Study On The Follicle Growth And Development Law During Estrus Of Xinjiang Donkey

Object:Donkeys are seasonal estrous animals with a long gestation period of an average of 360days,low fertility,generally 3 years and 2 births,long estrus duration of donkeys,large follicular growth,no rupture for several days,The estrogen secreted by granulosa cells causes estrus symptoms for 5-7 consecutive days,which makes it difficult to accurately judge the time of ovulation,breeding,and insemination of donkeys during production,resulting in a low conception rate of donkeys and slow breeding income.Understand the changes in the follicle development and corpus IUteum formation of Xinjiang donkeys during the breeding cycle,and determine the ovulation time and interval,so as to timely breed or artificial insemination,reduce the number of empty days,and give full play to the breeding capacity of the donkey.Therefore,in this study,the developmental dynamics of follicles and corpus iuteum were detected by veterinary B-ultrasound;the changes of FSH,LH,E₂and P₄in the serum during the estrous cycle were measured by ELISA;and the development of follicles was determined by RT-q PCR The infiuence of genes on follicle development in Xinjiang donkeys.Methods:（1）In this experiment,10 healthy non-reproductive female donkeys from Xinjiang were selected during the estrus season,numbered,and raised by special personnel.Use B-ultrasound to detect the dynamics of follicle size and number development and the growth and degeneration of the corpus luteum every day;use B-ultrasound and naked eyes to observe when the female donkey ovulates after estrus symptoms.（2）Daily（10:00）,collect 10 m L of blood in the jugular vein with a disposable vacuum tube,centrifuge and dispense it in a 1.5 m L centrifuge tube,detect the concentration of reproductive hormones FSH,LH,E2,P4 by ELISA,and draw Hormone change curve.（3）Through live egg collection and isolated ovary aspiration of follicular fluid in large follicles with a diameter of more than 30 mm and small follicles with a diameter of less than 20mm,the granular cells of the follicles are separated by centrifugation,and the granular cells of different sizes of follicles are compared and analyzed by fluorescence quantitative PCR.Different expressions of genes related to follicular development（FSHR,EGF,BCL2,BAX,CXCR4,St AR,CPEB1,PRKAG2 and AMH）.Results:（1）The estrous cycle of Xinjiang female donkeys（counted as 0 days on the last ovulation day）averaged 22.7±1.49 days,and the maximum follicle diameter on the first day of the estrous cycle was 9.3±0.7 mm;the number of follicles in Xinjiang was also within the estrous cycle Up to6.8±0.8,the follicle diameter is 25.5±1.3 mm as the follicle develops and grows to mid-estrus;the number of follicles drops to 4.66±0.7,and the female donkey has estrus symptoms 5-7 days before ovulation.All the remaining follicles were atresia and degenerated,and the dominant follicles entered the rapid growth stage,and their diameter grew at an average speed of 3.0±2.2 mm/d until the largest follicle diameter reached 40.7±1.8 mm and ovulated.The corpus IUteum appeared in Xinjiang donkeys after ovulation and continued to exist until 18±1.3 days.By the 15th day after ovulation,the corpus IUteum grew to a maximum diameter of 30.5±3.5 mm,after which the corpus IUteum began to rapidly degenerate.The duration from estrus to ovulation in Xinjiang donkeys is generally（2-9）days,and 69%of female donkeys have estrus symptoms from estrus to ovulation concentration to 4-6 days.（2）During the estrous period of the measured female donkey,there will be two peaks and one trough in the serum concentration of FSH.The concentrations are5.45±0.29 m IU/m L;5.24±0.31 m IU/m L;3.61±0.18 m IU/m L.The LH content in the measured female donkey serum reached its maximum after ovulation,and its concentration level was 16.48±1.22 m IU/m L.When the female donkey showed estrus symptoms,the E2 content increased rapidly until it reached its peak one day before ovulation and its content was 203.59±9.25 pg/m L;the P₄content reached its peak on the 15th day after ovulation and its content was50.06±4.57 ng/m L,then continued to decline.（3）Studies on follicle development-related genes in Xinjiang donkey follicular found that BAX,FSHR,CXCR4,STAR and PRKAG2 genes are highly expressed in Xinjiang donkey follicles;CPEB1,AMH and BCL2 are highly expressed in Xinjiang donkey follicles.Conclusion:（1）The estrus cycle of Xinjiang donkeys is 22.7±1.49 days,and the largest follicle diameter during ovulation is 40.7±1.8 mm;the maximum number of follicles that can be recruited is 6.8±0.8;the corpus luteum can last for 18±1.3 days in the estrus cycle,and the diameter reaches the 15th day The maximum is 30.5±3.5 mm;69%of female donkeys ovulate4-6 days after the appearance of estrus symptoms.（2）During the estrus cycle,the FSH concentration presents a double-peak pattern;the LH concentration presents a pulsed distribution;P4 and E2 are antagonistic to each other.The content of P4 in mid-estrus was greater than that in the other three stages;the overall trend was to increase first and then decrease.（3）There are significant differences in the expression of follicular development-related genes in the follicular fluid of Xinjiang donkeys.These results provide a theoretical basis for studying the regulation mechanism of Xinjiang donkey follicles,selection and treatment of related diseases.