Cattle are commonly fed soybean meal (SBM) and accidental intoxication sometimes occurs.
Cattle are commonly fed soybean meal (SBM) and accidental intoxication sometimes occurs.
To describe the biologic and clinical features of SBM intoxication.
Four steers with ruminal cannula.
Controlled experimental trial. SBM was administered once at 1 and 2% of body weight (BW) via cannula at 2-month intervals.
This study showed a 2-phase pathogenic course for 2% BW SBM intoxication. The 1st phase (until 10 hours post-administration) is restricted to ruminal modification with volatile fatty acid overproduction and moderate ruminal ammonia concentration. In the 2nd phase (12–22 hours post-administration), ruminal pH returned to initial values and marked ammonia accumulation occurred in blood, inducing severe metabolic alkalosis with hyperglycemia, hyperinsulinemia, and delayed aciduria (30–40 hours post-administration). Among the clinical signs, nervous signs were only observed during the period with increased plasma ammonia concentration. At 1% BW, ruminal and blood modifications were less pronounced than at 2% BW, and clinical signs were not observed.
Ammonia accumulation in blood during the second phase is the consequence of continued ammonia production, decreased carbohydrate fermentation, and overwhelming of hepatic detoxifying capacity. Because ammonia accumulation is associated with the clinical signs, treatment of SBM intoxication could be similar to treatment of urea intoxication, including rumenotomy, oral administration of cold water and vinegar, and measurement of ruminal pH.
plasma urea nitrogen
volatile fatty acid
Among the rumen fermentation disorders, some can be considered as true intoxication and are characterized by moderate to severe clinical signs. Acute ruminal lactic acidosis and ammonia toxicosis are the most common disorders. Acute ruminal acidosis is because of accidental ingestion of large amounts of readily fermentable carbohydrates inducing overproduction of volatile fatty acid (VFA) followed by D-L lactic acidosis. After absorption, lactic acid metabolism leads to acute ruminal lactic acidosis and severe clinical signs. Ammonia toxicosis occurs after ingestion and degradation of large amounts of nonprotein nitrogen (urea). The ruminal end-product ammonia is absorbed and exceeds the liver's capacity of detoxification. It leads to high plasma ammonia (P-ammonia) concentration and neurologic signs.[2-4]
Soybean meal (SBM) is commonly used to feed dairy cows and growing cattle because of its high energy and nitrogen content. In the field, accidental acute ingestion of a large quantity of SBM sometimes occurs in cattle. No data are available on the biologic and clinical consequences of this intoxication. As a result of the high energy and protein contents of SBM, ruminal and blood changes can be related either to acute ruminal lactic acidosis or urea toxicosis.
The aim of this study was to describe the clinical signs and ruminal and blood biochemical modifications after experimental SBM administration of 1 and 2% BW to steers.
Four Holstein steers with rumen cannulas were used in 2 experiments. The steers were housed in tied stalls equipped with mattress, but without straw. They could have visual, but no physical contact, and no other animals were present in the barn. Before SBM administration, they were fed ad libitum with grass hay and 3 kg of concentrate per day, in 2 equal meals (Table 1). The steers were 10 and 12 months old and weighed 366 ± 9 and 405 ± 9 kg, in experiments 1 and 2, respectively. SBM at 1% BW (3.5–3.7 kg as fed, experiment 1) or 2% BW (7.9–8.3 kg as fed, experiment 2) was administered once, via the cannula, in the morning (8 am). The last concentrate meal was provided 12 hours before the SBM challenges. After SBM challenges, hay and water were offered ad libitum to the animals. Before and after challenge, the quantity of hay offered and the quantity of hay not eaten before the next meal were weighed, so as to determine the actual ingestion of each animal. Water intake was not measured, and thirst was evaluated qualitatively by the operators for the 24 hours after SBM administration.
All times were indicated in reference to the SBM administration time (H0). In both experiments, the ruminal content and blood were sampled every 2, 4, or 8 hours for the H0–H32, H32–H40, and H40–H72 periods, respectively. Sampling also occurred every 8 hours for 40 hours before the challenge in experiment 2 (H−40–H0). Urine was collected by spontaneous urination for H0–H120 and H0–H250 in experiments 1 and 2, respectively. Ruminal content was sampled through the cannula by 1 operator in the cranio-ventral sac of the rumen. Five milliliters was immediately mixed with 0.5 mL of HgCl2 and frozen at −20°C. Fifty milliliters was used for pH measurement. Blood was sampled via a 2.7 × 80 mm catheter1 from jugular veins and placed in EDTA and heparin tubes, for immediate analysis (hematology, blood gas, and ammonia) or centrifuged (10 min; 4,000 × g; 25°C) before freezing plasma (−20°C).
General and specific examination was made before each sampling. Clinical examination included behavior and alertness, rectal temperature, dehydration, ocular mucosal coloration, frequency and type of respirations, cardiac and ruminal auscultation, flank palpation, and inspection of feces.
Biochemical analyses were made for all ruminal and blood samples. Microhematocrit was measured every 4 hours and blood counts, aspartate aminotransaminase, creatine kinase, and gamma-glutamyl tranferase were measured at H0, H24, H48, and H72. Ruminal pH was measured with a pH meter,2 averaging 2 successive measurements. Ruminal ammonia (R-ammonia) concentration was evaluated by the Berthelot method. Ten milliliter samples were centrifuged (20 minutes; 20,000 × g; 25°C) with 1 mL of phenol-pentacynonitrosyloferate (70 g 88% phenol, 0.25 g sodium nitroprusside per liter) added and 1 mL of alkaline hypochlorite solution (20 g NaOH and 43 mL 5.25%/NaOC1 per liter) added 5 minutes later. Absorbance was determined with a spectrophotometer3 at 625 nm, and concentration was calculated using standards. Total VFA concentration and C2, C3, and C4 concentrations were measured by gas chromatography. Samples were centrifuged (20 minutes; 4,000 × g; 25°C), treated for protein removal with meta-phosphoric acid and injected into a gas-chromatograph4 equipped with a flame ionization detector. The column used was a fused capillary5 (15 m × 0.53 mm; 1.2 μm film thickness). D-L lactic acid (experiment 2 exclusively) was determined by Enzytec acid D/L lactic kit.6 Blood was analyzed (VetStat Electrolyte and Blood Gaz Analyser7) immediately after sampling (Kasette Electrolyte 8 Plus7). Blood pH, blood partial CO2 pressure (pCO2), sodium, potassium, and chloride concentrations were measured, and bicarbonate (HCO3−) concentration was calculated. P-ammonia, plasma urea nitrogen (PUN), and glucose concentrations (Vitros 2508) were measured immediately after sampling. Microhematocrit (manual determination after centrifugation), blood count (QBC-VET Centrifuge7), and aspartate aminotransaminase, creatine kinase, and gamma-glutamyl tranferase activity (Vitros 2508) were evaluated immediately after sampling. Blood D and L lactic acid concentration was measured (Enzytec acid D/L lactic kit6) in plasma after deproteinization and freezing (−20°C). Insulin concentration was determined by a radioimmunol assay method (Insik Kit9). Urine pH was determined by a pH meter2 using 2 successive measurements. Urine was tested by urine dipstick tests (Combur test10).
The effects of sampling time and the effects of each animal were analyzed by ANOVA with the general linear model of R (version 2.9.1; 2009-06-26). Time and animal were considered as fixed effects. A Bonferroni correction was applied by multiplying the P value of each time and parameter by the number of times tested for this parameter. Correlations between parameters also were evaluated for the whole experiment or for particular periods.
Average ingestion of hay before SBM administration was 5.0 ± 0.5 and 5.5 ± 0.5 kg in experiments 1 and 2, respectively. After SBM administration, thirst increased slightly until H4–6 and H6–8, in experiments 1 and 2, respectively. After SBM administration, hay ingestion was stopped until H6–8 and H30 and then increased progressively to return to the initial amount (5.0 kg) after H24 and H72 for experiments 1 and 2, respectively.
Other clinical signs were observed only in experiment 2. Rectal temperature (+1°C) and respiratory frequency (from 40 to 60 respirations per minute) increased until H12. Abdominal distension, ruminal hypomotility, tachycardia, dyspnea, frequent urination and defecation, colic, successive lying down, and rising were observed until H12 in the 4 steers. From H14–18 to H24–30, more severe nervous signs appeared: dullness, disorientation, prolonged recumbency, and moderate muscle and skin tremors. Ruminal contraction frequency was decreased, and feces were soft. All clinical signs resolved at H30.
In experiment 1 (Fig 1), ruminal pH decreased (P < .05) from H2 to H6. Mild ruminal acidosis (pH = 5.5–5.8) was observed in 1 animal from H4 to H6. R-ammonia concentration increased (P < .01) from H2 to H20 and VFA concentration increased (P < .05) from H2 to H12. Between H2 and H26, C2 concentration decreased (from 72% to 65% of the VFA concentration at H0 and H10, respectively) and C3 concentration increased (from 15% to 18% of the VFA concentration at H0 and H10, respectively).
In experiment 2 (Fig 2), ruminal pH was decreased at H4, H6, and H10. Mild ruminal acidosis (pH = 5.5–5.8) was observed in all animals from H2 to H10. Compared with initial values (H0), ruminal pH increased from H20. Ruminal alkalosis (pH > 7) was detected in 3 animals. R-ammonia concentration increased significantly from H4 to H36, with a plateau between H4 and H12, a peak around H20, and a subsequent decrease from H18 to H36. The VFA concentration was increased (P < .05) from H2 to H22, with maximal concentrations between H2 and H6 and a plateau between H8 and H22. Between H2 and H52, C2 concentration decreased (from 70% to 66% of VFA concentration at H0 and H10, respectively) and C3 concentration increased (from 20% to 14% of VFA concentration at H0 and H10, respectively). Ruminal d-lactate concentration increased (P < .05) from 0.2 to 0.6 and 0.8 mM at H2 and H4, respectively. No variation was noticed for the ruminal l-lactate concentrations (0.005–0.01 mM).
In experiment 1 (Fig 3), P-ammonia concentration increased (P < 0.01) between H10 and H18, with a peak at H12–14. The PUN concentration increased (P < .01) from H8 to H30. The increase in PUN concentration was noticed as early as H2 and up to H36. Blood pH remained constant until H14, and then decreased slowly. Some individual pH values were ≥7.45 from H0 to H12. Bicarbonate concentration always remained within the reference range (data reported in Fig 7). No variation was noticed in pCO2. Insulin concentration increased from 30 mIU/L (H0) to 60 mIU/L (H6–H18, P < .05 from H10 to H18) and returned to initial concentrations at H24 Blood glucose concentration did not change in experiment 1.
In experiment 2 (Fig 4), P-ammonia concentration increased (P < .01) between H12 and H30. The PUN concentration increased slowly from H4 to H22, remained high from H24 to H32, and decreased from H36 to H72. Blood pH (Fig 5) increased (P < .05) from H4 to H28, with some values above 7.45, and decreased to initial values after H46. Plasma HCO3− concentration increased (P < .01) from H6 to H30. Glucose and insulin concentrations were increased from H14 to H36 and from H6 to H30, respectively (Fig 6). Blood l-lactate concentration (baseline concentration, 0.3–0.4 mM) peaked (1 mM; P < .05) from H8 to H12 and then reached a plateau (0.7 mM; P < .05) at H20–28. Blood d-lactate concentration increased slightly but significantly at H2 (0.05 mM).
Sodium, potassium, chloride, creatinine, aspartate aminotransaminase, creatine kinase, and gamma-glutamyl tranferase concentrations, microhematocrit, and hematologic results were normal during both experiments.
All results of urine dipstick tests and Heller tests were normal in both experiments. In experiment 1, urine pH decreased to 5.5 (P < .05) from H32 to H131 and returned to initial values (Fig 7). A similar pattern was observed in experiment 2, with an increase from H4 to H46, a subsequent decrease with a return to baseline values and a final increase from H216 to H240 (Fig 8).
Correlations between ruminal and blood parameters are reported in Tables 2-4. Correlations between ruminal pH and VFA concentrations were high for the overall period and for H0–H10, but low after H24. Most correlations between ruminal pH and R-ammonia concentrations were high (R ≥ 0.47). Correlations between blood pH and P-ammonia concentrations were low (R ≤ 0.41) in experiment 1, except for the H0–12 period, but high for experiment 2. Correlations between R-ammonia concentrations and PUN or R-ammonia concentrations and P-ammonia concentrations were always high (R ≥ 0.50). The correlations between blood pH and HCO3− concentrations or urine pH were very high (R ≥ 0.80), correlations between blood pH and l-lactate concentrations were high (R = 0.73), and correlations between blood pH and pCO2 were low.
|Period of Time||Ruminal pH/VFA Concentration||Ruminal pH/R-Ammonia Concentration||P-Ammonia Concentration/Blood pH|
|Experiment 1||Overall period||H0–H72||−0.89||−0.83||0.30|
|Experiment 2||Overall period||H0–H72||−0.83||0.12||0.62|
|Period of Time||R-Ammonia Concentration/PUN Concentration||R-Ammonia Concentration/P-ammonia Concentration|
|Experiment 1||Overall period||H0–H72||0.50||0.59|
|Experiment 2||Overall period||H0–H72||0.77||0.90|
|Period of Time||Blood pH/HCO3− Concentration||Blood pH/pCO2||Blood pH/Blood l-Lactate Concentration||Blood pH/Urine pH|
The present study showed a 2-phase pathogenic course of SBM intoxication, with mechanisms related to both urea intoxication and acute ruminal lactic acidosis. The 1st phase, occurring until H10, was restricted to ruminal modification with VFA overproduction and moderate R-ammonia concentrations. In the 2nd phase (H12–H22), ruminal pH returned to baseline, and marked ammonia accumulation occurred in blood, inducing severe alkalosis (experiment 2) and aciduria.
During the 1st phase (H0–H10), low ruminal pH arose from the increase in VFA concentration and moderate R-ammonia concentrations as suggested by the correlation between ruminal pH and VFA concentration. The carbohydrate and protein components of SBM are both reported to contribute to a rapid production of VFA after ingestion. The contribution of D and L lactic acid to the decrease in ruminal pH was low, as suggested by concentrations similar to those observed when fed diets based on hay or silage (0.2 mM). R-ammonia originated from the immediate and progressive degradation of SBM proteins. The moderate R-ammonia concentration described during the 1st phase could arise from 2 opposing mechanisms. First, produced ammonia could have been used for bacterial protein synthesis, because large amount of energy was available in the rumen. Second, ammonia absorption probably was decreased, because the ionized form which predominates at low ruminal pH (pKa of ammonia = 9.26) is less absorbed than the nonionized form.
In the 2nd phase (H12–H22), pH returned to pre-challenge values. In experiment 1, the increase in pH depended mostly on the decrease in VFA concentration (R = −0.91), despite the R-ammonia concentration decrease (R = −0.93). In experiment 2, pH depended more on the increase in R-ammonia concentration (R = +0.82) than on the decrease in VFA concentration (R = −0.16). The increase in R-ammonia concentration could originate from a relative lack of energy for bacterial synthesis and partly from the recycling of salivary urea[10, 11] in relation to the high PUN concentration.
The 3rd phase (>H24) observed in experiment 2 represented a return to the initial situation. The decrease in VFA and R-ammonia concentrations induced a ruminal pH of 6.5–6.9. All of the carbohydrate and protein components of SBM seem to have been digested.
In both experiments, the PUN concentration increase 2 hours after SBM administration probably was the result of the NH3 detoxifying hepatic mechanism. The correlation between the R-ammonia and PUN concentrations was high in the first 8 hours (experiment 1) and 22 hours (experiment 2) after SBM administration (R = 0.72–0.97). Substantial diffusion of ammonia across the ruminal wall despite low ruminal pH is likely to occur at high R-ammonia concentrations. Ammonia absorption seems to occur even at R-ammonia concentrations below the 500 mg/L threshold commonly cited. Saturation of hepatic capacity for ammonia removal as early as H8–12 is suggested by the increased P-ammonia concentration in both experiments, and by a high peak P-ammonia concentration at H14–16 in experiment 2. It is consistent with high correlations between R-ammonia and PUN concentrations (R = 0.78–0.72) before H8–12 and between R-ammonia and P-ammonia concentrations (R = 0.90–0.92) after H16 (experiment 1) and H24 (experiment 2).
The marked peak of P-ammonia concentration was only observed in experiment 2 at H14–16 First, the amount of SBM and R-ammonia absorption (because of increased ruminal pH) differed between experiments. Second, utilization of ammonia by the liver could be decreased because of a reduction in citrullogenesis during a severe increase in the propionate concentration in rumen.[13, 14]
For the H16–H72 (experiment 1) and H24–H72 (experiment 2 period), the decrease in R-ammonia concentration was associated with a return to the initial concentrations of P-ammonia and PUN, as suggested by the high correlations between the R-ammonia and PUN and between the R-ammonia and P-ammonia (R = 0.92).
A slight acidosis and marked alkalosis occurred in experiments 1 and 2, respectively. The high correlations between the HCO3− concentration and blood pH showed the metabolic component of the both acid–base disorders. Metabolic alkalosis was described during ammonia toxicosis.[2, 4, 15] The metabolic alkalosis in experiment 2 could have arisen from the severe increase in the P-ammonia concentration from H10–32. The pKa of ammonia is high (9.2) and ammonia is absorbed in the nonionized form (NH3) across the ruminal wall, but is in the ionized form (NH4+) in blood.[2, 4] This conclusion is consistent with high positive correlation between the P-ammonia concentration and blood pH until H10–12 and with the high P-ammonia concentrations from H10–12 to H24–26.
Terminal acidosis was described during urea intoxication and attributed to severe muscle tremors inducing a production of lactic acid. These findings were not observed here, probably because of the moderate clinical signs.
The correlation between urine pH and the HCO3− concentration was high (R = 0.80–0.90) and urine pH decreased to 5.5 in parallel with the HCO3− concentration. During blood alkalosis, increased filtration of HCO3− without modification of proton (H+) excretion is described, inducing alkaline urine. The decreased urine pH from H40 (experiment 1) or H52 (experiment 2) is consistent with acidic urine observed only 96 hours after blood alkalosis induced by duodenal obstruction in cattle.
In experiment 2, changes in insulin concentration were consistent with the changes in blood glucose concentrations. The decrease in insulin concentrations occurred parallel to the increase in P-ammonia (Fig 6). During urea intoxication in sheep, decreased insulin concentration and insulin resistance were reported. Ammonia could have an inhibitory effect on islet cells, and stimulation of insulin secretion by arginine is decreased.[20-22] The origin of the initial increase in the insulin concentration observed in experiment 2 remains unexplained.
Increased water intake and decreased hay intake probably contributed to the variations in ruminal and blood parameters. Increased water intake could have limited ammonia absorption across the ruminal wall, but precise evaluation of water intake was not performed. The lack of hay intake soon after SBM intoxication and for up to 3 days after SBM intoxication probably contributed to R-ammonia production. Compared with the situation described here, higher hay intake could allow higher VFA production and lower R-ammonia concentrations.
From H0 to H12, clinical signs were not specific and have been described in either ammonia intoxication (eg, loss of appetite, colic, and ruminal hypomotility) or acute lactic acidosis (abdominal distension, uneasiness, tachycardia, increased thirst, and dehydration with hypernatremia).
From H14 to H24–30, the neurologic signs were similar to those reported during ammonia intoxication (eg, prostration, ataxia, dullness, salivation, stiffening of the front legs, violent movements, hyperirritability, head shaking, and vocalization). The observed signs were nevertheless less severe than those usually reported during ammonia intoxication. Even if muscle and skin tremors were also present here, more severe clinical signs such as convulsions (described before death during ammonia intoxication) were not observed.[2, 25, 26]
In experiment 2, the maximal P-ammonia concentration (0.2–0.5 mM) detected during the clinical phase was in agreement with previous studies reporting clinical signs and death for blood ammonia concentrations of 0.5–1 mM and 1.2–2.2 mM[3, 4] respectively. The clinical signs can be attributed to a direct effect of ammonia on brain cells. During ammonia intoxication, the brain retains two-thirds of the arterial ammonia content. The inhibitory effect of ammonia on the citric acid cycle would induce a lack of energy in cells and neuronal degeneration. A high P-ammonia concentration is also reported to induce decreased ruminal motility and decreased appetite.[4, 29, 30]
Simultaneous nitrogen production and carbohydrate fermentation in the rumen probably decrease the ammonia absorption and explain the higher amount of feed needed to induce illness and the delayed onset of clinical signs for SBM intoxication compared with urea toxicosis. The minimal toxic dose for urea intoxication is 0.3–0.5 g urea/kg BW (ie, 0.14–0.23 gN/kg BW). It correlates with approximately 20 g SBM/kg BW (as fed; 147 gN/kg BW) for SBM intoxication in the present study. The lethal dose of urea is 1–1.5 g/kg BW (ie, 0.47–0.70 gN/kg BW) for urea intoxication. A mean ingestion of 40 g/kg BW (as fed; 294 gN/kg BW) in SBM in a dairy herd was associated with 20% mortality (D. Raboisson, personal observation). Clinical signs begin 10 minutes to 4 hours after urea intake and approximately 14 hours after SBM administration.
Because the pH decrease of the first pathogenic stage is not below 5.5 units, D-L lactic acid does not accumulate, and acidosis does not worsen. Nevertheless, the change in ruminal pH after SBM intoxication remains unknown when more than 2% BW of SBM is ingested.
This study was not performed to evaluate treatment of SBM intoxication, but it showed that the principles of treatment of SBM intoxication could be the same as those of urea toxicosis. The typical ruminal pH decrease noticed during acute ruminal lactic acidosis does not occur until a 2% BW SBM intoxication. On the contrary, clinical signs were associated with the increase in blood ammonia concentration. This observation suggests a therapeutic approach limiting the absorption of ammonia from the rumen.
In the few hours after SBM ingestion, a ruminotomy should be performed, in particular, if a high quantity of SBM is suspected to have been ingested by the animal. To decrease ammonia absorption, treatment during the clinical phase should at least include oral vinegar diluted in cold water or frequent oral administration of highly fermentable carbohydrates. As the quantity ingested and the precise times of ingestion often remain unknown, evaluation of rumen pH by rumenocentesis is likely to be useful. First, it could ensure that rumen pH is not <5.5, especially when a very large quantity is suspected to have been ingested. Second, it could allow adapting medical treatment to each case (frequency and quantity of oral vinegar, cold water, and fermentable carbohydrates).
Intraflon, Vygon, London, UK
pH meter TESTO 230, Hotek Technologies, Tacoma, WA
M330 spectrophotometer, Campsec, UK
Hewlett Packard 5890 Series II, Houston, TX
ECTM – 1000, Alltech, Deerfield, IL
Scil Diagnostics GmbH, Düsseldorf, Germany
Idexx, Westbrook, ME
Orto Clinical Chemistry, Issy les Moulineaux, France
Diasorin, Antony, France
Roche Diagnostics, Meylan, France