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EVIDENCE OF A LINK BETWEEN SUBCLINICAL VIRAL INFECTION AND POOR PERFORMANCE. Dr. Guillaume Fortier (DMV, MSci)

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HIGH PERFORMANCE HORSES HIGH PERFORMANCE HORSES SCIENTIFIC NEWS SCIENTIFIC sponsored NEWS by sponsored by

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SUMMARY ❚ A WORD FROM THE PRESIDENT

❚H ERPES VIRUSES AND SUBCLINICAL INFECTIONS OF HORSES IN TRAINING

❚ I N VITRO AND EX VIVO EVALUATION OF THE VIRUCIDAL AND ANTIVIRAL CAPACITIES OF A BIOFLAVONOID AGAINST EQUINE HERPES VIRUS TYPE 1

❚ AIDAN O’BRIEN : THE MASTER OF BALLYDOYLE

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❚ EFFECTS OF DIETARY EXTRACTS ON CHONDROCYTE PROLIFERATION, A SPRINGBOARD TO TISSUE RECONSTRUCTION

❚ T WYDIL® AROUND THE WORLD

❚ S TABILITY AND QUALITY

❚N EW PRESENTATIONS

❚U NEQUALLED ANTIDOPING PRECAUTIONS

❚N EW TWYDIL® DISTRIBUTORS

❚ T WYDIL® AT INTERNATIONAL EXHIBITIONS AND CONVENTIONS

❚ E NDURANCE HORSES IN TWYDIL® INVESTMENT IN APPLIED RESEARCH

❚M USCULAR TROUBLES

❚ I NVESTIGATION OF THE NEEDS OF AGED OR DEBILITATED HORSES

❚ T HE FULL STUD PROGRAMME

❚ I NVESTIGATION INTO THE RELATION BETWEEN SPORTS PERFORMANCE PARAMETERS AND OXIDATIVE STRESS MARKERS IN TROTTERS

❚ T HE TWYDIL® RANGE OF PRODUCTS

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A WORD FROM THE PRESIDENT In spite of an international market environment with a gloominess known to everybody, in 2008, once again, TWYDIL® achieved a sale increase of 12% worldwide. Do they not say that, in times of crisis, consumers think twice about every penny that they spend and therefore prefer reliability and quality?

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HERPES VIRUSES AND SUBCLINICAL INFECTIONS OF HORSES IN TRAINING Dr. Guillaume Fortier, DMV, MSc. Faculty of Veterinary Medicine of Liège, Belgium Laboratory Frank Duncombe, Caen, France

INTRODUCTION

he causes of poor performance among athlete horses – regardless of the discipline – have been under investigation with a number of research programmes for more than 25 years. Such research programmes have focussed mainly on the musculo-skeletal and/or the cardiorespiratory systems or on identifying and quantifying the markers of haematological disorders caused by the possibly harmful effects of training. So, quite naturally, the list of subclinical diseases (i.e. without a clear medical expression that can be identified by clinical examination) that can potentially lead to a lack of performance of the affected horse have gradually increased following technological progress and the contributions of applied research to medical study of equine effort. There are many symptoms that appear only when the horse is under exercise conditions (e.g. coughing, nose discharge, bleeding, lameness …).

tion of the respiratory system with tracheal or bronchoalveolar lavage have been in use, it is now known that diseases of the upper or lower respiratory tracts represent a major cause of poor performances among competition horses.

The introduction of tools such as high speed treadmills paved the way for investigations that previously could only be undertaken close to the race tracks. Also, since techniques of explora-

It has long been known that the upper respiratory tract is a favourite site for the establishment of infectious viruses. This is the case with equine flu (equine influenza virus) or rhinopneumonia (type 1

or type 4 equine herpesvirus) and some lesser known viruses such as the rhinoviruses (agents of cold or rhinitis especially in humans). These infectious diseases frequently affect horses and are easy to diagnose since they often show significant clinical symptoms ranging sometimes up to flu syndromes (nasal discharge, hyperthermia, exhaustion, excessive eye watering …). Both the upper or lower respiratory tracts have been studied in order to improve

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HERPESVIRUSES AND RESPIRATORY TRACT DISEASES

our understanding of a “devastating” syndrome in the performance of horses: airway inflammation. Numerous conferences, conventions and books have already been dedicated to these affections in an attempt to provide a definition for them or – recently – to find a consensus on the methods of diagnosis and on the remaining necessary progress ahead (Couetil et al., 2007). Recent works helped us under-

stand the necessity of a more precise determination of the role of viruses, notably in the trachea and the main bronchi that can be accessed by endoscopy and tracheal lavage (TL). Thus, the syndrome of tracheal inflammation is named without any knowledge of its real causes: allergens, bacteria, viruses… Since these air passages are a perfect anatomical and physiological “crossroad”, the coexistence of many favourable factors could be an explanatory option.

A horse can host 5 major herpesviruses of which 4 show a more or less intense “tropism” for the respiratory system (Table 1). Type 1 and type 4 equine alphaherpesviruses (EHV-1 and EHV-4) account worldwide for episodes of more or less severe respiratory and abortive diseases (EHV-1) or for acute pathologies of the respiratory tract (EHV-4). Type 2 and type 5 equine gammaherpesviruses (EHV-2 and EHV-5) have been less well studied and their pathogenic capacity is sometimes discussed, contrary to their homologues in humans like the infectious mononucleosis virus (Epstein Barr virus) or the human cytomegalovirus (HCMV). The main biological characteristic of herpesviruses is their capability of latency in the primo-infected animal. Similar to the labial herpes simplex in humans or to shingles (most of the time appearing as a herpes reactivation of the chickenpox from youth years), the virus may hide in the local ganglionic structures and be reactivated when favoured by various stress situa-

Table 1: Main herpesviruses of equidae (according to Davison et al., 2009) Natural host

Designation Family

Related to

Other designation

Horse Equine herpesvirus Alphaherpesvirinae / Equine abortive virus (EHV-1) EHV-2 Gammaherpesvirinae / EHV-3 Alphaherpesvirinae / Coital exanthema virus EHVE-4 Alphaherpesvirinae / Equine rhinopneumonia virus EHV-5 Gammaherpesvirinae / Donkey EHV-6 Alphaherpesvirinae HVE-3 Type 1 donkey herpesvirus EHV-7 Gammaherpesvirinae HVE-2 et 5 Type 2 donkey herpesvirus EHV-8 Alphaherpesvirinae HVE-1 Type 3 donkey herpesvirus AHV-4 Gammaherpesvirinae HVE-2 et 5 Type 4 donkey herpesvirus AHV-5 Gammaherpesvirinae HVE-2 et 5 Gazelle and zebra EHV-9 Alphaherpesvirinae HVE-1

Reference (example) Allen and Bryans, 1986 Studdert et al., 1996 Hartley et al., 1999 Patel and Heldens, 2005 Telford et al., 1995 Browning et al., 1988 Bell et al., 2008 Crabb et al., 1991 Kleiboeker et al., 2002 Kleiboeker et al., 2002 Borchers et al. 2005 Borchers et al. 2006

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Figure 1 : Normal ciliated epithelial cells (full arrows) in a tracheal lavage of a thoroughbred horse 3 years old (1000 x enlargement, May Grunwald Giemsa coloration). The ciliature is connected to the cell body through a zone that is slightly more refringent (dotted arrow).

tion. Horses are no exception to that herpetic rule and once a majority of horses have been infected during their raising by many of these viruses (EHV-1 to 5), they can suffer from reactivation in different forms at different moments of their lifes. Just like an athlete, a horse is exposed to various forms of stress in connection with transportation, exercise, competition and management of relaxation. The investigation of the influence of these forms of stress on humans and horses has just started. It appeared interesting to focus our study on the potential detection of these viruses in the upper or lower respiratory tract of horses and on their possible effects, by using direct “markers” like the detection of their genome through gene amplification (PCR) or indirect “markers” (cellular abnormalities in fluids from respiratory lavages).

HERPESVIRUSES AND RESPIRATORY SAMPLES

Questions like “how to take a sample” or “which sample to 8

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take?” are serious difficulties for veterinarians in everyday practice. The reasons lie in the sometimes complex interaction of laboratory and physiopathology techniques of investigation of these kinds of herpesvirosis combined with still partial knowledge about the diseases (neurological form of rhinopneumonia, antigenic variability of the “wild” strains, reactivation mechanism, and sensitivity of the methods ….). For 15 years, numerous methods of molecular biology like the PCR (polymerase chain reaction) have been developed and nowadays they enable the detection of these viruses even when the viral load is very low. This can happen in the following three main cases: -T he veterinary surgeon examines a horse that has already been sick for some days and consequently shows a decreasing quantity of detectable viruses. -T he horse has been vaccinated against this type of virus and its viral charge is accordingly low and temporary. -T he disease has just started to develop in the horse, so that the horse has not yet reached the acute viral load (the more contagious horses !). Facing the diversity of this family of viruses in horses and their implication in various equine diseases, detection methods could be developed to identify a common gene shared by mammal herpesviruses affecting a specific region of the genome (Leon et al., 2008). Such methods are very useful in current practice in equine specialized laboratories, because they provide practitioners a quick result about the presence of one of the herpesviruses (1, 2, 4 or 5) before proceeding to a more precise “identification” which would help

improve not only the treatment but also the prevention methods in raising and in training. The major studies about the correlation between herpesviruses and respiratory diseases in young horses under training have mainly used serological diagnosis tools (Wood et al., 2005a; Wood et al., 2005b). But Murray et al. (1998) show, however, that young horses experiencing reactivation do not necessarily develop a seroconversion with seroneutralisation methods of diagnosis. This limits also the interest of serologies even if done in pairs at intervals of 15 days during contagious periods. Hence, it is necessary to be cautious about diagnosing viral diseases with serologic tests, and this is all the more so when clinical signs have remained unclear. For all these reasons, some studies have been performed by means of direct detection of the viruses or of their genetic material in the fluids from the respiratory exploration of horses with or without clinical signs (Sutton et al., 1998; Fortier et al., 2009). To sum up, nasal-pharyngeal sampling remains the standard for any situation of contamination presenting an unambiguous clinical flu syndrome. It is easy and quick to carry out and must be stored cool to be sent to specialized laboratories in the best conditions with clear history of the suspected case. When clinical situation is less evident and/or a lavage of the respiratory tracts must be considered an option, it may be quite interesting to focus on the cytology of the fluid together with a PCR-investigation for herpesviruses. Table 2 summarizes the outcome of a systematic study of respiratory

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fluids of more than 700 horses by comparing a group of healthy horses to a population of poor performers. It shows that, contrary to bronchoalveolar fluids, tracheal lavages of the poor performance horses presented 3 times more EHV-2 viruses compared to the control group. By the way, this virus is the most common of the pathologic groups demonstrated in horses before EHV-4 which is the most common virus found in contagious and clinical episodes among groups of adult horses. Is it possible to link these EHV-2 findings to a particular cytological profile of the lavages?

VIRAL DISEASES AND INFLAMMATION OF RESPIRATORY TRACTS

The first replication sites called “initial” sites for these viruses are the epithelial cells of the nasalpharynx. This first phase accounts for nasal discharge, local adenitis, occasional cases of pharyngitis and fever signs. In the subsequent phases (whereby the second phase sometimes explains the fevers and the symptoms recurring after a short

transitional recovery also called flu “V”), the clinical signs are less evident just as for the respiratory exploration fluids which are, by the way, the only means of indication of suspect disorders. As a matter of fact, the target cells of these viruses in the trachea are the epithelial cells which appear in healthy horses as shown in Figure 1. Hence, it is normal to find these cells in low percentage in the tracheal lavage. Once the trachea has been experiencing some viral activity, these cells take over morphology as shown in Figure 2. Experiments could demonstrate such changes (Sutton et al.; 1998) for flu or for rhinopneumonia (EHV-4 and EHV-1) or among field samples in systematic examinations about poor performance or intolerance to effort (Fortier et al., 2009). These disorders in other (bacterial, inflammatory …) pathologies have not yet been fully clarified, which is a reason why it is necessary to be cautious about using only these cytological criteria for diagnosis. The virus investigation must hence

always be done simultaneously with the cytological analysis of the fluid – which has established itself as the “gold standard” for this kind of examinations. The tissue lesions caused by the replication of these viruses in the clinical or subclinical phases of the disease can account for the secondary inflammation and for the length of time this may take in horses suffering from inflammation of the upper or lower respiratory tracts (Moore et al., 1996). In 1992, Willoughby et al. demonstrated that the “tracheal clearance rate” is modified during flu infections and slightly so during infections with EHV-1. According to the authors, these results are due to a slight alteration of the respiratory mucous membrane and to an excessive secondary inflammatory response. This inflammatory response to a respiratory viral infection involving EHV-1 has been studied more deeply by Kydd et al. in 1992.❚

Table 2 : Detection of herpesviruses in the respiratory fluids of two groups of horses with one healthy and the other pathologic, according to Fortier et al. (2009).

Tracheal lavages (TL)

Bronchoalveolar lavages (BAL)

EHV-1 EHV-2 EHV-4 EHV-5

CTL (n=37) 0 [0%] 4 [11%] 1 [3%] 0 [0%]

CTL (n=25) 0 [0%] 9 [36%] 1 [4%] 0 [0%]

PAT (n=259) 4 [2%] b 85 [33%] 17 [7%] 8 [3%]

p-value > 0.05 0.006 * > 0.05 > 0.05

PAT (n=387) 4 [1%] 82 [21%] 16 [4%] 16 [4%]

p-value > 0.05 > 0.05 > 0.05 > 0.05

CTL : Control group – PAT: Pathologic group – n = number of samples p-value : statistically significant if < 0.05.

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Figure 2 : “Ciliocytophthoria” type picture of a bronchoalveolar lavage (1000 x enlargement, MGG coloration). The arrow shows an epithelial cell without its ciliature and with a cytoplasm and a core with inclusion. Another cytoplasm (dotted arrow) is deformed (rounded) and shows an apparently normal nucleus with an inclusion.

They demonstrated the significant rush of neutrophil polynuclear cells (PNN) as well as cellular profiles in the bronchoalveolar lavages (BAL) which confirm similar descriptions (Moore et al., 1996) in horses infected with inflammatory diseases of the respiratory tracts. This PNN increase has been described by Sutton et al. with a flu virus infection (equine influenza virus). These authors describe modifications of inflammatory profiles of the BAL over a more or less long period of time compared to the duration of infections with EHV-1 (up to 21 days post-challenge). Considering that these inflammatory diseases of the respiratory tract are suspected only in young horses and that their clinical manifestations are often unclear, the detection method for respiratory viruses based on nasal-pharyngeal swabs can be applied only to a minority of cases showing unambiguously an acute disease of the upper respiratory tract. We have been working for many years in the laboratory on the definition of reliable cytological profiles which would be representative of viral invasions of the respiratory system of poor performance horses by comparing the collected data 10

with results of systematic PCR on samples from routine examinations or research protocols. The analytical results of a significant number of fluid samples tend to follow the indications of some external works (Couétil et al., 2007). Subclinical viral pathologies (which are mainly herpesvirus infections) may be responsible for the cases of inflammatory diseases of the upper or lower respiratory tract and of the poor sport performance observed. Future experimental reproduction of these pathologies caused by the EHV-2 virus should help provide some answers in this regard.

CONCLUSIONS

The equine species is one of the most exposed to the herpersviridae family viruses among mammals. Just considering their economic consequences, both viruses EHV-1 and EHV-4 heavily affect animal farming by the high number of infected studs as well as by the types of diseases and complications that they cause in their most frequent expression which is the subclinical form. Such a disastrous observation has not yet been proved for EHV-2 or EHV-5, but it seems probable that these viruses also have some harmful effects

on horses, even if such a hypothesis must still be proved by future more systematic and experimental studies. Even though it remains simple and efficient for the study of diseases with a confirmed clinical expression, the use of nasal-pharynx swabs still represents just the “visible” part of the iceberg for the exploration of subclinical infections that can be detected only by endoscopies and exhaustive analysis of the lavages. The subclinical forms caused by these viruses are usually due to latency and reactivation phenomena – viral excretion, characteristics and biological features of herpesviruses. Molecular biology and increased knowledge about equine immunology supported significantly the progress made by researchers and industrial institutions in the last ten years. Even if it appears unlikely that specific antiviral therapy will be available in the near future (as a result of the expensiveness of the related costs and the instability of the effects achieved in the tests conducted so far), it seems reasonable to think that vaccines and immunization protocols in horses will improve quite quickly for the equine species (based on the presentation of the antigens and the administration mode). Molecular biology and the progress made since the first methods of gene amplification applied to the detection of these viruses

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have produced a fine-tuning of the field diagnosis of these diseases, a quicker intervention of veterinarians in the stud farms or training centers as well as a better understanding based on genome data about the virulence, the cellular tropism and the variability of the stumps confronting the horses in their life or career. This knowledge is consistent and complies with the ones acquired in the vaccine industry that has to cope with the heavy scientific and technical challenge of ensuring vaccinal efficiency against herpesviruses. The effect of both horse gammaherpesviruses that are least investigated at the moment must still be better determined in horse business. But the “silent” immunomodulation induced by this virus family in mammals may indicate interesting new directions of research. In case the syndrome of tracheal inflammation should include one of these viruses in its etiology, then they may certainly play a quite significant role in the process.❚

REFERENCES Couétil, L.L., Hoffman, A.M., Hodgson, J., Buechner-Maxwell, V., Viel, L., Wood, J.L., Lavoie, J.P., 2007. Inflammatory airway disease of horses. J.Vet.Intern.Med., 21, 356-361. Davison, A.J., Eberle, R., Ehlers, B., Hayward, G.S., McGeoch, D.J., Minson, A.C., Pellett, P.E., Roizman, B., Studdert, M.J., Thiry, E., 2009. The order Herpesvirales. Arch.Virol., 154, 171-177. Diallo, I.S., Hewitson, G.R., de, J.A., Kelly, M.A., Wright, D.J., Corney, B.G., Rodwell, B.J., 2008. Equine herpesvirus infections in yearlings in South-East Queensland. Arch.Virol., 153, 1643-1649. Fortier, G., Van, E.E., Fortier, C., Richard, E., Pottier, D., Pronost, S., Miszczak, F., Thiry, E., Lekeux, P., 2009. Herpesviruses in respiratory liquids of horses: Putative implication in airway inflammation and association with cytological features. Vet Microbiol. IN PRESS Kydd, J.H., Hannant, D., Mumford, J.A., 1996. Residence and recruitment of leucocytes to the equine lung after EHV-1 infection. Vet.Immunol.Immunopathol., 52, 15-26.

Leon, A., Fortier, G., Fortier, C., Freymuth, F., Tapprest, J., Leclercq, R., Pronost, S., 2008. Detection of equine herpesviruses in aborted foetuses by consensus PCR. Vet.Microbiol., 126, 20-29. Moore, B.R., Krakowka, S., Cummins, J.M., Robertson, J.T., 1996. Changes in airway inflammatory cell populations in standardbred racehorses after interferon-alpha administration. Vet. Immunol.Immunopathol., 49, 347-358. Murray, M.J., del, P.F., Jeffrey, S.C., Davis, M.S., Furr, M.O., Dubovi, E.J., Mayo, J.A., 1998. Neonatal equine herpesvirus type 1 infection on a thoroughbred breeding farm. J.Vet.Intern.Med., 12, 36-41. Sutton, G.A., Viel, L., Carman, P.S., Boag, B.L., 1998. Pathogenesis and clinical signs of equine herpesvirus-1 in experimentally infected ponies in vivo. Can.J.Vet.Res., 62, 49-55. Willoughby, R., Ecker, G., McKee, S., Riddolls, L., Vernaillen, C., Dubovi, E., Lein, D., Mahony, J.B., Chernesky, M., Nagy, E., ., 1992. The effects of equine rhinovirus, influenza virus and herpesvirus infection on tracheal clearance rate in horses. Can.J.Vet.Res., 56, 115-121. Wood, J.L., Newton, J.R., Chanter, N., Mumford, J.A., 2005a. Association between respiratory disease and bacterial and viral infections in British racehorses. J.Clin.Microbiol., 43, 120-126. Wood, J.L., Newton, J.R., Chanter, N., Mumford, J.A., 2005b. Inflammatory airway disease, nasal discharge and respiratory infections in young British racehorses. Equine Vet.J., 37, 236-242.w

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IN VITRO AND EX VIVO EVALUATION OF THE VIRUCIDAL AND ANTIVIRAL CAPACITIES OF A BIOFLAVONOID AGAINST EQUINE HERPES VIRUS TYPE 1 Alexandra Scipioni, Tatiana Art, Lorène Dams, Brieuc de Moffarts, Pierre Lekeux and Etienne Thiry

This text is a non-specialist public version of a research study partially presented at the Hippos conference of 2008. (Scipioni et al, 2008)

he study was undertaken under the authority of Prof. Etienne Thiry (Department of Virology of the Faculty of Veterinary Medicine of Liège) and was conducted in cooperation with Dr. Tatiana Art (Department of Physiology of the Faculty of Veterinary Medicine of Liège). Its aim was the evaluation of the virucidal and antiviral capacities of a bioflavonoid. This study was partially funded by TWYDIL®. This study showed: In vitro : : a significant virucidal activity of the bioflavonoid and some of its activity on the viral replication process that must still be confirmed by future investigations. Ex vivo : following oral administration of this bioflavonoid, biological fluids showed significant virucidal activity.

INTRODUCTION

Relations between viral pathologies, inflammation and performance are frequently studied in humans in sports as well as in horses. We should bear in mind that respira-

tory system diseases are the second most common cause of poor sports performance for horses. Among these diseases, viral infections are a major problem (for further information about these infections, see the previous article, p 4 - 9) where they were dealt with in part. We will review briefly the implications of herpes virus type 1 (EHV-1). EHV-1 (Alphaherpesvirinae family) is a major pathogenic virus in horses. It exists endemically in the world and causes abortion and myelo-encephalopathy; as for EHV-4,

it causes rhinopneumonia. Though vaccines (and their combined protection against EHV-4) exist, horses are not fully protected against these diseases. Many years ago, antiviral drugs were developed for the treatment of herpes virus infections in humans. Tests in vitro proved their efficiency against EHV-1 and were encouraging, but, unfortunately, results of tests ex vivo were disappointing. Accordingly, the research for chemicals able to help fight against viral infections in horses as well as pre13

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aimed at showing a possible effect of the bioflavonoid in the plasma and/or the bronchoalveolar lavage (BAL) after 7 days oral administration of the flavonoid to 6 horses kept under standardized conditions. vent them became more and more intensive. So far, various plant extracts rich in flavonoids have shown some activity against human viruses (HIV, herpes etc.). It should be remembered here that, whether clinical or subclinical, horse viral infections cause enormous economic losses that can even be catastrophic for the economy of a whole country (AQUIS report 2009).

PRESENTATION OF THE STUDY

The study took place in two phases: the first in vitro and the second ex vivo.

1. Study in vitro

Figure 1 : Cell culture (RK13) infected by EHV1 on decreased doses (left to right) (determination by DICC50 method).

fore infecting the cellular carpet. Different durations of contact (10 sec. to 30 min) were tested. The viral titre was measured after 3 days incubation using the method of infectious dose of the cells responsible for 50% lysis of the cell supports (DICC50). - Evaluation of the compoundâ&#x20AC;&#x2122;s effect on viral multiplication: The objective was to measure the compoundâ&#x20AC;&#x2122;s effect on the multiplication process of the EHV-1 in an RK13 cell culture. The cells were brought into contact with the compound at different degrees of concentration in the course of the viral infection. Two days later, the culture boxes were examined for signs of cytopathogenic effects that would be characteristic of these viruses (syncytium and lysis zone).

In order to avoid interferences between the effect of the supplement and the reaction of potential antibodies against EHV-1 (there was a possibility that the horses might be already naturely immunized against this virus), porcine herpes virus 1 (SuHV-1) from the same family as EHV-1 was used for this phase of the test. All the experiments done with EHV-1 were validated by SuHV-1. Thus, only the effects of the flavonoid were taken into consideration, to the exclusion of possible antibodies that might be present. Blood samples and bronchoalveolar lavages were carried out at three different times (D-1, D0 and D+7) and their virucidal effect tested.

DICC50

The activity of flavonoids comThe sampling was done following pound used on formulation of feed this plan: supplements was tested. Together with its effect on viral multiplica- D-1: blood and BAL samples as tion, its virucidal effect against control samples EHV-1 was evaluated for an RK13 - D0: blood sample (4 hours after cell lineage based on a reading of the first administration) the decrease of the viral titre. - D+7: blood and BAL samples (4 This work was done in several 2. Study ex vivo hours after the last administrastages: In the second phase, the study tion) - Producing a suspension of the Figure 2 : viral count reduction after in vitro contact of the compound using various sol- bioflavonoid with EHV-1 vents; - Measuring the cytotoxicity of the Significant reduction of solvents and of the compounds 1,60E+09 99.9998% dissolved in an RK13 cell culture; 1,20E+09 - Evaluation of the virucid50 al effect of the compounds. 8,00E+08 DICC Figure 2 : viral count The principle was a measurment reduction after in vitro of the possible virucidal effect of contact of the bioflavonoid the suspensions of flavonoids on with EHV-1 EHV-1 in an RK13 cell culture. 5000 For this purpose, the sample was 0 CONTROL BIOFLAVONOIDE added to the viral suspension be14

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Control

Figure 4 : viral count reduction after ex vivo contact of the broncho-alveolar lavage and EHV-1

Bioflavonoid

Figure 4 : viral count reduction after ex vivo contact of the broncho-alveolar lavage and EHV-1

10x Dilution of the bioflavonoid

Significant reduction of 56%

1000x Dilution of the bioflavonoid

The aim of the sampling was to answer the question whether – following the oral absorption –the flavonoid was still present in the horse’s blood and/or BAL and whether it was available in a concentration that was enough for a virucidal effect ex vivo. The operation was in every point similar to the tests in vitro including the usual controls.

RESULTS OF THE STUDY 1. Study in vitro

The verification revealed that the experimental conditions were appropriate. Figure 1 represents a reading plate for the EHV-1 in an RK13 cell culture after inoculation of the EHV-1 virus. The blue zones stand for cells still available and the white zones for lysed cells. The virucidal effect of the compound on the EHV-1 proved significant! Just 10 seconds contact with a concentration of 0.125µg/ ml was enough to reduce the viral titre by almost 100%. Figure 2 illustrates these results. The outcome of the test suggests a possible effect of the compound on the multiplication process of the virus. Figure 3 indicates an absence of cytopathogenic reaction of the EHV-1 on RK13 culture cells when the bioflavonoid is added to the culture. However, the virucidal effect could interfere with the effect on the process of viral multiplication.

1,20E+09 DICC50

Figure 3 : observation after 24 hours of the cytophatogens effect induced in cells culture pre-incubated by EHV-1 without bioflavonoid. MOI : multiplicity of infection, representing of viral particuls per cells.

8,00E+08 DICC 4,00E+08 0

day - 1

After 7 days of oral administration of the bioflavonoid

Figure 5 : viral count reduction after ex vivo contact of the plasma and EHV-1

Significant reduction of 50%

1,60E+09 DICC50

100x Dilution of the bioflavonoid

Figure 5 : viral count

reduction after ex vivo contact of the plasma and EHV-1

1,20E+09

8,00E+08 DICC 4,00E+08 0

day - 1

day 0

These results indicate clearly a virucidal effect and suggest an effect of the bioflavonoid on the viral multiplication process. Future additional studies must still investigate the latter in order to describe the corresponding behaviour.

2. Study ex vivo

Let us remember that the virucidal activity in the blood and the BAL was tested using the same methods as previously described, but by taking the porcine herpes virus 1 (SuHV-1) instead of EHV-1. The results of the readings were compared with those of the control. They are shown in figures 4 and 5. No significant virucidal effect could be demonstrated on D0 (i.e. 4 hrs after the first oral administration of the bioflavonoid). However, on D+7, a significant decrease of the viral titre was observed in the plasma as well as in the bronchoalveolar lavage. In this phase of the study, the use of SuHV-1 helped us avoid a bias that could result from the presence

after 7 days of oral administration of the bioflavonoid

of antibodies against EHV-1 in the horse. But another source of possible error is the presence of the EHV-1 virus itself! In this case, the virus could lyse the RK13 cells in its environment and thus lead to a false positive result. In order to eliminate this risk, the blood and BAL samples came first into contact with the RK13 cells without addition of the SuHV-1 virus. Subsequently, no cytopathogenic effect was observed, meaning that the animals were not in a phase of viral excretion. The researchers also tried to measure the flavonoid’s concentration in the physiologic fluids and, surprisingly, found that its concentration did not change significantly during the trial.❚ All these results suggest that the virucidal activity observed is due to the presence of the bioflavonoid’s metabolites in the blood, since they had not been specifically looked for during the study. However, the activity of the biological fluids in the presence of EHV-1 following oral administration was observed. 15

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GENERAL CONCLUSIONS With this study, it could be demonstrated: In vitro: that the bioflavonoid possesses a virucidal activity and that it probably has an effect on the process of viral multiplication. Ex vivo: that, following oral administration of the bioflavonoid, some biological fluids have shown a significant virucidal activity.

BIBLIOGRAPHY Song J.M. and Seong B.L. Tea Catechins as a potential alternative anti-infectious agent. 2007. Expert Rev. Anti Infect. Ther., 5; 497-506. Whalley J. M., Robertson G. R., Scott N. A., Hudson G. L., Bell C. W. and Woodworth, L. M. Identification and nucleotide sequenceof a gene in equine herpesvirus 1 analogous to the herpes simplexvirus gene encoding the major envelope glycoprotein B. 1989. J. Gen. Virol. 70; 383–394. Scipioni A., Dams L., de Moffart B., Thiry E.,: In vitro susceptibility of equine

QUESTIONS TO PROF. ETIENNE THIRY HPH: Are these results surprising? Is this type of activity a frequent discovery? Prof. E. Thiry : The observed virucidal effect in vitro is very important. Other studies showed this type of virucidal activity, but it is true that this type of effect had been mostly studied for bacteria and not for virus. These promising results allow us to expect an effect on horses infected by the EHV-1. HPH: Some antiviral activity has been demonstrated, but what is the chemical’s behaviour in your opinion? Prof. E. Thiry: It is premature to specify the antiviral mechanisms of this molecule, because certain results must be confirmed. Regarding the virucidal effect, the inactivation of the viruses is probably due to an effect of the bioflavonoids on the envelope surrounding the virus. HPH: Can this type of product be active against other viruses like, for example, the influenza virus or viruses in other animal species? Prof. E. Thiry: If the action is located at the level of the viral envelope, it would be possible that the bioflavonoid has an effect on other wrapped viruses, such as the influenza viruses. Particular studies are however necessary to confirm this hypothesis. HPH: Which perspectives can this type of research open? Prof. E. Thiry: At the moment, vaccination is still the only form of prevention for viral diseases affecting horses. Thus, it could be usefully complemented by the use of virucidal bioflavonoids.

herpesvirus type 1 to two feed additives containing flavonoids. In proceedings: Hippos congress, Liège, Belgium, 2008 (poster communication). de la Fentes R., Awan A.R. and Field H.J. The acyclic nucleoside analogue penciclovir is a potent inhibitor of equine herpesvirus type 1 (EHV-1) in tissue culture and in murine model. 1992. Antiv. Res., 18; 78-89. Barrandeguy M., Vissani A., Ortiz C., Becerra L., Miño S., Pereda A., Oriol J., Thiry E. Experimental reactivation of equid herpesvirus 3 following corticosteroid treatment. Equine Vet. J., 2008, 40, doi: 10.2746/042516408X333399. Fortier G., Pronost S., Miszczak F., Fortier C., Léon A., Richard E., Van Erck E., Thiry E., Lekeux P. Identification of equid herpesvirus 5 in respiratory liquids: a retrospective study of 785 samples taken in 2006-2007. Vet. J., 2008, doi:10.1016/j. tvjl.2008.07.004.

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Complementary feedingstuff containing notably vitamin C, prebiotics, Ginseng panax, Glycyrrhiza glabra and Hydrastis canadensis which helps support the natural defences of the body. Particularly indicated in the event of sudden cold, red mucosa, fatigue or lack of energy. •W ithdrawal time before competition: 48 hours. • Declared content guaranteed until expiry date.

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e-mail: info@twydil.com

HPH

08-09

AIDAN O’BRIEN: THE MASTER OF BALLYDOYLE Michael FORTUNE

FROM WILLING PUPIL TO RARE GENIUS

On Sunday, June 28th, 2009 Aidan O’Brien greeted Fame And Glory back to the traditional on-course winner’s enclosure at the Curragh after the son of Montjeu had raced to glory in the Dubai Duty Free Irish Derby. Fame And Glory had run out a very easy winner from his stable companion Golden Sword, thus emphasising the extraordinary genius of the man who took over the mantle of the late Vincent O’Brien at Ballydoyle. What the mere result of the 2009 Irish Derby doesn’t indicate is that this was the seventh Irish Derby winner trained by Aidan O’Brien, the man of youthful looks who, quite incredibly, was still four months short of his 40th birthday. Vincent O’Brien, the man who had built Ballydoyle to its longheld status of the world’s leading training centre, had trained six Irish Derby winners during a truly incredible career. But yet here was his unrelated successor of the same surname who had already surpassed that achievement at an age when most young men would only be thinking of taking up the training game.

AIDAN O’BRIEN – THE EARLY YEARS

There was always something very special about Aidan O’Brien, who had grown up in the midst of a 18

great sporting environment in Killegney in County Wexford. The main talk down there in his youth would have centred around hurling but his dad Denis, a farmer by profession, had a great interest in horses and rode many a point-topoint winner in his younger days. Hence, Aidan grew up with horses and after leaving school a little early he tried out various jobs before getting a chance to join trainer P.J. Finn on the Curragh. Within a few months that stable closed its doors and Aidan was lucky to get a job with leading trainer Jim Bolger, a fellow Wexfordman. It was the start of something quite extraordinary and under the tutelage of Bolger, the young O’Brien took the first steps towards becoming a legend in his own young lifetime. He quickly became the amateur rider for the stable and assistant to one of Ireland’s leading trainers. Jim Bolger believes in hard work and anybody involved with him would have to do likewise. In Aidan O’Brien he had a very willing pupil. The pupil learned fast. He rode his first winner when Galacto Boy won at Punchestown in January, 1989 and he became Champion Amateur Rider in the 1993/ 94 season. By now he was already married to Anne-Marie Crowley, daughter of famed Piltown trainer Joe Crowley and herself the leading amateur rider. A new dynasty was in the building process! Anne Marie

had created history when becoming the first ever female Champion N.H. Trainer in Ireland in 1992-93 and she then immediately handed over the reins of the Carriganog stables to her husband.

AIDAN O’BRIEN – THE NEW TRAINER

The racing world wasn’t prepared for what was to follow. On June

FAME AND GLORY

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Aidan O’Brien gives the thumbs up to his son Joseph after he had recorded his first victory aboard Johann Zoffany in the Kilmacud Handicap at Leopardstown on May 28th, 2009. Breeders’ Stakes at The Curragh, including the winner and runnerup. That day at the Curragh he had a total of four winners.

AIDAN O’BRIEN MASTER OF BALLYDOYLE

7th, 1993, Aidan O’Brien had his first winner as a trainer when Wandering Star won at Tralee and he went on to complete a double that day, courtesy of Tryarra. He finished his first season as Champion N.H. Trainer in succession to his wife and set a new prizemoney record. He also became the first trainer to turn out 100 winners in his first season. The following year he retained his championship and set a new record of 176 winners. In 1995 he was to smash that mark. That was the year that was to set Aidan O’Brien apart from all his rivals as he quite amazingly managed to turn out at least one winner per day for 23 consecutive racing days during the summer and had 20 winners in one particular week.

It was quite obvious there was a genius in our midst and it didn’t take long for John Magnier and his associates in the Ballydoyle/ Coolmore operation to become interested in this young man still in his mid twenties. It seemed a mere natural progression when Aidan O’Brien moved to Ballydoyle. It was an incredible

An historic occasion as Aidan O’Brien receives the trainer’s trophy from Queen Elizabeth following Yeats’ fourth successive Gold Cup victory at Royal Ascot on June 18th, 2009.

opportunity for such a young man and he grasped it with both hands. In those early years he mixed jumping with flat and it was during that period that he completed the Champion Hurdle hat-trick at Cheltenham with the marvellous Istabraq – and he would surely have achieved the four-in-a-row but for the intervention of Foot and Mouth in 2001. But it was already inevitable that he was growing too big for the jumping game – just like his great predecessor Vincent O’Brien – and the domination of the world of flat racing became the target of the highly ambitious team. The horse that signalled his arrival at the top table on the flat was Desert King, winner of the 1996 National Stakes before completing the Irish 2,000 Guineas and Irish Derby double in 1997. That year saw him do the Irish Guineas double as he also won the 1,000 with Classic Park. When Urubande won the Sun Alliance Hurdle at Cheltenham in 1996 the youthful O’Brien was turfed out of the Winner’s Enclosure by officials who just didn’t recognise him. Surely nobody so young could be a Cheltenham winning trainer!

Every jump trainer wants to win the famed Galway Plate but O’Brien wasn’t satisfied to train just the winner as he also supplied the runner-up and the third. But already he was making big waves on the flat and had four of the first five finishers in the Tattersalls 19

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However, the whole world knew who he was by 1998 when not only did Istabraq win the Champion Hurdle at Cheltenham, but O’Brien dared to take on the top flat trainers and win the 2,000 Guineas with King Of Kings and the Oaks with Shahtoush as well as many other top prizes. From there the rate of success became the stuff of fairytales. In two separate years O’Brien has trained the winners of 23 Group 1 races worldwide … in Ireland he has trained six winners of the 2,000 Guineas, 5 winners of the Champion Stakes, 7 winners of the Derby, 7 winners of the National Stakes, nine winners of the Phoenix Stakes, etc. etc.

A great family occasion as Aidan, Ann Marie and their three other children pose with Joseph and Johann Zoffany after his first ever success at Leopardstown.

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In the UK he has won the 2,000 Guineas five times, the Coronation Cup three times, the Derby twice, the Eclipse four times, the King George V1 & Queen Elizabeth Stakes three times, the Middle Park three times, the Oaks on three occasions, the St James’s Palace Stakes six times and the St Leger three times. But it doesn’t stop there …. In France he has won the 2,000 Guineas three times, the Criterium de Saint Cloud three times, the Prix Jean-Luc Lagardere seven times, the Prix Morny three times and the Prix de l’Arc once …. In America he has enjoyed three successes at the Breeder’s Cup while also winning the Arlington Million, the Secretariat Stakes and the Shadwell Turf Mile … In Italy he has landed the Gran Criterium twice and the Grand Premio del Jockey Club … In Canada he won the Canadian International. We could go on and on – what a record for a man still short of that 40th birthday!

AIDAN O’BRIEN – THE MAN

But despite it all, Aidan O’Brien remains the same modest, self effacing individual he was when he first set foot in the training world back in the ‘90s. Just listen to him after any big race success and do you hear him accepting all the plaudits on his own behalf? Not a bit of it. Each and every time his response will go something like this …. “This win is all down to the lads in the yard, they have given so much time to getting this horse right, they have done a fantastic job”. It’s always the lads and lassies in the yard that are singled out for special mention and he is meticulous in publicly acknowledging their work with his horses. He is also very much a family man. One of the most familiar sights on big race days is that of Aidan O’Brien on his mobile immediately after one of his horses has raced to Group 1 glory. Invariably you will find that on the other end of the phone is either his wife or his mum as they all share in the latest triumph. Aidan O’Brien is a quiet, unassuming man – whether you meet him in normal circumstances or in the aftermath of a massive Group 1 success. Back in the early days when he first made his mark he was also very shy and was far from being an interviewer’s dream subject! I first became really acquainted with Aidan back in 1994 after Dancing Sunset gave him his first Group success in the Royal Whip at the Curragh. My role was as interviewer for RTE Radio’s Saturday Sport programme and it was to become the first of hundreds of interviews we shared over the years. But I’ll never forget that first one.

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his horses – and that is just as it should be! His role as boss of Ballydoyle is to hone the talents of some of the choicest bred thoroughbreds in the world and to establish them as potential Champion Sires and Dams of the future. In that regard his record has been quite phenomenal. – and getting more phenomenal by the month.

THE FAMILY O’BRIEN

We commenced it just alongside the 1st Post in the Winner’s Enclosure and with each question I asked, Aidan would take a step backwards and I would follow. By the time the 2 minute interview was over, we were halfway down that long Curragh parade-ring! And, worse, it was like pulling teeth as this was a new experience for the young trainer and the answers were monosyllabic! But he learned fast. Yes, for about a year we would walk a few yards during each interview but the answers were flowing more easily all the time. For many years now he has been a gem and what’s more, he is an interviewer’s dream. At all times he is helpful and most obliging, no demands are too much unless it clashes with the welfare of

It must be virtually unique for two Champion Trainers to become husband and wife and it was probably inevitable that their kids should inherit their talents. Ann Marie had been the Leading Lady Rider while Aidan was Champion Amateur and then they won the National Hunt Trainers’ Championship in successive years. For Aidan it was the first of many, many years as Champion over hurdles and later on the flat. Not surprisingly, all four of their children have inherited the racing bug. Eldest son Joseph has already established himself as a very talented apprentice. Just sixteen years of age and getting tall for a flat jockey, he made a pretty instant impact when winning a handicap at Leopardstown on Johann Zoffany. He gave the three-year-old, trained by his dad, a peach of a ride, dictating the pace and comfortably holding the late challenges.

the kids have grown up with the horses and all four have aspirations to riding on the track. Joseph did some work experience with Jim Bolger but has a background in eventing and was on the Irish Under 16 team in the European Championships in Switzerland”. Next in line is Sarah, aged 14, and she also events and will be taking part in the RDS Show this summer before studying for the Junior Cert next year. Then there is 13 year old Anna and again she’s into eventing but, like Sarah, she rides out at home when not eventing. The youngster of the family is eleven year old Donncha and he is currently involved with ponies but with the mind very much set on following Joseph into the riding ranks. Sport in general has always played a prominent role in the lives of the young O’Briens with both girls involved in basketball while Donncha has also played football and hurling. One suspects a new dynasty has been established in Irish Racing.❚

That was on May 28th of this year and within a few weeks he has brought his record to 4 wins from 24 mounts. He had the rare experience for one so young in having the ride aboard the unplaced Byzantine in the Dubai Duty Free Irish Derby. Ann Marie admits she was very excited the evening Joseph rode his first winner. She says: “All 21

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EFFECTS OF DIETARY EXTRACTS ON CHONDROCYTE PROLIFERATION, A SPRINGBOARD TO TISSUE RECONSTRUCTION Marie Daix1, Brieuc de Moffarts², Samuel Schulsse1, Nathalie Kirschvink1 (1 Animal Physiology, Department of Veterinary Medicine, University of Namur, Belgium) (2 Pavesco AG-TWYDIL®, Switzerland)

Figure 1:

Chondrocytes

Structure of the articular cartilage

Inflammatory mediators IL-1-beta, NFKB…

INTRODUCTION

steoarticular pathologies represent a major cause of lameness in the equine species. They are also the first cause of performance regression. These diseases are characterised by signs of lesions on the articular cartilage of horses. The lesions can affect the chondrocytes (unique cells in the cartilage) or the extracellular matrix. This extracellular matrix is a supporting structure produced by the chondrocytes in order to furnish the cartilage with the resistance properties needed to absorb shocks occurring during locomotion. The pathologic process that leads to cartilage lesions is complex (Fig. 1). Many factors are accordingly involved in this destruction of the cartilage. The major ones seem to be oxidative stress, enzymatic stress and inflammatory mediators; their mutual influence leads to a serious cause of lesions of the cartilage. 22

Matrix metalloproteinases: increased activity

Damaging of the extracellular matrix

Chondrocytes Reactive Oxygen Species

Injuries of the DNA, of the membrane lipids and the proteins of the chondrocytes

Cellular ageing

Extracellular matrix

Cartilage damage

Oxidative stress is defined as excessive accumulation of free radicals following oxygen metabolism. The production of free radicals then exceeds the defence capacities of the metabolism, which ends up in damage to the cartilage cells [1] and particularly in inducing a cellular ageing that can cause osteoarthritis [2]. It also appears that the reactive oxygen species (ROS)

are involved in the triggering of the cascade leading to inflammatory reaction and to enzymatic stress [1]. Actually inflammatory reaction amplifies the process and the increasing cascade leads to a higher activity of the proteinases (notably the matrix metalloproteinases) which damage the extracellular

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matrix around the chondrocytes [3]. This phenomenon is called “enzymatic stress”. The result of this chain of occurrences is a weakening of the cells and of the extracellular matrix in the cartilage, which compromises its principal functions: mobility and absorption. The common treatment of articular troubles is based on the administration of steroid or non-steroid anti-inflammatory drugs which enable a “breaking” of this chain reaction. These drugs can hardly be used in a context of competition since they produce a doping effect, and moreover, they can induce significant side effects. Another important aspect is the reconstruction of the cartilage after the pathologic stage. The cartilage extracellular matrix produced by the chondrocytes must restore itself on the injured spots, which may take some time, particularly when the chondrocytes have been affected by cellular damage. This applies to most tissues, i.e. tissue repair or remodelling is crucial for a good restoration of the functions of the affected organ. Every tissue experiences daily aggression and injuries, and its cells are exposed to different weakening factors. This process would lead to a diminishing functional efficiency if tissue remodelling did not balance it out. Thus, this repair enables a “regeneration” of the tissue and a balance between tissue damage and tissue reconstruction. Under pathologic conditions, inflammatory reaction, oxidative stress, and very often enzymatic stress disturb this balance, which may possibly lead to excessive tissue damage. In this regard, nutraceuticals or functional foods, which are ther-

apeutic or preventative dietary supplements, have a threefold use: they possess an anti-inflammatory effect with minimal side effects on the one hand; on the other hand, few of them considered to be doping substances, and finally, since some of them are constituents of the extracellular matrix, they are able to stimulate tissue respiration.

OBJECTIVES OF THE PRESENT RESEARCH

The aim of this research is to investigate the effect of dietary extracts

on the proliferation of equine chondrocytes in vitro. A sample of articular cartilage has been taken from horse limbs from a slaughterhouse. The fetlock articulations were opened and cartilage samples were taken using a scalpel; then they were cut into pieces and subjected to enzymatic digestion in order to isolate the chondrocytes. The chondrocytes were then cultivated for 7 days in 12-well plates in a growth medium with or without (for control purposes) addition of dietary extracts based on glucosamine/chondroitin sulphate 23

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08-09 Figure 2:

* *

G/CS = glucosamine / chondroitin sulphate Hx = Hydroxylapatite CM = complex mineral

OUTCOME OF THE RESEARCH

The results of the cellular counts show that the Hx extract has a stimulating effect on the chondrocyte proliferation on the 3rd and the 5th day of cultivation (Fig. 2). Concerning the G/CS extract, it shows a stimulating effect on the 3rd day. By the 7th day, there is no further noticeable difference between the cultures with addi-

Control

* significantly different from the control sample p<0.05

Figure 3: ex vivo F2-isoprostanes in the chondrocyte supernate coming from horses first orally supplied with the feed complement F2-isoprostanes concentration in the chondrocyte supernate (ng/mL)

(G/CS), hydroxylapatite (Hx) or on a complex mineral (CM) made of carbonate, phosphate and calcium gluconate and sea salts. The chondrocyte proliferation was evaluated by counting the cells using trypan blue every second day. Each well was cultivated and measured in quadruplicate.

Number of chondrocytes per millilitre in percentage of D0

Equine chondrocyte proliferation in vitro with different dietary extracts

(with or without IL-1-beta for 6 or 24 hrs.)

Control

A = G/CS B = Placebo p<0.05

Data presented in average Âą standard error *Significantly different from the respective value in group A p < 0.05 *$ Significantly different from the respective control value

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tion of feed supplement and the control cultures. These very interesting results underline the possible effect of the extracts tested on tissue repair; an effect produced by stimulating proliferation of the chondrocytes. Results from a previous research about chondrocyte cultures coming from horses that had been orally provided with feed supplements show that the C/CS complement induces a decrease of the F2-isoprotanes (markers of oxidative stress) in the chondrocyte supernate, and it remains so even after a 6 or 24-hour stimulation with 1-β interleukins, which was intended to simulate an ex-vivo inflammation of the joints (Fig. 3). Consequently, it seems that the complement given orally to the horses provides the chondrocytes with some protection against oxidative stress. In addition, a modulating effect had been observed for the G/CS complement regarding its influence on oxidative stress after 6 weeks of oral use of this supplement [5]. Indeed, a decrease of the MMP9 activity (type 9 matrix metalloproteinase; Fig. 4; the MMP9 being the enzyme involved in the damaging of the cartilage) was observed together with a beginning increase of the MMP2 activity (type 2 matrix metalloproteinase; the MMP2 being the enzyme that is apparently more involved in the repair than in the destruction of the cartilage) [4]. This effect on the MMP2 has also been observed in a subsequent study in vitro during which the chondrocytes, which came from horses that had been orally supplied with the feed supplement, were cultivated and subjected to 1-β interleukins to provoke an inflammatory stimulation in them (Fig. 5).

Considering all these results, it seems that the G/CS supplement is able to modulate oxidative and enzymatic stress and to stimulate chondrocyte proliferation. Thus, this approach could help at two levels in the handling of articular pathologies: it could help decrease the cartilage destruction and stimulate its reconstruction. It would be interesting also to study the effect of these extracts on the tendons. In fact, tendon damage is most frequent and accounts for severe lameness and for a more or less extended interruption of training. In cases of tendon damage, observation reveals that the difficult tissue repair is the biggest problem. Actu-

Figure 4:

ally a tendon contains fewer cells than the extracellular matrix, while exactly the same cells deliver the constituents needed by the extracellular matrix composing most of the tendons. In many cases, tendon healing is long and the tendon never fully regains its original performance [6]. Accordingly it seems interesting to investigate the effect of the extracts on the test on tendinocyte proliferation. If the stimulation effect observed with the chondrocytes is confirmed for the tendinocytes, it would be a major outcome for the handling of tendinous regeneration.

Articular MMP9 activity in vivo after 6 weeks oral supply

MMP9 activity (arbitrary * unit)

Articular

Fore fetlock

Back fetlock

Stifle

tarsus

T0 = Before oral supply T6= After 6 weeks oral supply * Significant differences between group A and B, p<0.05

Figure 5:

MMP-2 activity ex vivo in the chondrocyte supernate coming from horses orally supplied with the feed complement (with or without inflammatory stimulant (IL-1-ß))

MMP9 activity (arbitrary unit)

Articular

CTL = control = without inflammatory stimulant IL-1-β = 1-beta interleukin = with inflammatory stimulant * Significantly different p<0.05

HPH

CONCLUSIONS AND PROSPECTS

The extracts tested here shows signs of a modulating effect on oxidative stress and on enzymatic stress. In addition, its apparent effect on chondrocyte proliferation could stand for a stimulation of the metabolism which is probably beneficial to the tissue respiration. The same effects have also been observed in tests of ex-vivo inflammatory stimulation, which suggests a preventative effect. Thus, not only are these molecules seen to be very promising in the management and the prevention of joint problems, but it would also be interesting to investigate their effects on tendons as well since tissue repair is the key to the horse’s healing in cases of tendon damage.❚

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REFERENCES 1. VALKO, M., LEIBFRITZ, D., MONCOL, J., CRONIN, M. T., MAZUR, M., and TELSER, J. (2007) Free radicals and antioxidants in normal physiological functions and human disease. Int. J Biochem. Cell Biol. 39, 44-84. 2. YUDOH, K., NGUYEN, T., NAKAMURA, H., HONGO-MASUKO, K., KATO, T., and NISHIOKA, K. (2005) Potential involvement of oxidative stress in cartilage senescence and development of osteoarthritis: oxidative stress induces chondrocyte telomere instability and downregulation of chondrocyte function. Arthritis Res. Ther. 7, R380-R391. 3. NAGASE, H., VISSE, R., and MURPHY, G. (2006) Structure and function of matrix metalloproteinases and TIMPs. Cardiovasc. Res. 69, 562-573.

4. GEPSTEIN, A, SHAPIRO, S, ARBEL, G, LAHAT, N, and LIVNE, E. (2002) Expression of matrix metalloproteinases in articular cartilage of temporomandibular and knee joints of mice during growth, maturation, and aging. Arthritis & Rheumatism 46, 3240-3250. 5. DAIX , M., BASTIN, JF. and KIRSCHVINK, N., Evaluation of an oral supplement enriched with glucosamine and chondroïtine sulphate on the joint enzymatic balance in young horses.( 2006/2007) HPH High Performance Horses, 4-8. 6. RICHARDSON, L.E., DUDHIA, J., CLEGG, P.D. and SMITH R. (2007). Stem cells in veterinary medicine - attempts at regenerating equine tendon after injury. Trends in Biotechnology, 25, 409-416.

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Dr de Moffarts visiting the endurance team AL-Shaqab in Doha, Qatar

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STABILITY AND QUALITY Dr Brieuc de Moffarts (Director Research & Development, TWYDIL®)

he research conducted by TWYDIL® to optimize its formulas would be vain and uninteresting if special attention was not paid to ensuring optimal stability of the components in every single product from manufacturing to best-before date. To achieve this goal, TWYDIL® not only carefully selects its ingredients, the type of packaging and of storage but also supervises the manufacturing process and permanently increases its controls.

1. Selection of the raw materials

By “selection of the raw materials” we mean not only the selection of the vitamins and dietary minerals but also the selection of plant extracts or probiotics and further additives in the composition of the products of the range.

The first criterion of selection is the standardisation the product’s concentration in active substances and the absence of contamination in it. The second criterion is the product’s intrinsic quality as a guarantee for long lasting efficiency. Let us take two examples: -A scorbic acid which is best known as vitamin C. Three types of vitamin C are used in the range: a first type suitable for packaging in sachets, (TWYDIL® VIGORADE, TWYDIL® PROTECT PLUS,…) ; a second, more stable type which is coated and hence allows pelletisation (TWYDIL® RACING, TWYDIL® BEBACK,…) and a third type which remains stable in aqueous environments (TWYDIL® ELECTROLYTES syringe). In all three cases, the intrinsic type of the molecule used is confirmed by results of a content control of vitamin C. - Standardised extract of Eleutheroccocus senticosus, Maxim. an essential component of TWYDIL® HIPPACAN+C. Its use requires special caution such as, for example, a verified absence of natural contamination in the extract batches. This control is done by the LCH that then issues a certificate of absence of contaminants such as caffeine, theophylline, morphine, atropine etc. Besides, this plant extract may be more or less concentrated in active substances depending on factors with specific influence on its growth such as the type of soil, the level of insolation and of rainfall. Consequently, it is crucial for TWYDIL® to be able

to standardise the concentrations of active substances in the components in order to ensure a constant homogeneity of the products. The same controls apply to all the plant extracts used for the whole range.

2. Production technique

The production technique used for the feed supplement (whether liquid, powder or granular) should not cause any destruction of the active substances. Variations in temperature, pressure or moisture are harmful to vitamins while oxidation phenomena must be avoided for some types of fatty acids (such as omega 3).

Determination by chromatography of the eleutherosides contained in TWYDIL® HIPPACAN+C

-T WYDIL® RACING is highly concentrated in vitamins, dietary minerals, magnesium and amino acids. The production of its granules requires meticulous control: a thermal probe in situ ensures a regulated speed of production and a temperature of production kept

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below 62°C. Addition of water is prohibited because water destroys some vitamins. -T WYDIL® OMEGADIL is heavily enriched with omega 3 fatty acids. Beyond a ratio of 5% of omega fatty acids in a product, oxidation phenomena occur and make it inefficient or even harmful. Consequently, TWYDIL® OMEGADIL is produced under air-free (nitrogen or vacuum) conditions from the refining of the fatty acids up to packaging.

3. A dequate packaging

The packaging must be chosen carefully! On the one hand it must ensure stability of the finished product and on the other hand it must make its administration easy. It may happen that both criteria are in conflict with each other (in terms of cost, comfort, stability etc.). The example of mouth syringes is an interesting one. On the one hand we have the electrolyte syringe designed to make product administration easy, and on the other hand we have TWYDIL® STOMACARE for which administration with a mouth syringe is a necessary condition for its maximal efficiency. The use of sealed aluminium polyester

The case of TWYDIL® OMEGADIL is even more interesting! As you already know, it is manufactured under nitrogen in order to prevent fatty acids from oxidising. Hence, the packaging must also protect the product against oxygen; this is achieved by giving the packaging a special lining with different protective films that keeps oxygen away. On top of this, the cap for daily doses is designed in such a way that it blocks air penetration during administration. Mind you, researchers have shown at the conference on Free Radical Biology and Medicine (FRBM) that the risk for men to suffer from arteriosclerosis or inflammation (tendinitis, arthrosis) is higher with the ingestion of oxidised fatty acids.

4. Storage

TWYDIL® products are stored in a spacious warehouse located about twenty kilometres from Basel. This warehouse complies with the strict regulations of Swiss authorities. The products are under

250

Standardized chromatography of TWYDIL® ARTRIDIL collected in pharmacy

200

1. 2. 3. 4. 5. 6.

150

(mV)

100

Chondroitin (RT = 3,15 min.) Excipient (RT = 3,40 min.) Saccharose Imp. Gluco Glucosamin Imp. Chondro

Intensity 50

5 4

0 0

Retention

times(min)

Standardized chromatography of a chondroprotector, collected in a pharmacy, presenting comparable labelling than the TWYDIL® ARTRIDIL

250

3 200

1. 2. 3. 4. 5. 6.

150

(mV) 100

Chondroitin (RT = 3,45 min.) Excipient (non detected) Saccharose Imp. Gluco Glucosamin Imp. Chondro

Intensity 50

0 0

Retention times

(min)

Influence of Gelbo cycles on oxygen permeability

02 perm/number of Gelbo cycles

Filling of sachets

sachets for individual doses has become a rule for a large part of the TWYDIL® range, with the advantage that they preserve the active substances in the products, which results in a longer shelf life. In this regard, TWYDIL® A RT R I D I L has been studied by the office of chemical analysis of the ULg where it has been compared to other chondroprotectors. It turned out that the other tested competing chondroprotectors did not meet the requirements of guarantee. One of the possible causes advanced by the university is the loose packaging of the products, which would not ensure the stability of glucosamine and other complex sugars.

Comparison of oxygen permeability of EVOH and PETMET films

constant supervision regarding not only their physical condition but also temperature and moisture. In addition, and in order to prevent any secondary contamination of the packages, coffee, tea or even chocolate are prohibited in the warehouse by the internal company rules of TWYDIL®. 31

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5. Quality control

Quality control at TWYDIL® is twofold: an anti-doping control and a conformity analysis of content and label for the components in every product. This control is carried out on a representative sample of the batch. The anti-doping certification remains the key advantage of TWYDIL® clients throughout the world. Every batch undergoes a threefold test by the LCH:

Responsible of storage and warehouse Christophe Muller

- Finished product test: the LCH investigates the presence of natural contaminants. - Urine test on a horse after an overdose with the product: the LCH carries out a “full screening” identical to the one undergone by a winning horse of the “Prix d’Amérique” or the “Prix de l’Arc de Triomphe”. - Blood test on a horse after an overdose with the product: the LCH undertakes here too a “full screening”. You can access all the certificates on www.twydil.com by entering

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the product batch number. Hence, all TWYDIL® products can be used until the day of competition without any risks*. The analysis of the constituents is essential to guarantee a constant quality of TWYDIL® products. The most sensitive additives are tested in every batch, and one or two less unstable elements are tested at random in the process of verification of the production. The analyses are carried out by independent laboratories with a CE certification and authorization or by the Swiss official control office. TWYDIL® has also tested the level of preservation of its products under extreme conditions of moisture and temperature (cf. HPH 06/07). The outcome was a high stability of the active substances. During the manufacturing process, we subject every active substance to an experiment of overdosage for the purpose of achieving a more satisfactory analysis result for the finished product, while considering simultaneously the coefficient of variation related to the inaccuracy of the technique of analysis.

Renal elimination of eleutheroside realised on 6 horses having received a over-dose of TWYDIL®HIPPACAN+C (%) 100 80 60

Photo representing a probiotic culture utilized in TWYDIL® productions

Picture of yeast in optical microscopy

6. Conclusion

Before being launched on the market, a TWYDIL® product must have passed the numerous necessary analyses (at the stage of raw material or after production). The launching means also that the product has received an anti-doping certification and has passed a quality control test in terms of long lasting efficiency and stability up to best before date.❚

content

GUARANTEED

0 2

Hours after the administration time (h)

Eleutheroside’s kinetics of elimination

* except TWYDIL® HIPPACAN+C, TWYDIL® MUCOPROTECT and TWYDIL® ARTRIDIL with Harpagophytum for which a withdrawal period of 48 hours must be allowed before any competition. This guarantee is not meant to substitute the regulations in force in the country of use.

content

en cont ido GARANTIZADO

GUARANTEED

cont

GARAN

NEW PRESENTATIONS Already available in pails of 3 kg, TWYDIL® GROWING is now also available in pails of 10 kg.

TWYDIL® ELECTROLYTES+C is now available in boxes of 10 sachets and cartons of 100 sachets.

TWYDIL® ARTRIDIL is available - except in Switzerland - with Harpagophytum (withdrawal period 48 h) and without Harpagophytum (no waiting time)

OBITUARY TWYDIL® is very sad to announce the sudden and unexpected death of Mr Jim Warnhoff on 12 March 2009. For many years Jim was the manager of PAVESCO USA and also responsible for the galenics of the TWYDIL® range of products. We always had a great respect for him as a biochemist and as a friend, a man of great humanity and compassion for others. We express our condolences and deepest sympathy to his family at this time of sorrow.

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UNEQUALLED ANTIDOPING PRECAUTIONS TWYDIL® PRODUCTS CAN BE USED WITHOUT RISK UP TO THE DAY OF COMPETITION* HOW IS EACH TWYDIL® BATCH OF FEED SUPPLEMENTS TESTED:

REPRESENTATIVE SAMPLE

During the manufacturing process samples are taken during the different stages of production and mixed, following a precise method, to obtain a final sample as representative as possible of the whole batch.

LCH SEARCHES FOR CONTAMINANTS IN THE FINAL PRODUCT

LCH checks that, even at the limits of detection, the final product is free from all the 9 natural contaminants (morphine, atropine, butofenine, caffeine, theophylline, theobromine, scopolamine, methylbufotenine, dimethyltriptamine) for which vigilance must be maximum because of their possible presence in feed ingredients.

A HORSE FED WITH AN OVERAGE OF THE FINAL PRODUCT

The Laboratory of Hormonology in Marloie, Belgium, directed by Dr Philippe Delahaut, follows a strict and precise protocol quality control experiment (specific for each product). Under this protocol, the Laboratory gives a horse 3 times the normal dosage of the product for a minimum of 3 days. Samples of urine and blood are taken and sent to LCH.

LCH ANALYSES THE URINE OF THE HORSE

LCH submits the urine to a complete screening for all the prohibited substances (stimulants, analgesics, narcotics, anabolic steroids, beta-blockers, diuretics and not just the likely contaminants).

LCH ANALYSES THE BLOOD OF THE HORSE

LCH submits the blood to a complete screening for all the prohibited substances (stimulants, analgesics, narcotics, anabolic steroids, beta-blockers, diuretics and not just the likely contaminants).

LCH ESTABLISHES A TRIPLE ANTI-DOPING CONTROL CERTIFICATE Certificate available on: www.twydil.com

SAMPLES SEALED

A sealed urine sample is kept refrigerated by the Laboratory of Marloie for one year beyond the expiry date of the product and TWYDIL® keeps in Switzerland a sealed sample of the final product for the same period.

* except for TWYDIL® HIPPACAN+C, TWYDIL® MUCOPROTECT and TWYDIL® ARTRIDIL with Harpagophytum, where a waiting period of 48 hours has been established by LCH. 34

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GUARANTEED

GARANTIZADO

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MARION FRANCOIS At the beginning of the year 2009 Marion FRANCOIS, a young veterinary delegate, 25 years old, joined our team. Marion has been travelling extensively across France as a representative of our products not only for equine veterinaries, pharmacists, trainers and horse breeders, but also wholesalers in all disciplines of equestrian sports. Since her childhood, our TWYDIL® agent has been experiencing the world of horses. She taught equitation for 1 year, and during her free time, she rides or harnesses trotters for training and treats young growing foals. In addition, the world of show jumping is not unknown to her since she has participated in such competitions and still regularly visits young horses’ events. As part of her professional experience before joining our team, our graduate veterinary delegate promoted some special drugs of the Pfizer laboratory and family health among general practitioners in the North of France. Hence, Marion has acquired an advanced scientific training in anatomy, physiology, pathology and drug regulation.

CLAUDIA LUCCHESE

On 1 April 2009, Claudia Lucchese took over distribution in Germany from Mr Günter Meyer who had been the TWYDIL® distributor for more than 25 years.

From now on, her responsibility is to provide the different agents of equestrian sport with reliable scientific information about our product range. Marion visits: - veterinaries and pharmacists since TWYDIL® products are exclusively available in clinics and pharmacies - trainers or horse breeders since these field agents are directly concerned by our products and generate the demand for them - wholesalers that store our products and provide clinics and pharmacies with them and thus significantly accelerate clients’ access to the products. Marion will be glad to advise and inform you about all our products.

Claudia completed her training as an industrial business management assistant in 1990 and then worked as Export Manager and Public Relations Officer abroad for 7 years. Due to her love of dogs and horses she then moved into the pharmaceutical industry back in Germany. She worked in the sales department of a big veterinary pharmaceutical wholesaler. For many years Claudia Lucchese had her own horse and was a passionate rider. In her free time she breeds dachshunds.

NEW

ENERGY REFUELLING ! AVAILABLE THROUGH YOUR VETERINARY SURGEON

TWYDIL® VIGORADE

With its new formula enriched with pineapple extracts and prebiotics, TWYDIL® VIGORADE which also contains 10 vitamins, 7 trace elements and a yeast, Saccharomyces cerevisiae, rich in chromium, is an ideal preparation before a competition. With one sachet per day for minimum 10 days prior to the event, TWYDIL® VIGORADE naturally favours muscular detoxification induced by effort, gives additional energy and brings the horse rapidly into condition. •O fficially certified (after controls on final product but also on urine and blood of a horse having received an overage of the batch): can be used without risk up to the day of the competition. • Declared content guaranteed until expiry date.

TWYDIL is used by most of the successful trainers in the world. HEAD OFFICE PAVESCO AG

CH-4010 Basel, Switzerland Tel. (41)(61)272 23 72 Fax (41)(61)272 23 88

PAVESCO U.K. LTD.

PAVESCO EQUINE HEALTH USA, LTD

321 N, 22nd Street 116, High Road Needham, Harleston, Norfolk IP20 9LG St.Louis, MO 63166, USA Tel. (314) 421 0300 Tel. (01379) 85 28 85 Fax (314) 421 3332 Fax (01379) 85 41 78

e-mail: info@twydil.com

TWYDIL® VIGORADE,

THE PREPARATION FOR COMPETITIONS ! Prebiotics allow balancing intestinal flora while Saccharomyces cerevisiae a living, high-chromium yeast, has beneficial effects on digestion and health. In summary, TWYDIL® VIGORADE’s new formula, given before a competition helps to bring the horse to its best physiological potential without risk.

Pineapple extract

Courbe théorique d'élimination des déchets cellulaires

Theoretical curve produits of elimination of cellular waste induced by effort par l'effort ou un trauma or trauma après after supplementation with pineapple extract. supplementation en bromelain 100

INDICATIONS

To prepare a horse for a specific competition.

80 70 60

COMPOSITION

Pineapple extract Bromelain

10 vitamins, 7 trace elements, pineapple extracts, Saccharomyces cerevisiae (a highchromium yeast), and prebiotics.

Control group placebo

40 30 20 10

HOW DOES IT WORK ?

Vitamins A, E, biotin and folic acid are the 4 key vitamins to guarantee maximum physiological potential especially for the 10 last days prior to competition. Most horses do not receive enough of these vitamins which are limiting factors in maximising performances. TWYDIL® VIGORADE also contains selenium which reinforces the action of vitamin E, vitamin B12 which participates and regulates iron metabolism, and also thiamine which helps cells to get rid of toxic metabolites. Some other nutrients (D3, K, B2, B6, niacin, C and also iron) work in synergy with the other ingredients. Recent studies showed that an extract of pineapple which is included in TWYDIL® VIGORADE, accelerates the elimination of toxins induced by effort.

temps Time

FEEDING INSTRUCTIONS

Mix one sachet per day with the feed for the last 10 days prior to competition.

PRESENTATION Box of 10 sachets of 50 g. Carton of 100 sachets of 50 g.

ANTIDOPING CERTIFICATE

OFFICIALLY CERTIFIED BY LCH (AFTER ANALYSIS ON FINAL PRODUCT, URINE AND BLOOD): CAN BE USED WITHOUT RISK UP TO THE DAY OF THE COMPETITION. Official certificates available on www.twydil.com after typing the batch number which is written on sachets.

Vigorade

AVAILABLE THROUGH YOUR VETERINARY SURGEON

TWYDIL® AT INTERNATIONAL

AL FARES (DUBAI) 2008 ▼

AVEF 2008, Reims (France) ▼ Seminary for the veterinarians of the different teams at the Olympic Games in Hong Kong 2008

10th congress of equine medicine and surgery, 2007, Geneva, Switzerland ▼ Equitana 2008, Melbourne (Australia) ▼

EXHIBITIONS AND CONVENTIONS

▲ TWYDIL® SCIENTIFIC EQUINE CLINICS; Berne (Switzerland) April 2009

Veterinary workshop 2008, Chantilly, France

Trotters exhibition, 2009, Vincennes, France▼

▲ XVIII. Congress on Equine Medicine within the Equitana Equestrian Sports World Fair, 2009, Essen, Germany

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ENDURANCE HORSES IN TWYDIL速 INVESTMENT IN APPLIED RESEARCH

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MUSCULAR TROUBLES Dr Brieuc de Moffarts Director Research & Development of TWYDIL速, Switzerland

id you know that 3 to 12% of all horses (depending on their discipline and their level of use) will suffer from muscular troubles during their career? These troubles, also called myopathies, are usually effort-related. The symptoms are stiffness, excessive perspiration and stubbornness of the horses that resist running or contracting their back. The causes of these symptoms are multiple and can be classified as follows:

- Overtraining or intense effort without suitable preparation - Heat exposure - Inappropriate feeding - Inappropriate dealing with electrolytes - Genetic factors (such an accumulation of glycogen disorders in the intracellular calcium metabolism of the horses) A simultaneous combination of some of these causes may lead to the development of myopathies in horses; whereby a lame or tense horse will show the pathology 41

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much earlier than healthier horses. Dealing with myopathies requires not only a clinical diagnosis but also an investigation of markers of muscular damage in the blood of the horses. Generally, these are enzymes such as CPK (creatine phosphokinase), AST (aspartate transaminase) and LDH (lactate dehydrogenase). These enzymes are found in muscles where they are necessary for normal function; but in cases of tissue lesion, they are released from the cells into the blood stream. Their ratio after effort gives more or less a precise idea of the extent of the muscular degeneration. In cases of uncontrollable recurrent myopathies, a biopsy diagnosis can be used. The figure opposite shows the evolution of the concentration of muscular enzymes in the blood after efforts leading to a moderate myopathy. The prevention of myopathies requires a flawless management of warm-ups and effort of horses, as well as appropriate handling of any lameness and a good genetic selection. However, all these measures are only efficient in combination with a balanced feeding of the horses! A horse needs daily (in addition to the whole range of vitamins and of basic micronutrients) at least 1,000 mg vitamin E, 3.5 mg selenium and 30 g electrolytes. For recurrent myopathies, feeds rich in fibres and fats but poor in cereals should be preferred. The following table shows a recapitulation of the nutritional needs of horses suffering from that type of myopathy.❚ 42

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Daily needs of a thoroughbred in training and suffering from muscular troubles: FAT SOLUBLE VITAMINS WATER SOLUBLE VITAMINS Vitamin A IU 60000 Thiamin (B1) mg 84 Vitamin D3 IU 6000 Riboflavin (B2) mg 35 Vitamin E IU 7000 Pyridoxine (B6) mg 30 Vitamin K mg 10 B12 mg 1.5 Niacin mg 100 + Beta carotene mg 300 Pantothenic acid mg 45 Biotin (H) mg 1.5 Folic acid mg 145 MICRONUTRIENTS Choline mg 800 Ascorbic acid (C) mg 10000 Copper mg 187 Iron mg 600 Manganese mg 300 MACRONUTRIENTS Zinc mg 770 Cobalt mg 3.75 Salt (NaCl) g 30 to 50 Iodine mg 5 Magnesium g 12 Selenium mg 5 Potassium g 55

TWYDIL® PROTECT PLUS has proved reliable for many years now as a feed supplement that can help to keep oxidative stress, as well as some risk factors caused by muscular failure of horses, under control. A combination of TWYDIL® ELECTROLYTES with TWYDIL® PROTECT PLUS will be an advantage since the protective effects will be enhanced through the beneficial influence of TWYDIL® ELECTROLYTES on the hydro-ionic balance.

Muscular enzymes curve evolution after a moderate myopathy on a horse in training (IU/L) 3500 3000 2500 2000

CPK AST LDH

1500 1000 500 0 1

Post-effort hours

BIBLIOGRAPHY : Valberg AAEP, 2006 de Moffarts et al., tVJ, 2005 McKenzie et al., J.Vet.Int.Med., 2003 Harris et al., ICEEP, 1991

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INVESTIGATION OF THE NEEDS OF AGED OR DEBILITATED HORSES Dr Brieuc de Moffarts (DMV, Ms, PhD) Director Research & Development of TWYDIL速

NEW PRODUCT: TWYDIL速 BEBACK

hough the needs of horses under intensive care, aged or debilitated are not entirely known, some general rules can be outlined about their basic nutrition. As a general rule, specialists are unanimous in saying that the needs of an aged but healthy horse with a light work load are

similar to those of an adult horse, while a horse aged but in increasingly poorer condition, with dental deficiency, must receive special appropriate feeding. As a rule, special attention must be paid to the health of horses in increasingly poor condition, whether due to a primary pathology (colic, diarrhoea, trauma resulting in a confinement) or simply to age (often during retirement).

1. An energetic provision based on regular body check scores

The factors of weight loss of such horses lie in the inversion of the anabolic balance (mass increase) into a catabolic one (mass decrease), and in an increase of their needs as well as in a regression of their ingestion process. The factors considered here are: ageing, cessation of training and lack of physical activity, progressive loss of the dental function, absorption trou43

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Figure 1 3

BODY CONDITION SCORE EVALUATION (ADAPTED FROM CARROLL, C.L. AND HUNTINGTON, P.J., BODY CONDITION SCORING AND WEIGHT ESTIMATION OF HORSES) bles, sickness or even parasitism. In such cases helping the horse requires an energy provision consisting of a feed rich in fat (paradoxically easily digested by Equidae). The advantage of such a feeding is that fat, which is highly concentrated in energy, needs no mastication. This fat must be progressively supplemented; some aged horses can ingest hundreds of millilitres of oil a day. It is generally advised to use a mixture of vegetable oil (maize, linseed, colza etc.). Cereals should not be neglected, since they can support the horseâ&#x20AC;&#x2122;s digestion when given in a treated form (as flakes, or ground etc.). 44

A regular body check, in case a regular weighing is not possible, can help spot the variations in the horsesâ&#x20AC;&#x2122; condition (cf. Figure 1). A score of 2.5 to 3 will be optimal for this type of horses.

2. A diversified provision of fibres of good quality

For most horses, grass of good quality is one of the best solutions; however, for horses that cannot masticate properly, the use of moistened sugar beet pulp will be necessary to balance their cellulose deficit. Horses showing intestinal troubles can also receive mucila-

ginous fibres (cooked linseed or bran) in the form of a mash. In addition, they can benefit from a feed supplementation with prebiotics and probiotics (Figure 2) which help stabilise their digestive tract by replenishing the microflora.

3. An adapted protein ratio and quality amino acids

Seriously debilitated horses in a state of rapid weight loss will draw energy from their protein reserves (the muscles); this phenomenon will make them cachectic. In such cases, some additional protein ratio of 14-15% in their feeding is

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necessary. In addition, aged horses with muscle loss due to inactivity can receive feeds rich in amino acids of good quality that, when combined with some just little work, will help keep their musculature in good shape (cf. HPH 02/03).

4. Vitamins and minerals to sustain the horses’ organic functions

As with all horse categories, the feeding of horses in this age group must also contain the 14 vitamins, the 7 dietary minerals, and magnesium. In addition, the calcium-phosphorus ratio (1.8 to 2:1) should be constantly checked, considering that this ratio is even more important for aged horses fed with bran (which is very rich in phosphorus). Horses with soft stool or with more or less serious dental deficiency need increased electrolyte provision due to the loss of water and electrolytes with the stool, and to the poor fermentation of fibres. It should be mentioned that the latter serves as a regulator of the hydro-

ionic balances. Please, plan a dose of 20 to 30g electrolytes a day to cover the horse’s needs. TWYDIL® BEBACK is a new feed supplement adapted for the needs of aged and debilitated horses since it provides them with the whole range of vitamins, trace elements, magnesium, amino acids and probiotics and prebiotics necessary for their well-being, convalescence and regeneration.❚

BIBLIOGRAPHY Carroll CL, Huntington PJ: Body Condition Scoring and Weight Estimation of Horses. Equine Veterinary Journal. 1988, 20, 41 - 45. Ralston SL: Clinical nutrition of adult horses. Vet Clin North Am Equine Pract. 1990, 6, 339-54. Siciliano PD: Nutrition and feeding of the geriatric horse. Vet Clin North Am Equine Pract. 2002, 18, 491-508. Dunkel BM, Wilkins PA: Nutrition and the critically ill horse. Vet Clin North Am Equine Pract. 2004, 20, 107-26. Julliand V: Impact of nutrition on microflora of the gastro-intestinal tract in horses. ENUCO, 2005, 85-103.

Figure 2

THE FULL STUD PROGRAMME BROODMARES, STALLIONS TWYDIL® STUD Supplies the needs and supports the intestinal flora of broodmares and stallions. Favours milk production and colostrum quality. Aids the nutritional balance of the developing foal. TWYDIL® MINERAL COMPLEX Mineral complement, highly concentrated in 3 sources of calcium.

FOALS, YEARLINGS TWYDIL® GROWING Contributes to optimal development and to the diversification of the intestinal flora of growing horses. TWYDIL® PMC For an optimal bone formation and for a good structural development of the horse.

AVAILABLE THROUGH YOUR VETERINARY SURGEON

TWYDIL is used by most of the successful breeders in the world. ®

HEAD OFFICE PAVESCO AG

CH-4010 Basel, Switzerland Tel. (41)(61)272 23 72 Fax (41)(61)272 23 88

PAVESCO U.K. LTD.

PAVESCO EQUINE HEALTH USA, LTD

321 N, 22nd Street 116, High Road Needham, Harleston, Norfolk IP20 9LG St.Louis, MO 63166, USA Tel. (314) 421 0300 Tel. (01379) 85 28 85 Fax (314) 421 3332 Fax (01379) 85 41 78

e-mail: info@twydil.com

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INVESTIGATION INTO THE RELATION BETWEEN SPORTS PERFORMANCE PARAMETERS AND OXIDATIVE STRESS MARKERS IN TROTTERS This article is a partial account of the study presented by Dr. Emmanuelle Van Erck at the 2008 conference of the Veterinary Comparative Respiratory Society (VCRS). The University of Liège and the University of Namur received support from TWYDIL®PROBIOX for this study.

QUESTIONS UNDER INVESTIGATION

Can the oxidative stress output of a racehorse be related to its performance parameters? Do horses affected by metabolic troubles of muscular origin show modified oxidative stress parameters? In their attempt to answer these questions, the scientists collected blood samples from more than 100 trotters during training sessions and after standardised tests of effort. The outcome of the study was that: 1.There is a relation between the factors that affect a horse’s sports performance and some of the oxidative stress markers. 2.Horses affected by muscular stiffness were less efficient and their oxidant profile was modified.

STATE OF THE KNOWLEDGE BEFORE THE STUDY

As was the case with several previous reports in this magazine (cf.

HPH 2002/2003 ; HPH 2004/2005, HPH 2006/2007), it is now confirmed that intensive exercise produces oxidative stress in horses (cf. : Kirschvink et al., 2008). Even though it has been demonstrated that a deficiency in antioxidants leads irrevocably to a decrease of performance and to malfunctions of the immune and/ or inflammatory systems of horses, the connection between oxidative stress and the performance parameters of horses has been studied only during the different phases of training (de Moffarts et. al., 2004). Various studies indicate that a feed

supplement corresponding to the age, the discipline and sometimes even to the sex of the horses is efficient and beneficial to their prooxidant-antioxidant balance (HPH 2002/2003 and HPH 2004/2005). Concerning the measurement of the key performance parameters – and though this is not easy to realise in practice – three variables must be analysed: 1. Changes in oxygen consumption which indicate how much oxygen a horse needs per kg (ml oxygen per kg). This consumption has a linear progression and reaches a plateau at the maximal 47

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consumption (cf. opposite figure). It is important to know that, unlike a human who can run 100 m almost in a state of apnea (i.e. without oxygen consumption during the sprint), a horse must cover 80% of its energy requirements with oxygen in order to run 1,600 m. The key parameter to consider is the speed at which the horse reaches its plateau of consumption (VO2max). Though many recent studies show that a measurement on the field may be an option, only the standardised test of effort on a treadmill is currently reliable. 2. Changes in the heart rate inform us about the level of effort of the horse, just like the revolution counter of a car tells us about the motor rating. The development of this parameter is linear and reaches a final plateau of maximal heart rate (cf. figure). There are three different ways to measure a horse’s performance by using its heart rate: the measure of the V180 (the speed at which the horse runs when it reaches a heart rate of 180), the measure of V200 (the speed at which the horse runs when it reaches a heart rate of 200), and the measure of Vfcmax (the speed at which the horse runs when it reaches its plateau). These parameters are reliable and can be used on the field. 3. The changes in lactic acid concentration in the horse’s blood, which is related to the anaerobic metabolism. As a matter of fact, during intense effort a horse needs a lot of energy that it immediately obtains without any oxygen consumption. This causes an accumulation of lactic acid in its muscle cells and in its blood. The evolution of this concentration is exponential (cf. figure). The key parameter is the speed at which the horse runs when the lactic acid concentration in its blood reaches 48

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4 mmol/L (Vla4). This speed may vary depending on the breed or on the training condition of the horse. This parameter is reliable and can be used on the field.

SOD (IU.Hb-1) 2400

r = 0.87 p < 0.05

2200 2000 1800 1600 1400

A STUDY ON 100 TROTTERS

1200 1000

The object of the study was to examine a large number of blood parameters and relate them to the key performance parameters. The whole study was conducted under standardised conditions on the same trotting track (racetrack of Wallonia) and in the same laboratory (TWYDIL®-PROBIOX®).

800 600 105

110

115

120

125

130

135

140

145

150

VO2max (mL.Kg-1.min-1)

A . Presentation

This study was based on blood sampling more than 100 horses from different horse trainers. All the horses were active and trained, and were presented for a sport test evaluation. It must be mentioned here that these horses were not necessarily part of a TWYDIL® supplementation programme. The markers of oxidative stress (i.e. the hydrophilic oxidant capacities ACW and the lipophilic ones ACL, the lipid peroxides POOL and the glutathione peroxidase GPx), the performance parameters (V200 and Vla4) and the markers of muscular damage (CPK and LDH) were investigated identically for all horses: at rest, during the standardised test effort and after the test.

B. Results 1. performance versus oxidative stress. The comparison of the parameters confirmed, as was already shown in HPH 06/07, the correlation between the ACL and the POOL. But even more surprisingly, significant correlations were found between the Vla4 and the POOL as well as between the V200 and

(VO2 ml/Kg/min)

200

trained

180 160 140

untrained

120

100

80 60 40 20 5

VO2 max

speed (Km/h)

HR (bit/min)

Maximum frequency

250 200 150 100 0

Speed (Km/h)

V180 V200

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Lactic acid (ᵑmol/L )

the ACL (cf. figure). In other words, without any obvious direct causal relationship, the horses with the worst sports performances had higher POOL values and less antioxidant reserves (ACL).

20 15 10

The interesting question is, on the one hand, how far the relation between the POOL and the Vla4 is relevant, knowing that the Vla4 reflects the ability of a cell to use energy; and, on the other hand, how far the relation between the ACL and the V200 is relevant, with the V200 being a marker of the level of use of the horse. It is still premature to draw any conclusion at this stage, and a reason why further experiments should be conducted in this direction.

5 0

VLa4

speed (Km/h)

trouble has a significantly higher oxidative stress compared to another horse without the same trouble.

ACL ( mol eq. Trolox/mL) 35

These results indicate that the parameters of oxygen consumption and the parameters of sports performance of a horse are interconnected, even though at various levels; and this tells us about the psychological state of the horse.

R = 0,36 p = 0.001

30 25 20 15 10 5 0

V200 (Km/h)

POOL (µmol/g) 120

Horses without significant CPK increase

Horses with significant CPK increase

2. muscular damage versus oxidative stress Some of the horses in the tests had initially normal values for their markers of muscular damage (CPK and LDH at rest), but these values were significantly higher than those of the rest of horses in the test. The horses with the higher values achieved a considerably poorer performance than the other horses during the standardised tests and there was a higher increase in their markers of muscular damage than for the other horses. The results indicated also that these horses with the muscular troubles had higher intrinsic POOL and a less important GPx activity that the rest of the test population. These results confirm that a horse suffering from muscular

GENERAL CONCLUSIONS

This study demonstrated that: There is a confirmed relation between the parameters of sports performance of horses and some of their oxidative stress markers. Horses affected by muscular stiffness were less efficient and their oxidant profile was modified.❚ 49

HPH

08-09

REFERENCES: de Moffarts B., Kirschvink N., Art T., Pincemail J., Michaux C., Cayeux K., Defraigne J-O., Lekeux P.Impact of training and exercise intensity on blood antioxidant markers in healthy standardbred Horses. ECEP 1 (3) 2004 211-220. Kirschvink N., de Moffarts B., Lekeux P. The oxidant/antioxidant equilibrium in horses. tVJ 177 (2008) 178–191. Van Erck E., de Moffarts B., Lekeux P., Kirschvink N. : Performance parameters but not oxidant-antioxidant equilibrium markers are modified in Standardbred horses with lower airway disease. In proceedings: 26th Annual Veterinary Comparative Respiratory Society Symposium, 2008, Oklahoma City, USA.

QUESTIONS TO DR BRIEUC DE MOFFARTS (Director Research & Development of TWYDIL®) HPH : We hear more and more about oxidative stress; is this anything new to TWYDIL® ? BdM : Even though this subject is new to the general public, TWYDIL® customers have been benefiting for more than 10 years from constant developments (most recent discoveries) in this regard. As a matter of fact, as you know, our laboratories have been collaborating with numerous partner laboratories and with Professor Montagnier (Nobel Prize for Medicine 2008) and this cooperation has supported TWYDIL® in the continuous improvement of the TWYDIL® PROTECT PLUS formula. Excellent scientific and field results are the outcome as regards the management of muscular problems similar to those mentioned in the article above. HPH : Some competitors of TWYDIL® boast that they have reinvented horse feeding and horse feeding supplementation by using the determination of the blood markers of oxidative stress of horses. What do you think about this ? BdM : We should bear in mind that oxidative stress is just one side of the problem. Our policy at TWYDIL® is to be in the lead of scientific research in different areas including oxidative stress but also enzymatic stress, inflammatory stress etc. That is what helps us to achieve a gradual development of the TWYDIL® supplements from a practical point of view.

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TWYDIL® PROTECT PLUS

Balanced formula containing vitamin C, E and ß-carotene, L-carnitine, glutathione precursors, branched amino-acids and more essential micronutrients. TWYDIL® PROTECT PLUS is designed to ensure a constantly optimal metabolic balance and to help maintain the muscle tone of horses. •O fficially certified (after controls on final product but also on urine and blood of a horse having received an overage of the batch): can be used without risk up to the day of the competition. • Declared content guaranteed until expiry date.

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THE TWYDIL ® RANGE OF PRODUCTS

Complete documentation on 1. COMPETITION LINE TWYDIL® ARTRIDIL With its unique formulation to help support healthy joints. With or without Harpagophytum procumbens. - WITH HARPAGOPHYTUM : WAITING TIME BEFORE COMPETITION 48 HOURS - W ITHOUT HARPAGOPHYTUM : OFFICIALLY CERTIFIED BY LCH (FOLLOWING CONTROLS ON FINAL PRODUCT, URINE AND BLOOD) CAN BE USED WITHOUT RISK UP TO THE DAY OF THE COMPETITION  DECLARED CONTENT GUARANTEED UNTIL EXPIRY DATE

NEW

TWYDIL® BEBACK TWYDIL® BEBACK helps to stabilize the intestinal flora and helps horses that have lost condition. - E ACH BATCH OFFICIALLY CERTIFIED BY LCH (FOLLOWING CONTROLS ON FINAL PRODUCT, URINE AND BLOOD) CAN BE USED WITHOUT RISK UP TO THE DAY OF THE COMPETITION D ECLARED CONTENT GUARANTEED UNTIL EXPIRY DATE

TWYDIL® CALMIN A scientific blend of selected vitamins, fructo-oligo-saccharides, trace elements and tryptophan to help manage excitability and stress, and to optimise digestive and muscular well being of the horse. - E ACH BATCH OFFICIALLY CERTIFIED BY LCH (FOLLOWING CONTROLS ON FINAL PRODUCT, URINE AND BLOOD) CAN BE USED WITHOUT RISK UP TO THE DAY OF THE COMPETITION D ECLARED CONTENT GUARANTEED UNTIL EXPIRY DATE

TWYDIL® COMPETITION An ideal daily supplement, highly concentrated (14 vitamins, 7 trace elements, 4 amino acids and magnesium) which covers the physiological needs of sport horses. - E ACH BATCH OFFICIALLY CERTIFIED BY LCH (FOLLOWING CONTROLS ON FINAL PRODUCT, URINE AND BLOOD) CAN BE USED WITHOUT RISK UP TO THE DAY OF THE COMPETITION D ECLARED CONTENT GUARANTEED UNTIL EXPIRY DATE 52

THE TWYDIL ® RANGE OF PRODUCTS

n w w w. t w y d i l . c o m TWYDIL® RACING

An ideal daily supplement, highly concentrated (14 vitamins, 7 trace elements, 4 amino acids and magnesium) which covers the physiological needs of high performance horses. - E ACH BATCH OFFICIALLY CERTIFIED BY LCH (FOLLOWING CONTROLS ON FINAL PRODUCT, URINE AND BLOOD) CAN BE USED WITHOUT RISK UP TO THE DAY OF THE COMPETITION D ECLARED CONTENT GUARANTEED UNTIL EXPIRY DATE

TWYDIL® ELECTROLYTES In pails

For the compensation of electrolyte loss after heavy sweating. - E ACH BATCH OFFICIALLY CERTIFIED BY LCH (FOLLOWING CONTROLS ON FINAL PRODUCT, URINE AND BLOOD) CAN BE USED WITHOUT RISK UP TO THE DAY OF THE COMPETITION D ECLARED CONTENT GUARANTEED UNTIL EXPIRY DATE

TWYDIL® ELECTROLYTES In mouth syringes

Oral paste for the compensation of electrolyte and vitamin C losses after heavy sweating. - E ACH BATCH OFFICIALLY CERTIFIED BY LCH (FOLLOWING CONTROLS ON FINAL PRODUCT, URINE AND BLOOD) CAN BE USED WITHOUT RISK UP TO THE DAY OF THE COMPETITION D ECLARED CONTENT GUARANTEED UNTIL EXPIRY DATE

TWYDIL

ELECTROLYTES+C

Compensation of electrolytes, vitamins and trace elements loss. - E ACH BATCH OFFICIALLY CERTIFIED BY LCH (FOLLOWING CONTROLS ON FINAL PRODUCT, URINE AND BLOOD) CAN BE USED WITHOUT RISK UP TO THE DAY OF THE COMPETITION D ECLARED CONTENT GUARANTEED UNTIL EXPIRY DATE

TWYDIL® HEMATINIC In mouth syringes and in bottles

Formulation (either in paste or liquid form) with key vitamins and trace elements to support haematological parameters. - E ACH BATCH OFFICIALLY CERTIFIED BY LCH (FOLLOWING CONTROLS ON FINAL PRODUCT, URINE AND BLOOD) CAN BE USED WITHOUT RISK UP TO THE DAY OF THE COMPETITION D ECLARED CONTENT GUARANTEED UNTIL EXPIRY DATE

C o m p l e t e d o c u m e n t a t i o n o n w w w. t w y d i l . c o m

TWYDIL® HEMOPAR Aids appetite. Helps maintain good digestive function. - E ACH BATCH OFFICIALLY CERTIFIED BY LCH (FOLLOWING CONTROLS ON FINAL PRODUCT, URINE AND BLOOD) CAN BE USED WITHOUT RISK UP TO THE DAY OF THE COMPETITION