Repeat Breeding in Buffaloes

Failure of pregnancy establishment referred to as repeat breeding (RB) is one of the biggest reproductive problems in female buffaloes [1-6], however; compared to cattle the incidence of repeat breeding in the buffalo is low (8.68% vs 18.79%) [7,8]. Pregnancy failure in buffaloes is common during the hot summer months [9-15]. The problem is exacerbated by poor nutrition [16-19], poor management [17,20-22] and poor housing [23-25]. Failure of buffaloes to conceive with 3 consecutive artificial inseminations (AI) or natural matings during the breeding season, is considered as repeat breeding [8,26,27]. In a recent review, spring and winter calvings, first parity, periparturient disease and lactation were considered as significant risk factors for RB in buffaloes [28]. The etiology of RB appears to be multifactorial and has been recently described under the headings of failure of fertilization and early embryonic deaths [28]. Clinical evaluations mention that endometritis [1,5-7,26,27,29,30] is the most frequent cause of failure of fertilization in RB buffaloes, whereas improper insemination and poor semen quality are the bull-side factors for fertilization failure in RB buffaloes [28]. Early embryonic deaths in buffaloes originate due to low luteal progesterone [31] and seasonal influence on corpus luteum (CL) development [32].

The exact diagnosis of failure of fertilization appears difficult yet the evaluation of females for pathologies of the tubular genital tract (such as endometritis) and ovaries (ovarian cysts, delayed ovulation) as well as the external genitalia is possible via visual inspection, transrectal palpation and transrectal ultrasonography [33]. In male buffaloes, semen is evaluated. The diagnosis of early embryonic deaths is possible by serial ultrasonographic evaluations of the embryonic vesicle from Day 20 to Day 45 as most embryonic deaths in buffaloes occur after Day 25 [34]. Plasma progesterone profiles can be an additional tool for evaluating embryonic deaths [35]. Therapeutic approaches for RB in buffaloes involves improved uterine health, correcting ovarian dysfunction/hypofunction and improving CL function; also important is the improvement in management and insemination procedures. In this chapter, the authors have addressed the incidence, risk factors, etiology, diagnosis and therapy for RB in buffaloes.

Incidence

Compared to cattle, the overall incidence of RB appears to be low in buffalo [7,8]. Two recent reviews analyzed the incidence of RB in buffaloes (from different reports), and found that the incidence varied from 0.32% to 55.4% in one study [36] and from 0.70% to 30.0% in the second study [28], however, these evaluations do not reflect the incidence of RB in buffalo herds. Evaluation of 1826 and 2318 lactation records from two Murrah buffalo herds (NDRI, Karnal and Military Dairy Farm Ambala, India) revealed that the incidence of RB was 20% in the NDRI herd where only AI was used for breeding whereas the incidence was only 5% at the military dairy farm where only natural service was used for breeding [37]. These results suggest that perhaps the failure to conceive was due to the AI technique used and to human factors. The incidence was higher in first calvers (heifers) compared to adult buffaloes in both herds. The heritability (0.092±0.14) and repeatability (0.11) was low for RB suggesting that RB is more affected by environmental and management factors [37]. In a field survey, the incidence of RB was highest in buffaloes kept by poor landless farmers [38] suggesting that perhaps the role of nutrition is important in RB in buffaloes.

Risk Factors

Similar to the analysis of risk factors for RB in dairy cows [39-41], a recent review analyzed the risk factors for RB in buffaloes [28]. Spring and winter calving, first parity, periparturient disease and lactation were considered significant risks for RB in buffaloes [28]. Autumn revealed the highest incidence in Murrah buffaloes in a recent study [42]. First parity buffaloes had a longer period to conception [15] and similar findings were recorded in another study [43], however, the incidence of RB was highest in the 2nd and 3rd parity in another study [38]. Shahzada et al., [44] found the highest incidence of RB in 3rd parity buffaloes and the lowest in 6th parity buffaloes and buffalo heifers. The maximum incidence of RB was recorded in heifers of Murrah and crossbred buffaloes in Nepal [27] and in Nili-Ravi buffaloes in Pakistan [45]. Rehman et al., [46] found a non-significant effect of parity on RB in buffaloes. Difficult births, postpartum metritis and endometritis significantly increased the services per conception in buffaloes [47,48]. In an analysis of 6064 calving from 10 farms, the maximum incidence of RB in buffaloes was observed during the dry hot summer months (20.62%) compared to the hot humid summer months (18.59%), autumn (12.66%) and winter (12.06%) [49]. Although it is known that there is a decline in fertility during the hot summer season [15], most buffaloes do not show estrus during this time, therefore the interpretation of RB should be done carefully. The conception rate (CR) with frozen semen in China in Murrah, Nili-Ravi and local buffaloes was 45.31%, 52.08% and 48.08% respectively [50]. The CR at organized buffalo farms in India is not very high: for Murrah and Nili-Ravi buffaloes the CR was 51.22% and 34.16% respectively [51]. The number of services per conception for Nili-Ravi buffaloes in Pakistan was 1.56±0.01 in one study that evaluated 9300 records [52] and 1.9±0.13 in another study that evaluated 451 records [15]. The highest number of services per conception (2.36±0.31) was recorded for fifth parity buffaloes [15]. In a study involving 2989 buffaloes from 518 farmers in 9 villages, the number of services per conception was 2.06±0.05 [53]. Therefore, it appears that the number of services per conception and hence RB is affected by the type of management. Differential fertility can also result from the buffalo bulls used [55,56]. As mentioned earlier [37] RB was commonly seen in buffaloes bred by AI and thus human factors should also be considered. CR can be affected by many confounding variables as explained previously [57,58].

Etiology

Recently the etiologies for RB have been classified under two headings: failure of fertilization and early embryonic deaths [28]. Failure of fertilization can occur due to problems with either the female or the male and poor breeding management. Female factors for fertilization failure include anatomic defects of the genital tract, infections of the genital tract, ovulatory disturbances and improper timing of insemination. The male factors include poor quality of semen, improper semen handling, infectious diseases and poor breeding management. Early embryonic deaths can result from defective function of the CL, infectious disease affecting the embryo and stress.

Fertilization Failure

Causes of failure of fertilization have been described in buffaloes recently [28], and include problems with the female, the bull (or the semen used in AI) and the breeding management often with overlapping effects.

Female Buffaloes

Failure of fertilization in female buffaloes can occur on account of congenital or acquired anatomic defects [5], ovulatory disturbances [59], genital infections such as endometritis [60,61] and defects of ova.

Anatomic defects have been described from abattoir [62,63] and clinical [5,30] studies in buffaloes. Anatomic defects present morphologic changes that prevent fertilization due to obstruction in the passage of the gametes. In one study, anatomic defects accounted for 10% and 9.4% cases of infertility in nondescript and Murrah-grade buffaloes [64]. The common anatomic defects found were poor genital development, ovario-bursal adhesions, uterine growths and oviductal lesions obstructing the lumen (Table 1). Some of the congenital defects of the genitalia described included double cervix, uterus didelphys and oviductal constrictions [62]. The most common acquired anatomic defect appears to be ovario-bursal adhesions which prevent the passage of ova into the oviduct. The incidence of this condition varies widely in different studies (Table 1). Another important genital anatomic defect reported in abattoir and clinical studies is the poor development of the genital tract (Table 1). Hemangioma was recorded in the uterus of a buffalo [65] and uterine tumor or abscesses were also recorded which can obliterate the uterine lumen preventing fertilization. The incidence of malformations of the cervix (kinked, abnormal shape) varies from 0.28% to 6.45% (Table 1). Oviductal pathologies that may be acquired or congenital were reviewed recently [59] and these include adhesions (1.5%-1.7%), congenital defects (0.2%), occlusions (0.29%-37.8%) and infectious conditions of the oviduct (salpingitis, hydrosalpinx, pyosalpinx). The overall incidence of oviductal abnormalities was 25.2 % in another study [66].

Ovulatory defects that can result in fertilization failure include delayed ovulation, anovulation and ovarian cysts [59,75,76]. However, clinical conditions involving delayed ovulation, ovarian cysts and anovulation are infrequent in buffalo [5,67,77,78]. The incidence of delayed ovulation and anovulation were 16.56% and 15.09% in one study [76]. Purohit [59] reviewed the various reports on ovarian abnormalities in buffaloes and found the incidence of ovarian cysts to range between 0.07% and 13.44%, for Parovarian cysts 0.19%-7.9% and for ovarian sclerosis 0.04%-2.47%. These conditions can cause failure of fertilization due to poor ovarian function.

The most common genital affection in buffalo that leads to failure of fertilization is endometritis [60,79-82]. Endometritis was significantly higher in 2nd parity buffaloes [83]. Endometritis is associated with accumulation of inflammatory debris in the uterus [84], increased oxidative stress [85,86] and increased polymorphonuclear leucocytes in the uterine lumen [26,87-90]. Endometritis results in a uterine environment unfavorable to the spermatozoa and the embryo. The endometrial morphological changes such as denudation of endometrial epithelium, and scanty uterine glands in buffaloes with endometritis [89,91,92] might prevent embryonic implantation and result in RB. Moreover clinical endometritis results in an alkaline pH that is detrimental to sperm and embryos [8]. The season of calving had a significant effect on the development of endometritis in buffaloes [93]. Significant association between uterine health and fertility was recorded in Murrah buffaloes [42]. Endometritis had a highly significant effect on all reproduction traits [94]. The most common predisposing factor for endometritis in buffaloes was retained placenta and the most prevalent bacteria in the uterine lumen of affected buffaloes were E. coli (13%), Arcanobacter pyogenes (13%) and Staphylococcus aureus (10%)[26,60]. The xanthine oxidase activity was significantly enhanced in postpartum Murrah buffaloes with bacterial infections, endometritis and mastitis compared to healthy buffaloes [95].

Buffalo Bull

Buffalo sires are known to affect pregnancy rates both in natural service and AI. The CR with frozen semen AI from different buffalo bulls varied from 17% to 84% [55]. Similarly fertility was known to vary between buffalo bulls in other studies [56,96-98]. Moreover the age of the bull and the season when semen was collected, affects the fertility with adult buffalo bulls producing the best quality semen during cooler months [57,94,99,100].

Breeding Management

Poor overt estrus expression in buffaloes [101] poses logistic problems for timing AI thus resulting in poor conception and RB. While this has been difficult to correct, other related problems such as time of insemination need attention. A recent review suggested that buffaloes should be inseminated 18 to 24 h after the onset of estrus as ovulation occurs relatively late (30 h) [102]. Similar suggestions were also mentioned in a previous study where buffaloes inseminated 18-24 h after estrus onset resulted in highest CR [103]. In another study when buffaloes were inseminated between 23-26 h, CR was 50% whereas CR was 40% when buffaloes were inseminated between 19-22 h after the onset of estrus [104]. When using frozen semen, sperm survival in the female tract was shown to be 24 h [105]. It has been shown by evaluation of plasma progesterone concentrations at the time of AI that more than 30% of buffaloes were inseminated at the wrong time [106]. Similarly in another study 26 RB buffaloes revealed plasma progesterone values of <1 nmoL/L and only 4 of these conceived [107]. These studies show that inseminating buffaloes at the wrong time increases conception failures and results in RB. Inseminator efficiency also affects the CR. CR was 31.57% in buffaloes inseminated artificially by veterinarians compared to 25.97% for buffaloes inseminated by technicians [103]. Significant variations were observed for CR in buffaloes inseminated by different inseminators [57]. The distance from the AI center to the farms appears to be important. As the distance from the AI center to the farm increased, the CR decreased [108]. Also, only 9.6% of rural farmers bred their buffaloes with AI and the rest preferred natural mating with buffalo bulls [22]. The buffalo herd size in rural areas in India in one study was only 1-5 animals with poor housing [20,22].

Early Embryonic Death

Early embryonic deaths (2-3 weeks) [109,110] account for 45% of pregnancy failures in dairy cows [111]. Luteal insufficiency (lower production of progesterone by the CL) appears to be the most common cause of early embryonic death in dairy cows [112] and buffaloes [35]. Optimal progesterone appears necessary for endometrial and embryonic signal exchanges necessary for maternal recognition of pregnancy and implantation [113]. High producing dairy cows have an increased metabolic clearance of progesterone with less availability for pregnancy [110]. Environmental or shipping stress [114] and presence of pathogenic microbes in the uterus are other causes of early embryonic death [115].

In buffaloes, embryonic death has been mentioned to occur between Day 25 to Day 50 of gestation [9,31,32,34,116]. In a study of 145 buffaloes examined ultrasonographically once a week from Day 23 to Day 50, ten (6.9%) embryonic deaths were recorded between Day 30-Day 36, four between Day 37-43 and three between Day 44 and Day 50 [117]. For buffaloes naturally mated during the periods of increasing daylight length embryonic death (8.8% to 21.8%) occurred between Day 28 to Day 60 [31,118,119] on account of low luteal progesterone and poor CL development [32,35,120]. Low luteal progesterone during summer months can originate due to high prolactin secretion that suppresses progesterone secretion [121] and disturbances in LH secretion [122]. It has been proposed that the late embryonic death in buffalo results from failure of the embryo to attach [35,123]. Two recent studies found that embryos undergoing mortality have retarded growth on Day 25 after mating and show altered proteomic profiles, gene expression and some deregulated functions that were associated with attachment to the uterine endometrium [123,124]. Changes in sera proteomes were also recorded during early pregnancy in buffalo in another recent study [125]. It thus appears that embryonic death in buffaloes probably occurs due to failure of implantation mechanisms. Embryonic death in buffaloes inseminated following an estrus synchronization program, during periods of declining reproductive function, is higher (20% to 40%) partly due to suboptimal CL function probably due to stress of hot summer months and high prolactin [9,34,126]. Embryonic death was not affected by age, parity or lactation of the dam [9,126]. Heat stress can contribute to embryonic death; rearing systems that provided adequate cooling during the hot summer months had significantly lower embryonic deaths [25].

Diagnostic Approaches

When RB is a herd problem, breeding management should be reviewed, as well as the buffalo bull and check for infectious diseases [8]. Before evaluating pregnancy failures in individual animals, the semen and the AI technique used should be evaluated. Fertilization failures often occur on account of pathologies in the genital tract and dysfunction of the ovaries [28].

A combination of visual estimates, transrectal palpation and transrectal ultrasonography are suggested for such evaluations [8,28]. More specialized techniques such as hysteroscopy [127,128], metabolic profiles, uterine biopsies and tubal patency testing suggested previously [8] should be reserved for pathologies in the genital tract not easily traceable. Ovarian and oviductal pathologies such as tumors, pyosalpinx and oviductal obstructions are often not detectable with routine techniques. Failure to regain fertility following medical or surgical therapy often limits their use [59] and the value of the affected animal.

Evaluating Genital Health

Visual inspection of cervico-vaginal mucus is often the first diagnostic method for finding infections in the genital tract of cows, however, due to the lower quantity of mucus secretions during estrus in buffaloes [129] and the tendency of buffaloes to show estrus during the night hours [102], visual appraisal of cervico-vaginal mucus appears difficult in the buffalo [28]. Manual massaging of the genital tract (uterus) is suggested for collection of cervico-vaginal mucus [130]. Alternatively, mucus can be retrieved from the vagina using sterile glass pipettes [131]. Cervico-vaginal mucus has been studied in terms of viscosity, and thin and thick consistency [130,132]. Mucus was found to be viscous in 38.33%, thin in 50.0% and thick in 11.67% of the buffaloes [130]. The percentage of conception was higher among buffaloes having thin cervical mucus consistency than those with a viscous or thick consistency [43,130,132]. Clear thin or stringy mucus showing typical fern pattern resulted in maximum RB buffaloes to conceive [133,134]. The pH of cervico-vaginal mucus of RB buffaloes was alkaline (8.02+0.11) [43,135]. Bacterial metabolites and inflammatory exudates change the pH towards the basic side [136].

Vaginoscopic examination is helpful in evaluating the health of the vagina and cervix and also of the uterine secretions accumulating in the vagina [26]. Such an examination can also help in diagnosing cervicitis or vaginitis [27,137] or changes in cervical morphology [30] which could be a possible cause of RB especially under natural mating conditions.

Transrectal palpation of the genital tract can determine gross enlargements, strictures and adhesions of the genital tract or ovarobursal adhesions [84] and the findings can be evaluated further by using transrectal ultrasonography [33].

A wide variety of microbes have been isolated from RB buffaloes including Salmonella, Staphylococcus, Corynebacteria, Pseudomonas and E. coli [90,138-143], however, their significance is important only when samples have been collected properly [8]. Often these organisms are present collectively with anaerobic bacteria like Fusoformis necrophorus and Bacteroids species [61]. Fungi are also sometimes present [144-146]. A high proportion of cervical mucus samples (62.76%) from infertile buffaloes were sterile without any bacteria [136] and E. coli, Staphylococcus aureus, Streptococcus epidermidis and Lactobacillus were considered normal microflora of the uterus [147]. One should not forget that the presence of bacteria in the uterus is common and does not necessarily mean that there is an infectious process under way. The use of uterine biopsies for evaluating endometrial status in endometritis affected buffaloes [65,148] has not become popular in live animals due to the invasive nature of the test. Uterine biopsies from RB buffaloes revealed marked neutrophilic infiltration in the epithelium, stroma and uterine glands [91,140,143,149]. In chronic endometritis, cystic dilatation of the glands and periglandular fibrosis was evident [91,140]. Endometrial biopsy can clearly indicate the future fertility of affected cows [150]. The presence of subclinical endometritis has been identified in buffaloes by indirect cytologic evaluation of uterine fluids collected by uterine lavage or cytobrush which shows increased proportions of neutrophils or polymorphonuclear leucocytes [87,89,90,151]. However, the lack of defined standards has hindered the popularity of this approach in diagnosis of endometritis [28,152]. Ultrasonographic examination of the uterus can confirm endometritis based on increased endometrial thickness, intrauterine fluid accumulation and presence of hyperechoic shades of pus flakes [33,84,152]. Sonographic examination can also identify other uterine pathologies such as hydrometra and mucometra [153] and ovarobursal adhesions [33] that can be a possible cause of RB. Reports on hysteroscopic visualization of abattoir derived bubaline uterus have shown uterine hemorrhages [127,128], however, neither their clinical significance was mentioned nor were the findings validated clinically. Newer markers for detection of endometritis are being developed. In a recent study, buffaloes with endometritis revealed significantly different toll-like receptors (TLR2 and TLR4) suggesting their likely use as detection markers for endometritis [148].

Evaluating Ovarian Function

The precise evaluation of ovarian function such as ovulation and CL formation is only possible with the use of transrectal ultrasonography because of the smaller dimensions of these structures in buffalo [59]. To monitor ovulations, frequent examinations (6-12 h after estrus) are required [8]. In one study, out of 122 transrectal CL diagnoses twelve (9.84%) were false positive and eight (6.55%) were false negative [154]. The greater development of the CL as observed by transrectal ultrasonography on Day 5 post-estrus is related to the increased likelihood of pregnancy in buffalo [155] and additional information on uterine blood flow can be obtained with color Doppler ultrasonography [120]. Compared to cattle, lower luteal function has been mentioned in buffaloes [156], however, plasma progesterone concentrations of greater than 1 ng/mL during the mid-luteal phase (Day 11-15) suggest proper functionality of the CL [157,158]. A problem that arises with the progesterone assay for pregnancy diagnosis in buffaloes appears to be the persistence of a CL without pregnancy, producing higher progesterone concentrations wrongly suggestive of pregnancy [159]. Therefore both ultrasound imaging and monitoring plasma progesterone should be used together to confirm gestation.

Embryonic Deaths

Evaluating embryonic death in buffaloes is possible through two approaches; the decline in progesterone concentrations [34] and the disappearance of the previously visualized fetus and its annexes as examined by transrectal ultrasonography [9,117,126,160,161]. Since most bubaline embryonic deaths occur after Day 25, weekly evaluations starting from Day 23 have been suggested [117]. Diagnosing embryonic deaths is helpful in rebreeding the non-pregnant animals as soon as possible [162]. Two recent studies have mentioned that embryonic width (evaluated by ultrasonography) of >2.7 mm on Day 25 is considered normal whereas embryonic width of <2.7 mm on Day 25 is considered retarded and such embryos have either died or are dying [123,124].

Serum Biochemical Profile

The uptake and utilization of minerals and energy decrease with the season in buffaloes. The breeding season was associated with a decrease in crude protein and mineral intake [163]. RB buffaloes had significantly lower serum calcium, phosphorous, magnesium, copper, iron and zinc [86,164-167]. Variations in circulating levels of biochemicals, macro and micro minerals were recorded in RB buffaloes (Table 2), however, the data cannot be used to predict potential fertility. These results could be used to predict deficiencies in representative samples from large herds [8].

Level of Oxidant and Antioxidant Markers

Increased presence of reactive oxygen species and free radicals such as nitric oxide affect fertility in domestic animals [170-172], therefore their evaluation could be helpful in predicting fertility. Markers for oxidative stress have been identified in buffalo serum [85,173,174]. In one study, buffaloes with endometritis showed increased malondialdehyde (MDA) and nitric oxide (NO) and decreased catalase (CAT), superoxide dismutase (SOD), ascorbic acid (ASCA), reduced glutathione (GSH-R) and total antioxidant capacity (TAC) [85,174,175] (Table 3).The significance of these antioxidants in relation to the level of lead in blood was stressed in one study on buffaloes [173] with significantly higher MDA, NO and lower SOD, glutathione and total antioxidant capacity in buffaloes with higher blood lead concentrations. Endometritis impairs the CL function and development and there is reduced luteal NO and ascorbic acid [175]. Lower levels of antioxidant vitamins are considered to be associated with poor fertility and milk production levels in ruminants [176] and possibly buffaloes.

Therapeutic Approaches

Therapies in a herd with suboptimal fertility include corrective measures to prevent/combat disease and/or deficiency and reducing stress [8]. Perhaps the easiest point to improve or correct is management. The temporary replacement/change of the bull may take care of infertility due to the bull. When using frozen semen, the storage and handling should be evaluated [8]. Strategies to control RB in individual buffaloes can be attempted using one or a combination of the following approaches.

1. Improving uterine health
2. Correcting ovarian malfunction
3. Maintaining luteal support
4. Improving management.

When applied correctly, alone or in concert, these therapeutic approaches should result in a decrease in RB in buffaloes. Altered uterine health, ovarian malfunction and poor management can have overlapping effects both on the fertilization and pregnancy maintenance.

Improving Uterine Health

Uterine infection that commonly occurs in RB buffaloes falls under the definition of subclinical endometritis with clinical signs showing at around 8 weeks postpartum, with a complete absence of cervical discharge [61] as was also mentioned for cows [177,178]. When a microbial infection in the uterus is suspected, there are many therapeutic approaches including intra-uterine infusion of antibiotics or immunomodulators and administration of hormones.

Intrauterine Infusion of Antimicrobial Compounds

The intrauterine infusion of different antibiotics is a traditional treatment [179], however, based on current evidence, this treatment is not always effective and may be harmful if irritant compounds are used [180]. It has been mentioned that the route of administration of antibiotics, such as intrauterine infusion or parenteral administration, has little effect on the outcome of endometritis [60,181,182]. Intrauterine treatment for endometritis is still widely used in practice, meeting the owner’s traditional expectations for treatment. The direct intrauterine administration of oxytetracycline produce immediate therapeutic concentrations in the caruncles of both healthy and affected cows [184] and oxytetracycline has been shown to be an effective therapy for endometritis in buffalo [60,136]. However, studies showed that the massive irritation of the uterine mucosa produced by oxytetracycline might have significant negative effects on uterine defense mechanism and on self-healing ability of the uterus [61,180,185]. The antibiotics commonly used for intrauterine infusion in therapy of bubaline endometritis have been recently summarized [84] and include penicillins, aminoglycosides and cephalosporins. It is suggested to administer non-spermicidal antibiotics such as aminoglycosides or penicillins 8-24 h after insemination [84,130] or for 3 days from the day of estrus followed by insemination during the subsequent estrus [84] if endometritis is diagnosed as a reason for RB. However, the routine intrauterine infusions of antibiotics suggested previously such as gentamicin [136,186], ciprofloxacin and tinidazole [187-190] or oxytetracycline plus tylosin [60] cannot be considered as optimal therapy for endometritis, particularly because of antibiotic resistance [191] and the possible residues in milk. Standards for milk withdrawal times after intrauterine infusion of antibiotics in buffaloes are neither followed in most buffalo rearing countries nor addressed in scientific studies.

Post insemination infusion of cephalexin 4 g and ceftriaxome 2 g given 24 h post AI resulted in improvement in CR in RB buffaloes [192]. Antiseptics such as lugol’s iodine (50 mL of 0.5%-1% solution) or povidone iodine (50-120 mL of 2% povidone) has been suggested for intrauterine infusion in endometritis affected buffaloes [27,86,193,194] with resulting improvement in CR (54.5% to 67.6%) in RB buffaloes. However, most of these studies utilized a very small number of animals (<15) and are thus not justified. Moreover, the hazards of such infusions particularly their potent irritating nature prohibit their intrauterine use.

The limitations of intrauterine therapy are development of drug resistance, inconsistent results and milk withdrawal after treatment that render such treatments uneconomical [195]. Moreover, the uterus seems to have a considerable capacity for spontaneous recovery, and a large proportion of animals probably do not require any therapy at all, especially since some treatments are ineffective and might even cause more harm than benefit [196]. When clinical or subclinical endometritis is suspected, because of their low cost, properly administered broad spectrum antibiotics such as aminoglycosides must be the clinician’s first choice [8]. If flakes of pus are present in the vagina or in cervico-vaginal mucus [80], flushing of the uterus with normal saline must be considered [197,198]. Third and fourth generation cephalosporins have shown efficacy against most uterine pathogens at low minimum inhibitory concentration values [199]. The first generation cephalosporin (cephapirin) is recommended for intrauterine use [200,201] as the drug of choice for subclinical endometritis [202]. RB buffaloes treated with an intrauterine infusion of Enrogil, which is particularly effective against E. coli, had a 71.6% pregnancy rate following treatment [203]. It has been suggested to combine the antibiotic with an imidazole derivative (metronidazole or tinidazole) to take care of anaerobic microbes and protozoa that might be present [189,190,204]. Without evaluating the type of microbes present the indiscriminate use of intrauterine antibioitics however, seems inappropriate. Addition of antifungal agents [145,205,206] must be considered when the endometritis turns out to be chronic after a prolonged therapy with antimicrobials. Intrauterine irrigation of 2-4% Lotagen solution increased CR up to 84% in RB buffaloes affected by fungal agents [145].

Use of Prostaglandins (PG) and Estradiol Benzoate

The uterus has an increased influx of PMNs, an increased blood supply, increased mucus production [207] and enhanced uterine production of leukotriene B4 during the estrus period because of an increase in pro-inflammatory cytokines stimulated by PG. The immune functions of the uterus are thus enhanced during estrus [208]. Therefore, returning buffaloes to estrus at short intervals should lead to endogenous clearance of microbes and resolution of the endometritis. This can usually be achieved by injecting a PG 5–10 days after onset of estrus [27,209-213]. Postpartum buffaloes treated with PG on Day 7 had less endometritis than non-treated buffaloes [214]. Mid cycle administration of PG to RB buffaloes resulted in 1st service CR of 50% in treated animals [215]. Similarly, the administration of PG on Day 6 of estrus or 2 PG injections 11 days apart starting from Day 6 of estrus in RB buffalo heifers resulted in 50% and 100% animals showing estrus and 80% and 100% CR respectively [212]. Uterine lavage plus PG resulted in better CR in RB dairy cows compared to uterine lavage alone [198]. In clinical practice it is sometimes recommended to keep the female buffalo indoors for 2–3 cycles (avoiding mating) in an effort to resolve the endometritis, however, the interval to conception is prolonged and therefore it is advised to use prostaglandins instead [8]. In an attempt to enhance the uterine clearance of inflammatory exudates some researchers used antibiotics and estradiol benzoate to treat endometritis but the results demonstrated that there is no beneficial effect on uterine infection or reproductive performance in dairy animals [60,88]. Injecting estradiol benzoate to lactating buffaloes could result in a sharp and irreversible decline in milk production, vaginal prolapse and the spread of the infection towards the oviducts [8] and should thus be discouraged.

Immunomodulators

Recently, several therapy alternatives to the use of antibiotics and hormones have been suggested for the treatment of endometritis. The intrauterine infusion of immunomodulators such as E. coli lipopolysaccharides (endotoxin, LPS) [214,216-221], oyster glycogen (OG) [180,218,222-225], infusion of serum, plasma or hyperimmune serum [180,221,226] or leukotriene B4 [227] has been reported widely. These immunomodulators act as a chemoattractant to the PMNs through stimulation of interleukins [228] produced by monocytes and macrophages. The PMNs, blood monocytes and macrophages are regarded as the professional phagocytes in cellular defenses against pathogenic micro-organisms [220]. After experimental intrauterine infection, the PMN population within the uterine lumen usually increases [229,230]. A single intrauterine infusion of 100 mg of E. coli LPS dissolved in 20 ml of phosphate-buffered saline (PBS) results in an increase in uterine neutrophils (of up to 80%) within 6 h, which remains elevated for 72 h [231,232]. Likewise, 0.1–10% oyster glycogen usually 500 mg dissolved in 60 ml of vehicle or 30 nmol/l of leukotriene B4, increase the PMN concentration within 12–24 h of administration [217,227]. Intrauterine infusion of aqueous extract of Tinospora cordifolia (3000 mg) [ an herbaceous vine of the family Menispermaceae indigenous to the tropical areas of India, Myanmar and Sri Lanka] or autologous plasma (150 mL) for 3 days starting from the day of estrus significantly reduced the bacterial population in buffaloes with uterine infections [226]. Using these treatments, the endometritis would usually be cured and animals can be inseminated at the subsequent estrus. However, LPS is known to suppress follicular growth, decrease estradiol production and delay the LH surge and ovulation [232,233], and thus the subsequent cycle may be delayed. Addition of a small amount of autologous serum or plasma (50–100 ml for 2–3 days) to uterine infusions, increases the opsonizing capacity and significantly enhances the phagocytic ability of PMNs [180].

Bacterial load has been used as an indicator for health status of an organ, including the uterus [234]. The possible antibacterial effect of both the immunomodulator substance Tinospora cordifolia and autologous plasma, induces leukocytosis and activates macrophages. The reduced bacterial population (80 to 83%) leads to reduced bacterial metabolites that reduce from 8.63 to 7.82 to near neutral level. In such cases, conception rates after AI on subsequent estrus shows better result [226].

Besides the use of immunomodulators, some other therapies suggested for resolving endometritis include the use of enzymes and antioxidants [8]. Enzymes like trypsin, chymotrypsin and papain when infused into the uterus resulted in a cure rate of 59.7% (confirmed by the absence of vaginal discharge at re-examination); however, conception rates were suboptimal [235]. Another enzyme that has been tried is lysozyme [218,236] with a good success rate. Other compounds like 4 mM taurine and 50 mM fructose in PBS [237] and ascorbic acid (vitamin C) have been used for intrauterine infusion in an effort to change uterine pH prior to insemination, and act as an antioxidant [8]. Antioxidants such as vitamins C and E are known to modulate oxidative stress and reduce the endometrial damage both at the biochemical and histological levels [238]. Moreover, buffalo semen has a low citric acid and ascorbic acid content [239] which is likely to convey a lower protection from the reactive oxygen species generated during the freeze-thaw process [240]. An infusion of antioxidants before AI may reduce the uterine luminal reactive oxygen species and the beta-endorphin that may reduce the functional competence of frozen spermatozoa [8]. The protection to reactive oxygen species may not be offered by the parenteral administration of antioxidants.

Correcting Ovarian Malfunction

Ovarian malfunctioning includes ovulatory disturbances. Ovulation in the buffalo is known to occur 10-14 h after the end of estrus [241,242]. Ovulatory disturbances resulting in RB buffaloes include delayed ovulation, anovulation and ovarian cysts [59]. The effects of these disturbances produce pregnancy failure and therefore RB [8]. Delayed ovulation results in poor fertility and repeat breeding [5] probably due to fertilization failure. The underlying physiology of anovulation seems to be a lack of a preovulatory LH surge in response to the high, concentrations of estradiol [243,244], presumably because of a lack of progesterone priming the hypothalamus or a multitude of other factors. Anovulation conditions with a clear growth of follicles not reaching ovulatory size may be due to undernutrition, suckling or disease. Another reason for anovulation may be the presence of suprabasal progesterone concentrations during estrus [103,107], producing an inhibitory effect on the positive feedback on the hypothalamus by high estradiol concentrations, resulting in a low LH pulse frequency affecting follicular growth [245]. Studies in RB buffaloes revealed higher progesterone concentrations (0.81±0.39 ng/mL) compared to normal buffaloes (0.64±0.35 ng/mL) on the day of estrus [246,247]. In cases of anovulation, there is some evidence for an absence or a deficient LH surge trigger mechanism. In such cases, GnRH treatment can stimulate ovulation and may result in increased pregnancy rates [27,248,249]. An improvement in pregnancy in 61.54 % cases of RB buffaloes was recorded [86] following treatment with GnRH. It thus appears that many anovulating events are primarily the result of deficient hypothalamic function and not of ovarian disorders. Clinically, therapies to stimulate ovulation in buffaloes with delayed ovulation/anovulation and ovarian cysts are essentially similar to those used in cattle [59]. These include the administration of either hCG (1500–3000 IU) at the time of insemination [5,76,86,250-254] or 10-50 μg of GnRH [27,86,212,248,255-259] or human menopausal gonadotropin (hMG) [260]. These therapies usually produce LH release [248,261], however, some researchers found that the CL formed by an hCG injection had a shorter life span [262,263] and 67% of the induced short cycles were followed by a return to acyclicity [264,265]. Intramuscular administration of 3000 IU of hCG to Nili-Ravi buffaloes at AI resulted in significantly higher mean plasma progesterone concentrations on Day 5 (1.90±0.08 ng/mL) and Day 12 (2.10±0.10 ng/mL) after insemination and improved first service CR to 48% compared to 17% in untreated controls [254]. Administration of a CIDR for 9 days and PG at removal followed by timed insemination in buffaloes at 48 and 72 h after withdrawal, improved CR in RB buffaloes [266].

Alternatives to these well-known therapies for ovarian dysfunction include the administration of glucose and insulin, prostaglandins, metformin (an oral diabetes medicine), antiprolactins and clomiphene [59]. The LH surge is known to be complex and affected by interplay of various endocrine, neurocrine, metabolic and cellular events. Low levels of glucose, insulin and insulin-like growth factors all affect the LH surge [267].

Luteal Insufficiency

Luteal insufficiency due to a diminished response to the circulating luteotrophic hormones [268] leads to insufficient progesterone production during the luteal phase, and could be the cause of embryonic death [269]. Serum progesterone is known to be altered in RB buffaloes [270,271]. In one study, serum progesterone during the mid-luteal phase was 1.44±0.39 ng/mL and 3.66±0.84 ng/mL in RB and normal buffaloes respectively [86], however, in another study there were non-significant differences in the plasma progesterone concentrations between normal cyclic and RB buffaloes [247]. The importance of progesterone concentrations during the first few weeks of pregnancy in reducing embryonic mortality has been demonstrated in buffaloes [271,273,274]. Early in the luteal phase, progesterone down-regulates oxytocin receptors (OTRs) for at least 10 days, thus preventing premature luteolysis [275]. The secretion of antiluteolysin factor IFN-t and bovine trophoblastic protein-1 (bTP-1) around Day 15–16 post ovulation mainly depends on progesterone concentrations around Day 4–5 post ovulation [276]. Moreover, a conceptus has to elongate and secrete IFN-t, and its growth is largely dependent on progesterone levels [277]. A low progesterone level has been shown to be significantly related to lowered production of IFN-t by bovine embryos recovered on Day 16 of pregnancy [278]. The most critical period for embryo survival in cattle may be around Day 5–6 post-insemination when the embryo descends from the oviduct and enters the uterine lumen. During this period, progesterone concentrations start rising and any delay in the rise and/or a low luteal phase progesterone concentration can cause a poor uterine environment for the embryo and poor embryo development and result in embryonic death [279]. Similarly, in buffaloes, the administration of GnRH on the day of insemination [86,249,257,258] and Day 5, and Days 11-13 [249,257,258,280] or an OvSynch protocol [281] all improved conception rates and plasma progesterone concentrations in RB buffaloes. Day 6 was considered as the growing phase of the first wave dominant follicle and the proportion of buffaloes that ovulated in response to exogenous GnRH administration on Day 6 was 75% compared to 16.67% when GnRH was administered on Day 10 of the estrus, which is considered as the regression phase of the first wave dominant follicle [282]. However, in one study administration of GnRH on the day of insemination or on Day 12 of estrus resulted in greater CL diameter and higher plasma progesterone and higher first service conception rates (52.9% in treated v/s 28.6% in untreated) RB buffaloes [283]. A greater development of the CL as early as Day 5 is likely related to pregnancy in buffaloes [155]. In buffaloes, a CL that show early development at Day 5 has a greater expression of vascular endothelial growth factor, increased vascularization and higher blood flow [155]. Some other studies have mentioned that due to delayed implantation [284], most bubaline embryonic deaths occur beyond Day 25 [160,285] thus luteal support in the form of hCG or GnRH is suggested on Day 5 [280] or Day 25 after insemination [31,116,126,271,273]. It thus appears that luteal profiles at Day 5 and Day 25 are both important and supplements should be considered at both these times. Administration of long acting bovine insulin (0.2 IU/kg body weight/day) for 3 days during mid luteal phase (Day 8-12) [286,287] or a single injection on Day 10 of estrus [288] resulted in an increase in plasma progesterone and conception rates. Such effects are due to the autocrine and paracrine roles of insulin on oviductal cells and embryonic development [278,289]. Administration of Ovsynch protocol to RB Egyptian buffaloes starting on the day of estrus resulted in CR of 80% and high progesterone concentrations in treated heifers [212]. Plasma prolactin and progesterone concentrations were negatively correlated during the summer estrous cycle in buffaloes, which indicates prolactin-induced suppression of progesterone secretion through poor luteal development [290]. Progesterone supplementation to prevent poor luteal function has also been suggested in the form of two to three 500 mg IM injections of progesterone in oil starting from Day 5 of estrus [8] or three injections (341 mg IM from Day 25 every 4 days) of progesterone [285] or combinations of progesterone vaginal implants (CIDR) with other hormones [88] with good success rates. Molecules to prevent PG production in early pregnant animals include COX-2 inhibitors, polyunsaturated fatty acids, oxytocin receptor antagonists and lypophosphatidic acid [291]. Administration of meloxicam (NSAID) (0.5 mg/Kg IM) on Day 13, 14 and 15 after inseminations [292,293] or flunixin meglumine (1.1 mg/kg IM) on Day 15 [294] to buffaloes resulted in higher plasma progesterone (Day 18, 21 and 24) and lower serum PGFM in treated buffaloes with a 20% higher conception rate [292,294]. Recombinant bovine somatotropin (500 mg SC) at the time of estrus and 10 days later significantly increased the conception rate because of an increase in circulating progesterone [295]. In a recent study on induction of estrus during the summer, buffaloes were treated with a single subcutaneous implant of melatonin (18 mg/50 kg body weight dissolved in corn oil) and inseminated with frozen semen. A significantly higher conception rate was obtained, when a SC injection of melatonin was given on Day 5 post-AI, compared to buffaloes that did not receive the melatonin injection post-AI [296]. The results suggest that the photoperiodic cues are important in conception and that the administration of melatonin can probably reduce embryonic mortality in buffaloes mated during the long daylight period [155].

Unconventional Therapies

Research studies in cows on less common therapies suggested that Omega-3 fatty acids decrease the secretion of PGF2α [297,298]. Eicosapentaeonic acid (EPA), dehydroascorbic acid (DHA) and alpha linolenic acid (an inhibitor of prostaglandin synthase enzyme) are natural sources of omega-3 fatty acids capable of decreasing the secretion of PGF2α and complement the antiluteolytic action of IFNt thereby improving pregnancy rates [299]. Feeding fish oil has also been suggested as it contains docosahexanoic acid (DHA) and eicosapentaenoic acid (EPA), both of which have antiluteolytic properties [300]. Feeding of 70 mL or 140 mL of fish oil daily to buffaloes for 49 days significantly improved the conjugated linoleic acid and omega 3 fatty acid concentrations in the milk [301]. Daily feeding of 250 gm fish meal to buffaloes for 55 days prior to estrus synchronization with an OvSynch protocol did not affect the size of the CL or plasma progesterone concentrations, yet significantly decreased PGFM metabolites (197.4±41.7 pg/mL) in response to an IV infusion of 100 IU of oxytocin on Day 15 of synchronized estrous cycle compared to buffaloes that were not supplemented with fish meal (326.3±33.5 pg/mL) [302]. Oral feeding of fish oil resulted in no abnormal smell or flavors in milk of cows [303] but similar effects were not mentioned in studies on buffaloes [301,302].

Management Factors

The overall management of buffaloes is important as it may affect fertility. Of consideration are nutrition, timing of insemination, periparturient disease, housing and reducing stress. Buffaloes managed under free stall housing with continuous exposure to a fertile buffalo bull had better conception rates compared to those managed in confined open-fronted tie-stall sheds [23].

Improving Nutritional Imbalances

The effects of nutrition on reproductive efficiency of buffaloes has been addressed in many studies [18,19,21,304,305] with high energy diets improving fertility [31,274] and mineral and vitamin supplements improving conception rates in RB buffaloes [86,164,167,306]. Poor nutrition during the dry and early postpartum period results in reduced glucose, insulin, insulin-like growth factor (IGF-1) and low LH pulse frequency with concomitant increases in beta-hydroxybutyrate, non-esterified fatty acids and negative energy balance, all having negative effects on the probability of conception [8]. Egyptian buffalo heifers with feed intake restriction had poor reproductive performance while heifers with a high feed intake required less number of services per conception and had low age at first service and an overall higher pregnancy rate [307]. Postpartum swamp buffaloes fed high or low levels of nutrition had average postpartum interval to conception of 222 and 169 days respectively [308]. Conversely, high nutrition can also increase the metabolic clearance rate of steroid hormones such as progesterone and estradiol, and high rumen degradable proteins can raise the blood urea nitrogen [8,21]. All these can impair conception and embryo survival. However, the impacts of nutrition on fertility appears to be complex, and recommendations for formulating effective dietary strategies to improve conception rates and prevent embryonic losses during the more crucial stages is difficult. A balanced feed during the dry period must therefore comprise a low-energy high-fiber ration containing high levels of chopped straw. Supplementation of mineral mixtures suggested in several previous studies [27,86,164,167] should be provided with caution keeping in mind the bioavailability, requirements and mineral interactions with other nutrients [309].

Improving the Timing and Technique of Insemination

Proper storage, thawing, and post-thaw handling of semen, correct insemination technique and appropriate with respect to estrus status are mandatory in achieving high success rate with AI [310]. The intensity of estrus is low in buffaloes. In one study, estrus signs were weak in 73.24%, moderate in 25.35% and intense in only 1.41% of buffaloes [311]. Primiparous and pluriparous buffaloes had significantly higher (77.28% and 72.88%) overt estrus compared to buffalo heifers (38.46%) [312]. Thus, buffaloes pose a greater difficulty in estrus detection and sub-estrus is frequent [313,314], therefore, timing inseminations becomes difficult [8]. Thrice daily estrus detection was suggested for improvement in conception rates at farm level [104] but this seems impractical. Based on sonographic evaluations it was shown that as high as 54.5 % buffaloes had silent ovulations [315]. Based on plasma progesterone profiles on the day of insemination it was found that many buffaloes are inseminated at the wrong time [316]. Vaginal electrical resistance measurements have been suggested for estrus detection and timing insemination in buffalo [131,317] with limited success. Likewise, the use of pedometers [318,319] and radio-telemetric devices [320] has been suggested to improve estrus detection and, hence, timing of insemination in cows. The transport of buffaloes to the insemination center affects fertility. As the distance to the AI center increased, conception rates decreased [108]. Higher CR was obtained when buffaloes were inseminated 11-14 h or 15-18 h after the onset of estrus [321]. Due to a longer window of ovulation in buffaloes it has been suggested that buffaloes should be inseminated twice at 12 h intervals, with the first insemination being performed 8-12 h after estrus onset [74,322]. A single insemination 20 h after the first observed estrus has also been suggested [323]. Zicarelli et al., [324] had contrarily suggested that due to the small size of the uterine body, the inadvertent deposition of semen in the uterine horns could be the reason for low conception rates. Moreover, according to Vale [325], a pregnancy rate higher than 50% can be considered good after insemination with frozen thawed buffalo bull semen. Deposition of semen in the body of the uterus offers a distinct advantage in improving the conception rates to insemination in buffaloes, compared with when it is deposited in the mid-cervix [8]. Errors in the preparation of the AI gun or in the upkeep of frozen/liquid semen can contribute to conception failures and must be avoided. A recent report informed over the improvement of conception rates to artificial insemination by 5–27% in several Asian countries, when the personnel involved in AI were given proper training [326]. Differential fertility of buffalo bulls [55,58] and seasonal influences on seminal quality of buffalo bulls [100,322] are some factors that need attention, especially for semen collection centers processing buffalo semen as they can inadvertently decrease fertility. Personnel involved in insemination should receive refresher courses from time to time to obtain optimum fertility.

Periparturient Care

Postpartum complications and metabolic disorders in the buffalo are less frequent compared to cows [327-329]. An important factor influencing fertility in buffaloes is periparturient hygiene. Prompt therapy of retained placenta and postpartum metritis should reduce the calving to conception interval and result in higher subsequent fertility [330].

Reducing Stress

Stress appears to play an important role in the modulation of various biological events including reproduction. The role of various types of stress caused by disease, inadequate nutrition, high production, social factors and environment on reproduction has been explained [331]. An important factor affecting buffaloes is heat stress that suppresses both male and female reproduction [332]. It is nearly impossible to avoid all forms of stress in buffaloes, but when animals require a higher number of services per conception, attempts must be made to minimize stress associated with environment by provision of wallowing, water sprinklers or shade [333]. The presence of a swimming pool in the paddock of lactating buffaloes, together with more space availability, improved fertility and calving to–conception intervals in buffaloes with days open shorter than 120 days [334,335]. Paddock overcrowding increased embryonic mortality in buffaloes [25]. The swimming pool presence reduced the NP (non-pregnant?)/CL ratio in buffaloes that calved between April and August but not during the January-March period. Clearly the swimming pool functioned as a tool against heat stress [25]. Provision of misting and wallowing during hot summer months significantly maintained physiologic, metabolic and endocrine homeostasis in Murrah buffaloes [336]. Short term cooling (before and after AI) resulted in low plasma cortisol and significantly improved CR in Murrah buffalo heifers [337]. Wallowing during hot parts of the day was considered beneficial compared to showering as shown by plasma cortisol, insulin and thyroid hormones [338]. Evaporative cooling with a high pressure fogger system with fans reduced stress, increased body comfort and milk yield in buffaloes [339]. Some other approaches to reduce heat stress in buffaloes include the feeding of electrolytes, ascorbic acid and zinc which reduced plasma cortisol and oxidants [340,341].