| There are two breeds of buffaloes（River and swamp）present in different parts of the world.Swamp buffaloes are more commonly found in China.This breed Characterized by low milk yield and poor reproductive efficiency as compared to River buffaloes.Therefore,the main objective of the current study was to find out new strategies to improve reproductive performance in Swamp buffaloes.Hybrid vigor is pivotal in improving existing performance of swamp buffaloes.Due to the difference in chromosome number（2n=48）of swamp buffaloes and（2n=50）of river buffaloes the aneuploidy（2n=49）was observed in the crossbred offspring,resulting in a lower reproduction rate.There are many reasons affect reproductive performance in water buffalo as small ovaries,irregular follicular development,irregular estrus and abnormal ovulation.Hence,it is very important to study the follicular and ovulatory control techniques development to enhance the reproduction rates by application of fix time artificial insemination which will ultimately improve the fertilization and pregnancy in buffalo thus speed up the breed improvement programs.In order to study the follicular dynamics,ultrasound diagnostic technology was applied to detect the ovarian follicles in different physiological periods and different estrous cycles,so a perfect technique can be established for the follicles development in crossbred buffaloes.A new technology（GPGMH）was developed for induction of estrus synchronization and timing artificial insemination of buffalo.Finally,the GPGMH method was further optimized by adding capsaicin or NCG to the diet according to the principle that capsaicin and NCG（n-glutamate）have the function of promoting blood circulation.The main research contents and results are as follows:1 Ovulatory follicle growth rules of crossbred buffalo during natural estrus1.1 Development rules of follicle at the puberty stage buffalo:To know when buffaloes start to alter follicular dynamics at the onset of puberty and body condition score,we selected crossbred buffaloes（n=28）with average age of 16 month to start the detection of follicular growth ultrasonically twice a day until arrive to first ovulation.In relation to the puberty and body condition score with reproductive performance,the results showed that the crossbred buffalos reach puberty at 23.59±0.4 months of age,the length of estrus cycle was 19.4±2.7 days and it had two follicles waves,the first wave appeared at 1.5±0.6days while the second wave appeared at 10.5±1.4 days with duration about 18.0±1.4 and10.6±0.3 days respectively.The maximum diameter of the dominant follicle was9.6±2.0mm during the beginning of season.On the other hand,the initial follicular diameter,maximum follicular diameter,follicular growth rate and estrus cycle length were5.4±0.9mm,9.2±2.2mm,0.76±0.06mm/day and 17.7±3.5 days respectively with multiple follicles development.1.2 Development rules of follicle in sexual maturity stage buffalo:To understand the estrus cycle of sexually mature buffalo,B-mode ultrasonic diagnosis technology was used to detect the follicular development,estrus and ovulation rules of 21 crossbred buffalos under natural estrus conditions.The results revealed that the length of estrus cycle at sexually mature buffaloes was 21.5±1.8 days,with 2 follicular waves in each estrus cycle.Two follicular waves were appeared,the first one at 1.0±0.5 days and the second one at 11.1±1.3 days with duration about 19.1±1.2 days and 10.2±1.7 days respectively.The maximum diameter of the dominant follicle was 10.6±0.5mm.In addition to,the initial follicular diameter,maximum follicular diameter,and follicular growth rate,estrus cycle length were 5.4±0.8mm,9.5±2.4mm,0.85±0.1 mm/day and21.6±2.0days respectively with multiple follicles development.1.3 Development rules of follicle and uterine regression of postpartum buffaloes:The B-mode ultrasonic diagnostic technique was used to track and detect the postpartum follicular development and uterine regression of 17 crossbred milk buffaloes.The results showed that the first,second and third ovulation periods of the healthy crossbred water buffaloes（n=10）were 27.7±7.1,48.5±8.4 and 65.0±13.8 days,respectively.The uterine cervix,pregnancy horn and empty horn were 33.6±2.4,36.8±3.4 and 31.6±1.8 days respectively.But,in cattle with uterine inflammation,the uterine cervix,pregnant horn and empty horn recuperation period were 43.1±5.2,45.6±6.8 and 39.6±5.7 days respectively and later than that in healthy cattle.The estrus cycle length of postpartum cows was 21.5±1.8 days,and 2 follicular waves dominated of each estrus cycle.The first and second waves occurred at 1.5±0.6 and 12.1±1.3 days respectively with a period about21.5±1.1 and 10.1±1.1 days,respectively,and the maximum diameter of the dominant follicle was 12.6±0.7mm.Also,the initial follicular diameter,maximum follicular diameter,follicular growth rate and estrus cycle length were 5.5±0.7mm,12.2±2.1mm,1.12±0.11 mm/day,and 21.9±2.1days respectively with multiple follicles development.2 Research on ovulatory regulation technology of crossbred buffalo in estrus2.1 Comparison of existing methods of estrus synchronization and timed insemination:In autumn,94 crossbred buffalo were selected and randomly and divided into 4 groups,which were treated with PmPgG（PMSG-PG-GnRH）,GPG（GnRH-PG-GnRH）,PGPE（PG-GnRH-PG-EB）and P4PmPgG（CIDR-PMSG-PG-GnRH）.The results showed that the GPG estrus synchronization protocols had the highest estrus rate（96.1%）,ovulation rate（84.6%）and ovulatory rate（30.8%）.2.2 Establishment of new GPGMH estrus synchronization protocols2.2.1 Determination of mifepristone dose in GPGMH method:In winter,65crossbred buffalo were selected,divided into four groups,all groups of buffalo on the basis of GPG estrus synchronization protocols,with the same time of the second injection of GnRH and body weight calculation,but injected 0（control）,0.3,0.4,and 0.5 mg/kg respectively of mifepristone at 24 hours after second injection of GnRH AI,then after 5days of AI injected the h CG 2000 IU（GPGMH method）was administrated.The optimal injection dose of mifepristone was 0.4mg/kg.Mifepristone results revealed that it had the highest estrus rate（82.4%）,ovulation rate（94.1%）and pregnancy rate（47.1%）.In contrast it had quiet estrus rate was about（11.8%）and follicular cyst lowest rates were about（5.8%）.2.2.2 Determination of mifepristone injection time in GPGMH method:In summer,52 crossbred buffaloes were selected and divided into three random groups.All of them were treated with GPGMH previously.The mifepristone was injected at the same time,4 hours before and 4 hours after the second injection of GnRH,to compare the effect of mifepristone on estrus synchronization protocols.The results showed that cows injected with mifepristone at the same time of the second injection of GnRH had an estrus rate of 100%,without follicular cyst.Although there was no significant difference in ovulation rate between the three groups,but the fertilization rate of cows injected with mifepristone at the second injection of GnRH was higher than that of other groups（p<0.05）.2.2.3 Determined of artificial insemination time in GPGMH method:In winter,100 crossbred buffaloes were selected and divided into 5 groups.All of them were treated with GPGMH previously.The time and frequency of insemination changed.The first 3groups received single artificial insemination injection at 20 hours（group 1）,24 hours（group 2）and 28 hours（group 3）after the second injection of GnRH.The other two groups received artificial insemination twice at 16,26 hours（4 groups）and 20,30 hours（5 groups）after the second injection of GnRH.The results showed that 24 hours after the second injection of GnRH was the best insemination time,and there was no significant difference between single and double insemination.2.2.4 The mechanism of action of progesterone and its antagonist mifepristone in GPGMH method:In summer,116 crossbred buffaloes were selected and divided into three groups,which were treated with GPG,GPGMH and P4GPG respectively.The results showed that there was no significant difference in estrus,ovulation and conception rate between the groups（P>0.05）.The GPGMH regime showed the lowest silent estrus and follicular cyst rates.The silent estrus rate was 6.7%and follicular cyst rate was 4.4%,indicating that the addition of progesterone in GPGMH method has a little significance,while the addition of progesterone antagonist mifepristone could reduce side effects.2.3 Optimization of GPGMH estrus synchronization protocols2.3.1 Optimization of GPGMH methods for young buffalo:In order to improve the effect of GPGMH method for young buffalo,the first GnRH injection was changed into PMSG on the basis of GPGMH method,and then the remaining steps in GPGMH method were followed.155 young crossbred buffalo were selected and allocated into four groups,at the same time rutting day 1000 IU per injected with PMSG,the 2nd day injected of chlorine forefront enol 0.5 mg,4th day injected of GnRH（including 200g/head）,during this time different dose of mifepristone 0,0.3,0.4 and 0.5 mg/kg body weight were injected respectively.At the 5th day injected of HCG 2000 IU（PmPGMH method）.The results showed that under the premise of replacing the first GnRH with PMSG,the estrus rate was（84.2%）,ovulation rate was（89.5%）and pregnancy rate was（47.4%）in the mifepristone 0.3mg/kg group were relatively higher than those in the other groups（P>0.05）,and the follicular cyst rate was lower than that in the other groups,but there was no significant difference（P>0.05）.Explanation:（1）PmPGMH method using PMSG instead of the first GnRH to treat the estrus conception rate of young cattle was better than GPGMH method;（2）when young buffalo were treated with PmPGMH,the optimal dose of mifepristone was 0.3mg/kg body weight.2.3.2 Improvement of GPGMH method of crossbred buffalo and cow with supplementation of capsaicin:In summer,101 crossbred buffaloes and 109 Holstein cows were selected and divided into 4 groups.The different supplementation doses（0,20,40 and 60mg）of capsaicin were added to the TMR diet per kilogram per day,respectively.After feeding for 1 month,GPGMH protocol was used for simultaneous estrus-timed sperm transfusion.The reproductive performance,milk yield,milk composition and serum biochemical indexes were determined.The estrus rate was（72.0%,18/25）,ovulation rate was（68.0%,16/25）,conception rate was（48.0%,12/25）,ovulatory follicle growth rate was（1.619 mm/day）and maximum follicle diameter was（13.7±1.5mm）in the crossbred buffaloes fed on diet had 40mg capsaicin extract per kg and there were relatively higher than other groups,but the difference between groups was not significant（p>0.05）.At the 20th and 25th days after the addition of capsaicin,the medium-dose group（40mg/kg）was significantly（P>0.05）improved the milk yield,the milk protein rate,milk fat rate,lactose rate and other indicators,and reduces the level of urea nitrogen in milk with increase the level of blood sugar.Especially,the milk yield was increased by16.61%.On the other hand,the estrus rate,ovulation rate,pregnancy rate and follicular development rate of Holstein cows fed on diet had 40mg/kg capsaicin added to each kg of diet were 78.6%,75.0%and 53.6%respectively.The maximum follicle diameter was13.8±0.4mm/day,and there were relatively higher than that of other groups,but the difference between groups was not significant（p>0.05）.At the 20th and 25th days after the addition of capsaicin,the medium-dose group（40mg/kg）was significantly（P>0.05）improve the milk yield,the milk protein rate,milk fat rate,lactose rate and other indicators,and reduces the level of urea nitrogen in milk with increase the level of blood sugar.Especially,the milk yield was increased by 26.67%.2.3.3 Improvement of GPGMH method in dairy cows with NCG:In summer,169 Holstein cows were selected and divided into four groups to investigate the effects of addition（NCG）20g/day supplement during heat stress.The first three groups were fed with NCG（20g/head）supplement for different duration time（15,30,45days）and the control group was fed with the basal diet and GPGMH regime was used for the treatment of estrus synchronization protocols.The animals group fed on 20g/45days of NCG showed the estrus rate was 82.5%,the ovulation rate was 80.0%,the conception rate was57.5%,follicle growth rate was 1.2337 mm/d with the largest follicle diameter was16.0±3.8 mm which relatively higher than other groups but the difference between groups was not significant（p>0.05）.It had the highest milk production from the 20th day after processing and increased by 29.54%higher than that of control group.Furthermore,blood glucose concentration and milk compositions such as milk protein rate,milk fat rate,lactose rate and other indicators increased but milk urea nitrogen level decreased. |
