Ergovaline does not alter the severity of ryegrass staggers induced by lolitrem B

AIM: To investigate a possible interaction between lolitrem B and ergovaline by comparing the incidence and severity of ryegrass staggers in sheep grazing ryegrass (Lolium perenne) containing lolitrem B or ryegrass containing both lolitrem B and ergovaline.

METHODS: Ninety lambs, aged approximately 6 months, were grazed on plots of perennial ryegrass infected with either AR98 endophyte (containing lolitrem B), standard endophyte (containing lolitrem B and ergovaline) or no endophyte, for up to 42 days from 2 February 2010. Ten lambs were grazed on three replicate plots per cultivar. Herbage samples were collected for alkaloid analysis and lambs were scored for ryegrass staggers (scores from 0–5) weekly during the study. Any animal which was scored ≥4 was removed from the study.

RESULTS: Concentrations of lolitrem B did not differ between AR98 and standard endophyte-infected pastures during the study period (p=0.26), and ergovaline was present only in standard endophyte pastures. Ryegrass staggers was observed in sheep grazing both the AR98 and standard endophyte plots, with median scores increasing in the third week of the study. Prior to the end of the 42-day grazing period, 22 and 17 animals were removed from the standard endophyte and AR98 plots, respectively, because their staggers scores were ≥4. The cumulative probability of lambs having scores ≥4 did not differ between animals grazing the two pasture types (p=0.41).

CONCLUSIONS AND CLINICAL RELEVANCE: There was no evidence for ergovaline increasing the severity of ryegrass staggers induced by lolitrem B. In situations where the severity of ryegrass staggers appears to be greater than that predicted on the basis of concentrations of lolitrem B, the presence of other tremorgenic alkaloids should be investigated.     

The evaluation of endophyte toxin residues in sheep fat

Abstract

AIM: To monitor changes in concentrations of lolitrem B and epoxy-janthitrems in the fat of sheep grazing perennial ryegrass infected with wild-type- and AR37-endophyte, respectively, during the time of year when ryegrass staggers would be expected to be observed.

METHODS: Ten 5-month-old lambs with no previous exposure to endophytes were grazed on either wild-type (containing lolitrem B, n = 5) or AR37 (containing epoxy-janthitrems, n = 5) endophyte-infected perennial ryegrass (Lolium perenne L.) pastures between October 2008 and June 2009. Animals were regularly assessed for ryegrass staggers using the Keogh scale (0 = no signs, 5 = severe tremors). When a score of > 3.5 was observed animals were removed from the treatment pastures for 1 month. Fat biopsy samples were taken from each animal at approximately monthly intervals and analysed for endophyte metabolites using high performance liquid chromatography (HPLC) methods developed during this study. Regular herbage samples were also taken and concentrations of endophyte metabolites measured.

RESULTS: Efficient and reproducible methods to analyse both lolitrem B and epoxy-janthitrems in fat were developed. Concentrations of lolitrem B and epoxy-janthitrems in herbage and in sheep fat increased from late November to peak in mid-February. Ryegrass staggers was observed in both groups of sheep at this time. Following 1 month of grazing non-infected pasture mean concentrations in fat of lolitrem B decreased by 43% from 61.8 to 35.3 ppb, and of epoxy-janthitrems by 38% from 1032.0 to 639.5 ppb. Maximum concentrations in herbage of epoxy-janthitrems (35.7 ppm) were higher than of lolitrem B (3.4 ppm), but signs of staggers were less severe in sheep grazing pasture containing the former compared with the latter (median Keogh scores in late February were 2 and 3, respectively), consistent with epoxy-janthitrems being low potency toxins.

CONCLUSION: This study demonstrated that concentrations of epoxy-janthitrems and lolitrem B in sheep fat increased quickly during the initial phase of the study when concentrations in pasture increased, and decreased when animals were removed from pastures containing these compounds. These data will be used in the risk assessment of the endophyte metabolites.     

Evaluation of perennial ryegrass straw as a forage source for ruminants

Two experiments were conducted to evaluate perennial ryegrass straw as a forage source for ruminants. Experiment 1 evaluated digestion and physiological variables in steers offered perennial ryegrass straw containing increasing levels of ergot alkaloid, lolitrem B. Sixteen ruminally cannulated Angus x Hereford steers (231+/-2 kg BW) were blocked by weight and assigned randomly to one of four treatments. Steers were provided perennial ryegrass straw at 120% of the previous 5-d average intake. Before straw feeding, soybean meal was provided (0.1% BW; CP basis) to meet the estimated requirement for degradable intake protein. Low (L) and high (H) lolitrem B straws (<100 and 1,550 ppb, respectively; DM basis) were used to formulate treatment diets: 100% L; 67% L:33% H; 33% L:67% H; 100% H (DM basis). Intake and digestibility of DM and OM, and ruminal pH, total VFA, and NH3-N were not affected by increasing lolitrem B concentration. Ruminal indigestible ADF (IADF) fill increased linearly (P = 0.01) and IADF passage rate decreased linearly (P = 0.04) as lolitrem B increased. Experiment 2 evaluated performance and production by 72 Angus x Hereford cows (539+/-5 kg BW) consuming perennial ryegrass straw containing increasing lolitrem B during the last third of gestation. Cows were blocked by body condition score and randomly assigned to one of three treatments. Cows were provided perennial ryegrass straw ad libitum and supplemented with soybean meal (0.1% BW; CP basis) to meet the estimated requirement for degradable intake protein. Mixtures of a L and H lolitrem B straw (467 and 2,017 ppb, respectively) were used to formulate treatment diets: 100% L, 50% L:50% H, 100% H (DM basis). Thirteen of 24 cows on the 100% H treatment exhibited signs of ryegrass staggers and were removed from the study; nevertheless, lolitrem B concentration did not influence pre- or postcalving weight or body condition score change. These data suggest that feeding perennial ryegrass straw containing up to 1,550 ppb lolitrem B (DM basis) did not adversely affect nutrient digestion or physiological response variables in steers. However, providing straw with a lolitrem B concentration of approximately 2,000 ppb (DM basis) resulted in 54% of cows exhibiting signs of ryegrass staggers. These data suggest that blending straws with a high (>2,000 ppb) and low (<500 ppb) concentration of lolitrem B can be a successful management practice.     

The effect of glyphosate, paraquat and paclobutrazol on lolitrem B levels in endophyte-infected perennial ryegrass

Two herbicides (glyphosate and paraquat) and a plant growth regulator (paclobutrazol) were applied to endophyteinfected (Acremonium lolii) perennial ryegrass swards. Subsamples of these swards were then chemically analysed at intervals up to 28 days later for lolitrem B, the compound responsible for perennial ryegrass staggers in domestic livestock. Glyphosate and paclobutrazol had no effect on lolitrem B concentrations. Paraquat applications decreased lolitrem B concentrations in the herbage. Because none of the chemicals tested increased the concentration of lolitrem B in the herbage, they are unlikely to be directly implicated in perennial ryegrass staggers in grazing animals.     

Genetic-diet interactions in the Hyperkalemic Periodic Paralysis syndrome in Quarter Horses fed varying amounts of potassium: I. Potassium and sodium balance, packed cell volume and plasma potassium and sodium concentrations

Six broodmares that were genetically tested to be heterozygous (H/N) and six broodmares that were tested to be homozygous negative (N/N) for Equine Hyperkalemic Periodic Paralysis (HYPP), that were descendents of the same stallion, were used in a replicated 3×3 Latin square experiment to determine the genetic-diet relationships in the HYPP syndrome.¹ The mares were fed rations consisting of 65% pelleted concentrate and 35% Coastal Bermuda grass hay that provided 1.1 (diet A), 1.9 (diet B) and 2.9% (diet C) potassium. The experimental periods were 14 days long, resulting in 28 meals per period. At meals 1 and 27, blood samples were taken every 30 minutes for 12 hours and every 10 minutes from 2 to 5 hours post-feeding. Total urine and fecal collections were made on the last 4 days of each period.Water, feed and fecal samples were analyzed for sodium and potassium content. Blood was analyzed for packed cell volume (PCV) and plasma potassium (K⁺) and sodium (Na⁺) concentrations.Apparent absorption of potassium was 99.8% across all diets, and potassium was excreted principally in the urine. More potassium was retained when the horses were fed diet B than diets A or C. Apparent absorption of sodium was 99.6% with no difference by diet. Neither potassium nor sodium balances were affected by HYPP status.There was no post-prandial pattern of plasma K⁺ concentration seen when the horses were fed the low potassium diet. When fed the higher potassium diets, they had greater plasma K⁺ values and exhibited a post-prandial peak at 2 to 5 hours after feeding. This peak occurred in all of the horses, meaning that plasma K⁺ cannot be used as a diagnostic aid for the presence of the HYPP mutation. By meal 27, some adaptation had occurred resulting in lower plasma K⁺ values when the horses were fed the higher potassium diets. The horses had increased plasma K⁺ concentrations throughout the post-prandial period while fed diet B compared to diet A, agreeing with the positive potassium balance seen in this experiment. The HYPP H/N horses had lower plasma Na⁺ concentrations in the early post-prandial hours and continuously greater PCV values than the HYPP N/N horses.     

Detection of endophyte toxins in the imported perennial ryegrass straw

From 1997 to 1999, 29 cases of disorders were detected in cattle and horses that had been fed ryegrass straw imported from the U.S.A. These animals showed symptoms resembling ryegrass staggers and the clinical signs disappeared after removal of the straw. Endophytic hyphae were detected in the seeds of all straw samples that were responsible for the clinical cases. Lolitrem B concentrations in the straw ranged between 972 and 3740 ppb. Ergovaline concentrations were between 355 and 1300 ppb. Even though the concentrations of lolitrem B were lower than the toxic threshold proposed by Oregon State University in better part of the cases, our observations suggest the possibility that lolitrem B lower than the proposed threshold can bring disorders to sensitive individuals.     

Correlation of ergovaline and lolitrem B levels in endophyte-infected perennial ryegrass (Lolium perenne).

The varieties of perennial ryegrass (Lolium perenne) infected with the endophytic fungus Neotiphodium lolii contain several classes of toxic alkaloids, including ergopeptide alkaloids and lolitrem alkaloids. Lolitrem B, a potent tremorgen, is generally considered to be the predominant alkaloid in endophyte-infected perennial ryegrass. Ergovaline, a vasoconstrictor normally associated with endophyte-infected tall fescue (Festuca arudinacea), is also present in endophyte infected perennial ryegrass. Clinical signs of animals ingesting endophyte-infected perennial ryegrass are consistent with the presence of lolitrem B. However, clinical signs normally associated with ergovaline poisoning are not usually observed in animals ingesting endophyte-infected perennial ryegrass. A survey was conducted to quantitate both lolitrem B and ergovaline in 459 perennial ryegrass straw samples received at the Oregon State University College of Veterinary Medicine. Samples were analyzed for each alkaloid using separate high-performance liquid chromatography analyses. A strong positive correlation between the 2 alkaloids (r2 = 0.7335) was observed, especially in the samples containing <3,000 ppb (ng/g) lolitrem B. The threshold levels above which clinical signs typically occur are 2,000 ppb lolitrem B and 300-400 ppb ergovaline. All of the samples analyzed contained <425 ppb ergovaline.     

Ryegrass Staggers: Isolation of Potent Neurotoxins Lolitrem a and Lolitrem B From Staggers-Producing Pastures

Sir, — We wish to report the isolation of two potent neurotoxins from herbage collected from pastures on which the disease of livestock known as ldryegrass staggers rd^(8)(11) occurred. These neurotoxins have been partially characterised by their mass spectral properties (see below) and appear to be new compounds not previously reported. We propose the general name lolitrems for the neurotoxins, based on their association with ryegrass (Lolium perenne L.) and on their ability to produce tremors in animals, and name the isolated compounds lolitrem A and lolitrem B.     

Ryegrass Staggers: The Presence of Lolitrem Neurotoxins in Perennial Ryegrass Seed

Abstract

Extract

Sir:- Recently, two potent neurotoxins named lolitrem A and lolitrem B were isolated from herbage collected from pastures on which the livestock disorder ryegrass staggers (RGS) occurred. et al.⁽¹⁾ Gallagher, R.T., White, E.P. and Mortimer, P.H. 1981. Ryegrass staggers: Isolation of potent neurotoxins lolitrem A and lolitrem B from staggers-producing pastures. N.Z. vet. J., 29: 189–190. [Taylor &; Francis Online], [Web of Science ®] , [Google Scholar] We now wish to report that samples of perennial rygrass (Loliun yeretzrze L.) seed obtained from several locations in New Zealand contained potent neurotoxins which include lolitrem A and lolitrem B. Further, when pellets made from lolitrem neurotoxin-containing seed were fed to sheep, symptoms indistinguishable from those of severe, natural RGS were induced in four sheep, and mild symptoms in three sheep.     

Effects of aerobic and anaerobic fluid collection on biochemical analysis of peritoneal fluid in healthy horses and horses with colic

Objective: To determine whether in healthy horses and those with colic, exposure of peritoneal fluid to room air affects values obtained on biochemical analysis. Study Design: Prospective study. Animals: Adult horses with a primary complaint of acute abdominal pain (n=29) and 12 healthy horses. Methods: Peritoneal fluid was aseptically collected under aerobic and anaerobic conditions. After collection, pH, PCO₂, PO₂, HCO₃^?, Na⁺, ionized Ca²⁺, K⁺, lactate, and glucose were immediately measured using a commercial blood gas analyzer. Biochemical variables were compared between aerobically and anaerobically obtained samples using a paired t‐test. Results: In healthy horses, peritoneal fluid samples collected under anaerobic conditions had higher PCO₂ and ionized Ca²⁺ and lower PO₂, HCO₃^?, and pH compared with samples exposed to air. No differences were observed for K⁺, Na⁺, glucose, and lactate. In horses with colic, samples collected anaerobically had higher PCO₂, ionized Ca²⁺, Na⁺, and glucose and lower PO₂, HCO₃^?, and pH value compared with samples exposed to air. No differences were observed for K⁺ and lactate. Conclusion: Exposure of peritoneal fluid to room air had a significant effect on pH, PCO₂, PO₂, and variables associated or dependent on changes in pH such as HCO₃^? and ionized Ca²⁺. Interpretation of biochemical analysis of peritoneal fluid may be influenced by sample collection method.     

Correlations between potassium, calcium and magnesium in blood and muscle of standardbred trotters

This study was conducted to investigate the relationships between the concentrations of potassium, sodium, calcium and magnesium of plasma and muscle in horses, as interactions between these ions can be important in the development of muscle cell injury. Blood samples and gluteal muscle biopsies were taken from 20 healthy Standardbreds to determine the potassium concentration in plasma (cK⁺(P)), red blood cells (cK⁺(RBC)), and muscle (cK⁺(M)), and the calcium and magnesium concentrations in plasma and muscle. No correlations were found between cK⁺(P), cK⁺(RBC), and cK⁺(M). Significant positive correlations were found between cK⁺(M) and muscle magnesium (p<0.01); and cK⁺(P) and plasma calcium (p<0.005). Significant negative correlations were found between cK⁺(M) and the percentage of muscle water (p<0.005); and the muscle magnesium and calcium concentrations (p<0.02).     

Effects of dietary cation-anion balance on blood PH, acid base parameters, serum and urine mineral levels, and parathyroidhormone (PTH) in weanling horses.

This study demonstrated that the feeding of treatment diets with calculated dietary cation-anion balances (DCAB) of +370.43 (H) and -25.69 (L) did not have significant effects on blood pH, pCO₂, and HCO₃-. Serum Ca²⁺, P, Na⁺, and Cl^- as well as plasma PTH did not differ (P > .05) between the two treatment groups. Serum K⁺ was higher (P< .05) in horses fed diet H rather than diet L. The DCAB of the diet significantly affected urinary Ca²⁺, P, Na⁺, K⁺, and Cl^- excretion in the young growing horse. Urine Ca²⁺ and Cl- levels were higher (P < .01) in horses fed diet H versus diet L. Furthermore, levels of P, Na⁺, and K⁺ in the urine were higher (P < .01) in horses on diet H as opposed to diet L. Results of this study indicate that horses were able to maintain acid-base status regardless of diet. However, these data imply that growing horses consuming diets low in DCAB may be predisposed to abnormal bone mineralization due to the increase in calcium excretion which could lead to a weakening of the skeletal system.     

Some Effects of a Low Sodium Diet High in Potassium on the Renin-Angiotensin System and Plasma Electrolyte Concentrations in Normal Dogs

Pedersen, H.D., J. Koch, A.L. Jensen, K. Poulsen, A. Flagstad: Some effects of a low sodium diet high in potassium on the renin-angiotensin system and plasma electolyte concentrations in normal dogs. Acta vet. scand. 1994, 35, 133-140. - Eight normal male Beagle dogs received 0.7 mmol Na⁺/kg/day for 5 weeks and 4.0 mmol Na⁺/kg/day in one 3 week control period preceding and another similar period following the low sodium period. The dogs received 6.8 mmol K⁺/kg/day throughout the study.The median plasma renin activity (PRA) and plasma aldosterone concentration (PAC) were higher in the low sodium period than in the following control period (0.67 versus 0.28 ng/ml/h, p < 0.0001) and (204 versus 31 pg/ml, p < 0.0001). PRA and PAC quickly stabilized on a new steady level in response to altered intake of sodium chloride. The angiotensin-converting enzyme (ACE) activity was not changed by the altered intake of sodium chloride.The plasma concentrations of sodium and chloride were increased during the low sodium period. This could be due to an indirect effect of the high potassium intake of the dogs. Potassium leads to an increased secretion of aldosterone and thereby to an increased retention of sodium and chloride in the kidney. The possible implications of a high potassium content in a low sodium diet are discussed.     

Ureteral ligation prevents the haemodynamic effect of frusemide in pentobarbitol anaesthetised horses

Frusemide reduces pulmonary vascular pressures in resting horses and attenuates exercise-induced increases in these pressures in exercising horses. The mechanism underlying these effects of frusemide is unclear. We tested the hypothesis that the haemodynamic effects of frusemide are dependent on diuresis by examining the effect of frusemide in anaesthetised horses in which diuresis was prevented by ligation of ureters. Twenty four horses were assigned randomly to one of 4 treatments: 1) frusemide (1 mg/kg bwt i.v.) and intact ureters; 2) frusemide and ligated ureters; 3) saline placebo and ligated ureters; and 4) frusemide and phenylbutazone (4.4 mg/kg bwt i.v. 12 h and 15 min before frusemide) and ligated ureters. Frusemide administration to anaesthetised horses with intact ureters increased plasma total protein concentration and reduced mean right atrial, pulmonary artery and aortic pressures. There was no significant effect of frusemide administration on haemodynamic variables or plasma total protein concentration in horses with ligated ureters. The combination of frusemide and phenylbutazone increased mean right atrial, pulmonary artery and aortic pressures in horses with ligated ureters. This study demonstrates that, in anaesthetised horses, the haemodynamic effect of frusemide is dependent upon diuresis. We interpret these results as providing further evidence that the haemodynamic effect of frusemide in horses is attributable to a reduction in plasma and blood volume.     

Rhinopneumonia or mycotoxin intoxication? Neurologic phenomena in horses from a riding school

In the course of several days most of the 40 riding-school horses turned out in paddocks developed ataxia of variable severity. Five of these horses showed severe ataxia and tremors, became paralyzed and were euthanized. Eleven privately-owned horses which were stabled on the same premises showed no clinical signs. The most likely diagnosis seemed to be the 'neurological form of EHV1', although the signs were not entirely typical. A few weeks later a second outbreak occurred among the riding-school horses and one of the privately-owned horses also showed signs of ataxia. In the meantime it had been shown that EHV1 titers in paired serum samples had not increased and that the cerebrospinal fluid of one of the severely affected horses was normal. Toxicological examination of hay, delivered just before the first outbreak and stored for the winter, showed a significantly increased concentration of lolitrem B mycotoxin (5-6 mg/kg). The hay appeared to have been made of ryegrass used for lawns and playing fields. Retrospectively it became probable that this hay occasionally been fed to the horses just before the onset of clinical problems. It is concluded that the horses showed the 'ryegrass-stagger syndrome'.     

The muscular dystrophic chicken is hypernatremic

1. The E3 ubiquitin protein ligase 1 (WWP1) gene, the mutation of which causes muscular dystrophy in chickens, is expressed not only in the pectoral muscle, but also in a number of tissues such as the kidney. Therefore, this study examined some parameters related to kidney function in muscular dystrophic (MD) chickens.

2. Plasma osmolality, Na⁺ and K⁺ concentrations, aldosterone levels, and the expression of aquaporin (AQP) 2, AQP3, and α subunits of the amiloride-sensitive epithelial sodium channel (αENaC) were analysed in the kidneys of 5-week-old MD chickens and White Leghorn (WL) chickens under physiological conditions or after one day of water deprivation.

3. Plasma osmolality, Na⁺ concentrations, and plasma aldosterone levels were significantly higher in MD chickens than in WL chickens. αENaC mRNA expression levels were lower in MD chickens than in WL chickens. AQP2 and AQP3 mRNA expression levels were similar in the two strains of chickens.

4. Plasma osmolality correlated with aldosterone levels and AQP2 and αENaC mRNA levels in WL chickens. In MD chickens, plasma osmolality correlated with AQP2 mRNA levels, but not with plasma aldosterone or αENaC mRNA levels.

5. These results suggest that neither water reabsorption nor the expression of AQP2 and AQP3 is impaired in MD chickens and that a WWP1 gene mutation may or may not directly induce an abnormality in Na⁺-reabsorption in the kidneys of MD chickens, potentially through αENaC.     

Influence of the dietary potassium content on transepithelial potassium transport in rat jejunum

In a recent study, we found that the distal rat jejunum is able to secrete K⁺ under in vitro conditions. The question therefore arises as to whether the small intestine might participate in K⁺ homeostasis. Consequently, this study examined the influence of the dietary K⁺ content on transepithelial K⁺ transport in rat jejunum. Rats were fed two diets differing in K⁺ content (control diet 4.0 g K⁺/kg, low K⁺ diet (LK) 0.27 g K⁺/kg). After a minimal feeding period of 7 days, distal jejunal sheets were mounted in Ussing chambers and unidirectional ⁸⁶Rb⁺ fluxes (as a marker for K⁺ transport) were measured under short‐circuit conditions. Jejunum obtained from rats fed the control diet showed a net K⁺ secretion of 200 nmol Rb⁺/h/cm². Unidirectional Rb⁺ fluxes were smaller in distal jejunum from rats fed the LK diet. In these tissues, glucose‐induced short‐circuit current and tissue conductance were also smaller than in controls. However, net Rb⁺ fluxes were not significantly different in small intestine from K⁺‐restricted rats compared with jejunum from control animals. Based on the observation that the dietary K⁺ content does not affect transepithelial net K⁺ transport, we conclude that transcellular K⁺ secretion by the small intestine is not involved in K⁺ homeostasis.     

Attenuation by phenylbutazone of the renal effects and excretion of frusemide in horses.

The objectives of this study were to determine the effect of phenylbutazone premedication on the pharmacokinetics and urinary excretion of frusemide in horses; and on frusemide-induced changes in urinary electrolyte excretion. Six Standardbred mares were used in a 3-way crossover design. The pharmacokinetics and renal effects of frusemide (1 mg/kg bwt i.v.) were studied with and without phenylbutazone premedication (8.8 mg/kg bwt per os 24 h before, followed by 4.4 mg/kg bwt i.v. 30 min before frusemide administration). A control (saline) treatment was also studied. Administration of frusemide without phenylbutazone led to diuresis, natriuresis, kaliuresis and chloruresis, and altered the ratio of sodium:chloride excretion from 0.4 to 1.0 in the first hour of diuresis. When frusemide and phenylbutazone were administered, sodium and chloride excretion in the first hour were significantly (P<0.05) reduced by 40 and 32%, respectively, when compared to frusemide administrationwithout phenylbutazone. The fractional clearance of sodium and chloride was also significantly reduced. Potassium excretion, potassium fractional clearance and the ratio of sodium to chloride excretion were not affected by administration of phenylbutazone. During peak diuresis, phenylbutazone did not affect the efficiency of frusemide with respect to electrolyte excretion. The plasma disposition of frusemide was not affected by phenylbutazone. However, the renal excretion of frusemide decreased by approximately 25%. We conclude that the decreased urinary excretion of frusemide by phenylbutazone led to an attenuation of frusemide-induced increases in urinary excretion of sodium and chloride. Since the efficiency of frusemide was not affected by phenylbutazone, we conclude that phenylbutazone attenuates the renal excretion of frusemide without inhibiting the intrarenal activity of frusemide in horses.     

Potassium concentration in equine red blood cells: Normal values and correlation with potassium levels in plasma

The concentration of potassium in plasma and in red blood cells was determined in 948 horses. The coefficient of correlation between the two parameters was low. In 436 of these horses, which were clinically healthy, the red blood cell potassium (RBCK⁺) levels did not fit within a normal distribution curve, but a bimodal distribution was observed with a section point at 90 mmol/litre. In 90 per cent of these normal horses, mean RBCK⁺ content was 97.5 mmol/litre. In the remaining 10 per cent, mean RBCK⁺ concentration was 93.8 mmol/litre. A subdivision into a ‘low potassium group’ and a ‘high potassium group’ was made. In 10 out of 15 horses in the ‘low potassium group’, bimonthly sampling over a period of one year showed that RBCK⁺ content remained low. In the remaining five horses an increase was observed.