Effect of feeding grain on ruminal acidosis in cattle: validation of an NIR acidosis index. Study Report

Transcription

1 Effect of feeding grain on ruminal acidosis in cattle: validation of an NIR acidosis index Study Report Ahmad Rabiee Ian Lean Bovine Research Australasia Bovine Research Australasia 2 Broughton St. P.O.Box 660 Camden NSW

2 Acidosis Grain Report/Dairy Australia Table of contents Executive summary Background and significance I. Pilot study 1. Objectives & experimental design 2. Study design & feeding during pilot study 3. Feeding regime II. Field study 1. Objectives 2. Study design 3. Sequence of trial events 4. Materials and Methods 4.1. Rumen sampling 4.2. Grass silage samples 4.3. Grain size 4.4. Analytical procedures 4.5. Statistical analysis Results 1.1. Pilot study & discussion 1.2. Field study 1.3. Correlation of in vivo finding with NIR Discussion References Appendices Appendix I- Strategic plans for ruminal sampling following the challenge (Pilot study) Appendix II - Grain offered and refusal Appendix III - Silage analysis results Appendix IV- Grain size Appendix V- Grain offered, refusal and sampling events Acknowledgement Disclaimer Bovine Research Australasia 2

3 Acidosis Grain Report/Dairy Australia Executive summary The objective of this study was to develop an index to reflect risk of acidosis created by feeding different amounts (Pilot study) and types (Field study) of grain to dairy cattle. A second goal was to compare the index developed with a NIR-based acidosis index to these developed in vivo. Two studies were conducted to explore these objectives: I. Pilot study Aims: A pilot study was conducted to determine the amount of grain (Triticale Jackie and mixed grain) that produces a moderate change in rumen function (one which would reflect a challenge typical to moderately severe in the dairy industry), following the oral administration of four different levels of Triticale Jackie and mixture of grains (n=20) (control). Methods: Eight young Holstein-Friesian heifers, less than 18 months of age, and 8 Holstein-Friesian dairy cows were randomly selected from a group of cattle and were allocated to 2 challenge diets, fed at four different levels. The feeding regime period was divided into 3 periods: Pre-adaptation period (7 days): Fed 1kg per head of rolled grain and ad libitum grass silage. Adaptation period (5 days): Fed 1 kg per head per day of rolled mixed grain (n= 8) (control; a blend of 20 test grains) or 1 kg rolled Triticale Jackie (n=8) and ad libitum grass silage. The Challenge: Cattle were withheld from all feed for a period of 14h and then fed challenge diets. The challenge diets provided the two test grains, mixed and Triticale Jackie, at 4 different levels to identify the amount of grain that may cause ruminal acidosis. Each of the two groups of four were fed as follows: 1. 0 kg/head (control) % of bodyweight (0.4 kg per 100 kg) % of bodyweight (0.8 kg per 100 kg) % of bodyweight (1.2 kg per 100 kg) Outcome measured: The following parameters were measured to assess the acidosis index of different amounts of grain. Rumen ph Rumen concentrations of Volatile Fatty Acids (VFAs) ÿ Acetate ÿ Propionate ÿ Isobutyrate ÿ Butyrate ÿ Isovalerate ÿ Valerate Bovine Research Australasia 3

4 Acidosis Grain Report/Dairy Australia ÿ Caproate Rumen concentrations of D-Lactate and L-lactate Rumen concentration of ammonia Grass silage and grain analyses Key findings: There was a significant reduction in rumen ph (P = 0.01) of cattle fed at the rate of 1.2% of their bodyweights. The concentrations of VFAs were higher in cattle fed at the rate 0.8% and 1.2% of bodyweights than the control. The concentrations of rumen valerate (P < 0.001) and ammonia (P = 0.04) were significantly greater in cattle fed at the rate of 1.2% of bodyweights. The rumen concentrations of valerate were significantly (P = 0.05) higher in cattle fed with Triticale Jackie than the mixed grain (Control). This may indicate that the difference in the acidosis index between different types of grain can be described more reliably with the rumen concentration of valerate than by other variables. Ranking procedures showed that cattle fed at 1.2% of bodyweight had a higher acidosis index compared with cattle fed grain at 0% (control), 0.4% and 0.8% of bodyweight. The acidosis index of Triticale Jackie grain was also greater than the mixed grain (Control). Conclusion: At the 1.2% of bodyweight challenge dose, cattle showed marked and rapid changes in ruminal VFAs and ammonia. There was some evidence at this dose (1.2%) of early and late accumulation of lactic acid. The 1.2% of bodyweight dose is consistent with that used in previous challenge studies in North America. Preliminary data evaluation, further data analysis using approaches developed by Bramley (2004), consistency with previous studies and lack of evidence of severe acidosis lead us to conclude that a 1.2% dose was satisfactory for a challenge model that could discriminate between grains. II. Field study Aims: The objectives of this study were to: i) Assess and compare the effect of different types of grain fed to the cattle on ruminal ph and ruminal fermentation products including volatile fatty acids (VFAs), lactate (D- and L-) and ammonia; ii) Determine the correlation between NIR derived estimates of acidotic risk of grains and the in vivo effect of the grains. Bovine Research Australasia 4

5 Acidosis Grain Report/Dairy Australia Methods: The design for this study formulated as row-column design with 40 rows (HEIFER) and column (RUN) with cattle split into 4 groups of 10 (GROUP). This design maxmised the number of treatments in each run and allowed for good estimation of the between grain treatment effects. Cattle were fed with mixed silage and rolled grain. The feeding regime period was divided into 6 periods: ÿ Pre-trial period: Fed mixed silage and 1 kg grain during this period. ÿ Adaptation period: The cattle were adapted to the test grain for a period of 4 days. All cattle were fed grass silage and 1 kg of test grain until the challenge day. ÿ Withholding period: All cattle had all feed withheld for 14 hours, and the n were fed with allocated test grain at the rate of 1.2% of their bodyweights. ÿ Experimental period: The amount of grain that was required to be fed at the experimental period was calculated to provide an appropriate amount of carbohydrate, as determined by the pilot study (1.2% bodyweight). Ruminal samples were collected immediately after the challenge, then 1h after challenge, and subsequent samples were collected every 45-50min, during the experimental period for a period of 5.0 h. ÿ Post-experimental period: All cattle were monitored for a period of 2 days following the experimental period. ÿ Wash out period: A 9-day period was considered as wash out period between phases of the study. Outcomes measures: Outcomes measured for the field study were identical to those in the pilot study. Key findings: The rumen ph significantly changed throughout the sampling period (4 hours) (P< 0.01). The rumen ph at Time 4 (4 hours after the challenge) was lower in cattle fed Barley2 (ID: 3864), Triticale3 (ID: 6824) and Wheat6 (ID: 1758), than the other grains. The rumen concentrations of propionate, butyrate, isobutyrate, valerate, isovalerate and D-Lactate changed significantly during the sampling period (P < 0.05). The rumen ph and concentrations of VFAs, except caproate, were significantly influenced by the day of sampling (P < 0.05). The rumen concentrations of propionate, isovalerate, valerate and ammonia were significantly different among different types of grain (P < 0.05). The ranking of acidosis index of different types of grain using discriminant analysis was similar to the mixed effect models ranking. Bovine Research Australasia 5

6 Acidosis Grain Report/Dairy Australia Conclusions: The rumen concentration of valerate was more discriminatory, between different types of grain, than the other rumen fermentation products. Consequently, these measures (rumen concentrations of valerate) were used to rank the acidosis index of different types of grain. The mixed effect model showed that triticale and wheats had the highest acidosis index. These rankings were similar to rankings derived from discriminant analysis. The correlation between the in vivo ranking results of Time 4 discriminant scores was highly correlated with the rankings of coefficients of rumen concentrations of valerate, derived from the mixed effect model (P < ). The coefficients of rumen concentration of valerate were correlated with the NIR results derived from the in vitro tests ranking (P = 0.033). The correlation between Time 4 discriminant scores and the NIR results derived from the in vitro tests were also highly significant (P = ). The only measures that are related to biological measures of acidosis are those based on the discriminant scores data. Limitations of the study: In this study cattle were fed grain at the rate of 1.2% of bodyweight, and there was a rapid and marked change in the rumen concentrations of fermentation products. However, we were unable to induce the clinical or sub-clinical forms of acidosis consistent with the studies of Bramley (2004) with the 1.2% dose rate. Cattle had ad libitum access to grass or mixed silage over the trial period. It appears that the fibre (silage) in the diet may have increased the buffering capacity of the rumen and prevented a more severe ruminal challenge. Recommendations: Future studies may be enhanced by use of forages that are lower in effective fibre. Some consideration should also be given to the role of sugars in the basal diet as these may play an important role in the pathogenesis of acidosis. Samples have been retained that would be suitable for meta-genomic studies. Dairy Australia should consider using these for characterisation of changes in bacterial populations during ruminal challenge from starch. Bovine Research Australasia 6

7 Acidosis Grain Report/Dairy Australia Background and significance Grains are subject to microbial fermentation in the rumino-reticulum. The microbial fermentation of starches contained in grains can proceed to a point that causes the rumen to become acidotic, a state reflecting increases in acid accumulation, changes to microbial populations and death of micro-organisms. The severity of acidosis may range from mild to life threatening. Lactic acidosis induced by feeding large amounts of rapidly fermentable substrate is an important clinical condition in the dairy herd that has been well documented and reviewed in the literature (Dirksen, 1970, Huber, 1976, Nocek, 1997, Owens et al., 1998, Slyter, 1976, Underwood, 1992). However, subclinical acidosis and secondary effects such as depressed milk production, milk fat content and laminitis have not been well documented, especially in predominantly pasture-fed animals until recently. Bramley (2004) developed a method to identify subclinical acidosis in cattle based on rumen concentrations of volatile fatty acids, ammonia, ph and lactic acid concentrations. The index developed was strongly influenced by rumen VFAs and ammonia, whereas lactic acid and ph concentrations, while significant were less important predictors. Bramley (2004) found that the ratio of NFC (Non Fibre Carbohydrate) to NDF (Neutral Detergent Fibre) was the best predictor of herds with a high prevalence of acidotic cows. Acidosis is difficult to measure in cattle and sub-acute acidosis is an even more insidious problem and more difficult to diagnose. It may not be possible to eliminate all acidosis and still maintain economic levels of production, but the condition must at least be managed and controlled. Ruminal lactic acidosis is a clinical disorder of cattle that can result in rumenitis, metabolic acidosis, lameness, hepatic abscessation, pneumonia and death. Of greater economic importance are losses that result from subclinical acidosis in dairy cattle, particularly those fed on pasture, that may result in decreased intake of pasture known as substitution and lower milk fat (Lean et al. 2000, Bramley et al. 2002, 2004). Bramley (2004) identified that approximately 10% of 800 cows, less than 100 days in lactation, had ruminal conditions that were associated with increased milk production, but lower milk fat content and higher risk of lameness (Bramley, 2004). It can be estimated that the lactational incidence of the condition would be considerably higher than 10%. Herds with a high prevalence of acidotic cows were at greater risk of having a high prevalence of lameness (Bramley, 2004). Lameness is a significant practical and animal welfare problem for the dairy industry. The findings of this study should assist us to identify the type of grains that increase the risk of acidosis. Therefore, by feeding appropriate grains to cattle we will be better able to control the ruminal fermentation products and prevent acidosis and, subsequently reduce the risk of laminitis in dairy cattle. While the true costs of acidosis are difficult to quantify, the impacts on health, lameness and fertility are likely to be substantial and exceed losses associated with the next most important disorder, mastitis. Further, effects on milk production are bidirectional, with increased risk of acidosis likely to have a quadratic effect on milk production (that is mild cases having increased production Bovine Research Australasia 7

8 Acidosis Grain Report/Dairy Australia and more severe, markedly decreased production) and a linear negative effect on milk fat. A preliminary (Pilot) study was designed to determine the amount of the grain predicted to have the highest acidosis index required to create a large, but not health-threatening, ruminal change following the oral administration of four different levels of grains. The findings of pilot study were used for the second phase of the study (Field study) The second study (Field study) was conducted to compare the effect of different types of grains on ruminal ph and ruminal fermentation products. The findings of this study were used to determine the concordance between NIR derived estimates of acidotic risk of grains and the in vivo effect of the grains. I. Pilot study 1. Objectives & experimental design This pilot study was used to determine the amount of grain (Triticale Jackie and mixed grain) that produces a moderate change in rumen function, following the oral administration of four different levels of Triticale Jackie and mixed grain (control). The reference grain (Triticale Jackie) was benchmarked against a standard mixture of the grains (n=20) selected for use in the study. The grain mix was used as the control grain throughout the study and it was expected that the amount selected in the pilot study would provide a low to moderate acidosis response. 2. Study design & feeding during pilot study Eight young Holstein-Friesian heifers, less than 18 months of age, and 8 Holstein- Friesian dairy cows were randomly selected from a group of cattle and were allocated to 2 challenge diets, fed at four different levels. The bodyweights of heifers and cows at entry ranged from 410 to 650 kg and 650 to 800 kg, respectively. 3. Feeding regime Two weeks prior to the challenge day cattle were accustomed to the feed pad, by feeding cattle with grass silage and grain at the feed pad. After a few days, cattle were head locked for a period of 40-60min every day, and fed with grain and grass silage. The feeding regime period was divided into 3 periods: i) Pre-adaptation period (7 days): All cattle were brought to the feed pad twice a day (am and pm) and fed 1kg of rolled grain (as fed), 0.5 kg in the morning and afternoon, and had ad libitum access to grass silage. ii) Adaptation period (5 days): During this period the basal diet was ryegrass silage. In order to adapt the cattle to the test grains, cattle were fed 1 kg per head per day of Bovine Research Australasia 8

9 Acidosis Grain Report/Dairy Australia rolled mixed grain (n = 8) (control; a blend of the 20 test grains) or 1 kg rolled Triticale Jackie (n = 8) in the morning. During the adaptation period cattle had free access to grass silage. A list of rolled grains blended for the mixed grain is included in Appendix IV. iii) The Challenge: Cattle were withheld from all feed for a period of 14h and then fed the challenge diets. Cattle were fed first with 1kg of ryegrass silage, and then challenged with the allocated diet to reduce the saliva contamination during the rumen sampling at time 0. The challenge diets provided the two test grains, mixed and Triticale Jackie, at 4 different levels to identify the amount of grain that may cause ruminal acidosis. Cattle were fed on the basis of a 500 kg cow being the base unit (Table 1). Each of the two groups of four were fed as follows: 1. 0 kg/head (control) gm per 100 kg ie 2 kg for 500 kg cow or heifer ie 0.4% of bodyweight gm per 100 kg ie 4 kg for 500 kg cow or heifer ie 0.8% of bodyweight kg per 100 kg ie 6 kg for 500 kg cow or heifer ie 1.2% of bodyweight. The feeding of the test grains in cattle receiving 1.2% bodyweight and ruminal sampling were performed according to our strategic plan as described in Appendix I. Table 1. Group allocation, actual amount of grain offered and refusal for the experimental cattle fed mixed grain or Triticale Jackie on challenge day Cow/Heifer ID Grain Allocated grain group Amount of grain offered Refusal (kg) (kg/hd) (kg/hd) 776 Mixed grain NA 1195 Mixed grain NA 1031 Mixed grain Mixed grain Mixed grain Mixed grain Mixed grain Mixed grain Triticale Jackie 0 0 NA 994 Triticale Jackie 0 0 NA 1202 Triticale Jackie * Triticale Jackie Triticale Jackie Triticale Jackie Triticale Jackie Triticale Jackie *1211 was a spare cow and fed mixed grain during the adaptation period, but was challenged with Triticale on sampling day Bovine Research Australasia 9

10 Acidosis Grain Report/Dairy Australia II. Field study 1. Objectives The objectives of this study were to: iii) iv) Assess and compare the effect of different types of grain fed to the cattle on ruminal ph and ruminal fermentation products including volatile fatty acids (VFAs), lactate (D- and L-) and ammonia; Determine the concordance between NIR derived estimates of acidotic risk of grains and the in vivo effect of the grains 2. Study design The design for this study was formulated as row-column design with 40 rows (HEIFER) and column (RUN) with the cattle split into 4 groups of 10 (GROUP). This design maxmised the number of treatments in each run and allowed for good estimation of the between grain treatment effects. The study design was a randomized controlled and partially blinded clinical trial. It was essential for the operators involved in the trial to have knowledge of the grains fed, but no knowledge of the NIR rankings of the cultivars. Statistical analysis was performed without prior knowledge of cattle diet allocation. Each individual heifer had equal chance to be selected in each RUN and group A, B, C or D (Table 2). Table 2. Row-column design for 40 heifers, in 4 groups of 10 and 2 runs Heifer ID Run 1 Run 2 A1 Wheat3 Barley1 A2 Oats2 Control A3 Triticale1 Oats3 A4 Wheat1 Triticale3 A5 Control Sorghum1 A6 Control Wheat4 A7 Barley4 Control A8 Triticale4 Barley2 A9 Sorghum2 Wheat5 A10 Barley3 Oats1 B1 Wheat2 Oats3 B2 Control Wheat5 B3 Wheat4 Triticale1 B4 Barley1 Sorghum2 B5 Sorghum3 Control B6 Oats1 Triticale2 B7 Control Barley2 B8 Barley3 Control B9 Triticale4 Wheat6 B10 Oats2 Wheat1 C1 Barley3 Wheat4 C2 Control Oats3 Bovine Research Australasia 10

11 Acidosis Grain Report/Dairy Australia C3 Oats1 Wheat3 C4 Wheat6 Sorghum3 C5 Control Triticale2 C6 Triticale3 Control C7 Sorghum2 Control C8 Wheat2 Barley1 C9 Triticale1 Barley4 C10 Sorghum1 Wheat1 D1 Control Wheat2 D2 Sorghum3 Triticale1 D3 Triticale4 Control D4 Wheat1 Barley2 D5 Wheat6 Control D6 Control Wheat3 D7 Wheat5 Triticale2 D8 Oats1 Sorghum1 D9 Barley4 Oats2 D10 Triticale3 Barley1 3. Feeding regime Two weeks prior to the commencement of the study, young cattle were accustomed to the feed pad by feeding cattle with mixed silage (lucerne, clover and ryegrass) and rolled grain at the feed pad. The intention was to feed cattle with ryegrass during the adaptation period, to be consistent with the protocol. However, due to the current drought it was not possible to provide good quality ryegrass during the entire trial, except for cattle in group A (A1-A10) during RUN1 which were fed ryegrass silage 2 days prior to the adaptation day. Cattle in other GROUPs and RUNs were fed with mixed silage during the adaptation and wash out periods Sequence of trial events The sequence of this study was assigned to enable to undertake a RUN every 14 days, consisting of a 4-day adaptation to the test grains; 1 day challenge and 9-day wash out period. The next group of grains allocated for the following RUN was tested with a similar procedure and study period. 1. Pre-trial period: All cattle received a physical examination for health status before being enrolled for this trial. Cattle were fed mixed silage and 1 kg grain during this period. Two days prior to the adaptation period, heifers received 0.5kg Triticale grain and 0.5kg test grain daily. This helped heifers to become accustomed to the test grains. 2. Adaptation period: Young cattle were assigned randomly to their IDs and to the feeding order according to Table 2. The cattle were adapted to the test grain for a period of 4 days. All cattle were fed grass silage and 1 kg of test grain until the challenge day. 3. Withholding period: All cattle had feed withheld for 14 hours, from 6.00pm to 8.00am, and were then fed the allocated grain at the rate of 1.2% of bodyweight. Bovine Research Australasia 11

12 Acidosis Grain Report/Dairy Australia 4. Experimental period: The amount of grain that was required to be fed for the duration of the experimental period was calculated to provide an appropriate amount of carbohydrate, as determined by the pilot study. The data obtained from the pilot study showed that at the 1.2% of bodyweight challenge dose, cattle showed marked and rapid change in ruminal VFAs and ammonia. The 1.2% of bodyweight dose is consistent with that used in previous challenge studies in North America. Preliminary data evaluation, further data analysis using approaches developed by Bramley (2004), consistency with previous studies and lack of evidence of severe acidosis led the investigators to conclude that a 1.2% dose was satisfactory for a challenge model that could discriminate between grains. The amount of grain refusal was also recorded. In this trial, ruminal change was induced by administering an appropriate amount of rolled milled grain per animal once by ingestion. Cattle were fed first with kg (wet matter) of ryegrass silage, and then challenged with the allocated test grains. The first ruminal sample was collected immediately after the challenge, then 1h after the challenge, and the subsequent samples were collected every 45 to 50min, during the experimental period for a period of 5.0h. There were occasions when an appropriate rumen sample was not obtained in this time frame, in these cases the ruminal sampling intervals were extended to 55min. The order of sampling from control or treatment cattle was similar for all grains and phases of the study ie a randomized sampling schedule determined a sampling regime that was replicated for all sampling days. 5. Post-experimental period: All cattle were monitored for a period of 2 days following the experimental period. Therapeutic interventions, if required, were based on our Standard Operating Procedure (SOP) for the treatment of lactic acidosis. 6. Wash out period: A 9-day period was considered as wash out period between phases of the study. During this period cattle were fed mixed silage and 1 kg grain. Bovine Research Australasia 12

13 Acidosis Grain Report/Dairy Australia 4. Materials and Methods 4.1. Rumen sampling Cattle were brought to the feed pad and head locked. Immediately after the head was locked, cattle were offered 1kg of grass silage (wet matter) for the Pilot study, and approximately 0.35 kg mixed silage (wet matter) for the Field study, and then cattle were offered the challenge test grain according to their allocated group. The rumen samples were obtained using a custom designed tube, a pump and a container. The tube was removed when approximately 50ml rumen fluid was collected in the container. Rumen fluid was tested for the presence of saliva contamination in the sample by placing a finger into the sample and withdrawing it. If there was a stringing effect (salvia contamination), the sample was discarded and a further sample taken. If three samples were taken and there was still saliva contamination present, the sample was kept and saliva contamination noted. Raw rumen fluid samples obtained by stomach tube were placed in 70ml containers in the field and kept on ice until samples were centrifuged after collection at 3,000 rpm for 15 minutes and aliquots of the supernatant were dispensed into polypropylene tubes and stored at -20 o C until analysis. Summary of data collected is defined in Table 3. The raw rumen fluid samples were also retained and stored in 5ml vials to allow for future investigation of rumen microbial populations using gene probe methods. The rumen samples were analysed for changes in volatile fatty acids (VFAs), rumen lactic acid (Land D-Lactate) and ammonia concentrations. These four measures, that is rumen ph, measures of rumen VFAs, lactic acid and ammonia concentrations, were used to determine the amount (Pilot study) and types of grain (Field study) at which acidosis can be induced. Table 3. Data collection summary for rumen samples Variable Sample Units Place of analysis preservation Rumen ph Raw rumen fluid In field Rumen VFAs Supernatant- frozen mmo/l Agriculture WA Rumen D-lactate Supernatant mmo/l Agriculture WA Rumen L-lactate Supernatant mmo/l Agriculture WA Rumen protozoa Raw rumen fluid 10 5 /ml Storage Spare rumen fluid Supernatant- frozen Storage Spare- raw Raw rumen fluid Storage Following the determination of the appropriate amount of grain for acidosis induction (Pilot study), intervention treatments were prepared to be able to control the ruminal acidosis in any cattle that may have shown evidence of acidosis (Appendix I). Bovine Research Australasia 13

14 Acidosis Grain Report/Dairy Australia 4.2. Grass silage samples Grass silage samples were collected from each bale during the adaptation period and challenge days. The silage samples were then transferred to labeled plastic bags, frozen at -20 o C until analysis at George Weston Technologies (Weston Animal Nutrition Laboratories; Sydney, Australia). Sample results are displayed in Appendix III Grain size In order to determine the proportion of different sizes of rolled grains, the test grains were screened using 3.35mm and 2.0mm sieves, and the fine particles less than 2.0mm were collected in a collection tray. The weights of rolled grains for each size sieve were measured and the approximate proportions of these weights were estimated (Figure 1). Data on the types of grain, IDs and percentages of different proportions of sizes are provided in Appendix IV. 3.35mm sieve mm sieve <2.0mm (Bottom tray) 80 Percentage (%) Control (Mixed) Barley1 (3863) Barley2 (3864) Barley3 (3862) Barley4 (3861) Oats1 (5817) Oats2 (5818) Oats3 (5816) Sorghum1 (7856) Sorghum2 (7857) Sorghum3 (7858) Triticale1 (6709) Triticale2 (6823) Triticale3 (6824) Triticale4 (6825) Wheat1 (1753) Wheat2 (1754) Wheat3 (1755) Wheat4 (1756) Wheat5 (1757) Wheat6 (1758) Figure 1. Approximate proportion of different particle sizes of rolled grains 4.4. Analytical procedures Rumen ph analysis. Rumen ph was measured immediately in samples of the raw rumen fluid obtained by stomach tube. The ph meter (ph Testr 30) was calibrated with buffers four, seven and 10 before the start of each sampling period. The probe was cleaned with tap water after each sample. The probe was inserted into the rumen fluid and the ph recorded. Bovine Research Australasia 14

15 Acidosis Grain Report/Dairy Australia Rumen volatile fatty acids (VFA) analysis. Rumen fluid was analysed for VFAs by the Department of Agriculture in Western Australia using a gas chromatograph (GC). The raw rumen samples were thawed to room temperature, mixed, and allowed to stand until settled. One half ml of the supernatant of each sample was placed into separate microcentrifuge tubes and centrifuged at rpm for 10 minutes. For each sample, 100 µl of the supernatant together with 1 ml of pre-made internal standard solution was placed into a GC vial. One hundred samples were run at once. With each run, a standard and blank were also run. The standard contained 1 ml internal standard solution and 100 µl working standard solution (pre-made). The blank contained 1 ml internal standard solution and 100 µl distilled water. The GC vials were then mixed following placement of lids and auto-analysed using the HP Capillary GC. Seven identical samples were analysed every 100 samples to ensure accuracy of the results. Concentrations (mm) of propionic, acetic, isobutyric, butyric, isovaleric, valeric and caproic acid were reported for each sample. The coefficients of variation for assay of propionic, acetic, isobutyric, butyric, isovaleric, valeric and caproic acids were 3.8%, 5.1%, 3.2%, 4.0%, 3.4%, 3.7% and 3.2% respectively. Rumen D-lactate and L-lactate. A Boehringer Manheim kit (Cat. No ) was used to analyse D-lactate. The oxidation of D-lactate is an enzymatic assay linked through D-lactate dehydrogenase by NAD +. The increase in NADH was monitored at 340 nm in a spectrophotometer. The coefficient of variation (CV) for assay of D-lactate also depended on the concentration of D-lactate; with a CV of 22.7% for concentrations up to 1 mm and 2% for concentrations greater than 1 mm. Rumen fluid samples were also analysed for L-lactate at Agriculture WA using the same kit used for D-lactate. The oxidation of L-lactic acid requires the presence of the enzyme L-lactate dehydrogenase with the increase in NADPH monitored at 340 nm in a spectrophotometer. The assay was completed using a Boehringer Manheim kit (Cat. No ). The coefficient of variation (CV) for assay of L-lactate also depended on the concentration of L-lactate; with a CV of 7.9% for concentrations up to 1 mm and 1.4% for concentrations greater than 1 mm. Rumen ammonia. Samples were analysed for ammonia using a Boehringer Mannheim kit (Cat. No ). The assay of ammonia is an enzymatic assay linked through glutamate dehydrogenase to the oxidation of NADPH. This oxidation of NADPH was monitored at 340 nm in a spectrophotometer. The coefficient of variation (CV) for ammonia assay depended on the concentration of ammonia; with a CV of 3.7% for concentrations up to 30 mg/l and 1.1% for concentrations greater than 30 mg/l up to approximately 50 mg/l Statistical analysis The data derived from the 5 consecutive samplings following the challenge time (time 0) were analysed. The effects of time, day of sampling (Field study), amount of grain (Pilot study only) and types of grain (Field study) on rumen ph, concentrations of VFAs, D- and L-lactate and ammonia and interactions between grains, time and day of Bovine Research Australasia 15

16 Acidosis Grain Report/Dairy Australia sampling were analysed using a Generalised Linear Model (GLM) with repeated measures (SPSS, Version 12.0 Apache Software). For the Pilot study the estimated marginal means derived from the GLM analysis were used to rank the acidosis index of different amount of grain fed to the cattle. For the Field study a mixed effect model was used to compute the coefficients for each variable using STATA 9 (StatCorp LP, USA). In this model heifer was the random effect term and each variable (measurement) of rumen function (such as ph, VFAs, ammonia and lactates) were the independent variables. A series of dichotomous (indicator) variables were generated for each grain leading to 21 grain contrast variables. Day of sampling was used as a covariate. The coefficients produced, using the mixed effect model, for each grain were used to rank the grains. Grains with the highest coefficients had the highest acidosis ranking; except for the rumen ph, where grains with the lowest coefficients had the highest acidosis ranking. Statistical analysis for group allocation was also performed using K-means cluster analysis and discriminant analysis (SPSS, Version 12.0 Apache Software). Predictive variables used in the cluster analysis were converted to standardized variates before clusters were assessed. Rumen data were then standardised (z score) and ruminal assessments (VFAs, ammonia, lactic acid) from each heifer were evaluated by discriminant analysis using cluster analysis algorithms previously developed (Bramley, 2004) and evaluated for concordance with acidotic group cattle. Both these methods were used to develop a single measure of the acidosis potential of a test grain with weighting being applied to the time taken for significant changes in rumen fermentation products. The discriminant scores of the standardized discriminant function coefficients were used to rank the different types of grain. The standardized discriminant function coefficients in Table 4 serve the same purpose as beta coefficients in multiple regression; these indicate the relative importance of the independent variables in predicting the dependent. These discriminant scores were calculated based on the standardised canonical discriminant function coefficients. Canonical discriminant function coefficients are used to compare the relative importance of the independent variables. The standardized discriminant function coefficients are also used to assess each independent variable's unique contribution to the discriminant function. Function 1 2 Zscore(Acetic) Zscore(Propionic) Zscore(Isobutytic) Zscore(Butyric) Zscore(Isovalerate) Zscore(Valerate) Zscore(pH) Zscore(Caproic) Zscore(D-lactate) Zscore(Ammonia) Table 4. Standardized canonical discriminant function coefficients Bovine Research Australasia 16

17 Acidosis Grain Report/Dairy Australia The level of agreement between the in vivo ranking of acidosis potential of a grain and the NIR ranking from the PGLP project were measured using Spearman's Coefficient of Rank Correlation. These correlations measured how rank orders were related. The Pearson correlations were also used to measure the correlation between the standardised values (z score) of rumen valerate concentrations and discriminant analysis category data. 5. Results 5.1. Pilot study No cow or heifer, except one, showed any sign of serious discomfort on challenge day. Rumen sampling was not successful in one of the cows in Triticale group (0.4%, 2kg); this heifer was subsequently replaced with a heifer, which was on mixed grain diet during the adaptation period. In most cases, rumen fluid was successfully obtained, and at least 5 rumen samples were collected following the initial sampling at time 0. There were three animals that the 4 th and 5 th rumen samplings were not successful or the rumen samples were contaminated with saliva. Figures 2a- 2l show changes in the rumen ph and the concentrations of VFAs, L- and D-Lactate and ammonia for cattle fed four different levels of grain. Figure 2a shows that cattle were fed with grains at 0.8% and 1.2% of bodyweights had a bigger drop in rumen ph than those cattle fed at 0% and 0.4% of bodyweights. (a) (b) Rumen ph Time_0 Time_1 Time_2 Time_3 Time_4 Rumen concentration of Acetate (mm) Time_0 Time_1 Time_2 Time_3 Time_ % of BW (2-3kg) 0.8% of BW (4-6kg) Groups 1.2% of BW (6-8kg) Control (0 kg) 0.4% of BW (2-3kg) 0.8% of BW (4-6kg) Groups 1.2% of BW (6-8kg) Control (0 kg) Bovine Research Australasia 17

18 Acidosis Grain Report/Dairy Australia (c) (d) Rumen concentration of Propionate (mm) Time-0 Time_1 Time_2 Time_3 Time_4 Rumen concentration of IsoButyrate (mm) Time_0 Time_1 Time_2 Time_3 Time_ % of BW (2-3kg) 0.8% of BW (4-6kg) Groups 1.2% of BW (6-8kg) Control (0 kg) 0.4% of BW (2-3kg) 0.8% of BW (4-6kg) Groups 1.2% of BW (6-8kg) Control (0 kg) (e) (f) Rumen concentration of Butyrate (mm) Time_0 Time_1 Time_2 Time_3 Time_4 Rumen concentration of IsoValerate (mm) Time_0 Time_1 Time_2 Time_3 Time_ % of BW (2-3kg) 0.8% of BW (4-6kg) Groups 1.2% of BW (6-8kg) Control (0 kg) 0.4% of BW (2-3kg) 0.8% of BW (4-6kg) Groups 1.2% of BW (6-8kg) Control (0 kg) Bovine Research Australasia 18

19 Acidosis Grain Report/Dairy Australia (g) (h) Rumen concentration of Valerate (mm) Time_0 Time_1 Time_2 Time_3 Time_4 Rumen concentration of Caproate acid (mm) Time_0 Time_1 Time_2 Time_3 Time_ % of BW (2-3kg) 0.8% of BW (4-6kg) Groups 1.2% of BW (6-8kg) Control (0 kg) 0.4% of BW (2-3kg) 0.8% of BW (4-6kg) Groups 1.2% of BW (6-8kg) Control (0 kg) (i) (j) Rumen concentration of L-Lactate (mm) Time_0 Time_1 Time_2 Time_3 Time_4 Rumen concentration of D-Lactate (mm) Time_0 Time_1 Time_2 Time_3 Time_ % of BW (2-3kg) 0.8% of BW (4-6kg) Groups 1.2% of BW (6-8kg) Control (0 kg) 0.4% of BW (2-3kg) 0.8% of BW (4-6kg) Groups 1.2% of BW (6-8kg) Control (0 kg) Bovine Research Australasia 19

20 Acidosis Grain Report/Dairy Australia (k) (l) Rumen concentration of Ammonia (mm) Time_0 Time_1 Time_2 Time_3 Time_4 Total VFA (Mm) Time_0 Time_1 Time_2 Time_3 Time_ % of BW (2-3kg) 0.8% of BW (4-6kg) Groups 1.2% of BW (6-8kg) Control (0kg) 0.4% of BW (2-3kg) 0.8% of BW (4-6kg) Groups 1.2% of BW (6-8kg) Control (0 kg) Figures 2a- 2l: Rumen ph and rumen concentrations of VFAs, D- and L-Lactate and ammonia in cattle fed four different levels of grain. The estimated marginal means produced for four different levels of grain (groups), using GLM with repeated measures methods, are presented in Table 5. There was a significant reduction in rumen ph (P = 0.01) of cattle fed at 1.2% of their bodyweights. The concentrations of VFAs were higher in cattle fed at the rate 0.8% and 1.2% of bodyweights. The concentrations of rumen valerate (P < 0.001) and ammonia (P = 0.04) were significantly greater in cattle fed at the rate of 1.2% of bodyweights (Table 6). The rumen concentrations of valerate were significantly (P = 0.05) higher in cattle fed with Triticale Jackie than the mixed grain (Control) (Tables 6). This may indicate that the difference in the acidosis index between different types of grain can be described more reliably using rumen concentrations of valerate than the other variables assessed. Table 5. Estimated marginal means of rumen fermentation products when cattle were fed at 4 different levels of grain (groups) Estimated Marginal Means Mean ± SD Quantities of grain fed Control (0 kg) 0.4% of BW (2-3kg) 0.8% of BW (4-6kg) 1.2% of BW (6-8kg) ph (Stomach tube) 7.30 ± ± ± ± 0.03 Total VFA (mm) ± ± ± ± 5.82 Acetate (mm) ± ± ± ± 4.43 Propionate (mm) 7.99 ± ± ± ± 0.78 Bovine Research Australasia 20

21 Acidosis Grain Report/Dairy Australia A/P ratio 5.67 ± ± ± ± 0.27 Butyrate (mm) 4.24 ± ± ± ± 0.79 Isobutyrate (mm) 0.53 ± ± ± ± 0.04 Isovalerate (mm) 0.76 ± ± ± ±0.11 Valerate (mm) 0.31 ± ± ± ± 0.07 Caproate (mm) 0.08 ± ± ± ± 0.05 L-Lactate (mm) 0.16 ± ± ± ± 0.04 D-Lactate (mm) 0.13 ± ± ± ± 0.04 Ammonia (mm) 3.96 ± ± ± ± 0.91 Estimated marginal means produced for two types of grain, using GLM with repeated measures methods, are presented in Table 6. Table 6. Estimated marginal means of rumen fermentation products when cattle were fed two different types of grain Estimated Marginal Means (Mean ± SE) Control Triticale Jackie (mixed grain) ph (Stomach tube) 7.07 ± ± 0.02 Total VFA (mm) ± ± 4.12 Acetate (mm) ± ± 3.13 Propionate (mm) ± ± 0.55 A/P ratio 4.80 ± ± 0.19 Butyrate (mm) 5.78 ± ± 0.56 Isobutyrate (mm) 0.58 ± ± 0.03 Isovalerate (mm) 0.93 ± ± 0.08 Valerate (mm) 0.61 ± ± 0.05 Caproate (mm) 0.13 ± ± 0.03 L-Lactate (mm) 0.06 ± ± 0.03 D-Lactate (mm) 0.13 ± ± 0.03 Ammonia (mm) 5.15 ± ± 0.68 Bovine Research Australasia 21

22 Acidosis Grain Report/Dairy Australia Table 7. Effect of time, group (quantity), types of grain (mixed vs Triticale) and their interactions on rumen fermentation products Time Rumen Ph F = 0.33 P = 0.77 Total VFA (mm) F = 0.41 P = 0.80 Acetate (mm) F = 0.95 P = 0.43 Propionate (mm) F = 0.06 P = 0.98 A/P ratio F = 1.54 P = 0.24 Butyrate (mm) F = 1.24 P = 0.32 Isobutyrate (mm) F = 0.10 P = 0.91 Isovalerate (mm) F = 0.17 P = 0.95 Valerate (mm) F = P < Caproate (mm) F = 0.33 P = 0.86 D-Lactate (mm) F = 4.25 P = 0.08 L-Lactate (mm) F = 5.18 P = 0.06 Ammonia (mm) F = 3.69 P = 0.04 GroupID (Quantity) F = 8.51 P = 0.01 F = 2.21 P = 0.18 F = 1.41 P = 0.32 F = 7.76 P = 0.01 F = 4.05 P = 0.06 F = 1.22 P = 0.37 F = 2.52 P = 0.14 F = 1.52 P = 0.29 F = P = F = 0.56 P = 0.66 F = 1.26 P = 0.40 F = 0.56 P = 0.66 F = 2.0 P = 0.20 GrainID (type of grain) F = 1.01 P = 0.35 F = 2.02 P = 0.20 F = 0.23 P = 0.20 F = 4.51 P = 0.07 F = 0.27 P = 0.62 F = 0.61 P = 0.46 F = P = 0.94 F = 0.03 P = 0.87 F = 5.94 P = 0.05 F = 0.12 P = 0.74 F = 0.05 P = 0.83 F = 0.22 P = 0.66 F = 1.41 P = 0.27 F values and probabilities (p) Time*GroupID Time*GrainID Time*GroupID*GrainID GroupID*GrainID F = 1.36 P = 0.28 F = 2.94 P = 0.02 F = 2.38 P = 0.06 F = 5.27 P = F = 2.07 P = 0.09 F = 3.09 P = 0.03 F = 0.85 P = 0.56 F = 4.43 P = F = 9.6 P < F = 1.44 P = 0.21 F = 1.29 P = 0.35 F = 0.47 P = 0.72 F = 4.32 P = F = 0.79 P = 0.50 F = 0.35 P = 0.78 F = 0.39 P = 0.74 F = 0.18 P = 0.92 F = 0.26 P = 0.84 F = 0.16 P = 0.89 F = 0.60 P = 0.57 F = 0.97 P = 0.41 F = 3.04 P = 0.05 F = 0.21 P = 0.76 F = 0.10 P = 0.77 F = 0.92 P = 0.37 F = 6.83 P = F = 0.42 P = 0.84 F = 1.12 P = 0.39 F = 1.25 P = 0.33 F = 0.91 P = 0.51 F = 0.96 P = 0.48 F = 0.69 P = 0.64 F = 1.38 P = 0.29 F = 0.58 P =0.70 F = 0.70 P = 0.66 F = 0.35 P = 0.80 F = 0.05 P = 0.95 F = 0.73 P = 0.52 F = 1.57 P = 0.23 F = 1.04 P = 0.40 F = 0.32 P = 0.74 F = 0.30 P = 0.75 F = 0.18 P = 0.84 F = 0.05 P = 0.95 F = 0.43 P = 0.60 F = 0.25 P = 0.78 F = 0.37 P = 0.70 F = 0.12 P = 0.89 F = 0.43 P = 0.67 F = 0.47 P = 0.64 F = 0.99 P = 0.42 F = 0.24 P = 0.79 Bovine Research Australasia 22

23 Acidosis Grain Report/Dairy Australia The estimated marginal means derived from the GLM analysis were used to rank the acidosis index of different amounts of grain. (Table 8, Figure 3). The estimated marginal means of rumen concentrations of VFAs, D- and L-lactates and ammonia were used to rank the acidosis index of different levels of grain (groups) and the two grains (mixed grain vs Triticale Jackie). Those groups with the highest concentrations of VFA, D- and L-Lactates and ammonia were ranked 1 and those with the lowest concentrations of these variables were ranked 4. The ranking method for the rumen ph was different from the other variables. Cattle with the lowest rumen ph were ranked 1 and those with the highest rumen ph were ranked 4. The weighted rank was also estimated using the ranking scores of rumen VFAs and ammonia (Table 8). These ranking methods showed that those cattle fed at the rate of 1.2% of bodyweight had a higher acidosis index compared with those cattle fed at the rates of 0% (control), 0.4% and 0.8% of bodyweight. Table 8. Ranking the acidosis index using the rumen ph, concentrations of VFAs, D- and L-lactate and ammonia ph Acetate Propionate Butyrate Isobutyrate Valerate Isovalerate Caproate L- Lactate D- lactate Ammonia Weighted rank (using VFAs & ammonia data) Control (0%) % of 3 BW % of 2 BW % of BW Bovine Research Australasia 23

24 Acidosis Grain Report/Dairy Australia Acidosis Ranking 5 4 Rank Control 0.4% of BW 2 0.8% of BW 1 1.2% of BW Figure 3. Ranking acidosis index of 4 different levels of grain using the estimated marginal means of rumen concentrations of VFAs and ammonia Bovine Research Australasia 24

25 Discriminant analysis Discriminant outputs (category membership) were used to rank the cattle fed at 4 different levels of grain. Group allocation using K-means cluster analysis also showed similar ranking on the acidosis index of different amounts of grain (groups). These data showed cattle fed at the rate of 1.2% of bodyweight had the highest rank, and those cattle in the control (0 kg grain) and 0.4% groups had the lowest rank (Table 9, Figure 4). Table 9. Summary of cluster analysis and ranking for each group and types of grain N Category membership Ranking (acidosis index) Groups Mean ± SE (allocated grain) 0% ± % ± % ± % ± Grain types Control (mixed) ± Triticale Jackie ± Acidosis Ranking using cluster analysis output 4 Ranks Control 0.4% of BW 0.8% of BW 1.2% of BW Figure 4. Ranking acidosis index of 4 different levels of grains using discriminant analysis category outputs Bovine Research Australasia 25

26 Discussion It is clear that at the 1.2% of bodyweight challenge dose, cattle showed marked and rapid change in ruminal VFA and ammonia concentrations. There was some evidence at this dose of early and late accumulation of lactic acid. The 1.2% of bodyweight dose is consistent with that used in previous challenge studies in North America. Preliminary data evaluation, further data analysis using approaches developed by Bramley (2004), consistency with previous studies and lack of evidence of severe acidosis lead us to conclude that a 1.2% dose was satisfactory for a challenge model that could discriminate between grains. Key indicators of the change in rumen function were rumen ph, propionate and valerate concentrations that varied with the amount of grain fed. Of these, only valerate also significantly differed between grain types (mixed vs Triticale Jackie), for time x group and grain type x time effects. Further, valerate had the highest F-test value of the amount of grain effects (F =12.2). Despite the marked changes in ruminal fermentation products with the 1.2% dose, we were unable to induce clinical or sub-clinical forms of acidosis with this dose. All challenged cattle were monitored for a period of two days post challenge, and no animal showed the clinical signs that can be related to ruminal acidosis. Rationale for the selection of indices used to determine acidosis risk: We concluded that two key indices should be used to determine risk of acidosis, specifically one based on Bramley (2004) and one based on ruminal concentrations of valerate. There were two primary sources of information used to develop these indices; ÿ The former index is based on the large dataset of Bramley (2004) and findings derived from this. Specifically, one hundred dairy herds were selected from lists supplied by veterinary practices in five areas of NSW and Victoria. Eight fresh cows (< 100 days in milk), consisting of three primipars and five multipars, were randomly selected from each herd. Rumen fluid was sampled from each cow by both rumenocentesis and stomach tube and samples were tested for ph. Stomach tube rumen fluid samples were analysed for VFA, ammonia and D-lactate concentration. Results from all assays detailed above were used to categorise cattle into groups using cluster and discriminant analysis. Three distinct groups of cows were identified. The random effect of herd was not a significant factor in the latter model indicating that cluster analysis could be used to define groups without the inclusion of the effect of herd. The percentages of animals in categories 1, 2 and 3 were 10.2%, 29.9% and 59.9%, respectively. Mean rumen ph for categories 1, 2 and 3 was 5.74(±0.47), Bovine Research Australasia 26

27 6.18(±0.44) and 6.33(±0.43), respectively. Biochemically, categories 1, 2 and 3 were characterised respectively as follows; mean total VFA concentration (mm) (±23.22), 94.79(±18.13) and 62.81(±15.65), mean ammonia concentration (mm) 2.46(±2.02), 7.79(±3.75) and 3.64 (±2.03), and mean D-Lactate concentration (mm) 0.34(±0.86), 0.28(±0.97) and 0.12(±0.51). The cows in category one had slightly higher milk production, but had significantly lower milk fat than cows in other categories. Herds with a higher prevalence of category one cows had a higher NFC: NDF ratio in the diet than herds with a high prevalence of category two or three cows. The data from the Bramley study (2004) and methods (discriminant analysis using the groups previously defined by cluster analysis) were used to classify samples obtained in the acidosis study. Rankings derived by this method have the advantages of i) using all the information on ruminal conditions obtained from the samplings ii) being related to biological outcomes at both a herd and individual animal level. ÿ The second assay was derived from the pilot study conducted to identify the optimal dose of grain for the study. As expected, increased concentrations of grain and the more reactive grain (Triticale Jackie) increased concentrations of most VFA. At the 1.2% of bodyweight inclusion rate with Triticale Jackie the following were found Variable Average increase to Percentage increase peak Propionate ~ 5 mmol/l ~ 50% Valerate ~ 0.8 mmol/l ~150% Ammonia ~ 2.3 mmol/l ~50% There was little evidence of accumulation of lactic acid, but rumen ph did decrease by units. It appears that, on a molar basis of the volatile fatty acid, increases in concentrations of propionate and valerate while not as large as that in acetate (an increase of approximately 20 mmol/l - less than 50% increase) were on a percentage basis larger. Biologically, it is critical that lactic acid does not accumulate in the rumen. This acid is 10 times stronger than the volatile fatty acids. The sodium salt of lactic acid is rapidly absorbed to clear this from the rumen. Both propionate and valerate are synthesised from lactic acid. While the pathways for propionate production are well described, those for valerate are not easily found. Annison and Lewis (1959) describe the following Bovine Research Australasia 27