Recovery of Oocytes Using Transvaginal Ultrasound in the Mare:
Current Equipment, Techniques and Applications

 

Abstract
Ultrasound-guided transvaginal oocyte aspiration provides an excellent non-invasive method for obtaining equine oocytes. The recovered oocytes can then be used in in-vitro fertilisation or other programmes or used for other purposes. This method of oocyte recovery is still relatively recent and there is a lack of detailed information available for those wishing to attempt the technique in the horse. The purpose of this paper is to provide a review of current techniques and equipment used and provide information on the actual 'mare-side' technique of ovum pick-up from someone relatively experienced in the aspiration procedure. The problem of comparing recovery rates between different workers is discussed and there is a brief comment on the applications of the technique in the mare.

Introduction
There is currently much interest in the so-called assisted reproductive technologies. The most established of these in the horse is embryo transfer that, despite problems such as poor ability to superovulate the mare, allows valuable mares and mares remaining in competition to produce foals. There are several reasons that a subfertile mare may be unable to provide an embryo for use in an embryo transfer programme. Although fertilisation rates are little different between normal or subfertile mares, embryonic losses between Days 2 to 4 and Day 14 post- ovulation are estimated at around 70% for subfertile mares compared with zero to 9% for normal mares (Ball et al 1989). Recent work by Carnevale and Ginther (1995) supports the conclusion that defective oocytes, oviductal embryos or impaired oviductal environment accounts for a large proportion of these losses. Subfertile mares with intraluminal fluid collections or a damaged cervix are poor candidates for embryo transfer and for such mares other techniques are necessary.

In-vitro fertilisation (IVF) programmes are well established in human medicine and a key part of the process is ultrasound-guided transvaginal oocyte aspiration (Cohen et al 1986; Kemeter and Feichtinger 1986) and this technique has superseded the more invasive laparoscopic techniques. In animals, including the mare, oocytes have traditionally been obtained from follicle aspiration of slaughterhouse ovaries or via a flank laparotomy technique (Vogelsang et al 1988). Slaughterhouse material has the obvious disadvantage of lack of repeatability and there is often considerable delay in the time between oocyte collection and placement in culture medium. Surgical laparotomy techniques have obvious disadvantages in terms of ease and repeatability.

Consequently it was an important finding when Pieterse and his co-workers at Utrecht University (Pieterse et al 1988), using cattle, were the first to describe the technique of oocyte aspiration during transvaginal ultrasound ovarian scanning in domestic animals. They coined the term 'ovum pick-up' (OPU) for the collection of the oocytes and OPU offered a repeatable, less invasive and less traumatic system to provide a source of oocytes. These oocytes could then be used in invitro maturation (IVM)/IVF programmes or gamete intrafallopian transfer (GIFT) studies.

The ability to perform non-surgical repeated OPU to obtain equine oocytes was an important step to allow progress in the horse. The first report of the technique in the horse was by Bruck et al (1992), although only four follicles were punctured in this report. More widespread use of the technique was reported by Cook et al (1992 & 1993) and Bracher et al (1993). Both groups of workers reported that the procedure was safe and well tolerated and the technique allowed for repeated collection of oocytes in vivo.

Since then there have been few reports of workers using the technique to obtain equine oocytes (Parlevliet et al 1993; Dippert et al 1994; Meintjes et al 1994) although at the recent International Equine Reproduction Symposium in Brazil, six papers from four groups of workers involved transvaginal aspiration of oocytes (Carnevale and Ginther 1995; Kanitz et al 1995; DuChamp et al 1995; Bezard et al 1995; Meintjes et al 1995; Li et al 1995)

Despite this current interest in oocyte aspiration, there are probably less than 10 groups of workers repeatedly aspirating equine oocytes using transvaginal ultrasound world-wide. It is, therefore, not surprising that gamete manipulation techniques in the horse are poorly advanced compared with other species. This not only reflects the relatively new stage of the techniques, but also certain difficulties innate to the horse as well as the economic structure of the equine breeding industry. Some of the difficulties such as in vitro oocyte maturation and sperm capacitation are outside the scope of this paper which will concentrate on technical aspects of transvaginal guided-ultrasound follicle aspiration.

The actual technique of ovum pick-up represents the key first step in whatever the aspirated oocytes are to be used for. Manipulating the equine ovary in vivo has many technical difficulties and this may be partly responsible for the generally low (20-30%) recovery rates found in the mare (Bracher et al 1993; Cook et al 1993). However, many factors such as size of follicle punctured (Kanitz et al 1995; DuChamp et al 1995) and stage of cycle/ pregnancy status of the mare (Meintjes et al 1995) affect recovery rate and so it is difficult to compare recovery rates between different groups of workers. This point will be addressed in more detail subsequently.

 

 

Follicle Aspiration in the Mare: Technique and Equipment Used

Mare Preparation and Restraint
Adequate preparation is essential to prevent movement and ensure adequate relaxation of the rectum. The most commonly used drug to achieve rectal relaxation is a hyoscine/dipyrone combination (Buscopan; Boehringer Ingelheim) at a dose rate of 20 mg Hyoscine per 100 Kg body weight given intravenously 10 minutes prior to the aspiration procedure. Other groups have used atropine (Meintjes et al 1995) and propantheline bromide (Carnevale and Ginther 1993) to achieve rectal relaxation. Following manual evacuation of faeces, 50 ml of 2% lidocaine infused into the rectum just before aspiration helps provide additional analgesia and relaxation. Sedation of the mare is also necessary and can be achieved by use of detomidine (Domosedan; Smith-Kline-Beecham) intravenously at a dose rate of 0.01 mg/kg given 5 minutes before starting. Concurrent administration of butorphanol (Torbugesic; C-Vet Ltd) helps provide additional analgesia.


Mare restrained in stocks with puncturing equipment on right hand side

The mare should be restrained in a set of stocks/crush which limit her movement. It is particularly important to limit lateral movement as this can make fixation of the ovary against the ultrasound transducer difficult. Bladder catheterisation and epidural anaesthetic, as commonly used in the cow (Pieterse et al 1988) are not generally used in the mare.

Ultrasound Equipment
Various ultrasound machines have been used, usually with a sector transducer of various frequencies: 5.0 MHz (Meintjes et al 1995); 6.0 MHz (Bruck et al 1992) or, most commonly, 7.5 MHz (Bracher et al 1993; DuChamp et al 1995). Use of a 5.0 MHz curvilinear transducer (Cook et al 1992; Cook et al 1993; Dippert et al 1994) and a 5.0 MHz linear-array transducer (Carnevale and Ginther 1993; Carnevale and Ginther 1995) have also been described.

Once experienced in the orientation of the sector transducer, the author's preference is for a 7.5 MHz sector transducer as this allows more possibility to manipulate the ovary to allow for more complete puncturing of all follicles. Our recent follicle puncturing studies have been done using a Scanner 200 Vet (Pie Medical Ltd; Maastricht, The Netherlands) with a 7.5 MHz mechanical sector annular array system with a multi angle transducer (150 / 90 / 60 scan- angles) (Pie Medical Ltd; Maastricht, The Netherlands). During 1995 the 90 or 150 scan- angle was used and this expanded ultrasound field facilitates the positioning of follicles within the puncture line.

Carnevale and Ginther (1993) point out that the advantage of their method using a linear- array transducer is that this is the commonest transducer used for routine mare gynaecological work. However, sector transducers have many applications in equine veterinary medicine and most facilities likely to be performing transvaginal follicle aspiration would be likely to have a sector transducer in the author's opinion.

Needle Guidance System
Some form of casing around the transducer is needed in order to guide the needle into the follicle to be aspirated. In the initial studies of Pieterse et al (1988 and 1991a & b) a 7.5 MHz sector transducer (Type SDR 1550; Phillips; Eindhoven, The Netherlands) was inserted into a 50 Cm long autoclavable stainless steel holder with a handle at one end to facilitate manipulation. The casing has a needle guide channel running the length of the casing. On the ultrasound image as displayed on the monitor there is a needle/biopsy guideline which corresponds to the location and direction of the aspiration needle, making it possible to predict the direction of passage of the needle on the monitor screen. This system was successfully applied for use in the equine (Bracher et al 1993). Other workers have either used similar devices, either designed themselves (Bruck et al 1992) or a commercially available casing (Follicular aspiration device, Tokyo Keiki Products) (Carnevale and Ginther 1993).


Needle guidance system before assembly

Assembled

Ovum Pick-Up Needle and Collection Systems
A variety of needles have been used of varying lengths and thickness’ and either specially designed ovum pick-up needles (Cook Veterinary Products; Queensland, Australia) or ordinary luer-lock disposable needles.

There are basically two types of ovum pick-up needle systems: double or single lumen needles with the double lumen needle design allowing both flushing and aspirations simultaneously. Some workers have used both single and double lumen needles (Bracher et al 1993; Cook et al 1993; DuChamp et al 1995) whilst others have used either single-lumen needles (Meintjes et al 1995) or double-lumen needles (Kanitz et al 1995).

It was agreed by the three groups of workers using both single and double lumen needles ((Bracher et al 1993; Cook et al 1993; DuChamp et al 1995) that the double lumen system provided better recovery rates. However, Meintjes et al (1995) using single-lumen needles reported a recovery rate of 75.8% which is higher than that reported by Bracher et al (1993), DuChamp et al (1995) and Kanitz et al (1995) all of whom used double lumen needles. However, Meintjes et al (1995) are the only group of workers to have aspirated oocytes from pregnant mares and this recovery rate of 75.8% was better that the 42.9% these workers obtained when puncturing pre-ovulatory follicles. It is clear that recovery rate is a confusing term as it depends how it is assessed and is also affected by many factors, not simply whether a single or double lumen needle is used (see later).

The length of the needle has varied from 50 Cm (Bruck et al 1992; Meintjes et al 1995) to the more usual 60 Cm (DuChamp et al 1995) with 60 Cm being the standard length supplied by Cook Veterinary Products, probably the main supplier of equine ovum pick-up needles. The gauge of the needle has varied from as thick as 12 gauge (Cook et al 1993; Meintjes et al 1995) to 15 gauge (Bracher et al 1993) or 16 gauge (Carnevale and Ginther 1993).

Throughout the repeated follicle puncturing attempts in our laboratory during 1995, a new disposable needle guidance system, based on that described by Bols et al (1995) in the cow, was used. In the system we used in 1995, an 18-gauge luer-lock disposable needle is attached via a stainless steel connector to sterile teflon tubing. The tubing was inside a hollow stainless steel tube and was then connected to a flushing syringe and a suction pump with a variable vacuum pressure normally set at 200 mm Hg. The stainless steel tube with the needle at the end is inserted into a guidance tube incorporated into the top of the OPU device. Care must be taken when inserting the needle into the guidance system that the needle goes smoothly through the hole at the end of the OPU device otherwise a rough edge may develop which can cause extra trauma during the puncture session. A marker on the metal needle guide indicates the extent the metallic needle guide and needle should be passed along the needle guide corridor.

 

Suction apparatus
Some form of suction is necessary to aspirate the follicles and recover the fluid containing the oocyte. Although two early equine follicle aspiration reports describe applying suction using a 50 ml syringe (Bruck et al 1992; Bracher et al 1993) all other workers have used a regulated vacuum pump. Pressures have ranged from 90 mm Hg (Meintjes et at 1995) to 300 mm Hg (Cook et al 1992) or a range from 230-300 mmHg (DuChamp et al 1995). For our studies in 1995 we used 200 mmHg.

 

Puncture technique
After emptying the rectum, bandaging the tail and administering the intra-rectal local anaesthetic and the sedation and uterine relaxant, the vulval and perineal area are thoroughly cleaned and disinfected. The transducer within its holder is covered with parafilm to prevent vaginal mucus and other debris entering the opening of the needle guide and then sterile lubricant applied. The transducer is gently inserted as far as possible to the left (when puncturing the left ovary) or the right (when puncturing the right ovary) of the external os of the cervix. By means of rectal manipulation, the ovary is positioned so that one or more follicle(s) are in the line of the needle by using the biopsy guide/puncture lines on the ultrasound monitor


Follicle imaged (Dotted lines indicate direction of needle)

On the instruction of the operator manipulating the ovary, the needle is then advanced beyond the needle guide, through the vaginal wall and into the follicle to be punctured. The instruction to advance the needle should only be given when the follicle has been steadily positioned on the puncture line. A distinct 'popping' sensation is felt when the follicle is entered and the echoic needle can be visualised within the follicle. As soon as the tip of the needle has been seen to enter the follicle, suction is immediately applied and the follicle, as imaged on the ultrasound screen, begins to collapse. Suction should be continued until the follicle appears to have completely collapsed. If flushing of the collapsed follicle is being performed, the flushing fluid can be visualised on the ultrasound screen re-filling the follicle and confirming the needle is still located in the correct place. During and after aspiration of the follicle fluid, the needle should be slowly and gently rotated in an attempt to curette the follicle wall. In addition the follicle can be gently manipulated per rectum. This should help dislodge any oocyte tightly attached to the follicle wall. This is important in the mare as recent work has shown considerable differences in equine and bovine oocyte-cumulus morphology within the ovarian follicle (Hawley et al 1995) including the presence of a 'thecal pad' in the mare which may at least partly explain the poorer recovery rates of the mare as compared with the cow. In any case, it makes thorough aspiration of the follicle important in the mare.

If a second follicle is seen adjacent to the punctured follicle, the direction of the transducer can be slightly adjusted so that the puncture line crosses the new follicle. Then the second follicle can be punctured and aspirated without withdrawing the needle from the ovary. This procedure should be repeated until all visible follicles have been aspirated. At the time of puncture, the size of the follicle can be estimated from the ultrasound screen. If information about recovery rates from different sized follicles is required, separate sterile plastic collection tubes should be used for each selected category of follicle. If significant blood (enough to be clearly visually obvious) the needle should be withdrawn and a new collection tube selected. When puncturing a large follicle it is probably better to puncture the follicle through the ovarian stroma rather than puncturing from the outside of the follicle. The later approach would seem to offer more potential for failure to recover the oocyte.

 

Flushing of Follicle
Although in 1995 an aspiration only system was used in our studies, prior to this follicles were flushed after aspiration using a previously reported method (Bracher et al 1993). Most workers report using flushing with modified Dulbecco's phosphate-buffered saline either with heparin (DuChamp et al 1995), heparin and calf-serum (Cook et al 1992) or heparin, calf-serum and a penicillin-G/streptomycin mixture (Meintjes et al 1995) , although Bracher et al (1993) simply used phosphate-buffered saline. If a double-lumen OPU needle with a bitubular connection between the needle and the vacuum system is used, then it is possible to use both flushing and aspiration of follicles simultaneously as reported by Kanitz et al (1995). Other workers prefer to interrupt aspiration to allow the follicle to fill again (DuChamp et al 1995). If a single-lumen needle is used then the follicles must be flushed by alternate filling and emptying of the follicle (Meintjes et al 1995).

The number of times the follicle is flushed depends on the size of the follicle: during dioestrus Cook et al (1992) simply aspirated follicles 5 mm. Larger follicles are flushed between 3 and 10 times using from 10-250 ml of flushing medium again depending on the size of the follicle (Bracher et al 1993; Cook et al 1992; DuChamp et al 1995; Kanitz et al 1995; Meintjes et al 1995).

 

After-care of mares
When this technique was first introduced mares were closely monitored following the aspiration procedure (Bruck et al 1995; Cook et al 1992; Bracher et al 1993). No abnormal clinical signs were detected. In many cases the mares used for our studies are ridden the following day and no problems have ever been reported. Some workers Some workers administer prophylactic penicillin either before (Meintjes et al 1995) or after aspiration (Cook et al 1992). Flunixin meglumine as an anti-inflammatory/ painkiller is also used by these workers either routinely before the procedure (Meintjes et al 1995) or after the procedure if any colic signs develop (Cook et al 1992).

 

Recovery and identification of oocytes
Briefly, in our studies, following collection into sterile plastic tubes, the follicular fluid is filtered through an embryo collection filter and the oocytes searched for under a stereomicroscope at x 120 magnification. Oocyte quality was judged on the number of cumulus layers, degeneration, transparency and homogeneity of the ooplasm and the presence of vacuoles (Parlevliet et al 1993). Evaluation of equine oocytes has been described in detail (Hinrichs 1991; Bezard and Palmer 1992; Bezard et al 1995; Torner and Alm 1995).

Recovery Rate
In this author's opinion meaningful comment on recovery rates between different groups of workers is impossible. However, recovery rate is generally quoted in all papers involving transvaginal ultrasound-guided needle follicle aspiration and the two most obvious features are an enormous variation in reported recovery rates of oocytes even by the same workers as illustrated by the 8% to 84% recovery rate found by Cook et al (1993) and , in general, rates are lower than would be expected for cattle where recovery rates of around 50% is typical (Kruip et al 1991; Moyo and Dobson 1995). It is very difficult to compare recovery rates between different groups of workers because recovery rate is dependent on several different factors including, but not limited to:

Stage of Oestrous Cycle
Cook et al (1993) report a 63% mean recovery rate for follicles punctured during oestrus and this figure falls to 22% during dioestrus. In contrast Bracher et al (1993) found no influence of stage of cycle on oocyte recovery rate (14.1% and 19.8% at oestrus and dioestrus respectively). Kanitz et al (1995) reported a difference in recovery rates between follicles aspirated at Day 5 or Day 12 of the cycle (26.3% versus 12.4% respectively). These workers also found that acyclic mares gave the highest recovery rates (40.4%). Meintjes et al (1995) also found that recovery rate from pregnant mares was higher than that from mares in oestrus (75.8% versus 42.9%).

Aspiration technique including flushing
Several aspects of the actual aspiration technique have been reported as influencing recovery rate. Using a wire loop device threaded through the aspiration needle and rotating the loop during the aspiration procedure resulted in a reduced recovery rate (51% versus 22%) (Cook et al 1993).

The use of a 12 gauge double-lumen needle rather than a 12 gauge single-lumen needle resulted in an increased recovery rate from pre-ovulatory follicles (84% versus 51%) (Cook et al 1993). Bracher et al (1993) reported a similar pattern, although overall lower, in recovery rate between follicles aspirated using a single-lumen (12.3%) or double-lumen (24.4%) needle. The lower overall recovery rate for these latter workers may be a reflection of the different follicles for puncture: the recovery rates for Cook et al (1993) where based on aspirating pre- ovulatory follicles whereas Bracher et al (1993) performed follicle aspiration as long as more than 3 follicles (size not specified) were seen at an ultrasound examination of the ovaries.

DuChamp et al (1995) also reported that recovery rate was higher with a two-way needle system then a one-way system (29% versus 22%). However both of these figures are lower than those reported by Meintjes et al (1995) using a single-lumen ; they reported recovery rates of 75.8% in pregnant mares.

The value of flushing has been evaluated for human IVF procedures and whilst some papers report no improvement in oocyte recovery rate with flushing (Scott et al 1989; Haines et al 1989) others suggest that flushing follicles with a double-channel needle can result in an increased recovery rate of oocytes (Waterstone and Parsons 1992).

Vacuum pressure.
In contrast to the situation in cattle, vacuum pressure does not seem to affect recovery rate (Kanitz et al 1995) who reported no difference in recovery rates between vacuum pressures of 0.2 or 0.4 bar. Vacuum pressure is partly dependent on the length of tubing from the needle to the pump device.

Follicle Size
Some workers report an effect of follicle size on recovery rate (Kanitz et al 1995; Meintjes et al 1995) . Somewhat confusingly the former reported maximal recovery rates with follicles less than 10 mm (38.9%) and lowest recovery rates for follicles between 21 and 30 mm (6.9%) whereas the latter found the greatest recovery rate in follicles 25 - 35 mm in diameter (68.8%) and this rate was not significantly different from the recovery rate for follicles 8 to 25 mm in diameter (43.5%). The lowest recovery rates for these workers were found in follicles above 35 mm (34.8%). Carnevale and Ginther (1993), aspirating only follicles above 30 mm during oestrus obtained a recovery rate of 89.2%.

To further complicate the situation others report no effect of follicle size on recovery rate (Bracher et al 1993; DuChamp et al 1995).

Hormone pre-treatment
Human chorionic gonadotrophin (hCG) is often given prior to follicular aspirations (Cook et al 1993; Carnevale and Ginther 1995; Meintjes et al 1995). Cook et al (1993) reported higher recovery rates in mares treated with hCG whereas Bracher et al (1993) found no such effect.

Needle size
Various needle sizes have been used to aspirate mare follicles and although most workers have used 12 gauge needles, one of the best recovery rates has been obtained with 16 gauge needles (Carnevale and Ginther 1993). Baltussen et al (1992) in cattle found no increase in recovery rate of oocytes could be obtained by increasing the diameter of the needle used to puncture the follicles and they suggested that a thin needle type may be the optimum to use in cattle.

In our '95 studies, a disposable 18 gauge needle system was used. The use of the disposable needles rather than the long specifically designed OPU needles has the advantage of always using a sharp needle as if the disposable needle becomes blunt it can easily and cheaply be replaced. Although the long OPU needles are able to be re-sharpened this can be difficult and the needle never regains its original sharpness. It remains to be seen if the recovery rates are as good with the disposable needles. Even if they prove acceptable for bovine work, the different features of the mare ovary means that the results from the bovine cannot be directly transposed.

Aspiration only with a disposable needle system has obvious economic and time advantages, but it is not yet possible to say if recovery rates could be improved by using a different system. To investigate this possibility we will be using both a 12 gauge disposable needle system without aspiration and comparing the recovery rates with the use of a double lumen 12 gauge equine OPU needle (Cook Veterinary Products Ltd). It was possible to bore out the existing metallic needle guide which used 18 gauge needles to take 12 gauge needles. The new needle guide can allow use of both disposable short needles and also the long 60 Cm OPU needles and so represents a versatile system.

To conclude this discussion of recovery rates, almost all workers reporting use of OPU in the equine have utilised long OPU needles and so the question of whether a disposable needle system can give acceptable recovery rates remains unanswered. There may be information on this based on our recovery rates for this current year (1996) using both systems. In any case, comparison of recovery rates has many difficulties. It is probably accurate to state that at this moment, anyone beginning equine OPU would be best to use a double-lumen 12 gauge needle system, aspirate and flush the follicle. Once acceptable recovery rates have been achieved, use of disposable systems may be tried and recovery rates compared.

 

 

Applications of Equine Ovum Pick-Up

1) Improvement of Fertility
The aim of equine breeders and veterinarians working in broodmare practice is to get as many live foals born from mares bred the previous year as possible. Obviously for the Thoroughbred industry this is limited to one foal per year as newer reproductive technologies such as embryo transfer are not currently permitted. It is, therefore, difficult to envisage the rapid acceptance of OPU techniques within the framework of Thoroughbred breeding

The non-Thoroughbred breeding population, which represents a large number of competition, show and other valuable animals, will represent the animals from which mares suitable for OPU will be chosen in the near future and until there is any acceptance of the technique by the relevant Thoroughbred authorities.

Equine embryo transfer has become widespread in the equine in certain countries such as USA. Within other countries such as the United Kingdom and the Netherlands there are very few foals born from embryo transfer. The reason for this poor uptake of embryo transfer in these countries is multifactorial with both innate equine physiological problems such as difficulty in achieving superovulation and economic reasons being involved. Nonetheless centres such as Colorado State University offering a commercial equine embryo transfer provide an opportunity for problem mares and mares remaining in competition to have foals and good pregnancy rates are obtained. Certain mares, however, can be in embryo transfer programmes for several years with no success. The most likely reasons for this are failure of the embryo to arrive in the uterus or early death of the embryo.

For such mare the assisted reproductive techniques of in vitro fertilisation (IVF), gamete intrafallopian transfer (GIFT) and a combination of IVF of in vitro matured (IVM) oocytes, perhaps also involving intracytoplasmic sperm injection (ICSI), may hold the key to the future. While the techniques have been found to work well in the bovine field progress in the equine has been slow. To date there has only been one IVF foal born in the world. However, by the time of this meeting, there will hopefully have been born a foal produced using a combination of IVM, IVF and ICSI techniques by Squires and his co-workers at Colorado State University. OPU has provided the bovine industry with an ideal way of increasing the number of gametes available for maturation, fertilisation and in vitro development which will in turn allow an increased production. The equine breeding industry has different criteria on which it is based and an enormous increase in production from one mare is not a realistic aim. Nonetheless it is possible to envisage that oocyte manipulation may become an option in the clinical situation for the problem, subfertile mare. It is likely that the development of transvaginal ultrasound-needle guided aspiration of oocytes will represent a very important link in this chain of events and the next few years should be exciting times for those of us involved in the field.

2) Research Tool
While much attention is focused on OPU as a future clinical tool for use in subfertile mares, it is important not to overlook other application s of the oocytes retrieved. For example in our laboratory, recovered oocytes from OPU have been used in an elegant series of experiments to develop a hemizona assay for stallion spermatozoa (Fazeli et al 1995). In addition recovered follicular fluid has been used to investigate sperm-oocyte interactions (Cheng et al 1996).

There would appear to be no doubt that refinement and continued development of the technique of OPU in the mare will have a major role in future equine breeding programmes.

 

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