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FEATURE - ARTICLE Advances In Assisted Reproductive Techniques For many years, artificial insemination using chilled and frozen semen has been the most widely used assisted reproductive technique(ART) in the horse-breeding industry. Assisted Reproductive Techniques: - AI Fresh, Chilled and Frozen - Embryo Transfer - Ovum Pickup (OPU) - OT, IVF, ICSI - & embryo sex determination - Cloning - Embryo and Oocyte Freezing Embryo transfer(ET) in cattle has been well established for well over twenty years as a way to increase the number of progeny per year from a genetically superior animal. The use of embryo transfer in horses has been much slower to develop, partly due to a lack of commercial/economic incentive, combined with a much lower yield of embryos. Cattle can be successfully super-ovulated (given a program of drugs similar to women having IVF) , producing up to 20-30 embryos per flush, compared with horses where only one embryo per flush is expected unless the mare has ovulated from both ovaries. The most relevant and useful application of ET is in the production of off spring from mares still competing which increases the ability and likelihood of improving the genetics of equine athletes. This seems like an ideal scenario when previously mares were only bred once they were older, retired and less fertile, reducing the chance of producing many offspring. This is not always as easy as it sounds, and sometimes owner expectations exceed what can be achieved in reality. Exercise, heat and stress can disrupt ovarian function while frequent joint injections have been seen to lower the overall fertility of mares and stallions. Furthermore, some disruption to exercise schedule should be expected, although every effort is made to plan breeding around the mare’s work schedule. The process of ET involves the donor mare being bred with either fresh, chilled or frozen semen; fertilization takes place within the mare’s fallopian tube and the developing embryo is present in the mare’s uterus by the sixth day following fertilization. Once the embryo is in the uterus it can be flushed out using special tubing through a cup with a filter; this is usually done on day 7-8 after ovulation depending on mare age, time of year and type of semen used. The gaps in the filter are smaller than the smallest embryo, therefore the cup will catch and retain the embryo during the flushing process. The contents of the cup is examined under a microscope and the embryo which is approximately the size of a spec of dust is located, washed and held in a special media until the recipient mare is prepared for transfer. It is ideal to select a couple of recipients for each donor mare flush to increase the chances of perfect synchronicity, and provides us which a choice so we can select the best recipient on the day and therefore increase the chances of the transfer being successful. Studies show more success with transfer when recipients have ovulated two days after the donor mare. ET is widely available throughout the UK although a high level of skill is needed to achieve high transfer and pregnancy rates. Embryos can be frozen using a method called vitrification to be stored indefinitely for the preservation of genetics or until recipients are available for transfer. It involves transferring a small, day 6 - 6.5 ‘morula’ stage embryo through varying concentrations of antifreeze, before submerging it in liquid nitrogen. Size of the embryo is critical for success, therefore it is important to have a back up plan (i.e. a recfipient on stand-by) if the embryo that is flushed is too big, as this will probably not survive the freezing and thawing processes. A decreased pregnancy rate following thawing and transfer is expected with every frozen embryo, however this can be limited by using an experienced technician. The next step in breeding technologies beyond ET needed to provide many advantages over embryo transfer to warrant the ensuing research needed for development and the extra costs involved in making it commercially viable. Ovum pick-up (OPU) ticks all the boxes! Advantages Of Opu/Icsi Over ET 1. Donor mares can have one-off visits to collection center when it suits work schedule. 2. OPU can be done outside the normal breeding season; in fact autumn and early spring are often the best times to get a high yield of oocytes. 3. Infertility issues in the mare where no embryos are retrieved using conventional ET e.g. inability to ovulate/ poor uterine environment/ cervical tearing do not affect the OPU process. 4. Embryos can be produced using semen from stallions with low fertility or very limited stocks of semen using ICSI once oocytes are collected using OPU. 5. Potentially more embryos can be produced per year (this will hopefully improve as the OPU/ICSI processes improve with practice and research). 6. Embryos are usually frozen after OPU/ICSI and can therefore be transferred into recipients when desired; synchronizing with donor unnecessary. OPU is where the oocytes (ova or eggs) are aspirated directly from the ovary, either transvaginally or transabdominally and identified under a microscope, washed and transferred into a special medium ready for further processing. Trans-vaginal OPU is the most commonly used technique now; a long rigid ultrasound probe, held within the vagina against the ovary, acts as a visual guide for an attached needle. Once each follicle is visualized the needle is advanced and the follicle is repeatedly flushed with a solution using a pump. 15-20 follicles may be flushed from the two ovaries during a single OPU session, yielding anything from 0-20 oocytes. A large dominant follicle which is close to ovulation yields a mature oocyte. As previously mentioned and similarly to ET, only 1-2 mature oocytes are available per flush if only dominant follicles are flushed. However, many immature oocytes can be harvested from multiple smaller follicles. Furthermore, immature follicles are much more stable and easier to transport for further processing. A donor mare in the transitional phase in early spring and late autumn have the ideal ovaries for harvesting as many oocytes as possible each flush. OPU can be carried out every 2-3 weeks. Oocyte transfer(OT) has been used in the place of ET for mares with reproductive issues which results in no embryo production. 46 | BRITISH BREEDER
It requires recovery of a mature oocyte using OPU from the donor mare. The recipient mare is synchronized with the donor, her pre-ovulatory follicle is flushed to remove her mature oocyte and the donor mare oocyte is placed in the fallopian tube of the recipient through a flank incision. The recipient is inseminated and the fertilzation of the donor oocyte takes place within the recipient’s fallopian tube. OT has not taken off over the years as it holds only a small advantage over ET and is more invasive for the recipients involved. Ten to fifteen years ago, much of the research effort was concentrated on IVF, the most widely used ART in humans. To this day only a couple of foals have been born using this technique. It requires the sperm to ‘swim up’ to the ovum in a petri dish for fertilization to take place. The equine sperm is usually unable to fertilize the ovum in these conditions. Intra-Cytoplasmic Sperm Injection 1. Immature oocytes matured in incubator. 2. One sperm selected and injected into cytoplasm of each oocyte using micromanipulator. 3. Fertilisation in vitro (in petri dish). 4. Incubation/ culture for 6-8 days - result = embryo! 5. Embryo transferred or frozen and keep our talented mares here. A very recent development which was reported in 2014 by Dr C Herrera at Zurich University is sex determination using material extracted from the embryo. Embryos produced by ICSI are biopsied and the genetic material Cloning 1. Cells taken from donor animal to be cloned - can be a gelding 2. Unfertilized oocyte from a random mare with DNA material removed therefore not transferring genetic influence to embryo produced 3. Cell nucleus from donor implanted into oocyte and an electric pulse results in fusion and the start of cell division 4. Resulting embryo transferred into recipient as normal 5. Cost around £100000 The practice has the back up of the RCVS Accredited Hospital. FEATURE - ARTICLE undergo genetic analysis using PCR. More research in this area may lead to improving our selection of individuals for phenotype, which is already commonplace in the cattle world. Interestingly the collapse of the embryo following biopsy also renders it more suitable for freezing at the blastocyst stage. Somatic cell nuclear transfer(SCNT) or cloning is the production of a population ofgenetically identical individuals. Dolly, the first animal clone was born in 1996, and the first horse clone born in 2003. Since then, Crestview genetics in the states has produced over 200 horses since 2009. Some highly respected reproductive specialists are concerned that if we rely too heavily on cloning we may not be developing and improving the genetics of our equine athletes, a sideways step so to speak? Since 2012, clones have been allowed to compete FEI, so no matter our personal view, cloning is here to stay. Brand-new facilities. Brand-new name. The Arundel Equine Hospital has now changed to Sussex Equine Hospital. It has become very evident at recent International ART conferences that all research has shifted towards OPU-ICSI as the way forward for production of embryos. An overwhelming amount of development has taken place in these areas over the last 6-8 years, and more equine reproductive clinics and labs are working towards providing these techniques commercially around the world. Intra-cytoplasmic sperm injection (ICSI) is a really exciting development in equine breeding, but requires expensive equipment in a purpose built lab run by highly skilled individuals that have dedicated most of their working life to the processes involved. However, it is possible for OPU, which requires less skill than ICSI and less equipment, to become available in the UK soon. This will allow mares resident in the UK to have oocytes harvested and shipped to an ICSI lab in Europe. The resultant embryos can either be transferred into recipients there and shipped back to the UK, or the embryos can be frozen and sent back to the UK, for transfer into the clients own recipients when desired. Its really important for the British equine breeding industry to keep up with the new developments in OPU/ICSI to enable us to become more competitive with our friends in Europe Sussex Equine Hospital Billingshurst Road Ashington, West Sussex RH20 3BB w e t Sussex Equine Hospital www.sussexequinehospital.co.uk info@sussexequinehospital.co.uk 01903 883050 REPRODUCTION Sussex Equine Hospital (RCVS Accredited) is still one of the oldest dedicated equine practices in the UK. A team of 20 experienced equine vets provide both routine and emergency care 24 hours a day, 365 days of the year throughout Sussex, Surrey and Hampshire. • Dedicated team of 5 experienced stud vets with further qualifications in stud medicine. • Thoroughbred mares managed for natural service. • Excellent fertility with AI - frozen and chilled. • Many years experience with Embryo Transfer. • Competitive packages available. • Stud vet only out of hours service - for routine and emergency work. • On-site laboratory - HBLB CEM accredited. • Full hospital backup for dealing with sick mares and foals. • Mares can be boarded for all types of breeding work. • Mare & stallion - poor fertility investigations carried out. For further information please contact us… SERVICES SUSSEX EQUINE H O S P I T A L - E S T 1 9 5 1 - We care for and about horses BRITISH BREEDER| 47
Page 1 and 2: BRITISH BREEDER May 2018 Launch of
Page 3 and 4: INSIDE this issue... Welcome As I w
Page 5 and 6: British Breeding MEET THE TEAM A gr
Page 7 and 8: BRITISH HORSES IN COMPETITION Briti
Page 9 and 10: NEWS - BRITISH BRED HORSES IN COMPE
Page 11 and 12: PREDATOR VIXEN, 1-2 HORSES, FROM £
Page 13 and 14: NEWS - FUTURITY GRADUATES EVENTING
Page 15 and 16: NEWS - 2018 FUTURITY By enhancing t
Page 17 and 18: STUDBOOK - UPDATES We are proud of
Page 19 and 20: ANGLO EUROPEAN STUDBOOK Elite Foal
Page 21 and 22: STUDBOOK - UPDATES Top horses carry
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Page 27 and 28: STUDBOOK - UPDATES The Mitsubishi M
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Page 31 and 32: STUDBOOK - UPDATES Some exciting 20
Page 33 and 34: The versatility of the Trakehner ho
Page 36 and 37: BREEDER - PROFILE BREEDER PROFILE C
Page 38: CLASSIC OPERA Sport Horse GB Graded
Page 41 and 42: Stud & Reproduction Services • Co
Page 43 and 44: Rosie De Courcy Rosie De Courcy has
Page 48 and 49: RIDER - INTERVIEW In conversation B
Page 50 and 51: FEATURE - STALLION SHOWS ROUNDUP CO
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Page 58 and 59: FEATURE - ASK THE EXPERT When will

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