12 December 2008

Embryo collection

The video clip I took along my visiting at Tarraweena Stud

Embryo collection
Embryos are usually collected from the donor 6-7 days after breeding. At that time embryos and any unfertilised ova are freely suspended in the small amount of fluid found in the horns of the uterus. Embryo collection in the small ruminant species is a surgical procedure although non-surgical techniques have been the subject of considerable research. The cervix of sheep and goats is very difficult to penetrate, especially 6-7 days after heat, and commercial success in non-surgical embryo collection is limited to goats.

Sedation and local anesthesia of the donor may provide adequate restraint but we prefer general anesthesia as it allows optimal control over the procedure. Once the donor is anesthetised she is placed on her back in a small tilt table and the underside of her abdomen is prepared for surgery. A laparoscope (a rigid fibre-optic device) is inserted through a small incision in the skin to allow evaluation of the response to super-ovulation. If there is evidence of a satisfactory response (several CL's should be obvious on each ovary) the uterus is identified and elevated through a small incision (7-10 cm.) on the midline. A soft rubber Foley catheter is introduced to the uterine horn and the cuff of the catheter inflated to seal off the uterus. A sterile solution is flushed through another small catheter placed at the tip of the uterine horn to carry the embryos out of the uterus, through the Foley catheter and into a small collection dish or filter. After one horn is flushed, the procedure is repeated with the other uterine horn. Following the flushing procedure, the incisions are closed, and the ewe is given prostaglandin to help her uterus evacuate any debris.

We take particular care to minimise the formation of adhesions (scar tissue) that can be stimulated during almost any abdominal surgery and can reduce fertility. While donors may be flushed 2-3 times in a season, allowing them to become pregnant after a few flushes lets the reproductive tract become stretched, and breaks down some of those adhesions.

The recovered flush fluid is examined under a microscope. Unfertilised eggs, degenerating embryos and good quality embryos may all be found in varying proportions. A quality grade (1 being excellent and 4 being very poor) and developmental stage is assigned to each of the recovered embryos. In most cases, excellent quality embryos result in the highest pregnancy rates. Only grade 1 embryos are eligible for export. Lower grade embryos (grades 2 and 3) may be transferred fresh and they can result in pregnancies. Depending upon the aims of the project, the embryos may be transferred into recipient ewes or frozen for use at a later time. The embryo has several biological features which spare it from infection by many serious viral and bacterial agents. Embryo transfer is an effective means by which a donor with a chronic illness - a ewe with OPP or a doe with CAE - may reproduce with no risk of disease transmission to her offspring. The washing of embryos in several changes of holding medium, and possible treatment with a protein dissolving enzyme (trypsin) for export purposes, means that there is virtually no risk of disease transmission to embryo recipients or to the developing fetus (assuming the recipients are free of the disease in question).

Recipient selection and care
A major determinant of the success of an ET program is the quality of the recipients. The pool of potential recipient animals should be in excellent health. All recipients should be sound. Well grown doelings or ewe lambs may be used but females with a history of good mothering are preferred. Animals with a history of mastitis, reproductive disorders or dystocia (difficulty giving birth) should be avoided. Recipients should be identified 4-8 weeks prior to sponging and penned by themselves. Each animal should be uniquely identified, preferably with an ear tag that can be read at a distance. Potential recipients should be treated with parasiticides appropriate to the geographic location and season. Vaccinations against Clostridial diseases and any screening blood tests for production limiting-diseases (CAE, OPP, Johne's disease etc) should be completed in advance of the program. The use of cull animals as recipients for an ET program will dramatically compromise the outcome. Even if these animals do become pregnant, entrusting them with the care of valuable offspring is ill-advised.

Donors and recipients
Lean but healthy recipients are desirable. Donors and recipients should be started on a plane of nutrition to allow a slight increase in body condition beginning two weeks prior to insertion of intra-vaginal devices. Salt and access to minerals should be provided. Where donors and recipients are fed stored forages, vitamin A and D supplementation is required. A plentiful supply of good quality hay with a moderate protein level (14-16%) is generally adequate and exclusively legume (alfalfa, clover) diets should be avoided. It is very important that the feeding levels of recipients should not be decreased, or dramatically altered, following ET as this may contribute to early embryonic or fetal loss. In all cases, every effort should be made to be minimise stress, including rough handling, sudden dietary switches and commingling with strange animals. Handling with dogs should be avoided. Donor and recipient cycle synchrony Ideally, the donor and the recipients should be in heat (estrus) and ovulate within 12 hours of each other to help ensure that at the time of ET the uterine environment of the recipient is very similar to that of the donor. While donor and recipients heats should be synchronised as closely as possible, asynchrony of plus or minus 24 hours still results in acceptable pregnancy rates. Synchronising the time of estrus in the recipients and donors usually involves the use of intra-vaginal sponges or other sustained release forms of progesterone. Prostaglandin (cloprostenol - Estrumate® or dinoprost -Lutalyse®) and PMSG (pregnant mare serum gonadotrophin also known as eCG equine chorionic gonadotrophin) and GnRH can also be incorporated to narrow the time interval during which ovulation occurs. Wherever possible, recipient females should be placed with teaser animals to enhance ovulations. Under no circumstances should recipients be allowed access to intact males as they will be bred and probably become pregnant. Pregnant recipients compromise the outcome of many programs. Despite hormonal manipulation of their reproductive cycle, some potential recipients do not synchronise or are found otherwise to be unfit as embryo recipients at the time of ET. To ensure adequate recipients are available, approximately 6 ewes or 10 does should be synchronised per donor. For frozen embryo programs an average of two embryos per recipient should be projected and 30% more recipients than will minimally fit the program should be synchronised. Where recipient availability is limited, up to three embryos of the same mating may be transferred into a recipient (assuming she is a relatively large animal). Where recipient numbers are very limited, surplus embryos may be frozen for transfer at a later place or time. Prior to embarking on an embryo transfer program, we consult with our clients to provide a schedule to optimise the synchronisation of their recipients and donors. All schedules, including recommended brand names, dosages, routes of administration, and timing of procedures are provided. Adherence to all details provided in the schedule is of considerable importance to the outcome of the program.

Embryo freezing
The purpose of freezing embryos is to hold the embryo in a state of suspended animation so that when the embryo is thawed for transfer at a later date or place, normal biochemical processes in the embryo may be resumed and the embryo can then go on to develop normally. Embryos of good to excellent quality can be frozen and thawed with a slight reduction (10-20%) in the pregnancy rate normally expected with fresh embryos. Sheep and goat embryos are generally frozen in 0.25 mL plastic straws, each straw containing 2-4 embryos of a similar developmental stage from the same flush.

Prior to freezing, the embryos are moved from their holding medium and placed in a cryo-protectant solution. This solution acts as an anti-freeze by replacing some of the water that is present in the cells of the embryo. This helps ensure that during the freezing process ice crystals do not form in the embryo's cells causing them to rupture and the viability of the embryo to be reduced. The embryos suspended in the cryo-protectant are then loaded into straws, the straws are sealed and labelled by convention as to their contents. The straws are then placed in an embryo freezer and then cooled at a controlled rate to between -30 and -35 C. At that point the embryos can then be plunged into liquid nitrogen (-196 C) for indefinite storage.

Most international movement of livestock genetics now occurs in the form of frozen or cryo-preserved embryos.

Thawing embryos
Frozen embryos must be handled to ensure that they stay frozen - improper handling and partial thawing and re-freezing is a potential cause of poor pregnancy rates with frozen embryos. They must remain immersed in liquid nitrogen in a storage tank, or transported in a special 'dry shipper', which holds the embryos in liquid nitrogen vapour.

Frozen embryos are thawed in a manner quite similar to frozen semen, and should be handled under carefully controlled conditions of time and temperature. Following a brief exposure to air, the straw containing the embryo(s) is immersed in warm water, the straw dried and the contents of the straw evacuated into a small sterile dish. The embryos are transferred from the freezing solution into a modified embryo holding solution. Depending upon the specific cryoprotectant used, step-wise removal of the cryoprotectant may be necessary prior to transferring the embryos into a recipient animal. This is an opportune time to re-evaluate the state of the embryo(s) prior to transfer into the recipients and to comment on the impact that freezing may have had upon them. The International Embryo Transfer Association has standardised codes which describe embryo grade (quality) and developmental stage (age) of the embryo and those should be indicated on the certificate of embryo transfer, along with embryo donor, sire and recipients identification.

Transfer of embryos
With the exception of the freeze-thaw cycle indicated above, the transfer of fresh or frozen embryos is essentially the same. Most embryo transfers are performed using the laparoscope, in a fashion similar to that used for AI. As transfers can be made quite quickly, local anesthesia and sedation are often sufficient. Once the animal is placed on the tilt table, the abdomen is prepared routinely and infiltrated with local anesthetic. The laparoscope is placed through the abdominal wall and the ovaries inspected for evidence of one or more CL's. A CL (corpus luteum) is a progesterone producing body on the ovary where an ovulation occurred. Usually in sheep and goat recipients more than one CL is observed. If a normal CL is identified, a small incision is made on the midline of the abdomen, forward of the udder. The tip of the uterine horn corresponding to the ovary bearing the CL is pulled out. A fine catheter is used to transfer the embryos into the uterine horn. The uterine horn is allowed to return to the abdomen and the small incisions are closed. In most cases, two embryos are transferred into each suitable recipient although this may range from one to three. Pregnancy rates are usually higher with fresh embryos than frozen embryos of a similar developmental stage. The steps involved in freezing can result in the destruction of some cells of the embryo and may reduce the ability of the embryo to develop normally.

An injection of long acting penicillin or tetracycline is given and depending upon the operator, anti-inflammatory products may be recommended. In the majority of cases, post-operative recovery is rapid. Donors and recipients should be monitored for at least a week for signs of illness and treatment begun immediately if deemed appropriate.

The majority of our small ruminant ET procedures are conducted on the farm. Efforts should be taken at all times to reduce stress to the recipient animals so maintaining them in their original groups on their own farm is preferable. While an absolutely sterile operating environment is impractical, a clean, dust free area is highly desirable to optimise the outcome for both surgery and embryo handling. Enclosed, well lit facilities with a solid well drained floor, preferably concrete, are desirable. Access to warm water and several power outlets (110/120v) will be required. Embryos are susceptible to injury and death under conditions of fluctuating temperature. It is very important that the area used for handling embryos and performing ET is draft free and can be maintained at a fairly stable 21 C. There should be no areas of very warm or cold air.

Ideally, the doors into and out of the ET area should not communicate directly to the outside as this often contributes to widely and wildly fluctuating temperatures and drafts. Animal movement through the ET area should flow in a single direction. Most importantly, animals about to undergo surgery should be kept on a dry, feed-free area. Their movement should be restricted so they may be caught and restrained with minimal anxiety. Animals should be allowed to recover in a warm, dry, deeply bedded area where they may be observed and allowed access to feed (hay) and water once they are standing. At least three assistants capable of handling and lifting sheep will be required. The ET team will be occupied with anesthesia, preparing animals for surgery, performing surgery, handling embryos and post-operative care. We cannot allow our ET team to be distracted from those responsibilities.

Expectations and caveats
Average Range
Efficiency of embryo collection 12 CLs 0-30
Transferable embryos 6 embryos 0-30
Embryos surviving to birth 70% 0-100%
Recipients pregnant 60% 0-100%

Approximately 25% of potential donors do not respond to super-ovulatory treatments. Pregnancy rates are generally, but not always, lower with frozen than with fresh transferred embryos.

Embryo collection and transfer in small ruminants involves general anethesia and surgical procedures. Although modern anesthetics and surgical techniques reduce risk to donor and recipient animals, it should be appreciated by owners that serious and potentially fatal complications, while rare, may occur. Regurgitation of stomach (rumen) contents under anesthesia is potentially fatal. To reduce the risk of aspiration occurring during anesthesia, we first induce anesthesia with an injection and then insert a cuffed, semi-rigid, rubber tube through the mouth and into the trachea (wind-pipe) of the animal This endo-tracheal tube is connected to an anesthetic machine which delivers oxygen and anesthetic gas at safe, controllable levels. Even with these precautions, it is critical that the donor and recipient animals not have access to any water or feed for at least 24 hours prior to the time of embryo transfer.

We make every effort to ensure that anesthesia and surgeries are performed safely but every client embarking on an ET project should be apprised of potential risks.

Pregnancy rates are variable and relate to donor, recipient, embryo and operator factors, many of which may interact, usually negatively, to influence the outcome. Clients thinking of an ET program should be prepared to follow directions explicitly. There are no guarantees and careful attention to detail is important to reduce the chances of a program being disappointing and increase the probability of it being remarkably rewarding.

The information contained herein is not all-encompassing. If questions arise prior to beginning or at any time during the program please contact us as soon as possible.

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