Reproductive Physiology of the Male and Female Buffalo

The reproductive physiology of the buffalo is similar to cattle in many aspects yet there are subtle differences in other aspects. Buffaloes exhibit marked influence of season on the expression of estrus, conception and calving in females [1,2] and depressed libido during hot summer months in males [2,3]. It has been mentioned that the timing of reproduction in buffalo species is highly variable. In sub-tropical and temperate countries, away from the equator, such as India, Pakistan and Italy [4,5], the breeding is seasonally dependent with breeding being favored during decreasing daylight length whereas in tropical countries near the equator the breeding is more dependent on the availability of feed [4]. Studies have demonstrated seasonal decline in sexual activity over many countries [6] in females [4,5,7-10] and males [11-14]. Therefore the breeding season for buffalo in the major buffalo rearing countries appear to extend from September to March [15-18]. Compared to cattle, buffaloes exhibit delayed puberty in both males [4,19] and females [1,8,20], poor estrus expression in a greater proportion of females [20,21], delayed postpartum estrus and prolonged inter-calving intervals [22]. Buffalo bulls have inherently lower libido [23], typically produce white colored semen [24] and buffalo bull semen has low contents of citric acid and fructose [25]. Female buffaloes inherently have lower population of primordial follicles in the ovaries [26,27] and evidence two to three follicular growth waves during the estrous cycle [28,29]. The estrus persists for 24-36 h and ovulation occurs 24-48 h after estrus onset [21]. Buffalo embryos have more lipid droplets and enter the uterus 96-108 h after ovulation [30,31] and rapidly develop into hatched blastocysts by Day 7 of ovulation [30]. Embryonic elongation is presumed to occur similar to cattle [32] and interferon-tau [33] and trophoblastic proteins are secreted during pregnancy that serve as molecules for placental remodeling, immune protection of the fetus and placenta and progesterone secretion [34].

The gestation period for the buffalo ranges from 310 to 330 days and the corpus luteum is presumed to be necessary for pregnancy maintenance in buffalo although the placenta also secretes some progesterone during later gestation [34]. Delayed postpartum estrus in the buffalo is presumed to be due to seasonal and management effects including nutrition [22]. Twinning is rare in the buffalo and the sex ratio of male to female births is 1.15:1 [35]. In this chapter the authors briefly describe the reproductive physiology of the male and female buffalo.

1. Reproductive Physiology of Male Buffalo

1.1.        Testicular Development and Onset of Spermatogenesis

Testes are the primary organs of the reproductive system in males and perform two major functions – spermatogenesis and steroidogenesis. These two functions are performed by two basic units of the testis – seminiferous tubules and interstitial (Leydig) cells. Seminiferous tubules produce viable and potentially fertilizing spermatozoa from the germinal epithelial layer by a series of cell divisions and the interstitial (Leydig) cells, which lie between the seminiferous tubules, produce androgens or the male sex hormone, testosterone. In the mature buffalo bull, seminiferous tubules occupy about 82% of the testis [36].

During fetal life, testes are intra-abdominal and start migrating into the inguinal region around Day 110 day of gestation. Testicular descent into the scrotal sac is almost complete by Day 243 of gestation [37,38]. The descent of the testes from the genital ridge in fetus to an extracorporeal location is a mandatory developmental process to ensure that the mature testis produces normal spermatogenesis.

Early in fetal life, seminiferous tubules develop as solid sex cords with no lumen. These solid sex cords contain peripheral primitive Sertoli cells and large centrally placed germ cells (gonocytes). An undifferentiated gonad was first observed in buffalo embryos at Day 43 as a nodular structure attached medial to mesonephros [39]. At around Day 47 the embryos developed tunica albuginea and testicular cord like structures [39]. Around Day 65 of fetal age, the seminiferous tubules are present at the gonadal periphery and a network of polygonal mesenchymal cells is seen in the centre of the testis. The pre-Sertoli cells are first observed at this stage in the periphery of seminiferous tubular epithelium whereas gonocytes can be observed in the centre of tubules at around Day 76 of fetal age. Fetal Leydig cells are also found at Day 65, however, after Day 92, the interstitium expands considerably due to the differentiation of mesenchymal cells into the Leydig cells. The number of seminiferous tubules as well as their diameter increase with the advancing age of the fetus [40]. The undifferentiated gonad of the male embryo is finally transformed into a testis at around 300 days of gestation [39].

At birth, seminiferous tubules are still solid and have pre-sertoli cells and gonocytes of recognizable size. Large intertubular spaces are present in between solid seminiferous tubules. With advancement of age, central gonocytes migrate towards the periphery of the sex cords and enter a series of mitotic divisions to form pre-spermatogonia that give rise to the future generations of spermatogonia. Rapid proliferation of tubular contents occurs at 12 months of age and fully differentiated Sertoli cells and spermatogonia are observed. The lumen of seminiferous tubules form at 12 months of age and Leydig cells in groups of two and three are also seen in the intertubular spaces that had become restricted due to enlargement of seminiferous tubules. The diameter of seminiferous tubules continues to increase further. At 18 months of age, round and elongated spermatids are clearly present in large numbers for the first time. Lumen formation is completed and single or clusters of Leydig cells are present. At 24 months of age, active spermatogenesis is evident in the majority of the seminiferous tubules. Therefore, the establishment of spermatogenesis is progressive from birth, and marked changes are observed at 18 and 24 months after birth [41].

1.2. Puberty

In males, puberty is defined as the age at which the ejaculate contains sufficient spermatozoa to impregnate a female. A buffalo bull is considered to attain puberty when an ejaculate contains 50 million sperm, of which at least 10% are motile [42]. In buffalo bulls, testicular spermatogenic cell divisions start at approximately 12 months of age and active spermatogenesis can be seen from 15 months. However, the ejaculate contains viable spermatozoa only after 24–30 months of age [41-43]. This indicates that the male buffalo matures more slowly than male cattle [44] and has a longer time lag between onset of spermatogenesis and reaching puberty. In general, riverine as well as swamp buffalo bull attain puberty at around 18–24 months of age and sexual maturity is achieved only after 2 years of age when a bull can be used for natural mating (Fig. 1) or semen collection in a breeding program [41,45,46]. The age at puberty and sexual maturity may vary with different breeds, plane of nutrition and environmental factors [47,48]. Buffalo bulls are capable of breeding throughout the year, but they show seasonal rise and fall in reproductive functions [49-51]. Seasonality in male buffaloes is evident by reduced testicular and epididymal functions [49,50]. However, season of birth does not influence age, body weight, or testicular volume at puberty [52].

Figure 1. An adult male Murrah buffalo.

Puberty in buffalo bull is preceded by a marked increase in body weight and rapid testicular growth from 8–15 months of age. There is a progressive rise of testosterone production from the testis. Testosterone concentrations which remain low from birth to 12 months of age (0.3 ± 0.1 ng/ml), rise sharply at 14 months of age (2.7 ± 0.9 ng/ml). This is followed by another peak at 18 months of age (3.3 ± 1.2 ng/mL) [52]. Testosterone levels in sexually mature bulls remain stable. Contacts with females exert a major stimulus for testicular androgen secretion in buffalo bulls; although other seasonal factors (climate, food intake) may affect control of gonadal activity [51]. Environmental factors may also affect the onset of puberty in buffalo bulls [23].

The luteinizing hormone (LH) values in sera of buffalo bull calves are high at birth (2.12 ± 0.47 ng/mL) and show an early rise in the first few weeks of life. Serum concentrations of LH decrease to a nadir (1.48 ± 0.45 ng/mL) at 3-4 months of age, followed by gradual increase as the age advances to reach a plateau (3.29-3.50 ng/mL) between 8 and 15 months of age. Thereafter, serum LH values decrease with advancing age and averages 2.14 ± 0.56 ng/mL in 17-19 month old buffalo bulls [53]. Similar to cattle, LH is secreted in a pulsatile pattern with an average of 0.6 pulses/h and testosterone levels in peripheral circulation follow the LH secretory pattern, with an average of 0.32 pulses/h [54]. Plasma LH levels do not vary significantly between different seasons [50].

1.3. Endocrine Regulation of Spermatogenesis

LH secretion in males is an important factor for early sexual development and onset of spermatogenesis. Suppression of LH secretion at this stage usually leads to delay in testicular development. At birth, the pituitary is unresponsive to exogenous GnRH. The testis of buffalo bulls become responsive to exogenous gonadotropins at the age of four months and high plasma testosterone concentrations are observed within 24 h of a PMSG injection [55]. Responsiveness to a gonadotropin releasing hormone (GnRH) injection, increases with sexual maturity as evidenced by an increase in testosterone secretion in buffalo bulls of >6 month age [56].

The exact endocrine mechanism that regulates semen production and sex libido in buffalo bulls is poorly described. Similar to cattle, reproductive function in males is regulated by complex interactions of hormones produced by hypothalamo-pituitary-testicular axis. GnRH is synthesized in the hypothalamus, and stimulates the secretion of LH and follicle stimulating hormone (FSH) from the anterior pituitary. Within the testis, Leydig cells and Sertoli cells are target cells for action of LH and FSH, respectively. Both cell types are involved in the complex endocrine and paracrine regulation of spermatogenesis. Leydig cells are stimulated by pulses of LH to secrete testosterone. Testosterone produced by Leydig cells diffuses into adjacent seminiferous tubules that support spermatogenesis and sperm transport. On the other hand, Sertoli cells are stimulated by FSH to secrete androgen binding protein (ABP), inhibin (INH) and convert testosterone into dihydrotestosterone (DHT) and estrogen. ABP forms a complex with androgens which helps in maintaining high concentration of androgens within testis for its normal function [57]. Testosterone released in blood, is responsible for maintenance and development of secondary sex characters, sex libido, accessory sex gland secretory activity and also to exert an anabolic effect. High levels of testosterone in blood suppress additional release of GnRH, LH and FSH from hypothalamus and the pituitary by negative feedback effect. On the other hand, inhibin suppress the FSH release without altering LH release and is responsible for the differential release of LH and FSH from the pituitary (Fig. 2A). The seasonal variations in plasma LH in male buffalo were significant [49]. A previous study on Murrah buffaloes [53] examined the circadian and pulsatile variations in plasma inhibin, LH, FSH and testosterone in adult Murrah buffalo bulls. A single FSH pulse was detected in 2 of the 6 buffalo bulls whereas pulsatility was detected in LH secretion with an average of 0.6 pulses/h. Testosterone levels in peripheral circulation followed the LH secretory pattern. The mean concentrations of FSH and LH over 24 h were 1.66+0.25ng/mL and 3.33+1.02 ng/mL respectively.

Unlike females, exogenous administration of GnRH in males does not enhance reproductive function. In one study, IM administration of 1000 IU of eCG daily for 6 days to four month old buffalo calves increased testis size, testosterone production and activated spermatogonia [54]. Concentrations of testosterone and oestradiol-17β increase after administration of GnRH in post pubertal buffalo bulls. However, the increased testosterone response does not have any significant correlation with sperm output characteristics [58,59]. A dose of either 8 or 12 μg Buserelin (GnRH) has been shown to increase testosterone secretion whereas a higher dose (16 μg, Buserelin) has a negative effect [60].

Figure 2. Mechanisms of endocrine regulation of steroidogenesis (A) and gametogenesis (B).

1.4. Spermatogenesis

Spermatogenesis is a complex biological process of cellular transformation of spermatogonial cells into spermatozoa within seminiferous tubules of the testis. The seminiferous epithelium consists of Sertoli cells and numerous concentric layers of germ cells known as spermatogonia in different stages of cellular transformation. Sertoli cells and spermatogonial stem cells are located along the basement membrane of the seminiferous tubules. As spermatogenesis proceeds, the developing gametes migrate from the basement membrane of the seminiferous tubules toward the lumen.

The cytoplasm of Sertoli cells extends around all germ cells to nurture and maintain the process of spermatogenesis. Sertoli cells have several important functions that include: provision of nutrition to differentiating germ cells, compartmentalization of seminiferous tubules (by tight junction of adjacent Sertoli cells for immunological protection of differentiated sperm cells), release of sperm from Sertoli cells into lumen of seminiferous tubules (spermiation), phagocytosis of degenerating germ cells and residual cytoplasmic body that remains from released sperm, secretion of fluid, several proteins, hormones and mediating the action of FSH and LH. In neonatal mammals, FSH stimulates Sertoli cells proliferation, producing a final number of cells that differentiate terminally during puberty. A single terminally differentiated Sertoli cell support only a limited number of germ cells; the number of Sertoli cells determine the final testis size in mammals [61].

The process by which spermatogonia develop in to mature spermatozoa (spermatogenesis) can be grouped into two distinct stages - spermatocytogenesis and spermiogenesis. Spermatocytogenesis involves mitotic germ cell division to produce stem cells and primary spermatocytes and then meiosis for duplication of chromosomes, exchange of genetic material, and two cell divisions that reduce the chromosome number and yield four spermatids. In spermiogenesis, spherical spermatids undergo a progressive series of structural and developmental changes to form spermatozoa. In buffalo seminiferous tubules occupy about 82% of the testis [36].

Spermatogenesis can be divided into 6 stages according to characteristic cellular associations in the seminiferous epithelium [36]. Heavy cell loss is observed in phase 4 and involves the spermatogonial fraction [62]. On the basis of morphological changes in the nuclear details, progressively dividing buffalo spermatogonia can be classified as type A (A0, A1, A2), Intermediate (In) and type B (B1, B2, B3) that ultimately differentiate to resting primary spermatocyte [63]. However, Bilaspuri and Guraya [61] classified spermatogonia as type A1, A2, A3, In, B1, and B2 before forming the primary spermatocyte. The spermatogonia of buffalo bull undergo 6 generations of successive mitotic divisions to form a primary spermatocyte [62]. A recent study on Nili Ravi buffalo demonstrated that differentiation of basal cells to Sertoli cells starts at 6 months of age and formation of Sertoli cells is completed at 12 months. Spermatocytes were first seen at 12 months whereas abundant spermatids were visible at 18 months of age [63].

1.5. Spermatogenesis Sequence within Seminiferous Tubules

Both ends of each seminiferous tubule (ST) are connected to rete testis and form a loop. Along the length of this tubular loop, the process of spermatogenesis is not uniform. Instead, a complete ST can be segmented into well-defined cellular associations of spermatogonia, spermatocytes and spermatids in different combinations that undergo cyclic developmental changes. As many as 8 distinct cellular associations or stages have been identified in buffalo bull that constitutes a wave of the seminiferous epithelium [64]. These cellular associations in the segment of tubules are arranged in a consecutive order of development and reappear in series with cyclic regularity. That is, stage I is followed by stage II, which is followed by stage III, etc. through stage VIII, which is again followed by stage I. The sequential order of the basic cellular associations or stages along the length of the tubule is known as the wave of seminiferous epithelium or spermatogenic wave. A loop of ST may contain several such spermatogenic waves along its length. The more advanced stages of the wave are located closer to rete testis whereas less advanced stage is located in the middle of the loop where a reversal site is typically found.

The cycle of the seminiferous epithelium is defined as a series of changes in a given area of seminiferous epithelium between two appearances of the same developmental stages (cellular associations). The duration between two successive appearances of the same cellular associations at a given location in the seminiferous tubules varies among domestic species. Duration of the seminiferous cycle is 8.6 days in buffalo bull [65]. The duration of spermatogenesis from the time of production of committed spermatogonia to spermiation is species-specific [65,66]. The total duration of buffalo spermatogenesis is constituted by 4.57 cycles of seminiferous epithelium [28]. The approximate duration of spermatogenesis is 38 days in buffalo bull [66]. Significant germ cell loss occurs during spermatogenesis in mammals and only 2-3 spermatozoa out of 10 are produced from each differentiated type A1 spermatogonia [62] (Fig. 3).

Figure 3. Schematic representation of spermatogenesis.

1.6. Sperm Maturation

Spermatozoa produced in the testis are immotile. After the process of spermiation in ST, released sperm are transported through the rete testis and vasa efferentia into the epididymis, where they are stored. This transit is aided by fluid secretions from Sertoli cells and rete testis, cilliary movement of efferent duct and contractile elements of the testis.

During this transit spermatozoa undergo a process known as sperm maturation in which sperm gain the potential ability to fertilize the egg and transfer the paternal genomic information to the next generation. Epididymal transit brings about several structural and functional changes in the sperm. There is progressive loss of water from sperm. Cytoplasmic droplet migrates distally and is eventually lost. Changes in sperm membrane lipid composition and surface proteins occur so as to acquire potential ability to fertilize the ova. These changes are a progressive phenomenon and brought about by epididymal secretions. Spermatozoa are transported through the epididymis in about 5.65 days in the buffalo bull [42] and remain stored in cauda epididymis until their release at ejaculation.

Sperm maturation is facilitated by seminal plasma proteins that are produced by the epididymis and the accessory sex glands that play an important role during the process. Some of these proteins bind to the surface of spermatozoa, where they might act as binding partners for surface structures in the female genital tract. Certain sperm surface proteins might be required for the modulation of the female immune system to suppress an adverse immune reaction directed against the spermatozoa and can be used as markers for male fertility [67,68]. Studies suggest that there are considerable species specific differences in the expression of seminal plasma proteins that contribute to sperm maturation [69]. Passage of spermatozoa from caput to cauda epididymis is associated with significant diminution of sperm head length, breadth and area with the head appearing more elongated [70,71].

1.7. Blood Testis Barrier

Spermatozoa produced within the ST are haploid and have heterogeneous population of X and Y chromosome bearing sperm cells. Therefore, these sperm cells are at risk of immunologic attack by the diploid body. This attack is protected by the presence of the blood testis barrier in which seminiferous tubules are not penetrated by blood or lymph vessels; and ST are compartmentalized by the tight junctions of adjacent Sertoli cells. The presence of S-100 beta (low-molecular-weight proteins found in vertebrates and characterized by two calcium-binding sites and implicated in a variety of intracellular and extracellular functions) in Sertoli cells is involved in establishing the blood testis barrier [72]. The adjacent Sertoli cells unite to form tight junctions and divide the seminiferous tubules into two distinct compartments; the basal and adluminal compartments [66]. The basal compartment is freely accessible to biological components that have previously penetrated the myoid layer of ST. This compartment is occupied by spermatogonia and preleptotene spermatocyte. These cells divide by mitosis to produce spermatocytes that are transferred to the ad-luminal compartment. The adluminal compartment contains the more advanced stages of spermatocyte and spermatids, which freely communicate with the lumen of tubule. The adluminal compartment is the site of meiosis and spermiogenesis, which comprise all the division and morphological changes that must occur to change round diploid spermatogonia into highly specialized motile, haploid spermatozoa.

1.8. Thermoregulation of the Testis

The testes are located outside of the body cavity in a specialized pouch called the scrotum. The main function of the scrotum is to support and protect the testes. The scrotum keeps the testicles 2-3 degree Celsius below normal body temperature, which is required for normal spermatogenesis [73]. Increasing the temperature of the testes, by insulation, results in a decrease in motility, increase in abnormal morphology and disruption of nuclear protamination [74]. Thin scrotal skin, absence of scrotal hair, presence of tunica dartos muscles, large numbers of sebaceous and sweat glands on the scrotum help in lowering the scrotal temperature. The pampiniform plexus of the testis also prevents excessive heating of the testis due to its angio-architecture [75]. During the hot season, tunica dartos muscles relax and hang the testes far away from the heat of the body. While during the cold season, the scrotal muscle contracts and retract the scrotum to bring the testes closer to the body. Semen quality in bull reflects the degree of normality of the function of their testes, ducti epididymides and the accessory sex glands.

1.9. Epididymis

The epididymis is a highly convoluted, tightly coiled tube that attaches to the exterior of the testes and connects the efferent ducts of the testicle to the vas deferens. The epididymis consists of three main segments: the caput (head), corpus (body) and the cauda (tail). The bovine epididymis can reach a length of 40 meters [57]. Epididymal transit time varies among bulls but averages 8 days, with a range of 4 to 15 days [76].

Cauda epididymis is the principal storage organ that stores more than 60% of the total epididymal sperm reserve. The main functions of the epididymides are sperm maturation, transport, concentration; protection and storage that results in a heterogeneous sperm population that become motile and capable of fertilizing oocytes [76]. The African buffalo epididymal sperm stored within the epididymides for 5 days at 4°C were compared for their motility [70]. A significant decrease in motility was noted in the first 8 hours of storage (60% to 50%) but this remained constant until 64 h of refrigerated epididymal storage (40% to 30%). Following 5 days of storage within the epididymides collection of motile sperm (10%) was still possible.

Epididymal sperm reserves in buffalo bulls range from 3.9 to 36.2 billion [77-79]. The percentage distribution of this reserve in different segments revealed that sperm were distributed between the caput, corpus and cauda epididymis in proportions of 28.82, 14.63 and 60.55, respectively [78]. The mean ± SEM epididymal transit time was 5.65 ± 0.24 d [42]. Passage of spermatozoa from caput to corpus and then to cauda epididymis is associated with significant reduction of sperm head length, breadth and area with the head appearing more elongated [71].

1.10. Accessory Sex Glands

The accessory sex glands of intact buffalo bulls vary significantly in the concentration of various elements except that the Fe, Ca, Cu and fructose are relatively more concentrated in the seminal vesicles [79]. The prostate has the highest concentration of Zn, while the highest concentrations of Na, K, Mg and P are found in the bulbo-urethral glands. The concentration of Cu decreases significantly in older bulls. Castration results in a highly significant reduction of Zn concentration in all accessory glands [79]. The distribution of noradrenergic and peptide-containing nerves during the mating season in male buffaloes revealed a dense innervation of the vas deferens as well as the other accessory genital organs compared to the innervation during the non-mating period [80].

1.11. Sperm Production in Buffalo Bull

Sperm are produced on a continual basis within the seminiferous tubules. Daily Sperm production per gram of testicular parenchyma is a measure of spermatogenic efficiency in sexually mature animals and is useful for species comparison. The efficiency of sperm production in swamp and riverine buffalo bull is quite uniform and average 13 to 14 x 106 sperm per gram of testicular parenchyma per day [42,46]. Actual production of spermatozoa is higher because all produced sperms cannot be collected. In buffalo bulls, a significant positive correlation between scrotal circumference and semen volume and concentration per ejaculate exist indicating that scrotal circumference is a useful indicator of potential sperm output and may serve as an important criterion for selecting young bulls as artificial insemination sires. The daily sperm output in Murrah buffalo bulls is nearly 45% lower compared to Holstein bulls of the same age, presumably due to their nearly 40% lower scrotal circumference [81].

1.12. Sexual Behavior

Buffalo bulls are generally quiet and easy to handle. They are rarely aggressive towards people but can be very aggressive towards one another. Free ranging breeding bulls show strong territory behavior. It is common practice in most Thai villages to castrate buffalo bulls to be used as draught animals when they reach the age of 3 years. Buffalo bulls, in general, are lethargic and have weak libido [24,82]. When a buffalo bull senses and notices a female in estrus, it straightens its body, elongates its neck and carries the head high followed by a "flehmen" response (Fig. 4). Bulls typically ejaculate instantaneously on the first intromission. The ejaculatory thrust and the forward leap at ejaculation are less marked as compared to that of the bull. Buffalo bulls are slow to mount on a dummy that is restrained in a chute compared to a freely moving dummy. Buffalo bulls spent more time sniffing the dummy before mounting. Reaction time of buffalo bulls varies between 29 to 105 seconds [24,83,84]. The libido did not vary between Murrah, Surti and local buffalo bulls in Sri Lanka [85].

Figure 4. Flehmen response shown by a buffalo bull.

1.13. Seminal Characteristics

Buffalo semen is milky white; never yellow. Its consistency depends on its content of spermatozoa and is affected, among other factors, by frequency of ejaculation. The average sperm concentration is about 800 million/mL. Values as high as 1500-2000 million sperm/ml and as low as 200 million sperm/mL have been recorded. First ejaculates contain higher number of spermatozoa per ml compared to second ones. According to several reports, sperm concentration is higher in summer [43-45] or spring [46]. Initial motility and live sperm percent are also optimal during winter [44,46]. Improvement of semen quality during winter and /or spring is consistent with higher conception rates of buffaloes observed during these seasons [47]. The percentage of abnormal spermatozoa in buffalo semen varies between 3 and 26% [48] being less frequent in winter [43]. Compared to Egyptian buffalo bulls, the quality of the Indian Murrah semen is optimal in spring [46]. Physical and biochemical characteristics of whole semen and seminal plasma of buffalo reveal variations (Table1). Buffalo bulls in regular use as semen donors at AI centers can be ejaculated thrice in rapid succession, two times a week without serious effect on their semen quality [47,82]. More detailed information on this subject can be found in Chapter 29 on Semen Characteristics and Artificial Insemination in the Buffalo in this book.

2. Reproductive Physiology of the Female Buffalo

2.1. Ovarian Development

Ovarian development in buffaloes has been recently described in detail [87]. It was mentioned that the 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 [88]. Studies in Egyptian and Murrah buffaloes demonstrated that at the 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 [89,90]. At 100 days, the left ovary started to descend towards the pelvic position while the right ovary was attached to the kidney [90]. 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 [89]. 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 [88,91]. Oogonia were positively identified in the 3rd and 4th month of gestation. By the 6th month, their numbers had decreased and few were visible [89]. 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) [34], a meiotic phase (4-6 months; primary oocytes are formed) [34,92] and an intense germ cells degeneration phase (7 month to the end of gestation) [89]. There are 3 waves of degeneration of germinal cells. The first wave affected the oogonia and peaked at the 4th month of gestation. The second and third waves affected oocytes and occurred at the 6th month of gestation and at term [89,90]. 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 [93].

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 [93]. 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 starting at the end of the period of oogenesis, some primordial follicles continuously begin to grow, however, up to puberty, the majority of these primordial follicles become atretic and disappear [93]. Probably, only one percent of the total oocytes attains maturity and is released through ovulation [92].

2.2. Ovarian Follicle Development

2.2.1. Ovogenesis

The ovum, the female gamete, originates from the primordial germ cells which develop during the early embryonic stage. These primordial germ cells migrate from the yolk sac to the genital ridge approximately by Day 35 of gestation in cattle and buffalo. These genital ridges differentiate into gonads and the primordial germ cells develop into oogonia. The oogonia multiply by mitosis after sexual differentiation and enter the prophase of the first meiotic division also called -primary oocytes (oogenesis). Oogenesis is completed before or shortly after birth in domestic animals. At this stage the ova are surrounded by a single layer of epithelial cells called follicles. Thus at birth, all female calves are born with their full complement of oocytes in primordial follicles which progressively decrease during the life of the animal. Buffalo ovaries have only 10,000–20,000 primordial follicles [27] compared to over 100,000 in cattle. Studies have shown that follicular turnover in buffaloes is similar to cattle [21].

After puberty, the oocytes resume development and undergo metaphase, anaphase and telophase of the first meiotic division and develop into secondary oocytes or undergo atresia. During this stage of meiotic division, the chromosomal number is reduced to half and the first polar body is extruded. Following ovulation prior to fertilization, the secondary oocyte undergoes another meiotic division and the second polar body is extruded before fertilization. Ovulation marks the beginning of the luteal phase, and is the culmination of a process called oogenesis, in which germ cells undergo maturation. Germ cells are present in follicles that contain receptors for FSH, which in turn stimulates the growth and maturation of responsive follicles.

2.2.2. Pre-Antral Follicle Development

After the end of oogenesis, the ovary consists of primordial follicles within a framework of interstitial tissue lined with germinal epithelium. The oocytes formed during the fetal and neonatal period are the only source of oocytes available during the entire sexual life [87]. The primordial follicle reserve either undergoes atresia or some primordial follicles begin to grow until puberty and subsequently undergo atresia. 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 [87]. 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 [94]. Based on in vitro studies the growth of bubaline preantral follicles to antral follicles is considered to require 3-4 months [95,96]. 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 [97,98]. The growth of preantral follicles is independent of gonadotrophins; however, the process appears to be more dependent on local intra-ovarian factors.

2.2.3. Antral Follicle Growth (Follicular Growth Waves)

The later stages of antral follicle development in buffaloes are characterized by two or three waves of follicular growth during each estrous cycle. The follicular dynamics in buffaloes has been recently described in detail [87]. Follicular growth waves are seen during prepubertal period, puberty, anestrus, pregnancy and the postpartum period [87]. 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 becomes the dominant follicle and the dominant follicle of the second or third wave ultimately is responsible for estrus and will ovulate.

2.2.4. Follicular Atresia

Follicular atresia is a common phenomenon reported in cattle and buffaloes and it appears to be greater in buffaloes compared to cattle [87]. Follicular atresia is the degeneration of follicles occurring in three steps. The first step of atresia is characterized by numerous pyknotic nuclei in the follicular fluid and in the granulosa layer of the follicular wall; the second step is characterized by changes in the granulosa layer alone, with few or no pyknotic nuclei in the antral fluid. The cumulus disappears and only the oocyte remains followed by the growth of connective tissue into the lumen. The last step results in the formation of a corpus atreticum. Studies in buffaloes revealed that there were twice as many atretic follicles as normal ones (31.7 vs. 14.6, respectively) in cycling animals and the average atresia frequency for buffaloes ranged from 76.6 to 82%, observed from ovaries collected at the slaughterhouse [99,100].

2.3. Puberty

The age at which estrus is first detected is referred to as puberty. Buffalo heifers attain puberty at about 24-30 months of age and at 225-275 kg body weight, i.e when animals attain 55–60% of their adult body weight [23,101], however, swamp buffaloes attain puberty at 21–24 months of age. Reaching puberty is more related to body weight than to age. However, individual’s genotype, nutrition, management, season of birth, climatic factors, occurrence of disease and the presence or absence of a mature male can influence the age at puberty [102].

Buffalo heifers are slower to reach puberty compared to cattle [5,103]. The age at puberty is difficult to establish because of difficulties in estrus detection in this species and most estimations appear to have been extrapolated from the age at first calving [1]. Swamp buffalo heifers are known to exhibit first estrus later (21 to 24 months) compared to the river type buffalo heifers (15 to 18 months), however, the first conception occurs at around 24 to 36 months of age [5,8]. The delay in puberty and the consequent delay in conception is one of the problems that lead to low reproductive efficiency in the buffalo species [1]. Many factors influence the age at puberty in buffalo such as breed, season, climate, nutrition and growth rate [5,19,21,23,104-106].

The reasons for the delayed onset of puberty in pre-pubertal buffalo heifers are only partly explained on the basis of low profiles of circulating thyroid hormones [107,108] low body fat [107] or the inherently suboptimal functioning of the hypothalamo-hypophyseal-gonadal axis and the consequently low circulating hormones [109,110]. A close association between growth hormone and LH has been found with regards to attainment of puberty [111]. The highest LH concentrations were recorded just 1 month before puberty was reached [111]. Pubertal heifers had follicular growth similar to adult buffaloes, however, growth rates were slower and the size of the dominant follicle was smaller in heifers [112]. There is a wide variation in the age at puberty and age at first calving in buffalo heifers of different breeds in different countries (Table 2). The age at first calving is influenced by many variables with being highest in rural buffaloes in India. The heritability estimates for this trait vary from 0.12 to 0.53 [113-116].

2.4. Endocrinology of Female Reproduction

The estrous cycle in buffaloes is regulated by the hypothalamic-pituitary-gonadal axis consisting of hypothalamus, pituitary, and the ovary. The hypothalamus produces GnRH in response to neuro-endocrine signals and circulating reproductive steroids. GnRH has a trophic action on the pituitary stimulating the production of gonadotropins; FSH and LH. These hormones stimulate ovarian follicles to grow and ovulate. Furthermore, upon ovulation the follicle transforms to form the corpus luteum (CL) under LH influence. The CL is responsible for progesterone production in cyclic and pregnant animals. The primary hormones produced by the ovary are estrogen and progesterone, in addition to other local hormones. These hormones are transported by the bloodstream to target tissues through sex steroid binding globulins. Estrogen, produced by the growing follicle exerts a positive feedback for stimulating pulsatile LH release [99,100]. It also influences the estrus behavior in buffaloes. In addition, estrogen initiates the uterine contractions necessary to transport sperm through the female reproductive tract. It also increases blood flow to the genital organs and production of mucus by glands in the cervix and vagina.

Progesterone produced by the CL prevents cyclicity by acting on the anterior pituitary in a negative feedback pattern, thereby, decreasing the release of FSH and LH. Progesterone primes the uterus for the reception of a fertilized egg and subsequent pregnancy. It also helps to maintain pregnancy by suppressing uterine contractions and promoting development of the uterine lining. Another hormone that has an important role in female reproductive function is prostaglandin F2α (PGF2α). This hormone is secreted by the endometrium of the uterus and helps in the initiation of ovulation and also causes the demise of the CL, which results in the withdrawal of progesterone’s negative feedback mechanism (Fig. 2B).

2.5. Physiology of the Estrous Cycle

2.5.1. Estrous Cycle

After reaching puberty, a rhythmic pattern of sexual activity known as estrous cycle is initiated in the female. The most clearly definable sign of this rhythmic pattern is estrus, a period of sexual receptivity, which recurs every 21 days (range 19-23 days). Broadly, the estrous cycle can be divided into four phases viz. proestrus (3 days), estrus (24 h), metaestrus (3-4 days) and diestrus (12-15 days). Proestrus and estrus phases are grouped under follicular phase whereas metaestrus and diestrus are classified under luteal phase of the estrous cycle [99,100,150].

In river buffaloes estrus lasts an average of 24 h (10-48 h) in comparison to a shorter time period in swamp buffaloes (19.9 ± 4 h). The female accepts the male for mating during this period [20]. During estrus, an ovum matures within the ovarian follicle under the influence of LH and ovulates approximately 11 h after disappearance of estrus signs in river buffaloes and 13.9 hrs in swamp buffaloes [151-153]. If the buffalo is mated or inseminated during estrus, the ovum is fertilized and reaches the uterus for further development. If pregnancy does not occur, then estrus recurs at the normal interestrus interval of about 21 days. Metaestrus bleeding does not occur in buffaloes. Under normal physiological conditions, cyclical activity remains absent before the onset of puberty, during pregnancy and for a short period after parturition.

2.5.2. Follicular Gowth Waves

Through daily visualization of ovarian follicles (Fig. 6A), it was observed that follicular growth occurs in a definite pattern in most domestic animals including buffaloes. Under the influence of hormones and other factors, the selection of follicles destined to grow occurs sequentially and a pool of follicles (5-20) grows for definite periods (2-3 times between two estrous cycles). This is referred to as follicular growth waves [100,154,155]. The first one or two follicular growth waves consist of a group of follicles being recruited and growing to 3-5 mm in diameter. These follicles then grow to 6-9 mm in diameter in response to FSH, and one of the follicles may even reach 12-15 mm [155-157]. From amongst this pool of growing follicles, one follicle within a growth wave will experience enhanced growth characteristics compared to the other growing follicles in its pool. This follicle is known as the dominant follicle (DF). However, follicles from the first wave in a two wave cycle or follicles from the first and second waves in a three wave cycle do not ovulate because of the inhibitory action of progesterone on the anterior pituitary production of FSH. Instead, the dominant and subordinate follicles undergo atresia (regression or death). The follicle selected to become the ovulatory follicle is the dominant follicle of the second or third follicular growth wave [1,158]. This follicle matures to a diameter of 12-15 mm and consists of an ovum or egg surrounded by many layers of theca and granulosa cells, around which forms a central cavity filled with fluid and encompassed by several thin cell layers. The buffalo oocyte has few peculiar features when compared to cattle. Oocyte mitochondria are present uniformly throughout in the cytoplasm. The cortical granules are present inside the oolemma. Other organelles viz. golgi apparatus, oval mitochondria, is also seen in the cytoplasm of buffalo oocytes. The most important feature of buffalo oocytes is the presence of a larger number of lipid droplets compared to cattle.

Post-ovulation after 14 days of progesterone influence, the uterus begins to release pulses of PGF2α into its venous drainage to the ovaries. Mean PGF2 alpha levels in buffaloes range from 200-250 pg/mL during estrous cycle and increase to peak values of 913 pg/mL 48 h before the onset of the next estrus [159]. Prostaglandin lyses the luteal tissue of the CL and causes its regression at the end of a non-fertile estrous cycle, resulting in a rapid decline of circulating progesterone and removal of the negative feedback of progesterone on the anterior pituitary. By Day 17, the luteal phase of the estrous cycle culminates.The action of prostaglandin is primarily through its G protein coupled receptor FPr, there is a marked reduction in the blood flow to the CL under the influence of prostaglandin. The FPr m-RNA expression, receptor number and affinity did not vary significantly within the luteal phase of the estrous cycle in the buffalo [160]. The follicular phase begins with the removal of the blocking action of progesterone, which allows for greater amplitude and frequency of GnRH pulses. Greater GnRH results in more FSH and LH production, which in turn supports follicular development of the dominant follicle in the follicular wave. This in turn stimulates the dominant follicle to produce increasing amounts of estrogen, which initiates positive feedback to the anterior pituitary. Once estrogen level reaches a threshold level, a surge of LH (at least 10 times greater than tonic levels) results in ovulation of the dominant follicle.

The CL in buffaloes is smaller when compared to cattle and another characteristic feature of CL growth in buffaloes is that it lacks a crown and is usually embedded into the ovarian stroma. These features hinder the accurate identification of ovarian structures by rectal palpation in buffalo as compared to cattle [159,160].

2.5.3. Endocrinology of the Estrous Cycle

Progesterone and Estrogen

During the estrous cycle, concentrations of progesterone and estrogen in blood and milk of buffaloes are similar to those in cattle; however, the peak concentrations are relatively lower in comparison to those in cattle [100,105,109]. Progesterone concentration in milk is below 0.3 ng/mL during the follicular phase of the estrous cycle and ranges from 3 to 12 nmol/L (~1–4 ng/ml) during the luteal phase and pregnancy [161]. Levels of Progesterone concentrations above 3 nmol/L indicate luteal function particularly 7 days after estrus. Concentrations of 4-5.1 ng/ml are reached by Day 5 and increase up to 12 ng/mL until Day 15 following estrus [162]. Progesterone levels below 0.3 ng/mL are interpreted as absence of luteal function. Estradiol plasma concentrations are at their highest during the peri-estrus phase (22.48+0.32 ng/mL), and eventually decline to 11.04+0.13 ng/ml during the mid-luteal phase [163,164]. Based on a gradual decline in circulating concentrations of progesterone in buffaloes during luteolysis, it has been proposed that CL regression is an extended process in the buffalo [165].

Gonadotrophic Hormones

In buffaloes, the FSH and LH concentration in blood show temporal patterns similar to those in cattle [164] with peak FSH levels (55-65 ng/mL) seen during the beginning of the estrous cycle compared to those observed during the luteal phase (24 ng/mL). In comparison, concentrations of LH that range between 25-35 ng/ml at the onset of estrus eventually decline after the end of estrus [166-168]. The interval from estradiol peak to LH peak has been reported to be 14.8 h and the duration of LH peak is considered to be 4.0 h [168]. Seasonal variations in plasma concentrations of FSH, LH, estradiol and progesterone have been recorded [109]. Seasonal increase in the concentrations of plasma prolactin in buffaloes has been attributed to the influence of photoperiod [164].

Other hormones which play a role in controlling the estrous cycle and pregnancy in buffaloes are melatonin, inhibin, activin, Pregnancy Associated Glycoproteins, prolactin and leptin [4,169,170].

2.5.4. Signs of Estrus in Buffaloes

Signs of estrus in buffalo are less overt than in the cow [100], homosexual behavior between females is rarely seen [27,169,171]. The main behavioral signs are restlessness, bellowing, tail raising, vulvar swelling, decreased feed intake and frequent voiding of urine [172]. The willingness of the female to stand for mating is regarded as a true sign of estrus [20]. During summer, estrus is exhibited only during the night or in the early morning hours. Silent heat is common during summer months [4]. The estrus signs are accompanied by changes in external genitalia viz. swelling of the vulva and reddening of the vestibular mucosa and changes in internal genitalia such as good uterine tone and coiling of the uterine horns. Due to vulval swelling, the horizontal wrinkles which are present on its external surface disappear in estrus animal [100]. Secretion of mucus from the cervix during estrus is less copious than in cattle and does not usually hang as strands from the vulva, [although a proportion of buffaloes may show mucus strands (Fig. 5)] but can be seen by transrectal back racking of genitalia or when the buffalo sits [20]. A few lactating buffaloes also exhibit Doka (temporary engorgement of teats without let down stimulus) 2-3 days prior to impending heat. However, individual variations are observed in the occurrence and the intensity of estrus signs in buffaloes.

Figure 5. A buffalo in estrus with cervico-vaginal mucus discharge.

2.5.5. Factors Controlling Estrus Behavior

The buffalo is regarded as seasonal breeder because the majority of buffaloes are cyclic only during the cooler months of the year [4]. High temperatures and long day lengths depress cyclicity and lead to suppression of ovarian function. High prolactin secretion has been identified as a factor contributing to acyclicity and poor fertility by lowering progesterone secretion during the summer months [4,169,170]. The seasonal effect on the reproductive function is governed by the pineal gland which secretes melatonin which in turn influences the circadian rhythm, and alerts the biological function of the hormones involved in the regulation of the reproductive function. Other factors which influence estrus behavior are genetic predisposition, age, uterine inflammation, the time of calving and their inter-relations [173].

2.6. Ovulation and CL Development

Ovulation is known to occur in buffaloes around 24-48 h (mean 34 h) after the onset of estrus [20] or 10 to 14 h after the end of estrus [17]. Ovulation is known to occur when the dominant follicle attains a diameter of 8.5 to 12.0 mm [29]. The size of the preovulatory follicle has been shown to have a positive impact on the size of the post ovulation CL and conception in buffaloes [174]. Due to a smaller ovarian size and a smaller follicle diameter, detecting ovulation by trans-rectal palpation in the buffalo seems difficult. The bubaline CL is smaller than that in cattle, often it does not protrude markedly from the surface of the ovary and sometimes it lacks a clear crown [20]. These characteristics make accurate identification of ovarian structures by transrectal palpation in buffalo more difficult than in cattle [22,175]. Ultrasonic imaging indicates that a mature CL in buffalo (Fig. 6) ranges in size from 1.2 to 1.7 cm in diameter [155-157] and weighs between 1.0 and 5.0 gm [176]. Compared to cattle, buffalo CLs have no yellow coloration at any stage of development due to lower amounts of beta carotene [176]. Under in vitro culture conditions, buffalo luteal cells grew constantly upto Day 7 with increased protein synthesis during development [176,177] and decreased prostaglandin synthesis with increasing days in culture suggesting that CL development induces protein synthesis. The functioning of bubaline CL appears to be similar to that in cattle with both large and small luteal cells being identified [178]. Progesterone concentration from bubaline CL increased gradually to peak around Day 7 and declined around Day 17-20 coinciding with CL regression [178]. However, peak concentrations were lower than those observed in cattle suggesting inherent luteal deficiency in the buffalo [178]. The total luteal cell population during pregnancy also increased subsequently and was maintained. The mechanism of spontaneous CL regression in the buffalo appears to be similar to that in cattle and involves MAP kinases that mediate PGF2-alpha induced apoptosis in the CL [179].

Figure 6. A Ultrasonographic appearance of a follicle. B A CL on the buffalo ovary and C Sonographic appearance of the buffalo CL.

2.7. Fertilization

Following natural service or artificial insemination, semen is deposited either in anterior vagina, cervix or in the body of the uterus. The movement of sperm through the cervix depends both on muscular activity in the female tract and sperm forward motility. In the buffalo, during the first 24 h, the cervix is the first sperm reservoir [180]. Subsequently, sperms are stored in the lower isthmus and the utero-tubular junction for 48 hrs thereby escaping from being phagocytized. Though spermatozoa have been found in the oviducts within 2-4 min after deposition of semen in the cervix in buffaloes and cows, sperms require at least 4-5 hrs in the female tract to complete the process of capacitation before being able to successfully fertilize the ovum [181]. In many studies on in vitro fertilization of buffalo oocytes, the time allowed for sperm capacitation with heparin was 4-6 h [182-185]. Only a small fraction of the ejaculate or deposited semen reaches the upper oviduct at the time of fertilization. The site of fertilization in buffaloes is the ampullary–isthmus junction of the oviducts tube [186]. Fertilization results in a zygote and cell division commences thereafter.

2.8. Embryonic Development

Following fertilization and fusion of gametes, the zygote undergoes subsequent mitotic divisions, which determines the formation of the blastomeres. The first cleavage of zygotes with appearance of a 2-cell embryo has been recorded on day 2-3 of estrus in buffalo [30,187]. Subsequent divisions continue sequentially. These cells, at least in the early stages of development, can be considered totipotent, because they have the ability of developing into two separate embryos. This characteristic has been demonstrated until the 8-cells stage in cattle [188], and it is thought to be similar in buffalo [32].

This stage of development (8-16 cells) is fundamental. In fact, in this period the activation of the embryonic genome that is essential for achieving implantation competency occurs. Once the embryonic genome is activated, the embryo grows rapidly to form a blastocyst. Some studies [31,39,189,190] indicate a faster rate of embryonic development in buffaloes compared to cattle. These results have been confirmed from further studies carried out in vitro demonstrating that buffalo embryos are 12 to 24 h more advanced than the bovine counterpart developing in parallel. Oocytes and embryos in buffaloes remain in the oviduct for a period varying from 74 and 100 hours post-fertilization [189] and reach the uterus 96-108 h (4.5-5 days) after fertilization. It seems that buffalo embryos are at morula stage when they reach the uterus [31] similar to that described in bovine at 120 h. In addition, in a previous study using abattoir derived buffalo reproductive tracts, 8-cell embryos were recovered from the uterine horns on Day 5 of estrus [187], suggesting an earlier transport of buffalo embryos into the uterus [30]. Some degree of transuterine migration of embryos has been recorded in the buffalo [182].

Although there are differences in the timing of these events and where they occur in the reproductive tract of the mother, blastocyst formation is generally initiated when the conceptus reaches the uterus. The rupture of the zona pellucida represents the event for a new stage of the embryo [32]. At this moment the hatched blastocyst survival is strictly dependent on the uterine environment and the conceptus exposes the other surface of trophectoderm directly to the uterine environment. Therefore, an adequate progesterone production and the responsiveness of the uterus to progesterone are considered necessary for embryo survival. Data on embryo development from blastocyst hatching to the implantation in buffalo species is scarce. It could be hypothesized that events similar to those recorded in other ruminants, such as cattle and sheep, [188] may occur. According to data described in these species, after hatching, a logarithmic growth and an elongation of the conceptus is observed [193]. In one previous study on abattoir buffalo reproductive tracts, the blastocysts recovered measured 112 x 108 mm on Day 10 and 328 x 170 mm on Day 15 suggesting a 3-time increase in length between Day 10 and Day 15 [187]. The filamentous embryo is able to occupy the contralateral horn since Day 18 of pregnancy in cattle. Since embryonic elongation has been observed in buffalo, it is likely that it occupies the non-pregnant horn similar to cattle. The progressive hyperplasia and expansion of trophoblast cells results in embryonic elongation, allowing the development of extra-embryonic membranes throughout the uterus. Through this mechanism, the embryo is able to block the synthesis of PGF2α and avoid luteolysis. In fact, it is known that in cattle, maternal recognition of pregnancy occurs between Day 16 and 19 post-insemination, and it is probably similar in buffalo [32]. This process is mediated by several molecules. The first messenger that has to be recorded is the Interferon-Tau (IFN-τ), which is produced by the elongated conceptus. The IFN-τ, has been recognized in many ruminant species [194] including buffalo [33] and plays a fundamental role in this process, by binding to the endometrium and the inhibition of oxytocin receptor synthesis. At the same time, IFN-τ is able to induce the production of several proteins, by binding to the apical portion of the uterine glands. The synthesis of these proteins improves the uterine environment and favors embryo survival [188].

2.9. Attachment and Implantation

A close cross talk between the conceptus and the mother is the basis of the implantation process. An adequate luteal activity, and consequently an adequate progesterone concentration, produces an appropriate uterine environment together with a sufficiently elongated embryo that is essential for implantation. Embryonic implantation is known to occur in 3 stages. The first stage is defined as pre-attachment period, during which the free floating blastocyst undergoes a significant elongation and also reaches the other uterine horn (Fig. 7). The second phase, defined as transitory attachment, is considered of primary importance in ruminants. The transitory attachment occurs between days 16-18 of pregnancy until Day 25-30 in various ruminants. A negative role, throughout this process, is played by a transmembrane glycoprotein called Mucine-one (MUC-1). MUC-1 has been described in several mammalian species, including buffalo [195]. The synthesis of this protein during the non-receptive period for the uterine epithelium is very high, whereas it shows a drastic reduction when the endometrium has undergone the action of progesterone. In fact, it has been demonstrated in cattle that the presence of progesterone on Day 8-10 is able to block the receptors on the endometrium and, consequently, the endometrial cells are not yet responsive to progesterone stimulation [193]. This process results in the block of MUC-1 synthesis for a negative feedback mechanism. Hence, the embryo is able to attach to the uterine epithelium by the interaction between some adhesive factors [193]. At this stage the conceptus projects developed villi-like structures into the crypts of uterine glands. The role of these structures favors complete attachment progression and furnishes a temporary anchor and adsorptive structures for the conceptus. Furthermore, these structures allow the absorption of the endometrial glandular secretions, a complex of histotrophic substances and proteins [196,199] (Fig. 8).

Figure 7. Schematic representation of embryonic elongation in ruminant species.

Figure 8. A 60-day bubaline fetus along with its membranes and cotyledons.

These growth factors, enzymes, cytokines, lymphokines, hormones, transport proteins and other substances, have a key role in embryonic nutrition and development, allowing the production of the first signals for maternal recognition of pregnancy [32]. Implantation in the buffalo is centric and non-invasive with increasing trophectoderm-uterine epithelial cell apposition and adhesion with no permanent erosion of uterine epithelium [197]. Within the chorionic villi, in ruminants including buffaloes; it is possible to distinguish two different cellular populations, which can be identified throughout pregnancy: the mononucleate trophoblast cells (MTC) and the binucleate trophoblast giant cells (BNCs). These cellular populations have different morphology and functions. The MTC are localized at the level of the basal lamina and are characterized by the presence of one irregularly shaped nucleus with dispersed chromatin. The number MTC in buffalo is definitely higher than that of BNCs, since they represent around 80% of the total number of trophectoderm cells [192]. However, they show cuboidal to columnar shape and smaller dimensions compared to BNCs. Buffalo binucleate cells migrate toward the maternal epithelium and fuse with a uterine epithelial cell to form trinucleate cells [192]. Larger syncytia, with more than three nuclei, are much less frequent than trinucleate cells in buffalo placentas [192].

The main morphological characteristic of these cells is the surface of their apical membrane, which is organized to constitute microvillar processes. The role of these villi is to get in contact with similar digitations that originate from the maternal uterine epithelial cells, constituting the attachment zones [32]. The main function of these cells is to guarantee nutrient exchanges between the embryo and the mother.

Pregnancy associated glycoproteins (PAG) form a diverse family of glycoproteins that are variably expressed at different stages of gestation. They are probably involved in the immunosuppression of the dam against the feto-maternal placentome. The PAG regulate progesterone production, by inducing the synthesis of prostaglandin E in luteal cells [200], and the release of granulocyte chemotactic protein-2 in the bovine endometrium [201]. This function is usually performed by the IFN-τ during the first stages of pregnancy. Therefore, it has been hypothesized that PAG can substitute the interferon during the late stages of pregnancy [32]. After the transitory attachment of the embryo to the endometrium surface, migration of BNCs and formation of syncytia and trinucleate cells, the formation of the placenta takes place. This is the third and final stage [202] that completes embryo attachment. In fact, prior to Day 16 in sheep and Day 25 in cattle, the placenta is essentially diffuse [188]. At this time (Day 25 in cattle) the chorion begins to attach to the caruncles of the uterus. It is likely that in buffalo species, embryo attachment and placenta formation starts later than in the bovine, probably around 30-35 days [32]. The presence of the products of binucleate cells in maternal circulation has also been correlated with placentogenesis and placental remodeling [203]. The chorioamnion and caruncles collected from pregnant buffaloes with embryos undergoing either normal or retarded development had different proteomic profiles that were associated with antioxidant protection, protease inhibition and protein folding [204]. Buffalo PAG have been isolated from placentae of pregnant buffaloes and had molecular weights ranging from 52 to 77 kDA [205,206]. Their appearance has been tested as marker molecules for pregnancy diagnosis with an accuracy of 90-100% from Day 19-31 post-breeding [207,208].

2.10. Placenta

The bubaline placenta is epitheliochorial and polycotyledonary [209]. The total number of placentomes increases from early pregnancy (Fig. 8) to mid pregnancy with a tendency to decrease towards the end of gestation [210]. The buffalo placentome has simple slightly conical villi branching less than in cattle, thus indicating different and less complex feto-maternal interdigitation in buffaloes [211]. Amniotic plaques are found from 79–220 days of gestation in the amnion of buffaloes [212].

2.11. Gestation and Parturition

Sequential growth characteristics of the bubaline uterus, fetal fluids and the fetus during pregnancy have been recently summarized [209]. Gestation length in buffaloes is nearly one month longer than that in cattle. Gestation length in the Indian buffalo varies between 300-320 days [15]. A slightly longer gestation length of 316 days for Egyptian and Bulgarian buffaloes was recorded whereas for the Thai and Philippine swamp buffalo the gestation length varied from 325-332 days [213]. The gestation length in buffaloes is known to be affected by the age of the dam, sex of the calf and environment [213].

The process of parturition was depicted and described in detail for buffaloes recently [214]. For more detailed information on this subject, the reader is referred to Chapter 14 on Parturition and Puerperium in the Buffalo in this book.

2.12. Postpartum Reproductive Events

The mean interval to complete uterine involution in the buffalo varies widely between 19 and 52 days [22]. Delayed resumption of postpartum ovarian activity in the buffalo is a major cause of economic loss for buffalo breeders in many countries [22]. In India, Pakistan and Egypt only 34-49% of animals resumed estrus during the first 90 days after calving while 31-40% remained in anestrus for more than 150 days. Calving intervals in buffaloes are generally considered as twice in three years. The rise in FSH and the growth of follicles is evident in buffalo during the early postpartum period [215], however, LH secretion continues to be low at this time [20]. Buffaloes at higher latitudes, which give birth during the period of increasing day length, may not resume cycling until the following period of decreasing day length [5]. Factors influencing postpartum cycling resumption in buffaloes were evaluated recently [214] and suckling, parity, nutrition and management were considered as the primary factors affecting postpartum cycling resumption besides housing management [216].

2.13. Service Period and Calving Intervals

The interval between calving to conception is known as service period, whereas the interval between subsequent calvings is known as calving interval. Both these traits of economic importance appear to be prolonged in buffaloes compared to cattle and are widely variable across the different breeds [186,217].

The average service period in Murrah buffaloes (Table 3) is 115 to 230 days with an overall average of 132 days [218-220], the average first service period is 201 days in Nili Ravi, 193 days in Bhadawari and 198 days in Egyptian buffaloes [221]. In general the service period in Murrah buffaloes at well-organized farms is much lower compared to that observed for other breeds [217]. The heritability estimates for service period are low and vary from 0.08 to 0.22 [186].

The reports on calving interval in the buffalo present wide variations (Table 3) and calving intervals of as long as 839.5 days were observed for Chinese swamp buffaloes [222]. It has been mentioned that Italian Mediterranean buffaloes with better nutritional management may have calving intervals as short as 400 days [138,217,223,224] compared to calving intervals of 420-504 days in buffaloes elsewhere. A large number of variables govern the calving intervals including season of calving, lactation yields and age at first calving [225]. The calving interval in Murrah, Nili Ravi and Egyptian buffaloes varies from 479 to 508 days [219], and calving intervals of as long as 583 days were recorded for Surti buffaloes [226].

The estimates for heritability (0.12 to 0.53) [186] and repeatability of calving interval in the buffalo suggest that the genetic component of this attribute is very small and hence selection for a shorter calving interval is not likely to be rewarding [217].

2.14. Twinning and Sex Ratio

Twins are rare in the buffalo species with only 0.01% of the pregnancies resulting in twins [230]. The incidence of twins in Egyptian [231], Nili Ravi [232] and Murrah [233] buffaloes were 0.2%, 0.3% and 0.062% respectively. The incidence of twinning in Indonesian and Malaysian swamp buffalo was cited to be 0.0002% [234]. The incidence of triplets [235] and quadruplets [236] is extremely rare in the buffalo. A Siamese twin was described in the buffalo [237] and a large number of abnormal twins in buffalo resulting in difficult births were reviewed recently [238]. Twinning was attempted in buffaloes by transfer of in vitro produced embryos with marginal success [234,239]. The sex ratio in buffaloes has a male bias. In Pakistan the sex ratio for 2903 Nili Ravi buffalo calves born was 1.36:1 (male: female) [136]. Beradar and Mallikarjunappa [240] observed the percentage of male calves as 49% in Surti buffaloes whereas for Murrah buffaloes the proportion of males was 55.64% [241] and 49.76% [242] and the females being 44.36% and 50.24% respectively. In Brazil, data from 232 pregnant buffalo genitalia revealed the proportion of male and female fetuses as 47.0% and 41.8% respectively [192]. In a recent evaluation of 34,911 calvings in Iranian buffaloes the proportion of male to female calf births were 53:47 (1.15:1) [35].