Cryopreservation and Sexing of In Vivo- and In Vitro-Produced Bovine Embryos for Their Practical Use

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1 Journal of Reproduction and Development, Vol. 50, No. 1, 2004 JSAR Innovative Technology Award Cryopreservation and Sexing of In Vivo- and In Vitro-Produced Bovine Embryos for Their Practical Use Keiichiro TOMINAGA 1) 1) Hyogo Agricultural Institute for Agriculture, Forestry and Fisheries, Kasai, Hyogo , Japan Abstract. My research awarded includes contributions to cryopreservation and sexing of bovine embryos produced in vitro and in vivo, as follows; (1) In vivo-derived morulae and blastocysts were cryopreserved in the presence of 10% glycerol, and the embryos were transferred into recipients after two-step dilution of glycerol in straw, with a practically acceptable pregnancy rate. (2) The survival rate of 16-cell stage embryos frozen in the medium with ethylene glycol was higher than that with DMSO or 1,2-propanediol. Addition of linoleic acid-albumin to culture medium enhanced the survival rate of post-thaw bovine 16-cell stage in vitro-produced (IVP) embryos. (3) Polarization of cytoplasmic lipid droplets by centrifugation of 2-cell stage embryos was found effective to increase freezing tolerance in 16-cell stage embryos developed from the centrifuged embryos, because blastomeres of 16-cell stage embryos were mostly lipid-free. (4) The usefulness of gel-loading tip (GL- Tip) as a container for ultra-rapid vitrification was demonstrated in IVP embryos from 2-cell to blastocyst stages, with a higher in vitro survival than the conventional two-step freezing. (5) PCR analysis for sexing of in vivo-derived Day-7 embryos indicated that male embryos developed faster and graded higher than female embryos. But such correlation between genetic sex and embryonic development was not found in IVP embryos obtained from individual cows. (6) Addition of % deproteinized hemodialysate product from calf blood to culture medium increased the producing efficiency of demi-embryos with good quality. Female embryos rather than male embryos required a longer time to repair after bisection. (7) In vivo-derived bovine embryos after biopsy for sexing by PCR analysis and subsequent vitrification using GL-Tips are available to practical use in the field. (8) Introduction of primer extension preamplification-pcr and purification of DNA product before standard sexing PCR of biopsy samples from Day 3 4 in vitro-derived embryos allowed accurate sex determination, and Day-7 blastocysts developed from Day 3 4 embryos were cryopreserved by GL- Tip vitrification without a loss of their viability. Thus the field application of bovine embryo transfer is in part supported by improvements of technologies in embryo cryopreservation and sex predetermination. Key words: Cryopreservation, Sexing, Bovine embryos, In vivo, In vitro (J. Reprod. Dev. 50: 29 38, 2004) ryopreservation and sexing of bovine embryos have been integrated into commercial embryo transfer technologies. Since 1970, I have devoted much time to develop and improve such techniques for field use of embryo transfer especially in Japanese Black cattle. In the present Accepted for publication: November 5, 2003 Correspondence: K. Tominaga ( tomi-@nike.eonet.ne.jp) paper, my research relating to cryopreservation and sexing of in vivo-derived and in vitro-produced (IVP) bovine embryos is summarized. Cryopreservation of In Vivo-Derived Embryos During slow cooling (0.3 C/min to 36 C)

2 30 TOMINAGA followed by rapid cooling (>1000 C/min to 196 C) and rapid warming (>360 C/min) of bovine embryos, 1.0 M glycerol played a role as a cryoprotective agent (CPA), resulting in birth of calves after non-surgical transfer of frozen embryos [1]. A stepwise dilution procedure to remove the glycerol from frozen embryos in laboratory was obviously the limiting factor for wide application of frozen embryos in the field. In 1982, solution containing a non-permeable sucrose, separately packed in plastic straws, has been reported to remove glycerol from post-thaw bovine embryos, allowing transfer into recipients of the embryos without expelling out from the straws [2]. Reduction in cytotoxicity of sucrose by glycerol We have investigated influences of concentration of sucrose, glycerol and sucrose-glycerol mixture on survival of frozen-thawed 8-cell mouse embryos [3]. The developmental rate of embryos was still low even when sucrose concentration in the medium was decreased to 0.08 M (Fig. 1-a). The developmental rate of embryos cultured in the medium with 5.0% glycerol was similar to that in the medium without glycerol (Fig. 1-b). Addition of glycerol to the sucrose solution delayed the occurrence of decrease in the developmental rate of embryos (Fig. 1-c), indicating that the cytotoxic effect of sucrose on embryos could be decreased by the presence of glycerol. From these results, it is clear that sucrose is more harmful than glycerol to the in vitro development of fresh mouse 8-cell embryos [4]. Embryo transfer after in-straw 2-step glycerol dilution Considering harmful effect of sucrose, we developed a two-step glycerol dilution method for cryopreserved bovine embryos within 0.25-mL straws. As shown in Fig. 2, top column with 1 volume of 10% glycerol, where embryos were loaded, is mixed with second column of 2 volumes of 0.6 M sucrose. Five minutes later, the glycerolsucrose mixture is mixed with 9 volumes of BMOC- 3 medium and kept for 5 min. The straw can be allowed for subsequent transfer into recipients without taking out of embryos [4, 5]. In , the pregnancy rate of cryopreserved embryos following 2-step glycerol dilution was 42.3% (60/ 142). This procedure, however, has not been widespread by the embryo transfer industries Fig. 1. Effect of sucrose (a), glycerol (b), and sucrose and glycerol (c) concentration in culture medium on the development of mouse developed in vivo 4 8-cell embryos. EB: early blastocyst, B: blastocyst, ExB: expanded blastocyst, HgB: hatching blastocyst, ( ): No. of tested embryos, a-c: Different letters denotes significant difference among the hatching rates (P<0.05). possibly because of the complex process of the dilution method or difficult yield of consistent pregnancy results. Therefore in the field condition, a direct transfer method of frozen-thawed embryos to recipients was used [6]. The embryos were frozen-thawed in the presence of 1.5 M ethylene glycol as a CPA, and the CPA was removed within the straws without using sucrose diluent. In 1992

3 CRYOPRESERVATION AND SEXING OF BOVINE EMBRYOS 31 Fig. 2. Two-step procedure for glycerol dilution from frozen embryos within straw. 1993, a field trial of embryo transfer in Hyogo prefecture was performed to compare the two methods (direct method using 1.8 M ethylene glycol at cooling rate of 0.5 C/min to 30 C [7] and two-step method using 10% glycerol at cooling rate of 0.3 C/min to 36 C). The pregnancy rate of frozen embryos following two-step glycerol dilution (59.3%, 16/27) was comparable to that of direct method (60.0%, 18/30) (unpublished data). Cryopreservation of In Vitro-Produced Embryos The IVP bovine embryos are more sensitive to the freezing-thawing procedure than those derived from in vivo [8]. It has been suggested that sensitivity to chilling of embryos depends on their developmental stage and on culture conditions [9], as well as on the presence of numerous lipid droplets in early stage IVP embryos [10]. As the post-thaw survival rate of 16-cell to morula stage embryos is very low [9 11], it is difficult to use frozen-thawed embryos earlier than blastocysts for commercial embryo transfer. Addition of linoleic acid-albumin (LAA) to culture medium enhanced the survival rate of IVP bovine morulae [12] and blastocysts [13] after cryopreservation. Ethylene glycol as a permeable cryoprotectant for freezing of 16-cell embryos Bovine embryos at 16-cell stage were frozen by a conventional two-step freezing method; embryos were cooled at a rate of 0.3 C/min to 30 C before being plunged into liquid nitrogen in the presence of 1.5 M DMSO, 1,2-propanediol or ethylene glycol, and 0.2 M trehalose. The survival rate of embryos frozen in the medium with ethylene glycol was higher than that with DMSO or 1,2-propanediol (Table 1). In addition, proportion of the blastocysts constituted from more than 150 cells was higher when ethylene glycol (33.3%, 10/30) was used rather than DMSO (0%, 0/13) or 1,2-propandiol (0%, 0/19). However, blastocyst development and quality of 16-cell stage embryos frozen even in ethylene glycol was still inferior to those of fresh embryos (67.3%, 37/55). When the 16-cell stage embryos were produced in CR1aa [14] containing 0.25 mg/ml LAA for 4 days after IVF, the postthaw survival of 16-cell stage embryos produced in LAA-containing medium was higher than that of those cultured without LAA [15]. Cytoplasmic lipids responsible for cryoinjury Nagashima et al. [16, 17] demonstrated that cryotolerance of 1 8 cell stage porcine embryos can be increased by removing cytoplasmic lipid droplets from centrifuged embryos. Moreover, post-thaw survival of bovine blastocysts was improved after removal of lipid droplets at an earlier stage [18 20]. However, Ushijima et al. [19] reported that cytochalasin treatment and centrifugation without micromanipulation did not

4 32 TOMINAGA Table 1. Development into blastocyst of in vitro-derived bovine 16-cell embryos after freezing in the presence of one of three permeable cryoprotectants with trehalose No. of % of No. of Total no. % Cryoprotectant cultured blastocysts stained of of embryos as surviving blastocysts cells a ICM a Propanediol cd ± 30.6 d 21.1 ± 7.8 DMSO d ± 30.6 d 22.7 ± 7.0 Ethylene glycol c ± 35.2 c 24.0 ± 8.6 Fresh b ± 36.5 b 25.4 ± 9.8 a Mean ± SD. 7 replicates. b-d Different superscripts within columns show significant difference (one-way ANOVA, P<0.05). Fig. 3. Cryosurvival of 16-cell stage bovine embryos that have been centrifuged at 1-, 2-, or 8-cell stage. Different letters above columns denote significant differences at P<0.05 by χ 2 test. Six replicates. ( ): No. of examined embryos. improve the cryotolerance of bovine zygotes or embryos. We have investigated the effect of polarization of lipid droplets on cryotolerance of 16-cell stage embryos without micromanipulation. The 16-cell stage embryos have been developed after centrifugation (15,500 g) at 1-, 2- or 8-cell stage in the medium containing cytochalasin-d. The embryos were classified into mostly or partially delipidated ones. The developmental rate into blastocysts after freezing of the mostly delipidated 16-cell embryos was higher when the embryos were centrifuged at the 2-cell stage rather than 1- cell stage (Fig. 3). The level of delipation was also important for avoiding cryoinjuries. These results demonstrate that high polarization of lipid droplets at the 2-cell stage by centrifugation without micromanipulation improved the cryosurvival of 16-cell stage embryos [20]. Ultra-rapid vitrification using GL-Tip as container In recent years, survival of bovine oocytes and younger embryos after cryopreservation has been improved by ultra-rapid vitrification procedures, using electron microscope grids [21] and openpulled straws (OPS) [22] as cryodevices. The postwarm survival rates of IVP bovine embryos at Day- 3 through Day-7 in the OPS were as high as those of fresh control embryos, whereas post-warm survival rates of Day-1 and Day-2 embryos were significantly impaired [23]. Another ultra-rapid vitrification methods were also reported for bovine oocytes or early-stage embryos, referred to as the minimum drop-size technique [24], micro-droplet method [25], and cryoloop technique [26]. However, it was difficult to control precisely the volume of vitrification solution, possibly explaining the poor reproducibility of these ultrarapid vitrification procedures.

5 CRYOPRESERVATION AND SEXING OF BOVINE EMBRYOS 33 Table 2. GL-Tip vitrification of in vitro-derived bovine embryos at various developmental stages Days Vitrifi- No. of No. (%) of P No. of Total no. % of after cation tested blastocysts stained of IVF (stages) embryos as surviving a value b blastocysts cells c ICM c (58.0) ± ± 5.3 (2-cell) (70.5) ± ± (68.3) ± ± 4.6 (4 8-cell) (75.9) ± ± (57.9) ± ± 5.9 (6 12-cell) (75.9) ± ± (62.5) ± ± 4.3 (8 16-cell) (77.8) ± ± (60.0) ± ± 3.8 (16-cell-M d ) (82.1) ± ± 6.1 7(B d ) (98.3) a The survival of Day-1 5 embryos was assessed on Day-7 and that of Day-7 was on Day-8. b Differences were analyzed by one-way ANOVA. Replicate of experiments: 5(Day-2, -3, -5 and -7), 6 (Day-4) or 7(Day-7). c Mean ± SD. d M: Morula, B: Blastocyst. It was examined whether commercially available gel-loading tips (GL-Tips) are available as containers for vitrification of IVP bovine embryos at various developmental stages [27], because the tip connected with micro-pipette allows the precise control of handling medium. Bovine embryos at Day-1, -2, -3, -4, -5, and-7 in µl of vitrification solution composed of 20% ethylene glycol, 20% DMSO, 0.6 M sucrose, TCM199 and 20% calf serum, were loaded into a GL-Tip, and then vitrified in liquid nitrogen. There was no significant difference in the survival rates between fresh and vitrified embryos at any developmental stages (Table 2). No significant differences were also found in the total cell numbers or the proportion of inner cell mass (ICM) cells between blastocysts developed from fresh versus vitrified embryos. A simultaneous comparison of in vitro assay indicated that the GL-Tip vitrification (97.8%, 45/46) was the better procedure than conventional two-step freezing (68.8%, 33/48) for IVP bovine Day-7 blastocysts (unpublished data). Embryo Sexing by PCR Analysis In vivo-derived bovine embryos have been transferred after sexing and cryopreservation in some embryo transfer units [28 30]. However, IVP embryos are more fragile for micromanipulation and more sensitive to cryopreservation than in vivoderived embryos [8, 9]. Factors affecting sex determination of in vivo-derived embryos Findings that male embryos develop faster than female embryos have been reported not only in mice [31] but also in cattle (in vivo-derived embryos [32] and IVP embryos [33, 34]). We have investigated the relationship between genetic sex and developmental stage, and/or the quality grade of Day-7 embryos of Japanese Black cattle [35]. Embryos were classified into 4 stages (late morula, early blastocyst, blastocyst, and expanded blastocyst) and 4 quality grades (excellent, good, fair, and poor) [36]. The sexing of 281 embryos by PCR analysis resulted in identification of 150 male (53.2%) and 132 female (46.8%) embryos. Among three sires examined (Y, N, U), the sire Y produced male embryos less than the sire N or U (Fig. 4). The percentages of male embryos at the early blastocyst and late morula stage by sire Y were lower than those by sire N (Fig. 4-a). The percentage of male embryos classified as excellent grade by sire Y was lower than that by sire N or U, and that as poor grade by sire Y was lower than that by sire U (Fig. 4-b). Thus, the rate of male embryos recovered 7 days after insemination was influenced by sires.

6 34 TOMINAGA Fig. 4. Relationship between proportion of male embryos and developmental stages (a) and quality grades (b) of Day-7 embryos recovered from cows that were inseminated semen from different sires. Significant differences (P<0.05) between developmental stage or quality grades of embryos and between sires were denoted by letters of a-c on columns, and between sires by letters of xy. ( ): No. of examined embryos. Factors affecting sex determination of IVP embryos Evidence that the first cleavage of embryos is related to the sex ratio has been reported in bovine embryos produced in vitro [37, 38], as male embryos reach to the 2-cell stage earlier than female embryos. Great variation in the development rate of embryos to the blastocyst stage among individual cows and heifers has been reported [39 41]. Therefore, we have examined the effect of oocyte donors on sex ratio of embryos derived from IVM-IVF-IVC, but no relationship between male predominance and the developmental rate of embryos was found [42]. Meanwhile, King et al. [43] reported that there was significant difference in the sex ratio of morulae bisected and cultured either in vitro or in vivo for h, probably due to that the environmental condition around bisected embryos resulted in a specific loss of female embryos. Keefer et al. [44] also reported a higher male to female ratio in bovine embryos biopsied for sexing by PCR analysis. Then, we have investigated the effect of various culture conditions for demi-embryos on their sex ratio [45]. Higher proportion of demi-embryos with good quality was obtained from splitting of Day blastocysts rather than Day blastocysts. In addition, the producing efficiency of good quality demi-embryos was improved when the blastocysts were bisected in the presence of % deproteinized hemodialysate product from calf blood (DHP-CB) ( % versus 21.1% in no DHP-CB), without changing the sex ratio. It may be concluded that female demi-embryos require more time than male ones for repairing their morphological characteristics such as re-expansion after bisection. Cryopreservation of sex-identified embryos An integrated sexing-vitrification experiment for in vivo-derived bovine embryos was conducted in collaboration with 14 Japanese prefectural livestock experiment stations (unpublished data). Day embryos were biopsied and then vitrified either by a GL-Tip method [27] or by a VSED-straw method [46] with minor modification. Pregnancy rate following transfer of sex-identified and cryopreserved embryos in GL-Tip vitrification method (46.9%, 61/130) was higher than that in modified VSED-straw method (37.2%, 119/320), and was similar to that of fresh embryos (51.4%, 73/142). This result indicates that in vivo-derived bovine embryos after biopsy for sexing by PCR analysis and subsequent vitrification using GL-Tips are available to their practical use in the field. Cell sampling by biopsy has been applied to IVP bovine embryos at the 16- to 32-cell stage [47, 48], morula-stage [49, 50], and blastocyst-stage [51, 52]. Since efficiency of sexing decreased significantly when less than five cells were subjected to PCR [52], the larger sampling size (8 10 cells) has been recommended for successful sexing of the embryos [53]. However, development of primer extension preamplification-pcr (PEP-PCR), which is an in vitro-system for amplifying a large fraction of the DNA sequence present in a single haploid cell [54, 55], made it possible to identify the sex of embryos using a single blastomere. The application of PEP- PCR to bovine embryo sexing resulted in a 91% sexidentification rate [56]. The IVP bovine embryos

7 CRYOPRESERVATION AND SEXING OF BOVINE EMBRYOS 35 Fig. 5. A successful example of calf production by transfer of sex pre-deterimined and vitrified embryo. (a) Day- 3, 8 16-cell stage embryo. (a ) Biopsied blastomere for sexing PCR. (b) Day-7 blastocyst developed from the biopsied embryo. (c) Blastocyst transferred after vitrification and warming. (d) Newborn calf with pre-determined sex. Scale bars show 100 µm. were biopsied on Day-3 or -4 and one or two blastomere samples were subjected to multiplex PCR using purified DNA after PEP-PCR. Developmental rates into Day-7 blastocysts of biopsied embryos were similar to those of nonbiopsied control embryos, and total cell numbers and the ICM ratio of blastocysts derived from the biopsied embryos were also comparable with those of control embryos. After vitrification in GL-Tips, high proportions of blastocysts derived from biopsied Day-3 or -4 embryos could develop to the hatching blastocyst stage. By transfer of a sexidentified and vitrified-warmed embryo, a female calf with a gestation length of 288 days and a birth weight of 21.7 kg (within the expected range for the Tajima strain of Japanese Black cattle) was born (Fig. 5). Conclusions In the last few decades, I have improved techniques for cryopreservation and sexing of in vivo- and in vitro-derived bovine embryos and simplified those for applying to field condition. Two useful cryopreservation methods have been developed; In-straw dilution method for in vivoderived embryos and following selection from cryoprotectants, addition LAA to the culture medium and lipid polarization by centrifugation in a conventional two-step freezing method, GL-Tip vitrification method for in vitro-derived embryos. PCR analysis of in vivo- and in vitro-derived embryos for sex determination suggested that bull individual was among factors influencing on the relationships between male embryo production and developmental kinetics or embryo grading, that IVP condition had little influence on male embryo productivity, and that female demiembryos require longer time for re-expansion after bisection than male demi-embryos. Introduction of PEP-PCR and DNA purification into standard sexing PCR made it possible the practical use of biopsied Day-3 or -4 IVP embryos for cryopreservation on Day-7. I hope these studies are found practically useful and contribute to development of cattle industries.

8 36 TOMINAGA Acknowledgements I wish to thank Dr. S. Kato (retired in 2002, Kobe University) for nomination of my studies for the Japanese Society for Animal Reproduction Award I also wish to thank Dr. H. Kanagawa (retired in 1998, Hokkaido University), Dr. A. Iritani (Kinki University), Dr. K. Utsumi (deceased in 1997, Kyoto University) and Dr. Y. Izaike (National Institute of Agrobiological Science) for technical teaching, scientific suggestions, and great encouragement. Dr. H. Imai (Kyoto University), Dr. T. Kojima (resigned in 1999, National Livestock Breeding Center) and Dr. S. Hochi (Shinshu University) are acknowledged for improving manuscripts and providing experimental advice. These works were supported by all the staffs at Biotechnology Laboratory in Hyogo Prefectural Agricultural Institute, Hyogo Prefectural Institute for Agriculture, Forestry and Fisheries, and staffs concerned in Hyogo Experimental Station of Animal Husbandry, and Hyogo Prefecture Livestock Hygiene Service Centers. Finally, I am grateful to Mrs. Y. Otsuka-Hamada (resigned in 2003, Hyogo Prefectural Institute for Agriculture, Forestry and Fisheries) for her great cooperation on recent experiments. References 1. Bilton RJ. Preservation of the large domestic species. Proc 9th Anim Reprod AI, Madrid 1980; Leibo SP, West AW, Perry B. A one-step method for direct nonsurgical transfer of frozen-thawed bovine embryos. Basic study. Cryobiology 1982; 19: Brinster RL. Cultivation of the mammalian embryo. In: Rothblatt GH and Cristofalo VJ (eds.), Growth Nutrition and Metabolism. New York and London Academic Press 1972; Tominaga K. Studies on freezing bovine embryos with special reference to the conventional glycerol dilution method. Special Bulletin Hyogo Prefectural Agricultural Institute 1991; 17: (In Japanese with English summary). 5. Tominaga K, Fukushima M, Hataya Y. Field trial of bovine frozen embryo transfer by two-step glycerol dilution method. J Jap Vet Med Assoc 1990; 43: (In Japanese with English summary). 6. Voelkel SA, Hu YX. Direct transfer of frozenthawed bovine embryos. Theriogenology 1992; 37: Dochi O, Yamamoto Y, Siga H, Yoshiba N, Kano N, Maeda J, Miyake K, Yamaguchi A, Tominaga K, Oda Y, Nakashima T, Inohae S. Direct transfer of bovine embryos frozen-thawed in the presence of propylene glycol or ethylene glycol under on-farm condition in an integrated embryo transfer program. Theriogenology 1998; 49: Hasler JF, Henderson WB, Hurtgen PJ, Jin ZQ, McCauley AD, Mower SA, NeeLy B, Shuey LS, Stokes JE, Trimmer SA. Production, freezing and transfer of bovine IVF embryos and subsequent calving results. Theriogenology 1995; 43: Leibo SP, Loskutoff NM. Cryobiology of in vitroderived bovine embryos. Theriogenology 1993; 39: Plante L, King WA. Light and electron microscopic analysis of bovine embryos derived by in vitro and in vivo fertilization. J Assist Reprod Genet 1994; 11: Hochi S, Semple E, Leibo SP. Effect of cooling and warming rates during cryopreservation on survival of in vitro-produced bovine embryos. Theriogenology 1996; 46: Hochi S, Kimura K, Hanada A. Effect of linoleic acid-albumin in the culture medium on freezing sensitivity of in vitro-produced bovine morulae. Theriogenology 1999; 52: Imai K, Kobayashi S, Goto Y, Dochi O, Shimohira O. Cryopreservation of bovine embryos obtained by in-vitro culture of IVM-IVF oocytes in the presence of linoleic-acid albumin. Theriogenology 1997; 45: 347 abstr. 14. Rosenkrans CF Jr, First NL. Effect of free amino acids and vitamins on cleavage and developmental rate of bovine zygotes in vitro. J Anim Sci 1993; 72: Tominaga K, Hamada Y, Ariyoshi T. Effect of linoleic acid-albumin on post-thaw survival of in vitro-produced bovine embryos. J Vet Med Sci 2000; 62: Nagashima H, Kashiwazaki N, Ashman RJ, Grupen CG, Seamark RF, Nottle MB. Removal of cytoplasmic lipid enhances the tolerance of porcine embryos to chilling. Biol Reprod 1994; 51: Nagashima H, Kashiwazaki N, Ashman RJ, Grupen CG, Nottle MB. Cryopreservation of porcine embryos. Nature 1995; 374: Diez C, Bourhis L, Heyman Y, Renard JP. Effect of partial lipid removal from in vitro produced bovine zygotes on further development in vitro and on the freezing tolerance of blastocysts. Theriogenology 1996; 45: 166 (abstr.)

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10 38 TOMINAGA Barandi Z, Vajta G. Biopsy and sex determination by PCR of IVF bovine embryos. J Reprod Fertil 1993; 98: Vajta G, Booth PG, Holm P, Greve T, Callesen H. Comparison of two manipulation methods to produce in vitro fertilized, biopsied and vitrified bovine embryos. Theriogenology 1997; 47: Shea BF. Determining the sex of bovine embryos using polymerase chain reaction results: A six-year retrospective study. Theriogenology 1999; 51: Hassun PA, Mello MRB, Porto LPC, Garcia JF. Bovine embryos sexing by primer extension preamplification polymerase chain reaxction (PEP- PCR). Theriogenology 1999; 51: 398 (abstr.) 51. Hasler JF, Cardey JE, Stokes JE, Bredbacka P. Nonelectrophoretic PCR-sexing of bovine embryos in a commercial environment. Theriogenology 2002; 58: Lacaze S, Lesclaux J, Cooupet H. The sexing of bovine embryos in the south-west of France. Part 1. Efficiency, accuracy and pregnancy rates after 3 years activity. Proceedings of the 12th Annual Meeting of AETE, vol. 156, Lyon, 1996 abstr. 53. Thibier M, Nibart M. The sexing of bovine embryos in the field. Theriogenology 1995; 43: Li H, Cui X, Arnheim N. Direct electrophoretic detection of the alleic state of single DNA molecules in human sperm by using the polymerase chain reaction. Proc Natl Acad Sci USA 1990; 87: Zang L, Cui X, Schmitt K, Hubert R, Navidi W. Whole genome amplification from a single cell: Implications for genetic analysis. Proc Natl Acad Sci USA 1992; 89: Tominaga K, Hamada Y. Efficient production of sex-identified and cryosurvived bovine in vitroproduced blastocysts. Theriogenology 2004 (in press.)