Development of rabbit zygotes into blastocysts in defined protein-free medium and offspring born following culture and embryo transfer

ELSEVIER

DEVELOPMENT OF RABBIT ZYGOTES INTO BLASTOCYSTS IN DEFINED PROTEIN-FREE MEDIUM AND OFFSPRING BORN FOLLOWING CULTURE AND EMBRYO TRANSFER J. Li, R.H. Foote,' Z. Liu and J.R. Giles Department of Animal Science, Cornell University, Ithaca, NY 14853 Received for publication: Accepted:

June 14, November

1996 5,

1996

ABSTRACT We report here on an improved, completely defined culture system for This system producing embryos in vitro which mimics development in vivo. avoids the confounding effects of the many unknowns introduced by the multivariate components of the serum or by unknowns attached to bovine serum albumin (BSA). Zygotes were obtained from superovulated rabbits and cultured in modified defined RPM1 1640:Dulbecco's MEM, 1:l (RD) medium. The effect of a novel and potentially ideal antioxidant, tempol, was tested (20 to 0.001 mM) but found to be either toxic or ineffective. In the presence of 2D% O,, 600 units of superoxide dismutase or 2.5 mN of taurine increased embryo hatching after 72 h of culture in RD medium to 75 and 763, respectively, compared with 46% in the control (Pt0.05). The need for antioxidants was reduced with 5% 0,. The beneficial effects of RD medium were demonstrated when 60 zygotes cultured for 48 h to the early blastocyst stage in this medium were transferred and resulted in 30 young (50%) compared with 35/60 (58%) young from uncultured control transfers. Only 12% of the young were obtained from slower developing morulae. Thus, high viability was established for rapidly growing embryos in culture, but fewer slow growing embryos survived after transfer. A further comparison of embryos cultured in RD medium with a high potassium simple, optimized, defined medium (KSOM), revealed that both yielded results approaching those of direct transfer without culture. Simple defined media may also be useful for the culture of embryos of other species. 0 1997 by Elsevier Science Inc. Key words: rabbit embryos, protein-free media, blastocysts, number of offspring INTRODUCTION Embryos from several species have been cultured successfully, often with BSA, serum and/or with a co-culture (1,7,9,14,15,22,23,39,43). Rabbit zygotes can be cultured to the blastocyst stage in a protein-free defined medium (6,18), but development of embryos cultured in vitro was retarded and the pregnancy rate reduced following culture and transfer (5). The complex culture Acknowledgments This study was part of the NICHHD National Cooperative Program on nonhuman in vitro fertilization and preimplantation development, and was partially funded by NICHHD through Cooperative Agreement HD21939. The LH was supplied by Vetrepharm and the Buserelin by Hoechst-Roussel Agri Vet Co. The authors thank M. Simkin and 6. A. Presicce for technical help, B. Bavister for Tanuscript review , and Deloris Bevins for manuscript preparation. To whom reprint requests should be addressed. Thenogenology47:1103-1113, 0 1997 by Elsevier Science

1997 Inc.

0093.691x/97/$17.00 PII SOOS3-691X(97)00067-6

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media may be deficient in certain components and/or contain substances present in amounts that depress embryo growth. It is important to develop completely defined media for successful embryo culture in order to establish the requirements for embryo growth and to avoid the variability and confounding that results when using different batches of serum or BSA (1). The gas atmosphere employed in the culture system can also appreciably affect embryo development. Although embryos often have been cultured in an atmosphere of 5% CO,:95% air, reduction of 0, from 20 to 5% has been shown to improve embryonic development in several species (2,18,20,27,37,40). The oxygen tension in the oviducts or uteri is approximately 5 to 8% (3,11,24). When higher concentrations of 0, are used, free radicals may be formed (13,34), and beneficial effects on cultured embryos have been reported with the addition of superoxide dismutase (SOD; 8,19,33,34) and taurine (B,lg,20,38). However, SOD has also been reported to have no beneficial effects on bovine embryos cultured in 5 or 20% 0, (20,23). It is not known if SOD crosses cell membranes or exerts its effect extracellularly. Mitchell et al. (30) identified tempol, a stable nitroxide which is highly soluble in water, membrane-permeable and metal-independent for transport. Tempo1 was nontoxic and more protective of Chinese hamster cells against oxidative damage than SOD; however, it had never been used for embryo culture. Because tempo1 is potentially an ideal antioxidant for embryo culture systems, it was hypothesized that it might protect the embryo against accumulation of both intracellular and extracellular toxic components. The objectives of the present study were twofold: 1) improve completely defined culture systems so that embryo development in vitro would mimic development in vivo and 2) to initiate tests comparing this basic system (6,lB) with a new simpler, completely defined new system recently found suitable for We tested the full the culture of mouse and bovine embryos (17,21,22). potential of both systems for producing offspring by culturing zygotes and transferring the resultant embryos to recipients, and recording the number of young produced. MATERIALS AND METHODS Medium The culture medium was similar to the medium of Carney and Foote (6) but with the low glucose content of Dulbecco's modified Eagle's medium (DMEM,' 1 g/L) mixed 1:l with RPM1 1640' (18), designated as RD medium. The final concentration of glucose was 1.5 g/L. Before use, 2.85 mg of sodium bicarbonate were added per milliliteg of RD medium, which was then equilibrated at 39-C overnight in 4-well dishes under the same gas conditions used for culturing the embryos.

'Gibco BRL, Grand Island, NY, USA. bNUNC, Kastrup, Denmark.

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1105

Animals and Zygotes Sexually mature Dutch-belted rabbits from our colony, maintained at 20X, were superovulated using a standard FSH and LH (FSH-LH) schedule (16). The rabbits were inseminated at the time of LH injection. After 19 h the oviducts were flushed with PBS containing 1 mg/ml polyvinyl alcoholC (PVA), and l-cell zygotes were obtained in an air atmosphere. These zygotes were washed twice with RD medium. Normal appearing zygotes from each donor rabbit were randomly placed into each culture medium treatment. The culture system consisted of RD medium with 120-~1 droplets under oil in Experiment 1, and 500~1 in each well of 4-well dishes in all other experiments. All processing was done as rapidly as possible to place the zygotes into the appropriate equilibrated media. Embryos were briefly evaluated after 24 h to determine if any that had been initially classified as zygotes were actually unfertilized oocytes. Embryo Culture and Evaluation The culture dishes with embryos were placed in humidified tissue culture The latter chambers. The chambers were sealed and incubated at 39X. The medium was temperature approximates body temperature of the rabbit. replaced with fresh equilibrated medium after 48 h. The chamber was regassed for 5 min with the appropriate gas whenever the chamber was opened. Embryos were evaluated for appearance and stage of development at the end of the culture period. Except in the embryo transfer experiment, this was always done after 62 h of culture, and in some experiments again after 72 and 92 h. The 62 h of culture is sufficient time for rabbit zygotes to develop into blastocysts. Design of Embryo Culture Experiments In Experiments 1 and 2, the effects of various levels of tempolC on embryo development were tested in a randomized block design. In Experiment 1, the embryos were cultured in RD medium with 0.0 II+!, 0.1 mM, 10 mM and 20 ml4tempol. Five to 15 embryos from each donor (12 rabbits) were placed in each 120-~1 droplet of culture medium in 35- x lo-mm Falcon Petri dishesd containing the different concentrations of tempol. These were covered with 4 ml of Corning 360 medical fluid.' The stage of embryo development was evaluated after 62 h of culture at 39'C in an atmosphere of 20% 0,:10x COp:70% Np. It was found in Experiment 1 that the tempo1 could migrate from one droplet to another through the overlying medical fluid, so Experiment 2 was designed to use culture dishes with separate wells for each treatment.

In Experiment 3, the effects of 600 units of SODC and 2.5 mM taurinec on development of embryos in 20% O,:lM CO,:70% N, were investigated. The stage of embryo development was recorded after 62 h and 72 h of culture at 39'C, and the percentage of degenerated embryos was recorded after 92 h of culture. 'Sigma Chemical Company, St. Louis, MO, USA. dFisher Scientific, Pittsburgh, PA, USA 'Dow Corning Company, Midland, MI, USA

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Experiment 4 was a test of the effects of SOD and taurine when the embryos were cultured with 5% 0, instead of 20% 0,. This was done to test the hypothesis that SOD and taurine exerted their effects as antioxidants and that the effects might be different when the 0, concentration was reduced from 20 to 5%. All other conditions were the same in the 2 experiments except a new set of embryo donors was used. Design of Embryo Transfer Experiments 5, 6 and 7 Embryo collection, processing and culture procedures were similar to those used in previous studies. Experiments 5 and 6 were a test of the full functional ability of embryos cultured in 4-well dishes for 24 or 48 h in the low glucose RD medium without antioxidants. The gaseous atmosphere was 20% 02:10% CO,:70% N, (10) as no embryo transfer experiments had been done previously with this medium and gaseous atmosphere. The success rate in Experiment 5 of transferring blastocysts cultured for 48 h from the zygote stage to blastocysts was very high. So Experiment 6 was planned to compare imnediate transfer of zygotes from donor to recipient versus an aliquot of these embryos cultured for 48 h before transfer. Thus the effects of culture plus transfer could be compared with the effects of transfer only. In Experiment 7, embryos were cultured for 0 or 48 h in RD medium with the antioxidant taurine (2.5 mM). These 2 treatments were compared with the simple new KSOM medium (17) that was successfully used for mouse embryos and with the modifications we successfully used for the culture of bovine embryos (20,Zl). No embryo transfer tests with modified KSOM have been reported previously for any species. The recipient animals were administered 1.2 pg, im Buserelin' to induce ovulation. This was administered 12 h after the injection of LH so that the rabbits would be better synchronized with the embryos, which are slightly delayed in development due to in vitro handling. All embryos were transferred to the oviducts of recipients. Statistical Analysis All culture experiments utilized a randomized complete block design, with the embryos from each donor representing one complete block. Embryos from each donor were divided equally and randomly assigned to treatment groups. Two-way analysis of variance was performed, using the general linear models procedure of the Statistical Analysis System (SAS Institute, Cary, NC). The arcsine transformation of the proportion of blastocysts formed in each droplet or each well was used for the analysis of percentage data. When significant mean differences were found by analysis of variance, these were compared using Duncan's new multiple range test. Chi-square analysis was used to test the proportions of transferred embryos that resulted in young.

'Hoechst Agri-Vet Company, Sommerville, NJ, USA.

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RESULTS Experiments 1 and 2 with Tempo1 In Experiment 1, there were 255 zygotes distributed among the 0.0, 0.1, 10.0 and 20.0 mM concentrations of tempol. The tempo1 was highly toxic (data not shown), and 86, 46, 0 and 0% of the zygotes develop to the 2 to 4-cell stage, respectively. No morulae were produced except in the control, and no blastocysts resulted. The culture arrangement of separate droplets of media, with several treatments per dish, was changed in subsequent experiments because it was obvious that the higher concentrations of tempo1 were being transferred through the silicone oil overlay to other treatments in the same culture dish.

In Experiment 2 the concentration of tempo1 was reduced and each treatment was in a separate well in each 4-well culture dish. After the zygotes had been equally distributed from 8 donor rabbits among 4 concentrations of tempo1 (0.0, 0.01, 0.1 and 1.0 mM), it was clear that the 1 mM concentration was toxic (Table 1). Therefore, embryos from donor Rabbits 9 to 16 (Table 1) were distributed into media with tempo1 covering an overlapping lower range of concentrations. Table 1.

Donors

1 to 8

9 to 16

Effect of tempo1 upon embryo development after culturing zygotes for 62 and 72 h in 4-well plates (Experiment 2) Tempo1 (mM)

No. of No. (%) of embryos deVelODinq in 62 hours Blastocysts zygotes 2-4-cell Morulae

0.0 0.01 0.1 1.0

48 47 49 46

0.0 0.001 0.01 0.10

39 38 39 45

albfcValues within (P
donors

(19)a (15)a (24)b (35)b

39 40 37 30

(81)a (85)a (76)a#b (65)b

4 (lo)a 3 (8)a 2 (5)a 4 (9)a

35 35 37 41

(90)a (92)8 (95)a (91)a

9 7 12 16

and columns with

different

In 72 hours

Hatching blastocysts

___ ___

superscripts differ

The results in Table 1 indicate that 1.0 mM tempo1 decreased the proportion of zygotes developing into blastocysts (Pt0.05). Zygotes from donors 9 to 16 appeared to be of higher quality, and a high proportion reached the blastocyst stage in all treatments. However by 72 hours of culture, the higher concentrations of tempo1 markedly reduced the hatching rate of blastocysts (P
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Experiments 3 and 4 Since tempo1 appeared to be of no value in the culture system, it was discontinued. Experiments 3 and 4 were conducted with presumed antioxidants (SOD and taurine) in a gas environment containing 20% 0, and later with 5% 0,. The results are shown in Table 2. Development into blastocysts was similar in Table 2.

Effects of SOD and taurine on development of embryos cultured under 20% 0, or 5% 0, with 10% CO,

Experiment

No. of zygotes

Treatment 3

4

20% 0, Control SOD Taurine

Number IX) of develoaed e b YOS cultured for 63 h at72mhr 2- to BlastoHatching Degenerated 4-cell Morulae cystsa blastocysts embryos

54 60 50

0 0 0

13 (24)b 41 (76)b 16 (27)b 44 (73)b 7 (14)C 43 (86)C

25 (46)b 45 (75)b 38 (76)'

29 (53)b 16 (26)' 8 (32)'

5% 0, Control

58

0

15 (26)b 43 (74)b

35 (60)b

23 (39)b

SOD Taurine

63 54

0 0

16 (25)b 47 (75)b

47 (74)b

11 (20)b 43 (80)b

42 (77)b

5

(7)c

9 (16)’

‘No hatching blastocysts at 62 hours. "CValues within experiments and columns with different superscripts differ (PcO.05). different treatments at 62 h. By 72 h more hatching blastocysts were formed in the presence of SOD and taurine in 20% 0, (PcO.O5), and by 92 h SOD and taurine were beneficial in decreasing the proportion of embryos degenerating at both 0, concentrations (PcO.05). However, with 5% O,, 60% of the zygotes developed into blastocysts within 72 h in the control, and the addition of SOD and taurine did not produce significant differences. By 92 h there was less degeneration of embryos cultured with SOD and taurine (Pt0.05). Embryo Transfer Experiments 5, 6 and 7 Experiments 5 and 6 involved the culture of embryos in low glucose RD medium with 20% O,:lO% COz:70% N,. The results are shown in Table 3. The encouraging results in Experiment 5 were the high pregnancy rates that were achieved following the transfer of early blastocysts after culture for 48 h from the zygote stage (75% in pregnant does). The 48-h culture period was tested further in Experiment 6 by comparing the pregnancy rate with a proportion of zygotes from each donor that were transferred inmediately, providing also a test of the transfer procedure. Of the 60 cultured zygotes

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Table 3.

Distribution of young following the transfer of rabbit embryos cultured in RD medium

Culture period (hours) Experiment 24 48 48 0 48 48

Embryo stage at transfer

No. of embryos

8 to 16 cells Morula Early blastocysts l-cell Morula Early blastocysts

No. pregnant No. of offspring per recipient

60

3/6

5

8a

17a

24 24

I/3 213

2 12

8a 5ob

25' 75b

60 24 36

4/5 I/2 213

35 4 18

58a 17u 5oa

73a 33b 75a

albValues within experiments with different superscripts differ (PcO.05).

36 became early blastocysts in 48 h, and 75% of these in pregnant does resulted in live young. This was not different (P>O.O5) from the 73% offspring for the embryos transferred without culture to does which became pregnant (Table 3).

In Experiment 7, one additional test of RD medium with taurine was conducted. Again the zygotes from each donor female were either all transferred imnediately to recipients or cultured for 48 h in RD medium and then transferred, or one-third were cultured for 48 h in a relatively new defined simple modified KSOM with Eagle's amino acids and taurine added. The results are not shown in tabular form, but 148 embryos distributed across the controls without culture versus embryos cultured for 48 h in RD medium and KSOM, respectively, resulted in 38, 30 and 28% young born following transfer (P>O.O5). DISCUSSION The media used in the present experiments contained no serum and no BSA, and no co-culture was used. Therefore, the requirements for 0, and various additives may be different than when large amounts of protein mixtures are present, such as are found when 10 to 20% serum is included in the culture system. With co-culture, a beneficial effect of 10 to 20% 0, was reported (42,43), and this beneficial effect of increasing the 0, to more than 5% was attributed to possible requirements of the co-culture rather than that of the embryos. Increasing the concentration of CO, from 5 to 10% had small but significant beneficial effects previously (4,10,12), so this was used in all of the culture studies with either 5 or 20% 0,. In the embryo transfer studies following

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In that final culture, 10% CO, also was used, except in Experiment 7. experiment, viability of the embryos was tested after culture with 5% CO, because this concentration was used for culturing bovine embryos in KSOM (21). Extensive studies by Mitchell et al. (30) indicated that the stable nitroxide, tempol, has properties that make it superior to SOD in protecting cultured cells from oxidative stress. This inexpensive water soluble compound at a concentration of 5 mM protected cells against damage normally induced by H,O, and hypoxanthine/xanthine oxidase. Tempo1 also prevents the formation of deleterious hydroxyl radicals. It was greatly superior to SOD in protecting somatic cells (30). The application of tempo1 to rabbit embryos caused death of the embryos until such low levels were reached that they were without any effect (Table 1). This was an unexpected result, but it is hoped that others will be aware of this potentially useful compound in other culture or coculture systems.

In Experiments 3 and 4 the beneficial effects of reducing 0, from 20 to 5% and the beneficial effects of SOD and taurine in the presence of 20% 0, are New consistent with the findings of previous reports (8,19,20,33,38). information on the beneficial effects of these compounds on hatching and preventing degeneration of embryos was obtained by culturing for 92 h. These findings support the concept that the high concentrations of SOD (32) and taurine (29) in the oviduct are important for normal embryo development. Furthermore these results indicate that SOD and taurine may be beneficial to embryos cultured under varying concentrations of 0,. Many embryos have been transferred following culture with serum in the medium. In our study, 48 h of culture produced 30% young, but this decreased to 14% after 62 h of culture (26). Further, when 2- to 8-cell embryos were collected for immediate transfer, often no more than 50% developed to term (25). In our current experiments, all the embryos were collected at the l-cell stage and were thus more difficult to culture than later stage embryos. All zygotes were cultured without serum or 8SA. Nevertheless 33% of the 108 zygotes cultured for 48 h developed into young and 50% of all zygotes developing to blastocysts in 48 h resulted in young; this is a marked improvement in the embryo culture systems. Future studies characterizing the slower growing embryos that became morulae versus those that became blastocysts and resulted in differences in the percentage of young (28,36,41) are warranted. The slow vs fast growing embryos may represent the kinds of oocytes that are fertilized in vivo but undergo early embryonic death. Conversely, some of these embryos may have survived had they remained in vivo rather than be collected, cultured and transferred. Thus even good culture systems are not equivalent to in vivo conditions. The importance of physiological synchrony between embryo development and the uterus of recipients also should be noted. The embryos were transferred to the oviducts of the recipients to obtain a mucin coat. Zygotes collected 19 h after the LH injection have a mucin coat 2 in thick (5), and this does not change during culture. Such embryos transferred directly to the uterus do not become implanted (26). Murakami and Imai (31) reported that rabbit embryos must have at least a 20-m thick mucus coat before transfer to the uterus or no young will be produced. By transferring embryos to the oviduct a sufficient

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mucin coat is obtained. With relatively rapid transfer to the uterus, blastocysts are in synchrony with the recipient females which temporally are 12 h behind the donors of zygotes. These experiments extend previous research on optimal conditions for culturing embryos using the rabbit model. This is the first report in which improved RD medium is used in embryo transfer, and demonstrates the high potential of this medium for the full development of embryos from cultured zygotes into normal young. The simple new defined medium (17), KSOM, modified for successful culture of bovine embryos (21), was compared for the first time with RD medium for their relative efficacy to produce blastocysts capable of developing into young. These results demonstrate that simple media optimized for several characteristics are suitable for embryo culture. They may also provide the framework upon which to test culture systems containing all the necessary components while excluding other components that may actually impede optimal development in vitro for embryos from different species. REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

11. 12.

Bavister 8D. Culture of preimplantation embryos: facts and artifacts. Human Reprod Update 1995;1:91-148. Influence of culture system and Bernardi ML, Flechon J-E, Doulis C. oxygen tension on the development of ovine zygotes matured and fertilized in vitro. J Reprod Fertil 1996;106:161-167. Bishop DW. Oxygen concentration in the rabbit genital tract. Proceedings of the Third International Congress of Animal Reproduction, Physiology. Cambridge. Brown, Knight and Truscott Ltd, 1956;53-55. Carney EW, Bavister BD. Regulation of hamster embryo development in vitro by carbon dioxide. Biol Reprod 1987;36:1155-1163. Carney EW, Foote RH. Effects of superovulation, embryo recovery, culture system and embryo transfer on development of rabbit embryos in vivo and in vitro. J Reprod Fertil 1990;89:543-551. Carney EW, Foote RH. Improved development of rabbit one-cell embryos to the hatching blastocyst stage by culture in a defined protein-free culture medium. J Reprod Fertil 1991;91:113-123. Chatot CL, Ziomek CA, Bavister BD, Lewis JL, Torres I. An improved culture medium supports development of random-bred l-cell embryos in vitro. J Reprod Fertil 1989;86:679-688. Dumoulin JCM, Evers JLH, Bras M, Pieters MHEC, Geraedts JPM. Positive effect of taurine on preimplantation mouse embryos in vitro. J Reprod Fertil 1992;94:373-380. Eyestone WH, First NL. Co-culture of early cattle embryos to the blastocyst stage with oviductal tissue or in conditioned medium. J Reprod Fertil 1989;85:715-720. Farrell P, Foote RH. Beneficial effects of culturing rabbit zygotes to blastocysts in 5% oxygen and 10% carbon dioxide. J Reprod Fertil 1995;103:127-130. Fischer 8, Bavister 8D. Oxygen tension in the oviduct and uterus of rhesus monkeys, hamsters and rabbits. J Reprod Fertil 1993;99:673-679. Hallden K, Li J, Carney EW, Foote RH. Increasing carbon dioxide from five percent to ten percent improves rabbit blastocyst development from cultured zygotes. Mol Reprod Dev 1992;33:276-280.

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13. Johnson MH, Nasr-Esfahani MH. Radical solutions and cultural problems: could free oxygen radicals be responsible for impaired development of preimplantation mammalian embryos in vitro? Bioessays 1994;16:31-38. 14. Kane MT. Minimal nutrient requirements for culture of one-cell rabbit embryos. Biol Reprod 1987;37:775-778. Factors affecting blastocyst expansion of rabbit 15. Kane MT, Foote RH. zygotes and young embryos in defined media. Biol Reprod 1971;4:41-47. 16. Kennelly JJ, Foote RH. Superovulatory response of pre- and post-pubertal J Reprod Fertil rabbits to commercially available gonadotrophins. 1965;9:177-188. 17. Lawitts JA, Biggers JO. Culture of preimplantation embryos methods in enzymology. In: Wassaman PM, DePamphilis MD (eds), Guide to Techniques in Mouse Development. San Diego: Academic Press; 1993;153-164. 18. Li J, Foote RH. Culture of rabbit zygotes into blastocysts in proteinJ Reprod Fertil free medium with one to twenty percent oxygen. 1993;98:163-167. Development of rabbit zygotes cultured in 19. Li 3, Foote RH, Simkin M. protein-free medium with catalase, taurine, or superoxide dismutase. Biol Reprod 1993;48:33-37. Development of bovine embryos in KSOM with added 20. Liu Z, Foote RH. superoxide dismutase and taurine and with five and twenty percent 0,. Biol Reprod 1995;53:786-790. 21. Liu, Z. and Foote RH. Effects of amino acids on the development of invitro matured/in-vitro fertilization bovine embryos in a simple proteinfree medium. Human Reprod 1995;10:2985-2991. Development of early bovine embryos in co22. Liu Z, Foote RH, Yang X. culture with KSOM and taurine, superoxide dismutase and insulin. Theriogenology 1995;44:741-750. Improvement in bovine embryo 23. Luvoni GC, Keskintepe L, Brackett BG. production in vitro by glutathione-containing culture media. Mol Reprod Dev 1996;43:437-443. Oxygen tension in the oviduct of 24. Maas DHA, Storey BT, Mastroianni L Jr. the rhesus monkey (Macaca mulatta). Fertil Steril 1976;27:1312-1317. Maternal ageing and embryonic mortality in the 25. Maurer RR, Foote RH. rabbit. I. Repeated superovulation, embryo culture and transfer. J Reprod Fertil 1971;25:329-341. Viability of cultured and transferred 26. Maurer RR, Onuma H, Foote RH. rabbit embryos. J Reprod Fertil 1970;21:417-422. 27. McKiernan SH, Bavister BD. Environmental variables influencing in vitro Biol development of hamster 2-cell embryos to the blastocyst stage. Reprod 1990;43:404-413. 28. McKiernan SH, Bavister BD. Timing of development is a critical parameter for predicting successful embryogenesis. Human Reprod 1994;12:22-24. 29. Miller JGO, Schultz GA. Amino acid content of preimplantation embryos and fluids of the reproductive tract. Biol Reprod 1987;36:125-129. 30. Mitchell JB, Samuni A, Krishna MC, DeGraff WG, Ann MS, Samuni U, Russo, A. Biologically active metal-independent superoxide dismutase mimics. Biochem 1990;29:2802-2807. 31. Murakami H, Imai H. Successful implantation of in vitro cultured rabbit Mol Reprod Dev embryos after uterine transfer: a role for mucin. 1996;43:167-170.

Theriogenology

1113

Narimoto K, Noda Y, Shiotani M, Natsuyama S, Mori T, Fujimoto K, Dgawa K, Role of superoxide dismutase in the fallopian tube function: Kim YC. Immunohistochemical assessment of superoxide dismutase in the fallopian tube. Acta Histochem Cytochem 1991;24:85-91. 33. Noda Y, Matsumoto H, Mori T. Superoxide dismutase overcomes 2-cell block in mouse embryos. Acta Obst Gynaec Jpn 1989;41:751-752. 34. Noda Y, Matsumoto H, LhnaokaY, Tatsumi K, Kishi J, Mori T. Involvement of Mol Reprod Dev superoxide radicals in the mouse two-cell block.

32.

35. 36. 37. 38. 39. 40. 41.

42. 43.

1991;28:356-360.

Onuma H, Maurer RR, Foote RH. In-vitro culture of rabbit ova from early cleavage stages to the blastocyst stage. J Reprod Fertil 1968;16:491-493. Plante L, King WA. Effect of time to first cleavage on hatching rate of bovine embryos in vitro. Theriogenology 1992;37:274. Quinn P, Harlow GM. The effect of oxygen on the development of preimplantation mouse embryos in vitro. J. Exp Zoo1 1978;206:73-80. In vitro culture of pig embryos. Reed ML, Illera MJ, Petters RM. Theriogenology 1992;37:95-109. Tervit HR, Whittingham DG, Rowson LEA. Successful culture in vitro of sheep and cattle ova. J Reprod Fertil 1972;30:493-497. Effect of Thompson JGE, Simpson AC, Pugh PA, Donnelly PE, Tervit HR. oxygen concentration on in vitro development of preimplantation sheep and cattle embryos. J Reprod Fertil 1990;89:573-578. Van Soom A, Van Vlaendern I, Mahmoudzadeh AR, Deluyker H, De Kruif A. Compaction rate of in vitro fertilized bovine embryos related to the interval from insemination to first cleavage. Theriogenology 1992;38:905919. Voekel SA, Hu YX. Effect of gas atmosphere on the development of one-cell bovine embryos in two culture systems. Theriogenology 1992;37:1117-1131. Yang BK, Yang X and Foote RH. Early development of IVM/IVF bovine embryos cultured with or without somatic cells in a simple serum-free medium with different concentrations of CO, and 0,. J Reprod Dev 1994;40:197-205.