Practice of Embryo Transfer

Transcription

1 Practice of Embryo Transfer Smith, A.K. BVM&S, DBR, MANCVS, MRCVS School of Animal and Veterinary Sciences, Charles Sturt University, Wagga Wagga, NSW 2678, Australia As humans we have domesticated and used animals to our own ends for centuries. The more we have learned about cellular makeup and function the surer we have become in our belief that we can control everything. This is probably true for many issues, and ET will be no different, but if we remember the important parts about caring for the animals and providing them with a husbandry/management system in which they can be contented then we are likely to achieve generally better, and certainly more consistent, results. Therefore forward planning is the key. Taking the time and effort to prepare the animals appropriately and paying attention to detail will improve success. Donor Selection Criteria Donor heifers: Body Condition Score (BCS) approx. 3*. Regular oestrous cycles, having achieved Critical Mating Weight ( Kg) Age: months preferably. It is possible to flush 13 month-old heifers but issues arise surrounding physically getting ones arm through the anus without causing damage, and catheterising the cervix. The combination of weight for age (above) plus BCS should provide a well-grown large-framed animal. Donor Cows: At least six weeks calved, but preferably eight weeks. No endometritis and returned to regular oestrous cycles. Preferably two natural heats since calving, but at least one. Maintaining or increasing liveweight (Body Condition Score 3). * Body Condition Score assessed over range 1-5 (Blackwood et al, 2013)) Nutrition It is now recognised that at least 60 days may be required to recruit and develop a primordial follicle through to an ovulatory follicle and the environment that the cow is in during that time can influence not only the vitality of that oocyte but also the subsequent embryo (Webb et al, 1999). Therefore nutrition becomes very important. Donors should be on a rising plane of nutrition for 6-8 weeks before flushing. All adjustments must be gradual without any sudden changes. Careful attention to mineral supplementation is also important. Superovulation Gonadotrophins such as equine Chorionic Gonadotrophin (ecg) and human Menopausal Gonadotrophin (hmg) have been used in the past to superovulate cows but due to the undesirable effects from extended activity of the luteinising hormone (LH) fraction they have been superseded by purified FSH. This is available as a freeze-dried product with diluent which when mixed produces a solution with controlled amounts of FSH & LH activity. These commercial FSH products have been historically sourced from ovine and porcine pituitaries. At present there is only one licensed FSH product available 28

2 in Australia: Folltropin (Bioniche, Canada). It is a porcine FSH product and is available through Minitube and Bioniche. It is best to start superovulation from a known reference heat so that the injections commence at a known point in the oestrous cycle. A superovulated response can be achieved starting anywhere in the cycle, given good control of the follicular wave, but the most repeatedly consistent results are achieved when the FSH injections commence between days 9-14, where oestrus is on day 0. The reference heat can be created by using any of the standard oestus synchrony protocols, but if one is reverse-planning treatment dates from a pre-planned calendar day for the flush-date then it would be prudent to use one that provides a narrow window for onset of heat. I always found using a simple progesterone-releasing device with prostaglandin 24 or 48 hours prior to device-removal gave adequate results. If a reference heat is not observed or for some reason there isn t sufficient time for the reference heat synchrony programme it is possible to use an intravaginal device to exogenously mimic the progesterone levels found mid-cycle. The results from this method are satisfactory but less consistent than starting from a known reference heat, however, can be useful if little or no behavioural signs are seen at the time of the reference heat. In the twice-daily injection regime the injection interval is ideally 12 hours but as long as the interval is no less than eight hours satisfactory results will still be achieved. Product manufacturers advocate that the total dose be split into eight equal injections but the use of a declining regime with higher dose volumes given at the start produces superior results (Broadbent et al, 1996). Responses to the same superovulation regime will vary from donor to donor and there are also breed differences in sensitivity to the treatment, with the Simmental breed being particularly sensitive. Additionally body condition, nutrition and environmental factors will all influence the response to treatment. A generalisation can be made that maiden heifers will respond satisfactorily to a lower total dosage of FSH than that required for a mature cow. Examples of a standard commercial programme used for maiden heifers and mature cows are shown in Table 4. Research is on-going in developing methods of delivering FSH in a single intramuscular, slow-release vehicle to reduce the need for so many multiple injections. Early results from a trial in Argentina involving 349 donors, of several different breeds, demonstrated no difference in superovulatory response or ova/embryos produced. Mean (+SEM) numbers of total ova/embryos, fertilized ova and transferable embryos were , and for the twice daily controls, and , and for the single injection group. In 2015 Trophogen,Inc.a US-based biotechnology company have signed an agreement with Zoetis to develop a long-acting recombinant modified bovine Follicle-Stimulating Hormone (rbfsh) analogue. Cleanliness at the time of injection is important as contamination of the stock solution can result in loss of potency of the FSH. New sterile syringes and needles should be used for each injection. In animals that have high body condition scores the superovulatory response is improved if the injections are given into the muscle of the neck. Insemination The number of straws of semen recommended can vary from operator to operator but given good quality semen usually three straws given in two insemination periods is adequate. Usually the afternoon of the day the donor is in oestrus and again the following morning. The precise timing of this action can be 29

3 dependent on the time of onset of oestrus. If the donor is in standing oestrus in the morning then an AI at mid-afternoon that day with two straws and again the following morning with one straw is sufficient. If the donor only comes to standing oestrus later in the day then the first A.I can be delayed. This is easily done if DIY A.I is used but may be awkward with a technician service. The time of onset of heat is usually directly proportional to the size of the superovulatory response. If a cow is in standing oestrus the night before she is expected then a large superovulatory response can be assumed and an extra A.I session using an additional two straws of semen should be planned for the morning of the first day of A.I. If through previous experience the semen is of known poor quality then extra straws may be used (e.g. Three sessions utilising 2, 2 and 1 straws of semen). A mix of semen from two different bulls can also be used when quality is questionable. This requires checking that Breed Society rules permit this practice and all progeny produced will need to be DNA-typed to confirm parentage. Natural service can also be used and the bull should be allowed to serve the donor as soon as she comes into oestrus. Remember that the owner should be advised that the bull will require DNA testing too. It is also worth remembering that for export purposes, embryos scheduled for export cannot have been produced as a result of natural service, only by the use of export-quality semen, by artificial insemination. Embryo Collection Epidural anaesthesia should be used to avoid continued rectal straining by the donor animal, and to avoid muscle exhaustion in the human. Cleanliness of the epidural site and the perineum is important and cotton wool swabs dampened with surgical spirit are ideal for this. All instruments, catheters, stylets, tubing and filters must be sterile. Metal instruments, silicone tubing, glass tubing and certain makes of catheter can be autoclaved. No disinfectants or harsh cleansing solutions that may persist on surfaces and be harmful to the embryos should be used. If any equipment is sterilised by ethylene oxide gas sterilisation, then it must be aired for a minimum of 7 days before use. A successful recovery rate requires good catheter position and good media flow. These conditions are easiest met if the epidural completely abolishes peristalsis. The ideal position for a 2-way catheter is to have the cuff inflated, and anchored, at least 5 cm past the level of the inter-cornual ligament (ICL) (Fig 3). Inflation of the cuff should be enough to anchor it in position without damaging the endometrium; usually 2mls of air and 4mls of water will be sufficient in most donors. Once the flushing media is infused through the catheter care must be taken not to over-distend the horn and aliquots of approximately 30 mls 50 mls will be sufficient. To aid successful drainage of the media down the catheter it is important to gently lift the tip of the uterine horn so that it is not curled under the body of the uterus. The bovine uterus is a fragile organ and any rough handling or forceful actions can result in damage to the endometrium or perforation of the wall. Best practice is to return the donor animals to normal oestrous cycles after the flush. This ensures termination of any skulking embryos and reduces opportunities for development of cystic ovarian disease after superovulation. This can be done either by an injection of PGF2α or by using a combined progesterone releasing intravaginal device with PGF2α. Generally a higher quantity of PGF2α beyond the standard dose is used, this would often be one-and-a-half or double the normal dose. If donors are to be repeat flushed they can be re-scheduled for a superovulation programme using the post-flush oestrus as the reference heat. In practice this translates to an approximate five-week interval between flushes. Alternately an additional oestrous cycle may be allowed giving an eight-week interval between flushes which may be helpful from a logistical, calendarisation, point-of-view. 30

4 Embryo Processing Examination of the embryos requires a good quality stereoscopic dissecting microscope with at least x10, x25 and x50 magnification. Disposable plastic Petri dishes, or welled dishes, should be used. Once retrieved from the flushing solution embryos should be held in embryo holding solution at a constant ambient temperature (approx. 20 C) and out of strong light until the next procedure. Ideally embryos should be frozen or transferred within three hours of collection. It is good practice to wash all embryos ten-times whether destined for freezing or fresh transfers. An outline of this procedure is given in Table 5 and a more detailed account can be found in the Manual of the International Embryo Transfer Society (IETS). The ten-times washing regime will remove a number of viruses from the zona pellucida whilst other more persistent ones can be removed by including a wash with trypsin in the procedure. Table 6 shows lists of viruses which can be successfully removed by standard washing procedures and trypsin washing. Embryo Freezing Prior to freezing the embryos are partially dehydrated by placing in cryoprotectant. Ready-to-use 10% glycerol and 1.5 M ethylene glycol are available commercially. These have the benefit of being quality controlled and screened for viruses. Worldwide 1.5M ethylene glycol is the most commonly used cryoprotectant. Prior to freezing the embryos need to be allowed time to equilibrate in the cryoprotectant. This state is achieved with 1.5M ethylene glycol after 5-10 minutes immersion but with 10% glycerol the embryo needs to be passaged step-wise through increasing concentrations of the solution. After the required equilibration time in cryoprotectant the embryos are loaded into straws (Fig 4), sealed, and labelled prior to being put in the freezing machine. At the appropriate time in the freezing protocol the cooling process is paused to allow seeding. Seeding is easily performed using a metal rod, such as a brass rod, supercooled in liquid nitrogen then gently and briefly touched against the straw. Alternatively tweezers can be used, but care must be taken as over time the metal will itself become supercooled making them uncomfortable to hold. The seeded media immediately becomes opaque as it nucleates. It is important not to seed at a point on the straw directly over the embryo. Many embryologists will seed on the media column adjacent to the one containing the embryo. Once all straws are seeded the cooling process continues and at the end the straws are swiftly plunged into liquid nitrogen. Straws containing embryos (especially the media column in the straw which contains the embryo) should be kept submerged in liquid nitrogen and should not be exposed to air until thawing. Handling of the straws should be done with tweezers not fingers. Placing the whole goblet containing the straws in a wide-mouthed insulated flask containing liquid nitrogen is the easiest method of checking identities of straw labels. This allows reading of labels/plugs whilst keeping the straws submerged. Vitrification An alternative to freezing embryos with a programmable freezer is the use of vitrification. This is an ultra rapid cryopreservation procedure that eliminates the formation of ice crystals in and around the embryo. The Syngro Bovine Vitrification kit is the first commercially-available bovine vitrification system. It was developed in conjunction with Colorado State University and Bioniche Animal Health, Canada, and is suitable for in-vivo derived bovine embryos. Although promoted as being a quick procedure, 10 minutes or less per embryo, the timings, volumes and column-lengths within the straw are critical; therefore, practice and manual dexterity are necessary. It is worth noting that IETS regulations mandate that vitrified bovine embryos be packaged with a light-blue coloured; label or seal, goblet, and cane tab (if applicable). 31

5 Embryo Transfers For successful results forward planning is essential. Recipient Selection Criteria Recipient heifers should be: At least 15 months old At least 370 kg (dairy heifers) At least 400kg (beef heifers) Of a suitable frame-size to carry to term and to calve naturally an embryo calf of the breed to be transferred. Body Condition Score 2½ -3*. Recipient Cows Ideally no more than 4 th calver. Good breeding history At least six weeks calved Clean uterus,and returned to normal oestrous cycles post-calving Maintaining or increasing liveweight Body Condition Score 3*. * Body Condition Score assessed over range 1-5 (Blackwood et al, 2013) Nutrition and Management It is important that recipients are on a rising plane of nutrition for six to eight weeks before transfer and that this continues for at least another six weeks post-transfer. The ration should be checked to confirm that it is providing an acceptable balance of energy and protein with sufficient levels of roughage. As with the donors a suitable supply of minerals is important. Purchasing recipients, mixing groups, and routine procedures, such as worming, bolusing, vaccination, freeze-branding and foot-trimming should all be completed at least 6-8 weeks before the transfer date to minimise stress. Disease Control A number of infective agents can severely affect the success of an ET programme. The principal culprits are Bovine Viral Diarrhoea Virus (BVDV) and leptospirosis spp., and more recently Neospora caninum. Vaccination is possible for the first two agents. If BVDV is known to be a problem on a farm then screening of the recipients prior to vaccination may be desirable to identify and remove any persistently infected animals before vaccination. The main route of spread for Neospora caninum is vertical transplacental transmission from an infected seropositive dam to the foetus during pregnancy. At present there is no information available to suggest that in normal circumstances embryos are exposed to the parasite Neospora caninum in the uterine cavity. Therefore it appears that the risk of transmission of N. caninum by ET is not related to the serological status of the embryo donors but is linked to the seropositivity of the recipients as transfer of embryos to seropositive recipients will lead to transplacental transmission of N. caninum resulting in a high incidence of abortions and will yield persistently infected calves at birth. Therefore it may be prudent to include screening for this agent in the preparation of recipients whilst appreciating that this can be equivocal in young maiden heifers, as the period when it is easiest to detect Neospora antibodies is when 32

6 the animal is pregnant!!. There is potential for false-negatives in the region of approximately 10% or more with ELISA tests. Synchronisation To successfully achieve pregnancy the recipients need to be synchronised to come into oestrus one week before the embryo is transferred. Therefore a tight synchrony period is required. A good method is to use a progesterone/progestagen releasing intra-vaginal devices in combination with an oestradiol treatment at the time of insertion, and a prostaglandin injection at the end. This gives a tight synchrony with the peak of activity hours after device removal. In the authors opinion a double prostaglandin injection protocol will not provide a tight enough synchrony to maximise recipient usage. It is best for stockman to observe recipients for oestrus the week before transfer. Observing should be performed at least three times a day for a minimum duration of twenty minutes per observation period. Observations should occur for the three days following the day the synchrony devices are removed. Transferring the Embryo Having checked for the presence of a corpus luteum and recorded which ovary it is on, the recipient is prepared for the embryo transfer. Epidurals should be given. Again cleanliness of the epidural site and the perineum is important. After traversing the cervix the transfer gun is guided along the uterine horn ipsilateral to the corpus luteum. Pregnancies can be achieved if the embryo is placed at the level of the ICL but results improve if the site of transfer is further up the horn. Ideally a position of 10cm or further past the ICL is desirable. It is important to be as gentle as possible and avoid manoeuvres which result in endometrial damage. Sometimes in fatter animals with large dependent tracts it is necessary to accept that the site of placement is not ideal in preference to over-handling the tract resulting in inflammation or physical injury to the endometrium. Sufficient lubricant and care should be used when dilating the anal ring. Apart from the obvious welfare aspects any repeat usage of a recipient is made a lot easier if the anal ring isn t narrowed and taut as a result of scarring from previous over-zealous manipulations. Figure 3: 2-way catheter Source: Bovine Medicine, 2 nd Edition Eds: Andrews, Blowey, Boyd & Eddy 33

7 Figure 4: Table 4: Examples of Commercial Superovulation Regimes for Cattle. Maiden heifer Mature cow day 12 am 2.25 mls FSH Solution 3.0 mls FSH Solution pm 2.25 mls 3.0 mls day 13 am 1.75 mls 2.5 mls pm 1.75 mls 2.5 mls day 14 am 1.25 mls & double dose of PGF mls & double dose of PGF2 pm 1.25 mls 2.0 mls day 15 am 0.75 mls 1.5 mls pm 0.75 mls 1.5 mls Total dose 12 mls 18 mls Notes: 34

8 1. At time of publication there is only one licensed FSH product in Australia. These examples are acceptable for use with Folltropin (Bioniche, Canada) 2. PGF2 = Prostaglandin F 2-alpha 3. These protocols are included as examples of regimes used in commercial embryo transfer practice. Alternative protocols do exist with variations in total volume, volume of individual injections and rate of decline of dosage regime. Table 5: Embryo Washing Procedure Only embryos from a single donor should be washed together. Ten or fewer embryos washed at one time. Only zona pellucida intact embryos washed. Only embryos free of adherent material washed. Minimum of ten washes ensuring gentle agitation in each wash. Use a new sterile micropipette each time embryos are moved from one wash to the next. Regulate volumes so that each wash is at least 100-fold dilution of previous wash, achieved by using 1 ml droplets of Dulbecco s PBS and a 10µl micropipette to move embryos. Source: IETS Manual 3 rd edition. Table 6: Pathogens not found after washing of embryos previously exposed to these agents. Normal Ten-times Washing: Akabane virus Bovine Leukaemia virus Bluetongue virus Bovine viral diarrhoea virus Foot-and-mouth disease virus Brucella abortus Trypsin Washing: Bovine herpesvirus 4 Infectious bovine rhinotracheitis virus Vesicular stomatitis virus Source: IETS Manual 3 rd edition. References BLACKWOOD, I.; EXTON,S.;LITTLER,B. & SIDDEL, J. (2013) A national guide to describing and managing beef cattle in low body condition. Meat & Livestock Australia. f Retrieved BROADBENT, P.J.; TRESGASKES, L.D.; DOLMAN, D.F. & SMITH, A.K. (1996) The effects of varying dose and pattern of administration of ovine FSH on the response to superovulation in performance tested, juvenile Simmental heifers. Animal Science 62: SMITH,A.K. (2004) "Embryo Transfer ", In Bovine Medicine, Blackwell Science, Oxford Editor : A H Andrews : chapter 40, pp WEBB, R.; GOSDEN, R G.; TELFER, E.E. & MOOR, R.M. (1999) Factors affecting folliculogenesis in ruminants. Animal Science 68: