In Vitro Fertilization (FIV) In Vitro Production of Equine Embryos and Preimplantation Genetic Testing Oocytes from or slaughterhouse ovaries Sperm sample Carolina Herrera, M Sc. Clinic for Reproductive Medicine, Vetsuisse - Faculty, Zurich In vitro maturation Sperm preparation 22-24h in microdroplets of IVFm under mineral oil 1 CO 2 incubator 2 In Vitro Maturation of oocytes 1978 Sir Robert Edwards y Louise Brown Nobel Prize in Medicine 2010 TCM199 + NaHCO3 + FSH + EGF + IGF + 10% SFB 38 C Saturated humidity 5% CO2 in air for 24 30 hs 3 4 MII oocytes SEMEN Sperm cells 5 6 Carolina Herrera - Zurich, 1
7 In Vitro culture for 6 to 8 days 5% CO2, 6% O2 8 : Factors affecting the success rate Clinical application of Donor mare.type of infertility Sperm.stallion.sample Laboratory setup.strict control of conditions Operator Subfertile stallions Subfertile mares Semen samples of poor quality, unsuitable for AI Limited amount of semen samples Mare or stallions that die unexpectedly Mares in training Young mares 9 10 Expected results Clinical application of 10 oocytes 7 mature oocytes 70 % IVM 65 % cleaved after 4.5 cleaved embryos 0.9 blastocysts 20% blastocyst rate 0.54 pregnancies 60 % pregnancy rate 11 Setting 1 Setting 2 Setting 3 12 Carolina Herrera - Zurich, 2
In vitro production of equine embryos and Ovum Pick Up () Intracytoplasmic Sperm Injection (). Equine Fertility Centre, Equine Medical Services, Missouri, Ovum Pick Up () Intracytoplasmic Sperm Injection (). Ghent, Belgium Utrecht, Honahlee, Oregon, ISME, Colorado State University, Equine Fertility Centre, Equine Medical Services, Missouri, Equigea, Florida, Colorado State University, Avantea, Italy InFoal-Inc, California, Ghent, Belgium Utrecht, Avantea, Italy InFoal-Inc, California, Texas A&M University, Equiembryo, Texas, Cecolfes, Colombia Zurich, Texas A&M University, Cecolfes, Colombia In Vitro Clonagem Brazil In Vitro Clonagem Brazil Commercial programs in 2016 Commercial programs in 2017 13 Ovum Pick Up () Intracytoplasmic Sperm Injection (). 14 Preimplantation Genetic Testing : - is performed throughout the year, every 14 days Biopsy sample Only one straw of semen can be used to fertilize more than 100 oocytes Embryo Cryopreservation Each straw can be thawed, diluted x 100 and re-frozen Cell/s Equine embryos produced by can be successfully cryopreserved Main challenges: Genetic analysis RESULT 1) Obtain a result after the genetic analysis, starting from a very small sample. 2) Maintain the viability of the embryo after biopsy and cryopreservation. 15 Samples 16 Materials and Methods Biopsy Metaphase II oocyte Polar Body I 2 PN Polar Body II 4-8 cell embryo Blastomere Morula Blastomeres Blastocyst TE Cells Blastocoele Fluid 17 Carolina Herrera - Zurich, 18 3
In vitro production of equine embryos and SRY Sex determination in males 429 pb (males) M MALE PRIMER Pregnancy rates at 25 days of gestation AMELOGENIN Protein involve in teeth enamel 184 bp (female) 160 y 200 bp (male) FEMALE SEQUENCE Embryos 5 -CTTAAGCTTCTGCTATGTCCAGAGTATCC-3 SRY-R 5 -GCGGTTTGTCACTTTTCTGTGGCATCTT-3 Non-Biopsied Hasegawa et al, 2000 AMEL-F 5 -CCAACCCAACACCACCAGCCAAACCTCCCT-3 n Pregnancy rate (%) 1 2h 58 34 / 58 (58.62) 6 10 h 46 29/46 (63.04) 1 2h 35 23/35 (65.71) 6 10 h 6 3 / 6 (50) 145 89 /145 (61.37) Biopsied REFERENCE SRY-F Time before ET TOTAL AMEL-R 5 -AGCATAGGGGGCAAGGGCTGCAAGGGGAAT-3 Herrera et al, Theriogenology 2014 Herrera et al, Theriogenology 2014 19 20 Preimplantation Genetic Diagnosis (PGD) Embryo biopsy vs. blastocoele fluid Results reported in n Amplified % Amplified Embryo biopsy 13 11 84.50% Blastocoele fluid 34 29 85.20% Total 47 40 85.10% - Argentina - Colombia - Germany - Spain - Sweden - Main application: - embryo sex determination Other applications: The collection of blastocoele fluid can be used in combination with vitrification of equine lasrge embryos - detect mutations associated to genetic disorders - select genes linked to certain phenotypes (eg: coat color) 21 22 Preimplantation Genetic Diagnosis (PGD) Transfer only embryos of the desired sex or carrying the desired genetic trait. Significantly reduce the number of recipient mares. WHY? Avoid the need to abort pregnancies of the undesired sex. 23 Carolina Herrera - Zurich, 24 4
100 embryos 50 female 50 male PCR (85%) 100 embryos 50 female 50 male Clinic of Reproductive Medicine Vetsuisse Faculty, Zrich 42.5 female 42.5 male 42.5 + 15 = 57.5 ETs 100 ETs 25 26 THANK YOU 27 Carolina Herrera - Zurich, 5