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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

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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 thuật 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 phương 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,

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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

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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 PGF2 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

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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

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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, PGF2-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

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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

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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

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