Voi 5 0 996): 509-514. In vitro production of cattle blastocysts in chemically defined medium with or without insulin supplementation Kristiina Bredbacka and Peter Bredbacka Agricultural Research Centre ofFinland, Institute ofAnimal Production, FIN-31600, Jokioinen, Finland, e-mail: kristiina.bredbacka@mtt.fi In this study we evaluated the use of a chemically defined medium in the production of blastocysts from bovine oocytes fertilized in vitro. As culture medium we used CRI-PVP, a modification of CRlaa medium with bovine serum albumin replaced by polyvinylpyrrolidone. After 168 h of culture (192 h after insemination) 8.7%, 10.5 and 12.8% of the cleaved embryos developed to the blastocyst stage in the presence of 0, 2 or 200 nM insulin, respectively. The supplementation of 200 nM insulin tended to increase cell numbers in morulae and blastocysts (P=0.I0). It is concluded that CRI-PVP can be used as a chemically defined medium in the production of blastocysts from bovine 1-cell embryos. However, further modifications are needed, and the insulin concentrations used may be below the optimum for blastocyst production. Key words', bovine, embryo culture, polyvinylpyrrolidone, cell number, Hoechst 33258 ntroduction Currently the in vitro production (IVP) of bo- vine embryos is most efficiently carried out us- ing serum- supplemented culture media and with co-culture with somatic cells (for review, see Brackett and Zuelke 1993). As serum consists of numerous unknown ingredients these media are termed undefined media. Improving such a medium by analysing the supplementation of various ingredients is complicated as the medi- um may already include these ingredients. Fur- thermore, the components tested may interact with ingredients present in the serum, and the effects may vary according to the batch of se- rum used. An alternative approach for studying the media component requirements of embryos is to use chemically defined media. Although still less supportive for growth than the best unde- fined media, such defined media have been shown to support embryonic development to the blastocyst stage (Pinyopummintr and Bavister 1991, Takagi et al. 1991, Seidel et al. 1991a, 1991b, Kimetal. 1993, Keskintepe et al. 1995) In recent years attention has been paid to the effect of supplementing media with growth fac- tors (Herrler et al. 1992,Flood et al. 1993, Hey- ner et al. 1993). Transcripts for receptors for in- sulin and the growth factors IGF-I, IGF-II and © Agricultural and Food Science in Finland Manuscript received June 1996 509 AGRICULTURAL AND FOOD SCIENCE IN FINLAND https://www.c-info.fi/en/info/?token=TQhwgmzFRzEdkKnH.wVh0TSwl_-vPe2xTeHJbNw.MTEziXL4ZQ-zWIjgH1539QQShmW8tZdn16Aa1RIKhO_hLD-lCgklP1DXjvhXrUENjcKSwxsM-4tY08IcS5i6x1QRoTeFFvdlm0_o4JZCFEwyK3TATQWIoQ_dwzclHV1b4gpb6YupfTsXwgzU6yW6Wc3ic2SBPX82i5QLrBdDqch5fSUYjYjZSZTnKhStRA-GsYD_lCFYFFvNqMF2Zq7yrpeuNI68UIOWn_fUBt12hpI42XrHC4v1w7tgvMeqMkj70yZUZ31acZTeBWcCnYT6tIbefN-_wQxUicLX Bredbacka, K. & Bredbacka, P. Bovine blastocyst production in defined medium PDGF-oc are detectable throughout bovine pre- implantation development (Watson et al. 1992), implying that the use of these growth factors in culture media could have beneficial effects on embryonic development. The effects of insulin on mouse embryo development have been char- acterized reasonably well (Rosenblum et al. 1986, Harvey and Kaye 1988, 1991, Rao et al. 1990), but relatively little is known about its ef- fect on bovine embryos. The objective of our study was to evaluate the use of a chemically defined bovine embryo culture medium with polyvinylpyrrolidone (PVP) as a macromolecular substitute for serum or serum albumin. Furthermore, using this me- dium, we examined the effect of insulin supple- mentation on bovine embryo development to the morula and blastocyst stage. Material and methods In vitro maturation Cattle ovaries were obtained from an abattoir and transported to the laboratory at room tempera- ture in 0,9% (w/v) NaCl supplemented with 100 IU/ml penicillin and 100 pg/ml streptomycin. The oocytes were aspirated from follicles with a diameter of 3-8 mm. Only oocytes surrounded by cumulus cells were selected for maturation. Oocyte-cumulus complexes were washed twice in TALP-HEPES (Parrish et al. 1988). Hepes was obtainedfrom Sigma (St. Louis, MO, USA). The maturation medium was TCMI99 (Sigma, Cat No. M 0148) supplemented with 0.25 mM sodi- um pyruvate, 2 mM L-glutamine, 100IU/ml pen- icillin, 100 pg/ml streptomycin, 2 pg/ml FSH (USDA-oESH-1 8; USDA, Beltsville, MD. USA), 10 pg/ml EH (USDA-bLH-B-6; USDA, Beltsville, MD, USA), I pg/ml oestradiol (Sig- ma), 5% (v/v) foetal calf serum and 5% (v/v) oestrous cow serum. In each 500-pl drop of me- dium, 40-50 oocyte-cumulus complexes were matured for 24 h in 5% CO, in air at 39°C. In vitro fertilization After maturation, oocyte-cumulus complexes were washed in TALP-HEPES and transfered to 100-pl fertilization drops covered with embryo- tested mineral oil (Sigma). The fertilization me- dium was fert-TALP (Parrish et al. 1988) sup- plemented with 2 pg/ml heparin, 2 pM penicilla- mine, 10 pM hypotaurine and 1 pM epinephrine (Sigma). Frozen-thawed spermatozoa were washed twice in sperm-TALP (Parrish et al. 1988), and 1.5 x 106/ml spermatozoa was added to each fertilization drop. After 24-h fertiliza- tion at 39°C in 5% C02 in air, the cumulus cells were removed by vortexing. In vitro culture The oocytes were washed in CRI+PVP, a modi- fication of the CRlaa medium (Rosenkrans and First 1991) including 4 mg/ml polyvinylpyrro- lidone (PVP; Sigma, Cat No. P 2307) instead of BSA. The oocytes were randomly selected and placed into one of three culture media, CRI+PVP, CRI+PVP supplemented with 2 nM insulin (Sigma, Cat No. I 1882) and CRI+PVP supplemented with 200 nM insulin. About 20 to 40 embryos were cultured in each 50-pl drop of medium covered with mineral oil at 39°C in a humidified atmosphere of 5% C02 in air. After 96 h ofculture, glucose (final concentration 5.56 mM) and fresh CRlaa medium were added to each culture drop (final volume 100 pi). The embryos were evaluated for development on Day 8 (fertilization = Day 0) using the criteria of Lindner and Wright (1983). However, embryos lower than Grade 2 were not classified as moru- lae or blastocysts. The cell numbers of the em- bryos were counted after staining with 3 pg/ml Hoechst stain 33258 in 15% of ethanol in PBS for 24 to 48 h at +4°C. The embryos were then washed and placed in a 1-pl drop of PBS into a 10 to 15 pi drop of a mixture of glycerol and water (5:1) on a glass slide. The embryo was then overlaid with a coverslip and examined under a Leitz Dialux 20 microscope using the Leitz A 510 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Vol. 5 (1996): 509-514. Table 1.Development of bovine embryos cultured for 7 days in CRI+PVP medium with different insulin concentrations. Insulin (nM) Oocytes % Cleaved % Morulae % Blastocysts' Cells 2 +blastocysts‘ (least square means) 0 168 75.6 11.0 8.7 56.9 2 172 72.1 16.9 10.5 58.3 200 167 79.6 14.3 12.8 71.2 ' Proportion of cleaved oocytes. 2 Hoechst 33258-stained nuclei in morulae and blastocysts. There were no significant differences (P >0.05) between treatments in cleavage rate, proportion of moru- lae and blastocysts, proportion of blastocysts (x2 -test) or in number of nuclei (ANOVA). filter block. The experiment was repeated 5 times. Statistical analysis Proportions (cleaved embryos of inseminated oocytes. Grade 1 and Grade 2 compacted moru- lae and blastocysts of cleaved embryos) were compared with the y 2 test. The cell numbers of embryos of transferable quality (Grade 1 and Grade 2 morulae and blastocysts) were compared using the GLM program of SAS (SAS Institute, Cary, NC, USA). The effect of thereplicates was included in the model. Results The results are summarized in Table 1. Of the cleaved embryos, 8.7% developed to the blasto- cyst stage in the absence ofproteins. Insulin sup- plementation did not significantly increase cleav- age rate or development to the morula and blas- tocyst stage, but supplementation with 200 nM insulin tended to increase cell proliferation in morulae and blastocysts (P=0.10). The highest cleavage rate, the highest proportion of blasto- cysts and the highest number of nuclei in moru- lae and blastocysts were achieved with the high- est concentration of insulin used. Discussion By extending the the previous observations of Pinyopummintr and Bavister (1991) and Takagi et al. (1991) we demonstrate in this study the possibility of culturing bovine zygotes to the blastocyst stage in a protein-free, chemically defined medium. The medium we used contained PVP as a macromolecular compound. Although we did not here compare it with other macromo- lecular ingredients, we have previously shown that PVP supports early cleavage better than does foetal calf serum (Bredbacka and Bredbacka 1995). PVP is of non-biological origin and we use it as a surfactant to facilitate subsequent embryo handlings. Takagi et al. (1991) produced 4% blastocysts per cleaved embryo using protein-free TCM-199 as the culture medium. Pinyopummintr and Bavister (1991) reported a success rate of 10- 15% blastocysts/cleaved oocyte in their protein- free culture system (either TCM 199 or HECM). Our success rate of 8.7% blastocysts in CRI+PVP medium is similar to the rates report- ed in the above two studies, although our cul- ture time was one day shorter than that used by Pinyopummintr and Bavister. Our cleavage rate (75.6%) was, however, significantly lower than theirs (about 95%). This difference may be due to differences in the in vitro fertilization proto- col rather than in the compositions of the cul- ture media. 511 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Bredbacka, K. & Bredbacka, P. Bovine blastocystproduction in defined medium Recent studies stress the importance of ami- no acids in the production of bovine blastocysts in protein-free media. Using a modified TLP medium, Kim et al. (1993) produced 0-1% blas- tocysts without amino acids as against 9-13% in the presence of amino acids. Keskintepe et al. (1995) produced 10.9-18.4% blastocysts in pro- tein-free SOF medium with an increase to 28.4- 42.3% with the addition of non-essential amino acids. The medium we used also contained ami- no acids, but probable not at optimal concentra- tions, as recent studies indicate that bovine em- bryo development is supported by increased con- centrations of glycine and alanine (Moore and Bondioli 1993). Using TCM-199 as control medium, Jiang et al. (1990) found no beneficial effect of insulin supplementation (10|ig/ml or 50 Rg/ml) on bo- vine morula and blastocyst development. How- ever, Seidel et al. (1991b) reported a beneficial effect of 5 IU insulin on blastocyst formation in chemically defined medium (CDM). The insu- lin supplemenation in our study tended to in- crease the proportions of morulae and blastocysts at the highest concentration used (200 nM). Sim- ilarly, the average cell number in morulae and blastocysts increased with the increase in the insulin concentration. Although neither of these trends was statistically significant, taken togeth- er, they may imply a supportive role of insulin. We decided to test very different concentrations because of the wide range ofconcentrations used in studies on biological effects reported in the literature. The lower concentration (2 nM) falls well in the range of 1.7 pM - 170 nM, which has been found to be stimulatory for protein synthe- sis in mouse embryos (Harvey and Kaye 1988). On the other hand, Seidel et al. (1991b) used 5 IU of insulin withbeneficial effects on the bo- vine. We used only about 0.003 units at the end of culture even with the higher concentration of insulin. It is possible that cattle embryos require considerably higher insulin concentrations than mouse embryos in an in vitro situation. Lower concentrations may act via insulin receptors, whereas high concentrations of insulin are re- quired for binding to insulin-like growth factor I (IGF-I) receptors. Transcripts for both recep- tors are present throughout bovine preimplanta- tion development (Watson et al. 1992). Whether insulin shows a beneficial effect or not may depend not only on the concentration of insulin, but also on the culture medium com- ponents used. Zhang and Armstrong (1990), for instance, observed that insulin was beneficial for the development of 8-cell rat embryos only in the presence of amino acids. The optimal con- centration of insulin may therefore differ from one medium to another. In conclusion, our study demonstrates the feasibility ofproducing bovine blastocysts from 1-cell stage embryos in a protein-free CRlaa medium supplemented with PVR Addition of 200 nM insulin tended to facilitate blastocyst formation. Higher insulin concentrations than those used here may be required for an accepta- ble quality and rate of morulae and blastocysts. Acknowledgements. The authors are grateful to T.-M. Nie- minen and J. Peippo for technical support. FSH and LH were kindly provided by D.J. Bolt (USDA Animal Hormone Program, Beltsville, MD, USA). References Brackett, B.G. & Zuelke, K.A. 1993. Analysis of factors involved in the in vitro production of bovine embryos. Theriogenology 39: 43-64. Bredbacka, K. & Bredbacka, P. 1995. Effect of polyvi- nylpyrrolidone, serum albumin and fetal calf serum on early cleavage of bovine embryos. Theriogenology 43: 174. (Abstract). Flood, M.R., Gage, T.L. & Bunch, T.D. 1993. Effect of various growth-promoting factors on preimplantation bo- vine embryo development in vitro. Theriogenology 39: 823-833. Harvey, M.B. & Kaye, P.L. 1988. Insulin stimulates pro- tein synthesis in compacted mouse embryos. Endocrinol- ogy 122: 1182-1184. 512 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Vol. 5 (1996): 509-514. - & Kaye, P.L. 1991. Mouse blastocysts respond meta- bolically to short-term stimulation by insulin and IGF-1 through the insulin receptor. Molecular Reproduction and Development 29: 253-258. Merrier, A., Lucas-Hahn, A. & Niemann, H. 1992. Ef- fects of insulin-like growth factor-1 on in-vitro production of bovine embryos, Theriogenology 37: 1213-1224. Heyner, S., Shah, N., Smith, R.M., Watson, A.J. & Schultz, G.A. 1993. The role of growth factors in em- bryo production. Theriogenology 39: 151-161. Jiang, H.S., Wang, W.L., Lu, K.H. & Gordon, I. 1990. Effects of PMSG, insulin, osmolarity and oestrus cow serum on development of IVF early bovine embryos cul- tured on granulosa cell monolayers. Theriogenology33: 258. (Abstract). Keskintepe, L., Burney, C.A. & Brackett B.G. 1995. Production of viable blastocysts in defined In vitro condi- tions. Biology of Reproduction 52: 1410-1417. Kim, J.-H., Niwa, K., Lim, J.-K. & Okuda, K. 1993. Ef- fects of phosphate, energy substrate, and amino acids on development of in Wfro-matured, in Wrro-fertilized bo- vine oocytes in a chemically defined, protein- free cul- ture medium. Biology of Reproduction 48: 1320-1325. Lindner, G.M. & Wright, R.W., Jr. 1983. Bovine embryo morphology and evaluation. Theriogenology20:407-416. Moore, K. & Bondioli, K.R. 1993. Glycine and alanine supplementation of culture medium enhances develop- ment of In vitro matured and fertilized cattle embryos. Biology of Reproduction 48: 833-840. Parrish, J.J., Susko-Parrish, J., Winer, M.A. & First, N.L. 1988. Capacitation of bovine sperm by heparin. Bi- ology of Reproduction 38: 1171-1180. Pinyopummintr, T. & Bavister, B.D. 1991. In vitro-ma- tured//n wfro-fertilized bovine oocytes can develop into morulae/blastocysts in chemically defined, protein-free culture media. Biology of Reproduction 45: 736-742. Rao, L.V., Wikarczuk, M.L. & Heyner, S. 1990. Func- tional roles of insulin and insulinlike growth factors in preimplantation mouse embryo development. In Vitro Cellular & Developmental Biology 26: 1043-1048. Rosenblum, 1.Y., Mattson, B.A. & Heyner, S. 1986. Stage-specific insulin binding in mouse preimplantation embryos. Developmental Biology 116: 261-263, Rosenkrans, C.F. & First, N.L. 1991. Culture of bovine zygotes to the blastocyst stage: effect of amino acids and vitamins. Theriogenology 35: 266. (Abstract). Seidel, G.E., Jr, Glass,T. & Olson, S.E. 1991a. Culture of 1-cell bovine embryos to blastocysts in chemically defined media. Biology of Reproduction 44, Supplement 1: 155. (Abstract). -, Nauta, W. & Olson, S.E. 1991b. Effects of myoinosi- tol, transferrin and insulin on culture of bovine embryos. Journal of Anirhal Science 69, Supplement 1: 403. (Ab- stract). Takagi,Y., Mori, K.,Tomizawa, M., Takahashi, T., Sug- awara, S. & Masaki, J. 1991. Development of bovine oocytes matured, fertilized and cultured in a serum-free, chemically defined medium. Theriogenology 35: 1197- 1207. Watson, A.J., Hogan, A., Hahnel, A., Wiemer, O.E. & Schultz, G.A. 1992. Expression of growth factor ligand and receptor genes in the preimplantation bovine em- bryo. Molecular Reproduction and Development 31: 87- 95. Zhang, X. & Armstrong, D.T. 1990. Presence of amino acids and insulin in a chemically defined medium im- proves development of 8-cell rat embryos in vitro and subsequent implantation in vivo. Biology of Reproduc- tion 42: 662-668. 513 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Bredbacka, K. & Bredbacka, P. Bovine blastocyst production in defined medium SELOSTUS Insuliinin vaikutus naudan blastokystien tuottamiseen in vitro kemiallisesti tunnetussa liuoksessa Kristiina Bredbacka ja Peter Bredbacka Maatalouden tutkimuskeskus Naudan alkion varhaiskehitystä tutkittiin viljelemäl- lä alkioita kemiallisesti tunnetussa viljelyliuoksessa. Kokeessa käytetty liuos CRI-PVP on muunnos CRlaa-liuoksesta, jonka proteiinilähde, naudan see- rumin albumiini, korvattiin 4mg/ml polyvinyylipyr- rolidonilla. Kokeessa käytetyt kolme insuliinipitoi- suutta olivat 0, 2 ja 200 nM. In v/fro-tuotetut alkiot jaettiin hedelmöityksen jälkeen kolmeen viljelyryh- mään: CRI-PVP, CRI- PVP + insuliini (2 nM) ja CRI-PVP + insuliini (200 nM). Alkioita viljeltiin seitsemän päivää, ja neljäntenä päivänä lisättiin tuo- retta viljelyliuosta ja glukoosia (lopullinen pitoisuus 5,56 mM). Viljelyn päättyessä alkiot arvioitiin kehi- tysvaiheen mukaan ja alkioiden solut laskettiin tuma- värjäyksen jälkeen. Proteiinittomassa viljelyliuoksessa saatiin blasto- kysteiksi kehittymään 8,7 %. Insuliinin lisäyksellä saatiin enemmän blastokystejä (12,8 %), mutta ei ti- lastollisesti merkitsevästi. Jakautumisprosentti, blas- tokystien osuus jakautuneista alkioista sekä morula/ blastokystivaiheen alkioiden tumalukumäärä (P=0,10) olivat korkeimmat ryhmässä, jossa insulii- nipitoisuus oli 200 nM. 514 AGRICULTURAL AND FOOD SCIENCE IN FINLAND