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Efecto del aceite de maiz sobre los acidos grasos volatiles en caballos con ulceras gastricas inducidas.

Effects of corn oil on the volatile fatty acids in horses with induced gastric ulcers

INTRODUCCION

El sindrome de ulcera gastrica equina (SUGE) es de naturaleza multifactorial e involucra caracteristicas anatomicas y eventos fisiologicos particulares que contribuyen a incrementar la presentacion de lesiones ulcerativas en el estomago de esta especie, incluyendo parte terminal del esofago, estomago proximal (mucosa escamosa), estomago distal (mucosa glandular) y duodeno proximal (1,2). La sanidad gastrica depende de la integridad de las areas de la mucosa (no glandular y glandular) que se genera por los mecanismos de proteccion a la constante exposicion a diferentes agentes gastrolesivos tanto endogenos como exogenos (1).

La accion caustica del acido clorhidrico es una causa primaria del SUGE (1). Sin embargo, el SUGE es un problema multifactorial que relaciona condiciones del animal (factores endogenos) y factores exogenos asociados a condiciones de ambiente, como el sistema de explotacion y manejo; ademas de los factores relacionados con el bienestar y el estado de salud. Existe una lista extensiva de factores o causas directas involucradas en el SUGE, donde se incluye entre los factores inherentes como el sexo, raza y temperamento. Entre las causas exogenas como los programas de entrenamiento, nivel de competicion, confinamiento, medicaciones frecuentes (particularmente, con los antiinflamatorios no esteroidales [AINEs]), inadecuado manejo alimentario y bacterias acidorresistentes; son descritos en la literatura como inductores de SUGE (1, 4).

Los acidos grasos volatiles (AGV) como el butirico, propionico, acetico y valerico, son derivados de procesos fermentativos de carbohidratos por bacterias gastricas; sin embargo, los AGV han sido relacionados a producir "in vitro" lesiones en la mucosa no glandular de equinos (5, 6). Sin embargo, en un estudio se determino variadas concentraciones de AGV en el contenido gastrico de caballos alimentados con dietas a base de heno o combinacion de heno y concentrado que no influyeron en la aparicion de ulceras gastricas (7). En este sentido, las lesiones ulcerativas en la mucosa no glandular se relacionan directamente por la accion acida y en la mucosa glandular son mas frecuentes por causas secundarias a los efectos toxicos sistemicos y locales de los AINEs, especialmente en caballos medicados con fenilbutazona (FBZ)(8). Los

efectos colaterales sistemicos de la FBZ son relacionados con la inhibicion inespecifica de la enzima ciclooxigenasa (COX), necesaria para la sintesis de prostaglandinas ([PGE.sub.2]) y oxido nitrico que favorecen la proteccion y perfusion normal de la mucosa glandular. Por otro lado, los AINEs (como la FBZ), puede inducir dano directo de las celulas epiteliales de la mucosa glandular (9).

Factores fisiologicos protectores de la mucosa glandular son la perfusion sanguinea gastrica, produccion de bicarbonato gastrico y secrecion de moco. Estos factores son principalmente influenciados por la produccion local de [PGE.sub.2]. Sin embargo, las [COX.sub.1-2] son inhibidas inespecificamente por AINEs como la FBZ, lo que induce a una disminucion de la [PGE.sub.2] y posible aparicion de ulceras en la mucosa glandular (10).

El aceite de maiz es una importante fuente de acido linoleico, el cual es un precursor del acido araquidonico, por lo que tiene la capacidad de aumentar la produccion de [PGE.sub.2] endogena (10,11). Sin embargo, existe poca informacion sobre los mecanismos del aceite de maiz en los procesos de reparacion de caballos con SUGE y como este podria afectar la concentracion de AGV en el jugo gastrico. Por lo tanto, se desarrollo un modelo de ulceracion gastrica inducido con FBZ en equinos, para: investigar la influencia de este aceite sobre las concentraciones AGV en el jugo gastrico de caballos con ulceras gastricas inducidas con FBZ, y correlacionar los hallazgos gastroscopicos con las concentraciones de los AGV.

MATERIAL Y METODOS

Animales. Un grupo de 15 equinos sin raza definida (11 hembras no prenadas y 4 machos castrados), con un rango de edad entre 5 y 20 anos y peso vivo entre 270-465 kg, fueron incluidos. Todos los caballos provenian de un sistema de explotacion a pasto. Durante el periodo de adaptacion de dos semanas, los animales fueron desparasitados y vacunados. Los caballos fueron alojados en pesebreras individuales y alimentados con heno de Coast cross (Cynodon dactylon), agua ad libitum y suplementados con concentrado comercial con 13% de proteina y administrado dos veces al dia en la proporcion del 1% del peso vivo.

Diseno del estudio. El diseno experimental fue en bloque completamente al azar y desarrollado en tres tiempos especificos. En el tiempo 0, todos los caballos fueron evaluados por laboratorio clinico y examen gastroscopico para obtener datos basales. El tiempo 1, fue considerado entre el primer y sexto dia, y comprendio el periodo de induccion. El tiempo 2, incluyo el periodo de evaluacion de cada tratamiento (tiempo de tratamiento), el cual comprendio el intervalo entre el septimo y el 21 dia. Este estudio fue realizado entre mayo y julio de 2012.

Grupos de Protocolo de induccion de ulcera gastrica con FBZ. Los 15 animales fueron distribuidos en tres grupos (cinco animales por grupo), de la siguiente manera: En el grupo I (grupo control), los animales recibieron 20 ml de agua via oral (vo)/s.i.d/cada 24 h por 6 dias; grupo II recibio una dosis unica de 13.2 mg/kg de FBZ diluida en 20 ml de agua/vo, en el sexto dia de iniciar el experimento; grupo III, fueron medicados con FBZ a dosis de 4.4 mg/kg/vo/s.i.d. (siempre diluido en 20 ml de agua), por cinco dias y en el sexto dia recibieron una dosis final de FBZ a 13.2 mg/kg, vo, diluido en 20 ml de agua. Este protocolo de induccion fue disenado para otros trabajos en el estudio de los fenomenos de gastroproteccion y gastroadaptacion a la FBZ en el equino, propuesto por Martinez Aranzales et al (12).

Grupos de tratamiento. Despues de seis dias de placebo y de FBZ para la induccion de las ulceras en los equinos de cada grupo, todos los grupos fueron tratados por dos semana de la siguiente forma: Grupo I recibio sucralfato a dosis de 30 mg/kg/vo/tid; grupo II recibio 70 ml/100kg/vo/tid de aceite de maiz y grupo III fue tratado tambien con aceite de maiz en dosis de 90 ml/100kg/vo/tid; el volumen de aceite de maiz utilizado en cada grupo, fueron extrapolados de trabajos realizados en la especie (10, 13). Las variables de sexo y edad se consideraron como bloques en este experimento.

Evaluacion gastroscopica. Valoracion por gastroscopia fueron realizadas en los dias 0, 7 y 21. Despues de un ayuno de solidos (12-14 horas) y de liquidos (4 horas) y sedacion (Detomidina 10 [micron]g/kg/iv, Dormiun V[R], Agener Uniao, Brasil), la mucosa gastrica de los equinos fue examinada a traves de videoendoscopio flexible (PortaScope[R], 1800PVS, United State) de 12 mm de diametro y de 3 m de longitud, introducido via nasogastrica. Las lesiones observadas fueron registradas en video y clasificadas segun el sistema de MacAllister et al (14), (Tabla 1).

Determinacion de la concentracion de AGV en fluido gastrico. Antes de cada gastroscopia y con la ayuda del endoscopio fueron recolectados 5 ml de fluido gastrico y depositados en recipiente de vidrio con 1 ml de acido metafosforico para medir posteriormente la concentracion de AGV (acetico, propionico, butirico y lactico) por cromatografia de gases (7), a traves de equipo de la referencia Shimadzu (Gas Chromatograph GC-17a /auto inyector AOC--20i Shimadzu).

Analisis estadistico. Los datos fueron analizados con el software SPSS 19.0 (IBM, SPSS Inc, USA). Se realizaron los test de Lilliefors y Barlett para evaluar normalidad y homocedasticidad en las variables estudiadas. Los datos del acido butirico y aletico fueron transformados a log+4. Los test de Tukey y t fueron utilizados para la comparacion de medias para el acido acetico y butirico respectivamente. El acido propionico no presento las caracteristicas evaluadas y junto al acido lactico, las medianas fueron comparadas por los test de Friedman y Kruskal-Wallis. Un p<0.05 se considero estadisticamente significativo para todos los test. El poder calculado para este estudio fue de 0.8 (80%), usando un tamano de muestra de cinco caballos por grupo experimental y un error alfa del 5%.

Este estudio experimental fue aprobado por el comite de etica para la experimentacion animal de la Universidad Federal de Minas Gerais Brasil, con el protocolo numero 234/09. El trabajo se desarrollo en la Escuela de Medicina Veterinaria de la Universidad Federal de Minas Gerais, en Belo Horizonte, Brasil; localizada en la latitud 19[grados]55' S y longitud 43[grados]56' W Gr; y una altitud de 832 m, con un clima tipo Cwa (segun la clasificacion de Kopper), caracterizado por un invierno predominantemente seco y un verano lluvioso, segun el Instituto Nacional de Meteorologia de Brasil.

RESULTADOS

Todos los animales finalizaron el periodo de estudio sin manifestacion de signos clinicos o de alteracion metabolica relacionada con enfermedad sistemica.

Hallazgos gastroscopicos. En La primera evaluacion gastroscopica (dia cero) no se reportaron lesiones. Posteriormente en el dia siete postinduccion, se pudo constatar que el protocolo a base de FBZ y el efecto del confinamiento ocasionaron lesiones en ambas mucosas del estomago. En general, las lesiones encontradas se clasificaron segun McAllister et al (14) en el rango de 1 a 3 tanto en numero como en severidad. Finalmente, al dia 21 postratamiento se pudo constatar que todos los tratamientos (aceite de maiz en las dos dosis y sucralfato), cicatrizaron las lesiones del area glandular de la mucosa gastrica. Sin embargo, ninguno de los dos tratamientos a base de aceite de maiz reparo las ulceras gastricas ubicadas en el area no glandular. Por tanto, una vez finalizada la fase experimental, estos animales fueron tratados con inhibidores de la bomba de protones (antiacido) en esquema tradicional para su recuperacion total.

Concentraciones de AGV en el fluido gastrico.

Las concentraciones de AGV determinadas en el contenido gastrico en las dos fases experimentales se describen en la tabla 2 y 3. La concentracion de los acidos acetico y butirico fueron las mas abundantes en el jugo gastrico (2.32-3.80 y 1.64-2.39 mmol/L respectivamente), donde el acido butirico represento la mayor parte de la concentracion total (Tabla 2). Los acidos propionico y lactico presentaron menor concentracion (0.0015-0.12 y 0.05-0.61 mmol/L respectivamente).

Las concentraciones basales o iniciales (Semana 0) del acido propionico presentaron diferencias significativas entre los grupos I y II. En la fase de tratamientos, la concentracion del acido propionico fue significativamente diferente en el grupo II entre la segunda y tercera semana. En la misma fase, la concentracion del acido lactico del grupo II tuvo diferencia significativa entre la primera y tercera semana (Tabla 3). Comparando las dos fases experimentales, la concentracion del acido propionico en el grupo I mostro diferencia significativa entre el valor basal y el periodo final; igualmente la concentracion del acido lactico de los grupos II y III en los mismos periodos. El butirico y el acetico no presentaron alteraciones significativas en todos los grupos y periodos evaluados durante el estudio (Tabla 2).

DISCUSION

El SUGE se caracteriza por la convergencia de varios factores predisponentes que contribuyen a las elevadas frecuencias de presentacion de esta patologia en el caballo. Igualmente, se conoce la susceptibilidad de cada una de las areas de la mucosa del estomago; en el caso de los AINEs y eventos que generan estres como el confinamiento, son comunmente relacionados con estas lesiones (15). Estos dos factores fueron considerados en el presente estudio para inducir ulceracion gastrica en los caballos estudiados. El protocolo empleado en este estudio, genero ulceras en ambas mucosas de la superficie gastrica. Situacion que permitio evaluar la dinamica de las concentraciones de AGV por efecto de la FBZ y la participacion de los AGV en la resolucion de las lesiones durante la administracion de aceite de maiz y sucralfato.

Los caballos de este estudio no presentaron signos clinicos y alteraciones en las variables del hemograma y de la bioquimica sanguinea. Estos hallazgos se debieron posiblemente al desarrollo de leves ulceraciones gastricas y con baja clasificacion tanto en numero como en severidad (14). Sin embargo, la severidad de una ulcera no siempre se relaciona con la intensidad de los signos clinicos (16). Lo que dificultaria determinar la relevancia clinica de los grados de ulceracion en algunos pacientes (15, 17). Ademas, es importante considerar la participacion de la oferta continua de alimento en el mantenimiento del gradiente del pH gastrico, que se traduce en alivio transitorio con poca expresion de signos clinicos.

La concentracion total de AGV en el contenido gastrico de los equinos de este trabajo, fue mayor a la reportada por Frank et al (13); en este estudio utilizaron preventivamente el aceite de maiz y de arroz para lesiones causadas por manejo alimentar. Sin embargo, la concentracion de AGV en este trabajo, fue menor a la reportada en dietas a base de concentrado y heno de alfalfa (7). No obstante, se debe considerar la naturaleza del presente estudio, una vez que se valoro el comportamiento de los AGV en presencia de ulceras.

Las diferencias en estos resultados pueden derivarse de varios factores que se deben considerar para su interpretacion. Fisiologicamente, la concentracion de AGV disminuye acentuadamente cinco horas despues de cada comida (18). En este estudio las toma de muestras fueron realizadas despues de 12-14 horas de ayuno. Por otro lado, es posible que el periodo de restriccion de agua antes de las gastroscopias, no fue suficiente para evitar el efecto diluyente. Ademas, la produccion de AGV depende del tipo, calidad y cantidad de la alimentacion (7), factor que varia ampliamente entre los trabajos realizados. Ademas, de la flora gastrica adaptada que participa en los procesos fermentativos tambien puede afectar la concentracion de los AGV (19).

Por otro lado, es probable que la concentracion AGV total encontrada, tambien haya sufrido modificacion por procesos de volatilizacion antes de la coleccion o de transformacion a dioxido de carbono, p-hidroxibutirato o acetoacetato que normalmente ocurren con estos acidos (5). Adicionalmente, los animales de este estudio fueron alimentados con una dieta baja en carbohidratos fermentables (aproximadamente 2.5kg de concentrado en promedio y distribuido dos veces/dia) a diferencia de otros trabajos (13).

A pesar de no reportarse diferencias significativas en las concentraciones de AGV entre los grupos en el periodo de induccion, los datos reflejaron leves alteraciones; no obstante, las reacciones derivadas del efecto del confinamiento posiblemente tuvieron participacion en la generacion de ulceraciones, tal como se evidencia en el grupo control que no recibieron FBZ en la induccion. Sin embargo, efectos ulcerogenicos importantes en el estomago por AGV (5, 7) y cambios en la produccion de AGV en la colitis dorsal derecha inducida con FBZ en equinos se han reportado contundentemente (20).

Estudios "in vitro" con camara de Ussing, demostraron el grado ulcerogenico de los principales AGV. En estas condiciones se expone una concentracion conocida y constante de cada AGV a la mucosa del estomago (5, 6); pero se excluye un ambiente gastrico natural que de cierta manera participa en la accion gastrolesiva. En contraste, un estudio "in vivo", engloba otros componentes como la motilidad, reflujo gastroduodenal, la condicion interdigestiva o basal, fluctuaciones del acido clorhidrico y flora gastrica, de importancia en la fisiopatologia del SUGE. Sin embargo, como en este estudio, algunas variables se caracterizan por ser complejas de controlar o cuantificar.

La participacion de los AGV en el desarrollo de las ulceras depende del pH en el lumen gastrico. Especificamente en pH <4 estos acidos acentuan las alteraciones en la area no glandular de la mucosa, especificamente en el area adyacente al margo plicatus en la curvatura menor (5, 6). En este trabajo, posiblemente la perpetuacion de las lesiones en la area no glandular de la mucosa fueron derivadas de la no ionizacion de los AGV analizados (acetico, butirico y propionico), por el pH bajo del contenido gastrico que oscilo entre 2.38 y 3.77 en los grupos tratados con aceite de maiz, donde no cicatrizaron las ulceras en su totalidad.

Los AGV modifican las propiedades bioelectricas de la mucosa al disminuir el transporte de sodio, lo que ocasiona diferentes niveles de alteracion en la permeabilidad segun el tipo de acido (5, 6, 21). El acido acetico presento la mayor concentracion (59%) en el contenido gastrico, dato similar a otros estudios (5, 6, 7), y menor que 85% determinado en caballos tratados tambien con aceite de maiz y de arroz (13). Sin embargo, este acido causa lesiones discretas a la mucosa gastrica.

Contrario a lo reportado por Frank et al (13), el acido butirico en los animales de este estudio, presento concentraciones de 40%. Estudios "in vitro" demostraron un mayor efecto lesivo del acido butirico sobre el area no glandular de la mucosa (5). Por las condiciones presentadas, posiblemente el acido butirico tuvo amplia participacion en las ulceraciones presentes en esta area de la mucosa de los caballos del presente trabajo.

El acido propionico tuvo un moderado efecto gastrolesivo (5). Sin embargo, en los jugos gastricos evaluados, su concentracion (0.0015 0.12 mmol/L) fue menor a las reportadas en la literatura; por consiguiente la participacion ulcerogenica fue minima en estos animales de este estudio. Las diferencias en la concentracion entre los trabajos, posiblemente obedece a los factores descritos anteriormente, es decir manejo alimentario y el prolongado periodo de ayuno.

Con respecto al acido lactico, trabajos describen abundante produccion en el estomago del equino (19), en especial inmediatamente despues del consumo del alimento (18). En contraste, las concentraciones del acido lactico halladas fueron minimas a las reportadas, tal vez el periodo de ayuno y el tipo y esquema de alimentacion influyeron en estos resultados. El poder ulcerogenico tambien es minimo en la mucosa no glandular segun estudios "in vitro" (21), siendo su efecto relacionado con la cantidad y el tiempo de exposicion a la mucosa; por consiguiente, se puede inferir una baja participacion en la presencia de las ulceras en este trabajo.

En conclusion, los tratamientos a base de aceite de maiz fueron similares al instaurado con sucralfato con relacion a la concentracion de los acidos acetico y butirico, pero con comportamiento diferente con el acido propionico y lactico, no obstante, estos ultimos por encontrarse en pequenas proporciones no se consideraron de relevancia tanto en la induccion como en los tratamientos. Los acidos en mayores concentraciones en especial el butirico al parecer perpetuo la lesion en el area no glandular de la mucosa, donde el aceite de maiz no mostro beneficios terapeuticos. Estos resultados comprobaron el efecto nocivo de algunos AGV sobre la mucosa no glandular; sin embargo, se debe considerar el efecto recuperador a traves de la induccion de prostaglandinas por cada tratamiento en la mucosa glandular.

Finalmente, los AGV deben ser medidos continuamente y en periodos de tiempo cortos para eliminar el efecto de la volatilidad, transformaciones metabolicas en el ambiente gastrico y las dinamicas de las fases de vaciamiento del estomago para disminuir el impacto en los resultados y poder comparar efectivamente con lo reportado en estudios "in vitro".

Agradecimientos

Los autores agradecen al personal del laboratorio de Nutricion Animal de la Universidad Federal de Minas Gerais (UFMG), Belo Horizonte, Brasil. Esta investigacion fue concedida por el CNPq, CAPES-PG, UFMG, Brasil, y el proyecto de sostenibilidad 2013-2014 de CODI Universidad de Antioquia, Medellin, Colombia.

INTRODUCTION

Equine gastric ulcer syndrome (EGUS) is a multifactorial problem that could involve one or several parts of the stomach of this species, including the terminal oesophagus, proximal (squamous mucosa) stomach, distal (glandular mucosa) stomach, and proximal duodenum (1,2). The health of the equine stomach depends on the integrity of the mucosal areas (nonglandular and glandular), which is generated by protective mechanisms that counteract the effects of constant exposure to both, endogenous and exogenous gastroerosive agents (1).

The caustic action of the gastric hydrochloric acid is a primary cause of EGUS (1). However, as mentioned, EGUS is a multifactorial problem linked to the conditions of the animal (endogenous factors) and exogenous factors related to environmental conditions, such as the husbandry systems management and factors related with the wellness and healthiness of the animals with this condition. There is an extensive list of factors or direct causes involved in EGUS; these could include inherent factors such as gender, breed, age, and temperament. However, exogenous causes like training programs, level of competition, confinement level, concurrent medication (particularly, with non-steroidal anti-inflammatory drugs [NSAID]), inadequate feed management, and acid-fast bacteria producing metabolites, among others, are described in the literature as EGUS inducers (1,4).

Volatile fatty acids (VFA) such as butyric, propionic, acetic and valeric acids are derived from carbohydrate fermentation processes directed by gastric bacteria. VFA have been found to produce "in vitro"lesions in the non-glandular mucosa of the equine stomach (5, 6). However, in an early study it was observed that varying concentrations of VFA in stomach contents of horses fed diets based on hay or hay plus concentrate did not influence the appearance of gastric ulcers in these animals (7).

On the other hand, ulcerative lesions in the non-glandular mucosa are directly related to the action of gastric acid on this stomach area. In contrast, glandular lesions are more commonly a secondary cause of the systemic and local toxic effects of NSAID, particularly in horses medicated with phenylbutazone (PBZ) (8). The systemic side effects of PBZ are related to nonspecific inhibition of the cyclooxygenase (COX) enzyme, which is necessary for the synthesis of prostaglandins ([PGE.sub.2]) and nitric oxide favoring protection and normal perfusion of the glandular mucosa. On the other hand, NSAID (e.g., PBZ) induce direct epithelial damage of glandular mucosa cells of the stomach (9).

Physiological protective factors related to the glandular mucosa are stomach blood perfusion, gastric bicarbonate production, and mucus secretion. These factors are mainly influenced by the local production of prostaglandin E2 ([PGE.sub.2]). However, once COX-1 and 2 are inhibited non specifically by NSAID, such as PBZ, the [PGE.sub.2] concentration declines and glandular ulcers could appear (10).

Corn oil is a very good source of linoleic acid which is a precursor of arachidonic acid, which has the ability to increase PGE2 production (10, 11). However, there is scarce information on how this substance could affect the healing process in horses with EGUS and how this could affect the concentration of VFA in gastric juice in these animals. Therefore, we developed an equine model of PBZ-induced gastric ulceration 1) to investigate the influence of this substance on the VFA concentrations in the gastric juice in the animals; and 2) to correlate the gastroscopic findings with the VFA concentrations.

MATERIAL AND METHODS

Animals. Fifteen clinically healthy crossbred horses (11 not pregnant mares and 4 geldings) with an age range between 5 to 20 years and weight range of 270 - 465 kg were included. All the horses came from a free-pasture husbandry system. Two weeks before starting the experiment, the animals were conditioned by deworming and vaccination. The horses were allocated in individual stalls and fed Coastal Bermuda hay and water ad libitum. Furthermore, they received a concentrate supplementation with 13% protein and administered twice daily in the proportion of 1% of body weight.

Study design. This randomized complete block study included three specific times. At time 0, all the horses were evaluated in clinical laboratory and gastroscopic tests to obtain basal data. Time 1 was considered between the 1st day and the 6th day, and included the induction time. Time 2 included the time of evaluation of each treatment (treatment time), which was between the 7th day and the 21st day. This study was performed between May and July of 2012.

Experimental PBZ gastric ulcer induction groups. The 15 horses were randomly allotted into one of three groups, as a follows: in group I (control group), the animals only received 20 ml of water per os (PO), s.i.d, each 24 hours for 6 days; group II received a single PBZ dose of 13.2 mg/kg diluted in 20 mL water on the 6th day of the start of the experiment; group III was medicated with a PBZ dose of 4.4 mg/kg, PO, s.i.d (always diluted in 20 mL of water) for 5 days and on the 6th day received a final PBZ dose of 13.2 mg/kg, PO, diluted in 20 mL water. This induction protocol was designed to study the phenomena of gastroprotection and gastroadaptation the PBZ in the horse, proposed by Martinez Aranzales et al (12).

Treatment groups. After 6 days of placebo (water) or PBZ gastric ulcer induction, the three groups of horses were treated as a follows: group I received 30 mg/kg sucralphate, PO, t.i.d, for 2 weeks; group II received 70 mL/100 kg of body weight corn oil, PO, twice a day, for 2 weeks; group III was treated with 90 mL/100 kg of body weight corn oil, PO, twice a day, for 2 weeks. The volume of corn oil used in each group, were extrapolated from work in the species (10, 13). The variables gender and age were blocked in this experiment.

Gastroscopy examination. Gastroscopy examination was performed on day 0, day 7 and day 21. After a solid fed fasting period of 12 hours and water deprivation time of 4 hours, the horses were sedated with detomidine (10 [micro]g/kg/ IV, Dormiun V, Agener Uniao, Brazil) and a 12 mm -width- and 3 m -long- flexible videoendoscope (PortaScope[R], 1800PVS, United State) was introduced into the stomach by nasogastric placement. The lesions observed were recorded and classified according to the scoring system of McAllister et al (14) (Table 1).

Determination of the concentration of VFA in gastric fluid. Prior to each endoscopy evaluation and with the help of a gastroscope, 5 mL gastric fluid (juice) were taken and deposited in glass flasks with 1 mL metaphosporic acid for later determination of the concentration of the VFA (acetic, propionic, butyric and lactic acids) by gas chromatography (7), through reference equipment Shimadzu (Shimadzu (Gas Chromatograph GC-17a /auto inyector AOC--20i Shimadzu).

Statistical analysis. Data were analyzed with the software SPSS 19.0 (IBM, SPSS Inc, USA). Lilliefors and Bartlett tests were used to assess normality and homoscedasticity in the studied variables. Therefore, data from butyric and acetic acids were transformed to log+4. The Tukey test and t test were used for comparison of means for acetic and butyric acid, respectively. Data from propionic and lactic acids did not present a normal distribution even after attempting several transformations. These data were compared by Friedman and Kruskal-Wallis tests. P <0.05 was considered statically significant for all the tests. The calculated power of this study was 0.8 (80%) using a sample size of 5 horses for experimental group and alpha error level of 5%.

This experimental study was approved by the local ethical committee for animal experimentation of the University Federal of Minas Gerais--Brazil, with protocol number 234/09. The study was conducted at the School of Veterinary Medicine at the University of Minas Gerais in Belo Horizonte, Brazil, located at latitude 19[degrees]55' S and longitude 43[degrees]56' W Gr, and an altitude of 832 m with kind of climate Cwa (according to Kopper classification), featuring winter predominantly dry summer rainy, according to the National Institute of Meteorology of Brazil.

RESULTS

All horses finished the period of the study without manifesting any clinical sign or metabolic alteration related to systemic illness.

Gastroscopy findings. At first gastroscopy assessment (zero day) no injuries were reported. Later on day seven post-induction, it was found that the protocol based in PBZ and even the effect of confinement produced lesions in the both non-glandular and glandular areas of the stomach. In general, these lesions presented a McAllister et al (14) degree for number and severity ranging from 1 to 3. Finally, at day 21 post-treatment it was found that all treatments (corn oil at two doses and sucralphate) produced healing of the injured the glandular area of the gastric mucosa. However, neither of the two corn oil treatments affected healing of the gastric ulcers located in the non-glandular area. Therefore, once the completed experimental phase, these animals were treated with proton-pump inhibitor (antacid) in traditional scheme for full recovery.

VFA concentrations in gastric fluid. The concentrations of VFA determined in the gastric contents in the two experimental phases are presented in tables 2 and 3. The concentrations of acetic and butyric acids were highest in the gastric juice (2.32-3.80 and 1.64-2.39 mmol/L, respectively), where butyric acid represented most of the total concentration of VFA (Table 2). Propionic and lactic acids were present in lower concentrations (0.0015-0.12 and 0.05-0.61 mmol/L, respectively).

The baseline concentration (t0) of propionic acid showed significant differences between groups I and II. In the treatment phase, propionic acid concentration was significantly different in group II, between the second and third weeks. In the same phase, lactic acid concentration of group III was significantly different between the first and third weeks (Table 3). When the two experimental phases were compared, the concentration of propionic acid in group I showed a significant difference between the baseline and the final measurement. The same was also observed for lactic acid in groups II and III during the same period of time. Butyric and acetic acids did not present significant alterations in any groups and period of time evaluated during the study (Table 2).

DISCUSSION

EGUS is characterized by several predisposing factors that contribute to the high frequency of presentation of this gastric pathology in horses. Furthermore, the susceptibility of each of the areas of the stomach zones is also known. Gastric lesions are produced in horses by the action of NSAID and by situations with potential for generating stress, like confinement (15)c. These two factors were considered in the present study for inducing gastric ulceration in the horses included. The protocol used in the present study generated ulcers in both, glandular and non-glandular mucosa. This situation allowed evaluation of the dynamics of the concentrations of the VFA under the effect of PBZ and participation of VFA in the healing of lesions during the administration of corn oil and sucralphate.

Horses in this study presented an absence of clinical signs and changes in their haemogram and biochemistry panel. These findings may be because these horses developed mild gastric ulcers with lower scores in number and severity(14). However, the severity of an ulcer is not always correlated with the intensity of the clinical signs (16). This situation prevents determination of the clinical relevance of the degree of ulceration in some equine patients (15,17). It is also important to consider the participation of the continued supply of food in maintaining gastric pH gradient, which results in temporary relief with little expression of clinical signs.

Total VFA concentration in the gastric contents of the horses in this study was higher compared with the concentration reported by Frank et al (13). In that study, corn oil and rice oil were added preventively to diminish gastric lesions associated with feed management. However, total VFA concentration of the present study was lower than those reported for diets based on concentrate and alfalfa hay (7). However, it is necessary to consider the nature of the present study, since the dynamics of VFA concentrations were measured in the presence of experimentally induced ulcers.

The differences found in the results of this study may derive from several factors that should be considered for their interpretation. In physiological conditions, the VFA concentration decreases markedly five hours after each meal (18). In the present study, sampling was performed after 12-14 hours of fasting. However, it is possible that the water restriction period was not enough to avoid its diluting effect on the gastric VFA concentration of the horses. On the other hand, the production of VFA is dependent on the type, quality, and quantity of food (7). This last factor varies widely in the different research studies. Furthermore, the bacterial flora adapted to the gastric environment is also involved in the fermentation processes; consequently, it may affect the VFA concentration (19).

Furthermore, it is likely that the total VFA concentration had also been changed by volatilization processes prior to the collection or by transformation to carbon dioxide, [beta]-hydroxybutyrate, and acetoacetate, which normally occur with these acids (5). In addition, the animals in this study were fed a diet low in fermentable carbohydrates (approximately 2.5 kg of concentrated and distributed on average twice/day), unlike other studies where these types of carbohydrates were used (13).

Although, in the gastric ulcer induction period of this study no significant differences in the VFA concentrations were observed between the groups of horses, the VFA data showed slight alterations. However, as shown in the control group, the effect of confinement may have had a greater action on the gastric ulceration in the horses than PBZ. However, it is also necessary to consider that VFA have ulcerogenic effects in the equine stomach (5, 7) and are altered in horses with right dorsal colitis induced by PBZ (20).

In vitro studies employing the Ussing chamber demonstrated the great ulcerogenic capacity of VFA. In this in vitro system, the concentration of each specific VFA is maintained in a constant fashion in the mucosa of the stomach (5, 6). However, this procedure excludes other natural components of the gastric environment that are involved in some way in its gastrolesive action. In contrast, an in vivo model includes all the components from the natural stomach environment, such as the motility, gastroduodenal reflux, interdigestive or basal condition, fluctuations in hydrochloric acid and gastric flora, amongst others, all of importance in the pathophysiology of EGUS. However, as in this study, some variables are characterized as being complicated to control or quantify.

The ulcerogenic effect of VFA depends on the pH in the gastric lumen. Specifically, at a pH lower than 4, VFA accentuate the alterations in the non-glandular mucosa area of the equine stomach, specifically in the area adjacent to the margo plicatus the lesser curvature (5, 6). In the present study, the perpetuating injuries focused in the non-glandular mucosa area were possibly derived due to the no ionization of the VFA assed (acetic, propionic and butyric acids) and, the low pH of gastric contents (2.38-3.77) in the groups treated with corn oil, which the ulcers did not completely heal.

It is know that VFA modify the bioelectric properties of the gastric mucosa by decreasing sodium transport. This results in different levels of change in the cell membrane permeability, according to the type of acid (5, 6, 21). Acetic acid showed the highest concentration (59%) in the gastric contents of the horses of this study. This result is similar to that of other studies (5, 6, 7), but it has been reported that in horses treated with corn and rice oils, this VFA may reach up to 85% of gastric contents (13). However, acetic acid causes discrete lesions in the gastric mucosa.

Contrary to what was reported by Frank et al (13), the gastric contents of this study showed butyric acid concentrations of 40%. "In vitro"studies have shown that butyric acid has a more damaging effect on the non-glandular mucosa area (5). The higher butyric acid concentrations reported in this study may be related to the extensive ulceration observed in the non-glandular mucosa area of the horses in this study.

Propionic acid has a moderate gastrolesive effect (5). However, the concentration of this VFA in the gastric juices evaluated was lower (0.0015-0.12 mmol/L) than those reported in the literature. Therefore, the ulcerogenic role of propionic acid was possibly minimal in the animals in this study. The differences in the concentration of propionic acid between different studies are possibly associated with the aforementioned factors, such as feed manipulation and prolonged fasting periods.

There are several studies that describe abundant production of lactic acid in the equine stomach (19), especially immediately after consumption of food (18). In contrast, the concentrations of lactic acid reported in the present study were lower in comparison with those of other studies. The fasting time and type and feeding scheme may have influenced these results. In vitro studies have demonstrated that VFA has minimal ulcerogenic capacity in the non-glandular mucosa of the horse (21). The effect of lactic acid is dependent on the amount and time of exposure to the mucosa; therefore, it is possible to infer that this VFA plays a poor role in the generation of gastric ulcers in the horses of this study.

In conclusion, the corn oil and sucralphate treatments did not lead to differences in the concentration of acetic acid and butyric acid in this study. In contrast, these treatments produced different gastric juice concentrations of propionic acid and lactic acid. However, the small quantity and proportion of these VFA with respect to acetic acid and butyric acid, let us to think that propionic acid and lactic acid were no relevant VFA during the induction and treatment periods in the horses of this study. Conversely, the gastric acids with the highest concentrations, in particular butyric acid, possibly perpetuated the lesions in the non-glandular mucosa area, where the corn oil showed no therapeutic benefit. These results verify the harmful effect of some AGV on non-glandular mucosa, however it should be considered restorative action through induction of prostaglandins per treatment in the glandular mucosa.

Finally, VFA must be continuously measured over short periods of time to eliminate the effect of volatility and metabolic transformations of these substances in the equine gastric environment. Furthermore, it is necessary to bear in mind the dynamics of the phases of emptying of the stomach to reduce their impact on the results and to effectively compare new results with reported "in vitro" studies.

Acknowledgments

The authors thank people from the laboratory of animal nutrition from the Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Brazil. This research was granted by CNPq, CAPES-PG, UFMG, Brazil, and sustainability project 2013-2014 from CODI--Universidad de Antioquia, Medellin, Colombia.

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(4.) Husted L, Sanchez LC, Olsen SN, Baptiste KE, Merritt AM. Effect of paddock vs. stall housing on 24 hour gastric pH within the proximal and ventral equine stomach. Equine Vet J 2008; 40(4):337-41.

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(6.) Nadeau JA, Andrews FM, Patton CS, Argenzio RA, Mathew AG, Saxton AM. Effects of hydrochloric, valeric and other volatile fatty acids on pathogenesis of ulcers in the nonglandular portion of the stomach of horses. Am J Vet Res 2003; 64(4):413-7.

(7.) Nadeau JA, Andrews FM, Mathew AG, Argenzio RA, Blackford JT, Sohtell M, et al. Evaluation of diet as a cause of gastric ulcers in horses. Am J Vet Res 2000; 61(7):784-90.

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(12.) Martinez Aranzales JR, Candido de Andrade BS, Silveira Alves GE. Orally administered phenylbutazone causes oxidative stress in the equine gastric mucosa. J Vet Parmacol Therap 2014; 38(3):257-264.

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(14.) McAllister CG, Andrews FM, Deegan E, Ruoff W, Olovson SG. A scoring system for gastric ulcers in the horse. Equine Vet J 1997; 29(6):430-3.

(15.) Luthersson N, Nielsen KH, Harris P, Parkin TDH. Risk factors associated with equine gastric ulceration syndrome (EGUS) in 201 horses in Denmark. Equine Vet J 2009; 41(7):625-30.

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DOI:10.21897/rmvz.830

Jose Martinez A, [1] * Ph.D, Jorge Carmona, [2] Ph.D, Geraldo Silveira A, [3] Ph.D.

[1] Universidad de Antioquia, Facultad de Ciencias Agrarias, Escuela de Medicina Veterinaria, Linea de Investigacion en Medicina y Cirugia Equina (LIMCE), Grupo Centauro, Carrera 75 No. 65-87. AA 1226. Medellin, Colombia. [2] Universidad de Caldas, Departamento de Salud Animal, Grupo de Investigacion Terapia Regenerativa, Calle 65 No 26-10, Manizales, Colombia. [3] Universidade Federal de Minas Gerais (UFMG), Departamento de Clinica e Cirurgia Veterinarias, Escola de veterinaria, Av. Antonio Carlos 6627, Pampulha, Belo Horizonte, Brasil. * Correspondence: jose.martinez@udea.edu.co

Received: July 2015; Accepted: February 2016.
Table 1. The scoring system of McAllister et al (14)
gastric lesion number and gastric lesion
severity.

Number     Description     Severity            Description
score                       score

0           0 lesions         0       No lesion

1         1-2 localized       1       Appears superficial (only the
             lesions                  mucosa is missing)

2         3-5 localized       2       Deeper than 1 and includes
             lesions                  deeper structures

3         6-10 lesions        3       Multiple lesions and variable
                                      severity (1, 2 and/or 4)

4        >10 lesions or       4       Same as 2 and has an
         diffuse lesions              active appearance (active =
                                      hyperaemic and/or darkened
                                      lesion crater)

                              5       Same as 4 plus active
                                      haemorrhage or adherent
                                      blood clot

Table 2. Means ([+ or -] standard deviations) of the
concentration of acetic acid and butyric acid
in gastric contents of the horses of the study
over a period of 21 days.

                                       Induction
Variable      Group.
                             Day 0                Day 7

Acetic acid     I      3.80 [+ or -] 1.27   2.82 [+ or -] 0.04
(mmol/L)        II     3.20 [+ or -] 0.09   2.79 [+ or -] 0.05
               III     3.44 [+ or -] 0.70   2.85 [+ or -] 0.05

Butyric         I      2.39 [+ or -] 0.04   2.00 [+ or -] 0.01
acid            II     1.61 [+ or -] 0.02   2.12 [+ or -] 0.01
(mmol/L)       III     1.98 [+ or -] 0.02   1.70 [+ or -] 0.01

                                       Treatment
Variable      Group.
                             Day 14               Day 21

Acetic acid     I      2.92 [+ or -] 0.10   2.32 [+ or -] 0.01
(mmol/L)        II     3.04 [+ or -] 0.02   2.39 [+ or -] 0.01
               III     2.72 [+ or -] 0.04   2.46 [+ or -] 0.02

Butyric         I      1.86 [+ or -] 0.01   1.83 [+ or -] 0.01
acid            II     1.88 [+ or -] 0.01   1.83 [+ or -] 0.02
(mmol/L)       III     1.64 [+ or -] 0.01   1.76 [+ or -] 0.01

Table 3. Medians (ranges) of the concentration of propionic acid
and lactic acid in gastric contents of the horses
of the study during 4 weeks.

                                          Induction
Variable        Group
                               Day 0                  Day 7

                I       0.12 (0.02-0.31) aA    0.02 (0.01-0.05) ab
Propionic acid  II      0.01 (0.01-0.05) abB   0.02 (0.01-0.02) ab
(mmol/L)*       III     0.07 (0.08-0.22) AB    0.01 (0.0010-0.05)

Lactic acid     I        0.08 (0.05-0.28)      0.05  (0.03-0.06)
(mmol/L)*       II       0.04 (0.03-0.06) b    0.61 (0.01-0.12) ab
                III      0.08 (0.02-0.40) a    0.05 (0.03-0.06) a

                                         Treatment
Variable        Group
                              Day 14                Day 21

                I       0.02 (0.01-0.05) ab    0.0015 (0-0.01) b
Propionic acid  II      0.03 (0.01-0.05) a      0.1 (0-0.01) b
(mmol/L)*       III     0.012 (0.01-0.13)       0.01 (0-0.041)

Lactic acid     I       0.06  (0.43-0.09)     0.05  (0.04-2.02)
(mmol/L)*       II      0.07 (0.06-0.09) ab   0.10 (0.06-0.13) a
                III     0.10 (0.06-0.27) ab   0.07 (0.06-0.104) b

* Medians followed by different lowercase letters represent significant
differences in same row (p<0.05). Medians followed by different capital
letters represent significant differences in the same column (p<0.05).
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Title Annotation:ORIGINAL
Author:Martinez A., Jose; Carmona, Jorge; Silveira A., Geraldo
Publication:Revista MVZ (Medicina Veterinaria y Zootecnia)
Date:Sep 1, 2016
Words:8015
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