OVARIAN FOLLICLE POPULATION AND FOLLICLE SIZE IN ANATOLIAN WATER BUFFALOES 111

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1 OVARIAN FOLLICLE POPULATION AND FOLLICLE SIZE IN ANATOLIAN WATER BUFFALOES 111 The relationship between ovarian follicle population and follicle size during different stages of estrous cycle in Anatolian Water buffaloes (Bubalus bubalis) O. YILMAZ 1 *, E. YAZICI 1, A. KAHRAMAN 2, E. OZENC 1, M. UCAR 1 1 Department of Obstetrics and Gynaecology, Faculty of Veterinary Medicine, Afyon Kocatepe University, Turkey 2 Department of Biochemistry, Faculty of Medicine, Afyon Kocatepe University, Turkey Corresponding author: oktayyilmaz@aku.edu.tr SUMMARY The objective of present study was designed to determine the relation of follicular growth patterns to stage of estrous cycle, using genital tracts from 137 Anatolian water buffaloes collected immediately after slaughter. The estrous cycle was classified as metestrous (days 1-4), early (days 5-10), and late diestrous (day 11-17) and follicular phase (days 18-21). The follicular population was counted and measured by water bath ultrasonography technique. The diameter of the follicles was divided into four stages: 12 mm; < 12-8 mm; < 8-5 mm and < 5 mm in diameter. Blood samples were taken for estimation of progesterone (P 4 ) and estradiol 17β (E 2 ) concentrations to confirm that buffaloes had normal ovarian function. The number of follicles <5 mm in diameter showed no significant difference throughout the estrous cycle, whereas follicles between 5 and 8 mm in diameter were fewer during late diestrous and follicular phase. The number of follicles between 8-12 mm in diameter was less during metestrous and follicular phase. Follicles at least 12 mm in diameter were more numerous in follicular phase. During early and late diestrous, totally 87 animals had follicles larger than 8 mm (96.6%; 87/90) and only three animals did not show follicles larger than 8 mm, but they presented at least one large follicle during the following stage. These findings suggest that the majority of Anatolian water buffaloes develops two patterns of follicular wave. Keywords : estradiol 17β, progesterone, follicle diameter, Anatolian water buffalo RÉSUMÉ Relation entre la population des follicules ovariens et leur taille au cours de différentes étapes du œstral cycle chez les buffles d eau d Anatolie (Bubalus bubalis) L objectif de l étude était d évaluer la population folliculaire au cours des différentes phases du cycle oestral des vaches, metestrus (jours 1-4), début (jours 5-10), fin du diestrus (jours 11-17) et phase folliculaire (jours18-21). à partir de 137 tractus génitaux collectés à l abattoir. Des échantillons de sang ont été collectés avant afin d évaluer les concentrations plasmatiques de progestérone et d oestradiol. Les diamètres des follicules ont été évalués par échographie dans un bain d eau. Les follicules ont été répértis en 4 stades en fonction de leur diamètre : supérieur ou égal à 12 mm; compris entre 8 et 12mm, compris entre 5 et 8mm et inférieur à 5mm. Le nombre de follicules de diamètre inférieure à 5mm n a pas varié au cours du cycle alors que le nombre de follicules de diamètre comprise entre 5 et 8 mm a été plus faible à la fin du dioestrus et au cours de la phase folliculaire. Le nombre de follicules de diamètre comprise entre 8 et 12 mm a été plus faible au cours du metoestrus et au cours de la phase folliculaire. Les follicules de diamètre au moins égal à 12 mm ont été plus nombreux au cours ed la phase folliculaire. Au cours du dioestrus, 87 vaches ont présenté des follicules de diamètre supérieur à 8mm (96.6%; 87/90), ce qui suggère que la majorité des vaches présente deux vagues de Mots-clés : 17β estradiol, progestérone, diamètre du follicule, Anatolie buffles d eau Introduction Buffaloes are well known domestic animals in Asia, Mediterranean and Latin American countries of the world for different breeding purposes such as milk, meat, draught and capital [23]. The buffalo has been regarded as poor reproductive efficiency and characterized by low conception rates [6, 22, 23]. Seasonality of buffalo breeding results in a milk supply that exceeds demand in summer and autumn and diminishes significantly in winter and spring [1]. Anatolian water buffalo (Bubalus bubalis) is a native breed and is commercially very important for the milk-cream industry in Turkey. It has been reported that calving and estrous activity occurs in a seasonal pattern in Anatolian buffaloes [1, 28]. However, poor reproductive management of dairy buffalo has resulted in various reproductive problems including unknown reproductive disorders causing infertility and thus economic losses in the milk industry [29]. The population of Anatolian water buffalo was more than 1 million head in the early 1980s, by 2010, there were less than 100,000 [30]. According to an international source, Turkey s buffalo population fell from 1,040,000 head in 1980 to 84,726 head in 2011 [12]. The reduction in the buffalo population over the 30-year period was thus 91.9%. Recently, government support continuously grows to improve commercial buffalo breeding in Turkey. The improving of fertility in female buffalo is in close relationship with the knowledge of follicular development. Since the ultrasound technology applied to animal reproduction, a large amount of information has been collected about ovarian follicular dynamics in domestic animals [13, 14, 24]. In cattle, follicular dynamics has been intensively studied [8, 16, 18]. First studies performed in buffaloes for follicular growth have been reported in Surti buffaloes by rectal palpation [26] and by histology of ovaries [9]. There are limited reports about follicular development in the Indian buffalo breeds, Murrah [20, 27], Egyptian [3] and Iraqi Northern buffaloes [5]. However, ovarian follicular

2 112 YILMAZ (O.) AND COLLABORATORS dynamics in Anatolian water buffalo has not yet been investigated. Therefore, the objective of present study was designed to test the hypothesis of a wave pattern of follicular growth in Anatolian water buffaloes by evaluating ex vivo the ovarian follicle population and follicle size throughout estrous cycle. Materials and methods The entire genital tracts of 137 clinically healthy, cycling Anatolian water buffaloes aged between 3 and 10 years, weighing kg, 94±18 days postpartum were collected at random intervals slaughtered at abattoirs in Afyonkarahisar province, Turkey. Following slaughtering, the non-pregnant genital tracts were separated from the carcass with the help of scissors and transported to the laboratory for examination grossly in order to exclude any specimen containing reproductive abnormality. The estrous cycle was assessed on the basis of ovarian luteal morphology. According to morphological appearance of the corpus luteum (CL), the estrous cycle was classified as being either in metestrous (days 1-4), early (days 5-10), and late diestrous (day 11-17) and proestrous or estrous (follicular phase) (days 18-21) as previously described by several authors [3, 5, 17, 31]. Briefly, metestrous (days 1 4) describes the interval between ovulation and the time when the epithelium is growing over the rupture point of the former follicle. In early diestrous (days 5 10), the CL is fully developed with visible vascularisation around its periphery, the apex is red or brown and the rest is greyish. Late diestrous (days 11 17) begins when the red or brown colour disappears showing the entire CL bright red or grey. In proestrous and estrous (days 18 21), regressed CL appears small, hard and bright. Prior to slaughter, blood samples were taken and sera were frozen at 20 C for later estimation of progesterone (P 4 ) and estradiol 17β (E 2 ) concentrations to confirm that buffaloes had normal ovarian function. This confirmed also the stage of the estrous cycle which cows were slaughtered. Moreover, if the concentration of P 4 did not correspond to morphological findings, the data were excluded from the study. Serum P 4 and E 2 concentrations were estimated by electrochemiluminiscence immunoassay (ECLIA) using a commercial test kit (Elecsys Progesterone II, Roche Diagnostics GmbH, Germany) in an immunologic test analyser (Cobas e 601, Roche Diagnostics GmbH, Germany). The standard curve of P 4 ranged from 0.03 to ng/ml and the sensitivity was 0.15 ng/ml, whereas the standard curve of E 2 ranged from pg/ml and the sensitivity was 12 pg/ ml. The average intra- and interassay coefficients of variation were kept under 20% for both investigated hormones. Crossreaction of the antibodies against P 4 estimation was 0.02% (testosterone) and against E 2 estimation, 0.51% (estrone). Immediately after slaughter, the follicular population was counted and measured by water bath ultrasonography technique to better understand the number and size of follicles. Ovaries were placed in a clean plastic bowl filled with water. The linear probe (Falco Vet 100, Maastrich, The Netherlands) was then placed in the water in close to ovary. The diameter and the number of the follicles were recorded and classified into four stages: 12 mm; < 12-8 mm; < 8-5 mm and < 5 in diameter. The dominant follicle (DOF) was defined morphologically the largest follicle and exceeding the diameter of all other follicles [19], whereas the subordinate follicle (SOF) was the second larger follicle of all other follicles. Differences between the numbers of follicles, diameters of follicles, E 2 and P 4 concentrations in blood during different stages of the estrous cycle were compared using the one-way analysis of variance (ANOVA) followed by Tukey test (SPSS 16.0). Data were considered to be significantly different at p < Results The ovarian luteal morphology and the estimation of P 4 and E 2 concentrations confirmed that all buffaloes had normal ovarian function and were thus cycling animals. The morphological appearance of ovaries allowed to determine the number of buffaloes at the different stages of the cycle: metestrous (n=16), early diestrous (n=27), late diestrous (n=63) and follicular phase (n=31). The mean blood P 4 concentration was the highest in late diestrous (p < 0.001), while E 2 concentration did not differ significantly throughout the estrous cycle (Table I). The average number of follicles different in size at the defined stages of the estrous cycle is presented in Table II. Accordingly, the number of follicles smaller than 5 mm in diameter was higher during metestrous and follicular phase but this discrepancy showed no significant difference (p > 0.05). There were fewer follicles between 5 and 8 mm in diameter during late diestrous and follicular phase than during metestrous and early diestrous (p < 0.01). The number of follicles between 8-12 mm in diameter was less during metestrous and follicular phase than early and late diestrous (p < 0.05). Follicles at least 12 mm in diameter were more numerous in follicular phase than other stages of estrous cycle (p < 0.001). During metestrous stage, only four animals (25%; 4/16) had follicles larger than 8 mm, whereas 25 animals in early diestrous and 62 animals in late diestrous, totally 87 animals, had follicles larger than 8 mm (96.6%; 87/90). It was observed that only three animals did not show follicles larger than 8 mm during both early and late diestrous stages, but all of buffaloes in follicular phase showed at least one follicle larger than 8 mm. The mean diameter of the largest follicle was the lowest in metestrous stage, whereas the highest diameter of the largest follicle was in follicular phase (p < 0.001). However, there was no significant difference between early and late diestrous stages. The mean diameter of subordinate follicles did not show any significant difference throughout the estrous cycle (Table III).

3 OVARIAN FOLLICLE POPULATION AND FOLLICLE SIZE IN ANATOLIAN WATER BUFFALOES 113 Discussion Ovarian follicular dynamics have been studied by several authors in buffaloes in vivo [4, 7, 21, 25, 26]. The value of ultrasound examination of ovarian follicle turnover in livestock is much greater than a static, instantaneous examination of follicle population. Besides, morphological studies are still common to test the hypothesis of wave patterns of follicular growth [3, 5]. The data obtained in this study is the first report that provided the follicular growth patterns in Anatolian water buffaloes in vitro. In our study, we found that follicles smaller than 5 mm in diameter were numerous during all stages of the estrous cycle. However, the number of these follicles were higher in metestrous and follicular phase than early and late diestrous, but this difference was not significant, statistically (Table II). Our results were inconsistent with the previous data reported by Ali et al. [3] that follicles smaller than 5 mm were numerous at metestrous and were fewer at other stages of estrous cycle in Egyptian buffalo cow. However, in another study described by Azawi et al. [5], follicles smaller than 5 mm was predominantly detected in metestrous and follicular phase, whereas these follicles were significantly fewer in early and late diestrous stages in Iraqi buffalo cow. Discrepancies in the above-mentioned observations indicate that the number of small follicles throughout estrous cycle may be different between breeds. It has been well documented that in cattle, the first follicular wave starts around the day of ovulation which is characterized by the presence of many small follicles at the same time [3, 5, Stages of estrous cycle P 4 (ng/ml) (min-max) E 2 (pg/ml) (min-max) Metestrous (n = 16) 0.73 ± 0.17 a ( ) 16.9 ± 1.02 ( ) Early diestrous (n = 27) 4.84 ± 0.60 b ( ) 16.7 ± 1.41 ( ) Late diestrous (n = 63) 7.37 ± 0.50 c ( ) 16.8 ± 0.71 ( ) Follicular (n = 31) 0.57 ± 0.11 a ( ) 18.4 ± 1.08 ( ) Columns with different superscripts differ significantly (p < 0.001). Table I: Mean, minimum and maximum blood progesterone (ng/ml) and estradiol 17β (ng/ml) concentrations during different stages of the estrous cycle in buffaloes (mean ± SEM). P 4 : Progesterone, E 2 : Estradiol 17β. Number of follicles (mean ± SEM) Stages of estrous cycle < 5 mm 5-8 mm** 8-12 mm*** >12 mm* Metestrous (n = 16) 7.3 ± 0.95 (n=16) 1.06 ± 0.21 a (n=11) 0.31 ± 0.15 a (n=4) 0.00 ± 0.00 a (n=0) Early diestrous (n = 27) 5.62 ± 0.79 (n=26) 0.96 ± 0.22 a (n=15) 0.88 ± 0.12 c (n=20) 0.40 ± 0.11 b (n=10) Late diestrous (n = 63) 5.74 ± 0.39 (n=62) 0.50 ± 0.08 b (n=26) 0.71 ± 0.08 bc (n=37) 0.44 ± 0.06 bc (n=28) Follicular (n = 31) 7.12 ± 0.79 (n=30) 0.32 ± 0.09 b (n=9) 0.51 ± 0.11 ab (n=14) 0.70 ± 0.10 c (n=20) Columns with different superscripts differ significantly (*** p < 0.05; ** p < 0.01; * p < 0.001). The number into brackets indicate the number of animals that possess follicles of the different sizes Table II: Mean number of follicles during different stages of the estrous cycle. Largest follicle Subordinate follicle Stages of estrous cycle Diameter (cm) Diameter (cm) Metestrous (n = 16) 0.70 ± 0.04 a 0.60 ± 0.03 Early diestrous (n = 27) 1.12 ± 0.04 b 0.66 ± 0.05 Late diestrous (n = 63) 1.17 ± 0.02 b 0.66 ± 0.03 Follicular (n = 31) 1.30 ± 0.05 c 0.70 ± 0.05 Columns with different superscripts differ significantly (p < 0.001). Table III: Mean diameter of largest and second larger (subordinate) follicles (mean ± SEM) during different stages of estrous cycle in buffaloes.

4 114 YILMAZ (O.) AND COLLABORATORS 7, 21, 26]. In buffaloes, a similar pattern for the first follicular wave has been reported that it appears approximately on the day of ovulation as detected by ultrasonography [7, 20, 26] and by morphological studies [3, 5]. In the present study, follicles smaller than 5 mm in diameter were numerous at the beginning of the estrous cycle (between days 1 and 4) postulating that the first follicular wave started during this period. It is suggested that other stages of estrous cycle may also have the same pool of small follicle capacity to initiate a wave pattern, since the follicles smaller than 5 mm in diameter did not show any significant difference between the stages of estrous cycle in the present study. Furthermore, in our study, we detected that there were fewer follicles between 5 and 8 mm in diameter during late diestrous and follicular phase than during metestrous and early diestrous (p < 0.01). Moreover, the number of these follicles was low at the end of the estrous cycle (between days 18 and 21) but increased significantly at the beginning of the cycle (between days 1 and 4), and then decreased slightly between days 5 and 10 and again between days 11 and 17. Moreover, it suggests that large number of follicles between 5 and 8 mm in diameter during metestrous (days 1 and 4) and early diestrous (days 5 and 10) might have been the consequence of growth of the large pool of small follicles present earlier in the cycle. Therefore, the fluctuation in development of this class of follicles in the present study indicates that those follicles might have grown in a wave-like pattern. It has been known that a follicular wave is characterized by the development of a group of follicle (5-7 follicles) synchronously [10]. In cattle, it has been reported that after the emergence of small follicles on day 1, they develop together until dominance of a DOF on day 4 or 5 [2]. Thereafter, a dominant follicle suppresses the development of other follicles in the same follicular wave [14, 15] between days 5 and 10, when another wave starts to develop [2]. Therefore, it may suggest that one wave initiates between days 1-4 and the other one between days 11-17, which is in agreement with other authors [3, 5]. It has been reported that a decreasing number of small follicles from the beginning to the end of the cycle is observed in only one pattern of follicular wave [3, 20, 26]. Nevertheless, in this study, we did not find this observation, since there was no significant difference. This may explain that most of the buffaloes in the current study show multiple follicular wave patterns rather than one-wave pattern. In the present study, the number of follicles between 8-12 mm in diameter was less during metestrous and follicular phase than early and late diestrous (p < 0.05). It was observed that the population of these follicles was higher at the midcycle period (between days 5 and 17), gradually decreased in follicular phase and less in metestrous stage of estrous cycle. In the current study, the development of follicles between 5-8 mm in diameter presented at the beginning of the cycle (between days 1 and 4) and early diestrous stage might have resulted in numerous follicles between 8 and 12 mm in diameter at the midcycle period. It has been reported that the presence of corpus luteum is responsible for atresia of the largest follicle due to negatively regulation of LH pulse by secretion of progesterone [11]. However, during the luteolysis process, progressive increase in diameter of the largest follicle starts and finally ovulation occurs [8]. In the present study, follicles 12 mm in diameter did not differ between late diestrous and follicular phase, whereas the number of the follicles at early and late diestrous was higher than those at metestrous stage of estrous cycle (p < 0.001) (Table II). Moreover, the mean largest follicle diameter was the highest in follicular stage and did not differ between early and late diestrous, while metestrous stage had the lowest follicle diameter (p < 0.001). Furthermore, the mean diameter of subordinate follicles did not differ between stages of estrous cycle (Table III). Overall observations support that the presence of follicles larger than 12 mm during follicular phase might have resulted from the follicles between 8 and 12 mm present earlier (days 11 and 17) in the cycle and second wave initiates between days Several authors have been reported different beginning days of follicular growth wave-like patterns that first follicular wave appeared approximately on day 1, second wave on day 7-11 and third wave on day [4, 7, 21, 26]. Baruselli et al. [7] have indicated the three-wave patterns of follicular growth in Murrah buffaloes and the incidence of follicular growth patterns as 3.3%, 63.3% and 33.3% in one, two and three-wave patterns, respectively. However, Manik et al. [21] revealed the predominance of two wave pattern (83%). In Mehsana buffaloes, single waves have also been reported [4]. In the current study, buffaloes that had follicles larger than 8 mm during early and late diestrous were 25 and 62 animals, respectively, totally 87 animals (96.6%; 87/90). It suggests that these buffaloes have developed two follicular waves in their cycle. In contrast, only three animals did not show follicles larger than 8 mm during both early and late diestrous stages, but all examined buffaloes presented at least one large follicle during the following stage. This suggests that these buffaloes have developed only one follicular wave in their cycle. In conclusion, this is the first report that describes follicular dynamics of the estrous cycles in Anatolian water buffaloes. Results of the present study depending on morphological investigation of the ovarian follicle population during the estrous cycle suggest the presence of two patterns of follicular growth in the majority of Anatolian water buffaloes. 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