8/5/16. Using Assisted Reproduc6ve Technologies to Preserve Yellowstone Bison Gene6cs

Transcription

1 Using Assisted Reproduc6ve Technologies to Preserve Yellowstone Bison Gene6cs Jennifer Barfield, PhD College of Veterinary Medicine and Biomedical Sciences Colorado State University Plains bison Bison bison bison Wood bison Bison bison athabascae! Bulls average 730 kg, cows 410 kg! Shorter neck! More rounded hump! Smaller and stockier! Chaps of long hair on foreleg! Cape is lighter in color! In summer, cape forms a dis6nct boundary from the rest of the body! Frizzy hair on head! Larger, longer beard! Bulls average 840 kg, cows 475 kg! Taller, squarer hump! Darker in color! Generally taller and larger! LiWle or no chap hair on forelegs! Cape does not form dis6nct barrier from the rest of the body! Long, straight hair sloping down on forehead! Smaller, more pointed beard Adapted from the AWCC, photos from hwps:// 1

2 Source: American Bison Status Survey and Conserva6on Guidelines 2010, IUCN By Unknown.Edited version by PawełMM (talk) - Burton Historical Collec6on, Detroit Public Library, Public Domain, hwps://commons.wikimedia.org/w/index.php?curid=

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4 Purebred beefalo are 3/8 bison and 5/8 cawle Photos from Texas Beefalo Associa6on Facebook Page 4

5 Assisted Reproduc6ve Technologies Ar6ficial insemina6on Embryo recovery and transfer In vitro embryo produc6on Embryo Cryopreserva6on Gonadal 6ssue preserva6on Estrus Synchroniza6on Sperm Injec6on Cloning How are offspring generated? * B+ Healthy * Denotes a cleaning step 5

6 Embryo washing First step contains trypsin Each dilu6on is 1:

7 How are offspring generated? * B+ Healthy * Denotes a cleaning step Cleaning the semen Cryopreserved with standard cawle protocol (8% glycerol) Post thaw Materials and Methods Frozen-thawed sperm used for IVF Standard IVF procedures used Media and embryos from each group cultured Posi9ve bull Nega9ve bull Posi6ve cow Nega6ve cow

8 Culture media for the embryos was nega6ve Four calves born via AI Calves are nega6ve for Brucellosis (2 years old now) Mothers remain nega6ve for brucellosis Results How are offspring generated? B+ or healthy * * + = Healthy * Denotes a cleaning step In Vitro Produc6on of Embryos Oocytes aspirated on site Shipped overnight to CSU Fer6lized upon arrival - Oocytes in groups of 20 - Mul6ple bulls per female when possible 8

9 Materials and methods In Vitro Produc6on of Embryos Day -1 Matura6on: Ship to CSU, h Day 0 Day 1 Day 3 Fer6liza6on: Co-incuba6on with sperm for 18 h Remove cumulus cells, transfer to CDM-1 for culture to 8-cell stage Assess development and transfer to CDM-2 for culture to blastocyst stage Day 4-8 Culture to blastocyst stage, embryo evalua6on (days 7 and 8) De la Torre Sanchez et al., 2006 Preserva6on of Embryos Ultra rapid cooling >2000 C/ min Embryo and fluid turn to glass! Can be held in definitely Materials and Methods Embryo Transfer CIDR μg GnRH 7 days Remove CIDR + 25 mg Lutalyse 10 days Transfer Embryos Embryos are put into the uterus of a female in the right stage of her reproduc6ve cycle 9

10 Results Immature Nonpregnant Adult Pregnant Adult Eggs collected 23 ± ± ± 10.5 Average embryos per 100 oocytes Spent media and degenerate embryos were all culture nega6ve One bull resulted in 0% blasts despite 30% cleavage rate No sta6s6cal differences (GLIMMIX, SAS) Superovula9on and Embryo Transfer We hypothesized that the two injec6on supers6mula6on protocol would result in the recovery of more embryos on average than a single embryo recovery protocol. Materials and Methods Superovula9on 25 mg PG 25 mg PG 12 d 12 d 25 mg PG 25 mg PG Presumed heat 3 d 3 d Presumed heat 266 mg FSH 8-12 d 48 h 7 d Embryos collected 134 mg FSH + PG 3 d Presumed heat 7 d Embryos collected Single embryo recovery 10

11 Materials and Methods Superovula9on 25 mg PG 25 mg PG 12 d 12 d 25 mg PG 25 mg PG Presumed heat 3 d 3 d Presumed heat 266 mg FSH 8-12 d 48 h 7 d Embryos collected 134 mg FSH + PG 3 d Presumed heat 7 d Embryos collected Single embryo recovery Goal = mul6ple ovula6ons Mul6ple ovula6ons = More embryos Results Structures recovered Superovula9on Single Ovula9on UFO and degen Transferrable embryos *Four bison did not respond to superovula6on: 3 were 3 y old, 1 was 15 y old. 11

12 Season 2011 Oct-Nov 2012 Aug Dec 2013 July Oct New recipients Discussion Performance of bulls New catheters, bewer recovery? When do they come in heat? Estrus alert patches do not work How to manage stress 12

13 New Protocol results Day 7! Post-breeding! Day 8! Post-breeding! Total embryonic structures" 4±4" 3±2.8" Viable Embryos" 2±3.4" 2±2.8" Unfertilized Ova" 3±1.7" 0±0" Degenerate Embryos" 0.3±0.6" 1±0" Furthest stage of embryo development recovered" Morula" Blastocyst" 13

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15 Source: Esri, DigitalGlobe, GeoEye, i-cubed, Earthstar Geographics, CNES/Airbus DS, USDA, USGS, AEX, Getmapping, Aerogrid, IGN, IGP, swisstopo, and the GIS User Community, Esri, HERE, DeLorme, MapmyIndia, OpenStreetMap contributors, Esri, HERE, DeLorme, Created TomTom, by City MapmyIndia, of Fort Collins OpenStreetMap Natural Areas - contributors 2015 and the GIS user community Fort Collins 8/5/16 SOAPSTONE PRAIRIE NA Bison Pastures Phase II RED MOUNTAIN OS MEADOW SPRINGS RANCH Bison Pastures Phase I RAWHIDE FLATS RD SSN/RMOS Bison Fencelines City of Fort Collins Natural Areas Larimer County DNR City of Fort Collins Utilities Project Area Larimer County Rawhide Energy Station ² Miles 15

16 Management Annual disease tes6ng (USDA) Vaccina9ons: Rabies Given in calxood or upon arrival to the designated holding facility and boosted yearly in all animals. 7-way or 8-way clostridial vaccines Given within 5 days of birth. Viral disease cocktail (e.g. Virashield gold 5, Zoe6s) Given to calves within 3 months of age followed by a booster 1 4 months later. Boosted yearly as an adult. RB51 brucellosis vaccine Given between 6 and 18 months to all calves born in the holding facility. All non-pregnant female bison will be vaccinated upon arrival to the holding facility. Pregnant females will be vaccinated 6 months azer calving. Ivermec9n Given during yearly examina6on to all bison. Eligibility for Reintroduc6on Category 1 - Animals resul6ng from assisted reproduc6ve technologies (ART including but not limited to ar6ficial insemina6on, embryo transfer, and in vitro-produced embryos) where the egg and sperm donor are free of Brucella abortus and Johne s disease. Calves monitored for 90 days Dam tested during pregnancy, d and 6 months post partum 3 neg tests for dam, 2 for calf Eligibility for Reintroduc6on Category 2 - Animals resul6ng from ART where the egg or sperm donor or both are posi6ve for a disease but the recipient female bison are nega6ve for disease. Calves born in facility monitored for 90 days Dam tested during pregnancy, d and 6 months post partum 3 neg tests for dam, 2 for calf Calves brought into facility for breeding at 3 years of age 16

17 Eligibility for Reintroduc6on Category 3 - Calves born to brucellosisnega6ve Yellowstone bison at a designated holding facility Pregnant females Deliver calf in facility, nega6ve tests 6 mos azer birth If dam is pos, calf must reach sexual maturity, calve, remain nega6ve for 6 mos., neg tests every 6 mos Nonpregnant females Bred to a bull, deliver calf, remain nega6ve 6 mos. Eligibility for Reintroduc6on Category 4 - Calves born to Brucella posi6ve Yellowstone bison at the Fort Collins USDA- APHIS facility Dam not eligible for reintroduc6on Calves Females tested every 6 mos, have calf, remain nega6ve 6 mos post partum Males neg tests (blood and semen) every 6 mos for 3 years (through first rut) 17

18 Photo credit: Kate Wilkins 18

19 Photo credit: Denver Zoo 19

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21 Team Bison Acknowledgements Donors from the community and beyond! 21

22 hwps:// DONATE HERE " hwps://advancing.colostate.edu/bison 22