Follicular Dynamics in Female Buffaloes

The mechanisms involved in the growth, development and ovulation/atresia of ovarian follicles are complex and are partially understood. A marked proliferation and differentiation of the somatic cell components of the follicle occurs during folliculogenesis which is regulated by gonadotrophins [1], intraovarian growth factors [2], local interactions and nutritional influences [3]. Identification of follicular growth continuum sequences (after birth) has resulted in the classification of follicles in the ovary as primordial follicles, primary follicles, preantral and antral follicles [3,4]. Folliculogenesis involves the continued growth of a group of primordial follicles on the ovary through a series of growth patterns that are regulated differently resulting in either ovulation of follicles or their regression/atresia. The time period for this transformation of primordial follicles into antral follicles is approximately 3 months [3]. The number of primordial [5], preantral [6] and antral follicles [7,8] in the buffalo ovary is smaller compared to cattle. Mechanisms regulating the activation and subsequent growth of the primordial follicles still remain poorly understood although some studies have depicted the role of growth factors [3,8,9].

Abattoir studies in buffalo species revealed that antral follicles of different sizes are present on the ovaries during different stages of the estrous cycle [7,10-14], however, similar to cattle, the daily ultrasonographic monitoring of antral follicles revealed that the later stages of antral follicle development is characterized by two or three waves of follicular growth during each estrous cycle [15,16,17]. Follicular waves appear to be fundamental and have been observed in young 5-9 month old buffalo heifers [18], prepubertal heifers [19], during pregnancy [20,21] and in anestrous buffaloes [22]. Each wave of follicular growth is preceded by a transient rise in FSH secretion [23] in cattle and similar changes do occur in buffaloes [18]. Based on temporal relationships of hormones, growth factors and other intra-ovarian factors, it has been mentioned that antral follicle growth is gonadotrophin and growth factor dependent [3]. The follicular growth of antral follicles progresses with the selection of antral follicles destined to grow leading to the dominance of one follicle and atresia of the others. The dominant follicle of the last wave is the ovulatory follicle [24,25] with estrus expression synchronous to luteolysis. However, the precise mechanism of selection and dominance remains to be fully elucidated. The mechanisms of follicular atresia in mammals have been reviewed. It has been suggested that in the buffalo, there is a decrease in the gap junction protein and in the area of vessels, which results in decreased cell proliferation [26]. The tools used to study antral follicles in buffaloes include selection of candidates for estrus synchronization [27,28], induction of ovulation [29,30], selection of stage for initiation of superovulatory treatment [31], and ultrasound guided ovum pick up [8,32] or selection of follicles from abattoir derived ovaries for in vitro fertilization [33,34].

This chapter describes the ovarian follicular growth in buffalo with respect to prenatal period, preantral and antral follicular growth during puberty, estrous cycle, pregnancy and in the postpartum period.

1.        Fetal Development of Ovaries and Follicles

1.1. Ovarian Development

Early studies on pregnant Egyptian buffaloes revealed that differentiation of the gonadal primordia occurred at a crown-rump length of 7 mm, sex differentiation was at 20 mm, and primordial follicles were first observed at 600 mm, continuing to develop until birth [35]. Subsequent studies in Egyptian and Murrah buffaloes demonstrated that at gestational age of 3 months, the ovaries appeared as oval or spindle-shaped symmetrical thickenings just cranial to the anterior end of the differentiating Müllerian ducts and attached to the caudo-lateral borders of the kidneys [36,37]. At 100 days, the left ovary started to descend towards the pelvic position while the right ovary was attached to the kidney [37]. The final pelvic position of the ovaries was reached by the end of the 6th month and the right ovary was slower to descend than the left [36].

Ovarian weights at different fetal stages increased from 27.0 ± 4.3 mg at 3 months to 82.8 ± 36.2 mg at 10 months of gestation [36]. The gross appearance of the prenatal ovaries in Surti buffalo revealed different shapes: almond, elliptical ovoid, ovoid and bean-shaped from 67 to 305 days of intrauterine life [36,38]. Similar descriptions have been recorded for the swamp buffalo ovaries [39].

The germinal ridge appeared at 36 days of gestation. The gonadal tissue differentiated into a peripheral cortex and central medulla by Day 62 [40]. At 3 months of age, the gonads were at a late stage of differentiation: the tunica albuginea was poorly differentiated but primitive germinal, granulosa, theca and stromal cells were present [36]. The medulla revealed an almost adult type appearance at Day 158. The ovary was covered by a flat type of surface epithelium mixed with cuboidal cells during 65 to 75 days of gestation which changed to columnar type at 158 days [40]. The germinal cells were mostly oogonia, many of which were in mitosis but some pyknosis was observed. A few oocytes were in early meiosis. By the 4th month, the germinal epithelium was invading the gonad to form epithelial cords and rete ovarii were apparent. The germinal cells were still at the oogonial stage and many were degenerating. During the 5th month, the ovarian stroma and tunica albuginea became more organized and sex cords were seen invading the stroma. The meiotic activity was apparent and formation of primordial follicles began and was seen until the 6th month of gestation [36]. The highest number of primordial and primary germ cells was recorded at 116-155 days [40].

During the 7th and 8th months of gestation, the tunica albuginea reached its maximum thickness (200 microns) and the cells of the germinal epithelium became flattened [36]. There were few oogonia, most of the germinal cells being oocytes. Several oocytes were undergoing degeneration. At the 9th and 10th months, hormonal activity was indicated by the formation of vesicular follicles which became atretic. The ovary was more clearly divided into cortical and medullary zones and germinal cells were found only in the cortical zone [36].

The number of germinal cells/mm3 of ovarian tissue remained fairly constant from the 5th month except during the 9th month. The enclosure of oocytes by granulosa cells to form the primordial follicle coincided with the first stages of meiotic prophase. The number of germinal cells/mm3 ovarian tissue was highest at the 7th month of gestation [35,36].

1.2. Oogenesis

At the time of sexual differentiation, the fetal female gonad is constituted of oogonia and somatic cells from the mesonephros. Oogonia develop from primordial germ cells that have migrated into the ovary early in embryogenesis [41]. Oogonia were positively identified in the 3rd and 4th month of gestation. By the 6th month, their numbers had decreased and few were visible [36]. Oocytes appeared sporadically as early as the 3rd month and progressively increased until birth. Schematically, oogenesis involves three phases: a prolific phase (0-3 months; the oogonia divide actively) [42], a meiotic phase (4-6 months; primary oocytes are formed) [42,43] and an intense germ cells degeneration phase (7 month to the end of gestation) [36]. There are 3 waves of degeneration of the germinal cells. The first affected the oogonia and was at a maximum at 4th month. The second and third waves affected oocytes and occurred at the 6th month and at term [36,44].

The oocytes that do not undergo degeneration, are arrested at the diplotene stage of the first meiotic division and are surrounded by a single layer of granulosa cells, constituting structures called primordial follicles. The meiotic phase occurs during the prenatal life in most mammals. In water buffaloes the formation of primordial follicles is completed before birth at 127.84 ± 11.55 days, when crown-rump length is 22.84 ± 4.74 cm [41].

At the end of oogenesis, the ovary encloses millions of primordial follicles within a framework of interstitial tissue and is lined with ovarian epithelium erroneously called germinal epithelium [41]. The oocytes formed during the fetal and neonatal period are the only source of oocytes available during the entire reproductive life. As soon as the primordial follicle reserve is constituted, it rapidly decreases by atresia and from the end of the period of oogenesis, some primordial follicles continuously begin to grow, but up to puberty, the majority of these disappear owing to atresia [41]. Probably, only one percent of the total oocytes reach maturity and are released through ovulation [43]. A histological section of the cortex of a reproductively active female reveals that ovarian follicles remain in a constant state of growth and maturation. The primary follicle stage is followed by a proliferation of granulosa cells surrounding the potential ovum giving rise to a secondary follicle. Later in the development, an antrum is formed by fluid collecting between the granulosa cells and separating them. At this stage the follicle is classified as a tertiary follicle, also termed as Graafian follicle [41].

The ovaries of post-pubertal buffalo heifers have a reservoir of only 10,000–20,000 primordial follicles [5] compared with over 100,000 in cattle. The mature ovaries are smaller than in cattle, weighing around 2.5 g when inactive and 4 g when active, with fewer tertiary follicles than in cattle [5,45]. The corpus luteum (CL) is smaller than that of cattle. The CL often does not protrude markedly from the surface of the ovary and sometimes lacks a clear crown. These characteristics make accurate identification of ovarian structures by transrectal palpation in buffalo more difficult than in cattle [2,5]. Ultrasonic imaging indicates mature follicles range in size from 1.3 to 1.6 cm in diameter and mature corpora lutea from 1.2 to 1.7 cm in diameter [10,29,46]. In one study, The accuracy to recognize fluid filled antral follicles of less than 4 mm using ultrasonography was low [47].

1.3. Historical Perspective

The earliest report on the study of ovarian follicular structures, appears to be those of Memon et al., 1971 [10] who recorded that antral follicles of different size are present on the ovaries during different stages of the estrous cycle. The earliest reports on the ultrasonographic appearance and growth of antral follicles on buffalo ovaries appear to be those of Taneja et al., (1996) on Murrah buffaloes [15] and Baruselli et al., (1997) on Brazilian buffaloes [16] and these workers recorded two and three wave patterns of follicular growth during estrous cycle. Many reports appeared thereafter on follicular dynamics of Mediterranean [24,48], Nili-Ravi [49], Mehsana [50], Egyptian [30,31] and Thai swamp [46] buffaloes. The pattern of follicular turnover has been reported during different reproductive status in prepubertal buffalo calves [18,19]; during postpartum period [51,52] and in buffalo heifers during early pregnancy [20].

1.4. Follicular Population

Follicular development in buffaloes has not been studied as much as in cattle. Singh et al., [53] delineated the pattern of development and atresia of large follicles (8 mm) on the surface of the ovaries of buffalo heifers. In 65% of the post-pubertal heifers, they found larger follicles at mid-cycle concluding that these findings complied with the theory of Rajakoski [54]. The follicles at mid-cycle become atretic and a new wave of follicles begins to grow about mid-cycle and gives rise to the follicle ovulating after estrus. The buffalo is characterized by a reduced follicle reservoir compared to that of cattle; the number of primordial follicles has been reported to be approximately 12,000-19,000 in riverine buffalo heifers [55]. Furthermore, through ovarian histological evaluations, studying the follicular system of cycling and non-cycling Surti buffalo heifers, Danell [5] reported 12,636 primordial follicles in cyclic buffalo heifers, and 10,132 primordial follicles in the non-cycling animals, with a range of 1,222–40,327 in an ovary pair. He observed more atresia in buffalo follicles (66.7%) than in bovine follicles (50%) and detected the same pattern of follicular dynamics in buffalo as observed in cattle [54]. The total number of surface follicles per ovary in abattoir buffalo ovaries at random stages of reproduction has been reported to range from 5.14 to 6.06 [7,57]. Many studies examined the preantral and primordial follicles in buffalo ovaries and reported that follicles were found to be lower in number in buffaloes than in cows’ ovaries [7,8,43,58,59].

Figure 1. Phases of follicular development.

1.5.        Preantral Follicle Growth

The primordial follicles formed in the ovary continue to grow sequentially throughout life and many of these processes are initiated even during fetal life in buffaloes [43]. Once primordial follicles begin to grow, the granulosa cells proliferate to form multilaminar structures known as preantral follicles. These preantral follicles grow with the subsequent formation of a fluid filled space (antrum) and a well differentiated theca cell layer. Follicles with an antrum are known as antral follicles. Mechanisms regulating the activation and subsequent growth of primordial follicles still remain poorly understood. However, their growth is considered to be independent of gonadotrophins but probably depends on the presence of oocyte/granulosa cell interactions and the secretions of local factors such as activins, inhibins and epidermal growth factors [3]. Based on in vitro studies the growth of bubaline preantral follicles to antral follicles is considered to require 3-4 months [60-64]. The diameter of preantral follicles varies from 100 to 200 µm at the start of culture and they grow to 600-800 μm after 80 days of in vitro culture [62,65]. The growth of preantral follicles is independent of the gonadotrophins, however, it appears to be more dependent on local intra-ovarian factors.

1.6.        Antral Follicle Growth

The later stages of antral follicle development in buffaloes are characterized by two or three waves of follicular growth during each estrous cycle. Follicular growth waves are seen during prepubertal period, puberty, anestrus, pregnancy and the postpartum period. Each wave of follicle growth is characterized by recruitment of a group of follicles which continue to grow to approximately 6 to 8 mm in diameter. From this pool of growing follicles, one follicle is selected to continue growing and become the dominant follicle. There are a number of terms that are particularly relevant when describing follicular waves based on ultrasonographic observations [66]. The day of a follicular wave emergence is the first day that a growing cohort of follicles is first detected using ultrasonography. Follicle recruitment is often used synonymously with emergence [67], however, it is more accurately defined as the growth of follicles that have become gonadotrophin-dependent [68]. This is usually evident when follicles are 4–6 mm in diameter in cattle and 2–4 mm in diameter in sheep. Follicle selection is the process that results in a decrease in the number of growing follicles in a wave to the species-specific number of follicles that ovulate. This ill-defined process probably occurs over a period of time and is thought to end when the dominant follicle(s) has been selected from the subordinate follicles as seen by a difference in follicle size. The point in time when there is a divergence in growth rates is referred to as deviation [67]. The dominant follicle is the one that continues to develop when the growth and development of other follicles in the same wave is inhibited. Subordinate follicles are the remaining follicles that regress in the presence of a dominant follicle(s).

1.7.        Follicular Dynamics during Estrous Cycle

The use of ultrasound technology in animal reproduction has paved the way in the collection of data regarding ovarian follicular dynamics in cattle [69-71]. These studies demonstrated that the follicular turnover occurs in waves during the estrous cycle, each wave being characterized by the synchronous development of a group of follicles. Ovarian follicular growth in buffaloes is similar to that observed in cattle and is characterized by waves of follicular recruitment, growth and regression [15,16,17,50]. These authors of this paper and many others (Table 1) have shown that buffaloes typically show two and three follicular waves during an estrous cycle, with the first wave beginning around Day 0 (day of ovulation). In each wave of follicular growth, one dominant follicle develops and suppresses the other follicles. The dominant follicle grows and reaches maximum diameter in the middle of the estrous cycle; concurrently, with high levels of progesterone during mid-cycle, ovulation does not occur; regression of the dominant follicle starts allowing a new wave of follicular growth to occur. The dominant follicle that develops during the last wave of follicular growth in each estrous cycle is the ovulatory follicle (Fig. 2).

Figure 2. Mean growth profile of the first and second dominant follicle of water buffaloes with a two-wave cycle [50].

Based on ultrasound analyses, most animals have one or two waves of follicular development during the luteal phase and a single wave of follicular development (ovulatory wave) during the follicular phase. Cohort refers to a group of similar sized nearly synchronously growing follicles. Emergence marks the beginning of a wave consisting of 3 to 6 follicles and is the first day a 4-5 mm follicle is observed in a new wave. The beginning of selection cannot be determined by ultrasound scanning, however, the end of selection occurs simultaneously with the onset of dominance.

Deviation is when the growth of one (dominant) follicle occurs at a faster rate than the other follicles (subordinate) in the cohort. Dominance occurs when the largest follicle in a wave is 1 to 2 mm larger than the next largest follicle and the growth of all subordinate follicles ceases and they all undergo atresia. Loss of dominance marks the end of a wave and is detected at the emergence of next follicular wave. The growth profile of a dominant follicle is divided in to three distinct phases, viz. growth, static and regression. The growth phase of the dominant follicle begins on the day of its emergence and ends the day in which the diameter of the follicle ceases to increase. The static phase is from the first day that follicle diameter ceases to increase (end of growth phase) until the preceding day when follicle diameter begins to decrease. The regression phase is the last day of the static phase until the follicle is no longer detectable, which is usually when it reaches four to five mm in diameter. In an estrous cycle with two follicular waves, the first wave begins at approximately Day one (Day 0 = day of ovulation) and the second wave begins at approximately on Day 11 [16,50]. The maximum size of each dominant follicle (15 mm) is attained on Days 9 and 21 of the estrous cycle for the first and second waves, respectively (Table 2). The deviation of follicles has been reported to occur at 2.6 ± 0.2 days after ovulation, and the diameters of the dominant follicle and the largest subordinate follicle at deviation did not differ during the first wave of follicular development in buffalo heifers [73]. It was further reported that the acquisition of ovulatory capacity occurred when the dominant follicle reached 8.5 mm in diameter after pLH treatment [73].

In a cycle with three waves, the waves emerge, on average, on Days 1, 9 and 16 [16,17,74]. There is no difference between second- and third-wave cycles with regards to the day of emergence of the first wave; the second wave appears earlier in an estrous cycle with three waves than with two waves. The average length of intervals to estrus and ovulation and the average length of luteal phase are greater in three-wave than in two-wave cycles [16,49]. In general, it is agreed that the number of follicular waves in each cycle is correlated with cycle length, which in turn is dependent on the life span of the CL. The onset of second wave begins 2–3 days after the beginning of the static phase of the first dominant follicle suggesting that the dominant follicle loses its dominance at the beginning of static phase. The dominant follicles are functionally active (E2-dominant; non-atretic) between 5 and 10 days of estrous cycle based on analyses for E2 and P4 level [75] suggesting that growth and static phase of dominant follicle of first wave lasts up to 10 days. The functional dominance is lost sometime between the early and late plateau phase, even though the follicles are still morphologically dominant [69]. The subordinate follicles of each follicular wave cease to grow after a few days and a new wave does not appear as long as a dominant follicle is in its growth phase or at the beginning of its static phase [50]. The end of the growth phase of the first dominant follicle is followed by a peak in FSH level [23] corresponding to the time of emergence of the second follicular wave. The FSH surge is related functionally to the recruitment and development of next wave.

Unlike cattle, water buffaloes occasionally show atypical follicular growth, which is characterized by one-wave of follicular growth (Fig. 3) during the estrous cycle [15,50]. Animals with a one-wave cycle have a significantly shorter inter-ovulatory interval and shorter length of estrous cycle compared to those with a two-wave cycle. The solitary wave in a one-wave cycle emerges significantly later than that of the first wave of a two-wave cycle. The dominant follicle of a one-wave cycle shows three distinct phases of growth i.e., growth phase, regression phase and re-growth phase culminating in ovulation. This pattern of follicular growth differs from the growth profile of the ovulatory dominant follicle of two- and three-wave cycles, which is characterized by continuous growth without any regression until ovulation at the end of cycle [16,17]. The dominant follicle of a single wave cycle regresses considerably around mid-cycle after initial growth, while still retaining its functional dominance and the same follicle becomes ovulatory after slow but steady growth from day 15 onwards. The linear growth rate of the solitary dominant follicle of a one-wave cycle is significantly higher during the growth phase compared to that of the re-growth phase. With greater size and under higher plasma progesterone concentration, the growth rate of the dominant ovulatory follicle is slower during the re-growth phase. There is no significant difference in size between the future dominant follicle and the largest subordinate follicle at the time of wave emergence in both one- and two-wave cycles. However, the dominant follicle grows at a faster rate and its diameter is significantly greater by Day 3 for the first dominant follicle of a two-wave cycle, and by Day 4 for both dominant follicles of a one-wave cycle and the ovulatory dominant follicle of two-wave cycle [50].

Figure 3. Mean growth profile of the dominant follicle in water buffaloes with a one-wave cycle.

Buffaloes showing atypical one-wave follicular growth during the estrous cycle may be attributed to

1. Lower number of primordial follicles in reservoir,
2. A delay in release of FSH at the time of regression of dominant follicle or
3. Inadequate amount of FSH that is responsible for emergence of a new follicular wave [18].
   It is possible that this characteristic feature may be breed-specific or hereditary.

The pre-ovulatory dominant follicle develops with equal frequency in ovaries with and without a CL [16]. Conversely, other report suggests that the pre-ovulatory dominant follicle develops on the ovary contralateral to the CL in two- follicular wave cycles [50]. The first dominant follicle usually develops more often on the ovary bearing the CL in cattle [76]. These observations suggest that there is a positive intra-ovarian influence of the CL on the growth of the dominant follicle of the first wave of a two-wave cycle (anovulatory) and a negative influence on the growth of the pre-ovulatory follicle of both one- and two-wave cycles. Some authors have failed to observe any apparent pattern of alternating sides of ovulation in cattle [77,78] and in buffaloes [15,16], whereas others have observed relationship between these structures [69]. There is little variation among the numbers of follicles in the different waves at the time of their emergence in buffaloes [16,50] suggesting that the number of follicles recruited depends on the individual animal. No information is available regarding the heritability of this characteristic; however, the selection of an animal as a donor for embryo transfer program based on the number of follicles per wave is a promising aspect. A positive correlation exists between the number of small follicles at the beginning of super-ovulatory treatment and the superovulatory response [79].

1.8.        Follicular Dynamics during Pre-Pubertal Period

The turnover of ovarian follicles is characterized by waves of follicular growth in pre-pubertal buffalo heifers between 5 and 9 months of age [18]. Two to six regular follicular waves have been reported during 50 days of daily observation through ultrasound scanning. The interval between two follicular waves was 9.9 ± 2.8 days and the largest diameter of the dominant follicle was 8.4 ± 1.2 mm. Growth rates of dominant follicles was significantly faster than for corresponding subordinate follicles. The static phase lasted longer in dominant follicles compared to subordinate follicles; however, the regressing phase was similar among dominant and subordinate follicles. Some short follicular waves lasting 6.1 ± 1.2 days were also observed. Similarly, a typical pattern of regular follicular development has been reported in a 20–24 months old pre-pubertal Murrah buffalo heifer [19], which supports the concept that the mechanisms of follicle dominance and wave-like pattern of follicular growth are operative during prepubertal life. On average, 3 complete waves per heifer were recorded during the 42 days observation period; however, no ovulation was recorded in these animals. The mean interval between emergence of 2 consecutive waves was 10.40 ± 0.86 days. The mean duration of follicular wave was 15.14 ± 1.08 days, the majority of waves (81%) persisted between 10–20 days and only 2 waves each of short (6–8 days) and long duration (22–24 days) were also observed suggesting that one-wave follicular growth also occurs during pre-pubertal period. The incidence of waves of shorter duration may suggest either immaturity of the hormonal regulation of wave emergence and follicular dominance, or the presence of a residual effect of gonadotrophin hormones in recruiting additional follicles for development although for a short time. Similarly, a wave of longer duration may reflect either a delay in release of FSH or its inadequate amount that is responsible for emergence of a new follicular wave [23]. However, Presicce et al., [18] reported that FSH release was less clearly associated with main wave emergence in buffalo calves and suggested that only a basal level of FSH in association with a regressing dominant follicle might be sufficient to trigger the development of a new follicular wave. A great variation exists in duration of follicular wave ranging from 9.9 ± 2.8 to 15.14 ± 1.08 days during pre-pubertal period in water buffaloes.

The size of a dominant follicle in female calves between 5-9 months old (8.4 ± 1.2 mm) and in pre-pubertal heifers between 20-24 months of age (9.55 ± 0.24 mm) is smaller [18,19] than in adult (range 13-15 mm) buffaloes [15,16,50]. This suggests that the maximum size of the dominant follicle increases gradually with age until it reaches the size equivalent to that of the ovulatory follicle. At the onset of puberty, the responsiveness of the hypophysis to GnRH secreted from hypothalamus increases resulting in pulsatile secretion of gonadotrophins, more importantly of the luteinizing hormone (LH), which is involved in the final maturation of dominant follicle. The mature follicle starts secreting more estradiol, which exerts a positive feedback effect on hypophysis for LH surge resulting in ovulation of the mature dominant follicle [80]. The mean number (8.48 ± 0.28) of small follicles recruited in a cohort is comparatively greater during pre-pubertal period than in adult animals (range 3-6) suggesting that the rate of follicular atresia is more elevated during pre-pubertal period than in adult animals.

1.9.        Follicular Dynamics during Postpartum Period

Resumption of cyclicity in postpartum buffaloes has been ascribed to an FSH plasma increase leading to new follicle recruitment and establishment of follicle dominance. Pituitary dysfunction is an important factor responsible for postpartum ovarian inactivity. Despite the ability of exogenous GnRH to release LH and FSH, the characteristic of a preovulatory surge is restored by Day 20 postpartum [81]. In addition, other factors are responsible for the resumption of cyclicity in buffaloes like dietary antioxidants supplementation [82], the light–darkness shift over the months together with cold or hot climatic conditions [83]. Postpartum resumption of ovarian activity and estrous cycle in buffaloes are conditioned by season and by reproductive maturity of the animals [84,85].

The ovaries are characterized by the growth and regression of several small (up to 5 mm) and medium sized (> 5 and < 10 mm in diameter) follicles until detection of first postpartum dominant (= 10 mm) and/or ovulatory follicle during the early postpartum period [52]. Follicles greater or equal to 3 mm in diameter register a gradual increase until the first 15–20 days after calving, thereafter, they show fluctuation caused by periodical selection of growing follicles from the available pool. Follicles greater or equal to 3 mm in diameter have been recorded significantly in higher number in the ovary contralateral to the previous gravid horn immediately after calving [51]. The interval from calving to the detection of first large follicle was significantly earlier (P < 0.01) on the ovary contralateral to the previous gravid horn during the first month of calving [86].

The first postpartum ovulation was equally distributed in the ovary ipsi-lateral (n=7) and contralateral (n=7) to previously gravid uterine horn between 14 and 28 Days postpartum; however, the first postpartum ovulation occurred significantly earlier (P < 0.05) on the ovary contralateral to the previous gravid uterine horn. Such a difference between the two ovaries persists for the first 3 weeks until a balance is reached toward the end of the first month after calving. Among primiparous (n=10) and pluriparous (n=10) Mediterranean Italian buffaloes, within the 60 Day period of transrectal ultrasound monitoring of the ovaries, first postpartum ovulation was recorded in 4 and 8 of them respectively [51]. The authors reported that the average calving to first postpartum ovulation interval was 25.5 ± 6.9 days in primiparous buffaloes and 15.5 ± 1.3 days in pluriparous buffaloes. Similarly, the average first postpartum ovulation interval was 23.1 ± 1.5 days and 14 of 20 animals ovulated by 28 days postpartum in suckled Mehsana buffaloes [44]. Conversely, calving to first postpartum ovulation has been reported later (52.67 ± 8.02 days) in 3 of 12 suckled Murrah buffaloes during two months postpartum [87]. The first postpartum ovulation is usually associated with the absence of overt signs of estrus in the majority of animals. The first postpartum ovulation occurred in 12 of 20 Mediterranean Italian buffaloes [51] and in 19 of 20 Mehsana buffaloes [52] by 60 days postpartum during the breeding season. The early return to active follicular development and occurrence of first postpartum ovulation in many animals by the end of first month postpartum, demonstrated the ability of the buffalo ovary to resume ovarian activity early after calving. This suggests that ovarian responsiveness may not be the major reason for the variable duration of the postpartum anestrus period commonly observed in buffaloes [52]. The mean interval from wave emergence to first postpartum ovulation has been reported to be 14.0 ± 1.6 days in Murrah buffaloes [88].

Following first postpartum ovulation, a new wave of follicular development leading to ovulation has been reported in buffaloes during the early postpartum period. Most of the animals display a short estrous cycle with a short luteal phase soon after parturition in Mediterranean Italian [51], Murrah [88], Mehsana [89] and Thai swamp buffaloes [46]. The length of such cycles varies between 8 and 12 days and is not affected by parity of animals. Short estrous cycle is characterized by a one follicular wave culminating in ovulation. Non-significant differences in the size of the ovulatory follicle have been reported between short and normal estrous cycles. The pattern of a one-wave follicular growth of a short estrous cycle is different to the growth pattern of a one-wave cycle of a normal length estrous cycle . The ovulatory follicle of a single wave of a short estrous cycle exhibits only a growth phase after its emergence before ovulation, whereas three distinct phases of follicular development are reported in buffaloes with one-wave cycles of normal estrous cycle length, viz. growth phase, regression phase and atypical re-growth phase [50]. With early demise of the CL, the first dominant follicle ovulates during a short estrous cycle because plasma progesterone concentrations declined with the regression of the CL. The animals that have a short estrous cycle, show a short luteal phase and have low plasma progesterone concentrations (< 1.0 ng/ml) indicating failure of proper formation of the corpus luteum. A short luteal phase has been reported at the onset of the estrous cycle after a postpartum anestrus in water buffaloes [90,91]. Following the first postpartum ovulation, plasma progesterone levels are correlated with morphologic and functional activity of the CL in buffaloes [92]. The early decline of plasma progesterone levels in such cycles may be attributed to a short life span of the CL [73]. It would appear that the corpora lutea associated with short estrous cycles have short life span as a result of (a) lack of luteotrophic support, (b) failure of luteal tissue to recognize a luteotropin and/or (c) enhanced secretion of luteolytic agent, mostly PGF2α, by involuting uterus during early postpartum period [93,94]. Some postpartum buffaloes display estrous cycles of normal length ranging from 19 to 22 days. Such animals show one- and two-wave follicular growth during the estrous cycle [51]. Buffaloes that never ovulated after calving up to 60 days postpartum, displayed nevertheless some degree of ovarian follicular activity [51,87]. Such activity is reported either in both ovaries or more predominantly in one of the two regardless of the site of the CL of gestation [51].

1.10.        Follicular Dynamics during Pregnancy

The follicular dynamics during early pregnancy in buffalo heifers (n=10) was studied from Day 18 to 60 of pregnancy [20]. The follicular development was characterized by waves of different patterns; three animals presented follicular waves of 9.67 ± 0.58 days in duration. The remaining seven heifers presented an irregular follicular development with three different patterns. The first irregular pattern was characterized by waves of 7.40 ± 1.26 days in duration, with the dominant follicle reaching maximum diameter of 11.4 mm. The second irregular pattern presented waves lasting 6.44 ± 1.81 days, with the dominant follicle a reaching maximum diameter of 9.0 mm. The third irregular pattern was characterized by periods of subsequent follicular growth without defined phases from 18 to 60 days of pregnancy.

The regular development of large follicles during pregnancy may not play a significant physiological role for the pregnancy, but they are present as safeguard in the event of early embryonic mortality. The presence of a large follicle would ensure a relatively fast return to estrus in two ways: (i) estradiol from a large follicle would be available for the estradiol-oxytocin-PGF2α cascade that is thought to cause luteolysis [95] and subsequently, (ii) a follicle would develop and be available for ovulation once progesterone had returned to basal concentration, thus preventing a prolonged anestrus period after embryonic mortality. A recent study [96] documented the presence of < 6 mm, 6-10 mm and >10 mm follicles both during 1-3 month and 3-6 months of pregnancy in abattoir derived genitalia of buffaloes in Saudi Arabia. Descriptions on follicular growth characteristics during the last trimester and near parturition in buffaloes are not available.

1.11. Temporal Relationship of Hormones, Growth Factors and Follicular Growth

Folliculogenesis can be divided into three separate stages: (1) recruitment, the stage during which a pool of growing follicles begin to grow rapidly; (2) selection, a process whereby follicles are selected for further growth; and (3) dominance, the process whereby the dominant follicle undergoes rapid development while the growth of subordinate follicles is suppressed. This pattern of follicle development is associated with changes in expression of mRNA encoding gonadotrophin receptors [97,98] and steroidogenic enzymes [99] and allows selected follicles, when exposed to the requisite hormonal environment, to ovulate in response to the preovulatory gonadotrophin surge. The intra-follicular concentrations of IGF-1 cause changes in the expression of genes associated with steroidogenesis and may thus significantly affect follicle development [98]. Endocrine signals (for example, gonadotrophins, inhibins and steroids), as well as locally produced growth factors, are responsible for the control and coordination of these processes. The temporal and spatial expression of many classes of growth factors and their receptors has been identified in many species during follicle development. In ruminants, 40 days are required for an early antral follicle to reach the preovulatory stage [100,101]. Schematically, antral follicular development involves two phases. In the first phase, follicle grows slowly, up to 3 to 4 mm in buffaloes, and follicular growth is directly related to the proliferation of granulosa cells. This phase is not dependent on gonadotrophin supply. The second phase corresponds to terminal development of antral follicles up to preovulatory stage. During this phase, follicular growth is rapid due essentially to the enlargement of the antrum. In addition, terminal follicular development is characterized by important increases in steroidogenic capacity and responsiveness of granulosa cells to FSH and LH. This phase is strictly dependent on gonadotrophin supply. In buffaloes, fluctuations of FSH concentrations are temporarily associated with waves of terminal follicular growth, suggesting a stimulatory effect of FSH on the emergence of each follicular wave [18]. However, the mechanisms leading to the selection of one dominant follicle and atresia of other gonadotrophin-dependent follicles of the same wave are not fully understood. Increasing evidence suggests that growth factors modulate folliculogenesis. The growth factors are ubiquitous peptides, acting in a paracrine and /or and endocrine way and are involved in regulation of cell proliferation, differentiation and survival. The different growth factors have been classed in to separate families, based on their structure and their biological activity. The epidermal growth factor (EGF) family, the fibroblast growth factor (FGF) family, the platelet derived growth factor (PDGF) family, the insulin like growth factor (IGF) family, the transforming growth factor β (TGF-β) family including inhibin and activin, and the hematopoietic growth factors (cytokines) have been discovered and identified. The intra-follicular concentrations of IGF-1 cause changes in the expression of genes associated with steroidogenesis and may thus significantly affect follicle development [98]. The growth of buffalo preantral follicles is enhanced during in vitro culture in presence of IGF, FGF and EGF [61,65,102]. However, knowledge of all the elements of these systems is complex and their exact role in folliculogenesis is still hypothetical as they belong to complex systems made up of factors themselves, their receptors and binding proteins [103].

From in vitro studies in various animal species, it is now established that growth factors modulate survival, proliferation, and differentiation of follicular cells, acting in interaction with gonadotrophins. The different biological roles of growth factors on follicular cells from domestic ruminants are summarized below:

Presumably, physiologic selection of the dominant follicle occurs 2 days prior to emergence of follicular wave [104] since the largest follicle contains almost 10 folds greater concentrations of estradiol in follicular fluid than the next largest (subordinate) follicle. Substantially, higher levels of estradiol in follicular fluid are associated with the first portion of wave divergence; as greater estradiol concentrations have been detected in the presumptive dominant follicle versus subordinate follicles on Day 2. The earliest intrafollicular change that differentiates a future dominant follicle from other growing follicles is the enhanced capacity to produce estradiol-17β [105]. The FSH receptor numbers are significantly lowered in subordinate follicles on Day 4 suggesting that estradiol concentration plays a role in the divergence in the growth rate between dominant and subordinate follicles. The estradiol level in follicular fluid may have a key role in determining the physiological fate of follicles during selection of the first wave dominant follicle and it can be used as a reliable marker to predict which follicle in the growing cohort of 5 to 8.5 mm follicles will become dominant [106].

The period of continued growth of the dominant follicle is associated temporarily with basal level of FSH in blood indicating that the dominant follicle may grow more readily than subordinate follicles under reduced FSH concentrations. A decrease in FSH receptor numbers per cell has been noted between Day 4 and 6, however, FSH receptor numbers per follicle and LH receptor numbers per follicle or per cell do not decrease. There is no difference in intrafollicular estradiol concentrations, progesterone concentrations or the estradiol: progesterone ratio between Days 2 and 6. Loss of functional dominance occurs by Day 10 as indicated by emergence of a new follicular wave [107]. There is a decrease in both FSH and LH receptor numbers per follicle, and a significant decrease in concentrations of estradiol in follicular fluid. The decreased estradiol concentration may indicate a reduction in aromatase activity in the dominant follicle. It has been shown that a decrease in estradiol concentration on Day 7 and 11 corresponds to a parallel decrease in intrafollicular androstenedione concentration. Therefore, a decrease in androgen as a substrate for estrogen biosynthesis may contribute to the regression of the dominant follicle of the first follicular wave [108].

Intrafollicular progesterone concentrations are higher in the dominant follicle on Day 10 than on Day 2. In addition, the estradiol to progesterone ratio decreases in the dominant follicle from Day 2 to Day 10. It has been demonstrated that lower estradiol concentrations and increased progesterone concentrations are associated with follicular atresia [109]. Interestingly, calf follicles more than 9 mm diameter have a low ability to produce estradiol (10 times less than in cow) despite a testosterone output by theca cells which is similar to that observed in cows [68].

The current hypothesis on the role of growth factors and their binding proteins in the development of ovarian follicles is summarized below.

Development of Small Antral Follicles

Development of small antral follicles is not strictly dependent on gonadotrophins for the initiation of preantral follicle growth in buffalo, since most in vitro studies did not supplement gonadotrophins to preantral follicles in culture [60,62,63,64,102]. These studies suggested that factors of the FGF, EGF and IGF families directly influence the follicular growth rate by enhancing granulosa cell proliferation. In these follicles, vascularization is poorly developed, suggesting that paracrine regulation might be of particular importance. For example, TGF-α and IGF-II, synthesized by theca cells, may play determinant roles in controlling proliferation of adjacent granulosa cells. However, an endocrine action of growth factors cannot be excluded. Administration of GH stimulates the growth of small antral follicles in vivo probably by an indirect mechanism involving IGF-I of endocrine origin; in addition, a stimulating effect of FSH on the growth of small antral follicles is well established in different animal species. Similarly, the acceleration of buffalo preantral follicle growth occurred in vitro where gonadotrophins were supplemented in culture system [61].

Apart from growth factors, somatic cells play important roles in the development of preantral follicles. The presence of antral follicles [102] and ovarian somatic cells like cumulus cells, granulosa cells or ovarian mesenchymal cells, enhanced the growth of preantral follicles during in vitro culture [62,63,64,102]. A recent study demonstrated the role of nitric oxide on the growth and survival of buffalo preantral follicles during in vitro culture [110]. The presence of a dominant follicle or stage of estrous cycle had no effects on the in vitro growth, survival and antrum formation rates in culture of small or large preantral follicles [9] confirming that the growth of preantral follicles is independent of the gonadotrophins, but seems to be more dependent on local intra-ovarian factors. Although many in vitro studies have been done to define the role of growth factors, developmental processes from primordial follicles to preantral and subsequently antral follicles in vivo are complex and require further studies.

Terminal Follicular Development

Terminal follicular growth is a strictly gonadotrophin-dependent process corresponding to the initiation of follicular waves, selection of a dominant follicle(s) and terminal maturation of the preovulatory follicle(s). It has been established that an increase in FSH plasma concentrations precedes each wave of follicular growth in buffalo and may determine its initiation [18], whereas a decrease in FSH concentrations coincides with the enhanced secretion of inhibin and estradiol by the dominant follicle(s), and it is associated with rapid regression of non-dominant follicle(s). During the follicular phase of the cycle, the strong increase in LH pulsatility, associated with a dramatic increase in LH receptors in granulosa cells of the dominant follicle(s), stimulate their terminal development up to the preovulatory stage [111]. By contrast, during the luteal phase, the low frequency of LH pulses cannot sustain terminal maturation of the dominant follicle(s), therefore it regresses and secretes decreasing amounts of estradiol and inhibin, allowing a rise in FSH concentrations and the initiation of a new follicular wave.

Growth factors have their specific regulatory roles in these processes. Firstly, factors of FGF, EGF and IGF families may control the growth of small antral follicles and, in such a way, participate with FSH in the initiation of follicular waves. Secondly, growth factors of the IGF family are likely to be chief players in the process of selection of dominant follicle(s). It is important to emphasize that terminal follicular growth is characterized by a strong increase in the sensitivity of granulosa cells to FSH [112]. In a study involving in vitro buffalo oocyte maturation (IVM), it has been reported that IGF-I and FSH seem to act synergistically as autocrine and paracrine regulators of granulosa cells and therefore together promote mitosis, steroidogenesis, and protein synthesis during follicular development in buffalo [113,114]. This high responsiveness of granulosa cells to FSH probably results from intrafollicular systems of amplification, involving estradiol and IGFs, since both potentiate the action of FSH on granulosa cells differentiation. The concentration of bioavailable IGFs strongly increase in large antral follicles during terminal development. It is thought that only the more “mature” follicle in a wave i.e., follicle with the highest intrafollicular concentrations of estradiol and bioavailable IGFs, will be able to develop in the presence of decreasing serum concentrations of FSH. Moreover, it is suggested that FSH and LH, in the early and late follicular phase, respectively, may contribute to increased concentrations of bioavailable IGFs in the dominant follicle by regulating the synthesis and proteolysis of IGF binding proteins, thereby reinforcing the amplificatory mechanism. In addition, the high intrafollicular concentrations of inhibin in the dominant follicle might enhance LH-stimulated androgen production by theca, thus contributing to increased estradiol synthesis by granulosa cells and, as a consequence, to sustained terminal maturation of this follicle. The members of the TGF family, TGFα and TGFβ1, inhibit the growth and survival of preantral follicles, which lead to induced oocyte apoptosis in buffalo’s preantral follicles in vitro[114]. Recently, a definite role of vascular endothelial growth factors (VEGF) and VEGF receptors has been reported during ovarian follicular growth, development and maturation in buffalo [115]. These authors concluded that the VEGF may contribute to the extensive capillary proliferation associated with the increase in size, selection, and maturation of the pre-ovulatory follicle. VEGF may facilitate follicle maturation by enhancing the supply of nutrients, hormones, and other essential blood-borne signals to the follicle.