Pre-pubertal, Postpartum and Summer Anestrus in Buffaloes

Reproductive efficiency in the buffalo appears to be limited on account of three distinct inherent problems, delayed puberty, prolonged postpartum anestrus and summer anestrus [1-3]. The seasons (environmental temperature, photoperiod, and feed availability) have overlapping effects on the postpartum estrus onset [4-6] which is also affected by suckling [7], uterine pathology and poor thermoregulatory mechanisms [6]. Anestrus is not a disease but a state of sexual inactivity with absence of estrus expression due to a variety of conditions. The incidence of anestrus (or lack of estrus expression varies from 9.09% to 69.4% in buffaloes [8,9]. A classification study on the type of anestrus revealed that around 50 to 60% of anestrus cases were subestrus, 30.5% to 40% were postpartum anestrus, 5.1% were summer anestrus and a high proportion of anestrus (26.6%) animals had genital infections [10]. A major problem in buffaloes appears to be the poor expression of overt estrus (silent estrus) which often complicates estrus detection in proportion to the number of buffaloes. Buffalo anestrus appears to be slightly different from that in cattle as seasonal and genetic influences often predominate.

Puberty in the buffalo is delayed presumably due to inherent slow growth, deficiencies of energy, protein, minerals and vitamins [4]. Climatic stress and poor nutrition delay puberty and increase the duration of postpartum anestrus [5,11]. Seasonal suppression of estrus in buffaloes probably originates as a result of hyperprolactinemia due to high environmental temperatures and a longer photoperiod [12] which suppresses the secretion of gonadotrophins and ovarian steroids. Concomitant to this, low levels of thyroxin are evident during high climatic temperatures, probably as a mechanism to keep the body cool and consequently lowering the metabolic turnover.

Puberty is probably initiated as a result of a series of complex developmental events that occur within the reproductive endocrine axis [13]. Puberty in buffaloes is delayed compared to cattle [14]. The age at puberty is difficult to establish because of difficulties in estrus detection in this species [15]. First conception occurs at an average body weight of 250-275 kg which is usually attained at 24- 36 months of age [16], 24.7 months at 310 kg body weight under common practices in the state farms of Egypt [17], and 15.4 months with average body weight of 271 kg under improved feeding system [18]. In Indian buffaloes, a large variation has been recorded in age at puberty occurring between 16 to 40 months in different breeds [15]. In Surti buffaloes, the first estrus and the first conception occur at 45.5 and 47.3 months, respectively whereas Murrah buffaloes kept under farm management reached puberty at 36.5 months and 355.8 kg body weight. Improving management and splashing buffaloes with water during the hot period, shortened the age at first estrus and conception [4].

Further, puberty has been evaluated in terms of a rise in progesterone concentrations, levels of Insulin like Growth Factor-1 (IGF-1) and Inhibin-A [19]. Buffalo heifers attained puberty when plasma progesterone levels exceeded 1ng/ml at about 20 months of age [15]. Plasma Inhibin–A concentration began to increase gradually a few weeks before the onset of puberty (1.883 μg/ml) and this increase continued throughout the pubertal period [19].

In the past, anestrus has been clinically evaluated by transrectal palpation of ovaries (no palpable CL) [20]. More recent evaluations suggest that the diagnosis be based on the absence of follicles and corpus luteum in both ovaries, as confirmed by ultrasonographic monitoring twice in an interval of 10 days and further validated by measurement of peripheral plasma progesterone concentration (≤ 0.50 ng/ml) [21].

Therapies and management strategies suggested for anestrus in buffaloes include supplemental feeding, administration of hormones and thermal amelioration measures, with encouraging results. In this chapter, anestrus in buffaloes has been described under the headings prepubertal, postpartum and summer anestrus.

1. Prepubertal Anestrus

1.1 Puberty

Mechanisms regulating puberty in mammalian species appear to be complex and only partly understood. The onset of puberty and initiation of the ovarian cycle in cattle appears to be the result of secretion of a gonadotrophin releasing hormone (GnRH) from the hypothalamus at the appropriate frequency and quantities to stimulate release of gonadotrophin hormones, such as luteinizing hormone (LH) and follicle stimulating hormone (FSH), from the anterior pituitary [13]. This increase in pulse frequency of GnRH is a key factor in increasing circulating LH, follicular development, and hence synthesis and secretion of steroid hormones, predominantly estradiol 17β (E2) [13]. Buffalo heifers are slower to reach puberty compared to cattle [22]. Studies evaluating puberty onset in buffalo heifers have mentioned a gradual change in various hormones such as a decrease in cortisol and T3 [23] and growth hormone [24] and puberty being signaled by prepubertal increase in circulating LH [24-25] and consequent increase in plasma progesterone (>1 ng/ml) [26]. In another study on buffalo heifers, it was found that plasma progesterone greater than 1 ng/ml for three consecutive days was defined as day of puberty [27]. Progesterone levels were low (0.20-0.30 ng/ml) during the prepubertal period [27]. There were two distinct elevations before the day of puberty onset. Plasma LH and GH concentrations increased a few months preceding puberty and were highest during the month before puberty [27].

Secretion of high amplitude GnRH and combination of neurohormones and peptides initiate the pubertal changes in heifers [13]. Some of the peptides recognized in cattle that play important roles in GnRH secretion and mediating steroid feedback communication include, neuropeptide Y and, agouti-related peptides [28], opioid peptides [29], kisspeptin and its receptors [30]. A few of these, such as opioid peptides [31] and kisspeptin [32] have also been recognized in buffaloes. The effect of naloxone (an opiate antagonist) administration to buffaloes during the luteal phase of the estrous cycle did not result in significant differences in the GnRH-induced peak of LH and FSH concentrations compared to the LH peak in buffaloes administered saline [31]. A single IV infusion of 1000 pmol/kg bw of kisspeptin-10 induced higher LH pulses (2.2±0.4) compared to a single injection of GnRH (10 μg IM), however, the GnRH treatment stimulated the ovulatory LH surge but kisspeptin-10 did not [32].

With sequential growth in heifers, the hormones and peptides interact with various internal metabolic signals, such as glucose, propionate, leptin, ghrelin, insulin-like growth factor-1, and its transport proteins that are recognized by receptors in the central nervous system [33]. Leptin has been proven to be involved in the regulation of the energy balance and reproduction of several mammalian species [34]. It seems to act not only like a mere signal but as an important neuroendocrine and metabolic hormone [34].Leptin is a product of white adipocytes and its receptors are localized in many different tissues and organs such as brain and pituitary, the key site for the control of appetite, reproduction and growth [35]. Buffalo leptins were similar to cattle and the nucleotide sequence variations observed in the leptin gene between Bubalus bubalis and Bos taurus species revealed 97% nucleotide identity [36-37]. Buffalo leptins play a significant role in promoting angiogenesis, steroidogenesis and CL formation [38].

1.2. Factors Affecting Age at Puberty

It has been mentioned that the onset of puberty in cows is related to GnRH, LH, ovarian E2, and the sensitivity of the hypothalamus to E2, and neurohormones [13]. The development of the hypothalamic-pituitary-ovarian axis in heifers occurs in a gradual fashion during the growth of the animal [13]. The age at puberty is influenced by genetic make-up of the animal, nutritional status, climatic changes and local management conditions. Puberty occurs slightly earlier in river buffaloes (24 to 30 months) compared to swamp (30-36 months) buffaloes [15]. The FSH concentration did not differ significantly from birth to 15-18 months of age; higher levels of serum FSH from 18-21 months of age to 24-30 months of age were recorded in female buffalo calves [23]. The higher concentration observed at 15-18 months suggests that a prepubertal increase in circulating LH concentrations is the critical event leading to onset of puberty in buffalo heifers [24]. Many factors, directly or indirectly affect this gradual development and may advance or delay the pubertal onset. These factors include genetic make-up of animal and breed, body weight and rate of body growth, body composition and nutritional status [22,39-42], feed, and certain environmental or social cues such as season [6,17], photoperiods [43], and presence or absence of bulls [44]. Some of these factors play more important roles than others. Understanding how these factors affect the onset of puberty is the key to evolve better management strategies and reduce the interval to puberty. A recent study on hormonal profiles at onset of puberty in buffalo heifers under intensive feeding system and pasture system revealed that heifers achieved puberty when plasma level of progesterone exceeded 1 ng/ml [27]. Plasma leptin concentration was maintained at lower levels until the onset of puberty in pasture system than in the intensive system (280 vs. 556 ng/ml) respectively [27]. The values of IGF-1 at puberty were 479 and 276 ng/ml in intensive feeding and pasture system respectively [3]. Plasma inhibin A concentration began to rise gradually 4 weeks before puberty (1883 and 1307 ng/ml, respectively) [19]. Thus the onset of puberty appears to be regulated by complex interactions of many hormonal, nutritional and metabolic signals.

1.2.1 Genetic Make-up of Animal

The age at puberty is highly variable within different breeds of buffalo. Nili-Ravi, Surti and Mehsana reach puberty at the age of 26, 28.2 and 30.1 months, respectively. Murrah buffaloes exhibit delayed maturity compared to other breeds [45]. According to Saini et al., [4] Murrah buffaloes kept under normal management, reach puberty at 36.5 months and 355.8 kg body weight, while improving management condition and splashing buffaloes with water during the hot period, shortens these periods. Sire and dam effects within a breed, and heterosis, also contribute to the genetic control of age at puberty in buffalo heifers [18].

1.2.2 Nutritional Status

In a study on Nili-Ravi buffalo heifers, it was found that age at puberty can be reduced by 8 months in heifers fed fodder plus concentrate compared to those that are fed fodder only [42]. In another study Chaudhary et al., [46] reported that age at puberty in Nili Ravi buffalo heifers could be reduced by one month through additional concentrate feeding for a few months before the onset of puberty. Chaudhary et al., [47] found daily weight gain of 780g , age at puberty at 24.60 months and weight at puberty at 393 kg when buffalo heifers were fed fodder plus concentrate at 1% of their body weight beyond 1 year age to puberty. Nutritional status of heifers would determine the metabolic hormones and metabolites [42]. Inadequate nutrition delays puberty and sexual maturity in buffalo heifers, reduces conception rate, and increases pregnancy losses [48]. From a practical standpoint, nutritional management involving proper rates of weight gain decreases the time interval to onset of puberty and increase reproductive efficiency in heifers [13].

Briefly, protein availability also affects age at puberty onset because replacement heifers poorly compensate for protein deficiencies. It is critical that protein be available, especially before 7 months of age [49]. Murrah buffalo heifers (116 Kg) fed 35.96% less crude protein attained sexual maturity 210 days later compared to those fed a standard diet [40]. Similar delays in attaining body weight and puberty were evident in Anatolian [50] and Egyptian [39] buffalo heifers. Bhatti et al., [51] consider protein and energy as the most critical nutrients affecting the weight and consequently the age at puberty in buffalo heifers. Buffaloes can survive on poor quality feed; however, this often compromises their growth, puberty and reproduction [8]. Thus good quality nutrition is a pre-requisite to attain good production and reproduction [52].

1.3 Manipulation of the Pre-Pubertal period

Puberty enhancement has been attempted in a number of ways such as by providing high energy and protein diets [39,48], mineral vitamin supplements [53], feeding of monensin [54] and hormonal supplements [55-57]. Studies concluded that the correlations of the body weight at 6, 12, 18, 24 and 30 months of age and age at first calving are highly significant (ranged from -0.46 ±0.11 to 0.86± 0.04) [56]. Buffaloes fed poor quality roughages exhibit late maturity thereby causing economic loss to the farmers. In a study on Nili-Ravi buffalo heifers, weighing 170 ± 8 kg and aged 12 ± 2 months, supplemented with 1 kg good concentrate for first 6 months and 1.5 kg during subsequent months /animal/day till puberty, exhibited puberty earlier compared to buffaloes fed only forages [42]. It was found that concentrate supplemented buffaloes could reach puberty at 23 ± 0.25 months compared to non-concentrate fed buffalo in which they reached the same stage at 35.8 ± 1.5 months. Thus, the type of feeding strategy showed a significant effect (P≤0.01) on age at puberty and resulted in a 55.5 % decrease in the time required until animals could be bred [42]. Similar results have been shown in other studies [50,59,60].

Supplementation of hormones such as growth hormone releasing factor [56,61,62] or bovine somatotropin [57] is known to enhance growth and puberty in buffalo heifers. Under a clinical setting, pubertal buffalo heifers can be induced to estrus employing a variety of hormonal approaches (Table 1) with moderate to good success rates. It is essential that heifers should have attained sufficient body weight and show proper genital structures on transrectal palpation before hormonal therapies are implemented. Buffalo heifers with poorly developed genitalia should first be supplemented with multivitamin mineral supplements and if possible kept on a high plane of nutrition before initiating hormonal therapies with GnRH, progesterone or their combinations. Progesterone and estradiol or progesterone and eCG combinations are the choices clinicians prefer [63,64] although treatments such as Ovsynch protocols have also yielded good results [65,66].

2. Postpartum Anestrus

Postpartum resumption of normal cyclic activity is based on re-establishment of an extremely coordinated hypothalamic–pituitary–ovarian–axis. To maintain a calving interval of 13-14 months in buffaloes, these events must be accomplished by 60-80 days after calving. Soon after parturition, failure of follicular development is primarily due to apparent lack of LH [5]. Resumption of LH pulsatility is limited by the depletion of anterior pituitary LH stores during early postpartum period (calving to 20 days postpartum) while it is suppressed during Day 20 to Day 35 postpartum due to suckling [7]. Buffaloes evidence prolonged calving intervals due to delayed postpartum estrus intervals [76-81]. An important factor appears to be the season at calving [81,82]. Buffaloes calving during the end of breeding season often suffer from prolonged postpartum anestrus due to effects of heat stress and prolonged photoperiod coupled with poor nutrition and management [,83]. The effects of photoperiod and heat stress often override the effects of better nutrition and management in countries away from the Equator [21]. The first postpartum ovulation is frequently followed by one or more short estrous cycles (≤ 18 days) [80]. Long anovulatory and anestrus periods due to a prolonged inter-luteal phase were reported to occur after short cycles. Also, long anestrus periods due to the cessation of cyclic activity (true anestrus) for three weeks or more and prolonged luteal activity for 28 days or more are reported in buffaloes after the first or second ovulation postpartum [83]. These cyclic irregularities present difficulties in estrus detection programs in postpartum buffaloes. Based on transrectal palpation of ovaries of 697 buffaloes, the first ovulation and first estrus averaged about 57.5 days and 78.5 days, respectively [79,80] where as in suckled buffaloes, the first ovulation and the first estrus was reported at 52 days and at 56.5 days in 73 animals, respectively [84,85]. The frequency of estrus at different intervals postpartum in buffaloes from Egypt has been reported with 34% presenting estrus within 90 days (n=883), 24% presenting estrus between 90-150 days (n=640) and 42% presenting estrus in more than 150 days (n=1114) [83]. The data reported from Pakistan regarding postpartum resumption of estrus was 49%, 20%, 31% in less than 90 days, 90-150 days and more than 150 days in 683, 290 and 437 animals, respectively [83]. In Swamp suckled buffaloes the first ovulation and the first estrus occurred in 127 days and 113 days based on progesterone hormone estimation [83]. In Swamp buffalo, follicular activity prior to first postpartum ovulation is characterized by a wave-like pattern, however, the maximum diameter of the follicle in the second, third and fourth cycle did not differ while that of the first ovulatory follicle was significantly smaller(14.31, 14.17, 14.0 vs 13.50 mm) [86].

2.1 Physiological Mechanisms Controlling Postpartum Anestrus

The hormonal and metabolic signals regulating the onset of postpartum estrus in buffaloes appear to be slightly different from cattle as these cues are often affected by environmental factors (Fig. 1). The elevated progesterone in pregnant buffaloes exerts a negative feedback effect on hypothalamic-pituitary axis leading to cessation of estrous cycles [87]. The follicular growth sequentially decreases with the increasing gestation period. The number of follicles (0-6 mm, >10 mm) on buffalo ovaries were lower during the 3-6 months of pregnancy compared to those during 1-3 months of pregnancy [88]. Plasma progesterone profiles are known to decline from 276-278 days of gestation to reach minimum levels on the day of parturition [89]. During the first two weeks postpartum decreased progesterone probably initiates GnRH secretion. The LH secretion is initiated by Day 16-29 postpartum in the river [89,90] and Day 30 in swamp buffaloes [90]. A substantial rise in FSH postpartum occurs at around Day 20 [91,92], however, it has been mentioned that FSH does not appear to be a limiting factor for resumption of postpartum follicular activity in the buffalo [90]. It appears that the capability of pituitary response to exogenous GnRH is restored by Day 20 postpartum in dairy buffaloes [92], however, Perera [93] state that the first postpartum ovulation is frequently followed by one or more short estrous cycles (<18 days) and cessation of estrous cyclicity after the first or second ovulation in about 25% of animals due to ovulatory failure or prolonged luteal activity.

Parturient buffaloes reveal less intense negative energy balance probably because of lower milk production [94,95]. Besides suckling, nutrition and management [90], the most important factor affecting the onset of estrus in postpartum buffaloes appears to be the calving season. Buffaloes calving towards the onset of summer, present prolonged anestrus probably due to the thermal stress arising out of poor thermoregulatory mechanisms and hyperprolactinemia [12], which in turn suppresses the secretion of gonadotrophins [96].

Plasma analysis of non-cyclic buffaloes indicated lower plasma concentrations of thyroid stimulating hormone and thyroid hormones compared to cyclic buffaloes. A subtle thyroid activity in lactating buffaloes may have impact on their fertility level [97]. Lower levels of vitamins such as Vitamin D and E might result in a high amount of free radicals affecting follicle growth. Studies confirmed that oral supplementation of 3000 mg of α-tocopherol per week per animal in anestrus buffaloes resulted in a progressive and significant decline in Erythrocytic Malonyl Dialdehyde (MDA) levels [98]. MDA is an index of lipid peroxidation in normal cycling animals. Supplementation of α-tocopherol improved their antioxidant status by alleviating the effects of oxidative stress, thus, the antioxidant status of buffaloes is also one of the factors indirectly controlling anestrus [98]. In another study it was found that the continuous exposure of female buffaloes during postpartum period to the bull, accelerates the resumption of ovarian cyclicity, reduces the incidence of silent ovulation and enhances the first service conception rate [99].

Figure 1. Possible factors for anestrus in a parturient buffalo (Courtesy of Prof. G. N. Purohit, Department of Veterinary Gynaecology and Obstetrics, College of Veterinary and Animal Sciences, Rajasthan University of Veterinary and Animal Sciences, Bikaner, Rajasthan, India.).

2.1.1 Nutrition, Season, Milk Yield, and Postpartum Anestrus

Nutritional deficiencies can affect the duration of postpartum anestrus in buffalo. A shorter postpartum anestrus period was observed for prepartum high energy fed buffaloes compared with those fed low energy [100]. Some authors found that a low plane of prepartum feeding significantly prolonged the acyclic and anestrous periods (53 vs. 75 days, respectively) compared with a high plane of prepartum feeding(42 vs. 66 days, respectively) [101]. Differences in the body condition score of buffaloes at calving and other environmental factors and management practices might account for some of these differences. The type of ration fed during postpartum period (green vs. dry) was reported to influence the length of acyclic and anestrous periods in favor of the green fodder [102].

In several studies, it was found that the season of calving influences the period of postpartum acyclicity in buffaloes. Significantly longer intervals were recorded of 84 ± 10, 47 ± 4 and 47 ± 4 days for hot season calving compared to 40 ± 4, 26± 4 and 36 ± 4 days for cold season calving buffaloes [103,104]. Also longer acyclic periods of 58± 6 days in summer calving buffaloes compared to 25± 4 days for autumn calving buffaloes [105] and 76± 15 days for summer calving buffaloes compared with 31±9 to 39±4 days for other seasons [106] were described in Italy and Egypt, respectively. In Brazil, it was reported that the acyclic period was significantly shorter (78-98 days) in buffaloes calving during January to March (rainy season) than for those calving during December (123 days) [107]. In Cuba, a longer acyclic period was noted in buffaloes calving during the rainy season (58 ±3 days) compared to those calving during the dry season (39 ±2 days) [108].

Temperature / humidity index (THI) plays an important role in the reproductive function of buffaloes and it is suggested that THI has a negative effect on reproductive performances of buffaloes [106].

A negative and non-significant correlation (r= 0.10) was found between daily milk yield and the interval to first postpartum estrus [109]. As regards to total milk yield, increased milk production was associated with lengthening of the interval to first postpartum estrus with a significant correlation between milk production and the appearance of first postpartum estrus [9]. It was also found that total milk yield over 120 days was significantly correlated with the interval to first ovulation (cr=0.34 for milked and 0.88 for suckled buffaloes) [110].

2.1.2 Blood Metabolites

Clinical evaluations revealed that many blood metabolites have an effect on the duration of postpartum anestrus in the buffalo [111]. In cattle, glucose is considered one of the most important metabolic substrates required for proper function of reproductive processes [13]. It has been hypothesized that low blood glucose, as a result of underfeeding and low energy diets, may be linked to reduced progesterone concentrations and lower fertility [112]. In contrast, during early lactation, the levels of glucose and calcium were fairly stable in buffaloes [113,114] and this is also evident by the exceptionally low incidence of milk fever and ketosis in buffaloes [115]. Clinical evaluations found that the blood glucose, total protein and total cholesterol was significantly higher in normal cyclic buffaloes compared to anestrus buffaloes (64.54 ± 1.40 g/dl, 6.65 ± 0.06 mg/dL , 82.15 ± 2.65 mg/dL vs 58.76 ± 1.62 g/dL, 6.37 ± 0.04 mg/dL and 62.87 ± 1.36 mg/dL , respectively) [111]. The micro nutrients, Zn, Fe, Cu, Co were significantly lower in anestrus buffaloes; however, there was no significant difference in the level of Mn and iodine [111]. The optimum ratio of serum Ca: P should be near 2: 1 for better estrus expression and conception [117]. In another study, it was found that there was significant reduction (p= 0.01) in the total cholesterol and phosphorus levels in anestrus buffaloes [118]. However, blood glucose, HDL cholesterol, triglycerides, Ca, Cu, Co, Mn and Zn levels did not differ between anestrus and regular cyclic buffaloes [118]. A significant role appears to be played by the levels of circulating plasma prolactin (PRL) in buffaloes. PRL was significantly higher in buffaloes that did not resume cyclicity within 90 days of calving compared to buffaloes that did regained cyclicity within the same period [96]. PRL did not show a significant correlation with plasma glucose and non-esterified fatty acids [96].

2.1.3 Insulin, IGF-I

A number of studies have shown the effect of insulin on reproduction, i.e. follicular growth is mediated through insulin [119-121]. Insulin has mitogenic effects at a lower concentration of 50 ng/ml for buffalo granulosa cells and also stimulates progesterone secretion at an approximately physiological concentration of 10 ng/ml and higher [122]. Moreover, insulin may affect synthesis and secretion of IGF-I, which also play a role in follicular development and implantation [123]. Insulin like growth factors (IGF-I and IGF-II) are expressed in embryos and in the reproductive tracts of several species. They are mitogenic and have endocrine, paracrine and autocrine function stimulating cell division, blastocyst formation, implantation and embryo growth resulting in a higher number of offsprings [123]. Clearly, the plane of nutrition can change the concentrations of IGF-1 and IGF binding proteins (IGFBP). Reduced nutrient intake resulted in decreased concentrations of IGF-I and plasma LH [123]. However, it has been mentioned that the levels of insulin are higher in buffaloes compared to cattle [124] suggesting slightly different roles in buffalo compared to cattle.

2.1.4 Leptin and Neurohormones

Leptin, is a 16-kD product of the ob-gene, and plays a major role in communicating nutritional status to the central nervous system and the hypothalamic-pituitary-gonadal reproductive axis of mammals [3,34]. The expression and secretion of leptin is highly correlated with body fat mass and acutely affected by changes in feed intake [3]. Moreover, exogenous leptin stimulated the secretion of LH in fasted, but not in normal-fed cows [125]. Research appears to indicate that leptin stimulates the hypothalamic–adenohypophyseal axis mainly in nutritionally stressed animals [3]. Assuming that cows are metabolically stressed during the early postpartum period and that cows lose body fat during this period, it is possible that leptin is one of the mediators of nutritional status to the hypothalamic-pituitary axis and hence may influence the duration of nutritional postpartum anestrus [13]. As already mentioned, buffalo leptins are similar to cattle leptins [36,37] and mediate steroidogenesis [38] therefore, the effects of leptins in buffaloes might be similar to cattle.

2.1.5 Suckling and Postpartum Anestrus

Suckling significantly prolonged the postpartum anestrus detected by either rectal palpation or progesterone assay in buffaloes [110,126,127]. A few authors reported a non-significant influence of suckling on postpartum acyclicity [128,129]. However, other studies [130,131] also mentioned that suckling significantly prolonged the postpartum anestrous period (75 ± 24 vs. 60 ± 25 and 59 ± 3 vs 29 ± 2 ) days , respectively. The prolongation of the postpartum anestrus period is due to failure of follicular development apparently due to a lack of LH [128]. Resumption of LH pulsatility is limited by the depletion of anterior pituitary LH stores during the early postpartum period (calving to 20 days postpartum), while it is suppressed during days 20-35 postpartum due to suckling [7].

Suckling also significantly prolonged the postpartum acyclic and anestrus periods in Swamp buffaloes. The interval to the first ovulation was significantly longer in free suckled than in restricted suckled Swamp buffaloes [85,132]. In another report, it was found that weaning at 30 days (17-32 days) postpartum induced estrus by 42 ± 8 days compared with 55± 10 days for suckled buffaloes. Temporary calf removal for 72 h in acyclic buffaloes 91-93 days postpartum induced ovarian cyclicity about 14 days earlier than in suckled animals [85]. It has been hypothesized that suckling induced suppression of GnRH/LH is mediated through a complex neuroendocrine system involving endogenous opioid peptides, i.e. β-endorphin in cattle [117,86]. Management practices [133] suggested to reduce the anestrus period include but are not limited to a) temporary calf removal (48-h calf removal) which should be combined with some method of estrus synchronization; b) once-daily suckling, which appears to be more beneficial with first-calf heifers [13]. In managing postpartum anestrus, all the other management alternatives should be considered and applied with before resorting to temporarily weaning the female [134].

2.1.6 Pregnancy

Pregnancy is a physiologic reason for anestrus. The secretion of progesterone by a functional CL suppresses estrus and ovulation. For 598 buffaloes, submitted for infertility problems, 16.38% were pregnant [135] and similarly for 155 buffaloes examined gynecologically, 15% were pregnant [136]. Thus buffaloes in anestrus should first be examined for pregnancy. The persistence of the CL without pregnancy is another physiologic reason for anestrus and many buffaloes exhibit this problem [135]. The reasons for such persistence are poorly understood, embryonic deaths are one possibility. A small proportion (13-14%) of pregnant buffaloes shows gestational estrus [137,138] one or more times between 22-309 days of gestation [137]. Buffaloes presented for AI should be carefully examined to rule out gestation before inseminating them

2.2 Sub-estrus (Silent Estrus)

Poor estrus expression is a reproductive problem inherent to buffaloes [139]. The problem is more evident during hot summer months [2,81,82,140] and during the first postpartum ovulation [93,130]. The overall incidence of sub-estrus varies from 7.2% to 70.6% [2,79,101,136,141-146]. The etiology of silent estrus appears to be complex and 37.2-46.2% of buffaloes with good body condition score show sub-estrus throughout the year reflecting a genetic predisposition [147]. Suckled postpartum buffaloes revealed a higher incidence of silent estrus compared to weaned buffaloes [125,148]. Hormonal evaluations in sub-estrus buffaloes revealed lower circulating FSH and progesterone [149,150], higher inhibin [151] and lower tri-iodothyronine and thyroxine [152]. Similarly lower plasma calcium and phosphorous were observed in sub-estrus buffaloes [153]. Lower FSH and higher inhibin, result in a slower growth of follicles and smaller diameter of ovulatory follicles [154] which probably fail to produce the sufficient amount of estradiol necessary to trigger the overt expression of estrus. The altered hormone secretion also appears to result from higher prolactin arising in response to high environmental temperatures [96] and prolonged photoperiods. The exposure of females to males appears to play some role in the overt expression of estrus, since silent estrus was lower (57.14%) in bull exposed postpartum buffaloes compared to non-bull exposed buffaloes (85.71%) [155].

The diagnosis of sub-estrus can be made by regular transrectal palpation and transrectal ultrasonography, however, milk progesterone assays validates ovulation and CL formation [156-158]. Plasma progesterone profiles can also be utilized for evaluation of CL function [159].

The therapy for sub-estrus involves the IM administration of prostaglandins [160,161], norgestomet + eCG [162]. The efficacy of a single IM administration of GnRH have been low [163] in buffaloes. Estrus synchronization approaches utilizing progesterone vaginal implants (CIDR) [164,165] or Ovsynch protocols [165] resulted in synchronized ovulation and pregnancy rates varying from 30% to 66.7% in sub estrus buffaloes during the low breeding season [164,165]. Intrauterine infusion of Lugol’s iodine (1:30) (50 ml) has been mentioned for therapy [166] but such therapies are neither used routinely nor suggested because of potential dangers or erosion of uterine endometrium. A combined strategy of improving the environment, nutrition and management is a prerequisite for hormonal manipulation in order to improve the conception rates during the summer anestrus and in sub-estrus buffaloes [167].

2.3 Pathologic Anestrus

Uterine pathologies such as metritis, endometritis and pyometra are frequent pathologic causes of anestrus although conditions such as fetal mummification, although rare, can produce the same result [168]. Ovarian luteal cysts are an uncommon cause of anestrus in the buffalo [169]. Metritis has a significant effect on the service period [170]. The inflamed endometrium is unable to secrete prostaglandins in a sufficient amount and that may be the reason why the animal remains in anestrus. Similar changes are more likely with pyometra wherein the uterus is filled with pus. The incidence of pyometra recorded for buffaloes varies from 4.11 to 7.56% [171,172]. The conditions mentioned above have been described in a previous chapter [173].

2.4 Therapies Adopted for Postpartum Anestrus in Buffaloes in Different Studies

Postpartum anestrus is the most critical period affecting the total economics of the livestock industry. Usually this period is prolonged in buffaloes for 30-90 days [93], however, only 45% of Indian buffaloes resume cyclicity within 90 days postpartum and the rest 55% remain in anestrus for about 150 days [90] probably due to combined effects of poor nutrition, seasonal stress and suckling. Many studies have been done in an effort to overcome the problem with various level of success (Table 2). These therapies are based on progesterone, GnRH, PG, bovine follicular fluid and some herbal formulations.

3. Summer Anestrus

Cattle and buffalo express apparent cessation of estrus cyclicity during extreme climatic conditions especially during hot and humid weather in some tropical and subtropical countries. Compared to cattle, buffaloes are more sensitive to such hot and humid conditions owing to their black skin color, a lower number of sweat glands per unit area and lower hair density on the skin resulting into poor thermoregulation that adversely affects normal physiological parameters. Domestic buffaloes have a tendency to breed seasonally showing a suspension of sexual activity during summer in many countries except those close to the Equator [11]. Authors from India and Pakistan attribute the decline of reproductive activity in buffaloes observed during summer to the heat stress [12]. During this period, buffaloes remain sexually inactive without any overt signs of estrus. This condition is popularly known as summer anestrus. Furthermore, a similar condition in Mediterranean buffaloes is observed during spring period and is thus, referred to as ‘spring anestrus’. The incidence of summer anestrus generally varies between 36.6% and 59.5% [191]. Furthermore, summer anestrus was reported to be higher in nomadic buffaloes (83.0%) than in housed rural ones (63.0%) [192], probably because nomadic buffaloes were more exposed to direct sunlight. The condition is characterized by inactive, smooth ovaries [193] and an abnormal hormonal profile. The heat stress causes hyperprolactinemia, reduced luteinizing hormone (LH) secretion, poor follicle maturation and decreased estradiol production in buffaloes [194,195] leading to ovarian inactivity and anestrus.

Seasonality in buffalo reproduction has been reported from Egypt, India, Italy, Pakistan and other countries [3,5,6]. Buffaloes become increasingly influenced by photoperiod as they are further away from the Equator [20,196].

3.1 Factors Responsible for Summer Anestrus

3.1.1 Environmental Factors

High environmental temperature stimulates the peripheral and core receptors to transmit nerve impulses to the specific centers in the hypothalamus to prevent the rise in body temperature. The specific center in the hypothalamus stimulates the evaporative cooling systems, while it suppresses the appetite center. The suppressive impulses transmitted to the appetite center cause a decrease in feed intake. Thus, less substance become available for enzymatic reaction, hormone synthesis and heat production, which help in cooling the body [6]. In tropical and subtropical areas, heat stress is the major constraint in animal productivity [197,198]. The effect of heat stress is aggravated when heat stress is accompanied with high ambient humidity [199]. In cows, heat stress is at the peak by midafternoon and cooling somewhat in the evening and early morning hours. During the day, cows in an unshaded environment have higher rectal temperatures and respiratory rates than shaded cows, but at night both measures were lower for cows with or without shade [200]. Dairy buffaloes maintained on shaded pastures, show a decrease in heart rate, pulse frequency, respiratory rate and rectal temperature, under tropical conditions [201]. When silvo-pastoral systems were used, improvements were described in animal comfort index, during intense rainfall or mild rainfall period. Another important factor determining the reproductive seasonality of buffaloes is the photoperiod [40,202]. Information regarding photoperiod is conveyed to the neuro-endocrine system by the circadian secretion of melatonin from the pineal gland. Melatonin, through its action on the hypothalamus may consequently regulate the seasonal variation in the plasma prolactin levels.

3.1.2 Endocrine Factors

Hormones play a pivotal role in the development of summer anestrus in buffaloes. Prolonged heat exposure suppresses the production of hormone releasing factors from the hypothalamic centers causing reductions in pituitary prolactin, somatotropin, thyrotropin, luteinizing hormone and insulin [195]. The decrease in substrates and hormones and rise in body temperature inhibit the enzymatic activities, which decrease the metabolism and consequently impair milk production, growth and reproduction. A few of the hormones known to be altered with the season in the buffalo [195] are described.

Prolactin

Prolactin is directly associated with the ambient temperature and may mediate the seasonal effects on reproduction in farm animals including buffaloes. Hyperprolactinemia has been proposed to be a possible cause of summer anestrus in buffaloes [12]. It presumably interferes with estrous cycle and fertility by exerting its effect both at hypothalamus [203] as well as at ovarian level [204]. Prolactin may block the hypothalamic mechanism responsible for episodic release of LH or may inhibit the positive feedback of estrogen on LH secretion. Besides, it affects ovarian steroidogenesis by altering the number of LH receptors. A high plasma concentration of prolactin makes the ovaries refractory to the influence of FSH and LH resulting in true anestrus [205]. This leads to anovulatory estrous cycle and consequently poor breeding performance during summer. The mean plasma prolactin concentrations in Murrah buffalo heifers during the winter months varied from 3.10 ± 0.48 to 9.17 ± 1.39 ng/ml whereas during the summer months, the plasma prolactin values were significantly higher and varied from 248.5 ± 16.03 to 369.63 ± 25.13 ng/ml. During summer, buffaloes had significantly low plasma progesterone suggesting the effect of prolactin on the suppression of ovarian activity [206]. Plasma prolactin concentrations on the day of insemination was about 200 ng/ml for primiparous and about 315 ng/ml for multiparous animals and fluctuated between 130 and 200 ng/ml for primiparous and between 250 and 345 ng/ml for multiparous pregnant animals. For the non-pregnant group, prolactin fluctuated between 145 and 240 ng/ml for primiparous and between 210 and 310 ng/ml for multiparous animals with minor elevations 1 to 2 days before estrus [203,204].

Melatonin

The role of melatonin in the regulation of reproductive seasonality is fairly well established in seasonal breeders such as sheep and mares, while only a few studies have been done to clarify the role of this hormone in buffalo reproduction [202,207]. Melatonin is secreted by the pineal gland and its pattern of secretion follows a circadian rhythm with significant levels only during the dark period. A distinct seasonal trend in melatonin secretion has been reported in Italian Mediterranean buffaloes with the highest concentration corresponding to the period of shorter day length [208]. The effects of melatonin administration were negligible in Brazil and 86% buffaloes evidenced cyclical activity during the long photoperiod [208].

Follicle Stimulating Hormone

Buffalo heifers show seasonal changes in the level of circulating FSH with the lowest FSH during hot summer months [209] that coincide with the low breeding period (March to June). In general, FSH levels in anestrus buffaloes remain low, when compared with the basal levels recorded in normal cycling buffaloes. Further, the pre-ovulatory FSH peaks synchronous to LH peaks were also reported to be absent in non-cycling buffaloes during the hot months [150]. The ratio of FSH to LH is lower in hot summer months compared to the peak during the breeding season [149,210].

Luteinizing Hormone

The secretion of LH was lower during the summer months compared to winter months [211]. Low LH results in reduced ovarian follicular activity in buffaloes during summer months [212]. Furthermore, the LH surge was also reported to be absent in anestrus buffaloes during summer [12]. The decrease in LH levels and absence of peaks is attributed to the inhibitory action of progesterone and high prolactin [12].

Thyroid Hormones

Anestrus associated with low thyroid function is common in buffaloes during the summer season [213]. It has been postulated that high ambient temperature leads to hypothyroidism, which results in reduced responsiveness of the ovaries to pituitary gonadotrophins causing summer infertility [214]. Thyroxine (T4) and tri-iodothyronine (T3) are biologically active, and T3 is several times more active than T4. T3 level was found to be at its maximum level in winter, decreasing in spring and dropping to its lowest level during summer in buffaloes. It takes T3, 72 h to drop to its minimum level after heat exposure [215]. The decline inT3 in the heat-stressed buffalo may be responsible for the decline in milk components during the hot months in Egypt [216] and the decline in daily body weight gain with elevated temperatures [217]. Thyroid stimulating hormones and thyroid hormones were lower in anestrus and non-pregnant buffaloes compared to cyclic or pregnant buffaloes [218].

Ovarian Hormones

The low reproductive efficiency in summer has been attributed to low luteal activity as anestrus buffaloes had progesterone levels below 1ng/ml. The peak P4 concentration was found to be much lower during the summer months (2.27 ng/ml) than during the winter months (3.96 ng/ml) [219].

Estradiol concentration is reported to be low in anestrus rural buffaloes during summer [220]. Since the expression of estrus and secretion of LH require an appropriate balance between estradiol and progesterone, the lower progesterone level during the hotter months in comparison to the cooler months is likely to be responsible for the inadequate expression of estrus during this period [194].

Cortisol

Blood cortisol levels increased due to an increase in ambient temperature from February to July. Cortisol values were 9.07 ng/ml and 12.53 ng/ml during February and July, respectively, in Egyptian buffaloes [211]. Exposure of non-pregnant female buffaloes to 2-3 h of solar radiation at 42.1°C increased plasma cortisol concentration rapidly for 30 minutes, followed by a gradual fall [221]. Increased level of corticosteroid secretion is known to inhibit GnRH and thus LH secretion [222] in cows. It has also been mentioned that when heat stress is prolonged it is likely that the secretion of gonadotrophins will be suppressed and reproduction will be inhibited [223]. However, cortisol appears to be one identified molecule and the effects of heat stress on the physiology of cattle [224] and presumably buffalo appear to be complex and partially understood.

3.1.3 Nutritional Factors

In general, anestrus is often attributed to nutritional factors in bovines. Buffaloes remain underfed due to poor availability of nutrients particularly protein, as tropical forages get lignified during summer months. Under such conditions, the quantity of consumed nutrients declines, dry matter intake including crude protein also declines and a negative nitrogen balance may occur [225]. Dry matter digestibility and protein / energy ratio were also found to decrease in heat stressed heifers [226]. Digestion and metabolism of non-pregnant buffaloes declined with exposure of 2-3 h to solar radiation at air temperature of 42°C [215]. In lactating Murrah buffaloes, digestibility coefficient value for each of dry matter and crude protein were significantly lower in summer (43.0 and 50.50 ± 0.7) than in winter (68.31 and 66.83 ± 0.05) [227]. Nutritional constraints combined with the reduction in voluntary feed intake that occurs during thermal stress, may cause a loss of body condition and lead to several reproductive limitations.

4. Diagnostic Approaches for Anestrus Buffaloes

Poor estrus detection due to silent ovulation is the most important cause of apparent anestrus in buffaloes; however, the percentage of the true anestrus is also quite high in postpartum buffaloes. Clinical surveys reported that about 60% of cases were true anestrus and 33% of cases were silent ovulation [67]. In another study 45% had inactive ovaries (true anestrus), while 55% had silent ovulation or missed overt estrus [228]. Ovarian cysts were diagnosed in only 5.2% of the cases of anestrus in buffalo [229]. A recent study mentioned four diagnostic approaches for anestrus in buffalo i.e., history, progesterone estimation, transrectal palpation and ultrasonography [9]. Poor estrus expression is common in buffaloes during hot summer months [230] thus sound estrus detection can exclude regular cyclic buffaloes. Estrus detection approaches should consider the body condition score (BCS) especially when applied to heifers [76]. BCS in buffaloes resuming postpartum estrus was constantly higher than those failing to resume ovarian activity [143].

4.1 History

Farmers often present their animals with a history of prolonged anestrus. The clinical history is the first step when suspecting anestrus [111,231,232]. However, the clinical history is not enough to make a diagnosis. Without further examination it is potentially dangerous to diagnose anestrus given that a proportion of buffaloes does not exhibit overt signs of estrus and might be pregnant. It is suggested to perform a transrectal examination with or without ultrasonography before selecting a therapy program. Buffaloes presented with uterine pathologies such as pyometra might be wrongly diagnosed if based only on the clinical history.

4.2 Transrectal Examination

In the past, transrectal examination of the uterus and ovaries [81,82,168,233,234] was the only means of diagnosis of anestrus based on palpation of ovarian structures and uterus. The technique continues to be the simplest approach for diagnosis of anestrus in buffalo [147,232,234-238]. The absence of uterine pathology and failure of palpation of follicles and CL in 2 examinations 10-12 days apart is considered anestrus [228,231]. The limitations of transrectal palpation for the diagnosis of ovarian structures and thus anestrus are i) failure to properly identify small sized follicles, ii) lesser projection of the CL on the buffalo ovary [169].

4.3 Ultrasonography

The ultrasonographic evaluation of ovaries has become increasingly popular for the diagnosis of anestrus in buffalo [147,228,235,237-240]. Two examinations 12 days apart are suggested [216]. Absence of a CL and lack of follicular growth over 2 examinations is considered true anestrus whereas the presence of CL and follicle growth without overt estrus is considered subestrus or silent estrus [228]. As high as 54.5% buffaloes had silent ovulation and 45.5% suffered from true anestrus with ovarian dysfunction [228].Ultrasonography can easily identify pregnancy or uterine pathology, both being possible reasons for anestrus [169]. A buffalo with no follicular activity at all would not show any follicular growth (Fig. 2) whereas a subestrus buffalo or a regular cycling buffalo would show follicular activity with the presence of small sized follicles (Fig. 3), a dominant follicle (Fig. 4) or a CL (Fig. 5). The appearance of these structures represents ovarian cyclicity. In a pregnant buffalo the presence of fetus and fetal fluids depending upon the stage of pregnancy (Fig. 6, Fig. 7) would confirm gestation and discard anestrus. Anestrus buffaloes with uterine pathologies such as a mummified fetus (Fig. 8) or ovarian cysts (Fig. 9) can be identified by ultrasonographic examinations.

Figure 2. Transrectal sonogram of an anestrus buffalo with no follicular growth.

Figure 3. Transrectal sonogram of an anestrus buffalo. A CL with central echogenic lumen and small follicle are visible.

Figure 4. Transrectal sonogram of a subestrus buffalo. A dominant follicle is visible.

Figure 5. Transrectal sonogram of an anestrus buffalo showing a completely echogenic CL.

Figure 6. Transrectal sonogram of a 40 days pregnant buffalo.

Figure 7. Transrectal ultrasonogram of a 4 month pregnant buffalo. The fetal head and the eye orbit are visible in this view.

Figure 8. Transrectal ultrasonogram of a buffalo with mummified fetus.

Figure 9. Transrectal ultrasonogram of a buffalo with a follicular ovarian cyst.

4.4 Hormone Assay

Studies on anestrus buffaloes have shown that they have lower levels of thyroxine [111,241], cortisol [242] and progesterone [111]. Subestrus buffaloes have lower levels of FSH [149,150], thyroxine [243] and progesterone [150] and elevated levels of inhibin [151]. However, the diagnostic significance of these findings is limited to progesterone assay. Progesterone levels continue to increase in animals that conceive but drop 3 days before the next estrus in those that fail to conceive [244]. Peripheral P4 concentrations may change during the seasons, lower P4 levels at estrus as well as during the mid-luteal phase in hotter (0.14±0.05 and 2.05±1.16 ng/ml, respectively) than during the cooler months (0.49±0.06 and 3.11±0.20 ng/ml, respectively) are believed to be responsible for the poor expression of estrus and low conception rate during summer [245]. Progesterone concentrations of ovarian follicular fluid have also been reported to be lowest during the monsoon season (Jul. to Oct.) and highest during summer (Mar. to Jun.) [246]. Studies on milk progesterone profiles [153,247] or serum/plasma progesterone [104,111,147,231,238,240] in buffaloes have shown that such evaluations are an useful indicator of ovarian function when performed at appropriate time intervals. Regular monitoring of milk progesterone is precise enough to detect buffaloes returning to estrus (progesterone declining below 1 ng/ml). A subsequent rise in progesterone (above 1 ng/ml) suggests ovulation and a functional CL. Thus buffaloes with silent estrus can be identified by monitoring progesterone levels (progesterone above 1ng/ml), however, a single measurement is not sufficient [156,158]. True anestrus buffaloes usually show consistent low progesterone (0.46±0.11 ng/ml) [111]. Progesterone estimations can validate the functionality of the CL observed by transrectal ultrasonography or palpated by transrectal palpation [169]. Low progesterone (below 0.50 ng/ml) in two consecutive samples collected at weekly intervals is considered as true anestrus [231]. Regular monitoring of postpartum Bulgarian Murrah buffaloes at 3 day intervals revealed that progesterone peaked at 19, 34 and 50 days postpartum in buffaloes that subsequently became pregnant whereas progesterone remained below 0.25 ng/ml up to Day 50 postpartum in buffaloes that did not become pregnant [240].

4.5 Biochemical Assays

Many studies have mentioned lower profiles of blood glucose, protein, cholesterol and serum calcium, phosphorous, magnesium, zinc and copper [111,248-254] in anestrus buffaloes; however, their diagnostic significance is limited to the evaluation of the nutritional status of buffalo herds for supplementation

5. Management of Summer Anestrus

Summer breeding of buffaloes can be successfully carried out by changing farm management practices. Protection from direct solar radiation is the principle of real management in the buffaloes during hot summer months [255]. Showering of water with air circulation can be helpful in reducing thermal stress.

5.1 Thermal Amelioration Measures

Provision of shade, housing system with sufficient space and application of water to the body surface by sprinkling/washing or providing wallowing facilities (ponds, swimming pools, etc.) during hottest parts of the day during summer can reduce the heat stress considerably, alleviating the adverse effects on buffalo fertility [255-258]. Proper extensive housing system helps in reducing the adverse effects of thermal stress on buffalo fertility [259]. Showering of buffaloes with water (2-4 times a day) during hot summer months (May and June) resulted in 83.33% of adult buffaloes and 57.8% of buffalo heifers showing estrus with 30% and 67.57% conception rates [260].

Air Movement

Apart from shifting animal to shaded airy place, fans or coolers should be installed for making the place airy. Air movement increases the rate of heat loss from the animal’s body surface as long as the air temperature is lower than the animal’s skin temperature. Water mist is another alternative to repeated sprinkling of water. Provision of sprinkling water and fan resulted in a comfortable environment for buffaloes during summer as evident by lower cortisol and higher blood glucose, protein, albumin, sodium and potassium in sprinkled buffaloes compared to values in buffaloes that were not provided sprinkling and fan during the same period [261].

5.2 Increasing Efficiency of Heat Detection

Improving estrus detection methods is one of the main factors that decrease the calving-conception interval of buffalo during the hot season of the year. The use of teaser bulls, tail head paint, the heat watch system, radio-telemetric pressure transducers and pedometers can improve estrus detection and translate into better fertility [255].

5.3 Nutritional Management

Feeding green fodder/silage/hay, provision for night feeding, grazing only in the morning and late in the afternoon will all help in reducing the excessive heat load [258].

Although the metabolic energy of buffalo increases in a hot environment, heat stress depresses feed intake [6], digestion [262] and nitrogen retention [263]. For this reason, it is important to increase the energy content of the diet in order to improve their energy intake under hot conditions. Fatty feed and calcium salts of fatty acids can be used as means of improving energy supply for buffaloes during summer [195]. Protein requirement increases and dry matter intake decreases in a hot environment, consequently, the protein supplied to lactating buffaloes during summer is not always sufficient [227]. By using fishmeal, which is bypass protein, fertility and milk yield can be maintained [264].

5.4 Hormonal Treatments

Various hormonal treatment regimens are being used to overcome summer anestrus. These treatments are primarily aimed either at stimulating ovarian activity, inducing/synchronizing behavioral estrus or controlling ovulation. A combined strategy with improved environment, nutrition and management is a prerequisite for hormonal management in order to achieve optimum results [167]. Progesterone-based treatment (PRID, CIDR, CRESTAR, progesterone injections) either alone [254] or in combination with gonadotrophins proved to be very effective in inducing ovarian activity in summer anestrus buffaloes (Table 3). The possible mechanism involved in the treatment with PRID seems to be the initiation of follicle turnover by increased serum progesterone before PRID removal. It is further hypothesized that the higher progesterone level in the blood sensitizes the hypothalamus and pituitary to the gonadal feedback. PRID in association with pregnant mare serum gonadotropin (PMSG) yielded better results than PRID alone, both in terms of estrus induction and conception (65.3% and 54.5 %, respectively) [265]. Another alternative is fixed time artificial insemination using PGF2α or its analogues [266]. The estrus induction rate achieved with this regimen was reported to be high with a relatively poor response in terms of conception (13.8%, [267]; 36% [266]) attributed to the difference in the interval between PGF2α administration and the commencement of estrus and ovulation [268,269]. Gonadotrophin releasing hormone and its analogues were also tested in an attempt to induce resumption of estrous cycle during summer in buffaloes with varying success i.e. 60-75 % conception rate [270,271]. Clomiphene citrate has also been used for treatment with a wide range of response (25-84 % conception [272,273]). Approaches using other drugs such as melatonin, bromocriptine and antioxidants have been used with varied success rates, 100, 56.2 and 22.2 % estrus induction, respectively [274-276]. Various nutritional supplements besides hormonal therapies were also used to overcome the negative effect of summer anestrus with variable success rates (Table 3).