Ovarian follicular dynamics and superovulation in cattle

Transcription

1 Ovarian follicular dynamics and superovulation in cattle John P Kastelic Professor of Theriogenology Head, Department of Production Animal Health University of Calgary Calgary, Alberta, Canada

2 Factors affecting ovarian response to superstimulation Extrinsic! Gonadotropins! Purity! Dose!! Route! Frequency! Site!!! Intrinsic! Nutritional status! Reproductive history! Age! Season! Breed! Ovarian status

3 Ovarian influences Superstimulation = rescue of subordinate follicles in a wave or recruitment, regardless of wave?! Timing of treatment! Wave 1 vs Wave 2! Predictability of response / repeatability! Follicles as small as 1 mm part of the wave

4 Predictive characteristics & repeatability of wave pattern 2-wave interovulatory interval 16 Follicle diameter (mm) Days after ovulation 3-wave interovulatory interval Follicle diameter (mm) Ov Luteal Phase Ov 40 LH 30 FSH Days after ovulation FSH (0.1 x ng/ml)lh (ng/ml) Luteal Phase Ov Ov 40 LH 30 FSH FSH (0.1 x ng/ml)lh (ng/ml) 91 interovulatory intervals (IOI)! Duration: 20 vs 23 d! If 21 d = 2-wave IOI! If 22 d = 3-wave IOI! Repeatable! Same more common (70%) than alternating (30%)! No seasonal effect Jaiswal et al., 2009

5 Superovulatory response to bfsh Small follicles (<6 mm) Medium follicles (6-10 mm) Large follicles (>10 mm) Number of days from ovulation Superstimulation = rescue of subordinates within a wave

6 Superstimulatory response and wave emergence Initiation of!!! No. follicles!! No. ovulations! treatment!! n! (>7 mm)!!!! Day -1!! 11! 22.0±3.4 a!! 9.5±2.3 b! Day 0!!! 7! 20.3±3.8 a!! 18.4±3.4 a! Day +1!! 6! 7.7±3.4 b!! 6.7±2.2 b! Day +2!! 6! 7.3±2.3 b!! 6.5±2.3 b!! ab Within columns, P<0.05!!! Day 0 = ovulation

7 (Expts 1 & 2 combined) No. heifers Superstimulatory response (Day 0=ovulation) Day - 1 Day 0 Day + 1 Day Ovulations per heifer No. follicles last day of treatment* 9.4 a 11.6 a 3.7 b 6.5 b 20.1 a 14.0 b 6.8 c 7.3 c Proportion that ovulated 14/16 a 15/16 a 9/12 a 6/6 a * follicles 7 mm a-c (P<0.05)

8 Wave 1 vs Wave 2? 2-wave vs 3-wave? High progesterone vs timing of progesterone?

9 Effect of progesterone Suppresses dominant follicle during growing phase by suppressing LH pulse-frequency! Low progesterone:! Persistent over-sized ( cystic ) follicles! Ovulation failure due to prolonged exposure during superstimulation - (Singh et al., 2007)! High progesterone:! Reduced ovulation rate in superstimulated Bos indicus given PGF at end vs beginning of CIDR treatment (Busarelli et al., Therio 2008)

10 Wave 1 vs Wave 2 & effects of progesterone Follicle diameter (mm) mg/day high med. low 150 mg/day 30 mg/day Days after wave emergence Adams et al., 1992

11 2-wave cycle 3-wave cycle CL CL CL CL CL CL CL Days Days Is fertility higher in 3-wave versus 2-wave animals?

12 Hypothesis A short period of progesterone exposure during the growing and early-static phase of the dominant follicle (ovulatory wave of 3-wave cycles) is associated with higher fertility than a long period of progesterone exposure (ovulatory wave of 2- wave cycles).

13 E2 + P4 + CIDR Wave emergence PGF + CIDR X LH + AI Pregnancy diagnosis Short P4 (n=181) D -4 D0 D3 D5 Wave emergence E2 + P4 + CIDR Long P4 (n=184) PGF + CIDR X LH + AI Pregnancy diagnosis D -4 D0 D6 D8 Goodale: n=40 heifers, n=116 cows; Kinsella: n=77 cows; Brandon: n=132 heifers Dias et al 2012

14 Pregnancy rate Short P4 Long P4 Cows 59/94 (63%) 58/99 (58%) Heifers 45/85 (53%) 41/87 (47%) P value P4 = 0.3 Parity = 0.6 P4*Parity = 0.9 Location = 0.6 Total 104/179 (58%) 99/186 (53%) Ovulation rate Cows 25/28 (89.3%) 26/29 (89.6%)! Heifers 16/19 (84.0%) 16/18 (88.9%) P value P4 = 0.9 Parity = 0.2 P4*Parity = 0.6 Dias et al 2012

15 Conclusions Longer P4 during ovulatory follicle growth:! Larger follicle at time of insemination! Larger subsequent CL (Pfiefer et al., 2009)! Both long and short P4 (analogous to 2- and 3-wave cycles) were similarly capable of ovulation and pregnancy (Dias et al., 2012)! Low P4 during growing phase of ovulatory follicle compensated for negative effect of short proestrus on pregnancy rate (Dadarwal et al., 2013)! All related to duration of high LH pulse-frequency

16 Superstimulatory response Note: half dose of FSH used Wave 1* Wave 2* No. heifers Day of initiation of tx 1 Emergence to start of tx 1 (Days) Ovulated No. ovulations per heifer ± ±0.1 18/19 6.6± ± ±0.1 20/21 8.2±1.7 * (NS) 1 (Day 0=ovulation)

17 Superstimulatory response Note: half dose of FSH used Wave 1* Wave 2* No. heifers No. ovulations per heifer No. CL per heifer Total embryos/ova recovered Transferrable embryos * (NS) ± ± ± ± ± ± ± ±0.7

18 Superovulatory response (Wave 1 vs Wave 2)!!!! CIDR! Control!!!!! (Day 1)!! (Days 8 or 9)!! N!o.!!! 22!!! 18! CL!!! 11.2!!! 8.1! Transferable! 7.3!! 6.7!!!!!!!!!!!!! Macmillan et al., 1996

19 Cycle & optimal response Dominant follicle depresses superovulatory response! Pierson et al, 1988, Guilbualt et al, 1991, Huhtinen et al, 1992, Adams et al, ! Period when dominant follicle not functional! 2-wave cycle: 25%; 3-wave cycle: 30%! Period when waves are emerging! 5 to 7 d (2-wave vs 3-wave IOI) = 25-30% of cycle! Therefore 70-75% of cycle not optimal for superstimulation

20 Can the superstimulatory response be predicted? Extreme variability in the superstimulatory response remains an enigma and a major limitation! 30% cows produce 70% embryos! 24% cows produce no embryos (Looney, 1986) Singh, Dominguez, Jaiswal & Adams, 2004

21 Hypotheses Superstimulatory response related to number of follicles at wave emergence! No. follicles recruited into successive waves consistent within individuals (repeatable)! Positive correlations will allow prediction of superstimulatory response

22 Materials & methods 1st synchronization (P + E): n=141 cows! Cows ranked according to no. follicles at wave emergence!! 2nd synchronization (follicle ablation)! Upper-ranked cows (n=16) and lower-ranked cows (n=20) selected! FSH 2x/day for 3 d, beginning day after ablation

23 Distribution based on antral follicle count (AFC) (n=141)

24 Predictability of superstimulatory response No. follicles at wave emergence repeatable within individuals and predictable (r = 0.77, P = )! Superstimulatory response can be predicted by no. follicles at wave emergence! High-end group had 2x response of low-end group! Response = 71% of number of follicles at time of wave emergence

25 Simple U/S test: no. follicles at wave emergence Realistic expectation of donor response! Selection of good donors (for oocyte retrieval or embryo transfer)! Anticipation of needs (i.e., recipients for embryo transfer, laboratory supplies for IVF, labor and time)

26 4-d versus 7-d FSH 7-d protocol had greater ovulatory response (P<0.05) and tended (P<0.1) to have more transferable embryos (Garcia Guerra et al., 2012; Dias et al., 2013a).! Using IVF, 7-d protocol:! greater number of follicles 9 mm (25 vs 11)! greater proportion of expanded COC! more transferable embryos (5.6 vs. 2.5 per heifer) 9 d after IVF Dias et al., 2013b! In addition, longer FSH treatment resulted in collection of greater proportion of mature oocytes (59 vs 22%, P<0.001) compared to standard FSH protocol (Dadarwal et al., 2012).

27 Summary of superstimulatory response response if treat in presence of large DF! response on Day 0 ± 1 d of wave emergence! Wave synchronization effective for scheduling superstimulation! Wave 1 = Wave 2! AFC at wave emergence predictive of response! Extending duration of FSH is beneficial

28 Superovulation and follicular wave dynamics Superstimulatory treatments must be initiated at the time of wave emergence to optimize response

29 Synchronization of follicle wave emergence for superstimulation Follicular ablation 1.5 d (2 largest follicles) Estradiol 4 d (2.5 mg E-17β or 1-2 mg EB or EV plus P4) GnRH/pLH 2 d (much variability)

30 Follicular ablation Ablation ~ 1.5. days Wave emergence Follicle Size FSH Days Bergfelt et al., 1994

31 Follicle wave emergence - GnRH or plh GnRH/pLH (Ovulation?) ~2.0. days Wave emergence Follicle Size Ovulation rate: ~60% in most cattle FSH Days Martinez et al., 1999

32 The dilemma Follicle ablation is difficult in the field! Estradiol is not available in many countries! GnRH does not synchronize follicle wave emergence consistently, and thus superovulatory response

33 Can we improve the ovulatory response to GnRH?

34 Ovulation of a persistent follicle induced by low progesterone pretreatment PGF Used P4 device GnRH 30 h >90% ovulation Day 0 Days 7-10 Small et al. (2009)

35 GnRH-induced ovulation of a persistent follicle PGF PGF P4 device GnRH Ovulation FSH P4 device

36 Improving the response to GnRH Does the P4 device have to be removed? PGF D4 pm and D5 am PGF GnRH FSH GnRH AI AI EmbryoC ollect P4 device D-7 PM D0 AMD1.5 PM D4 PM D5 AM D6 AM D6 PM D7 AM D13 7 d

37 The P4 device does not have to be removed! **>90% of the cows ovulated in response to GnRH Treatment Groups N Total ova/embryos Fertilized ova Transf. embryos With! P4 device! removal ± ± ±0.6 Without! P4 device! removal ± ± ±0.7 Carballo Guerrero et al., 2008

38 Superovulation with a single injection of Folltropin-V

39 Single subcutaneous injections of Lower results in dairy donors (body condition)! Comparable results in beef donors! but response was dependent on:! Site of injection (behind the shoulder)! Total dose of Folltropin-V (400 mg)! Methodology and needle used (short needle, avoid administering in the muscle)! Total volume administered (20 ml)! Experience of the operator Folltropin-V

40 Bioniche slow release technology (Hyaluronan = hyaluronic acid; HA) Objective: Determine superovulatory response of cattle treated with a single intramuscular injection of Folltropin-V diluted in 2% hyaluronic acid (HA)

41 Folltropin-V in HA

42 Mean follicle diameter (follicles 3 mm) changes during treatment 9,00 FSH Mean Follicle Diameter 2% HA Day: P<0,0001 Group: P = 0,9913 Day*Group P =0,0662 6,75 mm 4,50 1% HA 2,25 0,00 Day 0 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 Day 11 Day

43 Control 5 mg E-17β 50 mg P4 P4 device 300 mg FSH Superstimulatory treatments PGF2α plh FTAI Embryo collection % HA 5 mg E-17β 50 mg P4 PGF2α plh FTAI Embryo collection P4 device % HA 5 mg E-17β 50 mg P4 200 mg FSH in 5 ml 0.5% HA 200 mg FSH in 5 ml 1% HA 100 mg FSH in 2.5 ml 0.5% HA 100 mg FSH in 2.5 ml 0.5% HA PGF2α plh FTAI Embryo collection P4 device

44 Superovulatory response following a split intramuscular injection of Folltropin-V in 1% or 0.5% hyaluronic acid Treatment N Ova/embryos Fertilized ova Transferable embryos Control ± ± ± % HA ± ± ± 1.3 1% HA ± ± ± 0.9 P-value Tribulo et al., Theriogenology 2012

45 Pretreatment of donors with ecg to recruit additional follicles into the wave ecg EB+P4 FSH PGF AI Embryo Collection! P4 device Days !

46 Superovulatory response in Angus donors with history of a poor response Effect of pretreatment with 400 IU ecg Total Fert Grades Grades Tx n CL ova/emb ova ! No ecg ± ± ± ± ±0.2! ecg ± ± ± ± ±0.6! P-value Carballo Guerrero et al., 2007

47 Will a lengthened FSH treatment protocol result in recruitment of follicles into the wave and the time needed to reach an ovulatory size? 4-day protocol FA PGF2α plh 400 mg FSH Embryo P4 device FTAI Collectio n 11 7-day protocol FA PGF2α plh 400 mg FSH Embryo FTAI Collectio P4 device n

48 Superovulatory response in embryo donors treated with 4-day vs. 7-day protocol 4-d FSH - (FSH for 4 days following follicle ablation) 7-d FSH - (FSH for 7 days following follicle ablation) FSH Group 4-d (n=12) 7-d (n=12) Fol >9 mm 26.7±3.2 66%/36h 33.4±4.4 93%/36h CL Total ova/emb Fertilized ova Trans embryos 20.8± ± 5.6± ± ± ± ± ±1. ab P<0.05 Garcia Guerra et al., IETS 2012

49 OPU and IVF following 4- vs 7-d of FSH Endpoints recorded at 12 hr after LH treatment treatment 4-day FSH FSH Starvation 7-day FSH Total number of follicles ( 3 mm) 18.0± ± ±5.8 Number of follicles 9 mm 10.6± ± ±5.3 Number of follicles 6-8 mm 5.0± ± ±1.6 Number of follicles 3-5 mm 2.4± ± ±0.4 Number of follicles aspirated per animal 17.5± ± ±8.1 COC recovered per animal* 10.5± ± ±3.7 Number of COC collected/follicle aspirated (%) 85/144 (59.0) 74/157 (47.1) 125/243 (51.4) Dias et al., 2013

50 OPU and IVF following 4- vs 7 d treatment 4-day FSH FSH Starvation 7-day FSH Expanded 63/85 (74.1) 40/74 (54.1) 118/127 (92.9) Grade 1 and 2 36/85 (42.3) 16/74 (21.6) 77/127 (60.6) Grande 3 and 4 27/85 (31.8) 24/74 (32.4) 41/127 (32.3) Cleavage (%) 44/81 (54.3) 12/55 (21.8) 69/123 (56.1) Morula/blast 17/81 (21) 2/55 (3.6) 27/123 (21.9) 7-day FSH treatment resulted in: Prolongation of follicular growth More large follicles available for oocyte collection Collection of more COC that were fully expanded In 2.5 times more transferable embryos per heifer Dias et al., 2013

51 FSH must start at follicle wave emergence to optimize superovulatory response! Follicle ablation or E 2 will synchronize follicle wave emergence effectively for superstimulation, but! Can eliminate estrus detection in superstimulated donors and do fixed-time AI by extending P4 and using GnRH! GnRH is effective in synchronizing follicle wave emergence for superstimulation, if it induces ovulation! Response to GnRH can be improved by first inducing a persistent follicle

52 FSH can be mixed with HA and given as single im injection (2%), or 2 im injections (0.5 or 1%), 48 h apart! Can induce superstimulation with DF present by recruiting follicles and then causing them to reach an ovulatory size by administration of exogenous FSH! Extending the FSH treatment protocol will recruit follicles into the wave and provide the time required for those follicles to acquire the capacity to ovulate, while a 4-day protocol.