A Evaluation of the ruminal function of Belgian dairy cows suspected of subacute ruminal acidosis

Subacute ruminal acidosis (SARA) has been considered a major pathology in high producing dairy herds for years. These findings were corroborated by several studies in Europe. However, different feeding practices and herds’ production levels are found in Southern Belgium. This study aimed to ascertain whether dairy cows of several herds from the south of Belgium (Wallonia) with a suspicion of SARA really did present too low ruminal pH values. Twenty-four herds were visited and 172 cows were sampled using an oropharyngeal device to collect ruminal fluid, i.e. Geishauser probe. On the samples, three tests were performed: pH measurement, methylene blue reduction test and microscopic evaluation of protozoa vitality. Based on these analyses, no cows demonstrated pH values lower than 5.5 and, only ten cows could be considered at risk for SARA. By contrast, in eightteen cows, pH values higher than 7.0 were measured and ruminal inactivity was suspected. In this study, ruminal alkalosis appeared to be more frequently observed than SARA.


INTRODUCTION
For years, subacute ruminal acidosis (SARA) has been considered a major disease affecting high producing dairy herds.Nevertheless, this concept has been more and more questioned with the help of new technologies, which continuously record the rumi-BSTRACT Subacute ruminal acidosis (SARA) has been considered a major pathology in high producing dairy herds for years.These findings were corroborated by several studies in Europe.However, different feeding practices and herds' production levels are found in Southern Belgium.This study aimed to ascertain whether dairy cows of several herds from the south of Belgium (Wallonia) with a suspicion of SARA really did present too low ruminal pH values.Twenty-four herds were visited and 172 cows were sampled using an oropharyngeal device to collect ruminal fluid, i.e.Geishauser probe.On the samples, three tests were performed: pH measurement, methylene blue reduction test and microscopic evaluation of protozoa vitality.Based on these analyses, no cows demonstrated pH values lower than 5.5 and, only ten cows could be considered at risk for SARA.By contrast, in eightteen cows, pH values higher than 7.0 were measured and ruminal inactivity was suspected.In this study, ruminal alkalosis appeared to be more frequently observed than SARA.

Evaluation of the ruminal function of Belgian dairy cows suspected of subacute ruminal acidosis
Evaluatie van de ruminale functie bij Belgische melkkoeien verdacht van subacute pensacidose ration drop under 5.8 or 5.5 more important than a low pH value of short duration (Gozho et al., 2005;Al Zahal et al., 2007).On the other hand, field investigations cannot apprehend drop duration, so they consider SARA evidence when ruminal pH value is lower than 5.5, whatever the duration might be.Between 5.5 and 5.8, animals are considered at risk.Many studies have been published to assess SARA prevalence.In high producing herds, which receive high concentrate diets, SARA prevalence can reach 40% in one third of the studied herds in the United States (Garrett et al.,1997).In another study of Oetzel (2004), 20% of the sampled cows were detected as acidotic and 23% were considered being at risk.However, it must be kept in mind that feeding practices, which differ from one country to another, have a key impact on SARA development.In intensive dairy cattle industry in Italy, out of ten herds with a total of 3.490 cows producing more than 10,000 kg milk /year, three herds were considered to be demonstrating SARA and five other ones were diagnosed at risk (Morgante et al., 2007).These herds received a totally mixed ration (TMR), of which the composition was in compliance with NRC (2001) recommendations for crude protein, NDF and ADF concentrations.In a Dutch study based on 197 cows of 18 dairy herds producing 10,000 kg milk/ year, 13.6% of the sampled cows demonstrated pH values < 5.5 while 16.8% had pH values between 5.5 and 5.8 (Kleen et al., 2009).Those herds received a TMR composed of grass silage, maize silage and concentrates, providing nutrients in proportion considered secure regarding SARA.In only one herd, 38% of the animals was positive for SARA.In Ireland, ruminal fluid sampling was performed on 114 grazing cows, of which the average yearly milk production was 8,114 kg: 11 % demonstrated pH values lower than 5.5, while 42% had ruminal pH values between 5.5 and 5.8 (O'Grady et al., 2008).These cows were mainly fed spring grass supplemented 2 kg of concentrates maximum.In Southern Belgium, feeding practices and milk production are different from these published results; however, up till now, no study has been conducted to quantify SARA prevalence in Belgium.Despite this fact, in Belgium, it is very common to blindly complement dairy cows' diet with sodium bicarbonate to prevent ruminal acidosis.In this context, the present study aimed to verify whether dairy cows of several herds from the South of Belgium (Wallonia), where SARA was suspected, really did present low ruminal pH values indicating SARA, and whether sodium bicarbonate addition in their diet was really advisable.

Farm selection and cow production data
Twenty-four farms were investigated in Wallonia at the request of the farmers and/or the veterinarians who expected a potential risk of SARA based on lameness prevalence, poor milk production, low fat and low fat-to-protein ratio in milk.The milk yield on a 305-day basis was 8,898 ± 1,044 kg (mean ± SD) with 95 ± 43 cows per exploitation.Seven farms were equipped with an automatic milking system.Production, days in milk (DIM), milk yield (MY; kg), milk fat % (F), milk protein % (P), F/P ratio (F/P) and performances were obtained by the National Dairy Herd Improvement.

Ruminal fluid collection and analysis
Out of the 24 investigated farms, 172 cows (162 Holstein and 10 Brown Swiss) were sampled from 2011 to 2012 for evaluation of the ruminal function.At least five cows per herd were selected on basis of DIM (< 150), or low F (< 3.2%), or F/P ≤ 1 or at the farmer's request.
As the duration of low pH is impossible to estimate in field conditions, the pH determinations were completed with the Methylene Blue Reduction Time (MBRT) test and with the examination of rumen protozoa easily performed in farms.Furthermore, milk production and milk composition were recorded on the tested cows.
Ruminal fluid was sampled four to eight hours after the distribution of the TMR using a Geishauser oropharyngeal probe, preventing saliva contamination.The pH was immediately measured by a portable pH Meter (VWR, pH100, Liège, Belgium), and the values were reduced by 0.35 as proposed by Duffield et al. (2004) because of higher pH values in reticulum sampling than with rumenocentesis.The redox potential was evaluated by MBRT as described by Rosenberger (1981).The results were classified in three categories: discoloration within less than three minutes: high redox potential; three to six minutes: medium redox potential and low redox potential for discoloration taking more than six minutes.The protozoa number and mobility were assessed by optical microscopy.For standardization, it was decided to grade the microscopy results from 1 to 5 as detailed in Table 1.

Clinical Scoring
As SARA is usually considered a herd level pathology, health scores were assessed on several animals of the same herd.
The health scores were determined as described by Edmonson et al. (1989) for body condition (BCS) and by Zaaijer and Noordhuizen (2003) for ruminal fill (RF), fecal consistency (FC) and undigested fraction in feces (UF) in producing dairy cows.The locomotion scores (LS) were recorded according to Sprecher's grading scale (Sprecher, 1997) from 1 (normal gait) to 5 (severe lameness).All evaluations were made by the same operator.

Feed
The composition of the TMR allocated to the cows was transcribed from the balance on the feeder wagon.The concentrates allocated at milking were added.The composition of the feeds provided on the day of the visit was collected.The nutritional values based on NIRS analysis were compared to the production needs.Dutch units (VEM, DVE and OEB) were used to assess nutritional values.Silages were examined regarding their preservation (absence or presence of macroscopic moulds and temperature in the depth of the silage).The length of fibers was assessed using the Penn State Particle Separator (PSPS) as described by Kononoff and Heinrichs (2003).It was not possible to use the PSPS when liquid feeds were added to the TMR (nine farms).

Statistical Analysis
All results are presented as means ± SD.Descriptive statistical analysis (proc univariate) and t-test were performed using the SAS program (version 9.1-SAS Institute).The description of fed rations include mean ± SD, minimum and maximum values observed in the studied herds.
The t-test was used to compare the mean values of group 1 presenting pH values < 5.8 with group 2, of which the pH values were > 5.8.A second t-test was performed to evaluate the effect of adding bicarbonate by comparing farms using bicarbonate (BF) to prevent SARA and farms, which did not use it (NBF).A Chi-square test was performed to test the equality of distribution of MBRT values classified as normal (reduction time <6 minutes) and high (reduction time >6 minutes) in BF and NBF.

RESULTS
The mean characteristics of the rations are exposed in Table 2.The cows received 20.5 kg DM daily (minimum: 17.5 kg -maximum: 25.4 kg) on average.
Rations were mainly composed of forages including maize silage (mean: 6.5 kg DM/day, minimum: 0 kg; maximum: 9 kg), grass silage (mean: 5.6 kg DM/day; minimum: 0 kg; maximum: 8.7 kg) and beet pulp silage (mean: 2.4 kg DM/day; minimum: 0 kg; maximum: 3.5 kg) representing 14.5 kg DM.The rest of the diet was composed of concentrates (commercial mixtures, cereals, dry pulps, brewers, by-products).One farm (herd 17) did not include maize silage and another one (herd 8) did not include grass silage.The forage percentage in the TMR was calculated on all farms, considering the physical structure of the feed, e.g.brewers and dried beet pulps were recorded as concentrates, while fodder beets were recorded as forage.Based on this classification, the mean forage proportion was 70% with a minimum of 46% in herd 17 and a maximum of 82% in herd 13.An average quantity of concentrates of 6.4 kg (minimum: 3.6 kg -maximum: 11.2 kg) completed the diet.The average proportions of fibers evaluated by PSPS were the following: 53% in the first sieve (fiber length > 19 mm), 22% in the second one (fiber length > 8 mm) and 25% in the pan.Sodium bicarbonate (150 g per cow) was added to the ration of five herds (H2, 5, 9, 10 and 15).
Health scores were evaluated on average on 40% of the animals in each herd.The average production of the scored animals (DIM: 116 ± 77) was 30.4 ± 6.0 kg/cow/day, F%: 3.9 ± 0.6 and P%: 3.4 ± 0.2 (Table 3).The mean BCS, RF, CF, UF and LS were respectively 2.6 ± 0.4, 2.8 ± 0.4, 2.7 ± 0.3, 1.5 ± 0.4 and 2.0 ± 0.6 and were put in relationship with their production level.Of 24 herds, five (21%) recorded mean BCS >3 and four herds included overweight cows with a BCS higher than three despite DIM below 170 days.One herd showed BCS of 3.2 for DIM > 212.On the contrary, insufficient body condition was noticed in five farms with mean BCS < 2.5.In one herd, the animals were really lean (BCS = 1.6 ± 1.2) and produced 36.0 ± 7.6 kg milk, while receiving a TMR with nutritional characteristics allowing a production of 32 kg milk.Relative ruminal impaction was detected in eight herds (33 %) with RF > 3; poor fiber digestion was detected in ten herds (42%) with UF > 2 and lameness was detected in 14 farms with LS > 2.  The results of the individual samplings are presented in Table 4.Among the sampled animals, MY, F% and P% were 33.3 ± 8.9 kg, 3.5 ± 0.7 % and 3.3 ± 0.3%, respectively.The F/P ratio was 1.1 ± 0.2.The mean DIM and BCS were 106 ± 84 and 2.6 ± 0.6, respectively.
The mean ruminal pH value was 6.5 ± 0.4.Ten animals on 172 (5.8 %) had a pH < 5.8.No result was < 5.5.The distribution of low pH values was the following: 1/5 sampled animals in H18 and H20, 4/23 in H8, 1/7 in H3, 2/14 in H22 and 1/10 in H5.In other herds, no pH value lower than 5.8 was measured.Considering a herd positive for SARA when 25% of the cows presented ruminal pH < 5.5 or at risk for pH values < 5.8, no herd was positive or at risk.On the contrary, in 18 cows (10%), pH was higher than 7.In four out of the five herds supplemented with sodium bicarbonate, ruminal alkalosis (pH > 7) was reported in 25% of the sampled cows in H2, 10% in H5, 29% in H9 and 14% in H10.The relative proportions of low and high pH values within the herds are shown in Figure 1.The mean MBRT was 4.1 ± 3.0 minutes.In five out of 172 cows (3%), a value < 1 minute indicated a highly active bacterial flora.In 23 samples (14%), no reduction of MB occurred, demonstrating a relative bacterial inactivity.Results of MBRT were categorized in two classes: class 1 included values of MBRT <6 minutes while class 2 involved values of MBRT > 6 minutes.The relative distribution of MBRT within the herds is shown in Figure 2.
The mean microscopic score (MSc) was 1.35 ± 0.70.The disappearance of large protozoa was observed in seven samples (five samples from H8, one from H11 and one from H22).
In H8, on 23 sampled animals, abnormal findings in ruminal fluid were detected in nine animals (41%): four with low pH values (one combining low pH and high MSc, one low pH and no discoloration), one with no discoloration of MB and four cows with MSc equal to four and more.
In herds supplemented with sodium bicarbonate, the milk P % and the milk urea were significantly higher (3.5 ± 0.2 in BF vs 3.2 ± 0.3 in NBF; p<0.001; 270 ± 62 mg/L in BF vs 241 ± 71 mg/L in NBF; p<0.05).The discoloration of MB was slower in BF (5.7 ± 3.3 minutes versus 3.8 ± 2.9 minutes in NBF).A Chi-square test was performed to test the equality of the distribution of MBRT values classified as normal (reduction time <6 minutes) and high (reduction time >6 minutes) in BF and NBF.High MBRT-values were more frequent on BF-farms (p<0.01)(Figure 2).The pH also tended to be higher (6.60 ±0.46 in BF versus 6.47 ± 0.40 in NBF; p<0.1).

DISCUSSION
In this study, the evaluation of the ruminal function was required by the farmer or the veterinarian on the basis of suspected SARA.Low MY, low F% and low F/P in milk may be linked to SARA (Nocek, 1997;Enemark et al., 2004;Enjalbert, 2006;Mulligan et al., 2006;Toni et al., 2011).The sampled cows were selected on the basis of these criteria but no pH value was lower than 5.5.Ruminal acidosis could not be diagnosed in any herd.Only ten animals (5.8%) presented values between 5.5 and 5.8 and could be considered at risk for SARA.
Low milk yield was the usual complaint of the visited farmers.This relative lack of production could be due to several factors.The observed health scores suggest management failures.The body condition scores were not optimal for MY in eleven farms (in five farms: score > 3 and in six farms: score < 2.5).Ruminal impaction likely due to the long fiber proportion was observed in 33% of the visited farms.Poor digestion was noticed on ten farms out of 24.The high pH (> 7) largely observed in the sampled cows was probably linked to impaired digestion and ruminal dysfunction (Mouriño et al., 2001;Kozloski et al., 2008).Moreover, a poor silage quality regarding conservation and nutritional values was reported in five out of 24 farms and might have reduced the nutritional value of the diet.Lameness was a major problem as more than half of the herds presented high LS (LS > 2).This factor has been reported as impacting the productivity of animals (Juarez et al, 2003;Archer et al., 2010).The housing of the animals was therefore examined: the cowsheds demonstrated discomfort inducing competition between animals in some farms.Abnormal eating and rumination behavior are wellknown to be related to lameness and discomfort, and to influence ruminal fermentations and pH and hence, the production levels (Stone, 2004).
On the other hand, the addition of sodium bicarbonate influenced ruminal fermentation inducing a relative increase in pH and slowing down MBRT.High ruminal pH observed in the sampled cows could TMR: totally mixed ration (kg DM).DM: dry matter.KVEM: net energy (Dutch system), DVE and OEB respectively intestine and ruminal degradable proteins (Dutch system).MY expected: milk yield expected regarding the energy provided by the ration.Should the protein supply be the limiting factor, the milk yield based on the protein supply is indicated as a second figure .alter the ruminal function and impair the performance of the cows.An increase in P% was noticed in the BF.Similar effects of sodium bicarbonate supplementation on P% have not been reported in the literature (Erdman et al., 1982;Kennelly et al., 1999;Khorasani and Kennelly, 2001).However, in compliance with the results of this study, the association of high concentrate and buffer supplementation has been described as influencing ruminal fermentation pattern causing a decline in (acetate + butyrate) to propionate ratio (Khorasani and Kennelly, 2001).According to these authors, adding high concentrate and buffer supplementation has a moderate impact on lactose production, but also has an impact on the amino-acid production, which is privileged by the increased propionate concentration to the detriment of gluconeogenesis.As a result, the milk protein % is increased.
A similar process could be incriminated in the present study.The high urea level recorded on BF farms was possibly linked to the shift in bacterial population reflected by the high MBRT.
The herds involved could be considered efficient as their average milk yield (8,898 ±1,044 kg) was higher than the mean registered in Southern Belgium (7,638 kg per cow on a 305 days basis).Despite the high nutritional requirements linked to this production level, the feed was mainly composed of forages.Grass silage was included in all the rations but one (herd 8) and maize silage was another major component.A high proportion of fibers in the rations was confirmed by the results of PSPS with a majority of fibers (53%) measuring > 19 mm.Long fibers were mainly provided by grass silage.The concentrate level (on average 31%, minimum: 14% -maximum: 52%) was within the recommendations of NRC (2001) (maximum: 65%) in all the exploitations.These feeding practices prevent SARA (Stone, 2004;Mulligan et al., 2006).Therefore, the systematic addition of sodium bicarbonate in the cows' diet appeared inappropriate on the examined herds.
To improve the productivity of the visited herds, the main advice could be to improve the forage quality and to detect lame cows more efficiently.Introducing highly fermentable carbohydrates in the cows' diet could help improving ruminal fermentation.

CONCLUSION
Regarding these results, ruminal evaluation did not confirm SARA suspicion, while on the opposite, relative ruminal flora inactivity and high ruminal pH seemed far more common.
According to this study, the regular use of sodium bicarbonate in Walloon dairy farms is questioned.In case of low production or low fat syndrome, more attention should be paid to silage and housing quality as well as to the detection, prevention and treatment of lame cows.

Figure 2 .
Figure 2. Distribution of normal (reduction time < 6 minutes) and high (reduction time > 6 minutes) methylene blue reduction time in the 24 tested herds.

Table 4 . Ruminal parameters and body condition scores of sampled cows from the 24 tested herds.
Values ± SD.Abbreviations: N: number of sampled cows.DIM: days in milk; LN: lactation number, BCS: body condition score; MBRT: methylene blue reduction time; MS: ruminal microscopic score; MD: missing data.