Estrus Detection in Buffaloes

Estrus detection in the buffalo has been poor on account of some inherent problems such as poor expression of estrus [1,2], silent estrus, lack of vocalization and homosexual behavior by estrus females, peak sexual activity during night hours and profound negative effects of hot environmental temperatures on estrus expression [3-5]. Buffaloes located in countries away from the equator show seasonal breeding activities [6,7] and the majority of buffaloes show estrus during winter months compared to summer months [8-11]. Estrus often passes unnoticed in warmer climates and symptoms may be absent or very weak during the hot dry seasons [4,12-17]. In tropical and subtropical environments, estrus is mostly expressed during the late night or early morning hours [18].

The purpose of estrus detection for farmers continues to be identifying females in estrus to be covered by natural service by buffalo males (which is still the preferred mode of breeding). Farmers often detect buffaloes in estrus by vaginal discharge, bellowing, vulvar edema, restlessness and temporary teat engorgement [19]. Improper timing of insemination due to poor estrus detection appears to be one of the major factors affecting the success of artificial insemination in buffaloes [20-22].

Most estrus detection methods used in commercial cattle farms have yielded suboptimal results in the buffalo [23,24]. The traditional method of estrus detection is solely based on visual observation; still this method is very useful for estrus detection if practiced at-least 2-3 times a day for 30 minutes every time and this can successfully help to detect 80-90% of buffaloes in estrus in a herd [25,26].

Visual observations for estrus detection, however, require closer observations, and the inconsistency of behavioral signs renders them less efficient in buffaloes. Because more animals appear to show estrus during the late night and early morning hours in buffaloes [27] closer observations during this period of time would be necessary. The most reliable indicator for estrus in the buffalo appears to be mounting by the bull, thus the use of teaser animals [28-31] have shown promise for estrus detection and is routinely used at most organized buffalo farms. Clinical methods of estrus detection like transrectal palpation and transrectal ultrasonography and laboratory methods such as cervical mucus fern pattern are useful for farmers monitoring individual buffaloes yet impractical for herds as they require daily interventions.

Commercially available pressure activated heat mount detectors fitted on cows’ rump have been reported to be useful for estrus detection in cows [32], however, their use in buffaloes is questionable due to the lack of homosexual behavior and their wallowing habits. Physical activity meters (pedometers) relate well to estrus onset in cattle and thus useful for estrus detection, however, their efficiency in buffaloes has been poor [33,34]. Newer approaches such as the detection of salivary electrolytes [35] or the detection of chemical cues (pheromones) in the vaginal mucus, urine or feces [36-39] have shown promise for estrus detection in buffaloes and are likely to be available in an easy to use form in the near future. It has been suggested to combine a number of approaches for improving the estrus detection efficiency in the buffalo at organized buffalo farms [40]. In this chapter the salient features of estrus and methods of its detection in the buffalo are described.

1. Estrous Cycle in Buffaloes

River buffaloes are considered seasonally polyestrous animals in countries where their populations are the largest (India and Pakistan) and in many other countries away from the Equator (for example Egypt and Italy) whereas in countries near the Equator, buffaloes are polyestrous throughout the year when well fed [41]. Swamp buffaloes in Japan were polyestrous throughout the year [42] whereas in a report on swamp buffaloes from Thailand seasonal breeding was observed [43]. Similar to cattle, the length of the estrous cycle in river buffaloes is 21 days [44] although a wide variation does exist as reported in different studies (Table 1). The physiology of the estrous cycle in the buffalo has been described in a previous chapter in this book [44]. Ovarian follicular growth patterns during estrous cycle in buffaloes have been described in detail in a previous chapter in this book [45] and have revealed two to three follicular growth waves in buffaloes. Similar to cattle, the ovulatory follicle ovulates after the end of estrus in buffaloes. Exceptionally short estrous cycles of less than 16-19 days commonly indicate ovarian cysts in dairy cows. Although ovarian cysts can occur in buffaloes they are uncommon [46]. Mid-cycle estrus has been reported in buffaloes in the past [47-49] with an incidence of 5% to 12%. Increased production of estradiol by the dominant follicle of the first or second follicular wave probably results in such phenomenon. Most of the short cycles (11 days) occurred between the first and second estrus after calving [49]. Short estrous cycles can also result from intra-uterine infusion of irritant solutions. The etiologies of persistence of a corpus luteum and consequent longer estrous cycles (<30 days) are poorly understood. Early embryonic death has been identified as a frequent cause of longer estrous cycles [49].

The period of sexual receptivity (estrus period) in river buffaloes is 24 h (10-48 h) in comparison to a shorter time period (19.9±4 h) in swamp buffaloes (Table 1). The female accepts the male during this period and this is considered as the most reliable sign of estrus in buffaloes [41]. Ovulation of the mature follicle (under LH influence) occurs approximately 11 h after the disappearance of estrus signs in river buffaloes and 13.9 h in swamp buffaloes [30,51,52].

2. Signs of Estrus in Buffaloes

It has been mentioned that the signs of estrus are generally more evident in swamp buffaloes (especially the Philippines Carabao) than those of the river breeds [12]. Anatolian buffaloes sometimes become nervously aggressive [12]. Visual observations for estrus detection in buffaloes are far from perfect unless performed four times a day [85,86]. There appears to be a high variability in the most prominent visual signs of estrus in buffalo. While some reports mention vulvar edema as the most prominent sign [31,62,87], others found frequent urination and restlessness [88-90] or vaginal discharge in recumbent animals [85,91], and vaginal congestion [61,92] as the most prominent sign of estrus in buffaloes. In a few studies vulvar edema and vaginal discharge were found to occur with the same frequency in buffaloes in estrus [87,93,94].

Behavioral signs of estrus in buffaloes include restlessness, bellowing and frequent discharge of small quantities of urine (Fig. 1). In one study, buffaloes exhibited the behavioral signs of estrus during early morning and late evening with a peak in the early morning (more than 1/3rd of the total) and a lower rate at noon [95]. Behavioral signs such as vocalization (bellowing), tail raising and homosexual behavior are shown by a small proportion of buffaloes only [40,62,90]. In Mediterranean buffaloes estrus behavior was shown by only 37.5% of buffaloes whereas 62.5% had a silent estrus [96]. Segregation and bellowing confirmed estrus in 83.69 and 80.43% of Murrah buffaloes [85]. Sniffing, licking and restlessness has also been reported in buffaloes [62,97]. There is a wide difference in the expression of behavioral signs of estrus in buffaloes (Table 2).

Figure 1. A buffalo in estrus voiding a small quantity of urine and raising her tail to one side.

Parity is also known to affect estrus behavior. Significantly higher vocalization, micturition, tail raising in parous buffaloes were observed as compared to buffalo heifers, however, no significant difference was observed in behaviors like vulvar tumefaction, reddening of vulva, chin resting and flehmen reaction [89].

Estrus behavior is also considered to be less prominent during the postpartum period. Sniffing of the vulva, restlessness and cajoling were frequent signs while standing heat, mucous discharge, and chin resting and mounting were less frequent in Surti buffaloes during the post-partum period [98-101]. High estrus score buffaloes showed significantly higher estrogen concentration as compared to low estrus score buffaloes [31,102]. Behavior manifestation was better during the cooler part of the day i.e., during the early morning and late evening [90,99,103].

Acceptance of the male i.e., heterosexual behavior and mounting is regarded as the most reliable sign of estrus in buffaloes [104]. Homosexual behavior is less obvious in buffaloes as compared to cattle [23,81]. Vasectomized bulls can easily detect estrus in buffaloes when used twice a day [105].

2.1 Temporary Teat Engorgement

Buffalo farmers at many locations detect estrus in their animals by milk letdown in the teats in the absence of a calf or feed stimulus. This phenomenon has been termed the temporary teat engorgement (TTE). TTE is shown by 80% of buffaloes 2-5 days before estrus and is more common in pluriparous Murrah buffaloes [106]. In a study on 811 buffaloes the average duration of this phenomenon was found to vary from 2.8-3.4 days and estrus followed in a mean duration of 8.47±1.11 days after disappearance of TTE [107]. The reasons for this phenomenon continue to be poorly understood. It is possible that the ovarian oxytocin that induces a cascade of events leading to luteolysis somehow escapes the ovarian circulation to cause milk ejection in the teats [13], however, such a hypothesis need to be validated. The oxytocin concentration in bubaline corpora lutea was low (30.61±4.41 ng/g) in the early luteal phase (Day 4-6); it was significantly high (104.69±12.73 ng/g) in mid luteal phase (Day 10-12) and significantly low in late (Day 16-18) cycle luteal phase (49.87±5.95 ng/g). The oxytocin concentrations in CL obtained from pregnant buffaloes were significantly low compared to CL from non-pregnant buffaloes [108]. In one study the endometrial estrogen and oxytocin receptors were upregulated in the late luteal phase but peak of oxytocin receptors appeared before the estrogen receptors [109].

2.2 Standing Estrus in Buffaloes

The duration of standing estrus in buffalo is less than 24 h. Peak sexual activity is observed during night and early morning hours. LH surge lasts for about 9 h and spontaneous ovulation occurs on an average 24-29 hours after the LH Surge [44]. The interval between standing estrus and ovulation, which is very important for artificial insemination, was 30 hours in buffaloes [112].

2.3 Subestrus/Silent Estrus in Buffaloes

Occurrence of ovulation without behavioral manifestation of estrus is known as a silent estrus or sub-estrus [113]. The incidence of silent estrus is higher during the postpartum period [114] and during the summer months [115]. About 63% of buffaloes had silent ovulations without overt estrus behavior during the postpartum period [114]. This may be due to lack of on-time estrus detection in artificial breeding systems [116]. The reasons for subestrus are poorly known. Heat stress [117] and slower growth rate of follicles associated with lesser secretion of estradiol [118] are some of probable causes. Silent estrus is considered a major obstacle in estrus detection and in the use of assisted reproduction in buffaloes [119].

3. Factors Affecting Estrus in Buffaloes

Many factors can influence estrus onset and intensity in buffaloes, many of which have been described previously [120].

3.1 Breed

Variations in estrus behavior were observed between different breeds of buffaloes. Murrah-cross performance was found better as compared to pure Murrah, Surti and Nili-Ravi buffalo [121]. However, these differences were negligible and buffaloes are generally considered to have poor overt expression of estrus [2].

3.2 Ambient Temperature and Humidity

Under heat stress, the length of the estrous cycle increases and the estrus period decreases. Heat stress was shown to have detrimental effects on reproduction of buffaloes [112,123]. It was mentioned that hyperprolactinemia as a result of thermal stress inhibits the secretion of both FSH and LH [119]. High environmental temperature also decreases thyroid activity which leads to reduced reproductive efficiency in buffaloes [124]. High ambient temperature along with higher humidity affects biological functions in buffaloes [125] especially fertility [126] through increased production of free radicals and cortisol and decreased production of antioxidants. Significant decrease in reproductive performance in buffaloes was observed when THI (temperature humidity index) was more than 75 [127]. The effect of THI on productive and reproductive indices of Italian and crossbred buffaloes was evaluated in a recent study [128]. High THI reduced the estrus intensity and conception rates at first insemination when compared with low THI [128].

Heat stress has a direct effect on estrus behavior in dairy animals [129]. Estrus is known to be more frequent and stronger in the cooler season compared to hot season in buffaloes [130] and this is due to higher secretions of estradiol, progesterone and LH during the cooler months [131-134]. Hormonal estimations on serum and ovarian follicular fluids from 708 Egyptian buffaloes revealed significantly lower concentrations of serum and follicular estradiol, progesterone and cholesterol during summer and autumn compared to winter and spring [135].

High blood urea and hypoglycemia in extreme summer was associated with low fertility [63] including estrus behavior manifestation.

3.3 Season and Photoperiod

Many studies have mentioned seasonality in display of estrus, conception and calving in buffalo [3,120,136-138]. This is because of the effects of thermal stress, photoperiod and many other interactions [3]. Photoperiodic cues (duration and intensity of light) influence the pineal gland activity [139] and in turn the melatonin secretions [140] which govern the estrous cycle and estrus intensity of buffaloes in many countries [140,141] except those which are close to the Equator where photoperiod remains nearly similar throughout the year [6]. Complex interactions of photoperiod and environmental temperatures regulate estrus activity in buffaloes. Many studies have shown that a lower proportion of buffaloes exhibit estrus under long day length [3,142,143]. Reducing day light effects during longer days by melatonin injections have resulted in the resumption of ovarian cyclicity and stronger estrus behavior in buffaloes in Italy [144], India [145,146] and Egypt [147].

Melatonin is a hormone produced and stored in the pineal gland of the brain. Reproductive seasonality in buffaloes is regulated by melatonin secretion that is reduced during longer days lowering gonadotrophin secretion [140,148,149]. In buffaloes, a marked variation in melatonin secretion is observed between day and night (20 pg/ml during the day and 60pg/ml during the night) as compared to less seasonal breeders where melatonin remains the same during the day and night at 30-40pg/ml [14].

A melatonin implant induces estrus by alleviating oxidative and summer stress in anestrus buffaloes [146]. Summer anestrus in buffaloes can be controlled by treating animals with melatonin [150].

3.4 Postpartum Estrus

Postpartum resumption of estrus in buffaloes is dependent on a number of factors including nutrition, management and suckling [103,120,143,151,155], however, the most important factor appears to be season of calving. Significantly longer intervals of anestrus were observed for buffaloes calving during summer compared to those calving during autumn or winter [156-161]. Poor estrus expression and silent estrus is common during summer months in buffaloes [63,<160,161]. Thus a proportion of buffaloes are in estrus during the summer months also but are not detected. Suckled postpartum buffaloes revealed a higher incidence of silent estrus compared to weaned buffaloes [162,163].

3.5 Body Condition Score

Optimum body condition score (BCS) is essential for proper estrus expression. The BCS is assigned based on anatomical features and carcass fat reserves [164,165]. It was noticed that BCS and postpartum ovulation interval were correlated with metabolizable energy (ME) intake. Prepartum ME intake was higher in buffaloes that returned to estrus early postpartum compared to buffaloes that remained anestrus for prolonged periods postpartum [164]. Buffaloes that calved with a BCS score of 2 to 2.5 presented delayed ovulation and a longer service period compared to buffaloes that calved with a BCS of 3 to 4 [166,167]. Positive correlation was observed with the BCS of Murrah buffaloes at the time of AI to preovulatory follicle size and intensity of estrus in a recent study [168].

3.6 Nutrition

Nutritional effects on estrus expression are more evident in buffaloes raised in countries near the Equator where photoperiod is constant throughout the year [6]. Studies in the Amazon basin [169,170] found that 86% of female buffaloes did not express any seasonal anestrus in a long photoperiod season when they were well fed. However, nutrition is also important for buffaloes at other locations and has been addressed previously [164,171-174]. In particular estrus intensity is dependent on availability of energy [164], vitamin A, vitamin E and minerals [3].

3.7 Age and Body Weight

Young animals show shorter and abrupt estrus behavior as compared to mature animals. First estrus in buffaloes is generally of short duration and is less intense. Manifestation of estrus behavior improves with parity [174]. During puberty 50-70% of mature body weight should be achieved. Age at puberty in buffaloes is 16-40 month with an average of 30 months [175]. Estrus behavior is more related to body weight as compared to age.

3.8 Clinical Conditions

Lack of estrus expression (anestrus) in buffaloes can originate from clinical conditions including endometritis, pyometra, mummified fetus or hormonal deficiencies [143,176,177] and have been described in detail in another chapter [120].

3.9 Management

Management interventions have profound effects on estrus expression in cattle and buffaloes [178,179]. For proper estrus detection, thrice daily observations for at least 30 min each have been suggested [179]. Early morning and late evening visual observations, early weaning (7 days) and twice daily exposure to fertile bull improved the postpartum estrus onset and intensity of estrus behavior in Egyptian buffaloes raised in a free stall housing system [180]. In previous studies on Egyptian buffaloes, the beneficial effects of time of estrus detection and the bull exposure on estrus expression and detection have been mentioned [95,181]. Similar effects were also observed for Italian buffaloes [141,182]. Four times daily estrus detection has been suggested in a few studies [,31]. Besides estrus detection approaches, other management interventions [184] such as feeding and milking schedules and particularly of heat stress [185], provision of shade and wallowing space have profound effects on estrus. The effects of housing management [186] and space availability on reproductive behaviors of buffaloes were evaluated in two studies [187,188]. A space of 10 m2/head did not affect the reproductive efficiency of buffaloes. Also the provision of pools for wallowing or showering during hot summer months revealed significant improvement in reproductive behaviors including the expression of estrus [189-193].

3.10 Genetic Basis of Estrus

The duration and intensity of estrus behavior may be genetically influenced and is becoming increasingly important [194]. Genomic studies can help us in understanding the different regulatory mechanisms that lead to expression of estrus [195]. However, research reports on the involvement of a different set of genes in relation to estrus behavior in domestic animals are few. Boer et al., [195] mentioned that changes in systems that regulate estrus behavior could be manifested by altered gene expression. Estrogens in collaboration with other hormones cause up- or down- regulation in a number of genes at the hypothalamus of the brain (and at other locations) believed to be involved in estrus behavior in mammalian species [195]. Similarly it was mentioned that gene expression profile is altered in reproductive tissues at estrus in dairy cows [196,197]. A recent study in buffaloes found differential expression of ten candidate genes regulating prostaglandin action on reproductive tissues during estrous cycle and pregnancy [198]. The leptin gene and its receptor on CL of buffalo were identified in one study [199]. It has been estimated that around 269 genes undergo significant changes at transcript level during estrus [200]. Thus a coordination of different tissues and molecules up- or down- regulate different set of genes responsible for estrus expression. Identification of different set of genes that regulate estrus expression could be especially useful in species with inherently poor estrus expression such as the buffalo.

4. Approaches for Estrus Detection in Buffaloes

Female buffaloes are kept in small numbers (2-5) by majority of farmers in countries with the largest buffalo populations (India and Pakistan) and there are few commercial intensive farming herds (5-100 buffalo herd) or institutional farms [201-203]. The very purpose of estrus detection for a buffalo farmer is presenting their buffaloes to nearby buffalo bulls for natural mating or getting them inseminated. Natural mating continues to be the preferred manner of breeding in India and Pakistan with around 50% or more of buffaloes being bred by natural mating using buffalo bulls available in the vicinity [203-208]. In Egypt which has 5.32 million buffaloes, the proportion of buffaloes bred by artificial insemination (AI) is very low (1-2%) [209]. In European countries like Italy, Azerbaijan and Romania the proportion of buffaloes bred by AI is only 3-5% [210]. Similarly, low AI coverage was mentioned for Sri Lanka [211] and Bangladesh [212].

Although signs of estrus are less pronounced in buffaloes, the majority of farmers detect estrus based on visual observations [19,213-215]. When presented for AI, clinicians often base their diagnosis of estrus in buffaloes on transrectal evaluation of uterine tone, palpation of an ovulatory sized follicle and cervical status [216] and visualization of the ovulatory follicle and/or uterus when transrectal ultrasonography is available.

Estrus detection at organized buffalo farms should combine many approaches (including visual observations) to improve the estrus detection efficiency [40]. Studies comparing the efficiency of different estrus detection methods at organized farms have revealed that exposure to a vasectomized buffalo bull along with close visual observations are the best methods [105,217]. The methods for estrus detection include visual observations, clinical methods, laboratory methods, vaginal electrical resistance, pressure sensitive devices and physical activity detection systems and will be described next.

4.1 Visual Observations

Mucus discharge and bellowing are the most common signs observed by farmers for estrus detection in buffaloes [204,215,218-220]. Other common signs include restlessness, increased urination and temporary teat engorgement (2 days prior to estrus) [19,90,204]. Vulvar edema and congestion was a more prominent estrus sign in buffalo heifers [62,87]. A recent study reported difference in intensity of estrus signs in Murrah buffaloes based on parity [31,89]. Mohan et al., [31] have mentioned that vulvar edema is the first visible sign of estrus and this sign persisted for the longest time. The frequency of estrus detection by farmers is twice a day and commonly the duration is less than 5 min which leads to errors in estrus detection [19]. Since estrus activity is more concentrated during the early morning and late evening, twice to four times daily observations for 20-30 min each time are suggested for estrus detection at organized buffalo farms [31,86,94,105,221]. Closer observations are suggested during summer months as estrus signs tend to be weaker during this time [222].

The use of video surveillance alone could not detect buffaloes in estrus [207], however, when sexual activities were observed in Surti buffaloes by using low-light intensity camera and females were exposed to teaser bulls for 20-30 min thrice a day; estrus detection was efficient [90]. Dubey et al., [98] also observed different behavioral signs like sniffing and cajoling in buffaloes using a CCTV video recording system.

A recent study tried to classify the acoustic features of vocalization during different phases of the estrous cycle in Murrah buffaloes. They recorded the sounds using a microphone attached to a video camera. The duration of vocal signals (call duration) was significantly longer during proestrus and estrus phases and the intensity of vocal signals was significantly higher during proestrus whereas the pitch of vocal signals was significantly higher during estrus compared to other phases of estrous cycle. Significantly higher plasma estradiol was recorded in Murrah buffaloes during proestrus (29.46±0.65 pg/mL) when the intensity of vocal acoustic signals was highest compared to estrus (19.35±0.35 pg/mL) (when the pitch of vocal signals was high) or other phases (6.67±0.19 pg/mL at metaestrus and 5.43±0.13 pg/mL at diestrus) of the estrous cycle. The authors suggested that this method can be used for estrus detection in Murrah buffaloes [223] although such an approach needs to be validated further. Another study with Murrah buffaloes [224] reported that call duration was significantly higher during estrus (1.52±0.07 sec) as compared to diestrus (0.98±0.05 sec) along with other vocalizations. Call duration along with amplitude pitch and intensity was positively correlated with blood estradiol concentrations and negatively correlated with blood progesterone and LH. The above findings support the use of precise acoustic mapping in buffaloes as a tool for estrus detection in the near future.

Buffaloes in estrus may be show a different behavior in the milking parlor such as their entry habit, restlessness in the milking chute, kicking during teat cup fitting and a sudden drop in milk yield. Estrus behavior requires careful observation and perhaps demands a more experienced person watching the buffaloes while in the milking parlor. Under normal conditions buffaloes show an entrance order consistency (animals always come in in the same order) and they show a left side preference [225], however, this may change during estrus.

4.2 Clinical Methods

4.2.1 Transrectal Palpation

Manual transrectal palpation of the genital tract appears to be the oldest and most common clinical method for evaluating the status of estrus in buffaloes [90,226,230] presented for AI. The presence of a regressing corpus luteum and a large ovarian follicle on either of the ovaries along with increased turgidity (tone) of uterine horns and opening of the os externus are considered findings that indicate that the buffalo is in estrus [226,227,231]. The accuracy of estrus detection is dependent on the palpator’s experience. The accuracy to detect estrus in adult buffaloes and buffalo heifers was 77.78% and 100% respectively in one study [227]. Intense uterine tone with coiling of the uterine horns was recorded in 52.98% of buffalo heifers and 64.46% of adult buffaloes in estrus [227]. Palpable turgidity of uterine horns has been mentioned as a consistent sign of estrus when present in conjunction with vaginal congestion and vulvar edema [18,87,232].

Through transrectal palpation, buffaloes presented for infertility problems are often detected in estrus [231]. Buffaloes with silent estrus are also detected during such palpation. Transrectal palpation of 127 buffaloes in rural areas revealed that 1.27% of buffalo heifers and 2.05% of adult buffaloes were in estrus at presentation, and 9.35% of buffaloes with a history of anestrus were found to have had a silent estrus (based on palpation of a corpus luteum) [231]. Transrectal palpation of ovarian structures, however, has limitations of accuracy due to smaller size of the ovaries and the ovarian structures in buffaloes. Milk progesterone assay was considered more accurate in detecting the ovarian activity, especially the progesterone secreted by the CL [233,234]. Palpation of the ovulatory follicle must be done with great care as undue pressure may result in the rupture of the follicle. Transrectal palpation in buffaloes should be carried out with extreme care and extensive lubrication as the rectum of buffaloes is fragile and can bleed easily.

4.2.2 Transrectal Ultrasonography

Studies comparing transrectal palpation and transrectal ultrasonography (TRUS) for evaluation of genital structures in buffaloes, mentioned that TRUS is more precise [216,235]. In particular follicles >4 mm diameter can easily be detected by TRUS [236,237] but follicles and CL less than 9 mm could not be detected by transrectal palpation [216,238]. In fact, the studies on follicular growth dynamics in buffaloes utilized TRUS [45]. During estrus in buffaloes, the uterus examined by TRUS appeared enlarged with heterogeneous gray shades reflecting edema around the non-echogenic (black) central area representing the lumen [235]. During metaestrus the heterogeneous gray shades were reduced indicating reduction in edema. Using TRUS the follicles appear as dark non-echogenic structures surrounded by hyperechoic border of ovarian stroma [235]. However, findings of TRUS should supplement transrectal findings. Also since TRUS is not readily available at most buffalo AI centers, it has limited value in estrus detection programs in buffaloes.

4.3 Laboratory Methods

Any or all tests performed on cervico-vaginal mucus, urine, saliva, blood, milk and vaginal mucus membrane for estrus detection are considered under laboratory methods for estrus detection.

4.3.1 Cervico-Vaginal Mucus

The physical and biochemical properties of cervico-vaginal mucus in buffaloes have been evaluated in relation to conception rates at insemination [174,239-241] or in repeat breeding buffaloes [229,242,243]. The focus of these and other studies was the color, consistency; pH and arborization (fern) pattern of the cervico-vaginal mucus. The normal color and consistency of buffalo cervico-vaginal mucus at estrus is transparent and stringy. The normal pH of cervico-vaginal mucus in buffaloes is slightly alkaline (8.16±1.01 to 8.75±1.02) [106,244] but tends to be slightly less alkaline (7.49-7.82) in repeat breeding buffaloes [229,242]. Regarding the biochemical constituents of cervico-vaginal mucus, calcium, phosphorous, magnesium, sodium and potassium were found to be altered in one study on repeat breeding buffaloes [242] and another study mentioned that the chloride content of cervico-vaginal mucus of Egyptian buffaloes is highest at estrus (632.4mg/dL) and can be used as an approach for estrus detection even in silent estrus buffaloes [245]. High glucose content in cervico-vaginal mucus was associated with higher conception in buffaloes [230]. Treating buffaloes with prostaglandin resulted in changes in cervical mucus with decline in protein, alkaline phosphatase and peroxidase activities [246]. The ascorbic acid concentration in cervico vaginal mucus was lower in young buffaloes compared to older buffaloes [106]. However, the evaluation of different electrolyte components in cervical mucus appears to be difficult under field conditions and evaluation of arborization (crystallization) pattern is an indirect test for alterations in the electrolytes and is easy to perform.

Many studies [106,140,141,174,241,247,248] mentioned that the crystallization pattern of cervico-vaginal mucus (fern pattern) in smears (dried on glass slides) is an effective approach to detect estrus and time insemination in buffaloes. The crystallization appears on account of altered electrolytes in cervical secretions under the influence of altered circulating ovarian steroids and appears like a fern when observed under a microscope. Depending upon the appearance of branches that are visible the pattern has been described as typical (Clear appearance like a fern plant with many secondary and tertiary branches), atypical (when the secondary and tertiary branches are less marked) and nil (when there is no appearance of fern like structures) [58,229]. Buffaloes with typical fern pattern (Fig. 2) are in estrus and should be inseminated while those with atypical fern pattern can be considered and those with a nil fern pattern are not in estrus and should not be inseminated [58,240,241,249] as no pregnancy results. Scores of crystallization pattern of cervical mucus remained low during the entire diestrus period in Murrah buffalo heifers and increased with the regression of the CL, attaining a maximum score on the day of estrus [228]. The presence of cervical mucus at the time of AI indicated good pregnancy rates in buffaloes [232].

A recent report evaluating the different buffalo cervico-vaginal fluid proteins by SDS Page analysis found that the expression of heat shock protein-70 was higher during the estrus phase compared to the diestrus phase [250] and this can serve as a potential biomarker for estrus detection in buffaloes.

A practical problem with the detection and/or collection of cervico-vaginal mucus is the appearance of mucus only in a small proportion of buffaloes. Gill et al., [56] found that only 16.98% of estrus Murrah buffaloes had a free flowing mucus discharge. Likewise Rao and Kodagali, [92] observed mucus discharge only in 56.29% of Surti buffalo heifers in estrus. It was not possible to find the discharge even in sitting Surti buffalo heifers during the night time in one study [87]. Moreover, mucus discharge can occur 5 days before and 5 days after estrus in a large number of animals [85,88] further limiting the use of cervico-vaginal mucus for estrus detection in buffaloes. Studies evaluating the cervico-vaginal mucus discharge mention its collection by aspiration utilizing a glass pipette/AI gun or sheath [230,241,242,251].

Figure 2. Atypical (A) and typical (B) fern patterns of cervico vaginal mucus in buffaloes.

4.3.2 Milk and Plasma

Evaluations for estrus detection in buffaloes using milk can be done by assay of somatic cell counts or progesterone concentrations. Somatic cell counts and milk proteins increased significantly at estrus in buffaloes [252]. Progesterone concentrations in milk and plasma decrease significantly at estrus in buffaloes [87,253,254] and thus can be utilized for estrus detection. Plasma progesterone concentrations in Egyptian buffaloes on the day of estrus in two farms with different managements were 0.79 ng/mL and 0.18 ng/mL and increased to 1.78-2.04 ng/mL within 5-8 days post estrus [255] and similar findings were recorded in another study on Surti buffaloes [256]. Chauhan et al., [257] concluded from their study that buffaloes that have plasma progesterone concentrations of less than 0.50 ng/mL and more than 1.0 ng/mL were in estrus and luteal phase whereas those with plasma progesterone concentrations between 0.6ng/mL and 0.9 ng/mL are doubtful for either of the two stages (too late for estrus and too early for a real luteal phase). In a recent study on Murrah buffaloes the plasma progesterone concentration decreased from 0.30±0.23 ng/mL two days before estrus to 0.09±0.03 ng/mL on the day of estrus and then increased gradually to 0.27±0.03 ng/mL, two days after estrus [258]. Using such data the accuracy of estrus detection and insemination in buffaloes was evaluated in 2 studies by estimating the plasma progesterone concentration on the day of estrus and it was concluded that buffaloes were inseminated at the wrong time [259,260]. The conclusion is that inseminators and farmers need to be better trained in estrus detection.

Since plasma progesterone estimations require blood collection and involve high cost, milk progesterone assays have been utilized [261,262] basically for pregnancy diagnosis and partly for estrus detection [263-267]. Using radioimmunoassay to test progesterone concentrations in milk, it was found that 94 out of 100 Nili-Ravi buffaloes had progesterone concentrations ranging from 0.01ng/mL to 0.90 ng/mL on the day of estrus [265]. Progesterone concentrations in milk, serum and saliva of Nili-Ravi buffaloes on the day of estrus were 0.49±0.18 ng/mL, 0.23±0.03 ng/mL and 0.08±0.03 ng/mL respectively [264]. Milk progesterone concentrations determined by ELISA were 0.97±0.42 and less than 1.0 ng/mLin two studies [266,267]. Qualitative tests to determine progesterone in milk have been developed so that less time is required in obtaining the test results and lesser costs are incurred [268,269]. A recent study evaluated a commercially available qualitative progesterone test kit (Heat Aid, Ridgeway Sciences, UK) for pregnancy diagnosis in buffaloes and found that the test was 85.71% accurate in diagnosing pregnancy at 21 days post mating. The accuracy of detecting non-pregnant buffaloes returning to estrus at this time (21 days) was 100% [270].

A practical problem with progesterone assay for estrus detection is their efficacy only at certain time periods (only when the animal returns to estrus on Day 21 post-mating) and the false positive diagnosis of pregnancy. Thus, using such procedures on herds necessitate that the blood/milk samples be assayed regularly on a daily basis, which incurs heavy costs and does not seem logical or practical.

4.3.3 Salivary Evaluations

Salivary evaluations for estrus detection in buffaloes are recent concepts and are likely to develop in the future. Saliva can be collected from buffaloes suspected of being in estrus, from the posterior part of the tongue. Saliva is then smeared on a glass slide and dried naturally in air. The fern pattern observed on a dried slide under a microscope indicates that the animal is in estrus. Salivary fern pattern visualization is an unconventional indicator of estrus [97]. An increase in branching in the fern pattern shows the appropriate time for insemination in buffaloes.

The concentration of calcium, phosphorus, magnesium, sodium, potassium and chloride in saliva were significantly (p<0.01) higher during the estrus phase as compared to other stages of the cycle. The minerals were significantly positively and negatively correlated to plasma estrogen and progesterone concentrations respectively [35].p-Cresol was recently identified as estrus specific volatile compound in saliva of buffaloes [271] and has potential to be developed as a bio-marker for reproductive status evaluation.

While working on protein expression in saliva of buffaloes in estrus, 37 proteins were expressed in the estrus phase of cycle including β-enolase and TLR-4 considered as markers for estrus detection in buffaloes [272].

The dried salivary transcript analysis showed a significantly higher expression of heat shock protein-70 and toll-like receptors at estrus than at diestrus in buffaloes [273].

4.3.4 Chemical Cues in Urine/Feces

Flehmen response in buffalo bulls or teaser bulls is dependent on chemical cues (pheromones) secreted by the female buffaloes [274] and thus their identification can help in estrus detection in the buffalo. A few recent studies evaluated the presence of volatile compounds in the feces [38], vaginal mucus [37] and urine [251,275] of buffaloes in estrus. Twenty seven volatile compounds were found in the feces of buffaloes, of which 4-methyl phenol (4 mp) and trans-verbenol (tv) were found only in the feces of buffaloes in estrus. Buffalo bulls exhibited repeated flehmen when exposed to a combination of the 2 compounds (i.e 4 mp + tv) and thus the authors of the study concluded that these two compounds would be reliable indicators of estrus in buffaloes. Fourteen volatile compounds were identified from urine throughout the bubaline estrous cycle of which three compounds chlorooctane, 4 mp and 9-octadecenoic acid were only found on the day of estrus [275]. P-cresol and oleic acid were present in estrus urine of estrus synchronized buffaloes similar to urine of buffaloes in natural estrus [39]. Buffalo bulls were attracted and exhibited repeated flehmen behavior when exposed to 4 mp and displayed penile erection and mounting behavior when exposed to 9-octadecenoic acid. The compound 9-octadecenoic acid (oleic acid) was also specifically present in the vaginal fluid from estrus buffaloes [37]. These studies point out the presence of pheromones in buffaloes in estrus and these molecules could probably be used in the future for estrus detection in buffaloes. Based on the detection of estrus specific chemical compounds in bovine estrus feces [276], attempts have been made to electronically identify chemical compounds in gaseous phase by constructing an eNose with encouraging results [277]. An unreported yet common event in estrus rural buffaloes is the walking away from their location towards a male buffalo located nearby, sometimes a few kilometers away. This could be due to the detection of male pheromones by estrus females and therefore needs the attention of researchers. The urine levels of LH detected using bovine LH ELISA kits validated for serum [278] were evaluated in two recent studies on Murrah buffaloes as a predictor of estrus and ovulation [278,279]. Significant variation in LH was noticed during estrus compared to baseline values of 46.73±3.36 mIU/mL [278]. Urine LH levels of 105 mIU/mL was the cut off concentration which predicted the ovulation period [279].

4.3.5 Vaginal Cytology

Vaginal cytology at estrus is an uncommon and less efficient method of estrus detection in buffaloes [280,281]. Vaginal smears revealed that when animals are not in estrus there is a predominance of parabasal cells over intermediate and cornified cells [281]. Significantly more proportions of cornified (69.75%) and superficial cells (19.01%) were observed at estrus in Murrah buffaloes [280].

4.4 Teaser Buffalo Bulls

A teaser is a male animal that has undergone surgical alteration so that he is sterile yet maintains libido [282]. A teaser buffalo bull is the commonest approach for estrus detection in most institutional farms and farmers managing higher number of buffaloes (greater than 50). Some farms also use intact buffalo bulls for estrus detection [40,89,213,283]. The surgical approaches for preparation of teaser bulls have been described previously [282,284] and similar procedures have been used in preparation of teaser buffalo bulls [285,286]. Some of the surgical procedures including penectomy, preputial purse string, lateral deviation of the penis and insertion of bull chastity tube might have animal welfare implications and are not suggested. Bilateral vasectomy or caudal bilateral epididymectomy are the most common surgical techniques for preparation of teaser buffalo bulls [27,56,57,88,106,141,286-289].

Teaser buffalo bulls can detect buffaloes in estrus and a buffalo standing to be mounted is the most reliable sign of estrus. A wide variability has been observed between the onset of standing estrus and LH peak [287]. Buffaloes should be inseminated after the end of bull acceptance. Inseminating buffaloes after the end of estrus resulted in conception rates ranging from 40.4% to 60.7% [290]. Pregnancy rates were higher in buffaloes inseminated at estrus (spontaneous estrus or estrus induced using prostaglandins) in the presence of vasectomized buffalo bull [141]. The efficiency of estrus detection in buffalo herds can be increased when teaser buffalo bulls are paraded 2-4 times daily (especially early morning and late evening) and fitted with some marker devices such as chin ball markers [22,291,292]. Commercially available chin ball markers have straps and a steel ball with paint reservoir (Fig. 3). The marker is strapped in such a way that the round portion is underneath the chin of the male. When the teaser rides a female and presses his chin on the back of the female, the paint from the device is released and marks appear on the back of the female in standing estrus. Due to the black skin color of buffaloes, the color of the paint should be carefully selected. Colors that glow during the dark hours are especially useful as estrus activity in buffaloes is more concentrated during this time.

Figure 3. A commercially available chin ball marker device.

Using androgenized females as a teaser for estrus detection in the buffalo has been mentioned [293-295]. Non-pregnant, non-lactating female buffaloes were androgenized by twice weekly IM injections of 250 mg testosterone enanthate administered 9 times followed by IM administration of 250 mg testosterone enanthate once weekly [293]. Another approach is the initial IM administration of 2000 mg of testosterone propionate followed by 1000 mg every 15 days [296]. The efficiency of androgenized females in estrus detection in buffaloes was 69% [294] and 86.0% [293].

4.5 Vaginal Electrical Resistance (VER)

During estrus there is increased blood flow to the genital organs and under the influence of estrogen there is increased hydration of cervico-vaginal mucosa secreting viscid mucus. Due to such changes there is decreased resistance to the passage of low voltage current in the vagina. This can be measured by commercially available probes (Fig. 4). This has been used for estrus detection in dairy cows [255,297,298]. In buffaloes, two studies [299,300] recorded a decrease in VER at estrus. Later, Gupta and Purohit, [256] recorded VER in Surti buffaloes for several days and proved that VER can be successfully used to predict the stage of estrous cycle in buffaloes. VER decreased significantly at estrus (32.68±0.46 Ohms) and synchronously the plasma progesterone concentration also decreased [256]. After ovulation VER started rising to reach a peak (50.23±0.55 Ohms) during diestrus. When VER was recorded in buffaloes that were inseminated, higher conception rates (79.81% vs 20.15%) were obtained when buffaloes were inseminated at low VER (26-30 Ohms) compared to those inseminated at a high VER (36-40 Ohms) [301]. Kandiel et al., [302] recorded a significant drop in VER with a concomitant increase in estradiol on the day of estrus in Egyptian buffaloes. A recent study in Bangladesh also found significant decrease in vaginal electrical impedance at estrus in postpartum buffaloes treated with an Ovsynch protocol or left untreated [303]. Until simple automated systems are available, estrus detection using VER is of limited value due to the requirement of daily recording which is cumbersome.

Figure 4. Commercially available vaginal electrical resistance probes for measurement of VER in buffaloes.

4.6 Pressure Sensitive Devices

Cows in estrus are mounted by herd mates in proestrus, other cows in estrus or teaser bulls [304]. This behavior of estrus cows led to the development of pressure sensitive devices (Heat detectors) which can show which cow was in standing estrus and stood to be mounted. The earliest approach for detecting cows that were mounted was the daily application of paint on the tail head. When a tail painted cow is mounted, some of the paint is rubbed off indicating that the painted cows possibly stood in estrus while mounted by a herd mate [304].

Commercially available pressure sensitive devices (Kamar, Estrotect, Bovine Beacon) (Fig. 5, Fig. 6) are either small strips with a scratch off technique or a pressure sensitive dye releasing capsule. These are glued to the tail head (sacrum) of the cow and when an estrus female is mounted by a herd mate or bull, the frictional pressure from the brisket of the mounting animal scratches off the silver surface or the dye is released by the pressure creating a color (red, green, yellow) mark. The intensity of the color depends upon the time of contact. One strip is useful for one estrous cycle and if fixed properly and left undisturbed, it can remain in place for 8 weeks. The sensitivity and positive predictive value of pressure sensitive devices varies from 37% to 69% and 77% to 97% respectively, depending on whether cows are indoor or at pasture [305]. The pressure sensitive color changing devices are less useful for buffaloes because of limited mounting by herd mates and their wallowing habits. Some use can be expected if such devices are used along with twice or more daily parading of a teaser bull but this would increase the expenses and seems impractical.

Figure 5. Commercially available Kamar estrus detector for cattle.

Figure 6. Estrotect scratch card where the color changes indicating whether or not to breed cows based on the extent of the scratched surface.

Commercially available radiotelemetric devices (Heat Watch, DDX Inc, Colorado, USA) have a pressure sensitive battery powered transmitter fixed on the cow’s tail head. When activated by the pressure of the mounting cow, the transmitter emits a radio signal which is picked by a repeater or receiver and relayed to the personal computer (Fig. 7) where the information is stored [304]. Heat Watch classifies the standing estrus as a cow having 3 standing events in a 4 h period. A recent study induced Mediterranean buffalo heifers to estrus by prostaglandin injections. Heifers were fitted with a radio telemetric Heat Watch device and estrus was detected by androgenized female teaser buffalo cows. The beginning of estrus was considered when there were at least 2 mounts (≥2 sec duration) in one hour period and the end of estrus was determined by an interval of ≥4 h between two consecutive mounts [296]. The efficiency of radio telemetry in detecting estrus was 90%, and 10% of buffalo heifers exhibited silent ovulation without mounting by androgenized females. The duration of mounts throughout the 24 h period was uniform and similar findings were found in another study in Brazil [306]. The Heat Watch systems are readily available in some countries in costs within the approach of most dairy and beef producers. Radiotelemetric devices can be useful in the detection of estrus for buffaloes only when a teaser buffalo bull or androgenized female is available.

Figure 7. A radiotelemetric system.

4.7 Physical Activity Detecting Systems

The physical activity of animals in estrus increases significantly. Cows that are in estrus walk 2-4 times more than non-estrus cows [304]. Electronic devices designed to identify elevated physical activity have been available in some countries [307] for the past few years. Two types of devices are commonly available and used in dairy and beef cows- the neck mounted collars and the leg mounted pedometers [307]. Various kinds of receivers are available with the devices that analyze the data from these electronic devices. The recorded data from each animal is processed by a software program that analyzes the variation in physical activity of cows and those showing an increased activity are deemed to be in estrus. In cows, the efficiency of the neck-mounted collars was better in terms of percent positive predictive value compared to leg fixed pedometers [308,309].

Another approach tested in cows is the analysis of 3-dimensional position of cows using the ultra-wide-band technology (Thales Research and Technology, Reading, UK). The technique uses global navigation satellite system along with other location technologies. In a preliminary study, the system showed a high accuracy (9/10 cows were detected in estrus) [310].

The use of electronic devices for detecting physical activity in buffaloes is uncommon. The efficiency of pedometers in estrus-induced Egyptian buffaloes was 85%, whereas only 14% of buffaloes could be detected in estrus by visual observation alone [311]. Italian buffaloes fitted with a pedometer and kept with a teaser bull had an estrus detection efficiency of 85% and the accuracy of estrus detection was 75% [191]. The only study on neck collar activity tags in buffaloes was done in Sweden and could only detect one out of thirteen estruses in buffaloes [34]. It appears that due to complex seasonal effects and a frequent silent estrus in buffaloes, the use of electronic physical activity meters need further adjustments before they can be used for estrus detection in buffaloes.