Сельскохозяйственная биология, 2010, № 4, с. 59-67.

УДК 636.12/.13:577.125.33:591.133:577.15

SOME PARAMETERS OF ANTIOXIDANT PROTECTION SYSTEM IN SPORTING HORSES DURING VARIOUS INTENSITY TRAINING

A.V. Antonov

In triathlon horses of half-blooded races the author determined the content of the most important antioxidants in blood over a period of annual training cycle and at dynamics «rest—work—relaxation». It was shown, that by the end of annual cycle the catalase biosynthesis is reducing, but the ceruloplasmin biosynthesis is raising, the α-tocopherol mobilizes from tissues reserves, quantitative ratio between glutathione fractions changes for benefit of oxidated form, that suggests about the increase of load on antioxidant systems. The essential differences between stallions, mares and geldings the author doesn’t revealed.

Key words: horses, triathlon, antioxidants, α-tocopherol, catalase, caeruloplasmin, glutathione.

Self-sustaining chain reactions of free radical oxidation of lipids (or lipid peroxidation, LPO) are necessary for realization of certain biochemical reactions, but their severe intensity causes an adverse impact on animal organism. LPO processes in cells and tissues inhibit antioxidants - these include, firstly, complex enzyme ceruloplasmin (EC 1.16.3.1) and catalase (EC 1.11.1.6), whose prosthetic groups contain, respectively, copper ions and heme residues. Antioxidants also include tripeptide glutathione and the reduced form of vitamin E - α-tocopherol. Action mechanisms of these substances are diverse, but they all ultimately terminate the chain of peroxidation reactions.

Under training loads, LPO level in horses increases (1-3). Antioxidant system responses the changes (4-6), though, the nature and direction of this reaction are not well understood.

The purpose of this work was to evaluate the functional status of certain components of antioxidant protection system in sporting horses at various stages of winter training (preparatory) course and reactions of these systems to different load intensities.

Technique. Experiments were carried out in the training stable by L.A. Borisova (Divovo settlement, Rybnovskii region, Ryazan province). The subjects of the experiment were 12 triathlon half-breed horses aged from 4 to 8 years. Three groups of animals were formed: group I - 4 stallions, II - 4 mares, III - 4 gelding. The experiment included three periods. In the 1st period (the beginning of training season, October-November 2005), the exercise load included 15 min of trot run and 5 min Kenter, in the 2nd period (the middle of training season, December 2005-February 2006) - 15 minutes of trot and 10 min field gallop; in the 3rd period (the end of training season, March-April 2006) - 25 minutes of trot and 15 minutes of field gallop with 15 jumps over barriers.

During the experiment, animals’ health was regularly controlled: evaluations of exterior, behavior, consuming feed, as well as the measurements of respiration rates and heartbeat rates during a rest, before feeding and before work were performed.

In the end of each period, the blood samples were collected from jugular vein before exercise, just after exercise and 45 minutes later. The plasma composition was analyzed: ceruloplasmin activity - by oxidation of p-phenylenediamine (7), catalase activity – by inhibition of hydrogen peroxide reaction with ammonium molybdate (8), the content of α-tocopherol – by spectrophotometric method developed by authors jointly with V. G. Memedeykin (All-Russia Development and Research Institute of Horse Breeding, Ryazan province). To determine α-tocopherol, plasma lipoprotein complexes were destroyed with ethanol first, then lipids were extracted with hexane. In the hexane extract, the content of α-tocopherol was determined on UV absorption at the wavelength of 297 nm. In the whole blood, the concentration of total, reduced and oxidized glutathione was measured by iodometric titration (9).

The data were processed statistically using TBAS and Microsoft Excel software.

Results. Clinical and physiological monitoring of animals before the blood tests showed that heart rate in the 1st period was 32-64, in the 2nd - 32-48, in the 3rd - 32-36 beats per minute, respiratory rate – respectively, 16-32, 16-24 and 12-24 breaths per minute. These data indicated a good health of all studied horses and their improved fitness after the training season.

The data on antioxidant enzymes activity (Table 1) indicated the absence of any regularities and significant differences on these parameters between groups of animals. In the 2nd period, the initial activity of ceruloplasmin increased in stallions by 4,0%, in mares and geldings - decreased by 6,8 and 28,4%, respectively. Authors believe that these changes were not statistically significant and accidental in nature. In average for all the animals, the activity of ceruloplasmin measured before exercise load in the 2nd period was somewhat smaller (at 11,1%) compared with the 1st , while in the 3rd period it increased by 46,8% compared with the 2nd. In the 1st and 2nd periods, the level of this parameter was somewhat higher after work and then it decreased after relaxation; in the 3rd , it tended to increase both after work and after relaxation. However, these changes were not statistically reliable as well. It is known that ceruloplasmin is produced in the liver and its biosynthesis is regulated by cortisol (10). The authors’ data suggest that during the training season and the cycle "rest-work-relaxation", the intensity of ceruloplasmin biosynthesis didn’t decrease under the influence of more intense physical activity.

The activity of catalase before work in the 2nd period was higher than in the 1st by 146,8% (p <0,001), in the 3rd  - 42,9% lower (p <0,001) than in the 2nd. After work, this value increased in the 1st period and decreased in the 2nd and 3rd, while after relaxation it was always higher, but these changes were not reliable.

It is known for fast-allure horses, that catalase activity in the blood, as a rule, increases after work and decreases after relaxation (6). The author’s data indicate that this pattern can have some exclusions. It is obvious, that catalase activity can be limited by the intensity of its biosynthesis in tissues. In this experiment, the increased activity of catalase by the 2nd period was, obviously, manifested while the functional reserves were very close to exhaustion. In the 3rd period, the reserves of catalase were consumed and the leading role was occupied by other antioxidants such as ceruloplasmin. This fact is consistent with reports indicating that catalase is a labile link of the immediate response antioxidant defense system, in contrast to ceruloplasmin, which is called the late reactant (11, 12).

In the 1st and 2nd periods, no regular changes were detected in the glutathione system under the influence of work and a subsequent relaxation. This absence of visible reaction to exercise load can be explained by changes in activity of enzymes providing oxidation and reduction of glutathione. It has been found in sporting and racehorses after work load, that the activity of glutathione peroxidase (catalyst of glutathione oxidation) can raise in certain circumstances (5), and reduce - in others (1). Obviously, this should lead to the change in activity of glutathione reductase which reduces glutathione. For the contents of glutathione fractions, in the 3rd period after work, the ratio “oxidized / reduced forms” sharply increased and the ratio “reduced /total glutathione” – decreased, while in the 1st and 2nd periods these patterns were not observed. Apparently, the oxidation of glutathione during a work in the 3rd period proceeded most intensively and the activity of glutathione reductase was insufficient for refilling the supplies of reduced glutathione.

Differences between stallions, mares and geldings on the studied parameters were generally unreliable and irregular. In the same experiment, it was shown that the intensity of peroxidation in mares was always lower than that in stallions, as it has been reported earlier (17). In geldings, the intensity of LPO was maximum in the 1st and 2nd periods, in the 3rd - just slightly lower than in stallions (17). These facts suggest the role of other antioxidants (eg. steroid hormones) affecting the observed differences. It has been reported that estrogens inhibit peroxidation (18). Androgens have this property as well, but to a lesser extent (19).

So, a training course of sporting horses affects their antioxidant defense system. During a training season and while the corresponding adaptation of animals to stress, ceruloplasmin activity doesn’t reduce, but the reserves of catalase synthesis and its role decline to some extent owing to increase in participation of other antioxidants. The content of α-tocopherol in blood plasma also reduces, but its mobilization from tissue reserves starts by the end of training season in response to work load. The indicators of activity of glutathione system remain constant at low and medium loads, and only the hard load (the end of training season) disturbs this balance, which results in changes of the ratio between contents of total, oxidized and reduced glutathione. No significant differences between stallions, mares and geldings in the studied indicators were found, which suggests that the earlier revealed sex differences on LPO intensity (17) can be provided by antioxidant properties of sex hormones, especially estrogens.

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