Vol. 5(1996): 515-520. A simple culture system for time-lapse video recording of bovine embryos JaanaPeippo and Peter Bredbacka Agricultural Research Centre ofFinland, Institute ofAnimal Production, FIN-31600 Jokioinen, Finland, e-mail: jaana.peippo @ mll.fi Continuous observation of embryonic growth can improve understanding of the early developmental events and allow us to use parametric statistical analyses with time as a parameter. A cinematograph- ic study such as that reported here utilizes time-lapse video recording. Previously published methods for time-lapse video recording have involved building an incubator around a microscope, a process that is both expensive and laborious. Here we present a simplified method for time-lapse video re- cording of early bovine embryo development. The embryos were cultured during a 24-hour period in a standard pregassed tissue culture bottle, which was darkened and placed on the heating stage of an inverted microscope for recording through a red filter. The control embryos were cultured in a con- ventional C0 2 incubator. After 10 replicates we could not find a statistically significant difference between the cell numbers of these two treatments (P=0.95), suggesting that the culture setup is ap- propriate for continuous observation of early cleavage of the cattle embryo. Key words: cinematography, mini embryo culture device, cattle, development ntroduction Improvements in the in vitro production of em- bryos depend on well-designed embryo culture experiments. This is particularly important in domestic species, in which better culture condi- tions need to be defined. The traditional approach in performing embryo culture experiments in- cludes an end point, say, 7 days after insemina- tion. Typically the data from such experiments is expressed in categories, such as the propor- tion of blastocysts at the end of the culture. The choice of an appropriate end point can be a prob- lem. For instance, two culture treatments may appear to be equally successful with a certain end point, although a different outcome might have been obtained with an earlier or later end point. Continuous observation of embryo culture produces considerably more information than the end point approach. Abnormal events at the cleavage stages may not be recognized in em- bryos appearing as normal blastocysts after one © Agricultural and Food Science in Finland Manuscript received June 1996 515 AGRICULTURAL AND FOOD SCIENCE IN FINLAND https://www.c-info.fi/en/info/?token=iu33-KGipbh8prFu.391u5tETxcX1nuk4nUg0hw.ElCGjC8qCP7i29ebUaJkX1Vzj54MJYulHOmiRvoec_vV-1IRObA7JIDUiFYn8TI0GrYnKdhkIsotUHkJb0QzdbmvrgfPNBV884QPS8YuTDQR_goO6tMPR1R-kr0tWrtjmQs0NLrU-Q3hSZFQdmz6f5r9HpwcO4WuIJ19tBSntrtCw9XQv0Zrl74Ene_NYvlwmsRqt4-c-d26Wzc-7Sh2aHdwzK8PzUhbbQWigUBNcQE0kSUuFjut3INv0mVXSrhRLAFeUFe-o9msMrxr-XJRoiownQ Peippo, J. & Bredbacka, P. Time-lapse video recording ofembryos week of culture. The timing of early events can also be indicative of subsequent embryo viabil- ity (McKiernan and Bavister 1994). It has been demonstrated that bovine embryos reaching the 2-cell stage by 30-h post insemination (hpi) are the ones most likely to continue development and to give the highest rate of compacted morulae, blastocysts and hatching (Plante and King 1992, Miller et al. 1992, Van Soom et al. 1992). Fur- thermore, continuous observation facilitates more versatile use bf parametric statistical anal- yses, as time can be used as a parameter. In practice, continuous monitoring of embryo development makes use of time-lapse video re- cording. Several authors have demonstrated the potential of this approach (Mulnard 1967, Mas- sip and Mulnard 1980, Massip et al. 1982, 1983a and 1983b. Bavister 1988, Grisart et al. 1994, Gonzales et al. 1995). However, the setup has usually been based on a C02 incubator built around a microscope. This easily becomes too expensive for many laboratories. We present here a simple and inexpensive culture system de- signed for time-lapse video recording of early bovine embryo development. We compare the final cell numbers at the end of 24-h culture be- tween the time-lapse video recording and regu- lar C02 incubator environments. In vitro embryo culture After 20-h fertilization, the cumulus cells were removed by vortexing for 90 s, and about 12 pre- sumptive zygotes were placed in each 1-3 pi drop of preincubated culture medium. The cul- ture medium was CRI (Rosenkrans and First 1991) supplemented with 5.56 mM of glucose and covered with mineral oil either in a 100-ml tissue culture flask (Nunc) or 4-well culture dish (Nunc). The culture flask was darkened with black PVC tape at the top and on the sides leav- ing only a small window at the top for micro- scope observation (Fig. 1). After 2 h of equili- bration at 39°C in 5% C0,:95% air in an incu- bator, the culture flask was closed and placed on the heating stage (Linkam) of an inverted microscope; the control zygotes were placed in a 4-well culture dish and kept in the incubator. Using red light (> 620 nm) illumination caused by a filter placed over the window at the top of the flask, we recorded the culture period at 10 x magnification with a Hamamatsu C4200 CCD microscope camera and a time-lapse video re- corder (Fig. 2). At 44 hpi both embryo cultures were termi- nated and the cleavage rates and the cell num- bers per cleaved embryo were calculated in both groups. Material and methods In vitro embryo production The oocytes were aspirated from ovaries ofslaugh- tered cows and matured in TCM-199 supplement- ed with 5% foetal calf serum, 5% oestrous cow serum, 0.25 mM sodium pyruvate, 2 mM glutamine, 100 IU penicillin/ml, 100 pg strepto- mycin/ml, 2 pg FSH/ml (USDA-oFSH-18), 10 pg LH/ml (USDA-bLH-B-6) and 1 pg estradiol-17(3/ ml at 39°C in 5% C0,:95% air. After 24 h of mat- uration, the oocytes were inseminated with 1.5 x 106 spermatozoa/ml in fert-TALP medium (Parr- ish et al. 1988) for 20 h at 39°C in 5% CQ,:95% air. Statistical analysis The data (i.e. the average cell number per clbaved embryo) was analysed using Student’s ttest fol- lowing square-root transformation after combin- ing the data on all the replicates in both treat- ments. Results Ten replicates were completed and analysed. There was no statistically significant difference between the replicates in cell numbers 516 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Vol. 5 (1996): 515-520. (P=0.1350), thus the data on both treatments were combined for further analyses. A total of 120 zygotes were cultured in a culture flask (time-lapse) and 113 in a 4-well dish (incuba- tor). During the culture, 94 (78.3%) zygotes cleaved in the culture flask and 80 (70.8%) in the 4-well culture dish. We could not find a sta- tistically significant difference in the final cell numbers 44 hpi (Table 1) between these two treatments (P = 0.95). Discussion The time-lapse culture system presented in our study is convenient and comparable to culture in the traditional C02 incubator, at least when a 24-h culture period is used. This period covers three, sometimes four, cell cycles if it begins when the embryos are at the late 1-cell stage (20 hpi). In an ongoing study we transferred the Table I. Cleavage stage and mean values (± SEM) ofsquare-root transformed cell numbers after 24 h of culture in a time-lapse video recording enviroment and a standard C0 2 incubator. Position N Number of cells/embryo Mean(+SEM) 2 3-4 5-7 >8 cell no.’ time-lapse enviroment 94 20 46 22 6 1.99±0.04 co2 incubator 80 20 34 18 8 1.99±0.05 ’ Square root transformed cell data. 517 Fig. 1. For the video recording a standard tissue culture bottle (Nunc) was darkened at the top and on the sides leav- ing only a small window covered with a red filter at the top. (Photo: Jaana Peippo). Fig. 2. The setup used for time-lapse video recording of bovine embryo development. (Photo: Jaana Peippo). AGRICULTURAL AND FOOD SCIENCE IN FINLAND Peippo, J. & Bredbacka, P. Time-lapse video recording ofembryos embryos to the incubator after 24-h culture in the time-lapse environment. The rate of devel- opment to compacted morulae or blastocysts is at currently 24% (n=!4o), which is similar to that of control embryos cultured continuously in the incubator (26%, n=l37). In vitro culture exposes oocytes and embry- os to visible daylight during handling in the lab- oratory (320-740 nm, 1600 lx) (Schumacher and Fisher 1988) and to the more intensive light of the microscope (14 000 lx) (Nakayama et al. 1994). We are not aware of any studies report- ing the detrimental effects of light on bovine embryo development. In rabbit, daylight has been observed to have severe detrimental effects, especially at the 2-cell stage by causing cell de- generation and cell death (Schumacher and Fish- er 1988). The effect of short split-dose exposures (e.g. 4x 1 h) to visible light did not differ statis- tically from that of a single long exposure (4 h). In hamster and mouse, it has been suggested that the harmful effect of visible light is caused by an increase in hydrogen peroxide in the embry- os, the response being slower in mouse than in hamster embryos, but reaching the same level after 3 min exposure (Nakayama et al. 1994). In hamster embryos theresponse also depended on the 02 concentration and the culture medium used. In a 20% 02 atmosphere the increase of hydrogen peroxide levels occured earlier than in 5% Or In our study bovine embryos were subjected to 22 h of continuous light from the microscope through a red filter (> 620 nm). These long wave- lengths have been observed to be less harmful for embryonic cells in rabbit than 300-500 -nm wavelengths (Daniel 1964). Red filters were also used by Bavister (1988) and Gonzales et al. (1995) in their time-lapse recordings. Another way to minimize tha detrimental effect of visi- ble light was introduced by Grisart et al. (1994), who switched the microscope light on for only a few seconds before a single frame was exposed (every minute) and switched it off immediately after. Observations made by Schumacher and Fisher (1988) do not, however, indicate that this latter approach is any less harmful for embryon- ic development than other approaches. Jacques et al. (1987) showed that a small but significant proportion of the light spectrum of > 500 nm penetrates the uterine lumen of rats and guinea pigs transabdominally, and they suggest that this light may have direct and/or indirect beneficial effects on foetal development. The role of light in cattle embryo development is, however, ques- tionable as the reproductive tract of the cow is highly unlikely to be exposed to light this way due to the thickness of the skin. Another enviromental factor to be considered when embryos are cultured in vitro is tempera- ture. One way to solve the problem of tempera- ture variations is to keep the room and table sur- faces where embryos are handled constantly at over 30°C (Xu et al. 1987, 1992). In time-lapse studies temperature has been controlled by us- ing either heated and humified gas (Bavister 1988, Gonzales et al. 1995) or a standard hair- dryer to warm the culture enviroment (Grisart et al. 1994). In our study heating was carried out with a heating stage. The upper surface of the culture bottle was cooler than the surface against the heating stage, but the temperature of the heat- ing stage was adjusted so that a thermometer placed inside the culture drop at the opening of the heating stage showed 39°C. The gas atmosphere in our study was main- tained with a tightly closed conventional tissue culture bottle. According to Bavister (1988), it is important to have a constant gas flow as em- bryo metabolism can change the gas atmosphere in closed systems during prolonged culture. Our bottle was pregassed for 2 h before the addition of zygotes; at the end of the 24-h culture preser- vation of the gas atmosphere was checked with an indicator drop made ofTCM-199 medium and placed next to the culture drop under the oil. This control on pH was made to establish whetherany major pH changes were caused by leakage of CO, from the culture atmosphere. At least in our short-termculture the lack of constant gas flow did not seem to disturb embryo development. In long-term cultures, the culture bottles could be regassed daily to avoid the problems associated with a change in gas atmosphere. 518 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Vol. 5 (1996): 515-520. Our culture drops were smaller than those conventionally used, since we did not want em- bryos to change their positions. The small size of the culture drops may cause problems. For instance, accumulation of ammonium due to deg- radation of amino acids in culture medium can compromise embryonic development (Gardner and Lane 1993). Evaporation is another poten- tial problem ofculturing embryos in small micro- drops. We noticed that the drop becomes small- er with long-term culture; water may be absorbed by the oil even if the oil is equilibrated with 0.9% NaCl. In the method discussed here the upper surface of the culture bottle is colder than the bottom, thus facilitating evaporation ofH 2O from the oil and/or culture medium by condensation. This problem may be overcome by adding drops of medium to the flask. As the diameter of the opening in the middle of the microscope stage is 15 mm, the temperature of the stage is set at about 44°C to achieve a temperature of 39°C in the drop containing the embryos. The additional drops, which are in closer contact with the heat- ing stage, will then evaporate more readily and humidify the atmosphere of the culture flask, perhaps enough to prevent harmful evaporation of the drop containing the embryos. Another so- lution is to use larger drops and prevent the em- bryos from moving by placing them in small microwells produced at the bottom of the dish. In conclusion, we find the culture system outlined above convenient and suitable for the short-term time-lapse video recording of cattle embryos. Our intention is to evaluate the feasi- bility of this system for long-term (7-day) cul- ture, with modifications, if necessary. Acknowledgements. The authors thank K. Bredbacka, T. Hytti and T.-M. Nieminen for technical assistance. FSH and LH were kindly provided by D.J. Bolt (USDA Animal Hormone Program, Beltsville, MD). References Bavister, B.D. 1988. A minichamber device for maintain- ing a constant carbon dioxide in air atmosphere during prolonged culture of cells on the stage of an inverted microscope. In Vitro Cellular & Developmental Biology 24: 759-763. Daniel, J.C., Jr. 1964. Cleavage of mammalian ova in- hibited by visible light. Nature 201: 316-317. Gardner, D.K. & Lane, M. 1993. Amino acids and am- monium regulate mouse embryo development in culture. Biology of Reproduction 48: 377-385. Gonzales, D.S., Pinheiro, J.C. & Bavister, B.D, 1995. Prediction of the developmental potential of hamster embryos in vitro by precise timing of the third cell cycle. Journal of Reproduction and Fertility 105: 1-8. Grisart, 8., Massip, A. & Dessy, F. 1994. Cinemato- graphic analysis of bovine embryo development in se- rum-free oviduct- conditioned medium. Journal of Repro- duction and Fertility 101: 257-264. Jacques, S.L., Weaver, D.R. & Reppert, S.M. 1987. Penetration of light into the uterus of pregnant mammals. Photochemistry and Photobiology 45: 637-641. Massip, A. & Mulnard, J. 1980.Time-lapse cinemato- graphicanalysis of hatching of normal and frozen-thawed cow blastocysts. Journal of Reproduction and Fertility 58: 457-478. -, Mulnard , J., Van der Zwalmen, P., Hanzen, C. & Ectors, F. 1982. The behaviour of cow blastocyst in vit- ro: cinematographic and morphometry analysis. Journal of Anatomy 134: 399-405. -, Van der Zwalmen, P. & Zwijsen, W. 1983a. Atypical hatching of a cow blastocyst leading to separation of complete twin half blastocysts. Veterinary Record 112: 301. -, Zwijsen, W. & Mulnard, J. 1983b. Cinematographic analysis of the cleavage of the cow egg from 2-cell to 16-cell stage. Archives de Biologie (Bruxelles) 94: 99- 106. McKiernan, S.M. & Bavister, B.D. 1994. Timing of de- velopment is a critical parameter for predicting success- ful embryogenesis. Human Reproduction 9: 2123-2129. Miller, G.F., Gliedt, D.L., Lester, T.D., Pierson, J.N., Rakes, J.M. & Rorie, R.W. 1992. Addition of bovine ovi- ductal epithelial cells (BOEC) and/or penicillamine, hy- potaurine and epinephrine (PHE) to bovine in vitro fertili- zation (IVF) medium increases the subsequent embryo cleavage rate. Theriogenology 37: 259. (Abstract). Mulnard, J.G. 1967. Analyse microcinématographique du développementde I'ceuf de souris du stade II au blas- tocyte. Archives de Biologie, Liége 78: 107-138. Nakayama, T., Noda, Y., Goto, Y. & Mori, T. 1994. Ef- fects of visible light and other environmental factors on the production of oxygen radicals by hamster embryos. Theriogenology 41: 499-510. Parrish, J.J., Susko-Parrish, J., Winer, M.A. & First, 519 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Peippo, J. & Bredbacka, P. Time-lapse video recording ofembryos N.L. 1988. Capacitation of bovine sperm by heparin. Biol- ogy of Reproduction 38:1171-1180. Plante, L. & King, W.A. 1992. Effect of time to first cleav- age on hatching rate of bovine embryos in vitro. Theriog- enology 37: 274. (Abstract). Rosenkrans, C.F., Jr. & First, N.L. 1991. Culture of bo- vine zygotes to the blastocyst stage: Effect of amino ac- ids and vitamins. Theriogenology 35: 266. (Abstract). Schumacher, A. & Fischer, B. 1988, Influence of visi- ble light and room temperature on cell proliferation in preimplantationrabbit embryos. Journal of Reproduction and Fertility 84: 197-204. Van Soom, A., Van Vlaenderen, 1., Mahmoudzadeh, A.R., Deluyker, H. & de Kruif, A. 1992.Compaction rate of in vitro fertilized bovine embryos related to the inter- val from insemination to first cleavage. Theriogenology 38: 905-919, Xu, K.P., Greve,T., Callesen, H. & Hyttel, P. 1987. Preg- nancy resulting fromcattle oocytes matured and fertilized in vitro. Journal of Reproduction and Fertility 81: 501- 504. -,Yadav, 8.R., Rorie, R.W., Plante, L., Betteridge, K.J. & King, W.A. 1992. Development and viability of bovine embryos derived from oocytes matured and fertilized in vitro and co-cultured with bovine oviductal epithelial cells. Journal of Reproduction and Fertility 94: 33-43. SELOSTUS Yksinkertainen viljelymenetelmä naudan alkioiden aikaviivenauhoitusta varten Jaana Peippo ja Peter Bredbacka Maatalouden tutkimuskeskus Aikaviivenauhoituksen avulla naudan alkionviljelyä voidaan seurata yhtäjaksoisesti optimiolosuhteissa, joissa lämpötila ja kaasukehä pysyvät vakioina ja al- kiot eivät altistu valon lyhyille aallonpituuksille. Mikroskoopin ympärille on yleensä rakennettu pieni viljelykaappi, minkä takia menetelmä on ollut kallis ja työläs. Tässä kokeessa alkioita viljeltiin tavallises- sa soluviljelypullossa, joka peitettiin mustalla teipil- lä pientä päällä olevaa ikkunaa lukuunottamatta. Pul- lon sisään luotiin kaasukehä siten, että pulloa pidet- tiin viljelykaapissa kaksi tuntia sekä ennen alkioiden pulloon siirtämistä että siirtämisen jälkeen. Pullo sul- jettiin tiukasti ja viljely aikaviivenauhoitettiin ikku- nan päälle asetetun punaisen suodattimen läpi. Vil- jelypullo asetettiin käänteismikroskoopin alle lämpö- levylle, jonka lämpötila oli esikokeissa säädetty si- ten, että viljelypisaran lämpötila oli 39°C. Kokeessa tehtiin 10 toistoa. Niissä verrattiin aikaviivenauhoi- tettujen ja samanaikaisesti viljelykaapissa kasvanei- den kontrollialkioiden solulukumääriä 24 tunnin vil- jelyn jälkeen. Tulokset analysoitiin T-testillä yhdis- tämällä toistojen aineistot neliöjuurimuunnoksen jäl- keen, Aikaviivenauhoitettujen ja inkubaattorissa kas- vaneiden alkioiden välillä ei ollut eroa 24 h viljelyn jälkeen (p=0.95), joten kehittämämme menetelmä soveltuu ilmeisesti hyvin naudan alkioiden varhais- vaiheiden seurantaan. 520 AGRICULTURAL AND FOOD SCIENCE IN FINLAND