Kaatje Ducheyne was born in Belgium and was the daughter of an orthopaedic surgeon. She was fascinated by how vets could make a diagnosis just by using their senses and pursued her dream of following in their footsteps – qualifying as a vet at Ghent in Belgium. After finishing her master’s degree, she spent six weeks in Keros – the largest commercial embryo transfer (ET) centre in Europe. It was here that Kaatje found her passion for horse breeding.
Kaatje began an equine internship in Someren in the Netherlands, where she covered all aspects of equine medicine, surgery and radiology – along with reproduction. She realised she wanted to dedicate her career to breeding and took up a residency position at Ghent University. A year later and a research grant which focused on the effect of a mare’s age on her fertility gave Kaatje the chance to travel to Argentina, where she gained more experience at a large embryo transfer centre and later at the lab of Adrian Mutto in Buenos Aires.
After being introduced to cloning techniques and in vitro embryo production, Kaatje worked during the breeding seasons at Keros ET centre. For the last two years, Kaatje has been part of a team led by Tom Stout at Utrecht University where Kaatje was able to complete her PhD research.
Kaatje says her goal was always to go back to private practice. Once she had completed her ECAR Board exams and PhD, she took the opportunity to join the Sussex Equine Hospital at Ashington in West Sussex (Figures 1 to 3). “It was important to find a place where I felt I could grow together with the practice and I am convinced that we all have the same future vision in regard to horse breeding with a growing importance of assisted reproductive techniques and in special embryo transfer and ovum pick-up (OPU). The support the directors at the hospital have shown me is amazing and it has given me a rare opportunity to expand my work into the field.”
Advances in assisted reproduction
During the last 20 to 30 years, ET has gained an important role in the horse breeding industry. Kaatje explains, “With ET we refer to a technique where the embryo is flushed out of the uterus of the biological dam (embryo donor), who was inseminated and ovulated seven or eight days ago, and then transferred into the uterus of a synchronised recipient mare. The recipient mare will carry the embryo and raise the foal as if it was her own.
“Due to ET, breeders started to pay more attention to female genetics. Before the introduction of ET, competing mares could only produce offspring after they had finished their sporting careers. By then they were often already halfway through their teens and it is well known that the fertility of the mare reduces with increasing age. With ET, a mare can be inseminated while competing.”
It is often hard to match the reproductive cycle of the mare with her competition calendar. For those mares, in vitro embryos (“test tube foals”) can be the solution.
Ed Lyall, one of the practice directors, said, “We are very proud of the fact that Kaatje joined our practice to develop the ovum pick-up service. We have always tried to keep to the forefront of equine reproductive developments, enabling us to provide new services to our clients as they come along. We were one of the first practices, years ago, to offer artificial insemination, and then more recently we were one of the first to offer embryo transfer. Now we will be one of the first to offer ovum pick-up. Kaatje brings her knowledge and experience to an already excellent stud team based at the practice.”
The ovum pick-up technique
The embryos are produced with the combination of two techniques: OPU and intracytoplasmic sperm injection (ICSI). In OPU, also known as oocyte aspiration or oocyte collection, oocytes are collected by aspirating the follicular fluid. The OPU procedure is performed on the standing mare after sedation and administration of epidural anaesthesia.
OPU does not require hospital admission of the mare. Before scheduling an OPU appointment, the ovaries of the mare are assessed at home to determine if she is a suitable candidate. The ideal OPU candidate has a minimum of 15 follicles with a diameter at least 1cm. If there are not enough follicles present during this scan, the ovaries are reassessed two to three weeks later to allow enough time for follicular growth. So, in contrast to ET, where the mare needs to be in season, OPU can be performed year-round and without hormonal manipulation.
A single oocyte is present in every ovarian follicle. Unfortunately, oocytes by themselves are too small (125 to 150μm) to visualise via ultrasound. The follicles are visualised by inserting an ultrasound probe, equipped with a needle guide, into the vagina. The ovary is then grasped via the rectum and held against the vaginal wall. The fluid of each follicle of 1cm or bigger is aspirated and subsequently flushed several times while scraping the follicular wall to enhance oocyte recovery. After aspiration of all the follicles, the recovered fluid is filtered analogously to the recovered fluid after an embryo flush. This fluid is then searched for the presence of the oocytes. The oocytes are transferred into a transport medium and shipped to a specialised lab in Italy, without the loss of their fertility.
Once the oocytes have arrived at the laboratory, they are placed in a maturation medium for 24 hours. This is because the recovered oocytes are not yet ready to be fertilised; they need to finish the first meiotic division and arrest at the metaphase of the second meiotic division. In vivo, oocyte maturation happens while the follicle grows. Once the oocyte is mature, the follicle will rupture and ovulation takes place. The oocyte is then caught by the oviduct and ready to be fertilised.
Since the oocyte has been removed from the follicle, the conditions of the follicle need to be mimicked in the lab. This is the goal of the maturation medium, which contains the same kind of hormones that are present in the follicular fluid. After the 24 hours of incubation in the maturation medium, the oocytes are assessed under the microscope to evaluate whether or not they have successfully matured (ie extrusion of the first polar body can be identified). Only matured oocytes will be fertilised.
Fertilisation in the lab is done by ICSI, in which one spermatozoon is injected into the cytoplasm of the oocyte. The oocyte is then transferred into another medium to promote the development into an embryo. This development takes between seven and nine days. The fertilised oocytes that have successfully developed into a blastocyst will then be frozen and stored in liquid nitrogen. Those embryos are then shipped to the clinic. The frozen embryo can be thawed and transferred at any moment during the breeding season when there is a suitable recipient mare available. So, in contrast to ET, there is no stress with recipient mare synchronisation.
OPU not only offers solutions for competition mares but it also resolves certain male and female fertility issues. Furthermore, OPU-ICSI optimises the use of expensive or limited stocks of frozen semen. One 0.5ml straw of semen can, depending on the quality, be used for three to five ICSI sessions. Finally, OPU offers a last chance to produce offspring in case of sudden death of the mare.