A simple culture system for time-lapse video recording of bovine embryos
Jaana Peippo and Peter Bredbacka
AgriculturalResearch CentreofFinland, InstituteofAnimalProduction,FIN-31600Jokioinen, Finland, e-mail:jaana.peippo@mll.fi
Continuousobservation ofembryonic growthcanimprove understanding of theearly developmental eventsand allowustouseparametric statisticalanalyseswithtimeas aparameter. Acinematograph- icstudysuch asthatreportedhere utilizestime-lapse videorecording. Previously published methods for time-lapse videorecording have involvedbuilding anincubator arounda microscope,aprocess that is both expensiveand laborious. Here wepresenta simplifiedmethodfor time-lapsevideo re- cording ofearlybovineembryo development.Theembryoswereculturedduringa24-hourperiod in
astandardpregassed tissue culturebottle,whichwasdarkened and placed onthe heatingstage ofan invertedmicroscope forrecording throughared filter. The controlembryos werecultured ina con- ventional C02 incubator.After 10 replicates wecould not find astatistically significantdifference between the cell numbers of these two treatments (P=0.95), suggesting that the culture setup is ap- propriate forcontinuousobservation ofearly cleavage of the cattle embryo.
Key words:cinematography, mini embryoculturedevice, cattle,development
ntroduction
Improvements in the in vitro production ofem- bryos depend on well-designed embryo culture experiments. This is particularly important in domestic species, in which better culture condi- tions needtobe defined. The traditional approach in performing embryo culture experiments in- cludesan end point, say,7 days after insemina- tion. Typically the data from such experiments is expressed in categories, such as the propor-
tion of blastocystsatthe end of the culture. The choice ofanappropriate end pointcan beaprob- lem. For instance, two culturetreatments may appear to be equally successful with a certain end point, although a different outcome might have been obtained withan earlieror later end point.
Continuous observation of embryo culture produces considerablymoreinformation than the end point approach. Abnormal events at the cleavage stages may not be recognized inem- bryos appearingas normal blastocysts afterone
©Agriculturaland Food Science inFinland Manuscriptreceived June 1996
week of culture. The timing of earlyevents can also be indicative of subsequent embryo viabil- ity (McKiernan and Bavister 1994). It has been demonstrated that bovine embryos reaching the 2-cellstage by 30-hpost insemination (hpi) are theonesmostlikelytocontinue development and togive the highestrate of compacted morulae, blastocysts and hatching(Planteand King 1992, Miller etal. 1992, Van Soom etal. 1992). Fur- thermore, continuous observation facilitates moreversatileusebf parametric statistical anal- yses,astimecanbe used as a parameter.
In practice, continuous monitoring of embryo development makesuse of time-lapse videore- cording. Several authors have demonstrated the potential of this approach(Mulnard 1967, Mas- sip and Mulnard 1980,Massipetal. 1982, 1983
a
and 1983b. Bavister 1988, Grisartet al. 1994, Gonzaleset al. 1995).However, the setup has usually been based on a C02 incubator built arounda microscope. This easily becomes too expensive for many laboratories. Wepresenthere a simple and inexpensive culture system de- signed for time-lapse video recording of early bovine embryo development. We compare the final cell numbersatthe end of 24-h culture be-
tweenthe time-lapse video recording and regu- lar C02incubator environments.
In vitro embryo culture
After 20-hfertilization, the cumulus cells were removed by vortexing for 90s,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) or4-well culture dish (Nunc). The culture flask was darkened with black PVC tapeatthetopandonthe sides leav- ing only a small window at the top for micro- scope observation (Fig. 1). After 2 h of equili- brationat 39°C in 5%C0,:95% air inan incu- bator, the culture flask was closed and placed on the heating stage (Linkam) of an inverted microscope; the controlzygotes were placed in a4-well culture dish and kept in the incubator.
Using red light(>620 nm)illumination caused by afilter placed overthe windowatthetop of theflask, werecorded the culture periodat 10x magnification with a Hamamatsu C4200 CCD microscope camera and a time-lapse video re- corder (Fig. 2).
At44hpiboth embryocultures were termi-
nated and the cleavagerates and the cell num- bers per cleaved embryowerecalculated in both groups.
Material and methods
In vitro embryo production
Theoocytes wereaspirated from ovaries ofslaugh- teredcowsand matured in TCM-199 supplement- ed with5% 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/
mlat39°C in 5%C0,:95%air. After24 h ofmat- uration,theoocytes were inseminated with 1.5 x
106 spermatozoa/ml in fert-TALP medium(Parr- ishetal. 1988)for20 hat39°C in 5%CQ,:95%air.
Statistical analysis
The data(i.e. the average cell number per clbaved embryo) was analysed using Student’sttestfol-
lowingsquare-roottransformation after combin- ing the dataon all the replicates in bothtreat- ments.
Results
Ten replicates were completed and analysed.
Therewas no statistically significant difference between the replicates in cell numbers
(P=0.1350), thus the data on bothtreatments 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 notfind asta- tistically significant difference in the final cell numbers 44 hpi (Table 1) between these two treatments (P=0.95).
Discussion
The time-lapse culturesystem presented in our study is convenient and comparable toculture in the traditional C02 incubator, atleast when a 24-h culture period is used. This periodcovers three, sometimes four, cell cycles if it begins when the embryosareatthe late 1-cellstage (20 hpi). In an ongoing study we transferred the
TableI.Cleavagestage andmeanvalues(±SEM)of square-root transformed cell numbers after24 hof cultureinatime-lapsevideorecording enviroment andastandard C02incubator.
Position N Number ofcells/embryo Mean(+SEM)
2 3-4 5-7 >8 cellno.’
time-lapse
enviroment 94 20 46 22 6 1.99±0.04
co2
incubator 80 20 34 18 8 1.99±0.05
’ Squareroottransformed cell data.
Fig. 1.For the videorecording a standard tissue culture bottle (Nunc)wasdarkened at the top andonthe sides leav- ingonly asmall window covered with ared filter at the top.(Photo: JaanaPeippo).
Fig. 2.The setup used fortime-lapsevideorecording of bovine embryo development. (Photo: JaanaPeippo).
embryos to the incubator after 24-h culture in the time-lapse environment. The rate of devel- opment to compacted morulaeorblastocysts is atcurrently 24% (n=!4o), which is similartothat of control embryos cultured continuously in the incubator(26%, n=l37).
In vitro culture exposes oocytes and embry- ostovisible daylight during handling in the lab-
oratory(320-740nm, 1600lx) (Schumacherand Fisher 1988) and to themore intensive light of the microscope (14 000 lx) (Nakayama et al.
1994).We are not aware of any studiesreport- ing the detrimental effects of light on bovine embryo development. In rabbit, daylight has been observedtohaveseveredetrimentaleffects, especiallyatthe 2-cell stageby causing cell de- generation and cell death(Schumacherand Fish- er 1988).The effect of short split-dose exposures (e.g. 4x 1 h)tovisible light didnot differ statis- tically from that ofasingle long exposure(4 h).
In hamster andmouse, it has been suggested that the harmful effect of visible light is caused by anincrease in hydrogen peroxide in the embry- os, the response being slower inmouse than in hamster embryos, but reaching the same level after3 min exposure (Nakayama etal. 1994). In hamster embryos the response also dependedon 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
Inour study bovine embryoswere subjected
to22 h of continuous light from the microscope throughared filter(>620nm).These long wave- lengths have been observed to be less harmful for embryonic cells in rabbit than 300-500 -nm wavelengths(Daniel 1964).Red filterswerealso used by Bavister (1988) and Gonzales et al.
(1995) in their time-lapse recordings. Another way to minimize tha detrimental effect of visi- ble lightwasintroduced by Grisartetal.(1994), who switched the microscope lightonfor onlya few seconds beforea single frame wasexposed (every minute) and switched it off immediately after. Observations made by Schumacher and Fisher(1988)donot, however,indicate that this latter approach is any less harmful for embryon-
icdevelopment thanother approaches. Jacques etal. (1987) showed thatasmall but significant proportion of the light spectrum of> 500 nm penetrates the uterine lumen ofrats and guinea pigs transabdominally, and theysuggestthat this light may have direct and/or indirect beneficial effectsonfoetal development. The role of light in cattle embryo developmentis, however,ques- tionableas the reproductive tract of thecow is highly unlikely tobe exposed to light this way dueto the thickness of the skin.
Another enviromental factortobe considered when embryos arecultured in vitro is tempera- ture.One way to solve the problem oftempera- turevariations istokeep theroomand tablesur- faces where embryosare handled constantly at over30°C (Xu etal. 1987, 1992).In time-lapse studies temperature has been controlled by us- ing either heated and humified gas (Bavister
1988, Gonzalesetal. 1995)or a standard hair- dryertowarmthe culture enviroment (Grisartet al. 1994). In ourstudy heating was carried out witha heating stage. The upper surface of the culture bottlewascooler than the surface against the heatingstage,but thetemperature of the heat- ing stage was adjusted so that a thermometer placed inside the culture drop atthe opening of the heating stageshowed 39°C.
The gas atmosphere inour study was main- tained witha tightly closed conventional tissue culture bottle. According toBavister (1988), it is important tohave aconstant gas flow as em- bryo metabolismcanchange the gas atmosphere
in closed systemsduring prolonged culture. Our bottle waspregassed for2 h before the addition ofzygotes;atthe end of the 24-h culture preser- vation of the gas atmosphere was checked with anindicator drop made ofTCM-199 medium and placednext to the culture drop under the oil. This controlon pHwasmadetoestablish whether any major pH changes were caused by leakage of CO, from the culture atmosphere. At least inour
short-term culture the lack ofconstantgas flow didnotseemtodisturb embryo development.In long-termcultures, the culture bottles could be regassed dailytoavoid the problems associated withachange in gas atmosphere.
Our culture drops were smaller than those conventionallyused, since wedid not wantem- bryos to change their positions. The small size of the culture drops may cause problems. For instance,accumulation of ammonium duetodeg- 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- erwith long-termculture;watermay be absorbed by the oilevenif 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 ofH2O from the oil and/or culture medium by condensation.
This problem may beovercomeby adding drops of medium to the flask. As the diameter of the opening in the middle of the microscope stage is 15 mm, the temperatureof thestage is set at
about 44°Ctoachievea temperatureof 39°C in the drop containing the embryos. The additional drops, whicharein closercontactwith the heat-
ing stage, will thenevaporate morereadily and humidify the atmosphere of the culture flask, perhaps enough toprevent harmful evaporation of the drop containing the embryos. Anotherso- lution istouselarger drops andpreventthe em- bryos from moving by placing them in small microwells produced atthe 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 toevaluate the feasi- bility of this systemfor long-term (7-day) cul- ture,withmodifications,if necessary.
Acknowledgements. The authors thank K. Bredbacka, T.HyttiandT.-M.Nieminen for technical assistance. FSH andLHwerekindly provided byD.J. Bolt (USDA Animal HormoneProgram, Beltsville,MD).
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SELOSTUS
Yksinkertainen viljelymenetelmä naudan alkioiden aikaviivenauhoitusta varten
Jaana Peippo ja Peter Bredbacka
Maatalouden tutkimuskeskus
Aikaviivenauhoituksen avulla naudan alkionviljelyä voidaan seuratayhtäjaksoisesti optimiolosuhteissa, joissa lämpötila jakaasukehä pysyvätvakioinajaal- kiot eivät altistu valon lyhyille aallonpituuksille.
Mikroskoopin ympärille onyleensärakennettu pieni viljelykaappi, minkä takia menetelmä onollut kallis ja työläs.Tässä kokeessa alkioitaviljeltiin tavallises- sasoluviljelypullossa, joka peitettiin mustallateipil- läpientä päällä olevaa ikkunaa lukuunottamatta.Pul- lon sisään luotiin kaasukehä siten, että pulloa pidet- tiinviljelykaapissakaksi tuntia sekäennenalkioiden pulloon siirtämistäettäsiirtämisenjälkeen.Pullo sul- jettiintiukasti ja viljely aikaviivenauhoitettiinikku-
nanpäälle asetetunpunaisen suodattimen läpi. Vil-
jelypulloasetettiinkäänteismikroskoopin alle lämpö- levylle, jonka lämpötila oli esikokeissa säädetty si- ten,että viljelypisaran lämpötilaoli 39°C.Kokeessa
tehtiin 10toistoa. Niissä verrattiin aikaviivenauhoi- tettujen jasamanaikaisesti viljelykaapissakasvanei-
den kontrollialkioiden solulukumääriä 24tunninvil- jelyn jälkeen.Tulokset analysoitiin T-testillä yhdis- tämällätoistojenaineistotneliöjuurimuunnoksen jäl- keen,Aikaviivenauhoitettujen jainkubaattorissakas- vaneiden alkioiden välillä ei olluteroa24 h viljelyn jälkeen (p=0.95), joten kehittämämmemenetelmä soveltuu ilmeisesti hyvin naudan alkioiden varhais- vaiheiden seurantaan.
