Postpartum endocrinology and prospects for fertility improvement in the lactating riverine buffalo (Bubalus bubalis) and yak (Poephagus grunniens L.)

Livestock Production Science 98 (2005) 13 – 23 www.elsevier.com/locate/livprodsci

Postpartum endocrinology and prospects for fertility improvement in the lactating riverine buffalo (Bubalus bubalis) and yak (Poephagus grunniens L.) B.S. Prakash a,*, M. Sarkar b, Vijay Paul b, D.P. Mishra a, A. Mishra a, H.H.D. Meyer c a

National Dairy Research Institute, Karnal-132001, Haryana, India National Research Centre on Yak, Dirang, Arunachal Pradesh, India Lehrstuhl fu¨r Physiologie, Technische Universita¨t Mu¨nchen, D-85354 Freising-Weihenstephan, Germany b

c

Abstract In many Asian countries the riverine buffalo is the major milk producing animal besides contributing towards draught power and meat production. The animal however suffers from inherent reproductive problems such as poor estrus expression and long calving intervals which limits its lifetime production. The yak is a seasonal breeder and the mainstay of highlanders and tribal populations inhabiting the inhospitable terrains of the Himalayan region. The factors responsible for its seasonality include poor nutrition, harsh climate and high altitude. Very little information is available on postpartum endocrinology in riverine buffaloes and even less so in yaks in relation to milk yield and cyclicity commencement. Our recent investigations on endocrine changes associated with cyclicity commencement in buffaloes (Murrah breed) showed a positive correlation between plasma prolactin concentrations and delay in postpartum cyclicity commencement. A significant correlation of plasma GH concentration with milk yield was also obtained. Monitoring cyclicity through milk progesterone analysis in buffaloes postpartum indicated that the incidences of silent estrus was low in winter months and very high in summer months — the overall annual mean being 37%. Preovulatory LH surges post-estrus occurred at different times resulting in ovulations at 28 to 60 h after onset of spontaneous estrus in buffaloes. Progesterone profiles in some yaks indicated cyclic activity even during non-breeding season. The positive correlation between plasma prolactin and melatonin indicates valuable evidence for their role in reproduction in this animal. D 2005 Elsevier B.V. All rights reserved. Keywords: Postpartum; Buffalo; Yak; Endocrinology; Fertility

1. Introduction

* Corresponding author. Tel.: +91 184 2259071; fax: +91 184 2250042. E-mail address: [email protected] (B.S. Prakash). 0301-6226/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.livprodsci.2005.10.014

In many south and south-east Asian countries the riverine buffalo is the mainstay of milk production system and also contributes to the rural economy in terms of meat production and draught. The riverine buffalo is better adapted than cattle with respect to

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utilization of poor quality roughages and resistance to some of the tropical diseases (Chauhan, 1995). However, its reproductive efficiency is hampered by poor estrus expression especially in summer months contributing to distinct seasonal reproductive patterns as well as prolonged calving intervals. The yak is a unique bovine species domesticated by highlanders and tribal populations inhabiting the high altitude inhospitable terrains in the middle and inner Himalayan region, and survives only at high altitudes between 2500 and 6000 m. The world’s total yak population is estimated to be around 15 million of which more than 85% is accounted for in Tibet, China. India possesses about 60,000 yaks in the high reaches of the Himalayan region. The animal is a seasonal breeder and the factors which are considered to be responsible for seasonality in breeding include nutrition, climate, and altitude. Very little information is available on postpartum endocrinology in buffaloes especially in relation to milk production and cyclicity commencement. To the best of our knowledge there is no information on this aspect in yaks. In this paper we discuss our studies on the endocrinology of the riverine buffalo (Murrah breed) and yak with emphasis on the endocrine causes for postpartum delay in cyclicity in lactating buffaloes, and the endocrine changes in yaks during breeding and non-breeding seasons.

2. Postpartum buffalo endocrinology and reproduction 2.1. Endocrine changes in the buffalo postpartum After parturition, two events occur concurrently to contribute to lactation anestrous in bovines and buffaloes. These are (1) milk production, which causes increase in demands of nutrients for milk synthesis and (2) uterine involution, which is the time required for the recovery of the reproductive tract to its normal state after delivery. Postpartum anestrous remains a major reproductive limitation in buffalo. The calving to calving interval in 48 to 66% of buffalo is N 14 months as dictated by exposure to the environment and unpredictable management (Perera, 1999). Buffaloes are susceptible to stressors especially high temperature and nutritional deficiencies that

seriously affect reproductive efficiency. Suckling is encouraged in buffalo to enhance calf survival rate and facilitate milk let down, but unfortunately this practice also attenuates the neuroendocrine signals required for resumption of ovarian activity. Studies have been attempted for reducing postpartum anestrous in buffaloes but protocols still require refinement (Tiwari and Pathak, 1995). The causes for the long delay in cyclicity commencement with a mean of around 68 days in Murrah buffaloes as well as the variation among individual animals are not clear. In lactating bovines and buffaloes a complex interaction of several key metabolic hormones viz. GH, prolactin, thyroid hormones and glucocorticoids regulate availability of major substrates for milk synthesis, e.g. glucose, amino acid and fatty acids (Tucker, 1981). Prolactin while on the one hand is stated to be galactopoetic in nature (Tucker, 1981), it has also been attributed to be antigonadotropic (Tyson et al., 1972). GH is an anabolic hormone and has been seen to play a role in enhancing growth and early commencement of puberty in cattle and buffaloes (Simpson et al., 1991; Mondal and Prakash, 2003a,b, 2004; Haldar, 2004). Hence, the principal hormones which could influence the length of postpartum period for cyclicity commencement are GH and prolactin. In a recent study we found a significant ( P b 0.01) positive correlation between GH and milk yield. However, no significant correlation was found between GH concentration and days to cyclicity commencement. The plasma prolactin concentration in animals exhibiting delayed commencement of cyclicity (N 90 days) were significantly higher ( P b 0.01). Correlation of prolactin with days to commencement of cyclicity was highly significant ( P b 0.01; 0.9) which indicated that the hormone was inhibitory to early cyclicity commencement. There was however, no correlation of prolactin concentrations with milk yield. Further, we did not obtain a significant correlation ( P N 0.05) between GH and prolactin. Plasma glucose, NEFA and amino acids, were also not significantly correlated ( P N 0.05) either with milk yield or cyclicity commencement. Uterine prostaglandin F2a (PGF2a) is responsible for the cyclical regression of corpus luteum, initiation of parturition and resumption of ovarian activity in farm animals (Fairclough et al., 1975; Edqvist et al., 1978; McCracken et al., 1999). PGF2a is metabolized to 13,14-dihydro-15-keto prostaglandin F2a (PGFM)

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50

25

0 0

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Months (1-12) Fig. 1. Incidence of silent estrus in buffaloes during different months.

during the first passage through the lungs (Piper et al., 1970). We, therefore, developed a simple sensitive enzyme immunoassay (EIA) procedure for PGFM determination in buffalo blood plasma (Mishra et al., 2003). Plasma PGFM concentrations in buffaloes increase just prior to parturition and stay high for a varying period of time ranging from 15 to 30 days (Mishra et al., 2003). Uterine involution postpartum is associated with a gradual decline in peripheral PGFM concentrations. Reproductive tract infections in the buffalo were also associated with high concentrations of PGFM while the plasma progesterone concentrations stayed low. It is known that in animals with uterine infections, the presence of bacterial endotoxins acts as a powerful stimulus for the release of PGF2a (Edqvist et al., 1978). Subclinical uterine infections have been associated with delay in postpartum commencement of estrus and long service period in buffaloes (Usmani et al., 2001). 2.2. Estrus behavior Diurnal patterns of estrus behavior have been observed in most buffaloes. In Indian buffaloes, signs of estrus have been generally recorded between 6 PM and 6 AM. In a study conducted on Murrah buffaloes, 59% of estruses were recorded between 10 PM and 6 AM (Prakash, 2002). Confirmation of silent estrus occurrences in buffaloes by milk progesterone monitoring of Murrah buffaloes throughout the year (Prakash, 2002) indicated that out of a total of 292

estruses recorded by milk progesterone analysis, 108 estruses (37%) went unobserved. The incidence of silent heat was lowest in the month of December (10.5%) while the peak was seen in April (70%). There was a gradual decline in incidence of silent estrus occurrence from May onwards (Fig. 1). Due to the high incidence of silent heat large numbers of buffaloes are left unbred and substantially contribute to days open period in this animal. The overall mean days open period of 89 buffaloes was 139 days. Season of calving had a profound influence. The mean days open period of animals calving from December till June were more than 140 days and was significantly higher ( P b 0.01) than the mean of animals calving in July to November (b110 days). The long period in the former group of animals can be attributed to the high incidence of silent estruses which the animals would exhibit in the summer months once they commence cycling postpartum (Fig. 2; Prakash, 2002). From the hormone profiles obtained from 27 Murrah buffaloes sampled during the immediate postpartum period, mean period for commencement of cyclicity was found to be 68 days. 2.3. Timing of ovulation in Murrah buffaloes in relation to estrus and LH peak Accurate prediction of preovulatory events facilitates efficient reproductive management in artificial

250 27

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75

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100 50 0 Jan-Mar

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Periods (quarterly intervals) Fig. 2. The mean days open (mean F SEM) period for three months (quarterly intervals) of Murrah buffaloes (n = 89) calving from December till June. The value on top of each panel indicates the number of buffaloes observed for the service period.

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LH (ng/ml)

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Time Interval (hr) Fig. 3. Plasma LH (mean F SEM) profile and timing of ovulation after onset of spontaneous estrus in Murrah buffaloes (n = 10).

insemination and embryo transfer programs. Knowledge of the timing of ovulation, as monitored by rectal palpation (Paul and Prakash, in press), permits precise timing of AI. Ovulation occurred at 42.2 F 2.8 h with a range of 28 to 60 h after onset of spontaneous estrus in buffaloes. This large window for ovulations in buffaloes was due to the preovulatory LH surges occurring at different times (0 to 34 h) in individual buffaloes after onset of estrus (Fig. 3).

3. Yak endocrinology and reproduction 3.1. Postpartum cyclicity commencement and calving interval in yaks The period for postpartum cyclicity commencement in yak cows is highly variable and is influenced by a variety of factors including the age, parity, nutritive environment and season of calving. Wulin and Shengyu (1982) reported that the commencement of cyclicity postpartum was shorter (70.5 F 18.5 days) for yak in good condition than for those in poor condition (122.3 F 11.8 days). Cows calving between March and June come in estrus during the same year (Sarbagishev, 1989). However, only a few of the cows calving in later months return to heat in the same year. Magasch

(1990) also reported that the interval between calving and first postpartum estrus for Mongolian yak cows was longer for those calved after June than those calved earlier. The length of the gestation period in yaks is a month less than in cows. On an average a yak cow gives rise to 1 calf every 2 years or at the most 2 calves in 3 years. The average calving interval was 407 days and varied between 390 days for the sixth parity to 443 days for the second parity (Kalia et al., 1997). It was observed that if the yaks were bred immediately after calving in the same season then the calving interval would remain within the reasonable limits but for the yak cows calving later in the summers and those for the missed heats, the calving interval may be much higher. Magasch (1990) reported longer calving interval of 593 days (range 346 to 719 days) and the first postpartum estrus at 344 days (range 86 to 655). 3.2. Seasonal breeding in yaks Yaks are considered seasonal breeders. However, information about the breeding season is rather conflicting. The breeding season is affected by factors such as climate, grass growth, latitude and altitude. Following their long period of deprivation and weight loss over the winter the female yaks come into the breeding season when temperature and humidity start

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to rise and grass begins to grow which also improves their body condition. On the northwestern grasslands of Sichuan (China) the season begins around June (Hu et al., 1960), and at the higher elevation of Lhasa in Tibet the breeding season will not start until July. Similar observations are reported from Kirgizia where the annual onset of the breeding season started on May at an elevation of 1400 m and occur progressively later until at the altitude of 2700 m estrus started after June (Denisov, 1958). In Arunachal Pradesh, India the breeding season reaches its peak in July and August when temperature is at its highest and grass growth is at its best and lasts up to November. 3.3. Estrus behavior The average length of estrous cycle in yak has been reported to be 18 to 22 days and variation in the length is one of the problems in yak reproduction. Estrus in yaks is generally affected by the environment, and when the weather is unfavorable the onset of estrus is delayed. At the beginning of the breeding season but before normal estrus, yaks usually show estrus-like symptoms and some of them present a short cycle (Yu et al., 1993a). The duration of the estrus is not easily determined in the yak since the signs of estrus are not always clear (Cai and Wiener, 1995). Estimates from northwestern Sichuan (China) suggest that estrus lasts 12 to 16 h. In a study with 41 female yaks, 26 of them had an estrus lasting 24 h or less and 4 yaks showed estrus for up to 72 h. Purevzav and Beshlebnov (1967) recorded that among 54 Mongolian yaks, 26 were recorded in estrus for only 0.5 to 6.5 h, a further 17 females showed estrus between 6.5 and 12.5 h, 7 females showed estrus between 12.5 and 18.5 h, and only 4 females showed a longer estrus duration. Changes in the appearance of the reproductive organs at estrus are more obvious than behavioral changes. As in cattle, yak females in heat search out and ride other females and like to be approached by male yaks. However, these signs are less pronounced than in Bos taurus cattle. The most pronounced signs of estrus are bellowing and mounting by mature bulls, swollen vulva, reddening of vaginal mucosa and mucous discharge. Most yaks start to show estrus in the early morning or in the evening (Lei, 1964).

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3.4. Endocrine changes in yaks during breeding and non-breeding season Plasma progesterone was very low (b 0.2 ng/ml) at estrus, thereafter started to rise with a sharp increase during the late luteal phase and reached a peak at day 15–16 of the cycle, declining rapidly thereafter to basal levels at estrus. However, there exists species difference with respect to the day of the cycle at which plasma progesterone concentration reaches its peak (Fig. 4). Plasma progesterone concentration reaches its peak at day 10 or 11 of the cycle in Sahiwal and crossbred cows (Mondal and Prakash, 2003a,b). In Murrah buffaloes, plasma progesterone concentration reaches its peak at day 13 to day 15 of the cycle (Arora and Pandey, 1982). During non-breeding season, in 4 of 8 animals studied, plasma progesterone level was at basal level as anticipated. However, cyclic changes in plasma progesterone were seen in 3 yaks while the plasma progesterone level stayed high throughout the sampling period in 1 animal. There were clear indications of cyclic luteal activity in a large proportion of animals even during the non-breeding season, although estrus symptoms were not exhibited. Plasma total estrogen and estradiol-17h concentrations were high at estrus and then declined to basal level on day 2 of the cycle and another small elevation was found between days 8 and 12 of the cycle. This elevation of the hormones suggests the possibility of additional follicular waves in yaks, which could be usefully exploited for superovulatory response of yaks. In case of other bovines like buffalo, peak concentration of estradiol (30 to 35 pg/ml) were found on the day of estrus or one day before followed by a decline to 5 to 10 pg/ml within two days (Arora and Pandey, 1982). The relationship between prolactin and melatonin secretion in blood plasma during breeding (November) and non-breeding (February) months was investigated in yaks by Sarkar (2004). Circadian rhythm in circulatory melatonin concentrations was exhibited with low concentrations during daytime and high during night in both the periods under investigation. Season of sampling did not influence the melatonin profile probably because the day–night duration during the months of sampling was quite similar. A circadian rhythm of prolactin release was also seen with a maximum mean concentration at 0400 h and a minimum at 1400 h in breeding month and with a

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Progesterone (ng/ml)

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Days of cycle Fig. 4. Comparison of plasma progesterone profiles during estrous cycle in yak (mean F SEM), cattle (mean) and Murrah buffalo (mean) (from Mondal and Prakash, 2003a,b).

maximum value at 0000 h and a minimum at 1200 h in the non-breeding month. The observations on diurnal prolactin pattern in yaks seem to be in sharp contrast to those recorded in cattle and buffaloes where higher prolactin concentrations have been recorded during daytime (Singh and Madan, 1993; Gustafson, 1994). Diurnal rhythm in hormonal or biochemical constituents is influenced by many factors including metabolic rate, nutrition and environmental conditions. Positive relationship of prolactin secretion to stress related situations is also well known. Under extremely cold conditions, yaks are exposed to greater stress at night when there is a sharp drop in the night temperature. During the day, under the influence of sunlight and more comfortable conditions prevailing, the animals experience relief. This may be the reason for higher prolactin concentration during night times. Sarkar (2004) also observed that the overall plasma prolactin levels during day as well as nighttime were found to be more in non-breeding month than the corresponding time in breeding month (Fig. 5). This marked increase of prolactin level in non-breeding season may be stress related and associated with harsh environmental conditions particularly low temperature as well as nutritional stress during the non-breeding season. The positively correlated (0.8 in breeding season and 0.7 in

non-breeding season) circadian rhythms of plasma melatonin and prolactin are not clearly understood. Tindal (1974) reported that melatonin profoundly modulates prolactin release from the hypophysis. However, different species interpret the pineal gland’s hormonal signal in a fundamentally different manner. This preliminary investigation opens the gates for researchers to unravel the interrelationship of the two hormones for more comprehensive and detailed investigation. 3.5. Plasma oxytocin and prolactin profile during milk let down in yak Sarkar (2004) measured plasma oxytocin and prolactin profiles, before, during and after milking in two yaks (Fig. 6). Before start of udder stimulation the levels were low (3.5 to 7.5 pg/ml for oxytocin and 0.18 to 2.81 ng/ml for prolactin). Within a minute after udder stimulation there was a spurt release of oxytocin and prolactin release in both the animals. In one animal (Animal no. 27), the plasma oxytocin and prolactin level rose up to 57 and 4.2 ng/ml, respectively 3 min after start of udder stimulation and in the other animal (Animal no. 54) the oxytocin level reached 49 pg/ml after 2 min and prolactin level reached a peak value of

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19 100

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75 70

25 20 10

0

0 Br-MEL

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Br-PRL

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Fig. 5. Comparison of plasma melatonin and prolactin profile (mean F SEM) during day and night hours in breeding (Br) and non-breeding (Non-Br) seasons. In the breeding season, blood samples were collected from 8 yaks at 2 h intervals for 24 h in the month of November. In the non-breeding season, blood samples were collected from 8 yaks at 2 h intervals for 24 h in the month of February.

4.5 ng/ml after 4 min of start of udder stimulation. The higher levels of oxytocin and prolactin were maintained during milking in both the animals, falling sharply thereafter at the end of milking. The hormone concentrations stayed low thereafter till the conclusion of sampling 10 min after start of udder stimulation. The oxytocin profile before, during and after milking indicates the importance of udder stimulation in oxytocin release, which is known to trigger the milk ejection reflex. The observation that higher concentrations of oxytocin were maintained during milking indicates that there is a continual oxytocin requirement for providing the contraction stimulus to the myoepithelial cells during milking. However, the observation that oxytocin levels start declining just before the end of milking points to a possible role of the residual milk present in the udder as well as the relative evacuation of the alveoli sending neural signals for decreased oxytocin secretion during the latter half of milking. This milking behavior is quite similar to that of buffalo (Kumud and Prakash, 2001) but somewhat different from that seen in cows where the hormone levels stay relatively high till the end of milking (Prakash et al., 1998). Like oxytocin there was also quick release of prolactin shortly after udder stimulation in yak (Sarkar, 2004). In mammals, the most effective stimulus for the prolactin release is the activation of receptors at the base of the teat by suckling or milking (Tindal and Knaggs, 1970).

3.6. Timing of ovulation and plasma LH With the objective to determine the timing of ovulation in relation to onset of estrus and preovulatory LH peaks in yaks (Sarkar and Prakash, in press-a) it was observed that yaks ovulated at 30.5 + 0.8 h with a range of 28 to 34 h after the onset of spontaneous estrus and the onset of preovulatory LH surge occurred 3.0 F 0.7 h after the commencement of estrus signs with the surge lasting 7.3 F 0.6 h. Ovulation took place 20.3 F 1.0 h post LH surge with a range of 18 to 26 h. The occurrence of the LH peaks within a narrow time frame of 4–8 h post estrus in yaks could have contributed to the animals ovulating within a narrow time interval.

4. Endocrine applications for improvement of fertility in dairy buffaloes and yaks The basic goal of all production oriented animal reproduction technique is to increase the proportion of cycling females that get pregnant within a reasonable period of time. In the last two decades, considerable attention has been focused on understanding reproductive endocrinology and developing biotechniques for augmenting reproduction in animals.

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B.S. Prakash et al. / Livestock Production Science 98 (2005) 13–23 Start of Milking 5.0

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Time around milking (min) Fig. 6. Plasma oxytocin and prolactin profiles before, during and after milking in two yaks. Blood samples were taken at 2 min interval starting from 5 min before application of milking stimulus. During milking stimulus and milking, blood samples were taken at the time of milking stimulus and thereafter at 1 min intervals till 6 min after the end of milking.

4.1. Augmentation of fertility through progesterone The utility of progesterone assays in buffaloes for detecting ovulations (Kaul and Prakash, 1994a), diagnosing and treatment of reproductive disorders (Kaul et al., 1993) and non-pregnancy and pregnancy detection in buffaloes (Gupta and Prakash, 1990) has evolved considerable interest in simplifying the esti-

mation procedure by direct RIA and enzyme immunoassay (EIA) procedures (Prakash et al., 1990). 4.2. Pregnancy confirmation through estrone sulfate determination Estrone sulfate is quantitatively one of the major estrogens in the milk and blood plasma of pregnant

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and lactating cows and buffaloes. During the first half of pregnancy its concentration increases gradually so that after 100 days of pregnancy it is present in all milk samples whereas it is low or undetectable in nonpregnant animals (Hung and Prakash, 1990;). Hence, measurement of estrone sulfate in milk on a routine basis could serve as a viable test for pregnancy confirmation and detection of the presence of mummified fetuses (Prakash and Madan, 1994). 4.3. Estrus synchronization As in many domestic species of animals, including buffaloes, the duration of estrous cycle is controlled by the life span of corpus luteum (CL) in each cycle (Singh et al., 2000). Progesterone secreted by the CL prevents estrus and ovulation. If estrus and ovulations are desired, the luteal function must be terminated. Methods for regression of CL and induction of estrus were developed with their advantages and disadvantages. The effectiveness of these protocols is dependent upon the precision of estrus detection and the time of ovulation after the synchronization (Singh et al., 2000). The use of two PGF2a injections at intervals of 11 to 14 days is the most popular technique for estrus synchronization in buffaloes. The behavioral estrus signs expressed after synchronized estrus are weaker than those expressed after spontaneous estrus, which makes estrus detection more difficult after estrus synchronization. Consequently, buffaloes were fixed-time inseminated at 72 to 80 h after 2nd injection of PGF2a. A new estrus synchronization protocol called Ovsynch in cattle has been developed; it makes the use of a combination of GnRH–PGF2a–GnRH injections (Pursley et al., 1995). Ovulation was synchronized in cattle within an 8 h period from 24 to 32 h after the second injection of GnRH. This precise synchrony allows for successful AI without detection of estrus in dairy cows. Pregnancy rates per AI in cattle bred at detected estrus after synchronization of estrus with Ovsynch protocol were comparable to that after spontaneous estrus (Pursley et al., 1997). This estrus synchronization protocol will overcome the problem of estrus detection in buffaloes, hence, if found effective it can enhance the reproductive efficiency in this species. There are only three recent reports using this protocol in half-bred (Murrah  Mediterranean) buffaloes (Berber et al., 2002), Egyptian buffaloes (Bartolomeu et al., 2002) and in

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Murrah buffaloes (Paul and Prakash, in press). There is a general consensus that the new protocol improves the fertility rate in buffaloes and could be used for set time artificial insemination in this species. 4.4. Synchronized estrus with Ovsynch treatment in yaks Estrus synchronization treatment using the Ovsynch protocol has been recently investigated in yaks (Sarkar and Prakash, in press-b). As reported by Paul and Prakash (in press) in buffaloes the synchronized preovulatory LH surge post second GnRH administration in yaks resulted in the animals ovulating within a short interval of 20 to 34 h after the GnRH injection.

5. Concluding remarks The positive correlation obtained between plasma prolactin concentrations and delay in postpartum commencement of cyclicity in buffalo needs further investigation. The control of prolactin secretion could hold the key for reducing the postpartum interval to first estrus in this species. Although, a number of endocrine techniques have been developed for fertility improvement, these have to be adopted at field levels in a big scale to make an impact towards increasing milk production. The endocrine studies in yaks, particularly in relation to breeding and non-breeding seasons provide valuable clues for improving fertility in this animal. The relationship of prolactin and melatonin secretion during the breeding and non-breeding seasons also provides valuable evidence for their role in reproduction. The observation that some yaks were found to be cycling in the non-breeding season opens the possibility of exploiting the females throughout the year for fertility improvement. Our observations on the successful application of Ovsynch protocol for ovulation synchronization in this species holds potential for fertility improvement using endocrine techniques.

References Arora, R.C., Pandey, R.S., 1982. Pattern of plasma progesterone, estradiol-17h, luteinizing hormone and androgen in non-

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