Ovum pick up (opu)

Ovum pick up (opu)

1. Ovum Pick up (OPU)/Ovum Retrieval Method/Transvaginal Ovum Pick up Method/Ultrasonography Guided Ovum Pick up Method

2. Definition: The ovum pick up (OPU) method is a non-invasive and repeatable technique used for recovering large numbers of competent oocytes from antral follicles of live animals. OPU consists in the transvaginal removal of the oocytes by aspiration of the ovarian follicles with the aid of an ultrasound probe. OPU along with in vitro fertilization (IVF) allow for the removal of oocytes from ovaries of donor female animals, fertilizing these oocytes with IVF outside the body, and enabling large numbers of fertilized embryos to be available for implantation in recipient animals. First it was developed in the human to assist human infertility. When people realized its application prospect, considerable researches have been aimed at applying this technology in animals. In1988 OPU was first established in cattle by a Dutch team.

3. Advantages: OPU can be repeated many times twice a week. It is capable of generating more embryos per unit time than conventional multiple ovulation and embryo transfer. It has advantages to collect oocytes from animals with less invasiveness and the use of superior animals as oocyte donors in embryo transfer. Each group of oocytes taken can be fertilized with the seed of a different reproducer. Therefore it is possible to obtain embryo groups with different paternity from the same donor in a very short time. Ovum Pick Up does not require any preventive hormonal treatment and does not interfere with the normal physiology of the donor. Ovum Pick Up embryos can be produced by donors of any age, even during lactation and in the first 3 months of pregnancy without the possible side effects of superovulation: infertility, ovarian cysts, mastitis, traumas related to behavioral changes induced by superdosing hormones, loss of time and milk production.

4. Cont. For certain donors suffering from long-standing sterility, the Ovum Pick Up can even exert a therapeutic effect by removing the cystic formations that are often present on the ovaries of these animals, also as a consequence of repeated superovulations . All donors return to estrus 7-10 days after the last withdrawal and can be immediately fertilized. On request, the embryos produced can be sexed and the owner can selectively withdraw the embryos of the desired sex. Follicular aspiration also allows studying the molecular intricacy and the role of various cytokines during folliculogenesis . T here is no interference with the milk production since the lactating donor has no production loss and no effect on the breast or on the pregnancy.

5. Disadvantages: Vaginal bleeding, intra-abdominal bleeding, intra-peritoneal bleeding may be occur. Risk of injury of pelvic structures, pelvic infections. Discomfort and severe pain may be occurred even hospitalization may be needed. In some cases repeated OPU can cause acyclicity. Oocyte yield per ovary depends on different stages of estrus cycle. In case of hormonestimulated OPU the exogenous hormones disturbs donor’s endocrine, especially when exogenous hormones are used to the donor for a long time, which might lead to infertility of the donor.

6. Instruments: An OPU system consists of 3 major components: 1.An ultrasonographic scanner with an appropriate transducer (probe) Figure: Ovum pick up (OPU) ultrasound transducer

7. Cont. 2. An aspiration pump Figure: Ovum aspiration pump

8. Cont. 3. A needle connected to a ovum collected tube Figure: Ovum collecting needles with tube

9. Procedure: The transducer and the needle guide are commonly constructed as a single operational unit to enable accurate manipulation of the needle from outside the cow, while bringing the transducer into close contact with the ovaries. Insertion of the transducer and the puncture of needle can be visualized on the ultrasound screen when it is advanced into a follicle. To facilitate visualization, it is helpful to have a biopsy guide on the ultrasound screen. The needle is in turn connected to a vacuum pump by silicone or Teflon tubing such that follicular contents are aspirated as soon as aspiration pressure is applied via the vacuum pump.

10. Cont. Figure: Ovum pick up procedure

11. Cont. The follicular fluid and oocytes are collected into a collection device positioned between the needle and the pump. This oocyte collection device can be a regular embryo filter or a simple Falcon tube sealed with a stopper, into which an afferent tube delivers the follicle aspirate and from which an efferent line is connected to the vacuum pump that applies the aspiration pressure. Although not compulsory, prior to OPU cows can be sedated with detomidinehydrochloride and treated with hyoscine-N- butylbromide to induce relaxation of the intestines. Subsequently, the feces are removed from the rectum and epidural anesthesia is induced using 2% lidocaine to remove excessive straining during the trans-rectal manipulation. After the tail has been fixed to one side, the vulva and perineum are thoroughly cleaned and disinfected before the OPU device, containing the transducer and the needle guidance system, is inserted into the vagina.

12. Cont. While the OPU handle can be manipulated with one hand outside the cow, the head of the ultrasound transducer is positioned cranio-dorsally to the left or right of the cervix, depending on which ovary is to be collected from. Using the other hand per rectum, the operator fixes the ovary and positions it against the head of the transducer such that the ovary and follicles can be visualized on the ultrasound screen. A biopsy line programmed into the scanner’s software is displayed on the screen and indicates where the follicle needs to be positioned for successful puncture. The operator then advances the needle slowly forward until the vaginal wall is pierced and the needle is visualized entering the ultrasound field. By monitoring the needle’s position and simultaneously manipulating the ovary per rectum, the needle can be directed into a follicle. Once the needle enters the follicle, the aspiration pump is activated using the foot pedal and the follicular fluid and COCs (cumulus oocyte complexes) are collected into the embryo filter which contains some oocyte collection medium.

13. Cont. Subsequently, the filter contents are washed and transferred to a petri dish and the oocytes are identified using a stereomicroscope, captured using a glass pipette, and placed into maturation medium. After 24 hours of maturation, they will be fertilized and cultured for 7 days ‘in vitro’ to reach the blastocyst stage. The final outcome of OPU, in terms of numbers and quality of retrieved COCs, is influenced by both technical and biological factors.

14. Limitations: Sophisticated instruments and skilled individuals are necessary for carrying out this technique. The use of OPU in young donors is limited by the smaller dimensions of the pelvis which makes the intravaginal handling and transducer using difficult. T he major problem with pre-pubertal donors is the impaired in vitro developmental capacity of the recovered oocytes resulting in a low overall efficiency of the procedure. Embryo production from OPU oocytes is affected by age, season, follicle stimulating hormone (FSH) stimulation and can average 1-3 embryos developed from oocytes collected per session.

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