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Follicular dynamics, corpus luteum growth and regression in multiparous buffalo cows and buffalo heifers/Dinamica folicular, crecimiento y regresion del cuerpo luteo en bufalas multiparas y novillas.


Buffaloes have become a species of economic importance in developing countries in tropical and subtropical regions. They show an efficient conversion, are resistant and require relatively low maintenance costs in the tropics, where the constant availability of food is not always ideal (1). In addition, buffalo milk is of high quality and better paid by dairy processing plants; whereas for meat production, weight gains similar to and in some cases higher than those reported for bovine animals in the same conditions have been observed (1,2). This means that buffaloes are a good source of animal protein, both milk and meat (2,3).

In the past 30 years, the world's buffalo population (172, 263, 305) has increased 34%, while during this same period the world cattle inventory has only grown 12%. In addition, since the 1970s the world production of buffalo milk has increased 200% (4). In Colombia, the presence of large expanses of floodable lands with poor soil drainage, high rain seasonality and low fertility, where bovine production fails to be efficient, buffaloes have become in a promising species since it adapts to these conditions and manages efficient production parameters (5). Therefore, the interest in Buffalo production has notably increased in the last 20 years, with an increase in the number of heads and the establishment of an Association of Buffalo Producers (Aso Bufalos de Colombia; 1).

The productivity of the buffalo system is largely limited by the reproductive efficiency of female buffaloes. When compared to female bovines, the reproductive efficiency of the buffalo is affected by characteristics such as: the late onset of puberty (6-8); the poor presentation of signs of estrous that hinder the use of artificial insemination techniques when the estrous is detected (6.8); the long intervals of postpartum anestrus in females that give birth not during the mating station, mainly due to their condition of seasonally polyestrous and short-day breeders (7-9); a lower population of preantral follicles, approximately 10 times less than that in cows, affecting superovulation programs (7,9,10) and a longer gestation (7). However, it is worth highlighting that buffaloes have greater longevity and hardiness than cows and some authors have found that, using genotypes suitable for a specific production, handling and environment system, reproductive parameters can be satisfactory and be deemed a viable production alternative (11,12).

The knowledge and understanding of the physiological phenomena that occur in the bovine ovary allowed the development of reproductive biotechnologies that potentiate the exploitation of female gametes and the reproductive efficiency of cows (13,14). Despite the importance of buffaloes in the global and regional economy, there are still very few studies that deepen in the ovarian physiology of female buffaloes and to date no other experiment has been found where it has been proposed to determine the follicular dynamics of female buffaloes under the conditions of the Colombian tropics; further, comparing the follicular dynamic among multiparous buffaloes (BM) and buffaloes heifers (BN). The objective of this study was to determine the follicular dynamics and the growth and regression pattern of the corpus luteum in Murrah BM and BN subjected to Colombian tropical conditions.


Study site. The experimental phase took place in the facilities of the Centro Internacional de Formacion Agropecuaria (CIFA; north latitude 5[degrees]39.075" and west longitude 74[degrees]34.843"), located in the municipality of Puerto Salgar (Cundinamarca, Colombia). This municipality is located 195 km from Bogota D.C., 117 meters above sea level and has an average temperature of 27[degrees]C and a relative humidity between 75 and 80%.

Animals and grazing. Two experimental groups of 10 each were randomly selected from a population of 500 female buffalo cows. A group with virgin BN and another group with BM. The experimental units selection criteria was: at the time of starting the experiment all animals had regular estrous cycles and were clinically healthy with a body condition of 3 to 3.5 (scale of 1-5) and that all individuals belong to the Murrah breed. The BN cows had an age of 24.72 [+ or -] 1.45 months and BM of 81.97 [+ or -] 31.75 months with 2.7 [+ or -] 0.8 deliveries. To ensure that all animals were under the same conditions they were kept grazing in a experimentation pasture, where they remained until the end of the study. The pasture had Brachiaria mutica established and mineralized salt and water ad libitum.

Synchronization protocol. A transrectal, palpation and ultrasound examination was carried out prior to the start of the synchronization protocol to evaluate the structures present in the ovaries and select the animals that would enter the experiment. A fixed-time artificial insemination protocol was used (Sincrogest, Ourofino, Saude Animal, Brazil) with a progesterone-releasing intravaginal device on day zero, together with the application of 2 mg of estradiol benzoate (Sincrodiol, Ourofino Saude Animal, Brazil). Later on day 8, the intravaginal device was removed and PGF2 alfa was applied (Sincrocio, Ourofino Saude Animal, Brazil). Finally, 1 mg of estradiol benzoate was applied on day 9 (Sincrodiol, Ourofino Saude Animal, Brazil). Fifty-two hours after removing the intravaginal device a new transrectal examination was conducted to determine the response to the treatment by the presence of a preovulatory follicle. No artificial insemination was performed after the detection of estrous. Due to the low intensity of estrous behavior of buffaloes (6.8), all animals with a follicle larger than 10 mm, the presence of uterine tone and the presence of mucous vaginal discharge were considered in estrus (15).

Ultrasound examinations. Fifteen days after observing synchronized estrus, a new estrus detection began and daily ultrasound examinations was performed with an ultrasound device attached to a linear transducer of 7.5 MHz (Mindray DP 2200 Vet). The day of estrus was taken as day zero of the estrous cycle and thereafter, daily ultrasound monitoring was performed in order to make ovarian maps and assessing the number and diameter of the antral follicles and the diameter of the CL (16). After viewing the image of the ovary in the monitor of the ultrasound device, each ovary was scanned at various levels to ensure and capture the greatest amount of structures and determine their correct size, the data from the measurement of each structure were stored for further analysis. These ultrasound exam were carried out until a second estrus was evident and a second ovulation occurred in each one of the buffaloes.

A follicular wave was considered when finding the growth of a dominant follicle and its cohort and the day of emergence of the follicular wave was defined as the day when the first follicle reached a diameter of 4 mm (17). The deviation of the dominant follicle was determined as the moment in which the diameter of the largest follicle was at least two standard deviations above the mean of the follicles of its cohort (18). The dominant follicle was defined as that which grew at least 10 mm and its diameter was greater than that of the other members of its cohort (17,19).

Data processing and statistical analysis. The information from the ovarian map was entered in Excel 2010 spreadsheets, in order to organize the information corresponding to the following variables: length of the estrous cycle, number of follicular waves during the estrous cycle, day of emergence and number of follicles recruited in each wave, day and diameter of the deviation of the dominant follicle (18), maximum diameter reached by the preovulatory follicle and corpus luteum, day and diameter in which the luteal regression began (determined as the second consecutive day in which the diameter of the structure reduces; 18). Each of the variables analyzed was subject to descriptive statistics and t-Student tests were performed to compare the data from BM and BN and those from different follicular growth waves. A significant difference was considered when p<0.05. The data were analyzed in Excel (Microsoft Office) and SAS 9.0 (The SAS Institute Inc). All data were presented as mean [+ or -] standard deviation.


Response to the synchronization protocol.

Of the total number of females that initiated the protocol (10 BM and 10 BN) a preovulatory follicle was found in 85% (9 BM and 8 BN). The daily ultrasound exam began after 15 days.

Length of the estrous cycle and follicular dynamics. No significant differences were found for the length of the estrus cycle between BM and BN (p>0.05), it being 22.00 [+ or -] 4.50 and 22.00 [+ or -] 2.70 days, respectively. The follicular growth pattern occurred in one (n=1; 5.89%), two (n = 14; 82.35%) or three waves (n = 2; 11.76%; (Table 1).

For all the experimental units, the emergence of the first follicular growth wave occurred the day after ovulation, and an average of 8.33 [+ or -] 2.06 and 10.00 [+ or -] 2.72 follicles for BM and BN, respectively were observed on the ultrasound. Follicular deviation in the first wave took place at 4.00 [+ or -] 2.00 days for BM and 4.62 [+ or -] 1.84 days for BN, with a diameter of the dominant follicle of 10.00 [+ or -] 1.4 mm and 8.9 [+ or -] 1.7 mm, respectively. There were no significant differences between the groups for these variables (Table 1). The second wave of follicular growth began at 11.00 [+ or -] 2.00 days for BM and 10.50 [+ or -] 2.82 days for BN. An average of 8.37 [+ or -] 2.26 and 8.00 [+ or -] 1.51 follicular structures were ultrasonographically observed for BM and BN, respectively. On day 14.00 [+ or -] 2.13 and 14.50 [+ or -] 2.32 the deviation of the dominant follicle started in BM and BV, finding a diameter of 10.00 [+ or -] 1.50 and 9.10 [+ or -] 1.60 mm, respectively. The third wave of follicular growth started on day 16.21 [+ or -] 3.10 with 6.50 [+ or -] 1.70 follicles recruited. Only two multiparous BM showed three waves of follicular growth. The maximum diameter reached by the preovulatory follicle was 17.00 [+ or -] 4.60 mm for BM and 14.00 [+ or -] 2.90 mm for BN. No significant differences were found between the groups for these variables (Table 1).

When comparing the waves of follicular growth among themselves, regardless of the group, it was found that the interval since the emergence of the wave to the deviation of the dominant follicle was 4.29 [+ or -] 1.89, 3.29 [+ or -] 2.17 and 2.88 [+ or -] 1.30 days for the first, second and third wave, respectively; likewise, the number of follicles was 9.11 [+ or -] 2.47, 8.18 [+ or -] 1.86 and 6.50 [+ or -] 0.70. Finally, the diameter of the dominant follicle in the deviation was 9.56 [+ or -] 1.58 mm for wave one, 9.75 [+ or -] 1.67 mm for wave two and 9.89 [+ or -] 0.72 mm for wave three. There was no statistical difference between the waves of follicular growth (Table 2).

Corpus luteum diameter and growth. Through daily ultrasound exams it was to observe that the maximum luleal diameter was 19.58 [+ or -] 4.16 mm for BM and 17.74 [+ or -] 3.32 mm for BN. Luteal regression took place at 15.22 [+ or -] 5.26 and 17.62 [+ or -] 1.68 days of the estrous cycle for BM and BN, respectively. No significant differences were observed between the groups for these variables (Table 3).


The duration of the estrous cycle in this study showed no significant differences when comparing BN to BM nor when comparing the number of waves of follicular growth. Similar results have been obtained by other authors such as Presicce et al (16). They worked with Mediterranean BM and BN and found that for animals with two waves of follicular growth, the duration of the estrous cycle ranged from 20 to 26 days. However, they found a higher proportion of animals with a single wave of follicular growth, which had a shorter cycle duration (8 to 12 days). Baruselli et al (20), working with Murrah animals, also found 3.33, 66.66 and 33.33% of animals with one, two and three waves respectively. In this study, the duration of the estrous cycle of animals with one wave was 13 days. Awasthi et al (17) found a high proportion of animals with one wave (62.5%) in which the estrous cycle lasted 20.8 [+ or -] 0. 58 days. It is possible that the absence of significant differences in the duration of the estrous cycle in this study is due to the low proportion of animals with a single wave (n = 1) and to the duration of their cycle that was 18 days. The variables that influence the number of waves during the estrous cycle have not been accurately determined yet; however, it has been proposed that the breed, physiological and nutritional conditions, environmental conditions, among others, may affect this variable (19,21,22).

The results obtained in this and other studies are consistent with the fact that the first follicular wave emerges on day one of the estrous cycle (17.23). As to the emergence of the second wave, it occurred at 11 [+ or -] 2.00 days for BM and 10.5 [+ or -] 2. 82 days for BN. These data are similar to those found by other authors who have determined the emergence of the second wave around the day 10 for animals with two waves and around day 8 for animals with three waves (20,23,24). This study evidenced that the number of follicles recruited in the first wave was 8.33 [+ or -] 2.06 and 10 [+ or -] 2.72 and in the second wave 8.37 [+ or -] 2.26 and 8 [+ or -] 1.51, for BM and BN respectively, while for the third wave 6.5 [+ or -] 0.7 follicles were recruited, which showed no statistical differences when making a comparison between groups or waves. Similar results were found by Baruselli et al (20), who, working with Murrah BM, concluded that the number of follicles recruited in the first wave was 7.72 [+ or -] 4.64 and 7.50 [+ or -] 2.75 for animals with estrous cycles of two and three waves respectively, where there are no significant differences.

These results contrast with the experiment carried out in Egypt by Barkawi et al (23), who found a significant difference (p>0.05) in the number of follicles recruited in the first wave when comparing animals with two (6.3 [+ or -] 0.3) and three waves (7.8 [+ or -] 0.4). All the previous results contrast when compared with the number of follicles recruited in a wave of follicular growth in cows, where it has been estimated that: "the emergence of the follicular wave is characterized by the sudden growth of 8-41 small follicles" (25). It is likely that the marked difference between these species is a consequence of the lower number of primordial follicles of buffalo females with respect to bovine females (7,9,10).

The results obtained for the deviation of the dominant follicle showed that it occurs approximately four days after the emergence of the wave and when it has a diameter of 8-10 mm. Again, no differences were found when comparing between groups or waves. Sartori et al (26), working with Holstein cows, determined that the dominant follicle deviated when it reached 9.80 [+ or -] 0.30 mm in lactating cows and 8.30 [+ or -] 0.20 mm in heifers. In another study, Sartori et al (27) found that the diameter at which the dominant follicle deviate was 9.10 [+ or -] 0.40 mm in Holstein cows. Working with BN, Gimenes et al (28) found that the dominant follicle is diverted when it reached 7.20 [+ or -] 0.20 mm. This result is lower than that found in this study and could be explained by the method used to determine follicular deviation in both cases. In the present study, it was determined that follicular deviation was the moment when the diameter of the largest follicle was at least two standard deviations above the average of the follicles of its cohort (18), while, Gimenes et al (28) defined the beginning of the deviation as "the end of the common growth phase, when differences between the diameters of the two largest follicles were detected."

Gimenes et al (28,29) conducted experiments to characterize follicular deviation in bovine heifers and BN, respectively. They determined that the deviation in heifers occurs between 1.5-4 days after ovulation, when the follicle has a size of 5 to 7 mm (29), while in BN it was determined that the deviation occurred 2.6 [+ or -] 0.2 days postovulation at a size of 7.2 [+ or -] 0.2 (28).

The maximum diameter reached by the preovulatory follicle in the animals under study was 17 [+ or -] 4.6 and 14 [+ or -] 2.9 mm for BM and BN. Other studies report similar diameters when working with buffaloes and larger when working with cows. Baruselli et al (20) found that the preovulatory diameter of BM was 1.57, 1.55 [+ or -] 0.16 and 1.34 [+ or -] 0.13 cm for animals with one, two or three waves of follicular growth, respectively. Similarly, Awasthi et al (17) working with Mehsana buffaloes, found that the maximum diameter of the preovulatory follicle was 12.94 [+ or -] 0.59 and 16.03 [+ or -] 3.30 for animals with one or two waves. However, in a study comparing the pattern of follicular growth between Mediterranean BM and BN carried out in Italy by Presicce et al (16), significant differences were found (p>0.05) between the maximum diameter of the ovulatory follicle with 13.8 [+ or -] 0.6 and 11.0 [+ or -] 0.7. It is likely that the marked differences in the conditions under which the experiment of Presicce et al (16) was carried out and this study, are responsible for this discrepancy between results.

When assessing the characteristics of the CL, it was determined that the maximum diameter was 19.58 [+ or -] 4.16 mm for BM and 17.74 [+ or -] 3.32 mm for BN. Despite the apparent numerical difference in these results, no significant differences were found. In the study of Barkawi et al (23), the maximum diameter for the CL was found to be 15.00 [+ or -] 0.40 for BN in Egypt, while Di Francesco et al (30) working with Mediterranean buffaloes during the mating season, found that the CL at day 10 post insemination reached 18.6 [+ or -] 0.9 mm and 20.2 [+ or -] 0.6 mm in empty and pregnant buffaloes, respectively. They found no statistical differences when comparing the growth pattern of the CL in animals in the reproductive season and in the transition period to the next stadium. However, differences were evidenced in the synthesis of P4, where animals in the reproductive season showed higher concentrations of this hormone. In this study the regression of the CL was found from day 15.22 [+ or -] 5.26 for the BM group and 17.62 [+ or -] 1.68 for the BN group, data consistent with those previously reported by other authors. Satheshkumar et al (31) found that in Murrah BM the luteal regression began on day 16.20 [+ or -] 50.76; while Barkawi et al (23) determined that the half-life of the CL was 17.1 [+ or -] 0.8 days in buffaloes with normal estrous cycles, while in females with follicular cysts and persistent CL, this half-life increased to 24.80 [+ or -] 4.30 and 28.30 [+ or -] 6.10 days, respectively. In conclusion, the follicular dynamics and growth pattern and regression of the CL in BM and BN under Colombian tropical conditions are similar to what has been previously reported by other authors. However, it is necessary to conduct new studies with a larger number of animals involved.


To the Centro Internacional de Formacion Agropecuaria (CIFA) and its entire staff for their invaluable cooperation in the preparation of this study.


Los bufalos se han convertido en una especie de importancia economica en los paises en vias de desarrollo en regiones tropicales y subtropicales. Estos tienen una eficiente conversion, son resistentes y requieren de un relativo bajo costo de mantenimiento en las zonas tropicales, donde la disponibilidad constante de alimento no siempre es la ideal (1). Ademas, la leche de bufala es de alta calidad y es mejor pagada por las plantas procesadoras de lacteos; mientras que, para la produccion de carne, se han reportado ganancias de peso similares y en algunos casos superiores a las reportadas para los bovinos en las mismas condiciones (1,2). Esto ha hecho que los bufalos se constituyan en una buena fuente de proteina animal, tanto de leche como de carne (2,3).

En los ultimos 30 anos la poblacion mundial bufalina (172'263.305) se ha incrementado en 34%, mientras que en este mismo periodo el inventario bovino mundial solamente ha crecido en 12%. Adicionalmente, desde la decada de los setenta, la produccion mundial de leche de bufala ha aumentado el 200% (4). En Colombia, la presencia de grandes extensiones de tierras inundables, con mal drenaje de suelos, alta estacionalidad de lluvias y baja fertilidad, en donde la produccion bovina no logra ser eficiente, han hecho que los bufalos se conviertan en una especie promisoria ya que se adapta a estas condiciones y logra paramentos productivos eficientes (5). Por esto, en los ultimos 20 anos ha aumentado notablemente el interes por la produccion bufalina, viendose un aumento en el numero de cabezas y en la constitucion de una Asociacion de Productores de Bufalos (Aso bufalos de Colombia; 1).

La productividad del sistema bufalino esta en gran parte limitada por la eficiencia reproductiva de las bufalas. Al comparar con la hembra bovina, la eficiencia reproductiva de la bufala se ve afectada por caracteristicas como: la presentacion tardia de la pubertad (6-8); la pobre presentacion de los signos de celo que dificultan la utilizacion de la tecnica de inseminacion artificial a celo detectado (6,8); los largos intervalos de anestro postparto en hembras que paren por fuera de la estacion de apareamiento, debido principalmente a su condicion de poliestricas estacionales de dias cortos (7-9); una menor poblacion de foliculos preantrales, aproximadamente 10 veces menor que en la vaca, afectando los programas de superovulacion (7,9,10) y una gestacion mas larga (7). Sin embargo, cabe resaltar que las bufalas presentan una mayor longevidad y rusticidad que las vacas y que algunos autores han encontrado que, empleando genotipos adecuados para un sistema de produccion, manejo y ambiente determinados, los parametros reproductivos pueden mostrarse satisfactorios y considerarse una alternativa viable de produccion (11,12).

El conocimiento y entendimiento de los fenomenos fisiologicos que se presentan en el ovario bovino permitio el desarrollo de biotecnologias reproductivas que potencializan la explotacion de los gametos femeninos y la eficiencia reproductiva de las vacas (13,14). A pesar de la importancia que tienen los bufalos en la economia mundial y regional, existen aun pocos estudios que ahonden la fisiologia ovarica de la bufala y a la fecha no se ha encontrado otro experimento donde se intente determinar la dinamica folicular de la hembra bufalina en las condiciones del tropico bajo colombiano; aun mas, que comparen los eventos de la dinamica folicular entre bufalas multiparas (BM) y bufalas novillas (BN). El objetivo del presente estudio fue determinar la dinamica folicular, el patron de crecimiento y regresion del cuerpo luteo en BM y BN de la raza Murrah sometidas a condiciones del tropico bajo colombiano.


Sitio de estudio. La fase experimental se llevo a cabo en las instalaciones del Centro Internacional de Formacion Agropecuaria (CIFA; latitud norte 5[degrees]39.075" y longitud oeste 74[degrees]34.843"), ubicado en el municipio de Puerto Salgar (Cundinamarca, Colombia). Este municipio se encuentra a 195 km de Bogota DC, a 117 msnm y cuenta con una temperatura promedio de 27[degrees]C y una humedad relativa entre el 75 y el 80%.

Animales y pastoreo. De una poblacion de 500 hembras bufalinas, se seleccionaron aleatoriamente dos grupos experimentales de 10 individuos cada uno. Un grupo con BN virgenes y otro grupo con BM. Se tuvieron como criterios de seleccion de las unidades experimentales: que todos los individuos estuvieran ciclando y se encontraran clinicamente sanos y con una condicion corporal de 3 a 3.5 (escala de 1-5) al momento de iniciar el experimento y que todos los individuos fueran de la raza Murrah. Las BN tenian una edad de 24.72 [+ or -] 1.45 meses y las BM de 81.97 [+ or -] 31.75 meses con 2.7 [+ or -] 0.8 partos. Para garantizar que todos los animales se encontraban bajo las mismas condiciones fueron mantenidos en pastoreo en un potrero de experimentacion, donde permanecieron hasta finalizar el estudio. El potrero contaba con una pastura establecida de Brachiaria mutica y disposicion de sal mineralizada y agua ad libitum.

Protocolo de sincronizacion. Previo al inicio del protocolo de sincronizacion, se realizo un examen transrectal, de palpacion y ultra sonido con el fin de evaluar las estructuras presentes en los ovarios y realizar la seleccion de los animales que ingresarian al experimento. Fue empleado un protocolo de inseminacion artificial a tiempo fijo con dispositivo intravaginal de liberacion de progesterona (Sincrogest, Ourofino, Saude Animal, Brasil) al dia cero, junto con la aplicacion de 2 mg de benzoato de estradiol (Sincrodiol, Ourofino Saude Animal, Brasil). Posteriormente al dia 8, se retiro el dispositivo intravaginal y se aplico PGF2 alfa (Sincrocio, Ourofino Saude Animal, Brasil). Finalmente al dia 9 se aplico 1 mg de benzoato de estradiol (Sincrodiol, Ourofino Saude Animal, Brasil). Transcurridas 52 horas despues de haber retirado el dispositivo intravaginal, se realizo un nuevo examen transrectal para determinar la respuesta al tratamiento por la presencia de un foliculo preovulatorio Luego de realizarse la deteccion de celos, no se realizo la inseminacion artificial. Debido a la baja intensidad del comportamiento estral de las bufalas (6,8), todos los animales con presencia de un foliculo mayor a 10 mm, presencia de tono uterino y presencia de descarga vaginal mucosa fueron considerados en celo (15).

Seguimiento ultrasonografico. Quince dias despues de evidenciarse los celos sincronizados, se inicio una nueva deteccion de celos y al seguimiento ultrasonografico diario, con un equipo de ultrasonido acoplado a un transductor lineal de 7.5 MHz (Mindray DP 2200 Vet). El dia del celo fue tomado como el dia cero del ciclo estral y a partir de ese momento se realizaron seguimientos ultrasonograficos diarios con el fin de hacer mapeos ovaricos y evaluar el numero y diametro de los foliculos antrales y el diametro del CL (16). Luego de visualizar la imagen del ovario en el monitor del equipo de ultrasonido, cada ovario fue escaneado en varios planos para asegurar y capturar la mayor cantidad de estructuras y determinar su tamano correcto, los datos provenientes de la medicion de cada estructura se almacenaron para su analisis posterior. Estos seguimientos se realizaron hasta cuando se evidencio un segundo celo y una segunda ovulacion en cada una de las bufalas.

Una onda folicular fue considerada al encontrar el crecimiento de un foliculo dominante y de su cohorte y el dia de emergencia de la onda folicular se definio como el dia que el primer foliculo alcanzaba un diametro de 4mm (17). La desviacion del foliculo dominante se determino como el momento en el que el diametro del foliculo mayor era de al menos dos desviaciones estandar por encima de la media de los foliculos de su cohorte (18). El foliculo dominante se definio como aquel que crecio por lo menos 10 mm y su diametro fue mayor que el de los demas miembros de su cohorte (17,19).

Procesamiento de datos y analisis estadistico. La informacion proveniente del mapeo ovarico fue tabulada en hojas de calculo del programa Excel 2010, con el objetivo de organizar la informacion correspondiente a las siguientes variables: duracion del ciclo estral, numero de ondas foliculares durante el ciclo estral, dia de emergencia y numero de foliculos reclutados en cada onda, dia y diametro a la desviacion del foliculo dominante (18), diametro maximo alcanzado por el foliculo preovulatorio y por el cuerpo luteo, dia y diametro en los cuales se inicio la regresion luteal (determinada como el segundo dia consecutivo en el cual disminuye el diametro de la estructura; 18). Se realizo estadistica descriptiva a cada una de las variables analizadas y se realizaron pruebas T de student para comparar los datos provenientes de las BM y BN y los provenientes de las diferentes ondas de crecimiento folicular. Se considero diferencia significativa a partir de p<0.05. Los datos fueron analizados en los programas Excel (Microsoft Office) y SAS 9.0 (The SAS Institute Inc). Todos los datos son presentados como media [+ or -] desviacion estandar.


Respuesta al protocolo de sincronizacion.

Del total de las hembras que iniciaron el protocolo (10 BM y 10 BN) se encontro un foliculo preovulatorio en el 85% (9 BM y 8 BN). Luego de 15 dias se dio inicio al seguimiento ultrasonografico diario.

Duracion del ciclo estral y dinamica folicular.

No se encontraron diferencias significativas para la variable duracion del ciclo estral entre BM y BN (p > 0.05), siendo de 22.00 [+ or -] 4.50 y 22.00 [+ or -] 2.70 dias, respectivamente. El patron de crecimiento folicular ocurrio en una (n = 1; 5.89%), dos (n = 14; 82.35%) o tres ondas (n=2; 11.76%; Tabla 1).

Para el total de las unidades experimentales la emergencia de la primera onda de crecimiento folicular se presento al dia siguiente de evidenciarse la ovulacion, observandose a la ecografia un promedio de 8.33 [+ or -] 2.06 y 10.00 [+ or -] 2.72 foliculos para BM y BN respectivamente. La desviacion folicular en la primera onda ocurrio a los 4.00 [+ or -] 2.00 dias en BM y 4.62 [+ or -] 1.84 dias en BN, con un diametro del foliculo dominante de 10.00 [+ or -] 1.4 mm y 8.9 [+ or -] 1.7 mm respectivamente. No se encontraron diferencias significativas entre los grupos para estas variables (Tabla 1). La segunda onda de crecimiento folicular inicio a los 11.00 [+ or -] 2.00 dias en BM y 10.50 [+ or -] 2.82 dias en BN. Como resultado de la ecografia se observo un promedio de 8.37 [+ or -] 2.26 y 8.00 [+ or -] 1.51 estructuras foliculares para BM y BN, respectivamente. El dia 14.00 [+ or -] 2.13 y 14.50 [+ or -] 2.32 el foliculo dominante se desvio en BM y BV, encontrandose con un diametro de 10.00 [+ or -] 1.50 y 9.10 [+ or -] 1.60 mm, respectivamente. La tercera onda de crecimiento folicular inicio el dia 16.21 [+ or -] 3.10 con 6.50 [+ or -] 1.70 foliculos reclutados. Solamente dos BM multiparas presentaron tres ondas de crecimiento folicular. El diametro maximo alcanzado por el foliculo preovulatorio fue de 17.00 [+ or -] 4.60 mm para BM y 14.00 [+ or -] 2.90 mm para BN. No se encontraron diferencias significativas entre los grupos para estas variables (Tabla 1).

Al comparar las ondas de crecimiento folicular entre si, independientemente del grupo, se encontro que el intervalo desde la emergencia de la onda hasta la desviacion del foliculo dominante fue de 4.29 [+ or -] 1.89, 3.29 [+ or -] 2.17 y 2.88 [+ or -] 1.30 dias para la primera, la segunda y la tercera onda respectivamente; asi mismo, el numero de foliculos fue de 9.11 [+ or -] 2.47, 8.18 [+ or -] 1.86 y 6.50 [+ or -] 0.70. Finalmente el diametro del foliculo dominante en la desviacion fue de 9.56 [+ or -] 1.58 mm para la onda uno, 9.75 [+ or -] 1.67 mm para la onda dos y 9.89 [+ or -] 0.72 mm para la onda tres. No se encontraron diferencias estadisticas entre las ondas de crecimiento folicular (Tabla 2).

Diametro y regresion del cuerpo luteo.

Mediante los seguimientos ecograficos diarios se pudo observar que el diametro maximo alcanzado por el cuerpo luteo en las BM fue 19.58 [+ or -] 4.16 mm y en las BN de 17.74 [+ or -] 3.32 mm. La regresion luteal inicio a los 15.22 [+ or -] 5.26 y 17.62 [+ or -] 1.68 dias del ciclo estral, para BM y BN, respectivamente. No se observaron diferencias significativas entre los grupos para estas variables (Tabla 3).


La duracion del ciclo estral en el presente estudio, no presento diferencias significativas al comparar BM de BN ni al comparar el numero de ondas de crecimiento folicular. Resultados similares han sido obtenidos por otros autores como Presicce et al (16). Ellos trabajaron con BM y BN de la raza mediterranea y encontraron que para animales con dos ondas de crecimiento folicular, la duracion del ciclo estral variaba entre 20 y 26 dias. Sin embargo, ellos encontraron una mayor proporcion de animales con una sola onda de crecimiento folicular, quienes tenian una menor duracion del ciclo (8 a 12 dias). Tambien Baruselli et al (20), trabajando con animales de la raza Murrah encontraron 3.33, 66.66 y 33.33% de animales con una, dos y tres ondas respectivamente. En ese estudio, la duracion del ciclo estral de los animales de una onda fue de 13 dias. Awasthi et al (17) encontraron una alta proporcion de animales con una onda (62.5%) en los que el ciclo estral duro 20.8 [+ or -] 0.58 dias. Es posible que la ausencia de diferencias significativas en cuanto a la duracion del ciclo estral en el presente estudio, se deba a la baja proporcion de animales con una sola onda (n=1) y a que la duracion de su ciclo fue de 18 dias. Aun no se ha determinado con exactitud las variables que influyen en el numero de ondas durante el ciclo estral; sin embargo, se ha propuesto que la raza, el estado fisiologico y nutricional, condiciones medioambientales, entre otras, puedan afectar esta variable (19,21,22).

Los resultados obtenidos en este y los demas estudios analizados, concuerdan en que la primera onda folicular emerge en el dia uno del ciclo estral (17,23). En cuanto a la emergencia de la segunda onda, se presento a los 11 [+ or -] 2.00 dias para BM y 10.5 [+ or -] 2.82 dias para BN. Estos datos son similares a los encontrados por otros autores quienes han determinado la emergencia de la segunda onda alrededor del dia 10 para animales de dos ondas y alrededor del dia 8 para animales de tres ondas (20,23,24). El presente estudio evidencio que el numero de foliculos reclutados en la primera onda fue de 8.33 [+ or -] 2.06 y 10 [+ or -] 2.72 y en la segunda onda de 8.37 [+ or -] 2.26 y 8 [+ or -] 1.51, para BM y BN respectivamente, mientras que para la tercera onda se reclutaron 6.5 [+ or -] 0.7 foliculos, donde no existieron diferencias estadisticas al hacer la comparacion entre grupos ni entre ondas. Similares resultados fueron encontrados por Baruselli et al (20), quienes trabajando con BM de la raza Murrah concluyeron que el numero de foliculos reclutados en la primera onda fue de 7.72 [+ or -] 4.64 y 7.50 [+ or -] 2.75 para animales con ciclos estrales de dos y tres ondas respectivamente, donde no existen diferencias significativas.

Estos resultados contrastan con el experimento realizado en Egipto por Barkawi et al (23), quienes encontraron una diferencia significativa (p>0.05) en el numero de foliculos reclutados en la primera onda al comparar animales de dos (6.3 [+ or -] 0.3) y tres ondas (7.8 [+ or -] 0.4). Todos los resultados anteriores contrastan al comparase con el numero de foliculos que se recluta en una onda de crecimiento folicular en vacas, en las que se ha estimado que: "la emergencia de la onda folicular se caracteriza por el subito crecimiento de 8-41 foliculos pequenos" (25). Es probable que la marcada diferencia entre estas especies sea consecuencia del menor numero de foliculos primordiales con los que cuentan las hembras bufalinas con respecto a las bovinas (7,9,10).

Los resultados obtenidos para la desviacion del foliculo dominante demostraron que esta ocurre aproximadamente cuatro dias luego de la emergencia de la onda y cuando este tiene un diametro de 8-10 mm. Nuevamente no se encontraron diferencias al comparar entre grupos ni entre ondas. Sartori et al (26) trabajando con bovinos de la raza Holstein, determinaron que el foliculo dominante se desvio cuando tenia 9.80 [+ or -] 0.30 mm en vacas lactantes y 8.30 [+ or -] 0.20 mm en novillas. En otro estudio, Sartori et al (27) encontraron que el diametro en el cual el foliculo dominante se desviaba en 9.10 [+ or -] 0.40 mm en vacas Holstein. Trabajando con BN, Gimenes et al (28) encontraron que el foliculo dominante se desviaba cuando alcanzaba 7.20 [+ or -] 0.20mm. Este resultado, es inferior al encontrado en el presente estudio y podria explicarse por el metodo utilizado para determinar la desviacion folicular en ambos casos. En el presente estudio, se determino que la desviacion folicular era el momento en el que el diametro del foliculo mayor era de al menos dos desviaciones estandar por encima de la media de los foliculos de su cohorte (18), mientras que, Gimenes et al (28) definieron el inicio de la desviacion como "el fin de la fase de crecimiento comun, cuando se detectaron diferencias entre los diametros de los dos foliculos mayores".

Gimenes et al (28,29) realizaron experimentos para caracterizar la desviacion folicular en novillas bovinas y BN, respectivamente. Ellos determinaron que la desviacion en novillas ocurre entre 1.5-4 dias post ovulacion, cuando el foliculo tiene un tamano de 5 a 7 mm (29), mientras que en BN la desviacion se determino que sucedia 2.6 [+ or -] 0.2 dias post-ovulacion a un tamano de 7.2 [+ or -] 0.2 (28).

El diametro maximo alcanzado por el foliculo preovulatorio en los animales estudiados fue de 17 [+ or -] 4.6 y 14 [+ or -] 2.9 mm para BM y BN. Otros estudios reportan diametros similares al trabajar con bufalas y mayores al trabajar con vacas. Baruselli et al (20) encontraron que el diametro preovulatorio de BM fue de 1.57, 1.55 [+ or -] 0.16 y 1.34 [+ or -] 0.13 cm para animales con una, dos y tres ondas de crecimiento folicular respectivamente. Tambien, Awasthi et al (17) trabajando con bufalas de la raza Mehsana, encontraron que el diametro maximo del foliculo preovulatorio fue de 12.94 [+ or -] 0.59 y 16.03 [+ or -] 3.30 para animales de una y dos ondas. Sin embargo, en un estudio donde tambien se compara el patron de crecimiento folicular entre BM y BN de la raza Mediterranea realizado en Italia por Presicce et al (16) se encontraron diferencias significativas (p > 0.05) entre el diametro maximo del foliculo ovulatorio con 13.8 [+ or -] 0.6 y 11.0 [+ or -] 0.7. Es probable que, las marcadas diferencias en las condiciones en que se realizo el experimento de Presicce et al (16) y el presente estudio, sean responsables por esta discrepancia entre los resultados.

Al evaluar las caracteristicas del CL se determino que el diametro maximo fue de 19.58 [+ or -] 4.16 mm en BM y 17.74 [+ or -] 3.32 mm en BN. A pesar de la aparente diferencia numerica en estos resultados, no se encontraron diferencias significativas. En el estudio de Barkawi et al (23) se encontro un diametro maximo del CL de 15.00 [+ or -] 0.40 en BN en Egipto, mientras que, Di Francesco et al (30) trabajando con bufalas de la raza mediterranea durante la estacion de apareamientos, encontraron que el CL al dia 10 post inseminacion alcanzaba 18.6 [+ or -] 0.9 mm y 20.2 [+ or -] 0.6 mm en bufalas vacias y prenadas respectivamente. Ellos no encontraron diferencias estadisticas al comparar el patron de crecimiento del CL en animales en la estacion reproductiva y en el periodo de transicion hacia la estacion siguiente. Sin embargo, si lograron evidenciar diferencias en la sintesis de P4, donde los animales en la temporada reproductiva mostraron mayores concentraciones de esta hormona. En el presente estudio, la regresion del CL se encontro a partir del dia 15.22 [+ or -] 5.26 para el grupo BM y en 17.62 [+ or -] 1.68 para el grupo BN, datos que concuerdan con lo previamente reportado por otros autores. Satheshkumar et al (31) encontraron que en BM de la raza Murrah la regresion luteal iniciaba el dia 16.20 [+ or -] 50.76; mientras que, Barkawi et al (23), determinaron que la vida media del CL era de 17.1 [+ or -] 0.8 dias en bufalas con ciclos estrales normales, mientras que en hembras con presencia de quistes foliculares y CL persistentes, esta vida media incrementaba a 24.80 [+ or -] 4.30 y 28.30 [+ or -] 6.10 dias respectivamente. En conclusion, la dinamica folicular y el patron de crecimiento y regresion del CL de BM y BN ubicadas en condiciones de tropico bajo colombiano son similares a lo que ha sido previamente reportado por otros autores. Sin embargo, es necesario realizar nuevos estudios donde se aumente el numero de animales involucrados.


Al Centro Internacional de Formacion Agropecuario (CIFA) y la de todo su personal, por su invaluable colaboracion en la elaboracion de este estudio.


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Alejandro Ojeda R, [1] * Esp, Ricardo Londono O, [1] MVZ, Carlos Gutierrez R, [1] MV,

Angela Gonella-Diaza, [2] MSc.

(1)Fundacion Educativa Para La Equidad y el Desarrollo Rural. Carrera 1a A No 11-130 Oficina 505 Torre 1. Chia, Cundinamarca, Colombia. (2) Universidad Cooperativa de Colombia, Faculty of Veterinary Medicine and Animal Husbandry, Animal nutrition, toxicology and reproduction Research Group. Calle 30 No. 33--51. Bucaramanga, Colombia. * Correspondence:

Received: May 2013; Accepted: December 2013.
Table 1. Duration of the estrous cycle and follicular dynamics of
multiparous Murrah buffalo cows and heifers in the Colombian tropics.

                    Multiparous            Buffalo           P
Variable            Buffalo Cows           Heifers         valor

Duration of      22.00 [+ or -]4.50   22.00 [+ or -]2.70   0.74
the estrous
cycle (days)

Number of        2.00 [+ or -]0.50    2.12 [+ or -]0.35    0.56

Emergence of             1                    1             NC
wave 1
(day of the
estrous cycle)

Number of        8.33 [+ or -]2.06    10.00 [+ or -]2.72   0.17
follicles in
wave 1

Deviation of            4.00          4.62 [+ or -]1.84    0.42
wave 1

(day of the         [+ or -]2.00
estrous cycle)

Diameter of            10.00          8.90 [+ or -]1.70    0.13
the deviation
of wave 1

(mm)                [+ or -]1.40

Emergence of           11.00          10.50 [+ or -]2.82   0.68
wave 2

(day of the         [+ or -]2.00
estrous cycle)

Number of        8.37 [+ or -]2.26    8.00 [+ or -]1.51    0.70
follicles in
wave 2

Deviation of           14.00          14.50 [+ or -]2.32   0.66
wave 2

(day of the         [+ or -]2.13
estrous cycle)

Diameter of            10.00          9.10 [+ or -]1.60    0.11
the deviation
of wave 2

(mm)                [+ or -]1.50

Emergence of           16.21
wave 3

(day of the         [+ or -]3.10
estrous cycle)

Number of        6.50 [+ or -]0.70            -             ---
follicles in
wave 3

Deviation of           19.09                 ---            ---
wave 3

(day of the         [+ or -]0.99

Diameter of             9.89                 ---            ---
the deviation
of  wave 3

(mm)                [+ or -]0.72

Diameter               17.00          14.00 [+ or -]2.90   0.22
of the

(mm)                [+ or -]4.60

NC: Not calculated

Table 2. Number of follicles, diameter of deviation and interval of
the emergence of deviation in follicular waves of multiparous Murrah
buffalo cows and heifers in the Colombian tropics. colombiano.

Variable                   Wave 1         Wave 2         Wave 3

Number of follicles         9.11           8.18           6.5
                          [+ or -]2.47   [+ or -]1.86   [+ or -]0.70

Diameter of deviation       9.56           9.75           9.89
(mm)                      [+ or -]1.58   [+ or -]1.67   [+ or -]0.72

Interval of emergence of    4.29           3.29           2.88
deviation (days)          [+ or -]1.89   [+ or -]2.17   [+ or -]1.30

No significant differences were detected at 0.05

Table 3. Maximum diameter and luteal regression in
multiparous Murrah buffalo cows and heifers                     p
in the Colombian tropics.

Variable               Multiparous            Buffalo          valor
                         Buffalo              Heifers

Maximum diameter    19.58 [+ or -]4.16   17.74 [+ or -]3.32    0.33
of the corpus
luteum (mm)

Corpus luteum       15.22 [+ or -]5.26   17.62 [+ or -]1.68    0.23
(days of the
estrous cycle)
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Title Annotation:ORIGINAL
Author:Ojeda, Alejandro R.; Londono, Ricardo O.; Gutierrez, Carlos R.; Gonella-Diaza, Angela
Publication:Revista MVZ (Medicina Veterinaria y Zootecnia)
Date:May 1, 2014
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