Follicular dynamics , corpus luteum growth and regression in multiparous buffalo cows and buffalo heifers

Objective. Characterize the follicular dynamics and luteal growth and regression pattern of multiparous (MB) and heifer (BH) Murrah buffaloes in Colombian tropical conditions. Material and methods. Ten MB and ten BH were synchronized with a progesterone-releasing intravaginal device. No artificial insemination was performed during the estrous and daily ultrasound examinations were performed 15 days later to determine the number and diameter of the structures present in both ovaries in the subsequent natural estrous cycle. The Student’s T test was used to evaluate differences between MB and BH. All data are presented as mean ± standard deviation. Results. The length of the estrous cycle was 22.00±4.50 days for MB and 22.00±2.70 days for BH. Follicular growth occurs in one (n=1; 5.89%), two (n=14; 82.35%) or three waves (n=2; 11.76%). The first wave initiated the day after ovulation with the recruitment of 8.33±2.06 and 10.00±2.72 follicles in MB and BH, while the second wave started on day 11.00±2.00 and 10.50±2.82, presenting 8.37±2.26 and 8.00±1.51 follicles. The third wave began on day 16.21±3.10 showing 6.50±1.70 follicles, only BM had three waves. The maximum luteal diameter was 19.58±4.16 mm and 17.74±3.32 mm respectively. There were no significant differences between the groups for these variables. Conclusions. These results show that the follicular development in buffaloes occurs in waves, where two waves is the most common pattern, as previously reported by other authors.


INTRODUCTION
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)(7)(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
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.

MATERIALS AND METHODS
Study site.The experimental phase took place in the facilities of the Centro Internacional de Formacion Agropecuaria (CIFA; north latitude 5°39.075" and west longitude 74°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°C and a relative humidity between 75 and 80%.

Animales y pastoreo.
De una población de 500 hembras bufalinas, se seleccionaron aleatoriamente dos grupos experimentales de 10 individuos cada uno.Un grupo con BN vírgenes y otro grupo con 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 BM.Se tuvieron como criterios de selección de las unidades experimentales: que todos los individuos estuvieran ciclando y se encontraran clínicamente sanos y con una condición 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 tenían una edad de 24.72±1.45meses y las BM de 81.97±31.75meses con 2.7±0.8partos.Para garantizar que todos los animales se encontraban bajo las mismas condiciones fueron mantenidos en pastoreo en un potrero de experimentación, donde permanecieron hasta finalizar el estudio.El potrero contaba con una pastura establecida de Brachiaria mutica y disposición de sal mineralizada y agua ad libitum.Protocolo de sincronización.Previo al inicio del protocolo de sincronización, se realizó un examen transrectal, de palpación y ultra sonido con el fin de evaluar las estructuras presentes en los ovarios y realizar la selección de los animales que ingresarían al experimento.Fue empleado un protocolo de inseminación artificial a tiempo fijo con dispositivo intravaginal de liberación de progesterona (Sincrogest, Ourofino, Saúde Animal, Brasil) al día cero, junto con la aplicación de 2 mg de benzoato de estradiol (Sincrodiol, Ourofino Saúde Animal, Brasil).Posteriormente al día 8, se retiró el dispositivo intravaginal y se aplicó PGF2 alfa (Sincrocio, Ourofino Saúde Animal, Brasil).Finalmente al día 9 se aplicó 1 mg de benzoato de estradiol (Sincrodiol, Ourofino Saúde Animal, Brasil).Transcurridas 52 horas después de haber retirado el dispositivo intravaginal, se realizó un nuevo examen transrectal para determinar la respuesta al tratamiento por la presencia de un folículo preovulatorio Luego de realizarse la detección de celos, no se realizó la inseminación artificial.Debido a la baja intensidad del comportamiento estral de las búfalas (6,8), todos los animales con presencia de un folículo mayor a 10 mm, presencia de tono uterino y presencia de descarga vaginal mucosa fueron considerados en celo (15).Seguimiento ultrasonográfico.Quince días después de evidenciarse los celos sincronizados, se inició una nueva detección de celos y al seguimiento ultrasonográfico diario, con un equipo de ultrasonido acoplado a un transductor lineal de 7.5 MHz (Mindray DP 2200 Vet).El día del celo fue tomado como el día cero del ciclo estral y a partir de ese momento se realizaron seguimientos ultrasonográficos diarios con el fin de hacer mapeos ováricos y evaluar el número y diámetro de los folículos antrales y el diámetro del CL (16).Luego de visualizar la imagen del ovario en el monitor del equipo de ultrasonido, cada ovario fue escaneado en varios planos 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 ± 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.
For all the experimental units, the emergence of the first follicular growth wave occurred the day after ovulation, and an average of 8.33±2.06 and 10.00±2.72 follicles for BM and BN, respectively were observed on the ultrasound.Follicular deviation in the first wave took place at 4.00±2.00days for BM and 4.62±1.84days for BN, with a diameter of the dominant follicle of 10.00±1.4mm and 8.9±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±2.00days para asegurar y capturar la mayor cantidad de estructuras y determinar su tamaño correcto, los datos provenientes de la medición de cada estructura se almacenaron para su análisis posterior.Estos seguimientos se realizaron hasta cuando se evidenció un segundo celo y una segunda ovulación en cada una de las búfalas.

Duración del ciclo estral y dinámica folicular.
No se encontraron diferencias significativas para for BM and 10.50±2.82days for BN.An average of 8.37±2.26and 8.00±1.51follicular structures were ultrasonographically observed for BM and BN, respectively.On day 14.00±2.13and 14.50±2.32 the deviation of the dominant follicle started in BM and BV, finding a diameter of 10.00±1.50 and 9.10±1.60mm, respectively.The third wave of follicular growth started on day 16.21±3.10with 6.50±1.70follicles recruited.Only two multiparous BM showed three waves of follicular growth.The maximum diameter reached by the preovulatory follicle was 17.00±4.60mm for BM and 14.00±2.90mm for BN.No significant differences were found between the groups for these variables (Table 1).

DISCUSSION
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±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
Los resultados obtenidos en este y los demás estudios analizados, concuerdan en que la primera onda folicular emerge en el día uno del ciclo estral (17,23).En cuanto a la emergencia de la segunda onda, se presentó a los 11±2.00 días para BM y 10.5±2.82 días para BN.Estos datos son similares a los encontrados por otros autores quienes han determinado la emergencia de la segunda onda alrededor del día 10 para animales de dos ondas y alrededor del día 8 para animales de tres ondas (20,23,24).El presente estudio evidenció que el número de folículos reclutados en la primera onda fue de 8.33±2.06y 10±2.72 y  (17.23).As to the emergence of the second wave, it occurred at 11±2.00 days for BM and 10.5±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±2.06 and 10±2.72 and in the second wave 8.37±2.26and 8±1.51, for BM and BN respectively, while for the third wave 6.5±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±4.64and 7.50±2.75for 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±0.3) and three waves (7.8±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.(20), quienes trabajando con BM de la raza Murrah concluyeron que el número de folículos reclutados en la primera onda fue de 7.72±4.64y 7.50±2.75para animales con ciclos estrales de dos y tres ondas respectivamente, donde no existen diferencias significativas.
Ojeda -Follicular dynamics, corpus luteum growth and regression in buffalo 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±0.2days postovulation at a size of 7.2±0.2(28).
The maximum diameter reached by the preovulatory follicle in the animals under study was 17±4.6 and 14±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±0.16and 1.34±0.13cm 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±0.59and 16.03±3.30for 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±0.6 and 11.0±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±4.16mm for BM and 17.74±3.32mm 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±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±0.9mm and 20.2±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

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.

Table 3 .
Maximum diameter and luteal regression in multiparous Murrah buffalo cows and heifers in the Colombian tropics.
15.22±5.26for the BM group and 17.62±1.68for the BN group, data consistent with those previously También, Awasthi et al (17) trabajando con búfalas de la raza Mehsana, encontraron que el diámetro máximo del folículo preovulatorio fue de 12.94±0.59y 16.03±3.30para animales de una y dos ondas.Sin embargo, en un estudio donde también se compara el patrón de crecimiento folicular entre BM y BN de la raza Mediterránea realizado en Italia por Presicce et al (16) se encontraron diferencias significativas (p > 0.05) entre el diámetro máximo del folículo ovulatorio con 13.8±0.6y 11.0±0.7.Es probable que, las marcadas diferencias en las condiciones en que se realizó el experimento de Presicce et al (16) y el presente estudio, sean responsables por esta discrepancia entre los resultados.Al evaluar las características del CL se determinó que el diámetro máximo fue de 19.58±4.16mm en BM y 17.74±3.32mm en BN.A pesar de la aparente diferencia numérica en estos resultados, no se encontraron diferencias significativas.En el estudio de Barkawi et al (23) se encontró un diámetro máximo del CL de 15.00±0.40en BN en Egipto, mientras que, Di Francesco et al (30) trabajando con búfalas de la raza mediterránea durante la estación de apareamientos, encontraron que el CL al día 10 post inseminación alcanzaba 18.6±0.9mm y 20.2±0.6 mm en búfalas vacías y preñadas respectivamente.Ellos no encontraron diferencias estadísticas al comparar el patrón de crecimiento del CL en animales en la estación reproductiva y en el período de transición hacia la estación siguiente.Sin embargo, sí lograron evidenciar diferencias en la síntesis de P4, donde los animales en la temporada reproductiva mostraron mayores concentraciones de esta hormona.En el presente estudio, la regresión del CL se encontró a partir del día 15.22±5.26para el grupo BM y en 17.62±1.68para 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 regresión luteal iniciaba el día 16.20±50.76;mientras que, Barkawi et al (23), determinaron que la vida media del CL era de 17.1±0.8días en búfalas con ciclos estrales normales, mientras que en hembras con presencia de quistes foliculares y CL persistentes, esta vida media incrementaba a 24.80±4.30y 28.30±6.10días respectivamente.En conclusión, la dinámica folicular y el patrón de crecimiento y regresión del CL de BM y BN ubicadas en condiciones de trópico 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 número de animales involucrados.Agradecimientos A l C e n t r o I n t e r n a c i o n a l d e Fo r m a c i ó n Agropecuario (CIFA) y la de todo su personal, por su invaluable colaboración en la elaboración de este estudio.reported by other authors.Satheshkumar et al (31) found that in Murrah BM the luteal regression began on day 16.20±50.76;while Barkawi et al (23) determined that the half-life of the CL was 17.1±0.8days in buffaloes with normal estrous cycles, while in females with follicular cysts and persistent CL, this half-life increased to 24.80±4.30and 28.30±6.10days, 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.