1. Effects of Different Month, Semen and Parity on Bovine Superovulation Traits reporter ： Jia-Bao Zhang Xian-Feng Yu College of Animal Sciences, Jilin University National beef cattle industrial technology system

2. Content  Research background  Research materials and methods  Results and discussion

3. Research background  Assisted reproductive technology  Superovulation  Artificial insemination  Embryo transfer 。。。

4.  Superovulation Induction of ovarian follicle development and ovulation by Exogenous gonadotropins and the ovulated oocytes have fertilization ability. FSH Vitamin A,DE2 PG LHRH-A 3 Plug CIDR

5.  Artificial insemination (AI) Collecting the male semen with Specific instruments and then inseminating with the semen into estrus female reproductive tract after examination, dilution, preservation, etc. It is a kind of reproductive technology that can replace natural mating breeding. Nitrogen canisterWaterbathDry oven Insemination gun sheath for straws

6.  Embryo Transfer (ET) The process of assisted reproduction in which early embryos are transfer into the uterus of a estrus female with the intent to establish a pregnancy. Embryo transfer apparatus SheathSelection and loading into straw

7. Research object  TianYiGangShan black cows ， more than 500 recipients  Time ： From June to September  Place ： TianYiGangShan Livestock farm, Jiaohe, Jilin

8. Management of donor cow  Health, drying, suitable temperature of cowshed.  Keep stable environment and avoid stress  Meet the needs for clean drinking water of the donor cattle  Rational feedstuff formulation of donor cattle Example of Feed formulation of donor cattle CompositionAmount Compound feed3kg Yellow Storage13kg Leymus chinensis3kg Medicago sativa1.5kg Total20.5kg Feedstuff formulation ： corn 54.5% ； soybean 24% ； bran16% ； vitamin 0.5% ； stone dust 1.5% ； calcium bicarbonate 1.5% ； Feeding salt 1% ； premix 1% 。

9. Days01567891016 Reagents Vade 10 ml β-E 2 0.5 mg FSH 2 mg FSH 1.5 mg FSH 1 mg PGF2 α 0.6 mg FSH 0.5 mg Vade 10 ml LHRH-A 3 (if possible) 25 μg LHRH-A 3 (if possible) 25 μg Action Put in the plug Remove the plug Observe the estrous/Artif icial inseminatio n Artificial inseminatio n Colle cting embr yos Table 1 Super ovulation process ： CIDR+FSH+PG 16 days scheme Note: Vade: 10 ml Contains vitamins A 500 000 IU ， vitamins D 50 000 IU 。 LHRH-A 3 ： luteinizing hormone releasing hormone A 3 Drug delivery time:7 am and 7 pm. FSH and PG twice a day, The rest are once a day in the morning or in the afternoon.

10. Put in the plugInjection detection of oestrus Artificial inseminationCollecting embryos Identification of embryos embryo cryopreservation

11. Results  Effect of superovulation in different month  Effect of superovulation in different Semen  Effect of superovulation in different Donor cattle

12. The classification standard of embryo Embryo development stage ： 4 Morula ， 5 Early blastocyst ， 6 Blastocyst ， 7 Expanded blastocyst ， 8 hatched blastocyst.

15. June July August September Non fertilized embryo number1.74±0.3142.24±0.2821.51±0.2391.72±0.292 Non fertilized embryo rate0.154±0.02460.154±0.01620.103±0.01640.151±0.0196 Degenerated embryo number2.47±0.3383.09±0.3045.54±0.3212.61±0.251 Degenerated embryo rate0.209±0.02270.236±0.01830.238±0.01830.241±0.0218 Embryo number7.87±0.7718.68±0.5979.14±0.5747.15±0.556 Available embryo rate0.622±0.03320.613±0.02850.659±0.02280.608±0.0277 M1embryo number5.11±0.5776.88±0.5257.15±0.5105.85±0.457 M1 embryo rate0.451±0.0335 aA 0.573±0.0287 b 0.628±0.0231 B 0.601±0.0274 B M2 embryo number2.09±0.252 a 1.23±0.156 b 1.64±0.140 ab 1.20±0.177 b Blastocysts number0.46±0.133 a 0.52±0.128 a 0.41±0.123 ab 0.09±0.035 b Blastycysts rate0.030±0.0074 a 0.039±0.0098 a 0.031±0.0087 ab 0.007±0.0030 b Total embryo number12.08±0.95513.96±0.71414.18±0.74911.48±0.960 The representation of the data is :mean ± S.E.M. M1: Dead cells scattered around the morula cells<15% ， 15% <M2< 50% a ， b Indicate P<0.05 ， A ， B Indicate P<0.01 。 M1 embryo rate= （ M1 embryo number+ blastocyst number ） / Available embryo number ， Available embryo rate= Blastocyst number/ Blastula number Table 2. Effect of superovulation in different month

16. Figure 1 ： The average embryo numbers of M1 in July and August was higher than that in June and September, and June was the lowest( P ﹥ 0.05 ）。 Figure 2: The average embryo rate of M1 in August and September was significantly higher than that in June (P<0.01), and then in July was higher than that in June (P<0.05). Figure 3: The average blastocysts rate in June,July and August was significantly higher than that in September (P<0.05). Figure 4: The average embryo numbers in July and August was higher than that in June and September, and September was the lowest (P > 0.05).

17. ℃ mm Average number of sunshine hours per month h Figure 5:The average temperature in June,July and August was significantly higher than that in September, July and August are the highest Figure 6: The average precipitation in June,July and August was significantly higher than that in September. Figure 7:The average sunshine hours in July and August was significantly higher than that in September.The red part represents the time range of between the maximum and the minimum duration of sunshine.

18. 97007 2 2 Total embryo number12.52±1.10013.37±0.535 Non fertilized embryo number 1.84±0.351.66±0.160Non fertilized embryo rate 0.165±0.02830.133±0.0128 Degenerated embryo number 2.63±0.3893.06±0.219Degenerated embryo rate 0.219±0.02620.227±0.0139 Available embryo number 8.07±0.9028.68±0.453Available embryo rate0.617±0.03670.646±0.0204 M1embryo number5.25±0.672 A 6.89±0.395 B M1embryo rate0.437±0.0382 A 0.616±0.0202 B M2embryo number2.33±0.292 A 1.38±0.127 B Blastocyst number0.48±0.1550.41±0.091Blastocyst rate0.028±0.00800.030±0.0063 Table 3. Effect of Superovulation of different batches of semen The representation of the data :mean ± S.E.M. A ， B b Indicate P<0.01 。 M1 embryo rate=(M1embryo numbers+ Blastocyst numbers) / Available embryo numbers, Blastocyst rate= Blastocyst numbers / Available embryo numbers.

19. Figure 8: the average M1 embryo rate of no.2 bull’s semen by artificial insemination were significantly higher than that of no.97007 bull (P<0.01) Figure 9: The average M1 embryo number of no.2 bull’s semen by artificial insemination were significantly higher than that of no.97007 bull (P<0.01) Figure 10: The average M2 embryo number of no.2 bull’s semen by artificial insemination were significantly lower than that of no.97007 bull (P<0.01)

20. nonparous cattleparous cattle total embryo number11.83±0.503 A 14.44±0.580 B Non fertilized embryos number1.68±0.1611.96±0.239 Degenerated embryo number3.11±0.2042.81±0.231 Available embryo number7.04±0.369 A 9.69±0.494 B M1embryo number5.16±0.309 A 7.76±0.415 B Blastocyst number0.43±0.0850.30±0.070 Non fertilized embryos rate0.146±0.01310.131±0.0136 Degenerated embryo rate0.263±0.0141 A 0.197±0.0139 B Available embryo rate0.582±0.0180 A 0.679±0.0209 B M1embryo rate0.527±0.0182 A 0.627±0.0216 B Blastocyst rate0.034±0.0062 a 0.018±0.0044 b The representation of the data is :mean ± S.E.M. A ， B Indicate P<0.01. M1 embryo rate= （ M1embryo number+ Blastocyst number ） / Available embryo number, Blastocyst rate= Blastocyst number / Available embryo number Table 4. Difference of nonparous and parous cattle on superovulation

21. Indicate: P<0.01 Figure 11: The average total number of embryos in parous cattle was significantly higher than that of nonparous cattle. Figure 12: The average non fertilized embryos rate in parous cattle was lower than that of nonparous cattle. （ P ﹥ 0.05 ） Figure 13: The average non fertilized embryo numbers in parous cattle was higher than that of nonparous cattle. （ P ﹥ 0.05 ） Figure 14: The average Degenerated embryo number in parous cattle was lower than that of nonparous cattle. （ P ﹥ 0.05 ）

22. Figure 15: The average degenerated embryo rate in parous cattle was significantly lower than that of nonparous cattle. Figure 16: The average Available embryo rate in parous cattle was significantly higher than that of nonparous cattle. Figure 17: The average Available embryo number in parous cattle was significantly higher than that of nonparous cattle. Figure 18: The average M1embryo number in parous cattle was significantly higher than that of nonparous cattle. Indicate: P<0.01

23. Figure 19: The average M1embryo rate in parous cattle was significantly higher than that of nonparous cattle. Figure 20: The average blastocyst rate in parous cattle was lower than that of nonparous cattle. Figure 21: The average blastocysts number in parous cattle was lower than that of nonparous cattle. （ P ﹥ 0.05 ）。 Indicate: P<0.01 Indicate: P<0.05

24. Conclusion  The environmental temperature, humidity and semen can directly affect the quality of the embryo, but cannot affect the numbers of embryo. In the appropriate temperature and humidity and using motility sperm are essential to improve embryo quality.  Parous cow are more suitable to be donor than non-parous cow for superovulation  This study provides reference for the further application of assisted reproductive technology.

25. Thank You ! National beef cattle industrial technology system