Ovum Pick-Up and in vitro embryo production in buffalo and cattle: an Italian experience Bianca Gasparrini, DVM, PhD

Transcription

1 Ovum Pick-Up and in vitro embryo production in buffalo and cattle: an Italian experience Bianca Gasparrini, DVM, PhD 26 Congresso Annuale SIET International Event

2 IVM IVF IVC

3 OPU IVEP ET

4 Embryo yields with OPU-IVEP vs MOET in buffalo MOET OPU-IVEP TE/session Session interval TE/ d ET/ 180 d 5* 51

5 Limitations N. of oocytes recovered Poor oocyte quality Low viability of the of IVP embryos Low freezability Pregnancy to term Embryonic mortality (D25-D45)

6 N. oocytes recovered /OPU session/donor Bovine Buffalo Range Range India Italy Brazil COCs Grade A+B COCs

7 The % of good quality oocytes is lower than in other species Unsuitable C A+B Total abattoir-derived COCs = Data collected over a 4-year period (158 runs)

8 Physiological Factors Affecting the Recovery Rate BUFFALO BOVINE Ovary Primordial follicles Antral follicles

9 Is it possible to increase the number of oocytes recoverable? Selection of donors Pretreatment of donors?

10 Donor effect Gasparrini et al., 2014 Donor selection Follicular population AMH plasma levels?

11 Donor selection Correlation coefficient (r) values 27 replicates Time 0 n. n. A+B COC blastocysts SFL 0.73 TFL 0.80 COC Repeatibility (r) of parameters within 4 PS TFL 0.52 SFL 0.54 COC 0.60 A+B COC 0.57 Gasparrini et al., 2014

12 AMH intrafollicular concentration in good ( 12 FL; n=12) vs bad (< 12 FL; n =10) donors Liang et al. 2016

13 FSH priming DFR OPU OPU Control Day 0 Day 4 Day 7 OPU FSH FSH OPU FSH x 1,5 PRID Day 0 Day 2,5 Day 2 Day 5 OPU FSH FSH FSH OPU FSH x 3 PRID Day 0 Day 2 Day 3 Day 4 Day 6

14 Influence of FSH priming on follicular population (mean ± SE) Groups Total follicles Large follicles Medium follicles Small follicles Control 3.4 ± 0.4 A 0.2 ± 0.1 A 0.7 ± 0.2 A 2.5 ± 0.3 1,5 d. FSH 5.4 ± 0.7 Ba 0.3 ± 0.6 A 1.3 ± 0.1 B 3.8 ± d. FSH 7.0± 0.9 Bb 1.1 ± 0.1 B 2.6 ± 0.2 C 3.3 ± 0.8

15 Influence of FSH priming on the number of oocytes and embryos (mean ± SE) Groups Total COCs Total A+B COCs Total ABC COCs BL Control 2.4 ± 0.4 A 0.4 ± 0.2 A 1.3 ± 0.2 A 0.2 ± 0.1 A 1.5 d. FSH 2.4 ± 0.1 A 0.7 ± 0.1 A 1.7 ± 0.1 A 0.8 ± 0.03 B 3 d. FSH 4.4 ± 0.4 B 1.7 ± 0.3 B 3.3 ± 0.3 B 1.1 ± 0.2 B

16 Influence of season on the developmental competence of OPU-derived oocytes Di Francesco et al., 2012

17 Neglia et al., 2011 Long term OPU in buffalo I period (6 months, 34 PS) vs II period (3 months, 13 PS) Parameters I period Mean ± SE (%) II period Mean ± SE (%) Total follicles 5.06 ± 0.15 A 3.71 ± 0.20 B Recovery rate (49.21) A (40.16) B COCs 2.58 ± 0.14 A 1.56± 0.15 B Grade A+B COCs 0.70 ± 0.05 a (27.1) 0.50 ± 0.06 b (32.0) Blastocyst (Bl) 0.36 ± 0.05 A 0.00 B Bl/COCs (14.6) A (0.00 ) B Bl/Grade A+B COCs (22.2) A (0.00) B

18 Cattle in vitro system extrapolated to buffalo Is embryo physiology similar in the two species?

19 IVM: NUCLEAR MATURATION % M II h 18 h 21 h 24 h h 19 h 20 h 21 h 22 h 23 h 24 h % M II Gasparrini et al., 2008

20 Effect of ageing of buffalo oocytes on % cleavage % blastocyst , , h 21 h 24 h 27 h 30 h 0 18 h 21 h 24 h 27 h 30 h hours hours Gasparrini et al., 2008

21 Effects of cystine (added during IVM) in the presence of cysteamine on cleavage and tight morulae-blastocysts (TM-BL) rates 45 76,4 75, , ,5 25,5 73,75 67,7 67,5 22,5 61,3 17,3 17,7 61, Control 0.3 mm cystine 0.6 mm cystine 0.9 mm cystine TE/COC % Cleavage % Gasparrini et al., 2006

22 Kinetics of sperm penetration is correlated to in vitro fertility of buffalo (Bubalus bubalis) bulls 90 67, ,5 0 3 h 6 h 9 h 12 h 15 h 18 h Bull A BullB Bull C Bull D Bull E Bull F Rubessa et al., 2009

23 IVP embryos (n=8) that have been vitrified were transferred into synchronized heifers 3 pregnancies (37.5% PR) 2 pregnancies to term (25 %) Neglia et al., 2004

24 20 TE vitrified by cryotop were transferred into synchronized recipients (Ovsynch) 10 pregnancies were observed on D 30 (50%) 2 pregnancies on D 50 2 calves to term (10%)

25 ET 2011 Pregnancy rate after ET of fresh embryos 40, , , PR (d 25) PR to term 21,3 20 Pregnancy rate after ET of vitrified embryos 15,0 tot emb fast slow 50 45, ,8 19 PR (d 25) PR to term 10 tot emb fast slow Viable COCs/donor. 2.2; interval 3-4 d; 29.7% blastocyst

26 Current IVEP efficiency in buffalo Maturation rate (M II) = 90-95% Cleavage rate = 70% Blastocyst rate = 30-40% Pregnancy rate = 30%

27 Potential calves per buffalo per year with different technologies Technology AI MOET IVEP SESSION INTERVAL days 100 days 3-4 days n. sessions 1 2?? 52 N. VIABLE EMBRYOS Pregnancies D Pregnancies d Live calves to term

28 Ovum pick-up + IVEP in cattle Farm Breed Donors n. COC n. A+B COC n. Blastocyst % A Holstein ± B Holstein ± ± C Holstein ± ± D Holstein ± ± E Holstein ± ± F Holstein ± ± G Holstein 1* H Holstein ± ± ,4 I Holstein 1* L Limousine ± ± Total ± ±

29 Post-transfer development of fresh (n= 81) vs vitrified (n=32) bovine embryos 50 Fresh Vitrified 37,5 % 25 12,5 0 Pregnancy D 30 Pregnancy D 60 calvings

30 Post-transfer development of fast developing (n=36) vs slow developing (n=61) bovine embryos 80 77,1429 slow fast 65, ,2857 % 40 29, , , Pregnancy D 30 Pregnancy D 60 calvings

31 Pregnancy rates on Day 30 Pregnancy rates on Day ,5 52,5 % 45 % 35 22,5 17,5 0 TM ebl BL XBL-HBL Total 0 TM ebl BL XBL-HBL Total Calving rates 70 % 52, ,5 Fresh Vitrified 0 TM ebl BL XBL-HBL total

32 Gestation lenght and calf weight Type of embryo Gestation lenght (dd) Calf weight (Kg) Fresh embryos Slow developing ± ± 0.1 Fast developing ± ± 2.3 Total ± ± 1.7 Vitrified embryos Slow developing ± ± 2.3 Fast developing ± ± 0.8 Total ± ± 1.4

33 ET of IVP embryos produced with sexed (n=93) and non sexed (n=20) semen Non sexed Sexed Groups Cleavage Blastocyst Sexed semen 60.9 ± ± 7.3 Non sexed semen 54.2 ± ± 8.5 % Pregnancy rates on day ,1 40,9 27,3 0 Fresh Vitrified Total % Pregnancy rates on day Calving rates ,6 33,3 22,7 Fresh Vitrified Total % ,5 30, , ,5 0 Fresh Vitrified Total

34 Type of embryo n. calves Gestation lenght (dd) Calf weight (Kg) Female calves N (%) Non sexed semen for IVF Fresh ± ± (0.0) Vitrified ± ± (40.0) Total ± ± (22.2) Sexed semen for IVF Fresh ± ± (85.7) Vitrified ± ± (100.0) Total ± ± (88.0)

35 Embrionica S.r.l. Spin-off dell Università di Napoli Federico II - Prof.ssa Bianca Gasparrini, DMV PhD - Dott. Andrea Cammarano, Medico Veterinario - Dott.ssa Valentina Longobardi, PhD - Dott. Gianluigi Zullo, PhD - Dott. Giuseppe Albero, Tecnico Scientifico

36 Contacts EMBRIONICA S.r.l. Spin-off Università degli Studi di Napoli Federico II Sedi legale e operative Laboratorio di Biotecnologie Applicate all Allevamento Animale Dipartimento di Medicina Veterinaria e Produzioni Animali Via F. Delpino 1, Napoli Laboratorio Embrionica Via del Dopolavoro 161, Altavilla Silentina (SA) info@embrionica.com P.IVA :

37 Thank you for your attention Luigi Zicarelli Giuseppe Campanile Gianluca Neglia Giuseppe Albero Valentina Longobardi Gianluigi Zullo Carolina De Canditiis Angela Salzano