Change in blood antioxidant status of horses moved from a stable following diagnosis of equine motor neuron disease

Abstract

The antioxidant status of 10 horses living in stable 1 where 2 cases of equine motor neuron disease had previously been diagnosed was assessed before and 9 weeks after moving to another stable. Duration of residence in stable 1, subsequent moving, or both, significantly affected several parameters of the antioxidant status.

Alpha-tocopherol or vitamin E (Vit E) is a highly important liposoluble antioxidant element of the body and plays a key role in the prevention of oxidative stress. Lowered blood Vit E concentrations have been associated with equine motor neuron disease (EMND), a sporadic equine neurodegenerative disorder of unknown etiology (1). As a consequence of motor neuron degeneration, EMND is mainly characterized by weight loss, muscular atrophy, and generalized weakness (1). Oxidative stress is believed to play a role in the development of EMND (2). Besides Vit E, intrinsic, environmental, management, and nutritional factors are thought to be associated with EMND (3,4).

In the present study, 2 horses from the same stable (stable 1) had been histologically diagnosed as suffering from EMND within the last 3 y. However, EMND generally is considered to be a sporadic disease affecting only 1 horse in a stable (5). Therefore, horses housed in this stable were suspected of being at particularly high risk for developing EMND. As other horses from stable 1 were losing weight and showed exercise intolerance, the owner requested a measurement of plasma Vit E level of all the 23 horses [age 14 y, standard deviation (s) = 8 y] housed in this stable. Mean plasma Vit E concentration was 1.18, s = 0.57 mg/L (normal range, 1.70 to 9.50 mg/L), with values below the usual deficiency threshold (≤ 1.50 mg/L) (6) being observed in 17 of the 23 horses. Moreover, most of these horses were found to be in poor body condition [scores ranged from 2 to 5 (7)]. Stable 1 was located ~20 km west of Liege, Belgium, and ~7 km away from both the “Liege International Airport” and the “bassin industriel de la Meuse,” in an industrial area with a population density of 320 inhabitants/km². It was suspected that the location of the stable could potentially favor development of EMND, so the owner decided to move all 23 horses to a new stable (stable 2), while rigorously maintaining the same food regimen. Stable 2 was located ~30 km to the south of Liege in a well-wooded area with a population density of 125 habitants/km² and without industrial activities nearby. The distance between stable 1 and stable 2 was ~40 km. Daily feeding regimen invariably consisted of grass silage (4 kg/horse/d) and commercial molassed oats (2 kg/horse/d), and the horses were bedded on straw. They were not ridden, but they were allowed to move freely in a sandy paddock for ~1 h/d in both stables. The horses had no access to pasture at any time of the study. They were vaccinated regularly against the agents of tetanus and influenza, but not against those of rabies and herpes.

Ten horses (age 12, s = 6 y; Table 1) were randomly selected in stable 1, and their antioxidant status was assessed immediately before (T0) and 9 wk after (T1) they were moved from stable 1 to stable 2 (Table 2). In these horses, the plasma concentrations of Vit E, Vit A, and Vit C, as well as the activities of erythrocyte superoxide dismutase (SOD) and glutathione peroxidase (GPx), were determined. For statistical analyses, these horses were further divided into 2 groups according to their duration of residence in stable 1 before the study: Group A-horses that had lived in stable 1 for < 1 y (n = 4, age 12, s = 8 y); Group B-horses that had lived there for > 5 y (n = 6, age 12, s = 5 y). Nevertheless, none of the horses studied had been born in stable 1.

Table 1.

Characteristics of the 10 horses selected for the study

Horse	Age (years)	Gender	Group	Vit Ea (mg/L)
1	25	G	B	0.0
2	16	G	B	0.6
3	7	F	B	0.6
4	11	F	B	0.9
5	3	M	B	1
6	11	F	B	0.4
7	15	G	A	2.9
8	7	G	A	3.2
9	9	F	A	1.5
10	17	F	A	1.6

G — gelding, M — male, F — mare

^aVit E — plasma vitamin E concentrations measured at the 1st screening, 7 weeks before the start of the study (normal range, 1.7 to 9.5 mg/L). Vitamin E values at T0 (immediately before moving to stable 2) and T1 (9 weeks after moving to stable 2) are shown in Table 2

Table 2.

Plasma antioxidant concentrations determined in 10 horses immediately before (T0) and 9 weeks after (T1) changing from stable 1 to stable 2

		Time point of blood sample		Group effect	Stable effect
Marker (unit)	Groups	T0	T1	P-value	P-value
Vit E (mg/g chol)	A B	1.82, s = 0.54ac 0.52, s = 0.37bc	2.27, s = 0.56ac 1.49, s = 0.64bd	< 0.001	0.012
Vit A (IU/L)	A B	891, s = 98ac 936, s = 80ac	1471, s = 98ad 1411, s = 83ad	0.95	< 0.001
Vit C (mmol/L)	A B	2.35, s = 0.52ac 2.29, s = 0.55ac	1.98, s = 0.54ac 2.20, s = 0.42ac	0.87	0.65
SOD (IU/g Hb)	A B	1095, s = 153ac 1579, s = 109ac	912, s = 133ac 1211, s = 132ad	0.20	0.032
GPx (IU/g Hb)	A B	355, s = 33ac 285, s = 23ac	328, s = 29ac 279, s = 28ac	0.036	0.52

Vit E — vitamin E; Vit A — vitamin A; Vit C — vitamin C; SOD — erythrocyte superoxide dismutase activity, GPx — erythrocyte glutathione peroxidase activity; chol — cholesterol. P-values for group or stable effects are indicated in separate columns. Between-group differences (time of residence effect) at each sampling point are significant (P < 0.05) if the 1st superscript is different (a or b for within column comparisons), whereas within-group differences (stable effect) are significant (P < 0.05) if the 2nd superscript is different (c or d for within-line comparisons).

Horses of group A (n = 4) were living in stable 1 for less than 1 year and horses of group B (n = 6) for more than 5 years. Data are shown as means, s_χ̄.

Blood samples for assessment of antioxidant status were assayed as previously described (8). For standardization of the results, the Vit E/cholesterol ratio was calculated, and SOD and GPx activities were expressed per gram of hemoglobin (Hb).

Blood antioxidant concentrations measured at T0 and T1 were normally distributed and were analyzed by a mixed linear model for repeated measures (SAS proc mixed; SAS Institute, Cary, North Carolina, USA) allowing for the analysis of the effect of group (A versus B), of moving from stable 1 to stable 2 (T0 versus T1), as well as the group-stable change interaction. Differences were considered as significant when the P-value was lower than 0.05. Data are shown as mean values ± standard error of mean (s_χ̄).

Mean concentrations (s_χ̄ ) of blood antioxidant markers assessed at T0 and T1 in groups A and B are presented in Table 2.

At T0, plasma Vit A and Vit C concentrations were not statistically different between groups (P = 0.75 and P = 0.93, respectively). Contrastively, plasma Vit E concentration was significantly higher in group A than in group B (P = 0.002), while a trend towards lower erythrocyte SOD (P = 0.075) and higher GPx (P = 0.075) activities was observed in group A. Plasma Vit E values observed in group A were within normal limits for 3 out of 4 horses, while those values observed in group B were all deficient. The activity of SOD could have been upregulated in group B in response to an oxidative stress, as it is in response to increased workload in exercised horses (9). Nevertheless, the activity of GPx followed an opposite trend, with higher values observed in group A. Moreover, the overall group effect was significant for GPx (P = 0.036), even if this group effect at individual sampling times was not significant (P = 0.075 and 0.195 at T0 and T1, respectively), possibly due to the limited number of horses. These observations suggest that a GPx upregulation in response to an oxidative stress could exist, but it would be limited in time.

A significant stable-related difference was found for Vit E (P = 0.012). In group B, plasma Vit E concentration increased significantly (P = 0.009) between T0 and T1, while in group A, the increase was not significant (P = 0.25). Nevertheless, mean plasma Vit E concentration at T1 was still significantly higher in group A than in group B (P = 0.04). This was the only significant difference between groups at T1. No more trends were observed either in SOD (P = 0.996) or GPx (P = 0.195).

A significant increase between T0 and T1 was found for plasma Vit A in both groups (P = 0.002 and P = 0.003 in groups A and B, respectively), as well as a significant decrease of SOD in group B (P = 0.012). No stable effect was observed for Vit C (P = 0.65) or GPx (P = 0.52).

Clinically, all horses seemed to improve after changing stable. Although this appreciation was not based on systematic clinical examinations, the body scores tended to improve (range 3 to 6), the hair coat was slick and smooth, and the horses seemed to be more responsive. These improvements observed in both antioxidant status and clinical condition can be considered as spontaneous, as no nutritional or management factors were changed between stables 1 and 2. An oral supplementation in Vit E had been recommended for the 17 horses deficient in Vit E, but this was declined by the owner due to financial constraints. The commercial concentrated feed remained the same, and was given in the same amount by the same person. This was a popular sweet feed containing 62 IU Vit E/kg according to the manufacturer, and big differences in its composition with time are therefore unlikely. The grass silage was provided by the same manufacturer, and given in the same amount in both stables. The horses had no access to pasture, which is the best provider of Vit E in an equine food regimen. No access to pasture is known as an important risk factor for EMND, although recently some horses with regular access to pasture have been shown to suffer from EMND (10), suggesting that inadequate intake, but also abnormal bioavailability or excessive utilization, may lead to low Vit E status. Season could not have favored the plasma Vit E levels either, since moving occurred in November, and blood Vit E is known to decrease rather to increase during winter. Ideally, the Vit E content of the food should have been measured repeatedly during the study. However, grain and grass silage are not naturally rich in Vit E and their respective content was also expected to decrease with storage during winter.

The improvement in horses’ status could have been mediated by environmental factors. Stable 1 was located in a highly industrialized area. From the data, it could be suggested that a particularly high level of pro-oxidant elements were inhaled, ingested, or both, by the horses present in stable 1, resulting in an imbalance between pro- and anti-oxidants and a compromised antioxidant status. This, in turn, could have made the horses more at risk to develop EMND. Horses living for a longer period of time in stable 1 had a lower Vit E status before moving than had horses recently acquired. This further supports the hypothesis of an unusually high exposure to oxidative stress in stable 1. Moreover, improvement after moving was more marked in those horses whose antioxidant status was worse at T0 in regard to both Vit E and SOD. Duration of residence on a premises has been shown to be a risk factor for EMND (3).

It would also have been interesting to repeat the Vit E assessment at a later date in order to test whether horses of group B were able to recover a normal Vit E plasma concentration after a longer period of time. Furthermore, a thorough analysis of the food would have been interesting in order to correlate the intake of energy, minerals, vitamins, and trace-elements with the anti-oxidant status of the horses. Such a high rate of Vit E deficiency was not expected. Several mechanisms are proposed to lead to Vit E deficiency besides inadequate intake, such as interference of certain nutrients on Vit E availability (3), enhanced utilization in a pro-oxidant context or diminished intestinal absorption (10). It cannot be concluded by which mechanism the horses investigated in the present study increased their Vit E level. Even if each group included only a small number of horses, the gender and age partitioning was similar between groups, suggesting that these variables did not skew the results.

This report on potentially EMND-at risk horses suggests that the duration of residence in a stable where cases of EMND have occurred can affect the antioxidant status and especially plasma Vit E. The data also suggest that beside nutritional and management factors, environment might affect this status in horses. By changing only the environment of the horses of this study, a partial and spontaneous recovery of Vit E concentration and SOD activity, as well as an increase of Vit A, was observed after a short period of time.