High Performance Horses 2008/2009

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

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SUMMARY

❚ A WORD FROM THE PRESIDENT 3

❚ HERPES VIRUSES AND SUBCLINICAL INFECTIONS OF HORSES IN TRAINING 4

❚ IN VITRO AND EX VIVO EVALUATION OF THE VIRUCIDAL AND ANTIVIRAL

CAPACITIES OF A BIOFLAVONOID AGAINST EQUINE HERPES VIRUS TYPE 1 11

❚ AIDAN O’BRIEN : THE MASTER OF BALLYDOYLE 16

❚ EFFECTS OF DIETARY EXTRACTS ON CHONDROCYTE PROLIFERATION,

A SPRINGBOARD TO TISSUE RECONSTRUCTION 20

❚ TWYDIL® AROUND THE WORLD 26

❚ STABILITY AND QUALITY 28

❚ NEW PRESENTATIONS 31

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❚ NEW TWYDIL® DISTRIBUTORS 33

❚ TWYDIL® AT INTERNATIONAL EXHIBITIONS AND CONVENTIONS 36

❚ ENDURANCE HORSES IN TWYDIL®

INVESTMENT IN APPLIED RESEARCH 38

❚ MUSCULAR TROUBLES 39

❚ INVESTIGATION OF THE NEEDS OF AGED OR DEBILITATED HORSES 41

❚ THE FULL STUD PROGRAMME 44

❚ INVESTIGATION INTO THE RELATION BETWEEN SPORTS PERFORMANCE

PARAMETERS AND OXIDATIVE STRESS MARKERS IN TROTTERS 45

❚ THE TWYDIL® RANGE OF PRODUCTS 50

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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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Following 5 years of the most advanced scientific investigation, we are now offering our customers this product as the weapon that they did not had before and were looking for.

Valère HENRYPresident

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Dr. Guillaume Fortier, DMV, MSc.Faculty of Veterinary Medicine of Liège, Belgium

Laboratory Frank Duncombe, Caen, France

HERPES VIRUSES AND SUBCLINICAL INFECTIONS OF HORSES IN TRAINING

INTRODUCTION

The causes of poor per-formance among athlete horses – regardless of the discipline – have been un-

der investigation with a number of research programmes for more than 25 years. Such research pro-grammes have focussed mainly on the musculo-skeletal and/or the cardiorespiratory systems or on identifying and quantifying the markers of haematological disor-ders caused by the possibly harm-ful effects of training.

So, quite naturally, the list of sub-clinical diseases (i.e. without a clear medical expression that can be identified by clinical exami-nation) 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 con-ditions (e.g. coughing, nose dis-charge, bleeding, lameness …).

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

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 ma-jor cause of poor performances among competition horses.

It has long been known that the upper respiratory tract is a favou-rite 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 hu-mans). These infectious diseases frequently affect horses and are easy to diagnose since they often show significant clinical symp-toms ranging sometimes up to flu syndromes (nasal discharge, hy-perthermia, exhaustion, excessive eye watering …). Both the upper or lower respiratory tracts have been studied in order to improve

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our understanding of a “devastat-ing” syndrome in the performance of horses: airway inflammation. Numerous conferences, conven-tions and books have already been dedicated to these affections in an attempt to provide a defini-tion 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 pre-cise determination of the role of viruses, notably in the trachea and the main bronchi that can be ac-cessed by endoscopy and tracheal lavage (TL). Thus, the syndrome of tracheal inflammation is named without any knowledge of its real causes: allergens, bacteria, virus-es… Since these air passages are a perfect anatomical and physi-ological “crossroad”, the coexis-tence of many favourable factors could be an explanatory option.

HERPESVIRUSES AND RESPIRATORY TRACT DISEASESA horse can host 5 major herpes-viruses of which 4 show a more or less intense “tropism” for the respiratory system (Table 1). Type 1 and type 4 equine alphaherpesvi-ruses (EHV-1 and EHV-4) account worldwide for episodes of more or less severe respiratory and abor-tive 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 stud-ied 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 chick-enpox from youth years), the virus may hide in the local ganglionic structures and be reactivated when favoured by various stress situa-

Natural host Designation Family Related to Other designation Reference (example)Horse Equine herpesvirus Alphaherpesvirinae / Equine abortive virus Allen and Bryans, 1986 (EHV-1) EHV-2 Gammaherpesvirinae / Studdert et al., 1996 EHV-3 Alphaherpesvirinae / Coital exanthema virus Hartley et al., 1999 EHVE-4 Alphaherpesvirinae / Equine rhinopneumonia virus Patel and Heldens, 2005 Telford et al., 1995 EHV-5 Gammaherpesvirinae / Donkey EHV-6 Alphaherpesvirinae HVE-3 Type 1 donkey herpesvirus Browning et al., 1988 EHV-7 Gammaherpesvirinae HVE-2 et 5 Type 2 donkey herpesvirus Bell et al., 2008 EHV-8 Alphaherpesvirinae HVE-1 Type 3 donkey herpesvirus Crabb et al., 1991 AHV-4 Gammaherpesvirinae HVE-2 et 5 Type 4 donkey herpesvirus Kleiboeker et al., 2002 AHV-5 Gammaherpesvirinae HVE-2 et 5 Kleiboeker et al., 2002Gazelle and zebra EHV-9 Alphaherpesvirinae HVE-1 Borchers et al. 2005 Borchers et al. 2006

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

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tion. Horses are no exception to that herpetic rule and once a ma-jority of horses have been infected during their raising by many of these viruses (EHV-1 to 5), they can suffer from reactivation in dif-ferent forms at different moments of their lifes.

Just like an athlete, a horse is ex-posed to various forms of stress in connection with transportation, exercise, competition and manage-ment of relaxation. The investiga-tion 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 ef-fects, by using direct “markers” like the detection of their genome through gene amplification (PCR) or indirect “markers” (cellular ab-normalities in fluids from respira-tory lavages).

HERPESVIRUSES AND RESPIRATORY SAMPLESQuestions like “how to take a sample” or “which sample to

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 rhi-nopneumonia, antigenic variabil-ity of the “wild” strains, reactiva-tion 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:

- The veterinary surgeon examines a horse that has already been sick for some days and consequently shows a decreasing quantity of detectable viruses.

- The horse has been vaccinated against this type of virus and its viral charge is accordingly low and temporary.

- The disease has just started to develop in the horse, so that the horse has not yet reached the acute viral load (the more con-tagious horses !).

Facing the diversity of this fam-ily of viruses in horses and their implication in various equine dis-eases, detection methods could be developed to identify a common gene shared by mammal herpes-viruses affecting a specific region of the genome (Leon et al., 2008). Such methods are very useful in current practice in equine special-ized laboratories, because they provide practitioners a quick re-sult about the presence of one of the herpesviruses (1, 2, 4 or 5) be-fore 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 cor-relation 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 seroconver-sion with seroneutralisation meth-ods 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 cau-tious about diagnosing viral dis-eases 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 explo-ration of horses with or without clinical signs (Sutton et al., 1998; Fortier et al., 2009).

To sum up, nasal-pharyngeal sam-pling remains the standard for any situation of contamination present-ing an unambiguous clinical flu syndrome. It is easy and quick to carry out and must be stored cool to be sent to specialized labora-tories in the best conditions with clear history of the suspected case.

When clinical situation is less evi-dent and/or a lavage of the respira-tory 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

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).

Tracheal lavages (TL) Bronchoalveolar lavages (BAL)

CTL (n=37) PAT (n=259) p-value CTL (n=25) PAT (n=387) p-valueEHV-1 0 [0%] 4 [2%] b > 0.05 0 [0%] 4 [1%] > 0.05EHV-2 4 [11%] 85 [33%] 0.006 * 9 [36%] 82 [21%] > 0.05EHV-4 1 [3%] 17 [7%] > 0.05 1 [4%] 16 [4%] > 0.05EHV-5 0 [0%] 8 [3%] > 0.05 0 [0%] 16 [4%] > 0.05

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).

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

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fluids of more than 700 horses by comparing a group of healthy hors-es to a population of poor perform-ers. It shows that, contrary to bron-choalveolar fluids, tracheal lavages of the poor performance horses pre-sented 3 times more EHV-2 viruses compared to the control group. By the way, this virus is the most com-mon of the pathologic groups dem-onstrated 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 cy-tological profile of the lavages?

VIRAL DISEASES AND INFLAMMATION OF RESPIRATORY TRACTSThe first replication sites called “initial” sites for these viruses are the epithelial cells of the nasal-pharynx. 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 evi-dent just as for the respiratory ex-ploration 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 nor-mal to find these cells in low per-centage in the tracheal lavage.

Once the trachea has been expe-riencing 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 second-ary inflammation and for the length of time this may take in horses suf-fering from inflammation of the upper or lower respiratory tracts (Moore et al., 1996). In 1992, Wil-loughby 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 al-teration of the respiratory mucous membrane and to an excessive sec-ondary inflammatory response.

This inflammatory response to a respiratory viral infection involv-ing EHV-1 has been studied more deeply by Kydd et al. in 1992.❚

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They demonstrated the significant rush of neutrophil polynuclear cells (PNN) as well as cellular profiles in the bronchoalveolar la-vages (BAL) which confirm simi-lar descriptions (Moore et al., 1996) in horses infected with in-flammatory diseases of the respi-ratory tracts. This PNN increase has been described by Sutton et al. with a flu virus infection (equine influenza virus). These authors describe modifications of inflam-matory profiles of the BAL over a more or less long period of time compared to the duration of infec-tions with EHV-1 (up to 21 days post-challenge).

Considering that these inflammato-ry diseases of the respiratory tract are suspected only in young horses and that their clinical manifesta-tions are often unclear, the detec-tion 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 respi-ratory tract.

We have been working for many years in the laboratory on the defi-nition of reliable cytological pro-files which would be representative of viral invasions of the respiratory system of poor performance hors-es by comparing the collected data

with results of systematic PCR on samples from routine examina-tions or research protocols.

The analytical results of a signifi-cant number of fluid samples tend to follow the indications of some external works (Couétil et al., 2007). Subclinical viral patholo-gies (which are mainly herpesvi-rus infections) may be responsible for the cases of inflammatory dis-eases of the upper or lower respi-ratory tract and of the poor sport performance observed. Future ex-perimental reproduction of these pathologies caused by the EHV-2 virus should help provide some an-swers in this regard.

CONCLUSIONSThe equine species is one of the most exposed to the herpersviri-dae family viruses among mam-mals. Just considering their eco-nomic consequences, both viruses EHV-1 and EHV-4 heavily affect animal farming by the high num-ber of infected studs as well as by the types of diseases and com-plications that they cause in their most frequent expression which is the subclinical form. Such a disas-trous 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 hypoth-esis 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 expres-sion, the use of nasal-pharynx swabs still represents just the “vis-ible” part of the iceberg for the ex-ploration of subclinical infections that can be detected only by en-doscopies and exhaustive analysis of the lavages.

The subclinical forms caused by these viruses are usually due to latency and reactivation phenom-ena – viral excretion, character-istics and biological features of herpesviruses. Molecular biology and increased knowledge about equine immunology supported significantly the progress made by researchers and industrial institu-tions 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 spe-cies (based on the presentation of the antigens and the administra-tion mode).

Molecular biology and the prog-ress made since the first meth-ods of gene amplification applied to the detection of these viruses

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.

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have produced a fine-tuning of the field diagnosis of these diseases, a quicker intervention of veterinar-ians in the stud farms or training centers as well as a better under-standing 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 herpes-viruses. 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.❚

REFERENCESCouétil, L.L., Hoffman, A.M., Hodgson, J., Buechner-Maxwell, V., Viel, L., Wood, J.L., Lavoie, J.P., 2007. Inflammatory air-way disease of horses. J.Vet.Intern.Med., 21, 356-361.

Davison, A.J., Eberle, R., Ehlers, B., Hay-ward, 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. Herpesvi-ruses in respiratory liquids of horses: Puta-tive 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 leuco-cytes to the equine lung after EHV-1 infec-tion. 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 con-sensus 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., Da-vis, M.S., Furr, M.O., Dubovi, E.J., Mayo, J.A., 1998. Neonatal equine herpesvirus type 1 infection on a thoroughbred breed-ing 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 herpesvi-rus 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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Alexandra Scipioni, Tatiana Art, Lorène Dams, Brieuc de Moffarts, Pierre Lekeux and Etienne Thiry

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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

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

The 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 Vet-erinary Medicine of Liège). Its aim was the evaluation of the virucidal and antiviral capacities of a biofla-vonoid.This study was partially funded by TWYDIL®.

This study showed:In vitro : : a significant virucidal ac-tivity 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 administra-tion of this bioflavonoid, biological fluids showed significant virucidal activity.

INTRODUCTIONRelations between viral patholo-gies, 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 informa-tion about these infections, see the previous article, p 4 - 9) where they were dealt with in part. We will re-view briefly the implications of her-pes virus type 1 (EHV-1).

EHV-1 (Alphaherpesvirinae fam-ily) is a major pathogenic virus in horses. It exists endemically in the world and causes abortion and my-elo-encephalopathy; as for EHV-4,

it causes rhinopneumonia. Though vaccines (and their combined pro-tection 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 encourag-ing, but, unfortunately, results of tests ex vivo were disappointing. Accordingly, the research for chem-icals able to help fight against viral infections in horses as well as pre-

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Figure 1 : Cell culture (RK13) infected by EHV-1 on decreased doses (left to right) (determi-nation by DICC50 method).

Figure 2 : viral count reduction after in vitro contact of the bioflavonoid with EHV-1

DIC

C50

vent them became more and more intensive. So far, various plant extracts rich in flavonoids have shown some activity against hu-man viruses (HIV, herpes etc.).

It should be remembered here that, whether clinical or subclinical, horse viral infections cause enor-mous economic losses that can even be catastrophic for the econ-omy of a whole country (AQUIS report 2009).

PRESENTATION OF THE STUDYThe study took place in two phas-es: the first in vitro and the second ex vivo.

1. Study in vitroThe activity of flavonoids com-pound used on formulation of feed supplements was tested. Together with its effect on viral multiplica-tion, its virucidal effect against EHV-1 was evaluated for an RK13 cell lineage based on a reading of the decrease of the viral titre.This work was done in several stages: - Producing a suspension of the

compound using various sol-vents;

- Measuring the cytotoxicity of the solvents and of the compounds dissolved in an RK13 cell cul-ture;

- Evaluation of the virucid-al effect of the compounds. The principle was a measurment of the possible virucidal effect of the suspensions of flavonoids on EHV-1 in an RK13 cell culture. For this purpose, the sample was added to the viral suspension be-

fore infecting the cellular carpet. Different durations of contact (10 sec. to 30 min) were tested. The viral titre was measured af-ter 3 days incubation using the method of infectious dose of the cells responsible for 50% lysis of the cell supports (DICC50).

- Evaluation of the compound’s effect on viral multiplication:

The objective was to measure the compound’s effect on the multi-plication 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).

2. Study ex vivoIn the second phase, the study

aimed at showing a possible effect of the bioflavonoid in the plasma and/or the bronchoalveolar lavage (BAL) after 7 days oral adminis-tration of the flavonoid to 6 horses kept under standardized condi-tions.

In order to avoid interferences be-tween the effect of the supplement and the reaction of potential anti-bodies 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 experi-ments done with EHV-1 were vali-dated 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 bronchoalveo-lar lavages were carried out at three different times (D-1, D0 and D+7) and their virucidal effect tested.

The sampling was done following this plan:

- D-1: blood and BAL samples as control samples

- D0: blood sample (4 hours after the first administration)

- D+7: blood and BAL samples (4 hours after the last administra-tion)

0

8,00E+08

1,20E+09

1,60E+09

DICC

50

5000

CONTROL BIOFLAVONOIDE

Significant reduction of99.9998%

Figure 2 : viral count reduction after in vitro contact of thebioflavonoid with EHV-1

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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.

MOI 0,01

10x Dilution of the

bioflavonoid

100x Dilution of the

bioflavonoid

1000x Dilution of

the bioflavonoid

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

RESULTS OF THE STUDY

1. Study in vitroThe 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 com-pound on the EHV-1 proved sig-nificant! Just 10 seconds contact with a concentration of 0.125µg/ml was enough to reduce the viral titre by almost 100%. Figure 2 il-lustrates 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 reac-tion of the EHV-1 on RK13 cul-ture cells when the bioflavonoid is added to the culture. However, the virucidal effect could interfere with the effect on the process of viral multiplication.

These results indicate clearly a virucidal effect and suggest an ef-fect of the bioflavonoid on the viral multiplication process. Future ad-ditional studies must still investi-gate the latter in order to describe the corresponding behaviour.

2. Study ex vivoLet 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 com-pared 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 administra-tion of the bioflavonoid). Howev-er, on D+7, a significant decrease of the viral titre was observed in the plasma as well as in the bron-choalveolar lavage.

In this phase of the study, the use of SuHV-1 helped us avoid a bias that could result from the presence

0

4,00E+08

8,00E+08

1,20E+09

DICC

50

day - 1 After 7 days of oral administration ofthe bioflavonoid

Significant reductionof 56%

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

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

DIC

C50

0

4,00E+08

8,00E+08

1,20E+09

1,60E+09

DICC

50

Significant reductionof 50%

day - 1 day 0 after 7 days of oral administration ofthe bioflavonoid

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

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

DIC

C50

of antibodies against EHV-1 in the horse. But another source of possible error is the presence of the EHV-1 vi-rus itself! In this case, the virus could lyse the RK13 cells in its environ-ment 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 ef-fect 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 viru-cidal activity observed is due to the presence of the bioflavonoid’s metabo-lites in the blood, since they had not been specifically looked for during the study. However, the activity of the bio-logical fluids in the presence of EHV-1 following oral administration was ob-served.

Control Bioflavonoid

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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 mole-cule, 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 ex-ample, 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.

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 Wood-worth, 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 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 herpes-virus 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.

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.

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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 ge-nius 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 long-held status of the world’s leading training centre, had trained six Irish Derby winners during a tru-ly 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

great sporting environment in Kil-legney in County Wexford. The main talk down there in his youth would have centred around hurl-ing but his dad Denis, a farmer by profession, had a great interest in horses and rode many a point-to-point winner in his younger days. Hence, Aidan grew up with horses and after leaving school a little ear-ly 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 tute-lage of Bolger, the young O’Brien took the first steps towards becom-ing a legend in his own young life-time.

He quickly became the amateur rider for the stable and assistant to one of Ireland’s leading train-ers. Jim Bolger believes in hard work and anybody involved with him would have to do likewise. In Aidan O’Brien he had a very will-ing pupil.

The pupil learned fast. He rode his first winner when Galacto Boy won at Punchestown in January, 1989 and he became Champion Ama-teur Rider in the 1993/ 94 season. By now he was already married to Anne-Marie Crowley, daughter of famed Piltown trainer Joe Crow-ley and herself the leading ama-teur rider. A new dynasty was in the building process! Anne Marie

had created history when becom-ing 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

Michael FORTUNE

AIDAN O’BRIEN: THE MASTER OF BALLYDOYLE

FAME AND GLORY

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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 Cham-pion 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 win-ners.

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 particu-lar week.

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

Breeders’ Stakes at The Curragh, including the winner and runner-up. That day at the Curragh he had a total of four winners.

AIDAN O’BRIEN MASTER OF BALLYDOYLE 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 in-terested in this young man still in his mid twenties.

It seemed a mere natural progres-sion when Aidan O’Brien moved to Ballydoyle. It was an incredible

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 dur-ing 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 ar-rival 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 dou-ble as he also won the 1,000 with Classic Park.

When Urubande won the Sun Al-liance Hurdle at Cheltenham in 1996 the youthful O’Brien was turfed out of the Winner’s Enclo-sure by officials who just didn’t recognise him. Surely nobody so young could be a Cheltenham win-ning trainer!

Aidan O’Brien gives the thumbs up to his son Jo-seph after he had recorded his first victory aboard Jo-hann Zoffany in the Kilma-cud Handicap at Leopards-town on May 28th, 2009.

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

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However, the whole world knew who he was by 1998 when not only did Istabraq win the Cham-pion 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 Phoe-nix Stakes, etc. etc.

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 Guin-eas 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 win-ning 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!

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.

AIDAN O’BRIEN – THE MAN But despite it all, Aidan O’Brien remains the same modest, self ef-facing 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 ac-cepting 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 fam-ily man. One of the most familiar sights on big race days is that of Aidan O’Brien on his mobile im-mediately after one of his horses has raced to Group 1 glory. Invari-ably 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, unassum-ing man – whether you meet him in normal circumstances or in the af-termath of a massive Group 1 suc-cess. 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 in-terviewer for RTE Radio’s Satur-day Sport programme and it was to become the first of hundreds of in-terviews we shared over the years.But I’ll never forget that first one.

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We commenced it just alongside the 1st Post in the Winner’s En-closure 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 an-swers were monosyllabic!But he learned fast. Yes, for about a year we would walk a few yards during each interview but the an-swers 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 oblig-ing, no demands are too much un-less it clashes with the welfare of

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 po-tential Champion Sires and Dams of the future. In that regard his re-cord has been quite phenomenal. – and getting more phenomenal by the month.

THE FAMILY O’BRIEN It must be virtually unique for two Champion Trainers to be-come husband and wife and it was probably inevitable that their kids should inherit their talents. Ann Marie had been the Lead-ing Lady Rider while Aidan was Champion Amateur and then they won the National Hunt Train-ers’ 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 rac-ing 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 win-ning 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.

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 expe-rience for one so young in hav-ing 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

the kids have grown up with the horses and all four have aspira-tions 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 sum-mer 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 rid-ing 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.❚

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INTRODUCTION

Osteoarticular pathologies represent a major cause of lameness in the equine species. They are also the

first cause of performance regres-sion. These diseases are charac-terised 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 ex-tracellular matrix is a supporting structure produced by the chondro-cytes in order to furnish the carti-lage with the resistance properties needed to absorb shocks occurring during locomotion. The pathologic process that leads to cartilage le-sions is complex (Fig. 1). Many factors are accordingly involved in this destruction of the carti-lage. 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.

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

are involved in the triggering of the cascade leading to inflamma-tory reaction and to enzymatic stress [1].

Actually inflammatory reaction amplifies the process and the in-creasing cascade leads to a higher activity of the proteinases (nota-bly the matrix metalloproteinases) which damage the extracellular

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)

EFFECTS OF DIETARY EXTRACTS ON CHONDROCYTE PROLIFERATION,

A SPRINGBOARD TO TISSUE RECONSTRUCTION

Inflammatorymediators

IL-1-beta, NFKB…

ReactiveOxygenSpeciesMatrix metalloproteinases:

increased activity

Injuries of the DNA, ofthe membrane lipids andthe proteins of thechondrocytes

Cellular ageing

Damaging of theextracellularmatrix

Cartilage damage

Figure 1:

Chondrocytes

Extracellularmatrix

Structure of thearticular cartilage

Chondrocytes

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matrix around the chondrocytes [3]. This phenomenon is called “enzymatic stress”.

The result of this chain of occur-rences 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 adminis-tration 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 sig-nificant side effects.

Another important aspect is the reconstruction of the cartilage af-ter the pathologic stage. The carti-lage 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 di-minishing 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, inflamma-tory 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-inflam-matory effect with minimal side effects on the one hand; on the other hand, few of them consid-ered 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 RESEARCHThe aim of this research is to inves-tigate the effect of dietary extracts

on the proliferation of equine chon-drocytes in vitro.

A sample of articular cartilage has been taken from horse limbs from a slaughterhouse. The fetlock articu-lations 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 culti-vated for 7 days in 12-well plates in a growth medium with or with-out (for control purposes) addi-tion of dietary extracts based on glucosamine/chondroitin sulphate

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(G/CS), hydroxylapatite (Hx) or on a complex mineral (CM) made of carbonate, phosphate and cal-cium 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.

OUTCOME OF THE RESEARCHThe results of the cellular counts show that the Hx extract has a stimulating effect on the chondro-cyte 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-

G/CS = glucosamine / chondroitin sulphateHx = HydroxylapatiteCM = complex mineral * significantly different from the control sample p<0.05

* *

*

Figure 2:

Control

Equine chondrocyte proliferation in vitro with different dietaryextracts

Num

ber o

f cho

ndro

cyte

s pe

r mill

ilitr

e in

per

cent

age

of D

0

Figure 3:

A = G/CSB = Placebo$

$

*$

*

**

Data presented in average ± standard error

*Significantlydifferent from the respective value in group A p < 0.05

*$ Significantly different from the respective control value

p<0.05

ex vivo F2-isoprostanes in the chondrocyte supernate comingfrom horses first orally supplied with the feed complement

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

Control

F2-is

opro

stan

es c

once

ntra

tion

in th

e ch

ondr

ocyt

e su

pern

ate

(ng/

mL)

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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 articu-lar pathologies: it could help de-crease 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 ac-counts for severe lameness and for a more or less extended in-terruption of training. In cases of tendon damage, observation reveals that the difficult tissue re-pair is the biggest problem. Actu-

ally a tendon contains fewer cells than the extracellular matrix, while exactly the same cells de-liver the constituents needed by the extracellular matrix compos-ing most of the tendons. In many cases, tendon healing is long and the tendon never fully regains its original performance [6]. Ac-cordingly it seems interesting to investigate the effect of the extracts on the test on tendino-cyte proliferation. If the stimu-lation effect observed with the chondrocytes is confirmed for the tendinocytes, it would be a major outcome for the handling of ten-dinous regeneration.

tion of feed supplement and the control cultures. These very inter-esting results underline the possi-ble 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 com-ing from horses that had been orally provided with feed supple-ments show that the C/CS com-plement induces a decrease of the F2-isoprotanes (markers of oxi-dative 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 oxi-dative stress.

In addition, a modulating effect had been observed for the G/CS complement regarding its influ-ence on oxidative stress after 6 weeks of oral use of this supple-ment [5]. Indeed, a decrease of the MMP9 activity (type 9 ma-trix metalloproteinase; Fig. 4; the MMP9 being the enzyme involved in the damaging of the cartilage) was observed together with a be-ginning increase of the MMP2 activity (type 2 matrix metallo-proteinase; 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 sup-plied with the feed supplement, were cultivated and subjected to 1-β interleukins to provoke an inflammatory stimulation in them (Fig. 5).

* *

Figure 4:

T0 = Before oral supplyT6= After 6 weeks oral supply* Significant differences between group A and B, p<0.05

Articular

MMP9 activity (arbitrary unit)

Fore fetlock Back fetlock Stifle tarsusT0

Articular MMP9 activity in vivo after 6 weeks oral supply

Figure 5:

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

MMP-2 activity ex vivo in the chondrocytesupernate coming from horses orally supplied

with the feed complement(with or without inflammatory stimulant (IL-1-ß))

Articular

MMP9 activity (arbitrary unit)

β

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CONCLUSIONS AND PROSPECTSThe extracts tested here shows signs of a modulating effect on oxidative stress and on enzymatic stress. In addition, its apparent ef-fect on chondrocyte proliferation could stand for a stimulation of the metabolism which is probably ben-eficial to the tissue respiration. The same effects have also been ob-served in tests of ex-vivo inflamma-tory 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.❚

REFERENCES1. VALKO, M., LEIBFRITZ, D., MON-COL, J., CRONIN, M. T., MAZUR, M., and TELSER, J. (2007) Free radicals and antioxidants in normal physiological func-tions and human disease. Int. J Biochem. Cell Biol. 39, 44-84. 2. YUDOH, K., NGUYEN, T., NAKA-MURA, 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 MUR-PHY, 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 & Rheu-matism 46, 3240-3250.

5. DAIX , M., BASTIN, JF. and KIRSCH-VINK, N., Evaluation of an oral supple-ment enriched with glucosamine and chon-droïtine 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.

http

://w

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.twyd

il.co

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Used by most of the successful trainers and breeders in the world.

TWYDIL® PMCCovers the specific needs of growing horses. Assists the healthy development of osteoblasts – which form the bones, chondrocytes – which form the cartilage and fibroblasts – which influence tendons, ligaments and synovial fluid.

- Officially 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

Unchallenged excellence!





TWYDIL® AROUND THE WORLD

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

TWYDIL® distributor in South Korea with equine veterinarians of the National Stud.

Dr Stan Cosgrove, Manager of Moyglare Stud (Ireland) withDr de Moffarts

TWYDIL® AROUND THE WORLD

Mrs Bénédicte Maemoto, TWYDIL® agent in Japan, with Mr Sakiyama

TWYDIL® distri-butors in Saudi Arabia: Sheik Zaky and Mr Mustafa Hamza from the com-pany Al-Ghalia.

Valère Henry, TWYDIL® president, with the distributor in Greece

HPH 08-09

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The research conducted by TWYDIL® to optimize its formulas would be vain and uninteresting if special at-

tention was not paid to ensuring optimal stability of the compo-nents in every single product from manufacturing to best-before date. To achieve this goal, TWYDIL® not only carefully selects its ingre-dients, the type of packaging and of storage but also supervises the manufacturing process and perma-nently increases its controls.

1. Selection of the raw materialsBy “selection of the raw materials” we mean not only the selection of the vitamins and dietary miner-als 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 con-centration in active substances and the absence of contamination in it. The second criterion is the prod-uct’s intrinsic quality as a guaran-tee for long lasting efficiency. Let us take two examples:

- Ascorbic 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, (TWY-DIL® 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 aque-ous 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 Eleu-theroccocus senticosus, Max-im. an essential component of TWYDIL® HIPPACAN+C. Its use requires special caution such as, for example, a verified ab-sence of natural contamination in the extract batches. This control is done by the LCH that then is-sues 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 com-ponents in order to ensure a con-stant homogeneity of the prod-ucts. The same controls apply to all the plant extracts used for the whole range.

2. Production techniqueThe production technique used for the feed supplement (whether liq-uid, powder or granular) should not cause any destruction of the ac-tive substances. Variations in tem-perature, pressure or moisture are harmful to vitamins while oxida-tion phenomena must be avoided for some types of fatty acids (such as omega 3).

- TWYDIL® RACING is highly concentrated in vitamins, dietary minerals, magnesium and amino

acids. The production of its granules requires me-ticulous control: a ther-mal probe in situ ensures a regulated speed of pro-duction and a tempera-ture of production kept

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

STABILITY AND QUALITY

Determination by chromatography of the eleutherosides contained in TWYDIL®

HIPPACAN+C

HPH08-09

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below 62°C. Addition of water is prohibited because water destroys some vitamins.

- TWYDIL® OMEGADIL is heav-ily 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. Con-sequently, TWYDIL® OMEGADIL is pro-duced under air-free (nitrogen or vacuum) conditions from the refining of the fatty

acids up to packaging.

3. Adequate packagingThe 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 de-signed to make product administra-tion easy, and on the other hand we have TWYDIL® STOMACARE

for which adminis-tration with a mouth syringe is a neces-sary condition for its maximal efficiency.

The use of sealed aluminium polyester

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, T W Y D I L ® A RT R I D I L has been stud-ied by the office of chemical analy-sis of the ULg where it has been compared to other chondroprotec-tors. It turned out that the other tested competing chondroprotec-tors did not meet the requirements of guarantee. One of the possible causes advanced by the university is the loose packaging of the prod-ucts, which would not ensure the stability of glucosamine and other complex sugars.

The case of TWYDIL® OMEGA-DIL is even more interesting! As you already know, it is manufac-tured 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 pack-aging a special lining with differ-ent protective films that keeps oxy-gen 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 con-ference on Free Radical Biology and Medicine (FRBM) that the risk for men to suffer from arterioscle-rosis or inflammation (tendinitis, arthrosis) is higher with the inges-tion of oxidised fatty acids.

4. Storage TWYDIL® products are stored in a spacious warehouse located about twenty kilometres from Ba-sel. This warehouse complies with the strict regulations of Swiss au-thorities. The products are under

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 compa-ny rules of TWYDIL®.

Filling of sachets

Comparison of oxygen permeability of EVOH and PETMET films

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28

Retention times(min)

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Intensity

(mV) 1 2

3

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5

6

1. Chondroitin (RT = 3,15 min.)2. Excipient (RT = 3,40 min.)3. Saccharose4. Imp. Gluco5. Glucosamin6. Imp. Chondro

Standardized chromatography ofTWYDIL® ARTRIDIL collected in

pharmacy

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Standardized chromatography of achondroprotector, collected in a

pharmacy, presenting comparablelabelling than the TWYDIL® ARTRIDIL

1. Chondroitin (RT = 3,45 min.)2. Excipient (non detected)3. Saccharose4. Imp. Gluco5. Glucosamin6. Imp. Chondro

Influence of Gelbo cycles on oxygen permeability

02 p

erm

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HPH 08-09

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5. Quality controlQuality control at TWYDIL® is twofold: an anti-doping control and a conformity analysis of con-tent 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:

- Finished product test: the LCH investigates the presence of natu-ral contaminants.

- Urine test on a horse after an overdose with the product: the LCH carries out a “full screen-ing” identical to the one under-gone 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

the product batch number.Hence, all TWYDIL® products can be used until the day of competi-tion without any risks*.

The analysis of the constituents is essential to guarantee a con-stant quality of TWYDIL® prod-ucts. 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 inde-pendent 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 un-der 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 consider-ing simultaneously the coefficient of variation related to the inaccu-racy of the technique of analysis.

0

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2 6 11 18 24

Renal elimination of eleutheroside realised on 6horses having received a over-dose of

TWYDIL®HIPPACAN+C(%)

Hours after the administration time (h)

Eleutheroside’s kinetics of elimination

c

ontent

GUARANTEED

co

ntenido

GARANTIZADO

Photo representing a probio-tic culture utilized in TWYDIL®

productions

Responsible of storage and warehouse

Christophe Muller

Picture of yeast in optical microscopy

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

* 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.

c

ontent

GUARANTEED

co

ntenido

GARANTIZADO

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 andcartons of 100 sachets.

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

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.

OBITUARY

HPH 08-09

34

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

UNEQUALLED ANTIDOPING PRECAUTIONS

HPHHPH 08-09HPH 08-09

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 HORSELCH 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 CERTIFICATECertificate available on: www.twydil.com

SAMPLES SEALEDA 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.

c

ontent

GUARANTEED

co

ntenido

GARANTIZADO

HPH08-09

35

MARION FRANCOISAt 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 veteri-naries, pharmacists, trainers and horse breeders, but also wholesalers in all disciplines of equestrian sports.

Since her childhood, our TWYDIL® agent has been ex-periencing 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 ad-dition, 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. Hen-ce, Marion has acquired an advanced scientific training in anatomy, physiology, pathology and drug regulation.

From now on, her responsibility is to provide the different agents of equestrian sport with reliable scientific informa-tion 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 LUCCHESEOn 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.

Claudia completed her training as an industrial business management assistant in 1990 and then worked as Ex-port 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 phar-maceutical wholesaler.

For many years Claudia Lucchese had her own horse and was a passionate rider.

In her free time she breeds dachshunds.

ENERGY REFUELLING !


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.

• Officially 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.

NEW

NEW





TWYDIL is used by most of the successful trainers in the world.


TWYDIL® VIGORADE, THE PREPARATION FOR COMPETITIONS !

INDICATIONSTo prepare a horse for a specific competition.

COMPOSITION 10 vitamins, 7 trace elements, pineapple extracts, Saccharomyces cerevisiae (a high-chromium yeast), and prebiotics.

HOW DOES IT WORK ? Vitamins A, E, biotin and folic acid are the 4

key vitamins to guarantee maximum physio-logical 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 sele-nium 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® VIGO-RADE, accelerates the elimination of toxins induced by effort.

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.

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


Courbe théorique d'élimination des déchets cellulaires produits par l'effort ou un trauma

après supplementation en bromelain

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Theoretical curve of elimination of cellular waste induced by effort or trauma after supplementation with pineapple extract.

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TWYDIL® AT INTERNATIONAL EXHIBITIONS AND CONVENTIONS

AL FARES (DUBAI) 2008 ▼

Seminary for the veterinarians of the different teams at the Olympic Games in Hong Kong 2008

AVEF 2008, Reims (France) ▼

10th congress of equine medicine and surgery, 2007, Geneva, Switzerland▼

Equitana 2008, Melbourne (Australia) ▼

TWYDIL® AT INTERNATIONAL EXHIBITIONS AND CONVENTIONS

Veterinary workshop 2008, Chantilly, France

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

Trotters exhibition, 2009, Vincennes, France▼

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

HPH 08-09

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ENDURANCE HORSES IN TWYDIL®

INVESTMENT IN APPLIED RESEARCH

HPH

41

Did 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 wi-thout suitable preparation

- Heat exposure- Inappropriate feeding- Inappropriate dealing with elec-

trolytes- Genetic factors (such an accumu-

lation of glycogen disorders in the intracellular calcium metabo-lism 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

Dr Brieuc de Moffarts Director Research & Development of TWYDIL®, Switzerland

MUSCULAR TROUBLES

HPH08-09

HPH 08-09

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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 ef-fort gives more or less a precise idea of the extent of the muscular degeneration. In cases of uncon-trollable 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 re-quires a flawless management of warm-ups and effort of horses, as well as appropriate handling of any lameness and a good genetic selec-tion. However, all these measures are only efficient in combination with a balanced feeding of the hor-ses! A horse needs daily (in addi-tion 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 recapitu-lation of the nutritional needs of horses suffering from that type of myopathy.❚

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CPKASTLDH

Muscular enzymes curve evolution after a moderatemyopathy on a horse in training

(IU/L)

Post-effort hours

FAT SOLUBLE VITAMINS WATER SOLUBLE VITAMINS Vitamin A IU 60000 Thiamin (B1) mg 84Vitamin D3 IU 6000 Riboflavin (B2) mg 35Vitamin E IU 7000 Pyridoxine (B6) mg 30Vitamin 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 145MICRONUTRIENTS Choline mg 800 Ascorbic acid (C) mg 10000Copper mg 187 Iron mg 600 Manganese mg 300 MACRONUTRIENTS Zinc mg 770 Cobalt mg 3.75 Salt (NaCl) g 30 to 50Iodine mg 5 Magnesium g 12Selenium mg 5 Potassium g 55

Daily needs of a thoroughbred in training and suffering from muscular troubles:

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.

BIBLIOGRAPHY :

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

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Though the needs of horses un-der inten-

sive 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 heal-thy horse with a light work load are

similar to those of an adult horse, while a horse aged but in increasin-gly poorer condition, with dental deficiency, must receive special ap-propriate feeding.

As a rule, special attention must be paid to the health of horses in in-creasingly poor condition, whether due to a primary pathology (colic, diarrhoea, trauma resulting in a confinement) or simply to age (of-ten during retirement).

1. An energetic provision based on regular body check scoresThe factors of weight loss of such horses lie in the inversion of the anabolic balance (mass increase) into a catabolic one (mass de-crease), and in an increase of their needs as well as in a regression of their ingestion process. The factors considered here are: ageing, cessa-tion of training and lack of physi-cal activity, progressive loss of the dental function, absorption trou-

Dr Brieuc de Moffarts (DMV, Ms, PhD)Director Research & Development of TWYDIL®

INVESTIGATION OF THE NEEDS OF AGED OR DEBILITATED HORSES

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NEW PRODUCT: TWYDIL® BEBACK

HPH 08-09

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bles, sickness or even parasitism. In such cases helping the horse requi-res 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 supple-mented; some aged horses can in-gest 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 horse’s digestion when given in a treated form (as flakes, or ground etc.).

A regular body check, in case a regular weighing is not possible, can help spot the variations in the horses’ 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 qualityFor 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 ad-dition, 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 acidsSeriously 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 ra-tio of 14-15% in their feeding is

Figure 1

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BODY CONDITION SCORE EVALUATION (ADAPTED FROM CARROLL, C.L. AND HUNTINGTON, P.J., BODY CONDITION SCORING AND WEIGHT ESTIMATION OF HORSES)

HPH08-09

45

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 mus-culature in good shape (cf. HPH 02/03).

4. Vitamins and minerals to sustain the horses’ organic functionsAs 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 provi-sion 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 sin-ce it provides them with the whole range of vitamins, trace elements, magnesium, amino acids and pro-biotics and prebiotics necessary for their well-being, convalescen-ce 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

BROODMARES, STALLIONSTWYDIL® 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 cal-cium.

FOALS, YEARLINGSTWYDIL® 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 develop-ment of the horse.

THE FULL STUD PROGRAMME





TWYDIL® is used by most of the successful breeders in the world.AVAILABLE THROUGH YOUR VETERINARY SURGEON

HPH

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This article is a partial account of the study presented by Dr. Emma-nuelle Van Erck at the 2008 confe-rence of the Veterinary Compara-tive Respiratory Society (VCRS).The University of Liège and the University of Namur received sup-port from TWYDIL®PROBIOX for this study.

QUESTIONS UNDER INVESTIGATION Can the oxidative stress output of a racehorse be related to its perfor-mance parameters?Do horses affected by metabolic troubles of muscular origin show modified oxidative stress parame-ters?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 ef-fort.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 oxi-dative stress markers.2.Horses affected by muscular stif-fness were less efficient and their oxidant profile was modified.

STATE OF THE KNOWLEDGE BEFORE THE STUDYAs was the case with several pre-vious reports in this magazine (cf.

HPH 2002/2003 ; HPH 2004/2005, HPH 2006/2007), it is now confir-med that intensive exercise produ-ces oxidative stress in horses (cf. : Kirschvink et al., 2008).Even though it has been demons-trated that a deficiency in anti-oxidants 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 para-meters 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 ef-ficient and beneficial to their pro-oxidant-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 consump-tion which indicate how much oxygen a horse needs per kg (ml oxygen per kg). This consump-tion has a linear progression and reaches a plateau at the maximal

INVESTIGATION INTO THE RELATION BETWEEN SPORTS PERFORMANCE

PARAMETERS AND OXIDATIVE STRESS MARKERS IN TROTTERS

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consumption (cf. opposite figure). It is important to know that, un-like a human who can run 100 m almost in a state of apnea (i.e. wi-thout 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 in-form 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 rea-ches 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 rea-ches a heart rate of 200), and the measure of Vfcmax (the speed at which the horse runs when it rea-ches 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 mat-ter 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

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.

A STUDY ON 100 TROTTERSThe 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 un-der standardised conditions on the same trotting track (racetrack of Wallonia) and in the same labora-tory (TWYDIL®-PROBIOX®).

A . PresentationThis 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 ne-cessarily part of a TWYDIL® sup-plementation programme.

The markers of oxidative stress (i.e. the hydrophilic oxidant capa-cities 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 identical-ly 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, si-gnificant correlations were found between the Vla4 and the POOL as well as between the V200 and

20406080

100120

140160180

200

(VO2 ml/Kg/min)

VO2 max

trained

untrained

speed (Km/h)

5 15 25 35 45 55

0

150

200

100

Speed (Km/h) V200 5 15 25 35 45

HR (bit/min)

250

V180

Maximum frequency

600

800

1000

1200

1400

1600

1800

2000

2200

2400

105 110 115 120 125 130 135 140 145 150

r = 0.87

SOD (IU.Hb-1)

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

p < 0.05

HPH08-09

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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 an-tioxidant reserves (ACL).

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 conclu-sion at this stage, and a reason why further experiments should be conducted in this direction.

These results indicate that the pa-rameters of oxygen consumption and the parameters of sports per-formance of a horse are intercon-nected, even though at various levels; and this tells us about the psychological state of the horse.

2. muscular damage versus oxida-tive stressSome 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 tho-se 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 the-se horses with the muscular trou-bles 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

trouble has a significantly hi-gher oxidative stress compared to another horse without the same trouble.

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 stif-fness were less efficient and their oxidant profile was modified.❚

0

5

10

15

20

25

30

35

ACL ( mol eq. Trolox/mL)

28 30 32 34 36 38 40 42 44 46

V200 (Km/h)

R = 0,36 p = 0.001

0

40

80

120

POOL (µmol/g)

*

Horses withoutsignificant CPK

increase

Horses withsignificant CPKincrease

0

10

15

5

Lactic acid (ᵑmol/L )

VLa4VLa4

20

speed (Km/h)

5 15 25 35 45

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REFERENCES:de Moffarts B., Kirschvink N., Art T., Pincemail J., Michaux C., Cayeux K., De-fraigne J-O., Lekeux P.Impact of training and exercise intensity on blood antioxidant markers in healthy standardbredHorses. 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 Com-parative 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 any-thing 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 nume-rous 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 ma-nagement 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, inflam-matory stress etc. That is what helps us to achieve a gradual development of the TWYDIL® supplements from a practical point of view.


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.

• Officially 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.


FOR A SUPPLE AND TONIC EFFECT!

FAST ABSORPTION AND EFFICACY

PROVED





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1. COMPETITION LINE

TWYDIL® ARTRIDILWith its unique formulation to help support healthy joints. With or without Harpagophytum procumbens.

- WITH HARPAGOPHYTUM : WAITING TIME BEFORE COMPETITION 48 HOURS

- WITHOUT 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

TWYDIL® BEBACKTWYDIL® BEBACK helps to stabilize the intestinal flora and helps horses that have lost condition.

- EACH 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


TWYDIL® CALMINA scientific blend of selected vitamins, fructo-oligo-saccharides, trace ele-ments and tryptophan to help manage excitability and stress, and to optimise digestive and muscular well being of the horse.



TWYDIL® COMPETITIONAn ideal daily supplement, highly concentrated (14 vitamins, 7 trace ele-ments, 4 amino acids and magnesium) which covers the physiological needs of sport horses.



NEW

THE TWYDIL® RANGE OF PRODUCTS

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® RACINGAn ideal daily supplement, highly concentrated (14 vitamins, 7 trace ele-ments, 4 amino acids and magnesium) which covers the physiological needs of high performance horses.



TWYDIL® ELECTROLYTES In pails For the compensation of electrolyte loss after heavy sweating.



TWYDIL® ELECTROLYTES In mouth syringes Oral paste for the compensation of electrolyte and vitamin C losses after heavy sweating.



TWYDIL ® ELECTROLYTES+CCompensation of electrolytes, vitamins and trace elements loss. - EACH 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


TWYDIL® HEMATINIC In mouth syringes and in bottlesFormulation (either in paste or liquid form) with key vitamins and trace ele-ments to support haematological parameters.



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 THE TWYDIL® RANGE OF PRODUCTS

54

TWYDIL® HEMOPARAids appetite. Helps maintain good digestive function.



TWYDIL® HIPPACAN+CSupplement with bioflavonoids and vitamin C to minimise the effects of effort and help fortify the natural immune response system.

- WAITING TIME BEFORE COMPETITION : 48 HOURS.


TWYDIL® MUCOPROTECTSupports the natural defence system of the organism.

- WAITING TIME BEFORE COMPETITION : 48 HOURS.


TWYDIL® OMEGADIL Supports the microcirculation. Helps the body’s natural defences. Synergic action with other TWYDIL® supplements.



NEW


TWYDIL® PROTECT PLUS To provide optimum metabolic balance between antioxidants and oxidants, and give extra muscle protection.



TWYDIL® STOMACARE Specially formulated blend of refined oils, glucosamine fibres and magnesium which may help to soothe the stomach and coat its lining.



TWYDIL® TWYBLIDHelps to reinforce natural anti-viral defences, helps to maintain the integrity of ca-pillary blood vessels, and to maximise endurance and optimise the performance.



TWYDIL® VIGORADE To enable the horse to achieve its maximum potential.



NEW FORMULA


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2. BREEDING LINE

TWYDIL® GROWING Very sophisticated complementary feedingstuff, providing growing horses with vitamins, trace elements, diversified amino acids, pro- and pre-biotics, neces-sary for optimal development and for the diversification of the intestinal flora.



TWYDIL® PMCAssists the healthy development of osteoblasts which form the bones, chon-drocytes which form the cartilage, and fibroblasts which influence the ten-dons, ligaments and synovial fluid.



TWYDIL® MINERAL COMPLEX Appetising and bio-available mineral supplement with three different sources of calcium so that the total ration tends toward an ideal phosho-calcic ratio. -- EACH 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


TWYDIL® STUDSupplies the needs and supports the intestinal flora of broodmares and stallions. Favours milk production and colostrum quality.




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3. COSMETIC AND HYGIENIC PRODUCTS

TWYDIL® 4LEGS Cream for daily application to help soothe and relax tired legs.

- EACH BATCH OFFICIALLY CERTIFIED BY LCH CAN BE USED WITHOUT RISK UP TO THE DAY OF THE COMPETITION

TWYDIL® LEG GELLeg gel on iodine base to cover sore areas on the legs.


TWYDIL® LEG PAINTKeep your horse’s tendons and ligaments in good health.


TWYDIL® HOOFCARECream helping hoof growth and soothing pastern irritation.



PAVESCO AGHead OfficeCH-4010 Basel, SwitzerlandTel. (41) (61) 272 23 72Fax (41) (61) 272 23 88

PAVESCO U.K. LTD.116, High RoadNeedham, Harleston, Norfolk IP20 9LGTel. (01379) 85 28 85Fax (01379) 85 41 78

PAVESCO EQUINE HEALTH USA, Ltd.,321 N, 22nd StreetSt. Louis, MO 63166, U.S.A.Tel. (314) 421 0300Fax (314) 421 3332

Researchquality

andhorse sense

PAVESCO AGHead OfficeCH-4010 Basel, Switzerland

Tel. (41) (61) 272 23 72Fax (41) (61) 272 23 88

PAVESCO U.K. LTD.116, High RoadNeedham, Harleston, Norfolk IP20 9LG

Tel. (01379) 85 28 85Fax (01379) 85 41 78

PAVESCO EQUINE HEALTH USA, Ltd.,321 N, 22nd StreetSt. Louis, MO 63166, U.S.A.

Tel. (314) 421 0300Fax (314) 421 3332

TWYDIL® RACING/COURSE Vitamin supplementation Supplémentation vitaminique

TWYDIL® PROTECT PLUSAntioxidants, muscle protection

Raideurs musculaires

TWYDIL® MINERAL COMPLEXMineral supplementationSupplément minéral

TWYDIL® ELECTROLYTESRehydrationRehydratation

TWYDIL® ELECTROLYTES+CRehydration and recuperationRéhydratation et récupération

TWYDIL® HEMATINICTonic effect, increase of red cellsEffet tonique, globules rouges

TWYDIL® HEMOPARDigestion, appetite stimulantDigestion, stimulation de l’appétit

TWYDIL® HIPPACAN+CEndurance, stress

TWYDIL® ARTRIDILSupple joints Articulations

TWYDIL® CALMIN Nervous horses

Nervosité

TWYDIL® PMCGrowing horses

Qualité osseuse et structurelle

TWYDIL® 4LEGS Tired legsJambes fatiguées

TWYDIL® STOMACAREStomacal protection

Protection stomacale

TWYDIL® MUCOPROTECTImmune systemSystème immunitaire

ALWAYS A TWYDIL® SOLUTION

TWYDIL® HOOFCARE HoovesSabots

TWYDIL® OMEGADILImprovement of natural defences Omega-3

Micro-circulation, coeur

TWYDIL® GROWINGYoung horse’s development

Chevaux en croissance

TWYDIL® STUD/ELEVAGEBroodmares + stallionsPoulinières + étalons

TWYDIL® BEBACKAged horses

Chevaux d’âge

TWYDIL® VIGORADEBooster

TWYDIL® GEL MEMBRE/ TWYDIL® LIQUIDE MEMBRE

Tendons, ligaments

TWYDIL® TWYBLIDBleeders

Intégrité des vaisseaux sanguins

High Performance Horses 2008/2009

Documents

Transcript of High Performance Horses 2008/2009