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					      IN VITRO PRODUCTION AND CRYOPRESERVATION OF EQUINE
                   EMBRYOS: SUCCESSES AND LIMITATIONS

                               Fernanda da Cruz Landim-Alvarenga

  Department of Animal Reproduction and Veterinary Radiology, F.M.V.Z. – UNESP,
                         Botucatu, SP, 18618.000, Brazil
                            fernanda@fmvz.unesp.br


          In horses the progress of the study of the early events of fertilization has been
slow compared with that in other large domestic animals. Attempts to fertilize equine
oocytes in vitro have resulted in only limited progress. The only foals born following in
vitro fertilization (IVF) of horse oocytes were originated from an in vivo matured oocyte
inseminated in vitro with Ca2+ ionophore treated semen (PALMER et al., 1990) and from
in vitro matured oocytes inseminated by intracitoplasmic sperm injection (SQUIRES et
al., 1996; COCHRAN et al., 1998).
          Successful IVF depends on a number of factors such as: availability of healthy
immature oocytes, efficient and repeatable methods for IVM, physiological sperm
capacitation, and subsequent optimum embryo in vitro culture systems. The success rates
of individual steps involved in equine IVP are still far from allowing their use in routine
protocols like in bovines (PARRISH et al., 1986; XU et al., 1987).
       Oocytes utilized in IVP can be obtained in vitro from slaughterhouse ovaries or in
vivo using ultrasound guided transvaginal follicle aspiration.
       In slaughterhouse ovaries, oocytes can be collected by: follicle aspiration with or
without flushing the follicle, by dissection and rupture of the follicle with scraping to
recover the mural granulosa and by slicing the ovaries. Recovery of oocytes by aspiration
of the follicle with a needle and syringe, as done in cattle, results in a relatively low
recovery rate in the horse. With the aspiration technique 1.5 (OKOLSKI et al., 1987) to
2.7 (HINRICHS, 1991) oocytes per ovary can be collected. This number increases to 3.1
(ZHANG et al., 1990) when the follicular wall is scraped. Because of the low number of
oocytes obtained CHOI et al. (1993) sliced and washed the ovaries obtaining 4.14
oocytes per ovary. We use this method at IVF laboratory in Botucatu, Brazil obtaining in
average 5 oocytes per ovary.
       PALMER et al. (1987) were the first to describe a standing aspiration technique,
where the operator held the ovary via rectum and guided a needle through the flank
towards the follicle. The first millimeters of follicular fluid were reinjected to dislodge
the oocyte and the follicle was then rinsed with 20 ml of Dulbecco’s PBS + heparin. The
recovery rate reported in this pioneer work was 63%.
          The first group of researchers to describe the trans-vaginal ultrasound-guided
technique (TVA) in standing mares to harvest oocytes was BRÜCK et al. (1992). Based
on the same idea used in human and cattle IVF programs, they used a finger shaped
transducer connected to an ultrasound console that showed a puncture line on the screen,
to aspirate preovulatory follicles. COOK et al. (1993) performed a study comparing
single versus double lumen needles. The 12 g double lumen resulted in the highest
recovery rate (84%) of preovulatory follicles compared to the single lumen needle (52%).
The double lumen needle allowed the fluid to drip continuously into the follicle while
suction was being applied.


          OOCYTE MATURATION
       The changes that occur to the oocyte in preparation for fertilization are called
maturation. Oocyte maturation consists in the nuclear and cytoplasmic maturation. In
vivo, maturation is a process coinciding with follicular development, changing hormone
levels, and meiotic progression. The conditions for in vivo oocyte maturation in the mare
are somewhat different from those in other domestic mammals; the ovulatory LH surge
occurs as a progressive rise that takes several days, with a maximum concentration
occurring 1 day after ovulation (WHITMORE et al., 1973; IRVINE et al., 1994).
       Resumption of meiosis is marked by extrusion of the 1st polar body and formation
of a Metaphase II plate. In most species, the resumption of meiosis is mediated by LH-
receptor interaction in the cumulus cell membrane, mediating the fusion of a cyclin B and
a protein kinase-1 to form an active form of the protein Maturation Promoting Factor
(MPF) in the ooplasm (LEE & NURSE, 1988; CRAN & MOOR, 1990).
       Cytoplasmic maturation is characterized by several changes in the shape and
localization of the organelles. GRONDAHL et al. (1995) described a breakdown of the
intermediate junctions between the cumulus cell projections and the oolema with an
enlargement of the perivitelinic space. In the same paper he described the formation and
arrangement of a large number of cortical granules immediately beneath the plasmic
membrane and the structural change of the mitochondria to a round shape. The migration
of the cortical granules is believed to be an important step in cytoplasmic maturation
(CRAN, 1989) and can be used to assess the oocyte maturity (LONG et al., 1994;
GOUDET et al, 1997). The redistribution of the cortical granules provides the ovum the
capacity to initiate the block of polispermy and to induce sperm nucleus decondensation
after sperm penetration (CRAN, 1989).
       In comparison with other species, the in vitro maturation of equine oocytes is low.
In average 40 to 60% of the oocytes reach metaphase II (reviewed by Dell’AQUILA et
al., 1997) as compared to 97% for bovine oocytes matured in vitro (SHIOYA et al.,
1988). In association to this fact, comparisons between in vitro maturation procedures in
horses are difficult because each study has a different protocol.
       The majority of studies in equine oocyte IVM use TCM 199 as a culture medium
with the addition of LH, FSH and estradiol 17 (E2). However, the source and levels of
LH and FSH have not been optimized. Luteinizing hormone of ovine (Dell’AQUILA et
al., 1997), bovine (WILLIS et al., 1991; SHABPAREH et al., 1993; SQUIRES et al.,
1996), and equine origin (GOUDET et al., 1997; and BEZARD et al., 1997) have been
used. In an experiment to test the use of equine pituitary extract (EPE) in the maturation
media of equine oocytes we obtained 43.7% metaphase II after 36 hours of maturation
(LANDIM-ALVARENGA et al., 1999). Our low maturation rates can be explained by
IVM conditions and by the origin of the oocytes. Medium 199 containing serum,
estradiol and equine gonadotropins has been used by others (SQUIRES, 1996). However,
we used a crude equine pituitary extract containing 91% unknown products (DUCHAMP
et al., 1987), which could have a detrimental effect on the oocyte maturation.


       IN VITRO FERTILIZATION
       In the mare the fertilization process in vivo has been elegantly described
(BETTERIDGE et al., 1982; ENDERS et al., 1987; BEZARD et al., 1989; GRONDAHL
et al., 1993) and blastocyst formation and establishment of pregnancy have been achieved
following transfer of IVM oocytes to oviducts of mated recipient mares (ZHANG et al.,
1989). On the other hand, reports on conventional IVF of in vitro or in vivo matured
equine oocytes are few, and the data is difficult to interpret due to variations in
techniques used by different laboratories.
       The first cleavage after IVF of in vivo matured oocytes has been reported by
BEZARD et al. (1989). The fertilization rate was low, 26% of oocytes fertilized and only
18% cleaved. However, two successful pregnancies were obtained after 14 surgical
transfers of these fertilized oocytes (PALMER et al., 1990). The reason(s) for poor IVF
and subsequent development rates of equine oocytes remains unclear. Sperm cell
capacitation, oocyte maturation, and the quality of oocyte investment have all been
offered as possible reasons for the poor IVF rates.
       In order to increase the IVP rates in the horse, fertilization techniques such as
partial zona dissection (PZD) or removal (PZR), subzonal sperm injection (SUZI), zona
drilling (ZD), and intracytoplasmic sperm injection (ICSI) have been used.
       Recently, the intracytopasmic injection of a single spermatozoon (ICSI) has been
introduced in human IVF with great success (PALERMO et al., 1992). For ICSI, a direct
injection with a fine pipette of one spermatozoon, after crushing its tail, is performed into
the cytoplasm of a mature oocyte exhibiting the first polar body in the perivitelinic space.
ICSI has so far had only limited success in domestic animals (KEEFER et al., 1990;
IRITANI, 1991; SQUIRES et al., 1996; Dell’AQUILA et al., 1997), however
pregnancies and foals were obtained after fertilization of in vitro matured oocytes when
this technique was used. McKINNON et al (1998) reported pregnancies from fertile and
infertile stallion by ICSI of frozen/thawed spermatozoa into in vivo matured oocytes.
SQUIRES et al, (1996) obtained one foal after ICSI of a slaughterhouse equine oocyte
matured in vitro for 24-26 hours; COCHRAN et al., (1998) obtained three foals after
ICSI of oocytes collected from pregnant mares and matured in vitro. Dell’AQUILA et al.
(1997) published a study that compared IVF and ICSI on in vitro matured equine oocytes,
reporting higher fertilization rates with ICSI than with conventional IVF (24.5% vs 8.7%
respectively).
       IN VITRO DEVELOPMENT OF EQUINE EMBRYOS
       Successful IVP depends on efficient and repeatable methods, for in vitro oocyte
maturation, physiological sperm capacitation and fertilization methods and subsequent
adequate in vitro culture systems for the presumptive zygotes to perform at least three or
four cell cycles in vitro before being transferred or cryopreserved as 16- cell or morula-
stage embryos.
       The only equine pregnancies established and foals born after IVF have originated
from fertilized ova subjected to short-term in vitro culture and then surgically transferred
at early stages (2 to 6 cells) to the oviduct of synchronized recipient mares (PALMER et
al. 1990; SQUIRES et al., 1996; COCHRAN ET AL., 1998).                     In most domestic
mammals, early cleavage stage embryos undergo developmental blocks in culture: at the
8 to 16 cell stage for bovine and ovine embryos (BAVISTER, 1988). Equine embryos in
culture block after 4 days at 12 to 16 cell stage (BATTUT et al., 1991).
       Because of the poor results obtained with in vitro fertilization and in vitro culture
of equine embryos during early stages of development, we transferred equine gametes to
rabbit oviducts (LANDIM-ALVARENGA et al, unpublished data). Oocytes were
collected by ultrasound-guided trans-vaginal aspiration (TVA) from pregnant mares
between 50-60 days of gestation. In the same experiment we transferred bovine gametes
to the contro-lateral oviduct of the same rabbit as a control. The number of morulae
collected was 13 (22.4%) for cattle and 6 (14.6%) for horses. We concluded that bovine
and equine oocytes can be fertilized in the rabbit oviduct. While this is the first report of
successful fertilization and embryo development of equine ova in a heterologous
recipient, the number of morulae obtained was low.
       Successful equine embryo in vitro production is still very low, and even when
fertilization is achieved, the embryos hardly grow until the morula stage in vitro. This
condition is probably due to less then optimal conditions of sperm capacitation, as well as
problems in the oocyte in vitro maturation. For a better control of the in vitro system
further studies are needed regarding equine physiological sperm capacitation and
acrosome reaction as well as the metabolism of oocytes and the process of fertilization.
The control of the IVP in horses will have, for sure, a big impact on the horse industry,
since the use of animals with fertility problems as well as bad quality semen will became
reality.


           CRYOPRESERVATION OF EQUINE EMBRYOS
           The first pregnancy obtained from a cryopreserved equine embryo was reported
by GRIFFIN et al. (1981), and the first foal was born in 1982 (YAMAMOTO et al.,
1982). Since then several attempts have been made to cryopreserve equine embryos.
           Small embryos, in the early stages of development (morulaes and early
blastocysts), have      better survival rates after cryopreservation comparing to more
developed embryos (SLADE et al., 1985; SQUIRES et al., 1989; MEIRA et al., 1993).
Several hypothesis have been created to explain this unsatisfactory survival rate,
including insufficient dehydration of the blastocoele, lower permeability of the bigger
embryos to the cryoprotector due to the presence of the capsula, and toxicicity of the
cryoprotectors (YOUNG et al., 1997).
           The capsula, that develops soon after the embryo enters the uterus, is correlated
with changes in permeability to cryoprotectors. LEGRAND et al. (2000) analyzed the
relationship between the thickness of the capsula and the cellular damage in equine
embryos treated with cryoprotectors, showing that embryos with a thicker capsula are
more damaged during frozen-thawed
           Several methods have been proposed to freeze equine embryos using different
cryoprotectors and cooling rates. The pregnancy rates after transfer of frozen-thawed day
6 embryos, smaller than 300 m, is around 40% (MEIRA et al., 1993; LANDIM e
ALVARENGA et al., 1993). However the recovery rate of small embryos on days 6 is
smaller than the one obtained on day 7 or 8 (SLADE et al., 1985; SQUIRES, 1993;
FERREIRA et al., 1997). This may be related with the absence of embryo into the uterus
during the flushing especially when the moment of ovulation was not precisely
determined.
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