Gestation and Fetal Development in the Buffalo

According to the medical dictionary, the period of development of the young in viviparous animals from the time of fertilization of the ovum until birth is termed gestation. The word appears to have been derived from the Latin word "gestare" meaning "to bear". The mechanisms and molecules involved in the establishment and completion of pregnancy are complex and only partially understood. Gestation in the buffalo appears to be similar to cattle although one month longer in duration. Gestational lengths in buffaloes vary widely between the different breeds (305-315 days in river) [1-3] and types (320-340 in swamp) [4,5] of buffaloes. Pregnancy establishment in ruminants involves sequential fertilization, blastocyst elongation and hatching, formation of embryonic membranes, signals for pregnancy recognition, implantation, development of placenta and subsequent sequential growth of the fetus and the uterus till term. Many of these processes are complex and poorly understood in ruminants [6,7] and in buffalo our understanding is still lesser. Recent evaluations have mentioned that the molecules involved in pregnancy recognition and embryonic growth such as interferon-tau [8,9], maternal recognition of pregnancy [10], endometrial remodeling [11,12] and changes in endometrial proteomic profiles [13-15] embryonic elongation [16,17] in the buffalo, appear to be similar to those observed in cattle and yet some minor differences do exist such as one day earlier growth of embryos to blastocyst stage [18,19], comparative delayed implantation [20] and a longer gestation period. In this chapter, the information on gestation length, embryonic elongation, maternal recognition of pregnancy, implantation, fetal membranes and fluids, placental formation, placental proteins, endocrinological changes during gestation, changes in reproductive organs, prenatal development of fetal parts, and twinning and sex ratio is presented.

Gestation Lengths

The gestation length in buffaloes is nearly a month longer than in cows with a range from 299 to 346 days [21-23]. The mean gestation length for Sri Lankan buffaloes (316.3±6.5 days) is significantly longer than that for the Murrah (309.9±6.5 days) buffaloes [24]. In a recent study gestation lengths longer than 330 days were recorded for buffaloes in Sri Lanka [25]. The mean gestation length for buffaloes in India and Pakistan (305-308 days) [26,27] is 7-10 days shorter compared to that of Egyptian buffaloes (315-317 days) [21]. Ghanem et al., [28] observed a significant difference in the gestation length between paternal and maternal half-siblings and full-siblings. The heritability estimate for gestation length was 0.32+0.02 for Murrah [22], 0.06±0.03 for Nili-Ravi [29] and 0.32+0.13 for Egyptian [30] buffaloes. The repeatability estimates for gestation length was 0.59 [31] and the correlation coefficient between birth weight of calf and gestation length was 0.17 [31]. The gestation length for swamp buffalo is considerably longer than that of the river buffalo and can extend from 330 to 340 days [32,33].

Factors Affecting the Gestation Length

Within a narrow range, factors such as gender of calf, calving season, calf birth weight and parity of dam affect the gestation length of buffaloes. The most striking factor affecting the gestation length in buffaloes appears to be the breed (Table 1). Variations in the gestation lengths for the breeds due to their geographical location [34] and due to crossbreeding of swamp and river buffaloes [35-38] were marginal.

Gender of Calf

It has been mentioned in a large number of reports that male buffalo calves require slightly longer (1-2 days) time for completion of gestation compared to female river [39-43] and swamp [36,44-46] buffalo calves, although some reports did not find any difference between the gestation lengths of male and female buffalo calves [22,47-49] and a few observed longer gestation lengths in buffaloes carrying female calves compared to those carrying male calves [28,50,51].

Season of Calving

The effect of season of calving on the gestation length of buffaloes was appreciable [2]. Buffaloes calving in summer and autumn had significantly shorter gestation lengths compared to those calving in winter and spring [22,26-28,40,42,43,46,48,52-54]. The month of calving is known to affect the gestation length in cows. Holstein cows calving during July had a 2 day shorter gestation length compared to those calving in the month of October [55]. Ghanemi, 1998 [56] recorded that Iranian buffaloes calving between December and February had a longer gestation period (318.5 days) compared to those calving between September and November (314.5 days). The probable reasons for the shorter gestation periods during hot summer months could be environmental stress leading to release of corticosteroids and earlier induction of parturition [57].

Calf Birth Weight

Positive correlations between the gestation length and calf birth weight [26,58] suggests that a shorter gestation length would result in a reduced birth weight of calves. The birth weight of male buffalo calves (35.98±0.15 kg) is higher compared to female (30.45±0.14 kg) buffalo calves [5959,60] thus resulting in longer gestation in buffaloes carrying male calves [27,44]. Usmani et al. [27] noticed that Nili-Ravi buffalo calves weighing less than 25 kg and more than 45 kg at birth had longer gestation periods (312 days) compared to those weighing between 25 to 45 kg at birth (307 days). Other determinants to the weight of calf could be parity of dam, nutrition and season. A buffalo calf born to a first parity heifer was lighter in weight by 5.44 kg compared to those born to multiparous buffaloes [31]. The highest calf birth weight for Egyptian buffaloes was recorded for the fourth parity [31,61]. The differences in calf birth weights were also seen between two breeds of buffaloes (29.12±0.83 kg for Murrah and 25.46±0.42 kg for non-descript) with slightly different gestation lengths [41].

Parity

Analysis of differences in the gestation length of heifers and cows of various breeds of cattle revealed that heifers required slightly longer (1 to 3 days) period of gestation [55]. Primiparous Murrah buffaloes had a significantly longer gestation period (312.57 days) compared to pluriparous (309.60 days) buffaloes [26]. Similarly primiparous Nili-Ravi buffaloes had a longer gestation period compared to pluriparous buffaloes [62]. Although some reports [2,45-47,63] recorded no effects of parity on gestation length of buffaloes, a few other studies [28,31] recorded shorter gestation length in Egyptian buffaloes at first parity. Janakiraman [1] observed that the average length of gestation in Surti buffaloes in their 1st, 2nd and 3rd parity was 305, 307 and 310 days. A few studies [43,64] recorded that parity had a significant effect on the weight of the calf that increased from the first to the seventh parity with a slight increase in the gestation length (302.24±4.11 in first and 315.77 in the seventh parity).

Effect of Sire

The effect of sire on the length of gestation in buffaloes was not significant in a few studies [51,65]. However, studies on Murrah [41] and Nili-Ravi [27] buffaloes revealed significant effects of the sire on the gestation length of buffaloes. The interaction between the sire and the gender of the calf was significant (P<0.01), in addition to a significant difference in the gestation length of the male and female buffalo calves [41].

Fertilization and Embryo Formation

The features of fertilization, CL development and early embryonic development in buffaloes have been described in a previous chapter of this book (Chapter 2) [96]. Briefly, at mating the buffalo bull deposits semen in the anterior vagina [97]. Spermatozoa have been found in the oviducts within 2-4 min after deposition of semen in the cervix in buffaloes [98], however, the sperms must undergo capacitation before they become capable of fertilizing an ovum. In vitro studies have revealed that buffalo sperms require 4-6 h in capacitation [99,100]. A single capacitated sperm enters the oocyte and these results in the release of calcium within the oocyte till completion of fertilization and formation of pronuclei [101]. Upon fertilization of the oocyte, meiosis is completed and the cell cycle returns to a mitotic pattern [102]. The cleavage of in vitro fertilized buffalo oocytes is observed on Day 2-3 of fertilization [103,104]. On Day 4, 20.9% and 50.0% embryos were at 6-8 cell and 16-cell to morula stage [103]. The fertilized embryo descends in the uterus at around Day 4-5 in the buffalo [18,19]. The outer cells of the morula stage embryos then differentiate into an epithelium called the trophectoderm whereas the inner blastomeres become positioned at one pole of the embryo forming the inner cell mass (ICM) [102]. This is known as compaction and the resultant organized embryo is termed the blastocyst. The peaks of blastocyst were seen on Day 8 in buffaloes [103]. The blastocyst then accumulates fluid and expands eventually hatching out of the zona pellucida. Peaks of expanded and hatched blastocysts were observed on Day 9 and 10 respectively under conditions of in vitro fertilization in the buffalo [103]. Around the time of hatching the ICM differentiates into two cell populations: those facing the blastocyst cavity become flattened and delaminate forming an inner cell sheet referred to as hypoblast; the remaining cells form the multilayered epiblast [102]. The hypoblast and trophectoderm are extra-embryonic cell lineages that will participate in fetal membrane formation whereas derivatives of the epiblast will find all of the embryonic cell lineages [102]. The epiblast forms three germ layers: ectoderm, mesoderm and the endoderm [105] and a prominent derivative of the endoderm is the primitive gut which gives the name to the entire process as gastrulation [105]. Besides the 3 germ layers, gastrulation also establishes the primordial germ cells. The 3 layers ectoderm, endoderm and mesoderm were identified from embryonic stem cells derived from inner cell mass of buffalo embryos [106].

Pregnancy Establishment

Pregnancy establishment in ruminants involves four coordinated events: embryo elongation, maternal recognition of pregnancy, embryonic implantation and placentation.

Embryonic Elongation

The establishment of pregnancy in ruminant species including buffalo is initiated when the fertilized embryo enters the uterus at around 4.5 days (108 h) post-estrus [19] and is characterized by pregnancy recognition, implantation and placentation. Between 6.5-7.5 days, blastocyst stage embryos are predominant in the bubaline uterus [19]. The blastocyst has an inner cell mass, blastocele and an outer monolayer of trophectoderm. In cattle hatched blastocyst forms an ovoid conceptus between Day 12 to 14 that is only about 2 mm in length on Day 13 [107,108]. The bovine blastocyst then elongates and becomes filamentous. The bovine conceptus by Day 14 is about 6 mm and reaches a length of 60 mm by Day 16 and is 20 cm or more by Day 19 [107,108]. Conceptus elongation in buffaloes is poorly known. One study depicted the presence of thin and elongated chorion filament over the entire length of the pregnant uterine horn, with an elongated yolk sac between Days 10-15 of pregnancy [17]. In another study on abattoir derived buffalo reproductive tracts, the size of blastocysts was 112 x 108 mm on Day 10 and 328 x 170 mm on Day 15 suggesting a threefold increase in length between Day 10 and Day 15 [16]. The embryonic morphology observed in bubaline pregnancies between Days 17-19 [17] were similar to those observed in cattle. The elongation of the conceptus involves coordinated effects of ovarian progesterone, prostaglandins and cortisol secreted by the epithelial cells of the endometrium and the trophectoderm of the elongating conceptus [108]. When trophoblast elongation is completed on Day 16-18, the attachment between the conceptus and the uterine epithelium begins on Day 22 of gestation in the cow [109]. In ruminants, implantation is non-invasive (synepitheliochorial) and occurs at a predetermined site (caruncles) on the endometrium [109]. During the period of conceptus elongation, specialized intraepithelial binucleate cells appear within the trophectoderm and increase in numbers in the areas of the trophectoderm apposed to caruncular areas of the uterine epithelium [110].

Maternal Recognition of Pregnancy

In ruminant species, the presence of a viable embryo in the uterine lumen must signal the uterus of its presence in order to maintain the patency of the corpus luteum that will produce sufficient progesterone to allow the pregnancy to continue. In the absence of these signals, the endometrium would secrete prostaglandin around Day 16-17 in cattle and buffaloes which would result in the expulsion of the embryo and resumption of cyclicity. Interferon-tau (IFN-τ) is a type-1 interferon secreted from the ectoderm of the peri-implantation elongating blastocyst-stage embryo. IFN-τ has been implicated as the principal molecule involved in maternal recognition of the pregnancy in cattle [7] and buffaloes [8]. IFN-τ acts on the endometrium to abrogate the endometrium luteolytic mechanism by either inhibiting the transcription of estrogen and/or oxytocin receptors [111,112]. Another probable mode of action of IFN-τ appears to be the modulation of the pattern of prostaglandin synthesis by increasing luteotrophic PGE2 production rather than PGF2α [109]. The bubaline IFN-τ was identified by homology modelling from ovine IFN-τ [8]. The bubaline IFN-τ revealed 8 different isoforms that were expressed in the trophectoderm outgrowths during early bubaline pregnancy [9]. IFN-τ also appears to be involved in blastocyst development. The addition of recombinant bubaline Bu IFN-τ (2 μg/ml) significantly improved the rate of blastocyst production (45.5% against 31.0% for controls) [113] under in vitro culture conditions. Recombinant bovine IFN-τ stimulated the production of PGE2 by buffalo endometrial stromal cells in a dose dependent manner [10]. Bubaline endometrial stromal cells under in vitro culture revealed increased protein synthesis [11,12]. IFN-τ stimulates the transcription of a number of genes and activities of several enzymes implicated in the elongation and implantation of the conceptus [107,114]. Global transcriptome profile analysis of the Day 16 bovine conceptus and endometrial tissues revealed 87 ligands, 46 of which were conceptus specific and 34 endometrium specific [115] suggesting the importance of interactions between the conceptus and the endometrium in the maternal recognition of pregnancy. Changes in sera proteomes and proteomic profiles of the embryonic chorioamnion and uterine caruncles were also recorded during early pregnancy in buffaloes [14,15]. Besides IFN-τ, the presence of leukemia inhibitory factor (LIF), a cytokine from the interleukin-6 family, during different stages of buffalo embryo development from oocytes to the hatched blastocyst stage, indicated that it too plays a role in bubaline embryo development [116].

Implantation

Reciprocal signal exchanges between the conceptus and the uterine microenvironment maintain optimum progesterone production and an elongated conceptus that appear to be essential for implantation. The uterine luminal epithelium prepares itself for the attachment and implantation of the trophectoderm by alterations in the endometrial gene expression and secretory and functional activities [117]. Significant differences in the expressed genes were found during the transition of the blastocyst (Day 7) to an ovoid conceptus (Day 13) in cattle, however, the temporal gene expression in the endometrium are similar in pregnant and cyclic animals up to the time of maternal recognition of pregnancy (Day 14-16) when conceptus derived factors affect the expression of a large number of genes in pregnant animals [118]. Expression of a cascade of signaling molecules during maternal recognition of pregnancy is known to be affected by genes like interferon-τ, ubiquitin cross reactive proteins, ghrelin, aldo-keto reductase IB 5 and SERPINA 14 [118,119]. The ghrelin gene was identified from buffalo CL and signals of ghrelin protein were found to be higher during early pregnancy in buffaloes [120]. Changes in transcriptome profiles govern the implantation of embryos in cattle and buffaloes [121,122]. The major changes for uterine receptivity to implantation occur between Days 7 and 13 in response to ovarian progesterone irrespective of whether or not an embryo is present [123].

A large number of proteins were upregulated in buffalo embryos at Day 27 that developed normally [14,50]. Uterine luminal fluid analysis reflected that a wide variety of proteins of fetal and maternal origin do exist during the preimplantation period in cattle [123]. The uterine luminal fluids collectively known as histotrophs, governs the elongation of the conceptus via effects on trophectoderm proliferation and migration as well as attachment and adhesion to the endometrial luminal epithelium [124-126]. The histotrophs are derived from synthesis and secretion of substances by the endometrial luminal epithelium and the glandular epithelium and is a complex but poorly defined mixture of proteins, lipids, amino acids, sugars, ions and exosomes [127-130].

In a study on pregnant bovine heifers, endometrial transcriptome profile analysis revealed 109 mRNA’s with twofold higher abundance and 41 of these are known to be induced by interferons [131]. Weekly serum analysis of pregnant buffaloes (from day of insemination until Day 42) by 2-dimensional gel electrophoresis and densitometric assay, revealed significant changes in sera proteomes with at least 65 2-D gel spots showing upregulation, down-regulation or specific appearance during early pregnancy [15].

Subsequent to the elongation of the conceptus, establishment of uterine receptivity of the conceptus to the maternal endometrium is initiated [109]. Endometrial tissue remodeling is necessary for the formation of placental cotyledons and the development of angiogenesis in the uterine caruncle [109]. There is a fusion of fetal chorionic binucleate cells with those of the uterine epithelium forming the feto-maternal trinucleate cells, partial invasion of the trophoblast cells into the maternal endometrium and reorganization of the vessels [110].

When the attachments occur, cell contacts are established between the tips of the uterine microvilli and the trophoblastic cell membranes [109]. Integrins, which appear on the cell surface of the trophoblast and uterine epithelium, bond extra-cellular matrix (ECMs), followed by the production of tissue remodeling factors, matrix-metalloproteinases (MMPs) [109]. These MMPs degrade ECMs and help the conceptus to penetrate the uterine stroma. Distinct gelatinases and matrix metalloproteinases were present in uterine luminal fluid of early pregnant (43-65 days) buffaloes [132].

The attachment involves the blockage/down regulation of a transmembrane glycoprotein Mucine-1 (MUC-1) observed in ruminants including buffaloes [133] that has anti-adhesive properties. Transitory attachment occurs between Days 16-18 of pregnancy until Day 25-30 in various ruminants. The embryo sequentially attaches to the uterine epithelium by interactions of adhesive factors [134]. Development of villi-like projections between the conceptus and the uterine caruncular epithelium favors complete attachment that allows absorption of the endometrial glandular secretions the histotrophs [6,135].

POU-domain transcription factor (Pou5f1 (Oct-4) is involved in the transcriptional regulation during early embryo development and cell differentiation. The expression of Oct-4 was higher in inner cell mass of buffalo embryos compared to the trophectoderm [136]. This gene expression pattern is also dependent on the quality of oocytes [137]. Early embryonic development and implantation is considered to be supported by autocrine and paracrine effects of insulin as evident by insulin receptors within the embryo and oviductal cells [138]. This was shown in clinical experiments on buffaloes [139,140]. Buffaloes treated on Day 8-10 of breeding with insulin (0.2 IU/kg SC) had higher conception rates and higher plasma progesterone compared to untreated control.

Implantation in the buffalo appears to be similar to cattle that have a centric and non-invasive implantation with increasing trophectoderm-uterine epithelial cell apposition and adhesion with no permanent erosion of uterine epithelium [6]. Within the chorionic villi, in ruminants including buffaloes, it is possible to distinguish two different cellular populations, which can be identified throughout pregnancy: the mononuclear 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 of MTC in buffalo is definitely higher than that of BNCs, since they represent around 80% of the total number of trophectoderm cells [141]. 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 [141]. Larger syncytia, with more than three nuclei, are much less frequent than trinucleate cells in buffalo placentas [141]. Microvillar processes originating from the maternal uterine epithelial cells at the caruncula form the attachment zones that attach with specialized areas in the trophectoderm [20]. This begins the implantation and cell-to-cell communication.

Placenta

The mechanisms of placental development are complex. Placental formation starts after Day 18 to 19 of pregnancy in cattle, when the trophectoderm extends, and the cells of the trophectoderm establish themselves within the uterus. These cells play a key role in the growth of the placenta forming an intimate relationship with embryonic somatic cells or mesoderm to form the chorion, which thereafter becomes highly vascularized by fusing with the allantois to form the chorion-allantois [142,143]. The continuous growth and development of caruncles is a result of cell proliferation, and endometrial tissue modelling that increases the surface contact area for nutrient exchange and mechanical anchorage for effective embryo implantation and placental development [144]. It has been hypothesized that embryo attachment and placenta formation in buffalo species starts later than in the bovine, probably around 30-35 days [20]. The placental plates were first observed on the chorioallantoic membrane surrounding the bubaline embryo on Day 30 but the attachments were incomplete till Day 60 [145]. The primary crypts were observed in the maternal caruncle of buffaloes at Day 42 of gestation which gave rise to secondary branches from Day 53 onwards and firm feto-maternal appositions with tertiary villi were seen on Day 87 of gestation [146].

The bubaline placenta is epitheliochorial and polycotyledonary [147]. The total number of placentomes increases from early pregnancy to mid-pregnancy with a tendency to decrease towards the end of gestation [148]. The number of cotyledons in the pregnant and non-pregnant horns was 58.89 and 39.65 respectively [149].

The placentomes in gravid buffalo uteri were arranged in four rows with a maximum of 102 in the gravid and 96 in the non-gravid horn [150]. The maximum size of placentomes in the gravid uterine horn at up to 210 days of pregnancy was 10.0x4.6 cm and in the non-gravid horn was 6.6x3.5 cm [150]. The number of bubaline placentomes varied from 58 to 106 and increased between Day 55 to 95 of pregnancy [151]. The mean placental weight in Surti buffaloes was 2.89±0.10 kg (range 1.5 – 5.0 kg) and the mean number of cotyledons was 76±2.40 (range 38 to 105) [152].

The weight and volume of cotyledons, weight of fetus, fetal fluids and umbilical cord were strongly affected by the gestational age [153]. The buffalo placentome has a simple slightly conical villi branching, that appears to be smaller than in cattle, indicating a different and less complex feto-maternal interdigitation in buffaloes [154]. The microvascular architecture of buffalo fetal cotyledons studied from the 3rd to the 10th month of gestation revealed that the fetal vasculature of the buffalo placentomes was greatly increased from early pregnancy to near term as were the volume and the density of the capillary system of the terminal villi [155].

The umbilical cord in buffaloes was 11.8 cm and was formed by 2 thick arteries and 2 thin veins [156,157]. The diameter and thickness of the umbilical artery was larger than the veins up to Day 95 of gestation but subsequently there was no difference between their sizes [156]. The uterine blood flow in middle uterine arteries evaluated by color Doppler ultrasonography revealed a sequential increase in the diameter of both uterine arteries as gestation progressed, with a significant increase in the volume and velocity of blood flow during the last trimester of gestation [158]. The urachus, a tubular structure connecting the urinary bladder with the allantoic cavity used by the early fetus to pass urine directly into the allantoic cavity during the first half of gestation, disappears after birth due to the collapse of the umbilical arteries. The persistence of this structure after birth is rare in the buffalo [159,160].

During the first two thirds of gestation, the rate of growth of the placenta exceeds that of the fetus. During the last third of gestation, the situation is reversed probably because of increased nutritional demands of the rapidly growing fetus [97].

Fetal Fluids and Fetal Membranes

Three fetal membranes are seen in buffaloes the chorion, the allantoic sac and the amnion [161]. The yolk sac is an opaque membrane that disappears during gestation [161]. The sequential changes in the progression of uterine and fetal growth, and increase in fetal fluids and fetal membranes have been tabulated in a previous chapter [147] (Chapter 8). The chorion during the first 3 months of gestation was shown to have a simple layer of circular cells with spherical nucleus named trophoblasts; there was another cell type named trophoblast giant cells with 2 or more nuclei [161]. The allantois during the same period revealed many vessels filled with erythrocytes and elongated cells that form a stratified simple epithelium. The amnion was avascular and transparent consisting of stratified simple epithelium [161]. The yolk sac during the first 3 months of gestation was opaque and not in contact with the other membranes and disappeared during gestation [161].

Soft rubber-like flat irregular shaped (maximum size 2x2x1 cm to 8.5x5.5x2 cm) amniotic plaques were found towards the tips of white transparent amnion of parturient buffaloes with 66% of 106 buffaloes revealing 1 or more hippomanes [162]. They were observed between 79-220 days of gestation [150,163]. The mean concentrations of urea nitrogen, creatinine, protein and potassium were significantly higher in allantoic fluid, whereas the level of sodium was significantly higher in amniotic fluid during gestation with slight variations [164]. Sequential changes in the biochemical molecules in the allantoic and amniotic fluids were recorded specially from 55 to 165 days of gestation [164]. The evaluation of the volume, color, consistency and pH of allantoic and amniotic fluids during different stages of gestation in the buffalo revealed that up to 105 days and after 166 days of gestation, the quantity of allantoic fluid was more than the amniotic fluid whereas between Day 121-165 the quantity of amniotic fluid exceeded the quantity of allantoic fluid [165]. From mid-gestation the allantoic fluid was mucoid and straw colored whereas the amniotic fluid was amber to yellow colored and mucoid and the pH of both fluids was alkaline between Day 50 to 150 of gestation [165]. The concentrations of total protein, urea and creatinine increased in the amniotic fluid whereas the pH, density and calcium oscillated with advancing gestation. In the allantoic fluid, the density, albumin and creatinine increased whereas the pH and calcium decreased [166,167].

Placental Functions

The placenta performs the functions of nutrient, gaseous and waste product exchange between the mother and the fetus. Besides this, the placenta secretes and synthesizes some proteins, hormones and other molecules. Complex interactions between the mother and the fetus result in the remodeling of placental structures, blood supply and secretory function throughout gestation.

Placental Proteins

The placenta produces an array of proteins with distinct characteristics. Proteins of the bovine placental prolactin (PRL) family include bovine placental lactogen (PL) which is involved in partitioning of nutrients to maintain fetal development while other proteins of PRL family possess angiogenic properties [168]. Other important proteins such as pregnancy associated glycoproteins are a mixture of placental aspartic proteinase–like proteins. A large number of proteins, some of them showing immunoreactions, were extracted from placental extracts from buffalo [13]. The identified proteins include pregnancy specific protein-B [169] and pregnancy associated proteins [170]. In a study on pregnant Iraqi buffaloes, levels of pregnancy specific protein B were found to be higher in fetal serum (6 and 8 months) than in amniotic and allantoic fluids, whereas progesterone concentrations were higher in allantoic fluids from the 6th to the 8th month of pregnancy [169].

Pregnancy associated glycoproteins (PAG) are a diverse family of glycoproteins that are variably expressed at different stages of gestation. The ruminant placenta synthesizes pregnancy-associated glycoproteins during pregnancy. Two PAGs (PAG-7 and PAG-11) were identified from the bubaline placenta [171]. Proteins with molecular weights of 38, 56, 67, 75 and 85 kDa were detected in buffalo placental extracts during mid to late pregnancy [13,170,172,173]. Likewise the PAG-1 gene was present throughout gestation in buffalo [174]. Plasma PAG concentrations in buffaloes increased from Day 28 (4.48+0.92 ng/mL) until Day 41 (27.27+6.74 ng/mL) and remained high (20.71+9.20 ng/mL) until Day 103 in pregnant buffaloes that also had significantly higher plasma progesterone (3.51-4.80 ng/mL) [175]. PAG concentrations were determined from abattoir derived maternal and fetal plasma, allantoic and amniotic fluids obtained from swamp buffaloes. In the allantoic and amniotic fluids, the concentrations were 12.7+2.1 ng/mL and 24.0+7.3 ng/mL respectively. Maternal and fetal plasma concentrations of PAG varied from 15.5+1.4 ng/mL to 21.9+2.2 ng/mL for maternal and 16.1+1.8 ng/mL to 21.9+3.2 ng/mL for fetal concentrations using 3 different radioimmunoassay procedures [176]. PAGs were detectable at 6 weeks (3.9+1.3 ng/mL) and the concentration increased gradually until week 28 (39.6+4.0 ng/mL) [177]. A linear correlation between PAG concentrations in plasma and time of pregnancy was established [178]. Postpartum the concentrations decreased reaching very low levels (<1.0 ng/mL) at 5 to 8 weeks postpartum [177,179]. PAGs are synthesized by mono and binucleate trophoblastic cells [180]. Immunoreactivity in granules in binucleate placental cells (BNC) from buffalo was identified [181]. The presence of PAGs in the serum of buffaloes [182] has suggested that this molecule is a potential biomarker for early pregnancy detection in the buffalo [183] and has led to development of antisera [184] specific for detection of pregnancy by radioimmunoassay [185] with high accuracy (90-100%) between Day 19 to 55 [186,187]. More recently, an ELISA kit for the detection of PAG in buffaloes has been developed and validated for the purpose of pregnancy diagnosis [188].

It is believed that PAGs regulate progesterone production by inducing the synthesis of prostaglandin E in luteal cells and proteins in the endometrium [189,190] thus supporting pregnancy. The trophectoderm molecule IFN-τ is considered the chief molecule regulating these functions during the early stages of pregnancy [6]. It has been hypothesized that PAG probably takes over the functions of IFN-τ during the later stages of pregnancy [20]. The presence of the products of binucleate cells in maternal circulation has also been correlated with placentogenesis and placental remodeling [191]. 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 [14].

Placental Transfer

The placenta is a temporary maternal-fetal organ, whose principal function is to allow the controlled exchange of metabolites between mother and embryo/fetus during gestation [192]. Besides the secretion of various proteins, the exchange of nutrients across the placenta is important. The placenta mediates the transport of nutrients, gases, and waste products of their metabolism between the maternal and fetal circulation [193]. This involves complex interactions between the placental cells, their remodeling, secretion, and the fetal trophoblastic cells and the histotrophs secreted by the uterine endometrium [194]. During pregnancy, transport of nutrients from the mother to the fetus is predominantly dependent on uterine blood flow and many other interlinked factors [194]. The primary substrate for fetal growth appears to be glucose which is transferred via carriers and possibly by conversion to fructose by the placenta [195]. The transport and utilization of many substrates required for fetal growth such as lactate, fatty acids, ketone bodies, triglycerides, and amino acids were reviewed [195] and it was concluded by these authors that in ruminants, the energy requirements of the fetus are met by glucose, lactate and amino acid oxidation, and to a lower extent by acetate. Calcium concentrations in fetal blood are mainly regulated by fetal parathyroid hormone and plasma concentrations of vitamin D3 [196]. The transfer of iron in ruminant species is complex due to the lack of direct contact of maternal and fetal blood. Iron is hypothesized to be transferred to the fetus through trophoblastic erythrophagocytosis in the hematophagous area of the placenta and also in the endometrial glands [197]. Intercaruncular glandular regions in pregnant cattle and buffaloes are important sites of secretion and substance transfer from the dam to the fetus [198]. Hematophagous areas at the placentomes and the area at the interface between chorion and endometrial glands are important sites for iron transfer from mother to fetoplacental tissues throughout pregnancy in buffaloes [199,200]. The giant cells migration was first evident at 105 days of gestation with maximum migration at 143 days of gestation in buffaloes [201]. Numerous irregularly distributed placental hematomas were recorded in 7-10 month pregnant buffaloes along the feto-maternal interface and these contained a variable amount of leaked maternal blood [202].

Reproductive Organ Changes

Changes in Uterine Morphology and Function

Sequential changes in the fetal and uterine dimensions during gestation in the buffalo have been previously summarized [147]. The weight of the gravid and non-gravid uterus up to Day 60 of pregnancy was 815+60.48 g increasing to 16433.33+151.19 g between 166 to 210 days of pregnancy [203]; 58.33% of the pregnancies were in the right uterine horn [203]. Similar findings were previously recorded in Egyptian buffaloes [204]. Significant increase in the circumference of greater and lesser curvatures and weight of gravid horns were recorded during early gestation in Nili-Ravi buffaloes [205]. The mean weight of the fetus at 4 weeks was 0.68 g and the length of the gravid horn was 25.25 cm that increased to 241.12 g and 61.85 cm respectively at 14 weeks of gestation [206]. The increasing dimensions and weight of the uterus due to growth of fetus and fetal fluids, pushes the uterus cranially and the uterus descends into the abdominal cavity by the fourth month of pregnancy [147]. The descending uterus reaches the abdominal floor and then grows cranially towards the diaphragm. From the 7th month onward, uterine growth is upwards and the fetal parts are in the pelvic cavity from 8 month onwards [147]. The growth pattern of developing pregnant female genitalia in relation to fetal growth showed no consistent pattern [207]. Descriptions on sonographic evaluations in buffaloes have mentioned that the combined thickness of the uterus and the placenta (CTUP) increased from 2.5 mm at 2 months of gestation to 12 mm at full term [208].

The diameter of endometrial glands increased from 53.0 μ during early luteal phase to 60.8 μ at 100 days of gestation in buffaloes with sequential increase in the number of endometrial glands [145]. The epithelium in the intercaruncular area of the uterus was made up of pseudostratified columnar cells but at Day 55 of gestation, the caruncular part of the endometrium was devoid of columnar cells [209]. In the buffalo, at Day 77 of pregnancy, marked changes occurred in the sub-caruncular region and blood vessels were prominent with a thick wall [209]. The lining epithelium of both uterine horns demonstrated few muco-polysaccharide granules only during the first trimester and at the end of pregnancy [210].

Vaginal histological studies in buffaloes during different stages of gestation revealed non-significant differences in the number of cell layers and cell types. The vascular supply increased as the pregnancy progressed and during the last months of gestation, the submucosa was more vascular and edematous [211]. Epithelial cells did not change significantly in the external os of the cervix, however, the percentage of neutrophils and monocytes increased significantly as the pregnancy progressed [212].

Fetal Growth

Fetal growth and the associated growth in fetal membranes and fetal fluids revealed that the mean weight of the fetus at 4 weeks of gestation was 0.68 g and it increased to 241.12 g at 14 weeks [206]. The mean weight of the fetus in the second month was 5.64 g and it increased to 7150 g at seven months of gestation [213]. The volume of allantoic and amniotic fluids increased from 9.93 ml and 0.70 ml at 4 weeks, to 328.0 ml and 8.92 ml at 14 weeks of gestation in the buffalo [206]. The mean length of the fetus was 4.26-4.9 cm at 60 days of gestation increasing to 83.4 cm at 270 days of gestation [147]. Maximum fetal growth and development were observed from 240 to 305 days of gestation [207]. The prenatal development of the fetus in swamp buffaloes has been described previously and a linear correlation between gestational age of fetus and fetal size was observed [214].

Sonographic descriptions have been more precise in identifying fetal structures and their growth. The embryo and amniotic vesicle were detectable by the 4th and 5th week of gestation [215]. The fetal organization and ossification could be identified by the 8th and 10th week of gestation. Predictors of gestational age on the basis of fetal characters revealed high correlations for crown-rump length during early (8 weeks), biparietal diameter at the mid (18 weeks) and eye ball diameter during late gestation (28 weeks and onwards) [215]. The fetus adopted the final anterior presentation in buffaloes by the 30th week of gestation [215].

Ovarian Morphology and Function

A slight increase in the weight of the ovaries was observed from Day 60 to 210 of pregnancy in buffaloes [203]. The corpus luteum (CL) appeared brownish in color during 30 to 150 days of gestation. The size and weight of the CL increased up to 4 months of pregnancy and then remained unchanged up to 150 days of pregnancy [216]. Ovarian morphological changes in buffaloes, including ovarian and CL dimensions and weight, evaluated from abattoir specimens [205,217], and through transrectal ultrasonography [218], revealed an increase in the diameter and weight of the ovary ipsilateral to the gravid horn up to 4 months of pregnancy [218]. The increase was maintained up to 6 months of gestation [217]. Sequential increase in the dimensions of the CL and its weight were observed from 1 to 4 months [218], and up to 6 months [205,217] of gestation in buffaloes (Table 2). The ovaries were pulled towards the pelvic brim as the growing uterus descended into the pelvic cavity at around 3.5-5 months of pregnancy [147].

Ultrasonographic studies have revealed that the follicular growth on the ovaries continues during early gestation in the buffalo [219,220]. No influence of pregnancy on the number of waves or the time of onset of a new wave was seen [220]. However, during Days 30, 40, 50 and 60, follicular population on the ovary was reduced [221] and follicular waves became inconsistent with variable patterns [219]. There was no significant difference in the diameter of small and medium follicles on different days of pregnancy (20, 30, 40, 50 and 60), however, there was a significant decrease in the number of large follicles with the advancement of pregnancy [221]. These studies suggest that although follicles continue to grow during pregnancy in buffaloes, the high progesterone levels probably suppress the growth of follicles to ovulatory size and also suppress estrus during gestation. However, small proportions (1-13%) of buffaloes show gestational estrus [222,223], probably because of low luteal progesterone or a disruption of the mechanisms preventing follicle growth to the ovulatory stage however, the pregnancy continues normally until the dam is accidentally mated or inseminated.

Body Weight Changes of the Dam

With the advancement of pregnancy there are changes in the body of the pregnant dam. These changes include body weight, and at the terminal stages of gestation, there are important changes in the mammary gland. In dairy cows, 75% to 85% of the weight gain occurred during the last four months of gestation. The month to month weight gain was very uniform in first-calf heifers compared to older cows [224]. In Murrah buffaloes, there was no significant difference in body weight gain during the first three months of pregnancy, however, from the third month onwards, body weight showed more or less a uniform increase until parturition [41]. The increase in weight for pregnant Murrah buffalo heifers was 27.15 kg, whereas Murrah buffaloes gained 18.62 kg and non-descript (cross-breeds) buffaloes gained 21.03 kg [41]. For swamp buffaloes every two months of gestation from the onset, the increase of dam body weight were 6, 14, 43, 24 and 13 percent of the total increase (54.52 kg), respectively [225]. The body weight of Murrah buffaloes at calving varied from 491.70 kg at first calving to 607.5 kg at sixth calving [226]. Animals that were heavier at calving, lost more body weight during the first six months of lactation. The average body weight of pregnant swamp buffaloes increased from 444.5 kg to 477.6 kg from about 5 months prior to calving until parturition [227]. Lactating buffaloes lost body weight (12.2 kg) during the first 4 months postpartum [227]. Winter calving Murrah buffaloes were heavier compared to summer calving buffaloes 2 weeks prior to parturition however from parturition to 9 weeks postpartum both winter and summer calving buffaloes lost weight similarly (15%) [228].

Endocrinology of Gestation

Plasma Progesterone

Studies in buffaloes have shown that one day after insemination or mating, plasma progesterone starts rising and reaches its maximum level, both in river and swamp buffaloes [229-231], by Day 13. At Day 17, progesterone rises to a peak of 3.47 ng/ml in buffaloes that conceived [232,233]. Progesterone levels then continue to be elevated in pregnant buffaloes [232]. Elevated plasma progesterone during the entire gestation has been recorded in buffaloes [230,231,234]. During the first 2 months of pregnancy, plasma progesterone concentrations fluctuated between 1.9 and 3.8 ng/ml decreasing slightly to 2.9±0.8 ng/ml during the 3rd month [235] and constant levels were maintained with small variations until about 8 days prepartum [235] or 271-273 days of pregnancy [236]. Similarly, in swamp buffaloes plasma progesterone levels were fairly constant throughout the whole of gestation (average 1-5 ng/ml) [237-239]. Plasma progesterone concentrations decline slowly from 12-20 days prepartum [240-242] followed by an abrupt decline to basal levels at parturition [243,244]. Milk progesterone levels (8.5±0.8 ng/ml) in pregnant buffaloes were nearly 4 times higher than those in serum at Day 27 of pregnancy [245,246]. Progesterone in placental tissue was negligible during early pregnancy in the buffalo with slight increases between Days 97 and 250 [247]. Progesterone receptors have been observed in the binucleate trophoblast cells of buffalo placenta [181]. Prepartal decrease in progesterone starts from Days 276-278 [248]. A rapid decline in plasma progesterone has been observed 3 days prepartum [249].

Plasma Estradiol

The level of estradiol17β declines by one day post-insemination until Day 20 [250] and fluctuates within narrow limits throughout the first trimester of gestation in buffaloes [248]. Slight increases in mean plasma estradiol17β (14.8+2.1 ng/ml) were recorded during the first 4 months of pregnancy decreasing to basal values (<12 pg/ml) during the remaining months of pregnancy [235]. During the second trimester, although a plateau is maintained, the overall mean concentration is significantly higher compared to levels during the first trimester [248]. An increase in plasma estrone sulphate in maternal peripheral circulation was recorded from about 5-6 months of pregnancy in swamp [239] and river [235,252] buffaloes. High estradiol levels (108.2±9.1 ng/ml) were found in milk of pregnant buffaloes during the third trimester of pregnancy [253]. Estradiol levels start rising by Day 241-243 of gestation to reach the highest levels at parturition [234,235,248].

Plasma Corticosteroids

The concentrations of plasma corticosteroids remained fairly constant (1.7+0.3 pg/ml) throughout pregnancy [235]. A significant increase (5.3+1.8 ng/ml) was noticed on Day 12 prepartum in river buffaloes [235] and on Day 15-20 prepartum in swamp buffaloes [251] with peaks at 60h, 36h, 12h and 0h (16.8+3.2 ng/ml) prepartum [235].

Plasma Gonadotrophins and Pituitary Responsiveness to GnRH

There appears to be little change in plasma LH (average 1-5 ng/ml) throughout pregnancy in swamp buffaloes [251]. In river buffaloes, plasma LH levels at 2, 5 and 8 months of gestation were 2.01±0.11, 2.24±0.18 and 2.48±0.13 ng/ml respectively and were not significantly different from each other [254]. FSH levels at 2 months of pregnancy (15.66±1.09 ng/ml) were significantly higher compared to values at 8 months (13.62±0.17 ng/ml) suggesting a decrease in FSH concentrations [254]. The pituitary responsiveness to exogenous GnRH revealed a progressive decline during the advancement of pregnancy in buffaloes reflecting the effect of a negative feedback of increasing plasma progesterone during advancing gestation in buffaloes [254].

PG Metabolites

The peripheral plasma prostaglandin metabolite (PGFM) does not increase around Day 18-20 of mating if the buffalo is pregnant [255]. The plasma concentrations of PGFM ranged between 200-600 pg/ml during the first 9 months of pregnancy and increased thereafter (700-1000 pg/ml) 10 days prepartum [234,235]. A significant increase in PGFM concentrations was observed on Day 9 prepartum (2.2+0.2 ng/ml), at 54h (4.9+0.4 ng/mL) and at 6h (9.6+1.2 ng/ml) prepartum [235].

Thyroid Hormones

Based on studies on peripheral plasma thyroxine levels during advancing pregnancy in cattle and buffaloes, it was hypothesized that the T4 requirement of the fetal buffalo calf may be lower than that of the fetal cattle calf, since gestation in the buffalo is longer and the metabolic rate slower vis-à-vis cattle [256]. In pregnant buffaloes, peripheral plasma T4 levels fluctuated slightly throughout pregnancy with a minor increase at 8-9 months [257] whereas in cows, plasma T4 increased sharply during the first trimester reaching a peak in the third to fourth month of gestation, followed by a gradual decline until the last month of pregnancy [256,258,259]. In a study on pregnant Murrah buffaloes, the mean levels of T4 and T3 were 44.1±1.36 and 0.46±0.07 nmol/l which was significantly higher than those in non-pregnant buffaloes [260]. The peak levels of T4 and T3 were observed at the 4th and 8th week of gestation [260]. A seasonal increase in the plasma thyroxine levels (during the rainy season) has been observed in buffaloes [261]. The mean protein bound iodine concentrations (5.97 μg/100 ml) in Surti buffaloes were considerably higher during early gestation and started declining after Day 50 of gestation [262]. Supplementation of iodine (0.3 to 0.5 mg iodine per kg DM intake) during late pregnancy improved T3 and T4 in supplemented buffaloes and led to a significant subsequent decrease in calving intervals and days open after parturition [263].

Biochemicals and Electrolytes

Serum protein increased with stage of gestation (overall 8.78 g/dl in pregnant animals) up to Day 275 in buffaloes and then decreased significantly with a sharp decline 6h prepartum [264]. A considerable increase in plasma albumin occurred at Days 268-271 of gestation in buffaloes [265]. In pregnant buffaloes, protein requirement is 12% crude protein (CP) during the first 240-270 days of gestation whereas 14% CP is required at 270-308 days of gestation [266].

The mean blood glucose values evidenced slight decrease with increase in the gestational days in buffaloes [267]. Blood glucose levels were significantly lower and cholesterol decreased slightly on the day of parturition [268] compared to values one month prepartum. The energy requirement for pregnant buffaloes was 55.4% total digestible nutrients (TDN) (2.0 Mcal/kg ME) in dry matter between 240 and 270 days of gestation whereas TDN was 60.6% (2.19 Mcal/kg ME) between 270 and 308 days of gestation [266] reflecting increasing demands of the growing fetus during the last 2 months of gestation [269].

The electrolyte levels varied widely between pregnant buffaloes and a significant decrease was recorded after 6 months of pregnancy for serum calcium (Ca) and phosphorous (P) without any clinical disorders [270], however, these were not consistent as another study recorded non-significant changes in the Ca and P during the last 3 months of gestation in buffaloes [271]. During the 8th and 9th month of gestation in buffaloes, significantly lower concentrations of serum Ca and P were recorded in buffaloes with prepartum prolapse [272,273]. At 8th and 9th month of gestation the Ca and P requirements are 40 g and 35 g respectively, (Ca/P ratio 1:1) [269]. Buffaloes reared in areas with P-deficient soils revealed lower values of P [274]. Such deficiencies might result in pre-parturient hemoglobinuria [275]. The levels of iron decreased significantly in pregnant buffaloes at 9th month of gestation [272]. The concentrations of serum sodium, potassium and chloride remained unchanged during pregnancy in buffaloes whereas serum copper concentrations increased and zinc concentrations decreased with increasing gestational age because of the demands of the fetus [276]. Multiparous pregnant buffaloes had lower serum electrolyte levels compared to primiparous buffaloes and the concentrations decreased significantly during extreme hot and cold weather [277]. The reference values for the various electrolytes and biochemicals in pregnant buffaloes less than 6 month and more than 6 month pregnant have been mentioned recently [270], however, the range is too wide to be applicable practically. A slight anemia has been recorded during the terminal stages of gestation in river [265,278] and swamp [279] buffaloes with a significantly low total erythrocyte count [278].

Immunology

It has been recently mentioned that the conceptus and other endocrine mediators actively shape the maternal immune response during early gestation to facilitate the growth and development of a functional placenta [280]. Proteins from both cotyledon and non-cotyledon portions of buffalo placenta exhibited immunosuppressive activity [281]. The placenta is an active endocrine organ secreting numerous proteins and steroids and some of the placental products have immunomodulatory actions at the feto-maternal interface to prevent rejection of the fetus [282]. The bubaline placenta exhibits immunosuppressive activity under in vitro conditions [283]. A placental protein with a molecular weight of 65 kDa has been associated with immunosuppressive effects in buffaloes [283]. The non-rejection of the fetus involves B and T cells. Immunosuppressive effects of the 65 kDa buffalo placental protein (bPP65) on T [283] and B cells [282] were shown, suggesting that the primary antibody response involves both of these types of cells. Non-classical major histocompatibility complex (MHC) class I leukocyte antigens were expressed in the placenta of late pregnant buffaloes [284]. Placental transfer of immunity is not observed in ruminant and other domestic species in which the immunity is transferred via the colostrum at birth [192].

Embryology (Prenatal Organ Development)

Studies on the evaluation of organogenesis in the developing fetus have utilized abattoir material in assessing the morphology and histologic appearance of the cells and organs and their functionality in buffalo species. A limited number of studies have evaluated the development of the different organs and structures which are mentioned. The dimensions of the fetus have been used in many studies, namely the curved crown-rump length (CVRL) or the crown-rump length (CRL).

Heart

The heart is probably the first organ to form and function during development [285]. An ultrasonographic description in buffaloes mentions that the heartbeat first appears at 25-27 days of pregnancy [286,287]. Histomorphogenic studies on buffalo fetuses from abattoir revealed that at around 32 days of gestation or a CVRL of 0.9 cm, the heart was unseptated and tubular, and it was clearly divided into common atrial chamber dorsally, primitive ventricles ventrally, primitive outflow tract with bulbus cordis region proximally and aortic sac dorsally at CVRL of 1.2 cm [288]. The Septum primum, endocardial cushions, septum secundum and foramen ovale were observed at 3.0 cm CVRL [288]. The interventricular and four-chambered heart was recognized along with atrioventricular valve, chordae tendineae and papillary muscles at 8.7 cm CVRL (66 days) [288]. All the internal structures of the heart were well differentiated from 50 cm CVRL onwards [288].

Kidneys

Histomorphological studies have revealed that the development of fetal kidneys in buffaloes occurred in 3 stages: i.e., pronephros, mesonephros and metanephros [289]. The pronephros were observed at 3 cm CRL (42 days) which then started degenerating at 3.5 cm CRL (45 days) and complete degeneration occurred at 4.1 cm CRL (47 days) [289]. The pronephros was located at the level of the heart, cranial to the mesonephros with no glomeruli and duct system [289]. The metanephros started developing at 4.1 cm CRL (47 days) in the cortex of the kidney [290]. The cortex and medulla could be differentiated at 5.7 cm CRL (54 days) [289]. Three types of glomeruli i.e., the subcapsular, intermediate and juxtamedullary could be observed in the kidneys of buffalo fetuses at 10.3 cm CRL (75 days) onwards [290,291]. The proximal and distal convoluted tubules were seen in the cortex of the kidney at 4.1 to 5.7 cm CRL (54 days) [291]. Three types of glomeruli i.e., the subscapular, intermediate and juxtamedullary, could be observed in the kidney of buffalo fetuses at 10.3 cm CRL (75 days) onwards [291,292].

Pituitary

The histological sectioning of the hypophyseal cavity of buffalo fetuses revealed Rathke’s pouch at 30 mm CVRL lying parallel to the long axis of the fetus [293]. The marginal cell layer lining the hypophyseal cavity was first seen at 60 mm CRL and consisted of simple squamous epithelium [293]. The neurohypophysis differentiated at 60 mm CRL with the lumen disappearing at 80 mm [294]. The pars distalis, pars intermedia and hypophyseal cleft were seen enwrapping the neurohypophysis at 120 mm CRL and onward stages [294]. A recent study on the corticotrophs in fetal buffalo pituitary gland revealed that there was a significant increase in immune reactive ACTH during the 2nd trimester while immunoreactive proopiomelanocortin cells increased late during the 3rd trimester [295].

Tongue

At 1.2 cm CVRL (34 days) the tongue was lined by a single layer of cells that became 2 layered, a deep layer of cuboidal cells and a thin sheet of superficial cells at 2.5 cm CVRL (40 days) [305]. At 10.7 cm CVRL the lamina epithelialis was distinctly stratified and was divided into dark basal and light superficial zones [305,306]. At 21.4 cm CVRL (122 days), the stratified squamous epithelium was better differentiated at both the dorsal and ventral surfaces [305], with all the layers of epithelium well recognized at 29 cm CVRL (139 days). The first evidence of keratinization was observed in fetuses above 40 cm CVRL [305]. The first indication of the formation of circumvallate papillae was observed at 10.7 cm CVRL (27 days) [305,307], and at 21.4 CVRL (122 days), the two stacks of papillae invaginated deep into the tongue mesenchyma to form serous acini [308]. The primitive taste buds appeared on the apical surface of circumvallate papillae at 45.0 cm CVRL (175 days) and taste pores became apparent at 80.0 cm CVRL (254 days) [308]. The submandibular salivary glands revealed weak activity of phosphates and oxidoreductases at 11-19 cm CVRL (78-114 days) in the acinar cells and ductular epithelium which increased with advancing gestation. At 42-100 cm CVR (168 days to full term) the alkaline phosphates were found in the lumen of acini and along the intercellular canaliculi [309].

Esophagus

Up to 7.5 cm CRL the late lamina epithelialis was 2-layered having a basal layer of cuboidal cells and a superficial layer of columnar cells. The superficial cells became polygonal and polyhedral at 11.2 cm CRL [296]. The lamina muscularis mucosae had mesenchymal cells up to 11.2 cm CRL that differentiated into collagen and reticular fibers at 14.7 cm CRL with the skeletal muscle being observed at 11.2 cm CRL in the cranial end, and at 20 cm CRL in the caudal end [296]. A positive correlation was observed between the thickness of various esophageal tunics and subsequent CRL (22.4 to 62.0 cm CRL) of fetuses in another study [297].

Rumen

The lumina epithelia was stratified cuboidal and divisible into dark basal and light superficial zones at 5.5 cm CVRL (53 days) [298,299]. The ruminal papillae appeared at 19.6 cm CVRL (120 days) whereas the keratohyalin granules were observed in the epithelium at 38.5 cm CVRL (160 days) [299]. The collagen and reticular fibers [299] were well differentiated at 11.2 cm CVRL (79 days) [299]. The neutral mucopolysaccharides were increased in fetuses of 11.2 CRL and localized in the basal and middle cell layers of ruminal epithelium, however, at 22.4 to 28.0 cm CRL the neutral mucopolysaccharides showed a declining trend [300]. From 14.7 to 20.0 cm CRL, the acid mucopolysaccharides increased, probably providing strength to the connective tissue and epithelium [300].

Reticulum

The papillae of the lamina propria were initially observed at 14.7 cm CRL. The mucosal surface showed compartments characteristic of reticulum at 19.6 cm CRL. The reticular papillae exhibited muscle bundles and apical portions at 60.1 cm CRL and were comparable to those of an adult at this stage [301].

Duodenum

Histomorphologic studies on buffalo fetuses showed that the mucosal folds were observed at 6 cm CRL whereas the villi appeared at 8.0 cm CRL [302]. The same study reported that the epithelium transformed from stratified to simple columnar at 19.5 cm CRL and the enterochromaffin cells were observed at 14.5 cm CRL in duodenal villi. The primordial of intestinal glands are known to be present at 14.5 cm CVRL in the duodenum of Egyptian buffalo fetuses [303] and at 22.5 cm CRL in Murrah buffalo fetuses [302]. The duodenal glands were observed in the tunica submucosa at 22.5 cm CRL [302].

Large Intestine

The mucosal projection appeared in the large intestine of buffalo fetuses during the early stage of development whereas the villi were first observed at 19.5 cm CVRL (119 days) in the rectum [304]. The degeneration of villi started at 39.0 cm CVRL (161 days) in the rectum and villi disappeared at 88 cm CVRL (272 days) in all segments of the large intestine [304]. Goblet cells appeared earlier in the rectum (CVRL 31.0 cm, 144 days) compared to the cecum and the colon (CVRL 39.0 cm, 161 days). The intestinal glands were fully differentiated at 55.0 cm CVRL (198 days) and the lamina muscularis mucosae appeared in all segments of the large intestine at 60.2 cm CVRL (212 days) [304].

Liver

Histogenesis of the liver in buffalo fetuses revealed that the developing liver at 1.5 cm to 4.0 cm CVRL occupied a large part of the abdominal cavity and was surrounded by a single-layered connective tissue capsule [310]. The initial hepatic lobulation and constituents of the portal triad area were observed at 26.0 cm CVRL [310,311]. The capsule of the liver at 14.5 cm to 44.5 cm CVRL was made up of collagen and reticular fibers [310,311]. The typical arrangement of hepatocytes resembling the histological profile of the adult liver was seen at 45.5 cm CVRL (176 days) [311]. In the fetal liver at 10 cm CRL moderate to strong activity of histoenzymes was observed in scattered areas reflecting functionality [312].

Spleen

The spleen in a 51 day-old buffalo fetus was observed on the dorsolateral aspect of the stomach and was devoid of the typical splenic capsule [313]. The splenic stroma at this age consisted of mesenchymal tissue. At 74 days of gestation, the splenic capsule appeared as a thin fibrocellular layer made of immature collagen fibers with a thin layer of reticular fibers [313]. At 90 days of gestation, the trabeculae were seen emerging from the capsule. Well-developed fibromuscular capsule and trabeculae were observed in 262 day old fetus [313]. The parenchyma of the spleen showed development of arteriole and periarteriolar lymphoblasts in 90 day-old fetuses [313]. Hematopoietic activity was clearly observed in the red pulp in fetuses from 166 days onwards [313].

Thymus

At around 23.2 cm CRL, the parenchyma of the thymus was not divisible into the cortex and medulla [314]. Distinct trabeculae and septae were not found at this stage [315]. At 31.5 cm CRL some lobules of the thymus showed the formation of medulla while in others the parenchyma was well differentiated into cortex and medulla [314]. A well-developed medullary zone was observed in most lobules by 44.5 cm CRL which appeared lighter than the cortex [314]. Up to 44.5 cm CRL, the reticular fibers were confined to the peripheral zone of the medulla while these were observed in the entire medullary zone during later stages [314]. The differentiation of reticular fibers began at 63 mm CRL (57 days) from the fibroblasts and was distinct at 120 mm CRL (83 days) [316] in the thymus of a buffalo fetus. At 367 mm CRL (156 days) a fine network of fibers surrounded the major blood vessels of the interlobular septa. At 750 mm CRL (243 days) these fibers formed a double layered sheath around the blood vessels of the interlobular septa [316]. The presence of reticular fibers in Hassall’s corpuscles of thymus was seen in late gestation in buffalo fetuses [316]. The histogenesis of Hassall’s corpuscles began at 9.2 cm CRL (70 days) in the buffalo and at 13.5 cm CRL (90 days) distinct Hassal’s corpuscles were noted in the fetal thymic medulla [317]. The degeneration of Hassall’s corpuscles began at 6 months postnatal life and they disappeared completely by 2 years of age in buffaloes [317].

Adrenal

Micrometrical studies on prenatal buffalo fetuses showed sequential growth of the adrenal gland between 39 mm to 890 mm CRL [318]. The adrenal cortex and medulla were fully differentiated by 109 mm CRL, however, the adrenaline and noradrenaline secreting cells were identified by 210 mm to 325 mm CRL [319]. Fetal cortical cells (FC) were differentiated in the 10.9 cm CRL buffalo fetuses [320]. The nonspecific esterases activity was weak in the capsule, stroma and capillaries, strong to intense in the fetal cortex and weak to moderate in both zones of the medulla in buffalo fetuses with a CRL of 10.0 cm to 89.0 cm [320]. The distribution of neutral mucopolysaccharides and acid mucopolysaccharides varied in different regions of the prenatal bubaline adrenal glands (3.9 cm to 89.0 cm CRL) [321].

Mammary Gland

The mammary line was first observed at 1.2 cm CRL (34 days), the mammary hillock at 1.7 cm (37 days) and the mammary bud at 2.6 cm CRL (41 days) [322]. The epidermal cone was found at 6.7 cm CRL (58 days) whereas primary and secondary ducts were observed at 7.4 cm CRL (62 days) and 15 cm CRL (96 days) [322]. The mammary anlage appeared on the ventral abdominal wall, caudal to the umbilicus between the hind limbs between 90-109 days and consisted of centrally located primary sprout embedded in mesenchymal tissue and covered externally with epidermis [323]. At 120-146 days of age, the primary sprout formed canals and gave rise to secondary sprouts. At 152-182 days, the mammary gland showed primary and secondary sprouts with canals [323]. The differentiation of all the skin layers along with cornification was observed at 69 cm CRL (229 days) [322]. In buffaloes, hair follicles, sweat glands, pilomotor muscles and sebaceous glands in the mammary gland, developed at 102, 139, 142 and 158 days of gestation respectively [324].

Bony Skeleton

The earliest indication of skull development was first recognized as a mass of dense mesenchyma enveloping the cranial end of notochord at 27 days of gestation followed by the formation of the basal plate at 38 days. The formation of Meckel’s cartilage was first recognized at 41 days [325]. The ossification of the skull first appeared in the mandible, maxilla and molar bones at 45 days [326,327]. The basioccipital, exoccipital, and the lower part of the squamous occipital were formed from the basal plate [328]. Most of the chondrocranium was cartilaginous at 45 days, and began to ossify at 62 days in the basioccipital and exoccipital [326,329], and in the sphenoid and tympanic bulla [326]. The first indication of ossification in the mandible appeared at Day 45 in the incisive and molar parts and extended subsequently into the ramus [330]. Ossification in the coronoid part was observed at Day 61 and ossification was completed by Day 64 and the mandibular canal was distinct at Day 70 [330].

Most of the skull was ossified between Days 45 to 89th day of gestation in buffaloes [326] and the complete ossification of the squamous occipital, basioccipital and exoccipital was observed at 132 days [327,328]. The sphenoid bone developed from six ossification centres at 62 days [329]. Ossification was recognized first in the orbitosphenoid, the body of basisphenoid and the alisphenoid at 62 days [329]. The last bone of the skull to ossify was the dorsal turbinate at 193 days [327].

The differentiation of cervical, thoracic, sacral and coccygeal vertebrae started at 2.0 cm CVRL in buffalo fetuses [331]. The intervertebral discs were evident at 3.0 CVRL, being broader at the periphery and thin centrally [331]. The length of the cervical region decreased and that of the lumbar region increased with advancement of fetal age whereas the length of other regions remained unchanged [331].

Ovaries

The primordial germ cells appeared on the mesentery on Day 35 and in the gonadal ridge at Day 36 of gestation [332,333]. The gonadal tissues differentiated into a peripheral cortex and central medulla by Day 62 of gestation [334]. The formation of a spindle shaped indifferent gonad was recorded on the ventromedial aspect of the mesonephros at Day 47 [333]. A single layer of flat epithelium covered the indifferent gonad at Day 51 [333]. At 67-83 days, the ovaries were located on the caudolateral aspect of the metanephric kidneys [335]. At around Day 85-87, the ovaries detached from the nephric ends and migrated between 94 and 174 days [336] into the pelvic position that was completed at 180-305 days [207,337]. Ovarian germ cell degeneration occurs during the 4th, 6th, 8th and 10th month of gestation in buffaloes [337,338]. Most fetal ovaries were ovoid in shape during development and the maximum ovarian length and thickness was noticed between 158 to 305 days of gestation [339].

Oviducts

The first evidence of the formation of the oviducts was observed in 41 days fetuses as an invagination of the coelomic epithelium at the anterolateral aspect of the mesonephros-the open Müllerian groove [340]. The structure became tubular at 46 days of gestation and was located within the mesonephric fold. The primary, secondary and tertiary oviductal mucosal folds appeared at 94 days, 158 days and 191 days [340]. The mucosa, muscularis and serosa could be identified at 128 days and resembled that of an adult organ at 191 days of gestation [340].

Uterus

The first evidence of the formation of the uterus was observed in 61 days fetuses through the caudal fusion of the two Müllerian ducts [341]. The epithelium of the developing uterus was of pseudostratified ciliated columnar type in 75 Day fetuses [341]. A progressive condensation and differentiation of mesenchymal tissues and a serosal covering around the uterine portion was observed in 85 Day fetuses [341]. The uterine lining of the epithelium became folded at 116 days of fetal age and the endometrium, myometrium and perimetrium were clearly distinguishable in 130 day fetuses and resembled that of an adult uterus at 174 days of gestation [341]. The endometrial caruncles first appeared at 130-152 days [341,342] and the uterine glands at 191 to 280 [341,342] days of fetal age. The maximum mean length and width of the uterine horns occurred between 158 to 305 days of gestation [339].

Cervix and Vagina

The first evidence of the formation of the cervix in buffalo fetuses was observed at 67 days of gestation where 2 Müllerian tubes fused together caudally to form a common tubular structure which developed into the cervix [343]. The primary mucosal folds appeared between 85-121 days [342,343], the secondary mucosal folds appeared between 94 to 152 days [342,343] and tertiary folds at around 130 days of gestation [343]. The cervical lining epithelium varied from pseudostratified to simple columnar epithelium [207]. The vagina originates from the cervix. At 90 days, the cervico-vaginal junction was lined by stratified squamous epithelium and an extensive cellular mass in the lumen represented the part of the vaginal palate [344]. The maximum length and width of the cervix, vagina and vestibule occurred between 116 to 191, 158 to 191 and 240 to 305 days of fetal life [339].

Testis and Epididymis

The indifferent gonads were first observed in buffalo embryos as a nodular structure attached medial to the mesonephros at 43 days of gestation [345]. The formation of the tunica albuginea in differentiation of blastema into a testicular cord-like structure was evident at 47 days of gestation [345]. Both testicles were attached to the latero-ventral aspect of the kidneys and were intra-abdominal at the age of 8.0 cm CVR (65 days) and started migrating into the inguinal region at 18.2 CVR (110 days) with complete descent into the scrotal sac by 75.0 cm CVR (243 days) [346]. The indifferent gonads were finally transformed into testes at 300 days of gestation [345]. The differentiation between the efferent ductules and epididymal duct was observed at 74 days in male buffalo fetuses [347]. The epididymal duct was surrounded by 7-8 layers of peritubular mesenchymal cells which later on transformed to muscle cells at around 110 days of gestation [347]. Ciliated, non-ciliated and simple columnar cells were also observed in the ductus epididymis at this time [347].

Twinning and Sex Ratio

The incidence of twins in buffaloes is low. Earlier reports indicated an incidence of 0.01% to 0.2% [348,349]. The incidence of twin births in Indonesian and Malaysian buffaloes was cited to be still lower (0.0002%) [350]. Analysis of calving data from organized farms of various breeds of buffaloes revealed the incidence to vary from 0.057% to 0.63% (Table 3). Most buffalo twins were born from multiparous dams, on their third or higher gestation [351-353] suggesting an increase in ovarian activity beyond the 3rd gestation. Reports also indicated that most buffalo twins born were heterosexual (1 male and 1 female) [351,352,354], except in one report that indicated the birth of 2 pairs of male twins and 2 pairs of female twins besides 2 pairs of heterosexual twins [355], and one case report that indicated the delivery of 2 male twins [356]. The gestation length in buffaloes delivering twins may be shorter than in single births [352] or normal [351,352], yet the birth weight of the twin calves may be lower than the birth of a single born calf [352]. Twins may result in fetal death before or at parturition [352] and sometimes in difficult births [351,354,356]. It was not mentioned in any report whether twins of the same sex [355] were identical and originated out of a single zygote. Similarly, only one report mentioned the evidence of a freemartin (sex chromosome chimerism) in one female buffalo calf born co-twin to a male calf [352]. Three reports mentioned the birth of triplets in buffaloes [357-359] and one report mentioned the birth of quadruplets in buffaloes [360]. However, sex chromosome chimerism was mentioned only in two reports [357,358]. Freemartins were found by cytogenetic evaluation in Italian river buffaloes [361] presented with infertility; however, whether the freemartin was born with heterosex was not mentioned. It thus appears that the incidence of freemartin in buffaloes is low [362]. The prospects of induced twinning by transfer of in vitro produced embryos to buffaloes [350,363] are limited due to poor fetal growth and difficulty at delivery. A conjoined Siamese twin was described [364] and a large number of conjoined buffalo twins resulting in difficult births have been reviewed previously [365]. Twins were also recorded in swamp buffaloes in Thailand that had been treated for estrus synchronization [366].

The sex ratio of male: female births in the buffalo show a male bias (Table 4). The sex ratio of male births varied from 49.76% to 50.8% at two organized Murrah buffalo farms in one study [372] and was 55.64% in another study on Murrah buffaloes [373]. In Brazil, data from 232 pregnant buffalo abattoir derived genitalia revealed 47.0% male fetuses [141], whereas data from Italian buffaloes revealed 49.84% males [368]. The lowest and highest frequency of male births was recorded during summer [372] and rainy [374] or post monsoon [375] season. The second parity resulted in a higher proportion of male births in some studies [372,375], however, other studies revealed non-significant differences in the proportion of male: female births during different parities in buffaloes [60,374]. The repeatability of sex ratio was low (-0.038 to -0.064) [372,374] and the heritability estimates for male births were low to medium (0.124±0.12 to 0.43±0.19) for the two herds of Murrah buffaloes evaluated [372]. The effect of sire on the proportion of male births in buffaloes was marked [60,372].