J. Sci. Dev. 2010, 8 (Eng.Iss.1): 100 - 110 HA NOI UNIVERSITY OF AGRICULTURE 100 CHARACTERISTICS OF REPRODUCTION OF THE WATER BUFFALO AND TECHNIQUES USED TO IMPROVE THEIR REPRODUCTIVE PERFORMANCE Đặc điểm sinh sản và một số kỹ thuật ứng dụng nhằm cải thiện năng suất sinh sản của trâu Nguyen Hoai Nam Faculty of Veterinary Medicine, Hanoi University of Agriculture, Vietnam Corresponding author : hoainam26061982@yahoo.com. (066)874389148 TÓM TẮT Thành thục muộn, động dục thầm lặng, khoảng cách lứa đẻ dài và sinh sản theo mùa là những đặc điểm ảnh hưởng tới năng suất sinh sản của trâu. Nhiều biện pháp kỹ thuật đã được áp dụng nhằm cải thiện hiệu quả sinh sản của loài gia súc này. Sử dụng hoóc-môn gây động dục sau đó thụ tinh tại một thời điểm nhất định là kỹ thu ật được ứng dụng nhiều và cho một số kết quả khả quan. Gây rụng trứng nhiều hay hút trứng có thể thu được một số lượng lớn phôi, trứng trong một thời gian ngắn nhưng tỷ lệ trứng phát triển đến giai đoạn thụ tinh và tỷ lệ thụ tinh trong phòng thí nghiệm còn thấp. Cấy truyền phôi dù đạt được một số thành tựu nhưng hiệu quả của ph ương pháp này chưa ổn định. Nghiên cứu nhằm tăng năng suất sinh sản ở trâu nước là một vấn đề thiết yếu. Từ khóa: Sự thụ tinh, sự rụng trứng, sinh sản, trâu nước. SUMMARY Late puberty, silent heat, long calving interval and seasonal breeding are characteristics those hamper the reproductive performance of the water buffalo. Several techniques have been used to improve their reproductive efficiency. Ovulation program - fixed time artificial insemination (Ovsynch- TAI) has been applied as the most popular solution with prospective results. Superovulation demonstrates a fairly low number of embryos per buffalo in each session. Ovum pick-up (OPU) is superior to superovulation since it could yield quite a large amount of oocytes in a short time. Unfortunately, in vitro maturation (IVM) and in vitro fertilization (IVF) rates are still low. Embryo transfer has obtained some achievements. However, the efficacy of this technique is not really stable. Further studies on improving efficiency of reproduction in the water buffalo are essential. Key words: fertilization, ovulation, reproductive performance, water buffalo. 1. INTRODUCTION The water buffalo contributes substantially to the economy of many countries in tropic and subtropic regions including Indian sub-continent, China, South-East Asia, Mediterranean, South and Central America, Africa and Australia. They are classified into two distinct types as river and swamp buffaloes. While the river type is distributed largely in Indian sub-continent, Mediterranean regions, Caribbean, Africa and South America, the swamp buffalo is mostly found in China and South- East Asia (Kumar et al. 2007). The river buffalo is mainly used for milk, rather than the meat and draft purposes while the swamp buffalo is raised for power and meat as well. There is a genetic difference in the number of diploid chromosomes between river and swamp buffaloes, viz 50 in the former and 48 in the latter. Regardless of the difference, they could interbreed and produce fertile progenies (Bongso et al. 1983). The water buffalo is an ineffectively reproductive animal due to several features including late puberty, low population of ovarian follicles, silent heat, variable time of ovulation, Characteristics of reproduction of the water buffalo and techniques used to improve 101 seasonal breeding and long postpartum anestrus. To improve the poorly reproductive efficiency of the water buffalo, several techniques such as artificial insemination, superovulation, ovum pick-up, in vitro maturation, in vitro fertilization and embryo transfer have been applied with various achievements. The purpose of this article is to summarize the characteristics of reproduction in water buffaloes and solutions used to improve their reproductive performance. 2. CHARACTERISTICS OF REPRODUCTION IN THE WATER BUFFALO 2.1. Late puberty The age at puberty in buffaloes is much higher than that in cattle. In the male buffalo, the establishment of spermatogenesis is complete at the age of 24 months (McCool and Entwistle, 1989) compared with 4 months in cattle (Curtis and Amann, 1981). Though sperm production of swamp buffaloes is established at 12-15 months of age, they do not show in the ejaculation until buffaloes reach 2 years old (McCool and Entwistle, 1989). In the female swamp buffalo, puberty appears individually, seasonally, nutritionally and managerially different. According to a research in the Thai swamp buffalo, heifers reached puberty at the age of 3-4 years old when they were at 55-60% of their adult body weight (Bodhipaksha et al. 1987). In another research, the Thai swamp buffalo obtained puberty at around 24-25 months old (Kamonpatana et al. 1987). The swamp buffalo in the Philippines was reported to show the first estrus at the age of 26-29 months old while those in Cambodia reached puberty at around 3 years old and the swamp buffalo in Australia obtained puberty, even more early, at under 2 years old (Tulloch et al. 1981). In Vietnam, the female swamp buffalo reached puberty at about 30-36 months old (Cong et al. 1983). The age at puberty in river buffaloes also varies considerably. When the calves are fed sufficiently and sprayed with water during the hot season, their puberty was found to appear very early. Reports showed that puberty age ranged from 9.9 months to 24.7 months old in the Egyptian river buffaloes (Mohamed et al. 1980, Hafez et al. 1955, Salama et al. 1994, Barkawi et al. 1989). However, other breeds such as Murrah, Surti and Nili-Ravi were documented to obtain puberty later at 30-45.5 months old (Ishaq et al. 1972, Saini et al. 1998, Sule et al. 2001). 2.2. Less primary follicles The ovaries of buffaloes are smaller than those of cattle. There are less recruitable follicles at any given time in buffalo than those of the cattle. After birth, the ovaries of buffaloes have about 12,000 (Danell et al. 1987) primary follicles compared with 133,000 in cattle (Eriskson et al. 1966). The transition from the growing follicles to the secondary follicles stage is at a slow rate. The number of secondary follicles is only 7.56% of the growing follicles. Similarly, number of Graafian follicles > 1 mm in diameter is less while the follicular atresia is greater than that of cattle (Noakes et al, 2001). This may be a cause of poor reproductive performance in this type of animal. 2.3. Silent heat Due to the low level of blood oestradiol-17 beta, the expression of estrus in buffaloes is very poor. Silent heat is one of the deleterious features to the reproductive performance in the buffalo. Estrus often passes unoticedly, especially in the hot and dry seasons when grass, wallowing pools and shades are in deficiency which made the expression more dubious. A research in the Pakistani river buffalo showed that 51.5% of estrus was the silent heat (Qureshi and Ahmad, 2008). According to a study in the Egyptian river buffalo, there were two patterns of follicular waves in which the three follicular waves dominated the two follicular waves (Barkawi et al. 2009). In contrast, the one wave estrus cycles in the Thai swamp buffalo were depicted to be 22.7% while the two wave- patterns were 77.3% (Promdireg et al., 2004). Study of the follicular waves in the estrus cycles of the river buffalo showed that the one follicular wave patterns were quite usual (Awasthi et al. 2006). In one wave model, the ovulatory follicles persisted much longer than those in the 2 wave-pattern, and at the middle of the cycle there was a regression before a resurrection. The growth rate of ovulatory follicles was, therefore, slower than that in the 2 wave- patterns. The small size and slow growth rate of ovulatory follicles in the one follicular wave estrus were suggested to result in the low concentration of estrogen which was one of the causes of the silent heat in the river buffalo (Awasthi et al. 2007). Furthermore, the oocytes ovulated from the small sized follicles were hypothesized to have low quality which brought about the low fertility and conception Nguyen Hoai Nam 102 rate. For the proportion of silent heat much outnumbers the proportion of one follicular wave pattern and this pattern is only one of potential causes of silent heat, this suggests that there are various reasons rather than only follicular model. In the studies of using PGF 2 alpha to induce the luteolysis in buffaloes, the partial luteolysis was also pointed out as a probable reason of the silent heat (Dhaliwal et al. 1988, El-Belely et al. 1995). 2.4. Variable time of ovulation The estrus cycle of the swamp buffalo was reported to be between 11 and 38 days, with an average of 21,5± 4.7 days, 79,2% of the estrus being in the range of 17-26 days (Kanai and Shimizu, 1983). More than 20% of them were too long or too short. Duration of estrus in the swamp buffalo varies widely. It may range from 9-27 hours. The ovulation may occur during 6-21 hours after the end of estrus (Kanai and Shimizu, 1983). The short estrus was hypothetically induced by the one-wave cycle (Baruselli et al. 1996). This type of follicular wave cycle also generates variable durations of estrus which cause difficulties to the estimation of ovulation and appropriate time of artificial insemination. The time of ovulation was reported to depend on the protocols used to induce estrus (Warriach et al. 2008). In the PGF2 alpha and GnRH-induced estrus river buffaloes, the durations of standing estrus were 14.2±0.8 and 8.9±0.6 h, respectively. The intervals of ovulation after standing estrus were 30,6±1,5h and 15,0±0,8h in the PGF 2 alpha and GnRH–induced estrus buffaloes, respectively (Warriach et al. 2008). The ovulatory response of the river buffalo was found to be from 57.6%- 84.4% when eCG and CIRD were applied in ovulation synchronization (Murugavel et al. 2009). 2.5. Seasonal breeding The water buffalo is the multiestrus animal and the sexual activities can occur all round year. Nonetheless, the ovarian activities have shown to be characterized by the seasonal low breeding period during the hot season (Sule et al. 2001). The water buffalo is heat intolerant by nature (Chiu, 2003) they need shade and water or mud to get rid of the heat from the environment. In the summer, while the temperature is high, pools of water become disappear, grass is also scarce those factors contribute to a decrease in activities in the buffalo which results in weak libido in the male and poor reproductive performance in the female. The endocrine of both female and male buffalo changes throughout the year. In the low breeding season, the female river buffalo features a high concentration of prolactin and low concentration of progesterone and oestradiol-17beta (Roy and Prakash, 2007). This endocrine pattern may also be partially responsible for the low sexual activities and low fertility in the buffalo in low breeding season. The survival of embryo in the uterus is impaired due to the deficiency of progesterone in the hot season (Bahga and Gangwar, 1988). Hot season was also proved to adversely affect the number of oocytes collected per animal and the subsequent results of somatic cell nuclear transfer. In Vietnam, the optimum time of the year for working with buffalo oocytes was from January to April (Uoc et al. 2007). In male African buffaloes, the level of hormone testosterone and LH-receptors were higher in the breeding season compared to those in the low breeding season (Brown et al. 1991). In that study, when the high testosterone bulls were treated with GnRH or hCG the blood testosterone did not increase. This suggested that the sexual activities of those buffaloes in the breeding season were at peak. Moreover, the ejaculate volume, sperm motility, proportion of normal morphological spermatozoa was much higher in the breeding season than those in the low breeding season though the total amount of spermatozoa was the same. This result contradicted the conclusion that season did not have deleterious effects on the sperm quality in the swamp buffalo used as artificial insemination sires in Thailand (Koonjaenak et al. 2007). However, in that study, the experimented buffaloes were used in semen collecting station for artificial insemination where they were chosen, managed and taken care of very strictly and carefully and might not represent the buffaloes in the field. The seasonal reproductive characteristic in water buffalo also depends on melatonin excreted from pineal gland during the night and represents the endocrinal signal of the light-dark rhythm in the environment (Zicarelli et al. 1997, Di Palo et al. 1997). 2.6. Long postpartum anestrus The resumption of estrus postpartum is a critical factor to achieve a satisfactory production in buffaloes. In dairy cattle this period should be in about 60-80 days and conception must be obtained by 85-100 days post-calving to get desirable Characteristics of reproduction of the water buffalo and techniques used to improve 103 benefits. Unfortunately, postpartum estrus in buffaloes always comes much later than this figure. The long postpartum anestrus in the buffalo depends on several factors including: season of calving, uterine involution, suckling, milk yield, nutrition and body condition score at calving. The Australian swamp buffalo experienced a very wide range of postpartum anestrus period of 5.8 ± 3.3 months depending on the season of calving (McCool et al. 1987). The buffaloes calved in the spring seemed to have longer postpartum anestrus period than those calved during August to November. Significantly longer postpartum anestrus of 47 ± 4 and 84 ± 10 days in the hot season compared with 26 ± 4 and 40 ± 4 days in the cold season were documented by Khattab et al. (1995) and El-Sobhy et al. (1988), respectively. Longer acyclic postpartum period in the low breeding season than that in the breeding season was also described by Qureshi et al. (1998) and Perera et al. (1987). However, there were some contradictions exposing when seasons were approved to have no significant effects on the length of postpartum intervals (Qureshi et al. 1999, Patel et al. 1992, Mahdy et al. 2001). Uterine involution is also a key factor that impacts the time of postpartum anestrus in the water buffaloes. Normally, the involution of uteri would complete by 45 days post calving (Agrawal et al. 1978). Nevertheless, in any detrimental situations, this process is lengthened and results in the prolonged postpartum anestrus interval. Of all the uterine abnormalities, endometritis, perhaps, is the most prevalent disease (Azawi et al 2008). It was reported to be about 12.3% in a research on genital tracts of Iraqi buffaloes. Other types of metritis were also depicted however the occurrence was much lower with the prevalence of hydrometra, mucometra, pyometra were 0.2%, 0.7%, 0.49%, respectively (Azawi et al. 2008). Endometritis was also documented to be very high in Egyptian and Iranian buffaloes: 22.4%-47.9% (Al-Fahad et al., 2000; Alwan etal., 2001, Ghanem et al., 2002; Moghaddam et al. 2004; Moghami et al. 1996). Jainudeen et al. (1983) reported that uterine involution was at 28±6 days and 32% of the suckled Malaysian swamp buffaloes showed the first ovulation after calving 90 days, 68% of them were anestrus within 150 days postpartum. Subclinical uterine infection was supposed to slower the appearance of the first estrus post calving (El-Sheikh and Mahamed, 1976). Elongation of postpartum anestrus interval was found in the river buffaloes those produced more than 8kg of milk per day than those produced less than 8 kg of milk per day (El-Fadaly et al. 1980, El-Azab et al. 1984). Suckling obviously prolongs time of postpartum anestrus. Jainudeen et al. (1984) reported the weaned buffaloes at 30 days postpartum showed the first estrus earlier than those were suckled of 42 ± 8 and 55 ± 10 days, respectively. Similarly, milked buffaloes had longer acyclic interval of 72 ± 11 days compared with 44 ± 9 days in those who were weaned (Arya and Madan, 2001). Free suckled buffaloes had longest postpartum anestrus interval among restricted suckled and early weaned buffaloes in which those intervals were 82 ± 11 days, 69 ± 10 days and 50 ± 7 days, respectively (Nordin and Jainudeen., 1991). Nutrition plays a considerably important role in reproduction of water buffaloes. Shorter postpartum anestrus period achieved in those buffaloes who were fed high energy prepartum compared with those were not was proved by Salama et al. (1982) and Hegazy et al. (1994a). Nutrition has a close relation to body condition of the water buffaloes. In the buffaloes whose body condition score are low, the self tissues are used for the production of milk and other daily activities then they do not have enough energy stored for the next estrus cycle. This would elongate the interval of postpartum anestrus. The thin buffaloes were documented to be prolonged the time of acyclic postpartum than those had moderate body condition score; 63 days compared with 47 days (Hegazy et al. 1994b). In both river and swamp buffaloes, those had higher body condition were found to have shorter postpartum anestrus interval than those had lower body condition score (Baruselli et al., 2001). In addition, in a research on the India river buffalo by Palta and Madan, (1996), that the release of LH and FSH in response to exotic GnRH were progressively decreased with the advancement of gestation and low response after parturition regardless of the stable LH and FSH contents in the pituitary may suggest that this is one of the reasons of long postpartum anestrus in the buffalo. Reproduction of water buffaloes is greatly hampered by late attainment of puberty, seasonality of breeding, long postpartum anestrus. Moreover, silent heat and variable time of ovulation make the estrus detection very difficult. All of those mentioned characteristics bring about the poor performance of both reproduction and production of the water buffalo. Nguyen Hoai Nam 104 3. IMPROVEMENT OF REPREDUCTIVE PERFORMANCE IN THE SWAMP BUFFALO 3.1. Artificial insemination Artificial insemination (AI) has been used widely on dairy and beef cattle with satisfactorily stable conception rate whereas that in water buffaloes was reported to be various and humble. AI was mostly applied in buffaloes at fixed time following the ovulation synchronization programs (Ovsynch) which used several sexual hormones such as GnRH, progesterone, PGF 2 -alpha, PMSG, LH and estradiol-17 beta. The conception rates of water buffaloes were reported to range from 22.2% to 37.5% when PRID (contained progesterone and estradiol-17beta) and PMSG were used (Zicarelli et al. 1997, Barile et al. 2001, Pacelli et al, 2001). Neglia et al. (2003) found that the fertility rate of buffaloes induced estrus by GnRH and PMSG was from 28.2% to 36%. Similar results were also given by Paul and Prakash, (2005) and Chaikhun et al. (2009). According to these authors, when GnRH and PGF2alpha were applied in the Ovsynch, pregnancy rates of buffaloes were 30.7%-33.3% in the former research and 34.6%-34.9% in the latter research. In those studies, fixed time AI and AI at detected estrus gave similar results. Study comparing the effects of GnRH and LH in the Ovsynch conducted by de Araujo Berber et al. (2002) showed quite high fertility rates of 56.5% and 64.2%. By contrast, a very poor fertility rate was also documented of 12.5%-25%(Honnappagol and Patil.,1991). In the spontaneous estrus buffaloes, the conception rate varies depending on the time at which AI is performed. Kumaresan and Ansari, (2001) conducted AI at 6-12h, 12-18h and 18-24h on buffaloes after estrus, the pregnancy rates were shown to be 16.67%, 28.99% and 33.33%, respectively. Noncyclic buffaloes were announced to achieve lower fertility rate of 4.7%-30% compared with that of 35.7% - 51.5% in cyclic buffaloes (De Rensis et al. 2005). Low pregnancy rate in buffaloes might be explained by that the embryonic mortality rate between day 25 and day 40 post insemination was reported to be very high of 21%-50% (Campanile et al. 2005, Campanile et al. 2008, Vecchio et al. 2008). 3.2. Superovulation For the low amount of primordial follicles in the ovaries, the ovulation response in buffaloes is much less effective than that in cattle. Progesterone mare’s serum gonadotropin (PMSG) and follicle stimulating hormone (FSH) have been the most popular hormones used in superovulation programs. Due to a long half life (Schams and Himmler, 1978), PMSG induces a second follicular wave with anovulatory follicles after the first ovulation. These follicles secrete a large amount of oestradiol- 17beta that far exceeds the preovulatory concentration which results in an imbalance of progesterone:oestradiol-17beta ratio in the follicular fluid and unfavorable condition for the maturation and implantation of the oocytes and embryos in the oviduct and uterus (Schallenberger et al. 1990). The use of monoclonal antibodies against PMSG could reduce the peripheral inhibin of superovulated buffaloes and resulted in better results (Palta et al. 1996). Several efforts have been also made to increase the response of ovaries and production of embryos as well. The supplementation of GnRH at the standing heat and 8-12h after standing heat was conducted (Techakumphu et al., 2001). The numbers of embryos recovered from two treated and control groups were 2.33 ±2.24, 2.0±3.2, 1.91 ± 2.74, respectively. Although the production of embryos was not improved, the yield of transferable embryos in the group treated with GnRH at 8-12h after standing heat was highest. The use of oestradiol-17 beta and eCG as supplementation was reported to improve the ovarian stimulation in swamp buffaloes in Vietnam (Uoc et al. 1992, Nguyen et al. 1997). Number of recovered embryos and percentage of transferable embryos were documented to be of 3±1 and 75%, higher in the buffaloes supplemented with recombinant bovine somatotropin than those applied FSH alone, i.e. 0.8± 0.3 and 33 % (Songsasen et al. 1999). Vlakhov et al. (1986) demonstrated that the transferable embryos collected from each buffalo used FSH ranging between 2.1-2.6 embryos/ buffalo. A higher production was shown in the study by Misra et al. (1998). According to that research, by using FSH, those authors could recover 4.11 ± 2.46 embryos/ buffalo. 3.3. Ovum pick-up, in vitro maturation, in vitro fertilization and embryo transfer Ovum pick up in water buffaloes has demonstrated low efficiency. The number of oocytes collected in one session per animal is fairly low. Manjunatha et al. (2008) could recover 1.21 ± 0.07 oocytes/buffalo. Similarly, the same authors Characteristics of reproduction of the water buffalo and techniques used to improve 105 also collected 1.6±0.1 and 1.0±0.3 oocytes/buffalo in the high and low breeding season, respectively (Manjunatha et al. 2009). Researches on Thai swamp buffaloes showed a higher amount of 5.33 - 7.75 oocytes recovered from one buffalo (Techakumphu et al. 2004a; Techakumphu et al. 2004b). Those authors suggested that five repeat cycles of FSH and OPU did not influence the follicular response to the super-stimulation or the number of oocytes from the pre-pubertal and buffalo calves. Ovum pick-up was studied in buffaloes in different reproductive status with the similar results (Promdireg et al. 2005). The efficiency of PMSG used in OPU was found to be higher than that of FSH when the yields of oocytes collected were 8.3 ± 5 and 4.6±3.2, respectively (Techakumphu et al. 2000a). However, another study by the same authors gave a contradicted result when the application of FSH and GnRH tended to reproduce more oocytes collected/buffalo than those used PMSG and GnRH, i.e. 9.0±6.4 and 8.4±1.1, respectively (Techakumphu et al. 2000b). In vitro maturation and in vitro fertilization rates of buffalo oocytes are substantially various. A very low maturation rate of only 3% was announced by (Songsasen and Apimeteetumrong, 2002). This rate was improved by 23.5-42.5% in the study by Wani et al. (2004). Several reports showed that the maturation rate was ranged between 52.2-86.2% (Manjunatha et al. 2007, Techakumphu et al. 2000a, Uoc et al. 2007). Fertilization rate of buffalo oocytes is not as high as that in cattle. (Gasparrini et al., 2006) reported that the cleavage of oocytes was 55-78.4% while the rate of oocytes developed to the blastocyst stage was 17.1-30.9%. The same authors also found that the proportion of oocytes reached the stage of morulae–blastocyst was 25.7-32.6% (Gasparrini et al. 2004). In a research on Vietnamese swamp buffalo oocytes, the ratio of oocytes those could develop to blastocyst phase was 10.2-18.5% (Uoc et al. 2007). Embryo transfer in water buffalo has been conducted by several scientists with considerably variable results. The conception rate depends on the synchronization between the donors and receivers and fresh or frozen embryos. An overall pregnancy rate of 26.4% was established in the Indian buffalo. In that study, the conception rate was 40.7% in the receivers those had the same synchrony with the donors while the conception rates were 14.3% and 18.5% in the donors those were 12h deviatory estrus at either side from the receivers (Misra et al. 1999). Those authors also reported that the conception rates of buffalo transferred fresh and frozen-thawed embryos were 25.7% and 37.5%, respectively (Misra et al. 1990). These results contradicted what was found in a research on Chinese buffalo embryos. In that study, the conception rates of buffaloes received fresh sexed (26.5%) and unsexed (26.9%) embryos were higher than those received frozen-thawed sexed (11.6%) and unsexed (15.4%) embryos (Liang et al. 2008). In another Chinese study, twenty-nine swamp buffaloes were transferred fresh in vitro river and F1 buffalo cross (river x swamp) embryos, 41.4% recipients were pregnant and 10 calves were born accounting for 34.5% (Liang et al. 2007). The frozen effect on the conception rate was demonstrated by Techakumphu et al. (2001). Pregnancy rate in the fresh embryo transferred buffaloes was 35.7%, much higher than that in those transferred frozen-thawed embryos, i.e. 5.9%. Low conception and calving rates were also documented of 16.36% and 10.91%, respectively (Hufana-Duran et al. 2004). Certain of methods have been used to cope with reproductive aspect of water buffaloes. Artificial insemination, with some degree of success, is a preferable choice. However, other techniques such as superovulation, ovum pick-up, in vitro maturation and in vitro fertilization have limited application due to their low efficiency. 4. CONCLUSIONS The water buffalo is characterized by ineffectively reproductive performance. Late puberty reduces the duration of fertile life of this animal while silent heat and variable time of ovulation cause the difficulty to the estrus detection which results in either missing estrus unoticedly or obtaining low conception rate. In addition, seasonality of calving and long postpartum anestrus also detrimentally harm the efficiency of the buffalo’s reproduction. Several techniques have been employed to enhance the reproductive performance of the water buffaloes with certain level of success in each. However, no solution has been proved to be a highly efficient method. Artificial insemination needs the improvement related to the appropriate time of AI. Superovulation and ovum pick-up are obstructed by the low population of ovarian Nguyen Hoai Nam 106 follicles. Poor oocytes fertilization rate is the weak point of embryos in vitro production. In the future, studies are required to determine the appropriate time for fixed time artificial insemination, to improve the rates of in vitro maturation, in vitro fertilization and to ameliorate the pregnancy rate of embryo transfer techniques. Acknowledgement The author is grateful to Assoc. Prof. Suneerat Aiumlamai for her introducing the author into this topic, also for her encouragements and advices. REFERENCES Agrawal, K.P., Raizada, B.C., Pandey, M.D. (1978). Postparturient changes in the uterus of buffalo cows. Indian J. Anim. Sci. 47, 492–503. Al-Fahad TA, 2000. Morphological study of abnormal cases of female reproductive system of buffaloes in Basra Province. MSc Thesis, College Veterinary Medicine, Baghdad University, Baghdad, pp. 31–40. Alwan AF, Abdul-Hammed AN, Khammas DJ. (2001). A macroscopical study of abnormal genitalia of Iraqi female bualoes. Iraqi J Vet Sci, 14, 129–132. Arya, J.S. and Madan, M.L. (2001). Post partum reproductive cyclicity based on ovarian steroids in suckled and weaned buffaloes. Buffalo J., 17 (3): 361-369. Awasthi, M. K., Khare, A., Kavani, F. S., Siddiquee, G. M., Panchal, M. T., and Shah, R. R. (2006). Is one-wave follicular growth during the estrous cycle a usual phenomenon in water buffaloes (Bubalus bubalis)? Anim Reprod Sci 92, 241-53. Awasthi, M. K., Kavani, F. S., Siddiquee, G. M., Sarvaiya, N. P., and Derashri, H. J. (2007). Is slow follicular growth the cause of silent estrus in water buffaloes? Anim Reprod Sci 99, 258-68. Azawi.O.I, Ali.A.J, Lazim.E.H. (2008). Pathological and anatomical abnormalities affecting buffalo cows reproductive tracts in Mosul. Iraqi Journal of Veterinary Sciences. Vol.22.No.2,2008 (59-67). Bahga, C. S., and Gangwar, P. C. (1988). Seasonal variations in plasma hormones and reproductive efficiency in early postpartum buffalo. Theriogenology 30, 1209-23. Barile, V.L., Galasso, A., Marchiori, E., Pacelli, C., Montemurro, N. and Borghese, A. (2001). Effect of PRID treatment on conception rate in Mediterranean buffalo heifers. Livest. Prod.Sci., 68: 283-287. Barkawi, A.H., Mokhless, E.M. and Bedeir, L.H. (1989). Environmental factors affecting age at puberty in Egyptian buffaloes. Buffalo J., 5 (1): 71-78. Barkawi, A. H., Hafez, Y. M., Ibrahim, S. A., Ashour, G., El-Asheeri, A. K., and Ghanem, N. (2009). Characteristics of ovarian follicular dynamics throughout the estrous cycle of Egyptian buffaloes. Anim Reprod Sci 110, 326-34. Baruselli, P. S., Mucciolo, R. G., Visintin, J. A., Viana, W. G., Arruda, R. P., Madureira, E. H., and Molero-Filho, J. R. (1996). Ovarian follicular dynamics during the estrus cycle in buffalo (Bubalus bubalis). Preliminary research. Ann N Y Acad Sci 791, 408-11. Baruselli, P.S., Barnabe, V.H., Barnabe, R.C., Visintin, J.A., Molero-Filho, J.R. and Porto R. (2001). Effect of body condition score at calving on postpartum reproductive performance in buffalo. Buffalo J., 17 (1): 53-65. Bodhipaksha P.(1987). Physiology of female swamp buffalo reproduction. In swamp Buffalo Reproduction. Dept. Obstetrics Gynecology and Reproduction, Fac Vet Sci;Chulalongkorn University ed. 118p. Bongso, T. A., Hilmi, M., and Basrur, P. K. (1983). Testicular cells in hybrid water buffaloes (Bubalus bubalis). Res Vet Sci 35, 253-8. Brown, J. L., Wildt, D. E., Raath, J. R., de Vos, V., Howard, J. G., Janssen, D. L., Citino, S. B., and Bush, M. (1991). Impact of season on seminal characteristics and endocrine status of adult free- ranging African buffalo (Syncerus caffer). J Reprod Fertil 92, 47-57. Campanile, G., Neglia, G., Gasparrini, B., Galiero, G., Prandi, A., Di Palo, R., D'Occhio, M. J., and Zicarelli, L. (2005). Embryonic mortality in buffaloes synchronized and mated by AI during the seasonal decline in reproductive function. Theriogenology 63, 2334-40. Campanile, G., Vecchio, D., Di Palo, R., Neglia, G., Gasparrini, B., Prandi, A., Zicarelli, L., and D'Occhio, M. J. (2008). Delayed treatment with GnRH agonist, hCG and progesterone and reduced embryonic mortality in buffaloes. Theriogenology 70, 1544-9. Chaikhun.T, Tharasanit.T, Techakumphu.M, (2009). Efficiency of ovulation synchronization and fixed –time artificial insemination program Characteristics of reproduction of the water buffalo and techniques used to improve 107 in swamp bufflo in small holder farms. In: Proceeding of the Second FASAVA Congress, Bangkok, Thailand, pp 532-533. Chiu,Y.H. (2003). Image of Taiwan cattle.http://www.angrin.tlri.gov.tw/apec2003/C hapter2Cattle_2.pdf. Cited: 25/01/2010. Curtis, S. K., and Amann, R. P. (1981). Testicular Development and Establishment of Spermatogenesis in Holstein Bulls. J. Anim Sci. 53, 1645-1657. Danell, B., (1987). Oestrus behaviour, ovarian morphology and cyclical variation in the follicular system and endocrine pattern in water buffalo heifers. Ph.D Dissertation, Swedish University of Agricultural Sciences. Uppsala, Sweden. de Araujo Berber, R. C., Madureira, E. H., and Baruselli, P. S. (2002). Comparison of two Ovsynch protocols (GnRH versus LH) for fixed timed insemination in buffalo (Bubalus bubalis). Theriogenology 57, 1421-30. De Rensis, F., Ronci, G., Guarneri, P., Nguyen, B. X., Presicce, G. A., Huszenicza, G., and Scaramuzzi, R. J. (2005). Conception rate after fixed time insemination following ovsynch protocol with and without progesterone supplementation in cyclic and non-cyclic Mediterranean Italian buffaloes (Bubalus bubalis). Theriogenology 63, 1824-31. Dhaliwal et al., G.S. Dhaliwal, R.D. Sharma and G. Singh. (1988). Efficacy of prostaglandin F 2α administration for inducing estrus in buffaloes, Theriogenology 29 (1988), pp. 1401–1406. Di Palo, R., Parmeggiani, A., Spadetta, M., Campanile, G., Esposito, L., Seren, E. and Zicarelli,L. (1997). Influence of changing farm on repeatability of melatonin plasma level in Italian Mediterranean buffalo. In: Proc. Fifth World Buffalo Congress, Caserta, Italy, 13-16 October:758-761. Gasparrini, B., Boccia, L., Rosa, A. D., Palo, R. D., Campanile, G., and Zicarelli, L. (2004). Chemical activation of buffalo (Bubalus bubalis) oocytes by different methods: effects of aging on post-pathogenetic development. Theriogenology 62, 1627-37. Gasparrini, B., Boccia, L., Marchandise, J., Di Palo, R., George, F., Donnay, I., and Zicarelli, L. (2006). Enrichment of in vitro maturation medium for buffalo (Bubalus bubalis) oocytes with thiol compounds: effects of cystine on glutathione synthesis and embryo development. Theriogenology 65, 275-87. Ghanem M, Shalaby AH, Sharawy S, Saleh N, (2002). Factors leading to endometritis in Egypt with special reference to reproductive performance. J Reprod Sci 48, 371–375. El-Azab, E.A., Mansour, H., Heshmat, H., Shawki, G. (1984). The postpartum period and the future fertility of the Egyptian buffalo. In: Proceedings of the 10th International Congress on Animal Reproduction and AI, vol. III, No. 424, Urbana, p.3. El-Belely et al., M.S. El-Belely, H.M. Eissa, H.E. Omaima and I.M. Ghoneim. (1995).Assessment of fertility by monitoring changes in plasma concentration of progesterone, oestradiol 17-β, androgens and estrone sulphate in subestrus buffalo cows treated with prostaglandin F 2α , Anim. Reprod. Sci. 40 (1995), pp. 7–15. El-Fadaly, M.A., (1980). Effect of suckling and milking on the breeding efficiency of buffaloes. I. First postpartum estrus. Vet. Med. J. (Giza) 28, 399–404. El-Sheikh, A.S., Mohamed, A. (1976). First postpartum estrus in buffaloes. Indian J. Anim. Sci. 46, 580–583 El-Sobhy, H.E., Khalil, F.A., Abdelaal, A.E., El- Fouly, M.A. (1988). Use of progesterone levels in peripheral blood for studying reproductive patterns of Egyptian buffaloes. In: Optimizing Grazing Animal Productivity in Mediterranean and North African Regions With the Use of Nuclear Techniques. IAEA, Vienna, Austria, pp. 231–237. Erickson, B. H. (1966). Development and senescence of the postnatal bovine ovary. J. Anim. Sci. 25:800–805. Hafez, E.S.E. (1955). Puberty in the buffalo cow. J. Agr. Sci., 46: 137-142. Hegazy, M.A., El-Wishy, A.B., Youssef, A.H., Awadalla, S.A., Teleb, H.M. (1994a). Interrelationship between pre-and/or post-partum feeding levels, blood constituents and reproductive performance of buffaloes. In: Proceedings of the Fourth World Buffalo Congress, vol. III, San Paulo, Brazil, pp. 632–633. Hegazy, M.A., El-Essawy, S.A., El-Wishy, A.B., Youssef, A.H. (1994b). Effect of body condition score on reproductive performance of buffaloes. In: Proceedings of the Fourth World Buffalo Congress, vol. III, Sao Paulo, Brazil, pp. 630- 631. Honnappagol, S.S. and Patil, R.V. (1991). Oestrus synchronization and fertility in buffalo heifers Nguyen Hoai Nam 108 using Carboprost Tromethanine. Indian J. Anim. Reprod., 12 (2): 177-179. Hufana-Duran, D., Pedro, P. B., Venturina, H. V., Hufana, R. D., Salazar, A. L., Duran, P. G., and Cruz, L. C. (2004). Post-warming hatching and birth of live calves following transfer of in vitro- derived vitrified water buffalo (Bubalus bubalis) embryos. Theriogenology 61, 1429-39. Ishaq, S.M. (1972). Ninth Annual Report. Directorate of Livestock Farms, Punjab, Lahore. Jainudeen, M.R., Bongso, T.A., Tan, H.S., (1982/1983). Postpartum ovarian activity and uterine involution in the suckled swamp buffalo (Bubalus bubalis). Anim. Reprod. Sci. 5, 181-190. Jainudeen, M.R., Sharifuddin, W., Yap, K.C., Bakar Dahari, A., (1984). Postpartum anestrus in the swamp buffalo. In: The Use of Nuclear Techniques to Improve Domestic Buffalo Production in Asia. IAEA, Vienna, Austria, pp. 29-41. Kamonpatana, M., Pansin, C., Timsard, V., Limsakul, A., Vechabusakorn, A., Savasri, S.,Parnpai, R. and Pikultong, P. (1987). Indication of puberty as related to fertility based on bodyweight, age and profiles of reproductive hormones in swamp buffaloes. Buffalo J., 3 (2):181-194. Kanai, Y., and Shimizu, H. (1983). Characteristics of the estrous cycle of the swamp buffalo under temperate conditions. Theriogenology 19, 593-602. Khattab, R.M., El-Wardani, M.A., Enaam, M.M., Barkawi, A.H., (1995). Patterns of ovarian activity influencing calving interval of Egyptian buffaloes in relation to season of calving. J. Agric. Sci. Mansoura Univ. (Egypt) 20, 4331–4335. Koonjaenak, S., Chanatinart, V., Aiumlamai, S., Pinyopumimintr, T., and Rodriguez-Martinez, H. (2007). Seasonal variation in semen quality of swamp buffalo bulls (Bubalus bubalis) in Thailand. Asian J Androl 9, 92-101. Kumar, S., Nagarajan, M., Sandhu, J. S., Kumar, N., Behl, V., and Nishanth, G. (2007). Mitochondrial DNA analyses of Indian water buffalo support a distinct genetic origin of river and swamp buffalo. Anim Genet 38, 227-32. Kumaresan, A. and Ansari, M.R. (2001). Evaluation of conception rate in buffaloes (Bubalusbubalis) with reference to semen quality, stage of oestrus and inseminator. Indian J. Anim.Sci.,71 (2): 144-145. Le Xuan Cong, (1983). Performances in Vietnamese Swamp buffalo. Buffalo Bulletin, 2 (2):12-13. Liang, X., Zhang, X., Yang, B., Cheng, M., Huang, F., Pang, C., Qing, G., Liao, C., Wei, S., Senatore, E. M., Bella, A., and Presicce, G. A. (2007). Pregnancy and calving rates following transfer of in-vitro-produced river and F1 (river x swamp) buffalo (Bubalus bubalis) embryos in recipients on natural oestrus or synchronised for ovulation. Reprod Fertil Dev 19, 670-6. Liang, X. W., Lu, Y. Q., Chen, M. T., Zhang, X. F., Lu, S. S., Zhang, M., Pang, C. Y., Huang, F. X., and Lu, K. H. (2008). In vitro embryo production in buffalo (Bubalus bubalis) using sexed sperm and oocytes from ovum pick up. Theriogenology 69, 822-6. Mahdy, A.E., El-Shafie, O.M., Rigalaty, H.A., (2001). Relative importance of some factors affecting performance traits in a herd of Egyptian buffaloes. Alex. J. Agric. Res. 46, 1-18. Manjunatha, B. M., Gupta, P. S., Devaraj, M., Ravindra, J. P., and Nandi, S. (2007). Selection of developmentally competent buffalo oocytes by brilliant cresyl blue staining before IVM. Theriogenology 68, 1299-304. Manjunatha, B. M., Ravindra, J. P., Gupta, P. S., Devaraj, M., and Nandi, S. (2008). Oocyte recovery by ovum pick up and embryo production in river buffaloes (Bubalus bubalis). Reprod Domest Anim 43, 477-80. Manjunatha, B. M., Ravindra, J. P., Gupta, P. S., Devaraj, M., and Nandi, S. (2009). Effect of breeding season on in vivo oocyte recovery and embryo production in non-descriptive Indian river buffaloes (Bubalus bubalis) Anim Reprod Sci 111, 376-83. McCool, C.J., Townsend, M.P., Wolfe, S.G. and Entwistle, K.W. (1987). Endocrinological studies on pregnancy, post partum anoestrus and seasonal variation of ovarian activity in the Australian Swamp buffalo cow. Buffalo J.,1: 67-72. McCool, C. J., and Entwistle, K. W. (1989). The development of puberty and sexual maturity in the Australian Swamp buffalo bull. Theriogenology 32, 171-84. Misra, A. K., Joshi, B. V., Agrawala, P. L., Kasiraj, R., Sivaian, S., Rangareddi, N. S., and Siddiqui, M. U. (1990). Multiple ovulation and embryo transfer in Indian buffalo (Bubalus buoalis ). Theriogenology 33, 1131-41. Misra, A. K., Kasiraj, R., Rao, M. M., Rangareddy, N. S., Jaiswal, R. S., and Pant, H. C. (1998). Rate of transport and development of preimplantation embryo in the superovulated Characteristics of reproduction of the water buffalo and techniques used to improve 109 buffalo (Bubalus bubalis). Theriogenology 50, 637-49. Misra, A. K., Rao, M. M., Kasiraj, R., Reddy, N. S., and Pant, H. C. (1999). Factors affecting pregnancy rate following nonsurgical embryo transfer in buffalo (Bubalus bubalis): a retrospective study. Theriogenology 52, 1-10. Moghaddam AAI, Mamoei M, (2004). A survey on some of the reproductive and productive traits of the buffalo in Iran. 23rd World Buiatrics Congress, Quebec, Canada, Abstract 2910. Moghami SM, Saiyari M, Mayatri M, Sharma RN. (1996). Pathology of uterus in buffalo slaughtered in Ahwaz (Iran) abattoir. Buffalo J 12, 213–218. Mohamed, A.A., El-Ashry, M.A. and El-Serafy, A.M. (1980). Reproductive performance of buffalo heifers bred at a young age. Indian J. Anim. Sci., 50: 8. Murugavel, K., Antoine, D., Raju, M. S., and Lopez-Gatius, F. (2009). The effect of addition of equine chorionic gonadotropin to a progesterone-based estrous synchronization protocol in buffaloes (Bubalus bubalis) under tropical conditions. Theriogenology 71, 1120-6. Neglia, G., Gasparrini, B., Di Palo, R., De Rosa, C., Zicarelli, L., and Campanile, G. (2003). Comparison of pregnancy rates with two estrus synchronization protocols in Italian Mediterranean Buffalo cows. Theriogenology 60, 125-33. Nguyen, T. U., Duong, D. L., van Ty, L., Chupin, D., Renard, J. P., and Nguyen, B. X. (1997). Effects of estradiol-17 beta and hCG supplementation on superovulatory responses and embryo quality in swamp buffalo (Bubalus bubalis) implanted with norgestomet. Anim Reprod Sci 47, 181-7. Noakes,D.E., Parkinson,T.J., England, G.C.W. (2001). Arthur’s veterinary reproduction and obstetrics. W.B. Saunders press, 8th edition. p.789. Nordin, Y., Jainudeen, M.R. (1991). Effect of suckling frequency on postpartum reproductive performance of swamp buffaloes. In: Proceedings of the Third World Buffalo Congress, vol. II, Sofia, Bulgaria, pp. 737–743. Pacelli, C., Barile, V.L., Lenza, R., Terzano, M.G., Montemurro, N. and Borghese, A. (2001).Comparison of two different doses of PMSG on conception rate in Mediterranean buffalo heifers treated with PRID. In: Proc. Sixth World Buffalo Congress, Maracaibo, Venezuela, 20-30 May:160-165. Palta, P., and Madan, M. L. (1996). Effect of gestation on GnRH-induced LH and FSH release in buffalo (Bubalus bubalis). Theriogenology 46, 993-8. Patel, K.K., Ninan, J., Suthar, B.N., Kavani, F.S. (1992). Factors affecting fertile postpartum estrus in Mensoni buffaloes. Cheiron 21, 39–43. Perera, B.M.A.O., de Silva, L.N.A., Kuruwita, V.Y., Karunaratne, A.M. (1987). Postpartum ovarian activity, uterine involution and fertility in indigenous buffaloes at a selected village location in Sri Lanka. Anim. Reprod. Sci. 14, 115–127. Paul, V., and Prakash, B. S. (2005). Efficacy of the Ovsynch protocol for synchronization of ovulation and fixed-time artificial insemination in Murrah buffaloes (Bubalus bubalis). Theriogenology 64, 1049-60. Promdireg A , Na-Chiangmai A., Techakumphu M, (2004). Follicular dynamics in swamp buffalo cows (Bubalus bubalis). The 30th Veterinary Medicine and Livestock De- velopment Annual Conference, The Thai Veterinary Medical Association Under the Royal Patronage,Bangkok, p74 Promdireg, A., Adulyanubap, W., Singlor, J., Na- Chiengmai, A., and Techakumphu, M. (2005). Ovum pick-up in cycling and lactating postpartum swamp buffaloes (Bubalis bubalis). Reprod Domest Anim 40, 145-9. Qureshi, M.S., Samad, H.A., Nazir Ahmad, N., Habib, G., Anjun, A.D., Siddiqui, M.M., (1998). Reproductive performance of dairy buffaloes under peri-urban commercial farming in VWFP, Pakistan. Pakistan Vet. J. 18, 197–201. Qureshi, M.S., Samad, H.A., Habib, G., Usmani, R.H., Siddiqui, M.M., (1999). Study on factors leading to seasonality of reproduction in dairy buffaloes. I. Nutritional factors. Asian-Aus. J. Anim. Sci. 12, 1019–1024. Qureshi, M. S., and Ahmad, N. (2008). Interaction of calf suckling, use of oxytocin and milk yield with reproductive performance of dairy buffaloes. Anim Reprod Sci 106, 380-92. Roy, K. S., and Prakash, B. S. (2007). Seasonal variation and circadian rhythmicity of the prolactin profile during the summer months in repeat-breeding Murrah buffalo heifers. Reprod Fertil Dev 19, 569-75. . dài và sinh sản theo mùa là những đặc điểm ảnh hưởng tới năng suất sinh sản của trâu. Nhiều biện pháp kỹ thuật đã được áp dụng nhằm cải thiện hiệu quả sinh sản của loài gia súc này. Sử dụng. TECHNIQUES USED TO IMPROVE THEIR REPRODUCTIVE PERFORMANCE Đặc điểm sinh sản và một số kỹ thuật ứng dụng nhằm cải thiện năng suất sinh sản của trâu Nguyen Hoai Nam Faculty of Veterinary Medicine,. tinh tại một thời điểm nhất định là kỹ thu ật được ứng dụng nhiều và cho một số kết quả khả quan. Gây rụng trứng nhiều hay hút trứng có thể thu được một số lượng lớn phôi, trứng trong một thời