis (SARA) is a common and economically important problem in well managed dairy herds. Failure to maintain a consistent rumen pH in high yielding dairy cows may result in metabolic disorders and reduced production performance (Tajik and Nazifi, 2011). The rumen pH will fall when organic acids that are produced during fermentation by rumen microbes accumulate and rumen buffering is not sufficient to prevent the increase in acidity (Plaizier et al. 2008). Chemical buffers in diets for ruminants to provide rumen pH in a range that is optimal for the activity of cellulose-degrading organisms (pH=6-7) are used. The need for buffering agents in dairy cattle diets depends on the secretion of salivary buffers, the buffering capacity of feed and feed acidogenic value. Wadhwa et al. (2001) have extended a simple laboratory based technique for evaluating ruminal acid load from feedstuffs based on the dissolution of Ca from CaCO3. The acidogenic value (AV) of the feedstuffs different with the non-fiber carbohydrate (NFC) content, protein and fiber concentrations. The highest AV was for starch rich feeds, forages were intermediate and protein sources had the lowest AV. Rustomo et al. (2006) reported that fiber sources had intermediate AV and protein sources had the lowest AV, wheat straw and alfalfa hay, as a fiber sources in ration, had lower AV than alfalfa pellet or corn silage. Additionally, the AV of feed ingredients were positively correlated to changes in rumen fluid pH after incubation, suggesting that high AV feeds were expected to increase the risk of rumen acidosis in dairy cows than low AV feeds. Ruminal methanogenesis represents an alternative mechanism of reducing equivalent disposal for carbohydrate-fermenting bacteria, but interspecies hydrogen transfer is only exergonic at very low partial pressures of hydrogen (Wolin, 1975). If the methanogens are inhibited, hydrogen accumulates, the hydrogenases are inhibited and the carbohydrate-fermenting bacteria utilize other mechanisms of reducing equivalent disposal (e.g. the dehydrogenases of propionate production) (Gottschalk, 1986). Sauvant and Giger-Reverdin (2007) realize the relationship between methane production and proportion of concentrate in the diet to be curvilinear, with methane losses of 6-7% of gross energy (GE) being constant at 30- 40% concentrate levels in the diet and then decreasing to 2-3% of GE with a concentrate proportion of 80-90%. The objective of the present experiment was to determine the effect of buffering capacity (BC) of various mixed inorganic compounds on in vitro rumen acidogenic value and methane emission from diets containing various forage to concentrate ratios.

 

MATERIALS AND METHODS

Diets, chemical composition and inorganic buffering compounds

The experimental diets were designed to provide two different forage to concentrate ratios as 40:60 (FC40:60) and 30:70 (FC30:70), respectively. Ration ingredients and nutrient compositions are presented in Table 1. Inorganic mixtures were made of different compositions of NaHCO3, Na2CO3, CaCO3, MgCO3, MgO and bentonite Na as 500, 200, 90, 100, 100 and 10 g/kg (M1) or 300, 200, 200, 150, 100 and 50 g/kg (M2), respectively. This inorganic mixtures had the highest buffering capacity and obtained from Acros brand. A modification of the procedure of Evans and Ali (1967) was used to measure the buffering capacity. Approximately, 1 g DM sample of individual buffering compound or the mixtures of M1 and M2 was suspended in 100 mL distilled water and stirred continuously with a magnetic stir bar. Titrations were performed by addition of HCl (83.3 mL/L) or NaOH (40 g/L) (Merck brand) in variable increments until pH was decreased to 4 or increased to 9. Buffering capacity and initial pH of the individual and the compositions used in the present experimental diets are shown in Tables 2 and 3, respectively.

 

Table 1 Ingredient and chemical compositions of the experimental diets

 

 

Each composition (M1 or M2) were added to the diets of FC40:60 and FC30:70 at the rate of 0.0, 10 and 20 g/kg DM. Ingredients used in the diets were ground through a mill with a 1-mm sieve, then dried using air-forced oven (48 h, 65 ˚C). Nitrogen content of each ingredient was determined using Kjeldahl method (Kjeltec 2300 Autoanalyzer Foss Tecator AB, Hoganas, Sweden) and CP was calculated as N × 6.25. Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined according to Van Soest et al. (1991). Samples were also analyzed for ash by igniting the samples in muffle furnace at 525 ˚C for 8 h.

 

In vitro acidogenic value

In vitro technique used in this experiment adapted from Wadhwa et al. (2001). Appropriately, one-gram (DM) of each diet was placed into a 125-mL incubation bottle, then M1 or M2 was added as the experimental protocol, then bottles were held at 39 ˚C in a water-bath. The samples were incubated in a 3 run and quadruplicate with 30 mL of buffered rumen liquor comprising 60% buffer and 40% rumen liquor. The buffer (5.880 g/L NaHCO3; 5.580 g/L Na2HPO4; 0.282 g/L NaCl; 0.342 g/L KCl; 0.028 g/L CaCl2.2H2O and 0.036 g/L MgCl2) (Merck brand) was made up at 20% the strength of the Tilley and Terry (1963). Rumen fluid was collected from two rumen fistulated dairy cows fed corn silage, alfalfa hay and concentrates 25, 25 and 50%, DM, respectively; at 3 h after the morning feeding. Cysteine hydrochloride monohydrate (Merck brand) (0.025% wt/vol) was added just prior to the incubations.

 

Table 2 Buffering capacities and initial pH of the individual inorganic buffering inorganic compounds

  

The bottles were closed with gas release valves and shaken continuously. A set of bottles without feed sample was also incubated similarly which served as blank. After 24 of the incubation, the bottles were transferred to an ice bath to stop fermentation, and then opened to measure medium pH using a pH meter (Metrohm pH meter, model 691). Bottle contents were filtered and a 2 mL sample of the supernatant from each bottle was taken to analyze residual acidity (acidogenic value).

 

Table 3 Buffering capacities and initial pH of different composition of inorganic compounds

 

* M1 and M2 were made of from NaHCO3, Na2CO3, CaCO3, MgCO3, MgO and bentonite Na as 500, 200, 90, 100, 100 and 10 g/kg or 300, 200, 200, 150, 100 and 50 g/kg, respectively.

 

The filtrated residual was oven dried (75 ˚C for 48 h), weighted and used to calculate in vitro dry matter disappearance (IVDMD). The supernatant of each bottle was transferred into a 2 mL centrifuge tube containing excess amount of CaCO3 powder (Merck brand) (50 mg). The mixture was shaken manually for 5 s and then centrifuged at 4000 × g for 10 min. The supernatant Ca concentration was then immediately determined using an autoanalyzer (A15 Biosystem). A measurement of dissolution of Ca from insoluble CaCO3 powder makes it possible to assess residual acidity after fermentation of feeds.

 

In vitro rumen methane emission

An in vitro incubations were carried out as proposed by Menke and Steingass (1988). Appropriately, 200 mg (DM basis) of each experimental diet was placed into a 125 mL incubation bottle, then inorganic mixtures of M1 or M2 was introduced as rate of 0.0, 10 or 20 g/kg DM. A set of bottles without feed sample was also incubated similarly which served as blank. The bottles were incubated with 30 mL of buffered rumen fluid (artificial saliva to the rumen liquid in ratio of 2:1) and held at 39 ˚C in a water-bath. The artificial saliva was made up of 475 mL/L distilled water, 240 mL/L buffer solution (ammonium bicarbonate 4 g/L and sodium bicarbonate 35 g/L, Merck brand), 240 mL/L macromineral solution (5.7 g anhydrous Na2HPO4, 6.2 g anhydrous KH2PO4 and 0.6 g MgSO4.7H2O per liter, Merck brand), 0.12 mL/L micro-mineral solution (13.2 g CaCl2.2H2O, 10.0 g MnCl2.4H2O, 1 g CoCl2.6H2O and 8.0 g FeCl3.6H2O per 100 mL, Merck brand), 1.22 mL/L Resazurin aqueous (Merck brand) (1 mg/1 mL). The medium was then reduced by addition of reducing agent (47.5 mL distilled water, 2 mL 1 N NaOH and 336 mg Na2S.9H2O, Merck brand) per liter of medium. Rumen fluid was collected as described previously. The incubation was carried out in two set (run) and in triplicate. After 24 of incubation, the bottles were transferred to an ice bath to stop fermentation, then total gas was recorded by digital pressure indicator (model SEDPGB0015PG5) and methane emission was determined using a biological gas recorder (SR2-BIO).

 

Calculations and statistical analysis

The buffering capacity (BC, meq/L) was calculated by the following formula (Evans and Ali, 1967):

BC= [(milliliters of 1 N HCl) + (milliliters of 1 N NaOH)] × 103/30

In vitro acidogenic value (mg Ca/g DM) of each sample was calculated as the product of Ca concentration (mg/mL, from the analysis) and fluid volume (30 mL) divided by the sample weight (1 g). In vitro dry matter disappearance was calculated as follows (Jahani Azizabadi et al. 2011): 

IVDMD (%)= [(A-(B-C)) / A] × 100

Where:

A: dry weight of sample.

B: dry weight of residue after incubation.

C: dry weight of blank.

Data were analyzed as a completely randomized design to compare the diets and buffering composition in each experimental diet with replications using Dunnetts test (P<0.05) procedure in SAS (2002).

 

RESULTS AND DISCUSSION

The effect of inorganic mixtures supplementation on the in vitro medium pH, AV and IVDMD after 24 hours incubation within the experimental diets containing 40:60 or 30:70 forage to concentrate ratios are shown in Table 4 and Table 5, respectively.

 

Table 4 In vitro ruminal medium pH, acidogenic value [AV, (mg Ca/g DM)] and dry matter disappearance (IVDMD, %) of a dairy cow diet containing different forage: concentrate ratio as 40:60 which supplemented with different composition of inorganic buffering compounds (M1 and M2), after 24 h incubation

 

1 M1 and M2 were made of from NaHCO3, Na2CO3, CaCO3, MgCO3, MgO and bentonite Na as 500, 200, 90, 100, 100 and 10 g/kg or 300, 200, 200, 150, 100 and 50 g/kg, respectively.

2 FC40:60: a dairy cow diet containing 40% forage and 60% concentrate.

SEM: standard error of the means.

The means within the same column with at least one common letter, do not have significant difference (P>0.05).

 

Table 5 In vitro ruminal medium pH, acidogenic value [AV, (mg Ca/g DM)] and dry matter disappearance (IVDMD, %) of a dairy cow diet containing different forage: concentrate ratio as 30:70 which supplemented with different composition of inorganic buffering compounds (M1 and M2), after 24 h incubation

 

1 M1 and M2 were made of from NaHCO3, Na2CO3, CaCO3, MgCO3, MgO and bentonite Na as 500, 200, 90, 100, 100 and 10 g/kg or 300, 200, 200, 150, 100 and 50 g/kg, respectively.

2 FC30:70: a dairy cow diet containing 30% forage and 70% concentrate.

SEM: standard error of the means.

The means within the same column with at least one common letter, do not have significant difference (P>0.05).

 

Increased concentrate from 60 to 70%, due to increasing the level of rapidly fermentable carbohydrates caused a decrease in medium pH and a significant (P< 0.05) increase in AV. By increasing the amount of acid produced as a result of fermentation of carbohydrates, pH levels on the medium was reduced. High concentrate diets contain high amounts of non structural carbohydrates which are quickly fermented by ruminal microbes, resulting in a greater decline in ruminal pH (Kalscheur et al. 1997). Results of previous studies have shown a decline in ruminal pH when more rapidly fermentable carbohydrates were included in the diet (Krause et al. 2002b). Danesh Mesgaran et al. (2009) reported that there is a positive correlation between non fibrous carbohydrates in diet and acidogenic value, so that by increasing the non fibrous carbohydrates in diet, the AV enhanced. As described by Rustomo et al. (2006), energy feeds and fiber sources have the highest and intermediate AV, respectively. Results indicated that the adding of M1 at both rate and M2 at 20 mg/kg to FC40:60 caused a significant (P<0.05) increase in medium pH. Inorganic buffers are capable in preventing pH reduction in the medium through neutralizing acids produced by bacterial activities. The highest significant (P<0.05) level of medium pH was belonged to FC30:70 plus M1 and M2 which were added at the rate of 20 g/kg DM. Inorganic buffers enhance ruminal environmental conditions by modulating acidity of the ruminal contents, preventing severe drops in pH (Le Ruyet and Tucker, 1992). Tripathi et al. (2004) have also reported that NaHCO3 supplementation caused a linear enhancement in ruminal fluid pH. In experiment of West et al. (1987), the addition of various buffers to the diet also resulted in a significant increase in rumen pH. Santra et al. (2003) reported that dietary buffers prevent the reduction of rumen pH when animal fed a high levels of concentrate. With regard to AV (Tables 4 and 5), none of the M1 and M2 supplementation had significant (P>0.05) effect compared with the non-supplemented diet, which might express to this cause that the inorganic mixtures could not significantly affected the buffering capacity and ultimately the acid load created in the medium. Dietary buffers are widely used to improve the harmful effects of acidity in high concentrate diets (Coppock et al. 1986), but the response of buffer is variable and sometimes unpredictable. Erdman (1988) have also reported that the buffering agents that possess a pKa above the typical ruminal fluid pH will act as alkalinizing agents rather than simply as buffers to increase the resistance of the rumen to a change in pH. Present results indicated that the adding of the inorganic mixtures to the experimental diets, had no significant effect on IVDMD (Tables 4 and 5), which, probably indicated no effect of the inorganic mixtures on fermentation at the medium. Bodas et al. (2009) used sodium bicarbonate in the diet of lambs and did not obtain difference in dry matter digestibility. However, Mould and Qrskov (1983) reported that the addition of buffer due to maintenance of ruminal pH above the critical level might improve the DM digestibility. The effect of inorganic mixtures supplementation on the in vitro total gas, CH4 and CO2 emission from diets containing different forage to concentrate ratios as 40:60 and 30:70 are shown in Table 6 and Table 7, respectively. Increased concentrate from 60 to 70%, caused a significant decrease in both total gas and CO2 emission, which is probably due to the negative impact of rapid fermentation of carbohydrates on microorganisms. Rumen pH is one of the most critical determinants for rumen function as cellulolytic bacteria fail to grow below pH 6.0, while a slight increase in ruminal pH favors the activity of these bacteria (Santra et al. 2003).Within both the experimental diets, the supplementation with M1 and M2 alter total gas, methane and carbon dioxide produced in the medium significantly (P<0.05). The lowest levels of total gas were observed in FC40:60 containing M1 at 20 and M2 at both 10 and 20 g/kg DM. Methane emission was significantly (P<0.05) higher when M1 was added to FC40:60 at the rate of 10 g/kg DM and FC30:70 at the rate of 20 g/kg DM compared with the non-supplemented diets. Present results indicated that FC40:60 containing M1 at 20 g/kg DM, M2 at 10 and 20 g/kg DM and FC30:70 plus M1 at 10 g/kg DM had the lowest level CO2 emission (P<0.05). The pattern of the responses was influenced by the kind of inorganic composition and the concentration applied. Inorganic mixture M1 to M2, had the highest buffering capacity and by increasing the amount of M1 in the FC40:60, decreased total gas, methane and carbon dioxide production, while, with the increasing the amount of M2 in the diet, increased total gas, methane and carbon dioxide production. But the results in the FC30:70 against the results of the FC40:60.

 

Table 6 In vitro total gas (mL/0.20 g DM), CH4 (mL/0.20 g DM) and CO2 (mL/0.20 g DM) of a dairy cow diet containing different forage: concentrate ratio as 40:60 which supplemented with different composition of inorganic buffering compounds (M1 and M2), after 24 h incubation

 

1 M1 and M2 were made of from NaHCO3, Na2CO3, CaCO3, MgCO3, MgO and bentonite Na as 500, 200, 90, 100, 100 and 10 g/kg or 300, 200, 200, 150, 100 and 50 g/kg, respectively.

2 FC40:60: a dairy cow diet containing 40% forage and 60% concentrate.

SEM: standard error of the means.

The means within the same column with at least one common letter, do not have significant difference (P>0.05).

 

Table 7 In vitro total gas (mL/0.20 g DM), CH4 (mL/0.20 g DM) and CO2 (mL/0.20 g DM) of a dairy cow diet containing different forage: concentrate ratio as 30:70 which supplemented with different composition of inorganic buffering compounds (M1 and M2), after 24 h incubation

 

1 M1 and M2 were made of from NaHCO3, Na2CO3, CaCO3, MgCO3, MgO and bentonite Na as 500, 200, 90, 100, 100 and 10 g/kg or 300, 200, 200, 150, 100 and 50 g/kg, respectively.

2 FC30:70: a dairy cow diet containing 30% forage and 70% concentrate.

SEM: standard error of the means.

The means within the same column with at least one common letter, do not have significant difference (P>0.05).

 

It seems that increase the buffering capacity environment by adding the inorganic mixtures, as well as increasing its concentration with effects on pH, rumen bacterial fermentation performance is affected. Dietary buffers by increasing the buffering capacity of the medium might produce a situation by which a huge decrease in pH may prevent and thereby causing an increase in methane levels in the culture medium. It was reported that dietary buffer would prevent depression in rumen pH and improve rumen ecology associated with high concentrate feeding (Santra et al. 2003). The amount of decrease in pH after an increase in the fermentation rate will depend on the buffering capacity of the rumen fluid (Counotte et al. 1979). Supplementation of minerals in the diet of animals is known to increase the number of total ruminal bacteria especially the cellulolytic bacteria which contributed to better cellulose digestibility (Koul et al. 1998). However, diets high in cereals consequently reduce ruminal pH and cellulolytic activity (Franzolin and Dehority, 1996). It has long been recognized that the addition of cereal grains to ruminant diets causes a decrease in methane and an increase in propionate production (Czerkawski, 1986), but the cause of this fermentation shift was not clear.

 

CONCLUSION

It has been concluded that there is a chance to increase the buffering capacity of inorganic composition when different amount of the inorganic chemical compounds were used compared with the sodium bicarbonate. Adding inorganic compound had the highest buffering capacity, reducing the amount of the acid load and increased IVDMD, although the effect was not significant compared to the control that might be due to lack of impact of the inorganic compound on the buffering capacity of the medium. Increasing the concentration of mixture M1 in the diet, causes was reduced in the total gas, CH4 and CO2. But with regard to mixture M2, the opposite was observed may be due to the effect of buffering capacity created by this mixtures on pH medium and microbial activity.

 

ACKNOWLEDGEMENT

The authors wish to acknowledge the Ferdowsi University of Mashhad, Iran, for financial support.

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