1. The Role of Assisted Reproductive Techniques (ARTs) In Building a Competitive Livestock Industry Dr. Neil Van Zyl Director In Vitro Africa

2. Presentation Outline: Introduction ARTs Timeline Artificial Insemination Conventional Embryo Transfer In Vitro Fertilization Cloning In Vitro Africa Our Experience Future Directions 3. Hunger It is estimated that 827 million people were hungry in developing regions in 2011-13. This numbers has fallen by 169 million, or 17% in the past 2 decades 658 million Introduction: 4. Introduction: 5. Food security Biotechnologies have contributed immensely to increasing livestock productivity and can help to alleviate poverty and hunger, reduce the threats of diseases and ensure environmental sustainability in developing countries Introduction: 6. First generation Artificial Insemination and semen cryopreservation Second generation Conventional Embryo Transfer Third generation In Vitro Fertilization Fourth generation Cloning ARTs Timeline: 7. Artificial Insemination: Artificial Insemination (AI) was the first great biotechnology applied to improve reproduction and genetics of farm animals The acceptance of AI technology worldwide provided the impetus for developing other technologies, such as: Semen Sexing and Cryopreservation Estrus Cycle Regulation Embryo Harvesting, Freezing and Culture In Vitro Fertilization and Cloning 8. Artificial Insemination AI helps prevent the spread of infectious or contagious diseases Fast increase of genetic development and production gain It enables breeding between animals in different geographic locations Advantages AI is a powerful tool when linked to other reproductive biotechnologies, such as: Estrus Synchronization, Semen Sexing and Multiple Ovulation Embryo Transfer (MOET) 9. Conventional Embryo Transfer: Procedure 10. Conventional Embryo Transfer: The Advent of Superovulation Cover of Science Magazine, 1981 Betteridge,2003 11. Conventional Embryo Transfer Expected Results: 1 Flushing Session 6 Embryos 3 Pregnancies Expected 50% of pregnancy rate (6 cycles a year) 18 Pregnancies from 1 donor/ year One Flushing session each 60 days 12. Conventional Embryo Transfer Advantages Increased number of calves out of genetically superior cows Increased marketing opportunities through the sale of offspring, pregnancies, and embryos Generate more offspring from rare and valuable semen Larger numbers of offspring can help prove the genetic merits of a female at an earlier age in life Disadvantages High cost of superovulation program Requires a higher level of management Not all donors respond to the superovulation treatment = LOW REPEATABILITY 13. IVEP - In vitro production of embryos The IVEP technique started gaining popularity in 2006 14. In Vitro Fertilization (IVF): In Vitro Fertilization (IVF) is the process of producing embryos from oocytes by fertilizing them with semen in a Petri dish. Oocytes are first collected from the ovaries of donors by ultrasound-guided follicular aspiration. Oocytes are then placed in a incubator until they are mature to be fertilized the following day (with conventional, sexed-frozen, or reverse- sorted semen). 15. Ovum Pick Up (OPU): Ultrasound guided non invasive Allows the used of hormonal stimulated or non stimulated donors Highly repeatable Donors can be aspirated every two weeks Indicus breeds yield a high number of viable oocytes 16. Ovum Pick Up (OPU): 17. In Vitro Maturation (IVM) Source: The Big Book of Bovine Embryos , University of Florida, 2013 Cumulus Oocyte Complexes (COCs) after Ovum Pick Up (beginning of IVM) 18. In Vitro Fertilization (IVF): Source: In Vitro Africa 19. Embryo Culture: 2-4 Cells Embryos (2-3 days) 4-6 Cells Embryos (3-4 days) Source: In Vitro Africa Source: In Vitro Africa After IVF embryos undergo a seven days period of In Vitro Culture in a incubator with controlled atmosphere and temperature Early Development 20. Embryo Culture: Expanded Blastocyst (6-7 days) Hatching Blastocyst (7 days) Source: In Vitro Africa Source: In Vitro Africa Late Development 21. Embryo Vitrification: Vitrification refers to the fast freezing of IVF embryos and provides a higher survival rate, minimal deleterious effects on post-warming embryo morphology. Source: In Vitro Brasil Pregnancy rates after transfer of vitrified IVF embryos are similar from those achieved when transferring fresh embryos 22. In Vitro Fertilization: The possibility of using Sexed Semen represents one of the best advantages of the IVF Semen sexed prior to freeze, and semen sexed after it is frozen (reverse sorted), both work very well in an IVF program The ability to create up to 95% of the gender desired, eliminates the gestation costs of the unwanted gender Multiple donors (3+) can be fertilized with one straw of sexed semen = COST EFFECTIVE Optimizing the use of Sexed Semen 23. In Vitro Fertilization (IVF) Proven Results Source: In Vitro Africa 24. In Vitro Fertilization (IVF) Expected Results 1 OPU Session 20 Viable Oocytes 6 Embryos Expected 30% of production using conventional semen 3 Pregnancies Expected 50% of pregnancy rate (24 cycles a year) 72 Pregnancies from 1 donor/ year One OPU session each 15 days 25. Conventional ET x IVF: Conventional ET In Vitro Fertilization Lower initial investment doesnt require a fully equipped laboratory Requires equipped laboratory and highly trained personal Slight Pregnancy rate advantage More repeatable donors can be aspirated twice a month More cost effective - Single straw of semen to inseminate multiple donors (sexed semen = cost effective) Heifers and cows near the end of the reproductive life can be used as donors What is the best option? 26. In Vitro Africa: Our Experience Case Studies ALS BEEFMASTERS North of KwaZulu Natal, South Africa (2012) MATERIALS AND METHODS: Donors: 16 Stud registered ALS Beefmaster cows, aged 7 to 11 years of age OPU: 936 oocytes collected over 4 OPU sessions. IVF: 16 straws of different bulls were used to fertilize the 936 oocytes RESULTS: Embryo Culture: Total of 259 embryos were produced (28%) Embryo Transfer: 238 Embryos were transferred (a percentage of the embryos was frozen to act as a genetic bank) Pregnancies: 118 animals resulted pregnant after embryo transfer (49.6%) 27. In Vitro Africa Our Experience Case Studies ALS BEEFMASTERS North of KwaZulu Natal, South Africa (2012) CONCLUSIONS: Thirty four parentage combinations were achieved by mixing oocytes from the different donors with the semen from different bulls All the donors were mated after the 4 OPU sessions and ALL reconceived! 118 pregnancies were achieved using oocytes from 16 donors and 16 straws of semen 28. In Vitro Africa Our Experience Case Studies SMALL STOCK IVF Western Cape, South Africa (2014) MATERIALS AND METHODS: Donors: 4 Juvenile (pre-pubertal) Boer Goats OPU: 135 oocytes collected in one session (picked up by laparotomy) IVF: Refrigerated semen from a male of proven fertility. RESULTS: Embryo Culture: Total of 47 embryos were produced (34%) Embryo Transfer: 47 Embryos were transferred to 23 synchronized recipients (2-3 embryos per recipient) Pregnancies: 19 animals resulted pregnant after embryo transfer (80%) 29. Cloning: The Fourth Generation of ARTs Source: In Vitro Africa Cloning technology allows breeders to recreate the genotype of a superior animal, providing a source to extend their genetic influence 30. Cloning The Fourth Generation of ARTs Produce quantities of elite individuals for the purpose of large-scale, consistent genetic influence within a herd-building program Breeders most often look at cloning as a technology to: Produce a genetic twin of an elite animal no longer capable of producing embryos or semen (including steers) Produce a genetic twin of an elite animal that has died 31. In Vitro Africa: Our Experience Season 2013/14 43% 40% 34% 40% 32% 25% 24% 21% 22% 27% 30% 31% 0% 5% 10% 15% 20% 25% 30% 35% 40% 45% 50% Mrt Apr May June July Aug Sept Oct Nov Dec Jan Feb Row Labels Sum of ET/VITR Sum of Cultured Sum of = Mrt 425 981 43% Apr 82 205 40% May 92 271 34% June 32 80 40% July 129 400 32% Aug 416 1636 25% Sept 387 1626 24% Oct 556 2663 21% Nov 827 3824 22% Dec 553 2049 27% Jan 788 2669 30% Feb 342 1097 31% Grand Total 4629 17501 26% 32. Genetic Preservation In Vitro Africa Future Directions Establishment of an international embryo trading platform Consulting on reproduction and herd genetic improvement Genetic banking is the cryopreservation and storage of an animals genes in order to be available for eventual future use in cloning. Genetic banking is an affordable way to protect your investment, and preserve the option to multiply that investment later through cloning. If you lose an animal genetic banking is your insurance. The South African livestock population is of a very high quality and could be a reliable source of superior genetic material for genetic improvement projects in Africa. 33. Embryo Sex Determination Prior to Transfer In Vitro Africa Future Directions 2013 Thermo Fisher Scientific Inc. Pink fluorescence indicates presence of bovine Y chromosomal DNA in the sample. Female samples do not show fluorescence Embryo sexing prior to embryo transfer represents a powerful tool in modern cattle breeding. A small biopsy of a single embryo is taken and analyzed by PCR to determine the its sex. Pregnancies after transfer of biopsied embryos are not significantly different from the rates obtained using intact ones. 34. Improvement in Production Efficiency Figure 1. Genetic trend for WW205 in the Afrikaner Figure 2. Genetic trend for WW205 in the Bonsmara Proceedings, 10th World Congress of Genetics Applied to Livestock Production Did Genetic Change Improve Production Efficiency in Three Landrace Breeds of South Africa F.J. Jordaan, et al. 35. The value of genetic improvement In South Africa the value over the last ten years has been U$0,45 billion or U$ 4 per calf weaned - Breedplan $$ US 4 36. Concluding remarks 1. Livestock is the backbone of rural economy for millions of people in Africa Population sizes of livestock species in tropical Africa (1986). Cattle 161 135 000 (85 893 000) Sheep 121 388 000 (67 939 000) Goats 142 711 000 (69 620 000) 37. Concluding remarks 2. Monetary value of research, developmental work spend on cash crops vs. livestock 3. Nucleus breeding programs Jan Rendel 4. Value of ART in creating a competitive livestock industry THANK YOU