Voi 5 0 996): 535-540. Non-surgical transfer of 4’,6’-diamidino-2-phenylindole -stained equine embryos Mirja Huhtinen Agricultural Research Centre ofFinland, Equine Research, FIN-32100 Ypäjä, Finland, e-mail: mirja.huhtinen@mlt.fi Peter Bredbacka Agricultural Research Centre ofFinland, Institute ofAnimal Production, FIN-31600 Jokioinen, Finland 4’,6’-diamidino-2-phenylindole (DAPI) is a fluorescent dye that binds only to the DNA of dead blas- tomeres of an embryo. The use of DAPI for assessing viabilities of equine Day 6 to 7 embryos was studied. The pregnancy and normal development of the embryo until 20 days after transfer (day of pregnancy termination) was used as an indicator ofembryonic viability. Eleven embryos were stained with DAPI at room temperature for 15 min. They were then exposed to UV light to visualize staining, and cultured for 2 h before non-surgical transfer to recipient mares. Eight control embryos were cultured for 2 h before transfer to recipient mares. The recipient mares were scanned for pregnancies every other day starting 6 days after transfer. Twenty days after the transfer the pregnant recipient mares received luprostiol to induce abortion of the foetus. The pregnancy rate 6 days after transfer was 82% in the treatment group, and 75% in the control group (P>0.05). One DAPI-stained embryo resorbed and was no longer visible 12 days after transfer. However, this particular recipient mare had a uterine inflammation as shown by examination in the oestrus following embryo transfer. Although the number of embryos is insufficient to demonstrate lack of treatment effect, the high pregnancy rates following DAPI staining should encourage further studies on this subject. Key words: embryo quality, DAPI, vital staining ntroduction Morphological assessment of an embryo using a high-quality microscope is usually sufficient for deciding which embryos are worth transferring or freezing. There are situations, however, when the difference in quality cannot be distinguished by morphological criteria only. A quick and easy viability test would be desirable in practical sit- uations and also when new techniques in em- bryo technology, e.g. freezing, are developed. Metabolic tests measuring glucose, glutamine or oxygen consumption have been developed for © Agricultural and Food Science in Finland Manuscript received June 1996 535 AGRICULTURAL AND FOOD SCIENCE IN FINLAND https://www.c-info.fi/en/info/?token=NjN_0eut33-1y2Cb.8O2etdnMcuNxGxZZCzC_qQ.9H6eB-OuFSEVixA2lc0yuDaBAIPllMWmqrLKPq1pbkvQGMmwdPGzKMGsTfe_os_-40lSlQTu-JbSG7LAMmEvJqhRP6930umE8XaJWOJ4HtjBwR1ckHNzWS7bQdPCt0BRQUlMm506Im8l4LeDTUqdcutS6WblGKG5Z9dAVYJvyhszRdC04i8Utapa_OTFw2DJww_4mQsOmUq9Fi8CjFAhHZ4-DBUXRuV7K4v1m_cy0HxHalNNaFqDJgGEkGOFET3PzkcteVPLSnCBMXBK4PLRYYnBXmXSUQ Huhtinen, M. & Bredbacka, P. Non-surgical transfer ofDAPI-stained equine embryos horse and cattle embryos (Rieger et al. 1987, Rieger and Guay 1988, Briick and Hyland 1991, Overstrom et al. 1992). They seem to work un- der laboratory conditions, but are time consum- ing and require delicate analysis techniques. Less time and equipment are needed to evaluate em- bryo quality using vital stains. Several workers report using a vital stain to test the viability of an embryo. Examples ofnon-fluo- rescent dyes are neutral red (Kardymowicz 1972) and trypan blue (Hutzetal. 1985,Majumdar 1990). Some fluorescent dyes, e.g. fluorescein diacetate (FDA), stain live cells (Schilling and Doepke 1978, Schilling et al. 1979a, Mohr and Trounson 1980, Schilling etal. 1982,Pruitt etal. 1988);others, e.g. 4’,6’- diamidino-2-phenylindole (DAPI), dead cells of the embryo (Schilling et al. 1979b, Low et al. 1986). Rhodamine 123, a mitochondrion-specific fluorescent dye, may also be useful for assessing the metabolic integrity of mammalian embryos. This stain has, however, been shown to have cyto- toxic effects if exposed to UV light for more than 5 min (Petters and Lucy 1987). Only FDA (Pruitt et al. 1988, 1991) and DAPI (Huhtinen et al. 1995) have been used to assess the viability of equine embryos with flu- orescent stains. A high pregnancy rate was re- ported (Huhtinen et al. 1995) following transfer of whole equine embryos after treatment with cytochalasin B and nocodazole, and DAPI stain- ing. Transrectal ultrasonographies between Days 12 and 25 revealed that seven of eight whole embryos produced embryonic vesicles, but two of these exhibited growth retardation, and were expelled from the uterus as the recipient mares returned to oestrus. The retarded growth exhib- ited by two out of seven embryos in that group suggested possible detrimental effects due to the compounds used or exposure to UV light. As DAPI staining might be useful for diagnosing the viabilities of embryos after treatments such as freezing and thawing, the toxicity of that stain- ing alone would have to be examined. The aim ofour study was to establish whéther DAPI could be used for staining fresh Day 6 to 7 equine embryos before transfer, without compromising further development of the embryo. Material and methods The study was carried out in March and April 1994 with 35 Finnhorse and 23 Warmblood mares 3 to 18 years old (mean age 8.7 years). One to three cycles per mare were used in the experiment. Each mare served as donor or re- cipient or both. Two Finnhorse stallions of known fertility were used for inseminations. Oestrous cycles were synchronized by inject- ing 7.5 mg of luprostiol i.m. (Prosolvin, Intervet International 8.V., Boxmeer, Holland) in dioe- strus. Ovulations were confirmed by rectal pal- pation and daily transrectal ultrasonography. Freshly collected semen was diluted with Kenney’s skim milk/glucose extender. Insemi- nations were undertaken every other day before ovulation, using more than 500 million progres- sively motile spermatozoa per dose. The ovula- tions of recipients were induced, when neces- sary, by administration of 2500 IU hCG (Chor- io-Gonadon, Orion-Farmos, Turku, Finland) i.v. in oestrus. Embryos were recovered 6 days after detec- tion of ovulation by flushing the uterus three times with 1 L of PBS supplemented with BSA (2 g/L) and kanamycin (25 mg/L). Flushing me- dium recovered from the mare was passed through an embryo filter (Emcon - Immuno Sys- tems, Spring Valley, WI, USA), and the fluid in the filter cup was examined for the presence of embryos using a stereomicroscope. Embryos were washed and transferred at room tempera- ture to Hepes-buffered (25 mM) Ham’s FlO (Gibco BRL, Life Technologies, Paisley, Scot- land) supplemented with 10% foetal calf serum (Gibco BRL), 50 IU/ml of penicillin, and 50 (Ig/ ml of streptomycin. Embryos were classified according to McKinnon and Squires (1988). Di- ameters were measured using an eyepiece grati- cule. All the embryos were photographed on a black-and-white film and on a videotape before staining and/or culture. Nineteen randomly selected embryos were used in this experiment; other embryos were sub- mitted to another experiment. 536 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Vol. 5 (1996): 535-540. Table 1. Description of DAPI-stained embryos transferred in this study. number diameter, stage quality UV time, number of pregnancy |im grade sec stained cell 1 160 EB I 14 I 2 160 EB 1 12 2 +/- 3 170 EB 1 14 2 + 4 160 EB 2 12 3 + 5 200 B 2 10 1 6 160 EB 1 11 0 + 7 170 EB I II 2 + 8 170 EB 2 13 5 + 9 170 EB 2 12 3 + 10 220 B I 10 0 + 11 180 B I 13 0 + Stage: EB = early blastocyst, B = blastocyst. Quality grade: 1 = excellent, 2 =good. Pregnancy: + = pregnant, - = nonpregnant, +/- = early embryonic death. Treatment group: Embryos (n= 11) were placed in Hepes-buffered Ham’s FlO solution containing 1 (ig/ml DAPI (Sigma Chemical Co., St. Louis, MO), and kept at room temperature for 15 min. The staining was visualized using an inverted microscope (Olympus, IMT-2) with a dichroic mirror unit (IMT2-DMU: high pres- sure mercury burner, wavelength 334-365 nm, fluorescent radiation 420 nm and up) and a 20 x objective. Ultraviolet irradiation (10-14 s) of DAPI-stained embryos was recorded on a video- tape. Fluorescing cells were considered dead, and were counted from the videotape images. After staining, the embryos were washed three times and placed in bicarbonate-buffered Ham’s FlO (Flow Laboratories, Irvine, Scotland) supple- mented with 10% foetal calf serum (Gibco BRL), 2 mM ofL-glutamine (Gibco BRL), 50 IU/ml of penicillin, and 50 jig/ml of streptomycin. They were cultured in 5% CO, in air at + 37°C for 2 h before being transferred into uteri of recipient mares that had ovulated 0 to 2 days later than the donor. The embryos were loaded into 0.25-ml plastic straws and transferred via the cervix using a Cassou insemination instrument protected with an aseptic plastic shield. Control group: Embryos (n=B) were washed three times and placed in bicarbonate-buffered Ham’s FlO (Flow Laboratories) supplemented with 10% foetal calf serum (Gibco BRL), 2 mM ofL-glutamine (Gibco BRL), 50 IU/ml of peni- cillin, and 50 flg/ml of streptomycin. They were cultured in 5% C02 in air at + 37°C for 2 h be- fore being transferred nonsurgically into the uteri of recipient mares that had ovulated 0 to 2 days later than the donor. Uteri of the recipient mares were scanned every other day for embryonic vesicles starting 6 days after transfer. Twenty days after transfer the pregnant mares received 7.5 mg luprostiol (Prosolvin) i.m. to abort the foetuses. One-way analysis of variance was used for statistical analyses to compare sizes or morpho- logical grades of embryos, or times from em- bryo recovery to transfer between the two groups. % 2-test was used to compare pregnancy rates after transfer. Results The embryorecovery rate was 64% (35/55). Fif- ty-five flushes yielded 37 embryos (including two sets of twins). The embryos used in this ex- 537 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Huhtinen, M. & Bredbacka, P. Non-surgical transfer ofDAPI-stained equine embryos periment were randomly assigned to treatment and control groups. The mean diameter of em- bryos was 175 pm (range 160 to 220 pm) in the treatment groupand 213 pm (range 160 to 360 pm) in the control group. The diameters of embryos in the two groups were not significantly differ- ent (P>0.05). All embryos were classified as excellent or good. The quality grade of embryos did not have a significant effect on the survival of the embryo after transfer, nor was it statisti- cally different in the two groups (P>0.05). A more detailed description of DAPI-stained em- bryos is presented in Table 1. Ultraviolet exposure timewas 10-14 s (mean 12 s). The average number of dead cells was 1.7 (range 0-5). The number of dead cells did not have a significant effect on survival after trans- fer. The time from embryo recovery to transfer was 3.0 h (SD±O.4 h) in the treatment group and 2.7 h (SD±O.3 h) in the control group. This dif- ference was not statistically significant (P>0.05). Transrectal ultrasonography 6-20 days after embryo transfer revealed that 9of 11 DAPl- stained embryos produced embryonic vesicles and the heartbeat was observed in eight of them before the inductionof abortion. The embryo that did not develop further was no longer in the uter- us by the time of the fourth examination(12 days after transfer). A cytological smear from uterine swab taken from this particular recipient 19days after transfer revealed uterine inflammation. In the control group, six of eight embryos survived transfer and in all of them the heartbeat was ob- served before abortion induction 20 days after transfer. There was no difference in pregnancy rates 6 or 20 days after transfer between treat- ment and control groups (P>0.05). Discussion The embryorecovery rate for mares in this study was consistent with that previously reported (Squires et al. 1982, Woods et al. 1986). The pregnancy rates of DAPI-stained embryos and control embryos were also acceptable when com- pared with other reported nonsurgical embryo transfers (McKinnon et al. 1989, Pashen et al. 1993). Staining with DAPI could easily and unam- biguously be detected as cells fluoresced either brightly or not at all. Stain uptake of cells along the plane of bisection (Huhtinen et al. 1995) is further evidence for selective uptake of the dye. Moreover, cattle blastomeres lysed by means of bisection are stained with DAPI and propidium iodide in a similar fashion (Bredbacka 1995). Propidium iodide is widely used to selectively stain trophectoderm cells lysed with immuno- surgery. The embryonic resorption noted here was most likely caused by uterine inflammation of the recipient mare. In our previous study (Huhtinen et al. 1995), embryos were exposed to cytochalasin B and nocodazole in addition to DAPI staining. The reason for early embryonic deaths in that study may not have been caused by DAPI staining, but the combinedeffect of all treatments may have had adverse effects on the embryo. Since cytochalasin B and nocodazole have not been used extensively in horse embryo experiments, high sensitivity of horse embryos to these compounds cannot be excluded. On the other hand, the rate of embryonic deaths noted in this and our previous study does not signifi- cantly differ from the rate of early (2-4 weeks) embryonic losses in commercial brood mares reported by Woods et al. (1985). The embryos used here were of such good quality that by using morphological criteria or the number of DAPI-stained cells, we could not detect any difference between embryos that sur- vived transfer and those that did not. With the low number of DAPI-stained cells in our study, it is unlikely that a decreased survival rate would be observed with a small number of transfers unless the DAPI treatment itself were clearly toxic to the embryo. With this number of em- bryos and assuming that the pregnancy rate in the control group were constant, we should have obtained a pregnancy rate in the DAPI-stained group of 2/11 or less to be able to statistically 538 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Vol. 5 (1996): 535-540. demonstrate a negative effect of the treatment. The value of a DAPI test could be better demon- strated if embryos with a higher number of lysed cells were transferred. However, the DAPI treat- ment might then be harmful. The staining pro- cedure most likely facilitates the generation of reactive oxygen species with an adverse impact on non-lysed cells. Although the low number of embryos is the most common limiting factor in equine embryo research, and almost all embryos in private prac- tice are worth transferring regardless of their quality, situations may arise when a choice has to be made between the embryos to be trans- ferred. In the future, when equine in vitro em- bryo production technology is further refined and freezing of embryos becomes routine practice, such a choice might become more realistic. Staining with DAPI might reveal minor quality differences between embryos, since cell death may sometimes be indicative of degeneration of the embryo. Although the number of embryos available here was insufficient to demonstrate lack oftreat- ment effect, the high pregnancy rates following DAPI and UV treatment should encourage fur- ther studies on this subject. As the stained em- bryos in this study had only a few dead cells (five or fewer), testing the viability of DAPI-stained embryos with a higher number of dead cells would be important. References Bredbacka, P. 1995. Factors affecting cell viability dur- ing bisection of bovine embryos. Theriogenology44:159- 166. Bruck, I. & Hyland, J.H. 1991. Measurements of glu- cose metabolism in single equine embryos during early development. Journal of Reproduction and Fertilility, Sup- plement 44: 419-425. Huhtinen, M., Bredbacka, P. & Kotilainen,T. 1995, Mon- surgical transfer of DAPI-stained equine demi-embryos treated with cytochalasin B and nocodazole. Biology of Reproduction, Monograph Series No. 1: 325-328. Hutz, R.J., BeMayo, F.J. & Bukelow, W.R. 1985. The use of vital dyes to assess embryonic viability in the ham- ster mesocrietus auratus. Stain Technology 60:163-167. Kardymowicz, O. 1972. A method of vital staining for determining the viability of fertilized sheep ova stored in vitro. In; Proceedings of the 7th International Congress on Animal Reproduction and Artificial Insemination, Munich, I, Volume 1. p. 503-506. Low, 8.G., Takahashi, Y. & Kanagawa, H. 1986. Viabil- ity of frozen-thawed mouse blastocysts with different numbers of dead cells. Theriogenology25: 675-680. Majumdar, A.C. 1990.Embryo transfer in rabbit: assess- ment of viability of embryo using dye technique. Journal of Veterinary Physiology and Allied Science 9: 15-20. McKinnon, A.0., Carnevale, E.M., Squires, E.L., Car- ney, N.J. & Seidel, G.E., Jr. 1989. Bisection of equine embryos. Equine Veterinary Journal, Supplements: 129- 133. - & Squires, E.L. 1988. Morphologic assessment of the equine embryo. Journal of American Veterinary Medicine Association 192: 401-406. Mohr, L.R. & Trounson, A.O. 1980. The use of fluores cein diacetate to assess embryo viability in the mouse. Journal of Reproduction and Fertility 58: 189-196. Överström, E.W., Duby, R.T., Bobrinsky, J. & Roche, J.F. 1992. Viability and oxidative metabolism of the bo- vine blastocyst. Theriogenology 37: 269, (Abstract). Pashen, R.L., Lascombes, F.A. & Barrow, M.B. 1993. The application of embryo transfer to polo ponies in Ar- gentina. Equine Veterinary Journal, Supplement 15:119- 121. Petters, R.M. & Lucy, M.C. 1987. Rhodamine 123 as a cytoplasmic stain for mammalian zygotes. Theriogenol- ogy 28: 639-646. Pruitt, J.A.,Forrest, 8.W., Burghardt,R.C., Evans, J.W. & Kraemer, B.C. 1991. Viability and ultrastructure of equine embryos following culture in a static or dynamic system. Journal of Reproduction and Fertility, Supple- ment 44: 405-410. -, Wilson, J.M. & Kraemer, B.C. 1 988. Viability of equine embryos following fluorescein diacetate staining. Therio- genology 29: 291. (Abstract). Rieger, B. & Guay, P. 1988. Measurement of the metab- olism of energy substrates in individual bovine blasto- cysts. Journal of Reproduction and Fertility 83: 585-591. Lagneaux-Petit, B. & Palmer, E. 1987. Preliminary investigations of the metabolic activity of early horse embryos. Journal of Reproduction and Fertilility, Supple- ment 35: 699-700. Schilling, E. von & Boepke, H.-H. 1978. A rapid diag- nostic test for the viability of early cattle and rabbit em- bryos using Diacetyl-fluorescein. Naturwissenschaften 65: 658. -, Smidt, 8., Sach, B. & El Kaschab, S. 1979a. Diagno- sis of the viability of early bovine embryos by fluores- 539 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Huhtinen, M. & Bredbacka, P. Non-surgical transfer of DAPI-stained equine embryos cence microscopy. Annales de Biologie Animale, Bioche- mie, Biophysique 19; 1625-1629. -, Niemann, H., Cheng, S.P. & Doepke, H.-H. 1979b. DARI - a further fluorescence test for diagnosing the vi- ability of early cow and rabbit embryos. Zuchthygiene 14: 170-172. Niemann, H. & Smidt, D. 1982. Evaluation of fresh and frozen cattle embryos by fluorescence microscopy. In: Hafez, E.S.E. & Semm, K. (eds.). In Vitro Fertilization and Transfer, MTP Press, Falcon House, Lancaster, Eng- land. p. 349-355. Squires, E.L., Imel, K.J., luliano, M.F. & Shideler, R.K. 1982. Factors affecting reproductive efficiency in an equine transfer programme. Journal of Reproduction and Fertility, Supplement 32: 409-414. Woods, G.L., Baker, C.8., Hillman, R.B. & Schlafer, D.H. 1985. Recent studies relating to early embryonic death in the mare. Equine Veterinary Journal, Supple- ment 3: 104-107. Hillman, R.B. & Schlafer, D.H. 1986. Recovery and evaluation of embryos from normal and infertile mares, Cornell Veterinarian 76: 386-394. SELOSTUS Hevosen alkioiden värjääminen DAPhlla ennen vastaanottajaan siirtoa Mirja Huhtinen ja Peter Bredbacka Maatalouden tutkimuskeskus Kokeessa tutkittiin 6-7 päivän ikäisten hevosen al- kioiden elävyyttä 4’,6’-diamidino-2-phenylindole (DAPI):n avulla. DAPI on fluoresoiva väriaine, joka sitoutuu DNA;han ja värjää alkioista kuolleet solut. Yksitoista alkioita värjättiin 15 minuutin ajan DAPIdla huoneenlämmössä. Tämän jälkeen värjäys katsottiin UV-valon avulla, minkä jälkeen alkioita pidettiin lämpökaapissa (+37°C, 5% C0 2 ) kaksi tun- tia ennen ei-kirurgista siirtoa vastaanottajatammoi- hin. Kahdeksan kontrollialkiota käsiteltiin samoin, mutta niitä ei värjätty. Vastaanottajatammoille tehtiin tiineystarkastus ultraäänen avulla kuusi päivää alkion siirron jälkeen, ja siitä lähtien joka toinen päivä tiineyden keskeyttämiseen asti. Kun sikiön sydämen syke havaittiin, vastaanottajatammoille annettiin prostaglandiinia abortin aikaan saamiseksi. Vastaan- ottajista joihin siirrettiin DAPI-värjätty alkio tiineh- tyi 82 %. Vastaanottajista joihin siirrettiin värjäämä- tön alkio tiinehtyi 75 %. Yksi DAPI-värjätyistä alki- oista kuoli vastaanottajatamman kohdussa, eikä al- kiota voitu enää havaita 12 päivää siirron jälkeen. Kyseisellä tammalla todettiin seuraavan kiiman aika- na tehdyssä tutkimuksessa kohtutulehdus. Vaikka al- kioiden lukumäärä kokeessa oli liian pieni, jotta se todistaisi värjäyksen haitattomaksi, koeryhmän kor- kea tiinehtymisprosentti rohkaisee lisätutkimuksiin. 540 AGRICULTURAL AND FOOD SCIENCE IN FINLAND