FREE-ROAMING HORSE AND BURRO FERTILITY CONTROL WORKSHOP Albuquerque, NM November 8, 2018

Transcription

1 FREE-ROAMING HORSE AND BURRO FERTILITY CONTROL WORKSHOP Albuquerque, NM November 8, 2018

2 Current Contraceptive Use pzp GonaCon Porcine Zona Pellucida Antibodies to ZP3 Cons: Requires boosters Continuous estrous cycling Pros: Remote administration Safe in pregnant mares Will not enter food chain Highly effective EPA Approved Pesticide Antibodies to GnRH Cons: Requires boosters

3 Our Approach: Immunization against oocyte-specific growth factors Jason E. Bruemmer Doug Eckery

4 Atresia Primordial Primary Preantral Antral Preovulatory Quiescent Pregranulosa cells Single granulosa layer Multiple granulosa layers Thecal layer Fluid-filled antrum FSH receptors Dominant follicle LH receptors Ovulation Gonadotropin Independent Gonadotropin Dependent

5 GDF-9 BMP-15 GDF-9 BMP-15 GDF-9 BMP-15 GDF-9 BMP-15 Primordial Primary Preantral Antral Preovulatory Finite number of oocytes Contained within primordial follicles Development and maturation highly regulated Endocrine factors Oocyte-secreted factors

6 GDF-9 and BMP-15 TGF-β Superfamily TGF-β, activin/inhibin, GDF, BMP, AMH Cell growth and differentiation (Massague and Hata, 1997) Secreted by oocyte Exclusively in most species (Otsuka et al. 2000) Regulation Normal follicular development Ovulation rate (Dube et al., 1998; McGrath et al., 1995)

7 X-linked mutation Heterozygotes: fertility Twinning Homozygotes: Infertile Streak ovaries Heterozygotes: Infertile Streak ovaries BMP-15 Mutations BMP-15 knockout mice Heterozygotes: No effect Homozygotes: Decreased fertility Higher incidence of abnormal follicles (Bodin et al., 2007; Davis et al., 1991; Braw- Tal et al., 1993) (Di Pasquale et al., 2006, 2004; Dixit et al., 2006) (Yan et al. 2001)

8 Heterozygotes: fertility Twinning Homozygotes: Infertile Streak ovaries Limited research Mutation: Dizygotic twinning Infertility GDF-9 knockout mice Heterozygotes: No effect Homozygotes: Infertile No follicles past primary stage GDF-9 Mutations (Hanrahan et al., 2004; Nicol et al., 2009) (Palmer et el., 2006; Dixit et al., 2005) (Yan et al. 2001; Dong et al. 2009, Carabastos et al. 1998)

9 Immunization against BMP-15 and GDF-9 in other species

10 Immunization Against BMP-15 and GDF-9: Sheep Decreased ovulation rates Majority: anovulatory Minority: Increased ovulation rates Abnormal ovaries No follicles past primary stage Granulosa-cell clusters Unruptured luteinized follicles (McNatty et al., 2006; Juengel et al., 2002)

11 Immunization Against BMP-15 and GDF-9: Cattle GDF-9 Treatment No difference in ovulation rate Decrease in size and number of antral follicles BMP-15 Treatment: Increased or decreased ovulation rates Decrease in size and number of antral follicles (Juengel et al., 2009)

12 Immunization Against BMP-15 and GDF-9: Deer GDF-9 Treatment No difference in fertility in Year 1 Infertile in Year 2 and 3 BMP-15 Treatment: Increased fertility Higher incidence of 2 or 3 offspring (Eckery et al.,2014)

13 Our Study: Immunization Against BMP-15 and GDF-9 in Horses

14 Immunization Against BMP-15 and GDF-9: Horses Hypothesis: Vaccination against BMP- 15 or GDF-9 will prevent ovulation and/or accelerate the depletion of the oocyte reserve in the mare

15 Materials and Methods: Overview 30 mares 3 treatment groups GDF-9 BMP-15 Control Vaccinations Weeks 0, 6, 12, 18 Antibody responses Every 2 weeks Ovarian Activity 3 times per week Estrous Cycle Behavior 3 times per week

16 Materials and Methods: Immunization Protocol BMP-15 and GDF-9 Vaccines BMP-15 or GDF-9 Peptide Carrier protein: keyhole limpet hemocyanin (KLH) Adjuvant: Seppic Montanide Pet Gel A Control Vaccines Adjuvant: Seppic Montanide Pet Gel A Phosphate buffered saline (PBS)

17 Materials and Methods: Immunization Protocol BMP-15 Vaccine 24 AA of mature protein: QAGSMGSEVLGPSREREGPESNQC 100% homologous to horse 80% identical to sheep and deer GDF-9 Vaccine 14 AA of mature protein: SEYFKQFLFPQNEC 100% homologous to horse 100% identical to sheep and deer

18 Materials and Methods: Estrous Cycle Observation Transrectal ultrasound: Follicle growth and ovulation Three times a week Teasing: Stage of estrous cycle and behavior Normal Follicle Normal CL Hemorrhagic Follicle Anovulatory Follicle Hemorrhagic CL

19 OD 450NM OD 450NM Results: Antibody Responses BMP-15 Antibody Response GDF-9 Antibody Response T 1 T 2 T 3 T 4 T 1 T 2 T 3 T /4 3/17 4/28 6/9 7/21 9/1 BMP-15 Mean Response Threshold Control Mean /4 3/17 4/28 6/9 7/21 9/1 GDF-9 Mean Response Threshold Control Mean

20 Average CL Results: Ovulation Rates * BMP-15 Control GDF-9 BMP-15: Decrease compared to controls P= mares ovulated less than 3 times GDF-9: No change compared to controls P=0.66

21 Pre-ov Follicle Size (mm) Results: Follicle Size Before Ovulation * * Both BMP-15 and GDF-9 groups ovulated smaller follicles than controls P< BMP-15 Control GDF-9 BMP-15 Group: 50% of ovulations from small (<30mm diameter) follicles

22 Ovulations Results: Follicle Size Before Ovulation BMP-15 Control GDF-9 B-A B-B B-C B-D B-E B-F B-G B-H B-I B-J C-A C-B C-C C-D C-E C-F C-G C-H C-I C-J G-A G-B G-C G-D G-E G-F G-G G-H G-I G-J Horse ID Small OV Normal OV

23 MEAN ABNORMAL STRUCTURES Results: Abnormal Ovarian Structures Normal Follicle BMP-15 Group: Higher incidence of abnormal follicles * * Normal CL HAF and PAF 12 P=0.01 GDF-9 Group: Higher incidence of abnormal follicles HAF and PAF P= BMP-15 Control GDF-9 Hemorrhagic Follicle (HAF) Anovulatory Follicle (PAF) Ab. Follicles Ab. CL Hemorrhagic CL (HCL)

24 Results: Estrous Cycle Behavior GDF-9 treated mares No difference in total number of days observed in estrus BMP-15 treated mares Fewer days observed in estrus compared to controls after 1 st booster injection P=0.03

25 Discussion GDF-9 Treatment Altered ovarian function Decreased pre-ovulatory follicle size Increased abnormal follicles Altered ovulation rates in Year 2? BMP-15 Treatment Altered ovarian function Decreased pre-ovulatory follicle size Increased abnormal follicles Decreased ovulation rates Altered estrous behavior Fertility? Long-term efficacy?

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27 Immunization against GDF-9 and BMP-15 Year 2

28 Year 2 Same treatments Control GDF-9 BMP-15 Same mares 5/group unilateral ovariectomy 5 /group intact Same parameters Behavior Ovulations Follicular dynamics Antibody titer Steroid hormone

29 25 Ovulations - Year * 10 5 * 0 Control BMP-15 GDF-9 Ovulations Abnormal

30 45 Size at Ovulation * * Control BMP-15 GDF-9

31 Current (Year 3) Combined BMP15 and GDF9 Mares treated N = 10 Treatment Dec Teased 3 X week U/S Demonstrated estrus Once weekly P4 sample once weekly Antibody titer once weekly

32 Estrous behavior CONTROL Regular Multiple events Associated with ovulations All mares demonstrated all traits multiple times

33 Estrous behavior COMBINED VACCINE Irregular Multiple events Not with ovulations Only 1 mare displayed full estrus on a single day

34 RESULTS Combined vaccine 12 Average ovulations per mare CONTROL COMBINED

35 RESULTS Combined vaccine 12 Average ovulations per mare CONTROL * COMBINED

36 Future Research Combined BMP-15/GDF-9 vaccinationsingle dose Fertility trials Trials in wild horse herds Efficacy Effects on herd dynamics Remote delivery

37 THANKS! BLM L15AC00144 Graduate students Kelli Davis Krisitn Klohonatz Ashley Reisenauer Matthew McQuagge Hannah Twenter Bethany Leibig Technical Support Darcy Mora Peter Graham Abraham Equine