Vol. 5 11996): 503-508. Effect of glycine and alanine supplementation on development of cattle embryos cultured in CRlaa medium with or without cumulus cells Kristiina Bredbacka and Peter Bredbacka Agricultural Research Centre of Finland, Institute ofAnimal Production, FIN-31600 Jokioinen, Finland, e-mail: kristiina.bredbacka@mtt.fi The effect of alanine (I mM) and glycine (10 mM) supplementation on bovine embryo development in vitro was investigated. Presumptive bovine zygotes, produced by in vitro maturation and insemi- nation of oocytes, were cultured for 144 h in CRlaa medium in the absence (Experiments 1 and 2) or presence of cumulus cells (Experiment 3). In Experiment I, the proportion of morulae and blasto- cysts of cleaved embryos in glycine-supplemented medium was not different from that of the control medium (34% in both media); however, the cell numbers of morulae and blastocysts were signifi- cantly higher in the glycine-enriched medium (69.5 vs. 53.3, P = 0.016). In Experiment 2, addition of alanine did not improve the formation of morulae and blastocysts (13% vs. 21% in control medium), and the mean cell numbers in morulae and blastocysts were lower than those in the control group (34.3 vs. 68.7, P = 0.007). In the presence of cumulus cells, the combined supplementation of glycine and alanine increased the proportion of morulae and blastocysts over that in the control medium (31% vs. 14%, P = 0.003). Key words: bovine, morula, blastocyst, amino acids, in vitro production ntroduction Efficient in vitro production of cattle embryos suitable for embryo transfer is hampered by our limitedknowledge of the environmental require- ments of the preimplantation embryo. At present, only a minority of fertilized oocytes can be cul- tured to the morula or blastocyst stage (Brackett and Zuelke 1993). In general, the best results have been achieved when co-culture with somat- ic cells has been used in a complex culture me- dium. The reasons for the beneficial effect of somatic cells are unclear, but it might be attrib- uted to the addition ofembryotrophic factors into the medium and/or removal of harmful ingredi- ents from the culture system. Attempts to define such components have had limited success due to the complexity of the medium. Furthermore, some components in the culture medium may be beneficial for somatic cells but detrimental to embryos or vice versa. This could make inter- pretations of results in the optimization process difficult and perhaps misleading. © Agricultural and Food Science in Finland Manuscript received June 1996 503 AGRICULTURAL AND FOOD SCIENCE IN FINLAND https://www.c-info.fi/en/info/?token=0FETCz4WpSTJ1sHO.6fck1wNMmsScIAnLxBgcaQ.gHUsBNZBVfYCwPPlJ1rd3myAnlC_Yxx_V4S8LkLViTl18EofCAxSzNsqQiwaYRDjwHGsK7yvBwAbFHeYZgmKKBuH5PDv1WNU9pg0WrAXwC8cRq2GOtRRicgskwOeFaCBDlRAFkT3xYwLMkvHPzXGjmTKKU7yIN-fRcul84feHSASp4M5kXltM9MRxLoUYBTrfRvc05Ap4nSZ6pXwFmpguhsP9qOlEDr1c2QGdggMHnYfCthzO_JXEw4pDkjhkue5v-gVBb_bVRMGyBtC30oZRX6KPAzxWgJsN8uN Bredbacka, K. & Bredbacka, P. Bovine embryo culture in glycine and alanine The development of chemically definedcul- ture media represents an alternative approach for improving the consistency of culture conditions. Zygotes have been developed to the blastocyst stage in a number of defined media (Pinyopum- mintr and Bavister 1991, Rosenkrans and First 1991,Seidel et al. 1991,Takagi et al. 1991, Kim et al. 1993,Keskintepe et al. 1995). In some cas- es bovine serum albumin (BSA) has been used to replace serum. Due to undefinedcomponents in BSA, such media may more appropriately be termed semi-defined media. The amino acid requirement of embryos has been the focus of some recent studies (Bavister and Arlotto 1990, Moore and Bondioli 1993,Liu and Foote 1996, Partridge et al. 1996). Moore and Bondioli (1993) analysed bovine oviductal fluid and concluded that the predominant free amino acids were glycine and alanine. The au- thors went on to examine the effect of increased concentrations of alanine and glycine on bovine embryo development in BMOC or MBMOC medium. Their results indicated that in the pres- ence of oviductal cells, a combination of gly- cine and alanine improved embryonic develop- ment. Their study was supported by Bondioli et al. (1995), who added high concentrations of glycine and alanine in CRlaa-medium to co-cul- ture with beneficial effects. Our aim here was to further examine the ef- fects of increased levels of alanine and glycine on bovine embryo development with or without cumulus cells. As base medium we used a semi- defined medium, the widely applied CRlaa (Rosenkrans and First 1991). In addition to mor- phological evaluation we extended the embryo quality assay by counting nuclei of the embryos. Material and methods In vitro maturation Cattle ovaries were obtained from a slaughter- house and transported to the laboratory at room temperature in 0.9% NaCl containing antibiot- ics. The oocytes were aspirated from follicles (diameter 3-8 mm) and only cumulus-surround- ed oocytes were accepted. Oocyte-cumulus com- plexes were washed twice and matured in TCMI99 medium supplemented with 0.25 mM sodium pyruvate, 2.0 mM L-glutamine, 100 IU/ml penicillin, 100 pg/ml streptomycin, 2 pg/ml FSH (USDA-oFSH-1 8; USDA, Beltsville, MD, USA), 10 pg/ml LH (USDA-bLH-B-6; USDA, Beltsville, MD, USA), 1 pg/ml estradiol-17(3 (Sigma, St. Louis, MO, USA), 5% foetal calf serum and 5% oestrous cow serum. Forty to fif- ty oocytes were cultured for 24 h in a 500-pl drop of maturation medium in 5% CO, in air at39°C. In vitro fertilization After maturation the oocyte-cumulus complex- es were inseminated with 1.5 x 106/ml of washed frozen-thawed sperm at 39°C in 5% C0 2 under oil in 100 pi of fert-TALP (Parrish et al. 1988) containing 2 pg/ml heparin and 20 pmol/1 peni- cillamine, 10 pmol/1 hypotaurine and 1 pmol/1 epinephrine. After 24 h the cumulus cells were removed by vortexing (Experiments 1 and 2) or were left intact (Experiment 3). In vitro culture After insemination the oocytes were washed in the culture medium CRI aa (Rosenkrans and First 1991) and randomized into treatment groups. After 96 h of culture in 50 pl-drops of medium without glucose (120 h after insemination), 50 pi of fresh culture medium with glucose was added to bring the final glucose concentration to 5.56 mM. The embryos were cultured under mineral oil at 39°C in a humidifiedenvironment of 5% CO, in air. The chemicals were obtained from Merck (all salts) or from Sigma (BSA, glutamine, pyruvate, hemi-lactate, amino acids and gentamicin). In Experiment 1, embryo de- velopment in the control medium (CRlaa) was compared with that of the control medium sup- 504 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Vol. 5 (1996): 503-508. Table I. Effect of glycine supplementation on development of cattle embryos cultured in CRlaa without cumulus cells. Treatment Oocytes % Cleaved % M+B x(±S.E.) of cleaved nuclei in M+B CRlaa 180 56.7 34.3 53.3“ (±4.1) CRlaa + gly 176 55.1 34.0 69.5b (±5.1) “■b Values with different superscripts differ significantly (P<0.05) Abbreviations: gly = glycine (10 mM), M+B = morulae+blastocysts plemented with 10 mM glycine. In Experiment 2 the control medium was compared with the supplementation of alanine (1 mM). In Experi- ment 3, the control medium was compared with supplementation of glycine (10 mM), alanine (1 mM) and a combination of these amino acids. Each experiment was repeated at least five times. After 144 h of embryo culture (Day 7 after insemination) the embryos were examined for appearance and stage of development. The qual- ity of embryos was scored according to Lindner and Wright (1983), however, lower than Grade 2 embryos were not classified as morulae or blas- tocysts. The cell number was assessed by stain- ing the nuclei of blastomeres with 3 pg/ml Hoechst stain 33258 in 15% ethanol in PBS for 24 to 48 h at +4°C. After fixation and staining each embryo was individually washed and placed in a 1 pi drop of PBS into alO to 15 pi drop of a mixture of glycerol and water (5:1) on a glass slide. The embryo was then overlaid with a cov- erslip and the nuclei were counted under a Leitz Dialux 20 fluorescence microscope using the Leitz A filter block. Statistical analysis Proportions (cleaved embryos of inseminated oocytes, and Grade 1 and Grade 2 compacted morulae and blastocysts of cleaved embryos) were compared by analysis. The cell numbers of embryos of transferable quality (Grade I and Grade 2 morulae and blastocysts) were compared using the GLM program of SAS (SAS Institute, Cary, NC, USA). The effect of replicates was included in the model. Results Experiment I The addition of glycine (10 mM) had no effect on cleavage rate or development to the morula/ blastocyst stage (Table 1). However, the supple- mentation increased the cell proliferation in morulae and blastocysts when compared with the controls (P = 0.016). The cell number in moru- lae and blastocysts in glycine-supplemented CRlaa medium was 69.5 (±5.1), which is sig- nificantly higher (P = 0.016) than that in the con- trol CRI medium, 53.3 (±4.1). Experiment 2 Supplementation ofCRlaa medium with alanine did not significantly affect cleavage rate or de- velopment to the morula/blastocyst stage (Table 2). However, cell numbers of morulae and blas- tocysts were significantly lower (P = 0.007) in alanine-supplemented medium than in the con- trols (34.4 and 68.7, respectively). 505 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Bredbacka, K. & Bredbacka, P. Bovine embryo culture in glycine and alanine Table 2. Effect ofalanine supplementation on development of cattle embryos cultured in CRlaa without cumulus cells. Treatment Oocytes % Cleaved % M+B x(±S.E.) of cleaved nuclei in M+B CRlaa 185 54,6 20.8 68.7“ (±8.6) CRlaa + ala 185 55,7 12,6 34.4b(±s.B) ab Values with different superscripts differ significantly (pcO.Ol) Abbreviations: ala =alanine (I mM), M+B = morulae+blastocysts. Table 3. Effect of supplementation of glycine and/or alanine on development of cattle embryos cultured in CR I aa in coculture with cumulus cells. Treatment Oocytes % Cleaved % M+B Nuclei in M+B of cleaved (least square means) CRIaa+CC 182 55.5 13.9“ 94.1 CRI aa+CC+gly 182 58.8 20.6“ h 115.7 CRI aa+CC+ala 178 53.4 23.2ab 105.5 CR I aa+CC+gly+ala 177 57.6 31.4 b 95.3 ab Values with different superscripts differ significantly (P<0.05) Abbreviations: CC = cumulus cells, gly = glycine (10 mM), ala = alanine (1 mM), M+B = morulae + blastocysts. Experiment 3 As shown in Table 3, in cumulus cell co-culture the percentage of transferable embryos (Grade 1 and Grade 2 morulae and blastocysts) was sig- nificantly higher (P = 0.003) in CRlaa medium supplemented with both glycine (lOmM) and alanine (ImM) than in the absence of supple- mentation. Supplementation of only one of the amino acids gave morula/blastocyst rates inter- mediate between the nonsupplemented and dou- ble-supplemented group (N.S.). No effects of the amino acid supplementations on cleavage rate or cell number were observed. Discussion In our study the in vitro production of cattle morulae and blastocysts in CRlaa medium was increased by the combined supplementation of glycine and alanine when a cumulus cell co-cul- ture approach was used. The effect was due not to an increase in cleavage rate but to an increase in the development of cleaved zygotes. This in- crease was statistically significant only when both of these amino acids were added to the cul- ture medium. When either of the amino acids was used alone, the proportion of morulae and blas- tocysts was between that of the nonsupplement- ed treatment and the treatment in which both amino acids were added. Although the combined addition of these compounds did not significantly improve the morphological development when compared with supplementation of either glycine or alanine alone, a synergistic effect might be implicated. The study of Moore and Bondioli (1993) presents a rather similar picture. These authors used a modified Brinster’s oviduct culture me- dium and a one-day-longer culture period (7 days). In co-culture with oviduct cells, they re- 506 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Vol. 5 (1996): 503-508. ported the highest rate of morula/blastocyst de- velopment when alanine and glycine were used in combination. However, alanine alone was not sufficient to improve development. Bondioli et al. (1995) demonstrated increased development to the expanded blastocyst stage when CRlaa medium supplemented with foetal calf serum and buffalo rat liver cells was used with a combina- tion of glycine and alanine. Employing a com- plex medium (TCMI99), Shi et al. (1995) showed that supplementation of a combination of alanine (2.5 mM) and glycine (12.5 mM) im- proved blastocyst development in a 7 day co- culture with a granulosa cell monolayer when the serum concentration was 10%; at a lower serum concentration (2.5%), no effect of the add- ed amino acids was found. We did not observe an effect of glycine or alanine supplementation on morula/blastocyst development rate in a cell-free culture system. However, glycine supplementation improved the quality of morulae and blastocysts as measured by number of nuclei. By the same criteria, alanine supplementation alone diminished the quality of morulae and blastocysts. Using cell- free culture with CR2 as the base medium - a very similar culture system to the one used here - Suh et al. (1995) observed a higher proportion of blastocyst formation with glycine supplemen- tation than with both glycine and alanine sup- plementation. Thus, alanine would seem to have a favourable effect on bovine embryo develop- ment in co-culture with somatic cells, but a det- rimental effect in a cell-free system. It is there- fore possible that, in a co-culture, alanine im- proves embryo development only indirectly, per- haps by altering the function of somatic cells, as suggested by Moore and Bondioli (1993). Gly- cine, on the other hand, may have both direct and indirect effects on bovine development. Although our experiments were not designed to compare the cell-free system with the co-cul- ture system, a comparison of the number of nu- clei seems to indicate that the co-culture system better supports cell proliferation in morulae and blastocysts. However, the proportion of moru- lae and blastocysts does not appear to be signif- icantly affected by the co-culture. The cell count may be an important parameter in the quality assay of different embryo culture systems. In conclusion, we found that the combined use of glycine and alanine in a co-culture sys- tem with cumulus cells is promotes bovine em- bryo development, confirming previous reports. Furthermore, cell numbers in morulae and blas- tocysts were increased by the addition of gly- cine and decreased by the addition of alanine in a cell-free culture system. Acknowledgements. The authors are grateful to T.-M. Nie- minen and J. Peippo for technical assistance. FSH and LH were kindly provided by D.J. Bolt (USDA Animal Hor- mone Program, Beltsville, MD, USA). References Bavister, B.D. & Arlotto, T. 1990. Influence of single amino acids on the development of hamster one-cell embryos in vitro. Molecular Reproduction and Develop- ment 25: 45-51. Bondioli, K.R., Hawk, H.W. & Wall, R.J. 1995. Effect of glycine and alanine on co-culture of bovine blastocysts. Theriogenology 43: 170. (Abstract). Brackett, B.G. & Zuelke, K.A. 1993. Analysis of factors involved in the in vitro production of bovine embryos. Theriogenology 39: 43-64. 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 v/fro-matured, in Wfro-fertilized bo- vine oocytes in a chemically defined, protein-free culture medium. Biology of Reproduction 48: 1320-1325. Lindner, G.M. & Wright, R.W., Jr. 1983. Bovine embryo morphology and evaluation.Theriogenology 20:407-416. Liu, Z. & Foote, R.H. 1996. Effects of amino acids on development of IVM/IVF bovine embryos in a simple pro- tein-free medium. Theriogenology 45: 169. (Abstract). Moore, K. & Bondioli, K.R. 1993. Glycine and alanine supplementation of culture medium enhances develop- ment of in vitro matured and fertilized cattle embryos. 507 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Bredbacka, K. & Bredbacka, P. Bovine embryo culture in glycine and alanine 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. Biol- ogy of Reproduction 38: 1171-1180. Partridge, R.J., Pullar, D.. Wrathall, A.E. & Leese, H.J. 1996. Consumption of amino acids by in vivo and in vit- ro-derived bovine embryos. Theriogenology 45:181. (Ab- stract). 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. 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. 1991. Culture of 1 -cell bovine embryos to blastocysts in chemically defined media. Biology of Reproduction 44, Supplement 1: 155. (Abstract). Shi, D.S., Tan, S.J., Huang, F.L. & Lu, K.H. 1995. Ef- fects of glycine and alanine on the in vitro development of early bovine embryos derived from IVM/IVF. Therio- genology 43: 323. (Abstract). Suh, T.K., White, K.L., Bunch, T.D., Spendlove, R. & Wilkinson, R. 1995. Effect of glycine, alanine and calf plasma in serum free culture medium on bovine embry- onic development in vitro. Theriogenology 43: 328. (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, SELOSTUS Glysiinin ja alaniinin vaikutus CRlaa-liuoksessa viljeltyyn kumulussolullisen ja -soluttoman naudanalkion kehitykseen Kristiina Bredbacka jaPeter Bredbacka Maatalouden tutkimuskeskus Glysiinin (10 mM) ja alaniinin (1 mM) vaikutusta naudan alkion varhaiskehitykselle tutkittiin kolmes- sa viljelykokeessa. Kumulussoluttoraia alkioita vil- jeltiin hedelmöityksen jälkeen CRlaa-liuoksessa, jo- hon oli lisätty glysiiniä (koe 1) ja alaniinia (koe 2). Kokeessa 3 käytettiin alkioita, joista ei poistettu ku- mulussoluja ja ne jaettiin neljään eri viljelyliuokseen: CRlaa + glysiini (10 mM), CRlaa + alaniini (1 mM), CRlaa + glysiini + alaniini sekä kontrolliliuos, joka oli kaikissa kokeissa CRlaa. Alkioita viljeltiin yh- teensä kuusi päivää. Neljäntenä päivänä viljelytippoi- hin lisättiin tuoretta viljelyliuosta ja glukoosia (lopul- linen pitoisuus 5,56 mM). Viljelyn päätyttyä alkiot luokiteltiin kehitysvaiheen mukaisesti ja niiden so- lujen lukumäärä laskettiin värjäämällä fiksoitujen al- kioiden tumat Hoechst-värillä, Kokeessa 1 glysiinin lisäyksellä ei ollut vaikutus- ta alkioiden kehittymiseen morula/blastokystivaihee- seen. Morula/blastokystialkioiden solujen lukumää- rä oli kuitenkin merkitsevästi korkeampi glysiiniryh- mässä (69,5) kuin kontrolliryhmässä (53,3). Kokeessa 2 alaniinilla ei todettu merkitsevää vai- kutusta morula/blastokystien määrään, mutta moru- la/blastokystivaiheen alkioiden solujen lukumäärä oli merkitsevästi alhaisempi alaniiniryhmässä (34,4) kuin kontrolliryhmässä (68,7). Kumulussolullisia alkioita viljeltäessä (koe 3) aminohappojen lisäyksellä ei havaittu vaikutusta so- lujen lukumääriin. Sen sijaan morula/blastokystivai- heen alkioita saatiin kontrolliryhmään verrattuna merkitsevästi enemmän kun viljelyliuokseen lisättiin sekä glysiiniä että alaniinia. 508 AGRICULTURAL AND FOOD SCIENCE IN FINLAND