Cryopreservation of Follicular Oocytes and Preimplantation Embryos in Cattle and Horses

Journal of Reproduction and Development, Vol. 49, No. 1, 2003 Japanese Society for Animal Reproduction: Award for Outstanding Research 2002 Cryopreservation of Follicular Oocytes and Preimplantation Embryos in Cattle and Horses Shinichi HOCHI 1) 1) Faculty of Textile Science and Technology, Shinshu University, Ueda, Nagano 386 8567, Japan Abstract. Factors affecting sensitivity of preimplantation embryos and follicular oocytes to cryopreservation were analyzed in the equine and bovine species. (1) Survival of equine blastocysts after two-step freezing in the presence of glycerol as the cryoprotective agent (CPA) was influenced by development of the embryonic capsule. The use of ethylene glycol (EG) with sucrose as CPAs improved the post-thaw survival of blastocysts and made it possible to transfer the embryos into recipient mares without removing the CPAs. In addition, early blastocysts cryopreserved by vitrification could develop both in vitro and in vivo when the embryos were exposed to vitrification solution in a stepwise manner. The vitrification procedure was also applied to the relatively large expanded blastocysts. (2) Bovine embryos produced in vitro have been considered to be highly sensitive to the process of cryopreservation. To solve this problem, Day-7 blastocysts produced in a serum-free system were cooled at 0.3 C/min rather than 0.6 C/min before being plunged into liquid nitrogen, resulting in no loss of the post-thaw viability. The supplementation of LAA in IVM/IVF media or IVC medium was effective in producing pronuclear-stage zygotes or morula-stage embryos relatively tolerable to freezing, respectively. (3) Transmission electron microscopic observation of immature equine oocytes showed that cellular injury occurred near the sites of gap-junctions between cumulus cells and the oocyte. In cattle, higher fertilization rates of oocytes were obtained when the oocytes were subjected to cryopreservation at an intermediate stage during IVM (GVBD for freezing, Met-I for vitrification). Vitrification of bovine Met-II oocytes in open-pulled glass capillaries, characterized by an ultra-rapid cooling rate (3,000 5,000 C/min), was found to avoid any harmful influence of vitrification and warming. Key words: Cryopreservation, Oocytes, Embryos, Cattle, Horses (J. Reprod. Dev. 49: 13 21, 2003) ithin a few decades many substantial improvements have been made in procedures routinely used for cryopreservation of domestic embryos. Recent renewed interest in details of cryopreservation methods is due in part to differences in the freezing sensitivity of in vitroproduced bovine embryos and their in vivoproduced counterparts, as well as in the practical Accepted for publication: September 20, 2002 Correspondence: S. Hochi application of vitrification procedures as an alternative approach to conventional freezing. Although oocytes have been shown to be much more difficult to cryopreserve than zygotes or later stage embryos, there is considerable interest in the capability to cryopreserve oocytes, since this would permit establishment of banks of cryopreserved oocytes. The purpose of this paper is to review our findings in the cryopreservation of preimplantation embryos and follicular oocytes, with special

14 HOCHI reference to cattle and horses. Embryos Equine blastocysts There are many unique and interesting features in equine embryogenesis. For example, fertilized ova are retained at the oviductal ampullaryisthmus junction for as long as 120 hours postovulation [1], and late morula- to early blastocyststage embryos are in transit from the ampulla to the uterus for up to 132 hours post-ovulation [2]. After the rapid transport of equine embryos through the oviductal isthmus, the mucin-like embryonic coat, the so-called capsule, is deposited on the inner surface of the zona pellucida of early blastocysts coincident with expansion of the blastocoele [3]. The capsule surrounds the embryo and maintains its spherical form until the fourth week of pregnancy [4]. Although the fundamental role of the capsule during the early phase of pregnancy in horses remains to be determined, it is possible to speculate that the capsule serves as a protective barrier against any harmful environment for embryos in the uterus of mares. On the other hand, the presence of the capsule in equine blastocysts may contribute to the slow progress in developing methods for their successful cryopreservation (the first successful report of embryo cryopreservation in horses was not published until 1982 [5], 10 years after Whittingham et al. [6] had reported the successful freezing of mouse embryos). It is well established that smaller Day 6 embryos are more resistant to freezing injury than larger Day 7 embryos. The size-dependent sensitivity of equine blastocysts to freezing injury has been explained by the different permeability to glycerol [7]. Development of the embryonic capsule during the expansion of equine blastocysts may be responsible for limiting permeation of the cryoprotective agent (CPA). The use of a more permeable solute, ethylene glycol (EG), did not result in an improved pregnancy rate after transfer of cryopreserved embryos [8]. In contrast, when a small amount (0.1 M) of sucrose was added to the cryoprotective medium consisting of 1.8 M EG, all post-thaw embryos developed in vitro. Such frozen embryos could be transferred directly into the uterus of recipient mares after in-straw dilution of the CPAs with physiological saline. Because of the lower permeability to CPAs of equine embryos compared with bovine embryos, vitrification of equine embryos has been considered to be difficult, but successful vitrification has been achieved with unexpanded blastocysts after pretreatment with EG and brief exposure to an EGbased vitrification solution [9]. Non-surgical transfer of 5 vitrified-warmed embryos resulted in 2 pregnancies and the delivery of foals (Fig. 1). In conventional freezing, equine blastocysts 200 300 µm in diameter were more sensitive to freezing injuries than the smaller blastocysts [7], but with vitrification, equine blastocysts 200 to 300 µm in diameter survived cryopreservation as well as blastocysts that were <200 µm in diameter [10]. Since no culture system for equine blastocysts to support capsule formation has yet been established [11 13], post-thaw embryos should be transferred into the recipient uterus immediately after shortterm culture in vitro. Bovine IVP morulae & blastocysts Because of the economic importance of cattle, much experimental effort has been directed to the cryopreservation of bovine embryos, resulting in the successful slow-freezing [14], two-step freezing [15] or vitrification [16] at relatively early stages. Large numbers of preimplantation stage bovine embryos are now produced by the IVM/IVF/IVC system, but in vitro-produced embryos have been reported to be more sensitive to chilling and cryopreservation than in vivo-produced embryos [17]. Nevertheless, IVP blastocysts have been reported to survive freezing, and the transfer of frozen-thawed embryos has yielded live calves. Previously, the morphology of bovine embryos produced in vitro was different from that produced in vivo. IVP morulae have been reported to be dark with poor compaction and a small perivitelline space. Such blastocysts often retain a dark appearance and irregular shape without a clearly defined ICM. A higher ratio of lipid to proteins has been hypothesized to explain the darkness of the cytoplasm, and to be a factor influencing their postthaw survival. Recently it has been shown that removal of serum from the medium for culturing presumptive zygotes improves the resistance of blastocysts to cryopreservation [18]. Such morulae grown in serum-free medium had undergone compaction and had a distinct perivitelline space, and blastocysts possessed a well-defined ICM,

EMBRYO AND OOCYTE CRYOPRESERVATION 15 Fig. 1. The first pregnancy and new-born foal derived from a vitrifiedwarmed equine embryo. (A) Post-warm Day-6 embryo transferred. (B) Ultrasonographic image of the conceptus at Day 15. (C) The fetus at Day 60, with detectable heart-beat. (D) A Hokkaido-Native-Pony foal and the Thoroughbred recipient mother on 3 September, 1994. Fig. 2. Effects of cooling rates during cryopreservation on survival of in vitro-produced bovine embryos (P<0.05 between a and b, and among w to z). characteristics similar to in vivo-produced embryos. Theoretically, the extent of cell dehydration is determined both by the cooling rate and by the temperature at which slow cooling is terminated. Previously, an optimum cooling rate of 0.6 C/min until 35 C had been reported for bovine blastocysts produced in vitro and in vivo [17], but our results indicate that the optimum cooling rate for bovine blastocysts produced in a serum-free system is slower (Fig. 2) [19]. Very high survival of post-thaw blastocysts (96%) is obtained by a combination of cooling at 0.3 C/min with rapid warming, suggesting a difference in membrane permeability between blastocysts produced in vitro and in vivo. Nevertheless, only 42% of bovine morulae produced in a serum-free co-culture system survived cryopreservation even at an optimum cooling rate. This suggests the presence of a mechanism other than that caused by intracytoplasmic lipid droplets.

16 HOCHI It has been reported by Imai et al. [20] that the addition of an unsaturated fatty acid, linoleic acid, in the form bound to BSA to the medium for culturing IVF bovine zygotes improved the survival of the blastocysts after two-step freezing. Therefore, the effect of different linoleic acidalbumin (LAA) concentrations during IVC on postthaw survival of resultant morulae was examined [21]. The highest survival (60%) was from those cultured in 0.1% LAA, which was significantly different from the post-thaw survival of the morulae produced in 0 and 0.3% LAA (Fig. 3A). Next, the effect of the timing of exposure to LAA on the post-thaw survival of IVP morulae was examined. The post-thaw survival of morulae that had been exposed to LAA from 20 to 90 hours postinsemination (hpi) or from 90 to 138 hpi was higher than that of morulae cultured without LAA from 20 to 138 hpi (Fig. 3B). These survival rates were lower than that of morulae cultured with LAA over a period of 20 to 138 hpi (76%). The results indicate that culture of IVF zygotes in 0.1% LAA produces morula-stage embryos relatively tolerant to the process of freezing and thawing. Bovine pronuclear zygotes The one-cell stage zygote, the biggest single cell, is used as a volumetric indicator of cells in hyperosmotic cryoprotective solution. Practically, cryopreservation of pronuclear-stage zygotes is beneficial in the production of transgenic cattle by DNA microinjecton, as already reported in mice [22] and rats [23]. But the post-thaw developmental potential of pronuclear-stage bovine zygotes derived from IVM and IVF has been reported to be extremely low. As mentioned in the previous paragraph, we have confirmed that the supplementation of the IVC medium with 0.1% LAA is effective in improving the post-thaw survival of the embryos. Because the positive action of LAA could be derived from 48 h coincubation, the effect of LAA supplementation in 24 h for IVM plus 20 h for IVF on freezing sensitivity of pronuclear-stage bovine zygotes was examined [24]. More than half the zygotes appeared to be morphologically normal after cryopreservation, and cultured in modified synthetic oviduct fluid medium to assess their subsequent development. The post-thaw development of LAA-treated zygotes (5 14%) was higher than that of non LAAtreated zygotes (1 2%). In particular, the inclusion of a small amount of sucrose in the EG-containing solution was effective in promoting subsequent development of the post-thaw zygotes. In contrast, there was no positive effect of the LAA on cryosurvival of pronuclear-stage zygotes after vitrification in EFS40 and VS14 solutions. The positive effect of LAA on the freezing sensitivity of IVP bovine embryos may be explained by increased membrane fluidity, due to direct incorporation of linoleic acid into the lipid bilayer. In addition, membrane fluidity may be increased by depletion of membrane cholesterol. Fig. 3. Production of freeze-tolerable bovine morulae by co-incubation with linoleic acid-albumin. Effects of LAA concentrations (A; *P<0.05, **P<0.01 vs. 0% LAA) and exposure timing (B; a vs. b vs. c P<0.05).

EMBRYO AND OOCYTE CRYOPRESERVATION 17 Fig. 4. Injury pattern frequently observed in immature equine oocytes after vitrification. (A) Fragments in the perivitelline space (arrows) and vacuoles in the periphery of the ooplasm. Scale bar=5 µm. (B) Cumulus cell process ending and gap junction in a fragment. Scale bar=1 µm. The albumin from bovine serum is the factor involving cholesterol extraction from sperm cells as a water-soluble lipid carrier [25]. Therefore, it may be possible to speculate that affinity of linoleic acid in LAA for the lipid bilayer of the plasma membrane facilitates access of the bound albumin to membrane cholesterol. Oocytes Equine GV oocytes The in vitro production of equine embryos has not been very successful, although there has been only one full report of a foaling after IVF in 1991 and this had been accomplished after in vivo maturation of the oocytes [26]. The slow progress in IVF research in this species is due in part to the insufficient recovery rate of equine oocytes (because of the lack of an established regimen for superovulation of mares as well as the anatomical characteristics of the ovary). Furthermore, stallion spermatozoa are relatively sensitive to cryopreservation. In our study, ionophore A23187 had a striking effect in inducing capacitation of stallion spermatozoa [27]. The use of zona-free equine oocytes for a sperm penetration assay, however, did not always reflect the extent of sperm capacitation. This may be explained by the short life span of capacitated/acrosome-reacted stallion spermatozoa. As for IVF experiments, the rate of sperm penetration of zona-intact equine oocytes was very low unless a part of the zona pellucida was removed [28, 29]. More recently the rate of fertilization of equine oocytes has been improved by applying intracytoplasmic sperm injection (ICSI) [30, 31], and there have been two foals produced by ICSI in two separate laboratories [32, 33]. Under the geographic limitation to transporting equine ovaries to the laboratory, development of a cryopreservation method for equine oocytes seemed to be important. We have established an evaluation system for the viability of equine GV-stage oocytes after cryopreservation; 60% of fresh oocytes reach the metaphase-ii stage after 32 h culture [34]. When EG was used as a CPA, 16% of the frozen-thawed oocytes in the GV-stage matured in vitro [35]. As for vitrification, 17% of the post-warm GV-stage oocytes matured in vitro. Ultrastructural evaluation of the GV-stage oocytes exposed to a highly concentrated environment has suggested that freezing damage is associated with the destruction related to intercellular communication between cumulus cells and the oocyte, probably due to severe dehydration (Fig. 4) [36]. Bovine oocytes during IVM Many problems were found to be associated with chilling and freezing of in vitro-matured or ovulated oocytes, including spindle disorganization, and loss or clumping of

18 HOCHI Fig. 5. Effects of cooling rates during vitrification on survival of in vitro-matured bovine oocytes (*P<0.05, **P<0.01 vs. non-treated controls). microtubules [37]. Such changes may result in some scattering of chromosomes, or increased ploidy due to incomplete second meiosis at fertilization [38]. In addition, CPAs may induce a premature release of cortical granules, resulting in hardening of the zona pellucida and a decrease in fertilization rates. With GV-stage oocytes, mechanical destruction near the sites of intercellular communication between cumulus cell process endings and the oocyte may be harmful for subsequent development of the cryopreserved oocytes. Cryoinjuries occurring in oocytes therefore seem to be specific to the stage of maturation. Since the intercellular communication through gap junctions between cumulus cells and oocyte can be dissociated after the resumption of meiosis [39, 40], cryosensitivity of the oocytes may be affected by their nuclear stage. From the time-dependent shift in nuclear maturation, oocytes at 0 (GV), 6 (GVBD), 12 (Met-I), and 24 h (Met-II) of IVM were selected for comparison. In the experiment with two-step freezing [41], in vitro-matured Met-II oocytes became fertilized by multiple spermatozoa, whereas in vitro maturing GVBD/Met-I oocytes were fertilized normally like fresh control oocytes. Mechanical damage caused by freezing occurred less frequently in maturing and matured oocytes than in immature oocytes. The highest normal fertilization rate of frozen-thawed oocytes was 25% in the 6 h IVM group. With vitrification [42], fertilization rates of oocytes vitrified at any IVM stage were not significantly different from those of fresh control oocytes, and polyspermic fertilization was frequently observed in vitrified matured oocytes. Vitrification of maturing oocytes (12 h IVM group) generally was superior to that of immature or matured oocytes, with an overall normal fertilization rate of 36% in contrast to 56% for the fresh control group. Normal fertilization of oocytes after cryopreservation will be a prerequisite for the production of transferable blastocysts and live calves, but the proportion of vitrified-warmed oocytes that developed to blastocysts was as low as 5%. Bovine Met-II oocytes Recent advances in cryopreservation of bovine oocytes have been the development of vitrification procedures characterized by extremely rapid cooling rates. In 1996, Martino et al. [43] reported that in vitro-matured bovine oocytes can be cryopreserved on electron microscope grids, with a blastocyst development rate of 15%. Vajta et al. [44] reported an alternative way of ultra-rapid cooling for vitrification of bovine embryos at various developmental stages as well as in vitro-matured oocytes. When the oocytes were aspirated into open-pulled ministraws (OPS) and cooled by directly plunging into liquid nitrogen, 25% of the post-warm oocytes could develop into blastocysts after IVF and IVC. The OPS method has been

EMBRYO AND OOCYTE CRYOPRESERVATION 19 improved to use open-pulled glass capillaries [45, 46] or commercially available gel-loading tips [47]. Other types of containers so far reported for ultrarapid cooling are the cryoloop [48] and cryotop [49]. Complete containerless methods have also been reported by two independent laboratories [50, 51]. Our modified OPS system with heat-pulled glass capillaries [45] made it possible to examine the relationship between cooling rates during vitrification and post-warm survival of in vitromatured bovine oocytes. The morphological survival of oocytes cooled at 3,000 or 5,000 C/min was not significantly different from that of exposed control oocytes, but the survival and/or sperm penetration rate of the oocytes cooled at 2,000, 8,000, and 12,000 C/min was more or less influenced by the vitrification procedures. Within penetrated oocytes, the examination of normal fertilization rates resulted in a slightly lower outcome in the groups with very high cooling rates. The best result was obtained with the oocytes vitrified at 3,000 C/min: an 86% morphological survival rate, 79% sperm penetration rate and 69% normal fertilization rate. It remains to be determined for the precise action of the rapid cooling on revivability of bovine oocytes, their subsequent developmental potential in vitro and in vivo, and difference from the data from others in which a higher cooling rate is desired for oocyte vitrification. Conclusions Equine blastocysts with developing embryonic capsules are able to survive two-step freezing and vitrification, if a permeable CPA such as EG is used with some modifications. Membrane permeability of bovine IVP embryos is probably inferior to that of in vivo-produced embryos, and a slower cooling rate or treatment with LAA is useful for accelerating water dehydration and improving cryosurvival. In experiments with both horse and cattle oocytes, cryoinjuries occurring in the oocytes are found to be specific to the stage of nuclear maturation. Vitrification procedures characterized by extremely rapid cooling rates are now promising for cryopreservation of oocytes. Acknowledgements I wish to thank Dr. A. Hanada (retired in 2002; Shinshu University) for his nomination of S.H. for the 2002 Research Award from the Japanese Society of Animal Reproduction. The financial support of the Laboratory of Horse Production, Obihiro University of Agriculture and Veterinary Medicine, where most of my equine research was conducted, was obtained from the Japan Racing Association. Dr. N. Oguri (Obihiro University of Agriculture and Veterinary Medicine) and Dr. J.W. Braun (Muenchen University, Germany) are acknowledged for their skillful assistance on equine embryo recovery/transfer and helpful discussion. Research with bovine oocytes and embryos conducted in Shinshu University was supported by grants from the Japanese Government, the Ito Foundation, YS New Technology Institute Inc. and Japan Livestock Technology Association. Dr. M. Hirabayashi (National Institute for Physiological Sciences), and Dr. K. Ito (Sankyo, Co.) are acknowledged for their cooperation on the bovine experiments. During and after my short stay in Guelph, continuous support and encouragement have been provided by Dr. S.P. Leibo (University of New Orleans, USA) and Dr. K.J. Betteridge (University of Guelph, Canada). Finally, I wish to express my deepest appreciation to Dr. K. Utsumi (deceased in 1997; Kyoto University) for guiding me to work as a scientist at these Universities, with his warmest and thoughtful consideration. References 1. Betteridge KJ, Mitchell D. Direct evidence of retention of unfertilized ova in the oviduct of the mare. J Reprod Fertil 1974; 39: 145 148. 2. Weber JA, Freeman DA, Vanderwall DK, Woods GL. Prostaglandin E2 secretion by oviduct transport-stage equine embryos. Biol Reprod 1991; 45: 540 543. 3. Bosquet D, Guillomot M, Betteridge KJ. Equine zona pellucida and capsule: some physicochemical and antigenic properties. Gamete Res 1987; 16: 121

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