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TECHNOLOGIES FOR ASSI STED CONCEPTION/ EMBRYO TRANSFER IN AGRIC=T~ AD VET=IN=Y PRACTICE Neal L. First INTRODUCTION The natural reproductive processes of domestic animals have been controlled or assisted largely for the purpose of producing a larger number of offspring from individuals of superior meat or milk production. -For some technologies a secondary benefit has also been increased reproductive efficiency. Thus, the purpose for development of methods for assisting natural reproduction has been different between animal agriculture and human medicine where enhancement of reproduction has been the focus. The developed and developing technologies to be discussed here are artificial insemination, superovulation, embryo transfer, freezing of embryos, sexing of embryos, multiplication of embryos by bisection and by cloning, production of embryos in vitro and modification of embryos by gene transfer. Artificial Insemination The oldest and thus far most used biotechnology is artificial insemination. More than 60% of the 10 million United States dairy cows and nearly all West European dairy cattle are presently mated by artificial insemination (Betteridge, 1986~. The strength of this tool comes from the ability to statistically identify and select the very best bulls in the country in terms of genes for milk production and the ability to extend each ejaculate to produce more than 500 inseminations. Artificial insemination has been standard practice in the dairy industry for more than 30 years. During this period its use has contributed heavily to a doubling of the milk production of each cow and reduced the number of cows consuming the nation's grain and forage resources by 50% (Reid, 19781. It has been available but little used in other species except in limited economic situations of low labor or high animal value. Superovulation Superovulation by injection of a follicle stimulating hormone has been utilized for many years to increase by 5- to 10-fold the ovulation rate of cattle, sheep and swine (Pineda and Bowen, 1980~. While this treatment is common practice in the cattle embryo transfer industry as well as for human in vitro fertilization, better methods are needed. The ovulatory response 96

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is unpredictable ranging from 1 to 20 or more in cattle (Massey and Oden, 1984~. Ovulation does not always occur (Moor et al., 1984) and an increase in frequency of oocytes with abnormal chromatin occurs for all species superovulated and studied thus far (First and Eyestone, 1987~. For cattle and sheep some increase in yield of fertilizable oocytes has been achieved by use of an early luteal priming dose of follicle stimulating hormone in addition to the usual midiuteal treatments (Ware et al., 1988~. A more uniform superovulation and increased frequency of high quality transferable bovine embryos has also been reported from the use of recombinant derived pure ESH material (Chappel et al., 1988~. Embryo Transfer More recently superovulation and embryo transfer have been used to multiply scarce exotic breeds of cattle and to accelerate genetic improvement by expanding the maximum number of offspring possible from genomic combinations of the best females and progeny tested sires. The embryos are collected from cows induced by the use of hormones to ovulate -5 to 15 eggs. The embryos are then transferred nonsurgically to other recipient cows whose estrous cycles are synchronous with the donor cow (Seidel, 1981~. Bovine embryos are commonly stored frozen if more embryos are harvested than the number of recipient cows available (Massip et al., 1987) or bisected to double their number if insufficient embryos are harvested (Baker and Shea, 1985; Leibo, 1988~. These latter procedures are currently in use in the embryo bans f er industry. The use o f embryo bans f er has resulted in collection of the best dairy cows into a small number of herds designed to produce and sell embryos. In 1987 this commercial embryo transfer industry performed approximately 150,000 transfers in the USA and 250,000 in the world, of which approximately 30% were with frozen embryos (Seidel, personal communication). So far neither artificial insemination nor embryo transfer is widely practiced in domestic species other than cattle. This is either for reasons of low economic payback or technical difficulties such as low semen extension, inability to freeze semen effectively or absence of nonsurgical transfer methods. Nevertheless these technical problems are being solved. For example, the first method for nonsurgical transfer of swine embryos was recently reported (Sims and First, 1987~. In spite of their past impact on the dairy industry, the tools of artificial insemination and embryo transfer as practiced are slower than desired in effecting productivity changes, and are restricted to the gene pool of existing livestock breeds. Artificial insemination allows genetic change only through the male side of the pedigree and embryo transfer results in a cohort of embryos after superovulation, mating and recovery that are no more alike than siblings. Because heritabilities are low for - 97 -

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most desired production traits, methods are needed for producing large numbers of duplicate copies of high performance individuals or embryos. Freezing of Embryos Freezing embryos for storage, cryopreservation, is a valuable technology important in facilitating embryo transfer and in preserving rare genetic traits. Embryos of cattle can be frozen and, after thawing and transfer, produce pregnancies with nearly the same efficiency as fresh embryos (i.e., 45-60%; Massip et al., 1987~. The embryo transfer industry often stores frozen cattle embryos to preserve surplus; in 1986, for instance, it froze 30% of the cattle embryos it later transferred (Seidel, unpublished). Frozen cattle embryos are routinely thawed by a one-step method that allows nonsurgical embryo transfer directly from the straw, as with artificial insemination (Leibo, 1982; Renard et al., 1982; Chupin et al., 1984; Massip and van der Zwalmen, 1984~. Following freezing and thawing procedures, 50-80% of livestock embryos survive, including cattle (Lehn-Jensen et al., 1981; Kennedy et al., 1983; Renard et al., 1983; Pettit, 1985), sheep (Ware and Boland, 1987), goat (Chemineau et al., 1986) and horse embryos (Slade et al., 1984, 1985; Takeda et al., 1984~. Effectiveness of embryo freezing varies with mammalian species. Pig embryonic blastomeres have a relatively high lipid content (Edidin and Petit, 1977) that impedes freezing (Polge et al., 1974~. Because each species possesses a physically unique embryo, a universal cryoprotection scheme has not been possible. Sexing of Embryos Sexing of embryos before transfer is especially sought by the dairy cattle industry where females are the desired milk producing unit. To be useful sexing techniques must be accurate, efficient, rapid and without detrimental effects on the embryos. The three most successful approaches have been 1) cytogenetic karyotyping of the cells of the blastocyst, 2) immunological detection of a male specific antigen on male embryos and 3) use of DNA hybridization probes to identify Y (male) chromosome specific DNA. - Sexing by Karyotyping. Cytogenetic methods are highly accurate and also allow identification of aberrant chromosomal karyotypes. However, karyotyping requires a large number of coicemid arrested cells to insure a few readable metaphase chromosome displays (King, 1984~. Embryonic biopsies of sufficient size to yield sufficient cells may be damaging to the embryo. One approach to providing sufficient cells has been to bisect the embryo followed 98 _

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by cytogenetic analysis of the smaller half and transfer of the larger. In one experiment this resulted in a normal pregnancy rate after transfer of the half embryo but sex could be determined for only 60% of the half embryos subjected to cytogenetic analysis. Sex when identified was predicted with 100% accuracy (Picard et al., 1984~. Recent research has resulted in methods to increase the frequency of-metaphase spreads (Kent and First, 1988). Nevertheless, a large number of blastomeres, 15-20, is needed for accurate assay. Cytogenetic analysis does provide an accurate standard for quick evaluation (less than 15 hr) of other methods. Sexing by Immunological Methods. The sex of mammalian embryos is determined by the presence or absence of the Y chromosome derived from the father since males are the heterogametic sex and can produce either X or Y bearing sperm cells while oocytes from the female contain only X chromosomes. Antigens which are coded from Y specific genes are found in and on male but not female cells. Male specific antigens are expressed as early as the 8-cell stare in mice. One such antigen Goldberg expressed as early as the 8-cell has been called H-Y (Krco and , 1976; Epstein et al., 1980; reviewed by Haseltine, 1983~. Fluorescent labeled antibodies to one or more male specific antigen provide a way to recognize individuals expressing male specific genes. Except for cases of chromatin translocations which often cause intersexuality, the presence of a Y chromosome antigen specifically identifies a male. Male specific antigens such as H-Y antigen are known to be highly conserved across species and present on cells of at least 70 species of vertebrates including mice, rats, cattle, dogs, goats, donkeys, horses, pigs and humans as well as on cells of the female, the heterogametic sex, of birds (Wachtel, 1984a). Both polyclonal (Krco and Goldberg, 1976; Epstein et al., 1980; White et al., 1982) and monoclonal (Koo and Varano, 1981) antibodies have been prepared against the serological H-Y antigen. The H-Y antigen has been detected on preimplantation murine embryos by incubation with H-Y antiserum and complement (Krco and Goldberg, 1976; Epstein et al., 1980; White et al., 1982; reviewed by Wachtel, 1984a,b). Cell lysis occurred in approximately 50% of the embryos which were exposed to murine H-Y antiserum and complement from guinea pigs; but embryos cultured in medium with H-Y antiserum or complement alone were not damaged. Karyotypes of embryos that were unaffected by culture with antiserum and complement showed that 92% were female (Epstein et al., 1980~. When unaffected embryos were transferred to pseudopregnant recipients, 86% of the pups born were female (White et al., 1983~. The obvious disadvantage of this complement dependent cytolytic method for detection of male specific antigen is that survival of male embryos is reduced. When a male specific antibody and fluorescent labeled second antibody were utilized together the male specific antigen on male embryos was identified without toxicity to embryos of either sex _ 99 _

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(White et al., 1983~. Removal of complement and use of a fluorescent labeled second antibody for detection of binding of the first antibody have resulted in identification of both male and female embryos without cytotoxic death of the male embryos (White et al., 1984~. These studies in mice suggest that it should be possible to sex embryos of bovine and other species by use of an antibody to one or more male specific antigens. The accuracy of White et al. (1984, 1987a,b,c) in predicting each sex from application of this assay to several species is shown in Table 1. As indicated in Table 1 for the bovine the assay was nearly perfect for identification of female embryos (89%~. However, only 80% of male embryos were identified. This reduced efficiency for males appeared to occur because dead cells of some embryos exhibited an autofluorescence thus when embryos with dead cells were female the accuracy of male identification was reduced. It is likely that this problem could be eliminated by choosing a fluorescent label of a different wave length. A perfect prediction of sex may not occur from this method since females with a fragment of Y chromosome transiocated to an X chromosome may test as males by this test or by DNA hybridization. Overall, the accuracy for bovine was 86%. Using a similar method Wachtel et al. (1988) report an accuracy in bovine embryos of approximately 85%. While large scale testing of this antisera and second antibody system will be essential it would appear that with minor adjustments in the method such as indicated above, a method for determining the sex of embryos of domestic animals is at hand and ready for use. Its wide scale use will depend on the availability of antisera recognizing male cells. - 100 -

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Table 1. Accuracy of immunological detection of embryonic H-Y antigen in various speciesa "b~so,~-.e ~ -. , Sexed Species Male Female References Murine 78% 83% White et al., '983 67% 80% Piedrahita and Anderson, 1985 Bovine 80% 89% White et al., 1987a Ovine 88% 82% White et al., 1987b Porcine 77% 86% White et al., 1987c aFrom White, 1988. - 101 -

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Sexing by Y Chromosome Specific DNA Hybridization The third method is based on the isolation, cloning and subsequent labeling of unique and repetitive DNA sequences from the Y chromosome. Most of the unit e and transcribed sequences coding for critical male specific substances such as H-Y antigens are located at or near the pseudoautosomal (X pairing region) of the Y whereas repetitive sequences such as GATA-GATA or GACA-GACA tend to be dispersed over the length of the y'/2. These Y specific fragments are used as probes to locate homologous sequences present in DNA from blastomeres, trophoblasts, amniocytes and other cell types (Gosden et al., 1984; Moyzis et al., 1987; West et al., 1987~. As fewer three to five blastomeres can be biopsied from embryos and using an oligonucleotide pol~erase chain reaction for signal amplification, embryonic sex can be determined in from 1 to 3 hr (Grey and Langlois, 1987; Ou et al., 1987~. Numerous Y specific probes are currently available for sex selection in humans (Page et al., 1987; Kent and First, 1988), bovine (Leonard et al., 1987; Ellis et al., 1988; Popescu et al., 1988) and equine (Kent et al., 1988a,b). Multiplication of Embryos The ability to produce multiple copies of an individual or embryo is of interest not only to researchers, but also to the livestock industry. Genetic identicals provide the perfect control for experimental conditions thus reducing the genetic variation in experiments to zero. A large number of genetically identical embryos provides a means for embryo phenotypic selection wherein clonal lines descendent from one embryo are selected by progeny test for clonal multiplication to large numbers. This system approaches phenotypic selection and could permit rapid change in selected characteristics such as meat or milk production. Two methods of embryo multiplication will be discussed here. They are embryo bisection and nuclear transplantation. Embryo bisection is a procedure whereby an early embryo (2-cell through the blastocyst stage) is bisected to yield either 2 cells as with a 2-cell embryo, or 2 or more cell masses as with a morula or blastocyst stage embryo. This procedure results in identical offspring in sheep (Willadsen, 1982), pigs (Willadsen, 1982; Rorie et al., 1985) and cattle (Ozil et al., 1982; Baker and Shea, 1985; Deibo, 1988~. Since this procedure is successful it can be concluder] that these cells are totipotent. However, the number of identicals produced by this method is limited. If the embryo is divided more than twice survival to offspring is reduced. This is likely due to the requirement of a minimum cell ~ number at the time of blastulation. In the mouse this minimum is 8-16 cells. If blastulation occurs with fewer cells, a trophoblastic vesicle will form without an inner cell mass ~ 102 -

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(Tarkowski and Wroblewolsa, 1967). Therefore, the limit to the number of identicals produced by splitting is maximally four and efficiently two (Robl and First, 1985). This procedure is commonly used in the cattle embryo transfer industry and results in a pregnancy rate nearly equivalent to the whole embryo with the number of offspring nearly doubled (Leibo' 1988). Nuclear Transfer The second method for producing multiple copies of an embryo is by nuclear transplantation. A nuclear transplantation procedure has recently been shown successful in producing cloned embryos and offspring in cattle (Prather et al., 1987), sheep (Willadsen, 1986; Smith and Wilmut, 1988) and rabbits (Stice and Robl, IgSS). This procedure is a modification of a procedure developed for the frog in 1952 (Briggs and King, 1952). As shown in Fig. 1 the procedure involves transfer of a blastomere or nucleus from a valuable embryo of a multicellular stage into an enucleated metaphase II oocyte with subsequent development to a multiple cell stage and use as a donor in a serial recloning. This procedure is being developed in private industry as well as by the cited authors. Collectively in the USA and Canada several hundred pregnancies have been produced in cattle and recloning has been performed. Thus far the largest number of calves cloned from one embryo has been seven. These were born at Granada Genetics in 1987. A system for cloning of embryos useful to the livestock industry depends on the ability to produce offspring from donor embryos of large cell number and the ability to reclone as the clones develop to advanced cell number or to multiply donor ceils in culture. Studies with sheep at Edinburgh, Scotland, suggest this should be possible. The frequency of development to blastocyst after use of donor cells from blastocyst inner cell mass was 56% and pregnancies resulted (Smith and Wilmut, 1988~. In Vitro Production of Embryos The production of embryos in vitro from abattoir recovered oocytes is best developed for cattle (Lu et al., 1987; Eyestone and First, 1988~; although offspring have been produced from in vitro fertilization of in vitro developed oocytes in sheep (Crozet et al., 1987) and swine (Cheng et al., 1986~. There are at least three reasons for producing embryos of cattle in vitro. First, this technique provides large numbers of embryos for commercial transfer and calf production. The value of dairy calves is sufficiently low relative to beef calves in Europe and Japan that there are economic incentives for transfer of in vitro produced beef embryos into dairy cow recipients, particularly - 103 -

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Figure 1 ^1- ~ . _- ~ I,.' LN ~ it'' ~ DONOR EMBRYO I RECLONE CATTLE EMBRYO CLONING MODEL Transfer each donor cell to separate egg RECIPIENT EGG ~ ~ (~ OCR for page 96
with the goal of inducing twinning. In Ireland and Japan large commercial ventures have been established for in vitro production of cattle embryos. -Second, in vitro produced embryos are highly valuable for research where large numbers or precise timing of fertilization and development are needed. Third, the economic feasibility of embryo cloning by nuclear transfer requires that the enucleated oocytes be produced in vitro from abattoir recovered ovaries and that the new zygote be developed in vitro to a stage suitable for recloning. In vitro production of embryos requires development of technology in three areas, oocyte development and maturation, in vitro fertilization and in vitro embryo development. Oocyte Maturation In domestic species oocytes recovered from follicles matured in viva either with or without superovulation can be fertilized and proceed through embryo development with good success (cattle: Leibfried-Rutledge et al., 1987; swine: Cheng et al., 1986~. However, oocytes recovered from small follicles (1-5 mm) many of which have not completed growth and development produce zygotes which fail to complete embryo development (sheep: Moor and Trounson, 1977; Crosby et al., 1981; cattle: Leibfried-Rutiedge et al., 1987~. Development is enhanced when the underdeveloped oocytes undergo in vitro maturation in the presence of hormone stimulated granulosa cells (Staigmiller and Moor, 1984; Critser et al., 1986a; Lu et al., 1987) and to a lesser extent with cumulus cells confined in a small volume of medium (Critser et - al., 1986b; Sirard et al., 1988~. The embryo developmental signals developed during this co-culture or the beneficial material from the granulosa cells are unknown. At present the frequency of bovine blastocysts developing from in vitro fertilization of in viva matured oocytes is approximately >45 (Brackett et al., 1982; Sirard et al., 1985, 1986; Leitfried-Rutledge et al., 1987), from immature oocytes co-cultured with granulosa cells it is approximately 23 to 63% (Critser et al., 1986a; Lu et al., 1987; Xu et al., 1987; Fukui and Ono, 1988), from culture with significant cumulus contribution per volume of medium it is 20 to 30% (Critser et al., 1986b; Goto et al., lg88; Sirard et al., lg88) and from immature oocytes cultured so a "helper cell" effect is lost <20% (Critser et al., 1986b and unpublished; Sirard et al., 1988~. In Vitro Fertilization The second part of production of embryos in vitro is the sperm capacitation and fertilization system. Here numerous capacitation systems have been used including high ionic strength media and glycosaminoglycans such as heparin-sulfate and fucose sulfate, aging, pH shift, calcium ionophores and caffeine - iOS

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(reviewed by First and Parrish, 1987, 1988). In general any agent which causes Ca++ entry into the sperm acrosome and causes a pH increase within the sperm causes capacitation (reviewed by First and Parrish, 1988~. From this and with incubation in serum-free medium at body temperature for a given species in vitro fertilization has been successful in cattle, sheep, swine and goats (First and Parrish, 1987~. Development of Embryos In Vitro Embryos of each domestic species can be developed with good efficiency to the blastocyst stage or later by transfer at the 1-cell or 2-cell stage into the oviduct of the respective species. For cattle the embryos can also be successfully developed in the oviduct of the sheep or rabbit (Sirard et al., ~ 9 8 6; Eyestone et a ~ . , 19 ~ 7 ~ . Embryos of a ~ ~ Moment i c spec i es do not develop to morulae or blastocysts when cultured in any of the common culture media (Wright and Bondioli, 1981~. Their development is blocked at the transition from maternal to zygotic control of development (Barnes, 1988; First and Barnes, 1988~. The embryos remain alive but with cleavage arrested and in the resting phase (Eyestone and First, 1989b). Recently bovine (Eyestone and First, 1987, 1988) and ovine embryos (Gandolfi and Moor, 1987) have been cultured through the period of blocked development and to the blastocyst stage with good efficiency by co-culture with oviduct epithelial cells or media conditioned by cultured oviduct cells (Eyestone and First, 1988~. In the bovine the essential oviduct material is in the protein fraction but its identity is unknown. In the sheep the essential oviduct component is believed to be a protein of 92.5 KO or its combination with a 46 KO protein. The 92.5 KO fucose rich glycoprotein increases greatly in the oviduct just before the block period, transiocates to the zone and embryo and disappears by the blastocyst stage (Gandolfi and Moor, 1988~. Whether the bovine oviduct factor is the same is unknown. A protein fraction from trophoblast cells also has similar activity in enhancing embryo development (Heyman et al., 1987~. The nature of these embryotrophic compounds needs elucidation as well as their mode of action and the way in which the blocked development relates to initiation of embryonic transcription and the transition from short to long cell cycles (Barnes, 1988; First and Barnes, 19887. In spite of these gaps in our . knowledge, supplementation of embryo cultures with frozen oviduct cell conditioned media has provided an in vitro method for development of bovine embryos which has resulted in an approximately normal pregnancy rate (50%) after transfer into cows (Eyestone and First, 1989a). - 106

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Gene Transfer The first production of transgenic mice by Gordon and Ruddle (1981) and the evidence that mice transgenic for rat (Palmiter et al., 1982) or human (Palmiter et al., 1983) growth hormone grew to nearly twice normal size greatly excited animal scientists with hopes of producing transgenic livestock. In the ensuing years more than 400 strains of transgenic mice have been produced for use in studying problems of biology, medicine and animal agriculture. However, only a few new transgenic lines of domestic animals have been produced and in many cases the expected performance has not been achieved. The slow progress has been largely due to the low efficiency of production of transgenics by microinjection of DNA into pronuclei and to the high economic value of each egg microinjected. At present transgenic swine have been produced in at least five different laboratories, transgenic sheep in at least three and transgenic cattle embryos, fetuses or offspring in three laboratories (Rexroad and Pursel, 1988; Murray et al., 1988~. A second problem has been failure of expression of the desired response (i.e., growth) or failure of expression at the desired time or in the desired tissue (Rexroad and Pursel, 1988~. These problems are being resolved as more is learned about the promoter and enhancer sequences used with the gene of interest Especially exciting are the possibilities for targeting gene expression exclusively to skeletal muscle for alteration of the meat product (Shani et al., 1987) or to the mammary gland for alteration of the composition of milk or for production of pharmaceutical proteins in milk (Simons et al., 1987~. Conclusions A large array of gamete and embryo biotechnologies have been developed for use in animal agriculture. Older technologies with high economic value such as artificial insemination, embryo transfer, freezing and splitting of embryos have over time developed to high efficiency. Newer technologies such as embryo sexing, in vitro production of embryos, embryo cloning and gene transfer show promise for commercial use but require research to become more efficient. Rapid development of each technology is enhanced in a given species by availability of gametes and embryos as well as the existence of supporting technology such as nonsurgical embryo transfer and economic incentives. - 107

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REFERENCES Baker, R.D. and B.F. Shea. 1985. Commercial splitting of bovine embryos. Theriogenology 23:3-12. Barnes, F.L. 1988. Characterization of the onset of embryonic control and early development in the bovine embryo. Ph.D. Thesis, University of Wisconsin-Madison. Betteridge, K.~. 1986. Increasing productivity in farm animals. In: Reproduction in Mammals: Manipulating Reproduction (Austin and Short, eds.), Cambridge University Press, Cambridge, pp. 1-47. Brackett, B.G., D. Bousquet, M.L. Boice, W.J. Donawick, J.F. Evans and M.A. Dressel. 1982. Normal development following in vitro fertilization in the cow. Biol. Reprod. 27:147-158. Briggs, R. and T.J. King. 1952. Transplantation of living nuclei from blastula cells into enucleated frogs' eggs. Zoology 38:455-463. Chappel, S., C. Looney and K. Bondioli. 1988. Bovine FSH produced by recombinant DNA technology. Theriogenology 29:235a. Chemineau, P., R. Procureur, Y. Cognie, P.C. LeFeure, A. Lacatelli and D. Chupin. 1986. Production, freezing and transfer of embryos from a blue tongue infected goat herd without blue tongue transmission. Theriogenology 26:279-290. Cheng, W.T.K., R.M. Moor and C. Polge. 1986. In vitro fertilization of pig and sheep oocytes matured in vitro and in vivo. Theriogenology 25:146. Chupin, D., B. Florin and R. Procureur. 1984. Comparison of two methods for one-stem in straw thawing and direct transfer of cattle blastocysts. Theriogenology 21:455-459. Critser, E.S., M.L. LeiLfried-Rutledge, W.H. Eyestone, D.L. Northey and N.L. First. 1986a. Acquisition of developmental competence during maturation in vitro. Theriogenology 25:150 (abstract). Critser, E.S., M.L. Leibfried-Rutledge and N.L. First. 1986b. Influence of cumulus cell association during in vitro maturation of bovine oocytes on embryonic development. Biol. Reprod 34(Suppl 1):192 (abstract). ~ 08

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Crosby, I.M., J.C. Osborn and R.M. Moor. 1981. Follicle cell regulation of protein synthesis and developmental competence in sheep oocytes. J. Reprod. Fertil. 62:575-582. Crozet, N., D. Huneau, V. Desmedt, M.C. Theron, D. Szollosi, S. Torries and C. Sevellec. 1987. In vitro fertilization of sheep ova. Gamete Res. 16:159-170. DiBerardino, M.A. 1980. Genetic stability and modulation of metazoan nuclei into eggs and oocytes. Differentiation 17:17-30. Edidin, M. and V.A. Petit. 1977. The effect of temperature on the lateral diffusion of plasma membrane proteins. In: The Freezing of Mammalian Embryos, Ciba Foundation Symp. 52, Elsevier, Excerpta Medica, North Holland, Amsterdam, pp. 155-164. Ellis, S.B., K.R. Bondioli, M.E. Williams, J.H. Pryor and M.M. Harpold. 1988. Sex determination of bovine embryos using male-specific DNA probes. Theriogenology 29: 242a. Epstein C.~., S. Smith and B. Travis. 1980. Expression of H-Y antigen on preimplantation mouse embryos. Tissue Antigens 15:63-67. Eyestone, W.H., J. Vignieri and N.L. First. 1987 Co-culture of early bovine embryos with oviductal epithelium. Theriogenology 27:228. Eyestone, W.H. and N.~. First. 1988. Co-culture of early bovine embryos with oviductal tissue. Proc. lith Intern. Congr. AI and Anim. Reprod., Dublin. Eyestone, W.H. and N.~. First. 1989a. Co-culture of early cattle embryos to the blastocyst stage with oviductal tissue or in conditioned medium. J. Reprod. Fertil. 85:715-720. Eyestone, W.H. and N.~. First. 1989b. Characterization of development arrest in early bovine embryos cultured in vitro. J. Reprod. Fertil. (submitted). First, N.~. and J.J. Parrish. 1987. In vitro fertilization of ruminants. Second International Symposium on Reproductive in Domestic R~minants. J. Reprod. Fertil. 34:151-165. First, N.~. and W.H. Eyestone. 1987. Reproductive efficiency in domestic animals. "Proceedings Vth WorId Congress of In Vitro Fertilization and Embryo Transfer. Norfolk, VA. First, N.L. and J.J. Parrish. 1988. Sperm maturation and in vitro fertilization. Ilth International Congress on Animal Reproduction and Artificial Insemination, Dublin. 109

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