There is very little information currently available about ascorbic acid (AA) supplementation in horses. Most domestic animals, including horses, have the ability to synthesize vitamin C from D-glucose or D-galactose via the glucoronic pathway [1,2]. However, the limited AA stores in the adrenal gland are mobilized by prolonged stressors such as transportation in horses and hepatic synthesis may be inadequate to maintain adequate plasma concentrations [3]. Supplementation of AA orally during training and following stress or illness in racehorses [4] and in competitive endurance horses [5] has proven to be an effective method for increasing plasma AA concentration. However, it has also been documented to be ineffective in increasing plasma AA concentrations in unstressed horses [6]. In a preliminary study in this laboratory, prolonged supplementation (10 days) of AA to weanling horses that had been transported for 36 hours resulted in significant suppression of plasma AA for over 4 weeks after supplementation was stopped [7].

Transportation in a horse trailer evokes stress responses in horses indicated by increased heart rates, increased plasma cortisol concentrations, and decreased plasma AA [3,8,9,10]. The physiologic responses are present throughout the duration of transport even if the horses are not exhibiting obvious behavioral signs of distress [10]. Most indices of stress return to baseline values following a post-transport recovery period of 24 hrs [10], however, Laegreid et al. [11] observed increased plasma cortisol concentrations in horses for up to a one-week period following 36 hours of transport. The degree of stress generated by transport is apparently greater in young, inexperienced horses than in older, experienced animals [12,13].

An important result of stress is an increased demand for ascorbic acid [14]. Both physical and behavioral stressors cause increased secretion of adrenocorticotrophic hormone (ACTH) and glucocorticoids such as cortisol [15]. Increased plasma cortisol causes release of AA from the adrenal gland of mammals [16,17] and depletion of body stores, and is associated with a reduction in immune competence [2,11,18]. It has been hypothesized that supplementation with AA might reduce the incidence of stress related diseases following transport such as “shipping fever” in horses [7,19]. Shipping fever is a common term for pleuropneumonia, a bacterial infection of the lungs causing nasal discharge, coughing, elevated rectal temperatures and inappetance [19]. A major risk factor for the development of pleuropneumonia is transportation greater than 500 miles [20,21]. Supplementation of AA to aged horses that had pituitary dysfunction and high plasma cortisol with depressed plasma vitamin C concentrations improved antibody response to vaccines [22].

In preliminary studies conducted in 1999 and 2000 [7] weanlings were supplemented with AA for 5 days (1999) and 10 days (2000) following prolonged (>36 hrs) transportation stress. Results suggested that supplementing horses with vitamin C for 5 days may be beneficial in reduction of the incidence of “shipping fever”. In the first study, 4/5 unsupplemented weanlings had elevated rectal temperatures, nasal discharge, coughs and inappetance 1 to 2 weeks after arrival whereas only 1/5 of the weanlings supplemented with 5.0 g AA twice a day orally for 5 days after arrival had similar clinical signs. In that study none of the horses had any handling other than processing through chutes for vaccines and anthelminthic administration. The weanlings were highly resistant to being handled in the first week after arrival and most were stressed for the first 5 days. In the second preliminary study (2000) [7], none of the horses (n = 12) developed clinical signs of illness. That group had received 5 days of pre-transport handling by the authors and their students and only foals that calmly accepted handling were acquired for the study. The weanlings that year were noticeably calmer upon arrival and quickly accepted the routine. Following cessation of 10 days supplementation of 5.0 g AA twice daily, the supplemented horses had decreased (PP 4], though the statistical analyses of the decrease was not provided in the results. In humans prolonged AA supplementation reportedly increases the rate of urinary excretion [19]. There is also the possibility that hepatic synthesis of AA may be down-regulated with prolonged exogenous supplementation in species such as the horse where this occurs, but it has not been reported previously, to the authors’ knowledge.

The hypotheses for the current study therefore were (1) supplementation of AA for 5 days following prolonged transport (>36 h) would increase plasma AA and response to vaccination in naïve weanling horses relative to naïve weanling horses that did not receive AA supplementation, but were otherwise handled in the same fashion, (2) prolonged (10 day) supplementation of AA to weanlings under the same conditions would result in significant and prolonged decreases in plasma AA concentrations following cessation of the supplementation, and (3) supplementation of AA to weanlings that had low plasma AA would improve immune response to vaccines.

Due to technical errors, plasma AA data for day 3 and week 5 are not available. By day 5 all but one of the horses were calm and showing no behavioral signs of stress other than resistance to venipuncture. Plasma AA results are presented in Table 1 and Figure 1. None of the weanlings had clinical signs of disease or elevated rectal temperatures during the study. At day 5 there was a only a numerical trend for horses in treatment 1 to have a higher plasma AA (PP P P P P

There were no differences (P > 0.2) among treatment groups with respect to responses to the vaccines, though the vaccine titers only had increased (PTable 2). However, the previously vaccinated ND weanlings (n = 6) had a higher antibody response to WEE and EEE (PTable 3).

* Treatments differ at that time point, (P

The plasma AA concentrations reported in this study are somewhat higher than those reported in some other studies of normal, exercised and stressed horses [4,5,25,26] where values varied between 0.2 to 0.8 mg/dL (5 to 40 µmol/L). Plasma AA concentrations in the current study varied between 0.5 to 1.2 mg/dL, as reported by Jaeschke et al. [2] and previous studies by the author [3,7]. This may have been due to differences in analytic techniques, since high performance liquid chromatography was used in the more recent studies versus the colorimetric assays used by the authors. However the ranges did overlap and the relatively low inter-assay coefficient of variation gives confidence in the validity of the results. There were no differences (P > 0.3) in mean plasma AA concentrations in this study when compared to data obtained previously from weanlings from the same ranch in North Dakota and handled under the same conditions [7].

Ascorbic acid supplementation tended to increase plasma AA concentrations relative to unsupplemented weanlings following prolonged transport, though the increases were not statistically significant. There have been similar observations of response to supplementation in horses affected with recurrent airway obstruction [26], in race training [4] and during and after a 140 km endurance race [25]. Loscher et al. [6] stated that plasma AA concentration showed no significant increase when administered orally to unstressed horses, and it is presumed that AA in excess of need is excreted in the urine [1]. On the other hand, Snow et al. [4] found that repeated oral administration of AA to horses in strenuous training (stress condition) resulted in significant increases in plasma AA. The lack of clear significance in this study could have been due to variability in the weanlings’ responses to handling and training, which due to the relatively small sample size (n = 4 for each tx) may have obscured differences Supplementation of AA before transportation would be of no benefit since it would presumably be merely excreted. Short term stressors (

The horses had been weaned two weeks prior to transportation, which is a stressor that can cause two-fold increases in plasma cortisol concentrations for up to four weeks in young horses after separation from their dams [27,28]. It has also been shown that there is a correlation between stress and low plasma AA concentrations in young horses following weaning [29]. Therefore the weanlings potentially were experiencing post-weaning stress in addition to transportation stress in the first two weeks after arrival. However, though decreased appetite and increased reactivity to noises and handling was noted upon arrival in all of the weanlings, these behavioral indications of psychological stress had subsided after 5 days in all but one of the horses.

Supplementation of AA did not affect immune response to vaccines administered 6 weeks after transport of the weanlings. The delay in administering the vaccines was beyond our control and probablyhave contributed to the lack of effect. When the booster vaccinations were given, the treatment groups all had plasma AA concentrations within the normal ranges seen in previous studies and did not differ between treatment groups (data not shown). There were differences in antibody response between weanlings that had been previously vaccinated and those that had not. The increased titers observed in Canadian horses for the pre-vaccine sample could have been due to the presence of maternal antibodies that also could have interfered with subsequent vaccine responses [30].

The average half-life of AA is thought to be between 16 and 20 days [31] and the water-soluble properties of AA cause urinary excretion of the vitamin [18,31]. The prolonged decrease in plasma AA concentration could have been due to reduced endogenous synthesis and/or enhanced excretion of AA. Previous studies performed on humans have suggested that there is enhanced excretion of AA following increased vitamin C supplementation [19]. However, the possibility of reduced endogenous synthesis in horses following prolonged supplementation has not been reported and should be investigated further. Additional studies are required to assess the effect of prolonged AA supplementation and the effect of cessation of supplementation on the endogenous synthesis and excretion of AA. It would be of interest to evaluate urinary excretion of dehydroascorbate, the metabolite of AA, and to do labeling studies with C-14 labeled glucose to evaluate synthesis rates before, during and after supplementation of AA to both normal and stressed horses.

Based on the results of this and previous studies, it would appear that AA supplementation for 5 days following prolonged transportation in weanling horses may be appropriate. However, prolonged supplementation of AA after adaptation to a new environment is not recommended.