- Email: [email protected]
In vitro culture, freezing, thawing, and transfer of bovine embryos versus transfer of fresh embryos from the same collection: Preliminary results
THERIOGENOLOGY
IN
VITROCIJLlURE,PPEEZIIG,
WI8G,ANDTRANSFl3R OPBOVIIH BWRYOS OF FPESH l3HBRYOSWapI llllE SAHg COLLHCTION: PmLIMImmYRBsuLTS
VgaSDS WSPEB
W.A. Garcia,l H.L. Fahning,l,2 and E.P. Graha3 1CryovaTech International Inc. 3, Box 322, iluy35 S. Budson, Wisconsin
bute
54016
2Deparbrent of Large Animal Clinical Sciences College of Veterinary Medicine, University of Minnesota St. Paul, Minnesota 55108 3Department of Animal Science, University of Minnesota St. Paul, Kinnesota 55108
Received
for
publication: Accepted:
January October
11, 15,
1985 1986
ABSTRACT The collection of ten good embryos from a donor cow is reported Five of the embryos were transferred surgically to recipients here. immediately after collection, resulting in three pregnancies (60%). The remaining five morula embryos developed to blastocysts after being then frozen and stored in liquid placed in culture for 30 h and were only four embryos were judged to be nitrogen for 1 wk. After thawing, of the quality to transfer and resulted in two pregnancies (50%). The case illustrates that embryos can be cultured in vitro for up to 30 h prior to freezing and still result in pregnancies. Key words:
bovine,
culture,
freezing,
embryo
transfer
INlILODUCfION
Several reports concerning the effects of embryo storage at various temperatures above freezing prior to transfer have been published. The transfer of freshly collected bovine embryos has been widely practiced since the early 1970’s. The use of frozen embryos has also increased in recent year 8. Bovine embryos are commonly cultured in vitro under controlled laboratory conditions (pH, osmotic pressure, humidity, gas pressure) as a research tool to evaluate their viability after different experimental treatments. Schenk (1) mammalian embryos, yr
(2).
Brinster
DECEMBER
conducted but little (3)
was
1986 VOL.
the first progress the
first
26 NO. 6
studies on the cultivation was made until the last 10 to
report
in
vitro
culture
to
of 15 and
803
I-HERIOGENOLOGY
An extensive review of the factors influencing hatching of mouse ova. (2). culture and development of mammalian embryos was made by Brinster Today, bovine embryos are cultured in vitro to hatching under standard However, transfer of in vitro-cultured bovine embryos conditions (4-6). Whittingham (7,8) to synchronized recipients has had limited success. reported that except for the mouse, the exposure of most mammalian embryos to prolonged periods in culture during preimplantation development appeared to be detrimental to further development after Tervit et al. (9) indicated that cattle embryos develop trausf er. during short periods in culture but, as the period was extended, therr Elsden (10) stated that after 24 h of in viability decreased rapidly. the viability of bovine embryos &creased rapidly and vitro culture, immediate freezing was recasmended in cases when a period this long was required prior to transfer. Trounson et al. (11) recovered 26 bovine embryos on Days 5, 6, and 7 and cultured them for 48 h to the blastocyst stage in Dulbecco’s Eight of 13 modified (PBS) with fetal calf serum prior to transfer. recipients were carrying 13 normal fetuses at slaughter 3 to 18 wk after These researchers indicated that unlike earlier cleavage transfer. cow blastocysts tolerated cooling to OoC and retained viability stages, after storage for 48 h (12). Peters et al. (13) reported no significant differences between in vitro-cultured and noncultured embryos after transfer to recipients and reported 59.4% and 74.3% pregnancy rates, respectively. These researchers cultured embryos for 24, 48, and 72 h and after nonsurgical transfer obtained if 5, 113, and O/4 pregnant recipients, respectively. However, Beyman (14) obtained signif icant differences (P < 0.05) in 90-d frozen, or cultured embryos (from 24 to 48 pregnancy rates when fresh, h) were transferred to recipients. lo statistical differences were obtained between pregnancies with fresh or frozen embryos (55.4 and of cultured embryosyielded a 46.8x;, respectively); however, the transfer significantly lower pregnancy rate (33.3%) at 90 d. The low temperature preservation of bovine embryos is a relatively new technique, and much research is needed to improve the embryo postthaw survival. Lehn-Jensen (15) reported that the rate and temperature at which the cryoprotectant is added to the medium and also the length of equilbration are not critical for the survival of bovine This report agrees with those of Schneider and Mazur (16) and embryos. The latter authors demonstrated that bovine Chupin and Procureur (17). blastocysts can be equilibrated in 1.4 M glycerol in only one step of 10 to 30 min. However, slow removal of the cryoprotectant from the embryo after thawing is important. Direct transfers of nonfrozen embryos in glycerolated PBS (15) as well as of frozen embryos without glycerol removal have given poor results (18). In most cases, the concentration of cryoprotectant in the medium is increased at room temperature in 0.5 M steps every 10 min until the final concentration is attained. The remwal of cryoprotectant after thawing is accomplished by gradually reducing its concentration, generally in 0.25 M steps until the embryos are back in pure PBS (16,191.
804
DECEMBER 1986 VOL. 26 NO. 6
THERIOGENOLOGY
The freezing of cultured bovine embryos and their subsequent transfer to recipients has not been previously reported. This report demonstrates that Day 7 bovine embryos can be cultured in vitro for an extended period prior to freezing and can result in a satisfactory pregnancy rate after transfer. MATERIALS MD
HEmODS
An adult Holstein ccw was superovulated with follicle stimulating hormone (FSH-P, Burns Biotech, Omaha, NE) according to a treatment schedule previously described (15,201. The nonsurgical collection procedure used to recover the embryos has been previously described (20). The recovered collection medium was examined under a stereoscope to locate and evaluate recovered embryos. Embryos were evaluated at a magnification of 70x. Those judged to be of good quality according to the criteria of Linder and Wright (21) were transferred to Dulbecco’s PBS (22) with 15% heat-inactivated fetal calf serum (HIFCS) and held in a 370C incubator until transfer or freezing. Five nonfrozen embryos were surgically transferred into five Holstein heifers within 3 h after collection. The recipients had been The recipients were placed under in estrus within +- 24 h of the donor. general anesthesia using sodium thiamithal to induce and fluethane to A mid-ventral incision just anterior to the udder maintain anesthesia. was used to expose the uterus. A single embryo was transferred to the uterine horn ipsilateral to the ovary containing the corpus luteum. Emryos that were to be frozen were incubated at 370C in modified was delayed because the Dulbecco’s PBS with 15% HIFCS. (The freezing After the embryos had been freezing equipment was being serviced.) incubated for 30 h, they were prepared for freezing by transferring them through a series of solutions of modified PBS with increasing molarities The final glycerol concentration was of glycerol as the cryoprotectant. 1.5 M and was added in six steps (0.25, 0.5, 0.75, 1.0, 1.25, and 1.5 M) at lo-min intervals. Individual embryos were placed in 0.5-ml French straws and frozen at a rate of loC/min from room temperature to -60C. They were then ice-nucleated and held at that temperature for 10 min. Next, they were cooled at 0.3oClmin to -350C and at O.loC/min to -380C before being plunged into liquid nitrogen. Following storage in were thawed by placing the the straw were placed in a The glycerol evaluated.
DECEMBER
liquid nitrogen for 1 wk, the frozen embryos straw in a 220C water bath. The contents of petri dish and the embryos were located and was removed by passing the embryos through
1986 VOL. 26 NO. 6
805
I’HERIOGENOLOGY
seven PBS solutions of decreasing glycerol concentrations (in reverse The seventh solution order of its addition) at IO-min intervals. Five embryos were contained PBS with 15% BTFCS and no cryoprotectant. incubated, frozen, and thawed, but only four were judgel to be of sufficiently high quality for transfer. Pregnancy examination was made on all recipients 35 to 40 d after transfer and confirmation of pregnancy was done at 90 d of gestation.
RESULTS
The five embryos transferred immediately after collection 1) were evaluated as being of very good quality and resulted pregnancies (60%) which were confirmed at 90 d of gestation.
(Figure in three
The cultured embryos (Figures 2 and 3) devefoped from the morula to These were cultured for 30 h prior to freezing. the blastocyst stage. After storage in liquid nitrogen, thawing, and deglycerolization, the embryos were again evaluated. One embryo was judged to be of poor quality and was not transferred. The four ramaining embryos (Figure 4) and transferred to four recipients. were deglycerolated, photographed, Two of the four frozen-thawed embryos transferred resulted in confirmed 90-d pregnancies (50%).
DISCUSSIOR
Earlier reports indicate that the pregnancy rates obtained after transfer of cultured bovine embryos are lower than those rates obtained after transfer of freshly collected bovine embryos. Wright (23) reported pregnancy rates of less than 10% after keeping the embryos for 14 h from the time of collection to onset of freezing. The pregnancy rates of embryos freshly callected and immediately transferred surgically has reached 60 to 65% (20). With nonsurgical techniques, the pregnancy rates obtained are very similar (Fahning and Garcia, unpublished data). The techniques for freezing embryos as well as those for handling frozen-thawed embryos have been improved in the last years; however, additional work is needed to improve embryo survival rates of postthawed embryos and their pregnancy rates. The latter range from 30 to 56% (24-26). Embryos should be frozen as soon as possible after collection to achieve higher pregnancy rates. These results illustrate that it is possible to culture bovine embryos prior to freezing and achieve an acceptable pregnancy rate. Tbe sample size was very small and at this point we do not suggest that embryos routinely be cultured prior to freezing. However, cul tur ing may be desirable or necessary if freezing equipment is not available at the time or place of embryo collection. Culturing also maybe desirable for embryos of questionable quality to evaluate their progressive development to assess which embryos are to be frozen.
DECEMBER
1986 VOL. 26 NO. 6
THERIOGENOLOGY
A
Figure 1,
Nonfrozen embryos transferred to recipients within 3 h after collection.
DECEMBER 1986 VOL. 26 NO. 6
807
I’HERIOGENOLOGY
Figure
808
2.
Embryos cultured in vitro for 30 h in Dulbecco's phosphate buffered saline with 15% heat-inactivated fetal calf serum at 37OC. prior to freezing. Embryos A and B resulted in pregnancies. Embryo E was not transferred to a recipient after thawing.
DECEMBER
1986 VOL. 26 NO. 6
THERIOGENOLOGY
Figure
3.
DECEMBER
Embryos cultured in vitro for 30 h in Dulbecco’s phosphate buffered saline with 15% heat-inactivated fetal calf serum at 37oC, prior to freezing. Embryos C and D did not result in pregnancies after their transfer to recipients.
1986 VOL.
26 NO. 6
809
THERIOGENOLOGY
Figure 4.
810
Post-thaw embryos that were cultured in vitro for 30 h prior to freezing. At 90 d after transfer recipients of EanbryosA. B, C. and D were pregnant. pregnant, nonpregnant, and nonpregnant, respectively.
DECEMBER 1986 VOL. 26 NO. 6
THERIOGENOLOGY
REFERENCES
1.
Das Saugethieri Kunstlick befruchtet ausserhalb des Schenk.S.L. Mutter-tieres. In: Rothblat. G.H. and Cristofalo. V.J. (eds.). New York and London Academic Metabolism. Growth, Nutritionand Press, 1972, pp. 251-286.
2.
Brinster. R.L. Cultivation of the mammalian embryo. In: Rothblatt. G.H. and Cristofalo. V.J. (eds.). Growth. Nutrition zd Metabolism. New York and London Academic Press, 1972. pp. 251-286.
3.
Brinster, R.L. A method for in vitro cultivation of mouse ova from two-cell to blatocyst. Exp. Cell. Res. z:205-208 (1963).
4.
Wright, R.W.. Anderson. G.B.. Cupps. P.T. and Drost. M. Blasto-cyst J. Anim. expansion and hatching of bwine ova cultured in vitro. Sci. 42:170-174 (1976).
5.
Wright. R.W.. Anderson, G.B. and Drost. M. Culture of bovine wa in various media. Proc. 8th Ann. Meeting, Sot. Study of Reprod.. Fort Collins. co. p.73 (1975).
6.
Thibault. C. Le culture in vitro de I'oeuf de vache. Anim. Biochim. Biophys. -lo:141 abstr. (1970).
7.
Whittingham. D.G. Fertilization. early development and storage of mammalian wa in vitro. In: Balls, M. and Wild, A.E. (eds.). The Cambridge Univ. Press. 1975. pp. Early Development of Mammals. l-24.
a.
Whittingham, D.G. General aspects of egg culture and preservation. In: Rowson. L.E.A. ted.). Egg Transfer in Cattle. Commission of fT;eEuropean Committies. Luxembourg, Europe. 1976, pp. 101-113.
9.
Tervit. H.R.. Whittingham. D.G. and Rowson. L.E.A. Successful culture in vitro of sheep and cattle ova. J. Reprod. Fertil. 30:493-497 (1972). -
10.
Elsden. R.P. Conventional techniques of bovine embryo transfer. Paper presented at February 28-29. USDA Vet. Serv. Embryo Transfer Seminar, Washington. D.C., 1984. pp. l-9.
11.
Trounson. A.O.. Willadsen. S.M. and Rowson. L.E.A. The influence of in vitro culture and cooling on the survival and development of 47:367-370 (1976). cow embryos. J. Reprod. Fertil. -
Annal. Biol.
12. Trounson. A.O., Willadsen. S.M.. Rowson. L.E.A. and Newcomb. R. The storane of cow eaes at room temperature and at low -temperatures. J. Reprod,-Fertil. c:173-178 (1976). 13.
Peters, D.F., Anderson, G.B.. BonDurant. R.. Cupps. P.T. and Drost, M. Transfer of cultured bovine embryos. Theriogenology -10:337-342 (1978).
DECEMBER
1986 VOL. 26 NO. 6
811
THERIOGENOLOGY
14. Heyman. Y. Embryonic loss in cattle after transfer of fresh. frozen or cultured embryos. 10th Internat. Congress on Anim. Reprod. and Artif. Insem.. Urbana-Champaign. IL.1984. p. 228. H. Bovine egg transplantation. 15. Lehn-Jensen. embryos. Nord. Vet. Med. 3: 523-532 (1980). 16.
Preservation
of
Schneider, J. and Mazur. P. Osmotic consequences of cryoprotectant permeability and its relation to the survival of frozen thawed embryos. Theriogenology z:68-71 (1984).
17. Chupin. D. and Procureur. R. Glycerol equilibration for deep freezing of cattle blastocysts: effect of number of steps and of total duration. Theriogenology -21:230 abstr. (1984). 18. Willadsen, S.M.. Polge. C. and Rowson. L.E.A. The viability of deep frozen cow embryos. J. Reprod. Fertil. -52:391-393 (1978). 19. Lehn-Jensen. H., Grave. T. and Perez, A. Two step freezing of cow embryos in 1.4 M glycerol. Theriogenology -15:427-432 (1981). 20.
Fahning. M.L. Ova transfer in cattle. Proc. 77th Ann. Meeting North Dakota Vet. Med. Assoc., 1982. pp. 1-15.
21.
G. and Wright, R.W. Lindner. Bovine embryo morphology evaluation. Theriogenology %:407-416 (1982).
and
22. Whittingham. D.G. and Wales, R.G. Survival of mouse embryo after (1971). freezing and thawing. Nature (Land.) =:125-126 23. Wright, J.M. Commercial freezing of bovine Theriogenology 2~17-30 (1985). 24.
embryos in straws.
Leibo. S.P. Commercial production of pregnancies from one-step diluted frozen-thawed bovine embryos. Theriogenology -25:166 abstr. (1986).
25. Lehn-Jensen. H. and Rail. W.F. Cryomicroscopic observations of cattle embryos during freezing and thawing. Theriogenology 2:263-277 (1983). 26. Rail. W.F. Embryo cryopreservation. Current application. Prospects for the future. Proc. Ann. Conf. Artifical Insemination and Embryo Transfer in Beef Cattle. Denver, CO. 1986. pp. 48-52.
812
DECEMBER
1986 VOL. 26 NO. 6
IN
VITROCIJLlURE,PPEEZIIG,
WI8G,ANDTRANSFl3R OPBOVIIH BWRYOS OF FPESH l3HBRYOSWapI llllE SAHg COLLHCTION: PmLIMImmYRBsuLTS
VgaSDS WSPEB
W.A. Garcia,l H.L. Fahning,l,2 and E.P. Graha3 1CryovaTech International Inc. 3, Box 322, iluy35 S. Budson, Wisconsin
bute
54016
2Deparbrent of Large Animal Clinical Sciences College of Veterinary Medicine, University of Minnesota St. Paul, Minnesota 55108 3Department of Animal Science, University of Minnesota St. Paul, Kinnesota 55108
Received
for
publication: Accepted:
January October
11, 15,
1985 1986
ABSTRACT The collection of ten good embryos from a donor cow is reported Five of the embryos were transferred surgically to recipients here. immediately after collection, resulting in three pregnancies (60%). The remaining five morula embryos developed to blastocysts after being then frozen and stored in liquid placed in culture for 30 h and were only four embryos were judged to be nitrogen for 1 wk. After thawing, of the quality to transfer and resulted in two pregnancies (50%). The case illustrates that embryos can be cultured in vitro for up to 30 h prior to freezing and still result in pregnancies. Key words:
bovine,
culture,
freezing,
embryo
transfer
INlILODUCfION
Several reports concerning the effects of embryo storage at various temperatures above freezing prior to transfer have been published. The transfer of freshly collected bovine embryos has been widely practiced since the early 1970’s. The use of frozen embryos has also increased in recent year 8. Bovine embryos are commonly cultured in vitro under controlled laboratory conditions (pH, osmotic pressure, humidity, gas pressure) as a research tool to evaluate their viability after different experimental treatments. Schenk (1) mammalian embryos, yr
(2).
Brinster
DECEMBER
conducted but little (3)
was
1986 VOL.
the first progress the
first
26 NO. 6
studies on the cultivation was made until the last 10 to
report
in
vitro
culture
to
of 15 and
803
I-HERIOGENOLOGY
An extensive review of the factors influencing hatching of mouse ova. (2). culture and development of mammalian embryos was made by Brinster Today, bovine embryos are cultured in vitro to hatching under standard However, transfer of in vitro-cultured bovine embryos conditions (4-6). Whittingham (7,8) to synchronized recipients has had limited success. reported that except for the mouse, the exposure of most mammalian embryos to prolonged periods in culture during preimplantation development appeared to be detrimental to further development after Tervit et al. (9) indicated that cattle embryos develop trausf er. during short periods in culture but, as the period was extended, therr Elsden (10) stated that after 24 h of in viability decreased rapidly. the viability of bovine embryos &creased rapidly and vitro culture, immediate freezing was recasmended in cases when a period this long was required prior to transfer. Trounson et al. (11) recovered 26 bovine embryos on Days 5, 6, and 7 and cultured them for 48 h to the blastocyst stage in Dulbecco’s Eight of 13 modified (PBS) with fetal calf serum prior to transfer. recipients were carrying 13 normal fetuses at slaughter 3 to 18 wk after These researchers indicated that unlike earlier cleavage transfer. cow blastocysts tolerated cooling to OoC and retained viability stages, after storage for 48 h (12). Peters et al. (13) reported no significant differences between in vitro-cultured and noncultured embryos after transfer to recipients and reported 59.4% and 74.3% pregnancy rates, respectively. These researchers cultured embryos for 24, 48, and 72 h and after nonsurgical transfer obtained if 5, 113, and O/4 pregnant recipients, respectively. However, Beyman (14) obtained signif icant differences (P < 0.05) in 90-d frozen, or cultured embryos (from 24 to 48 pregnancy rates when fresh, h) were transferred to recipients. lo statistical differences were obtained between pregnancies with fresh or frozen embryos (55.4 and of cultured embryosyielded a 46.8x;, respectively); however, the transfer significantly lower pregnancy rate (33.3%) at 90 d. The low temperature preservation of bovine embryos is a relatively new technique, and much research is needed to improve the embryo postthaw survival. Lehn-Jensen (15) reported that the rate and temperature at which the cryoprotectant is added to the medium and also the length of equilbration are not critical for the survival of bovine This report agrees with those of Schneider and Mazur (16) and embryos. The latter authors demonstrated that bovine Chupin and Procureur (17). blastocysts can be equilibrated in 1.4 M glycerol in only one step of 10 to 30 min. However, slow removal of the cryoprotectant from the embryo after thawing is important. Direct transfers of nonfrozen embryos in glycerolated PBS (15) as well as of frozen embryos without glycerol removal have given poor results (18). In most cases, the concentration of cryoprotectant in the medium is increased at room temperature in 0.5 M steps every 10 min until the final concentration is attained. The remwal of cryoprotectant after thawing is accomplished by gradually reducing its concentration, generally in 0.25 M steps until the embryos are back in pure PBS (16,191.
804
DECEMBER 1986 VOL. 26 NO. 6
THERIOGENOLOGY
The freezing of cultured bovine embryos and their subsequent transfer to recipients has not been previously reported. This report demonstrates that Day 7 bovine embryos can be cultured in vitro for an extended period prior to freezing and can result in a satisfactory pregnancy rate after transfer. MATERIALS MD
HEmODS
An adult Holstein ccw was superovulated with follicle stimulating hormone (FSH-P, Burns Biotech, Omaha, NE) according to a treatment schedule previously described (15,201. The nonsurgical collection procedure used to recover the embryos has been previously described (20). The recovered collection medium was examined under a stereoscope to locate and evaluate recovered embryos. Embryos were evaluated at a magnification of 70x. Those judged to be of good quality according to the criteria of Linder and Wright (21) were transferred to Dulbecco’s PBS (22) with 15% heat-inactivated fetal calf serum (HIFCS) and held in a 370C incubator until transfer or freezing. Five nonfrozen embryos were surgically transferred into five Holstein heifers within 3 h after collection. The recipients had been The recipients were placed under in estrus within +- 24 h of the donor. general anesthesia using sodium thiamithal to induce and fluethane to A mid-ventral incision just anterior to the udder maintain anesthesia. was used to expose the uterus. A single embryo was transferred to the uterine horn ipsilateral to the ovary containing the corpus luteum. Emryos that were to be frozen were incubated at 370C in modified was delayed because the Dulbecco’s PBS with 15% HIFCS. (The freezing After the embryos had been freezing equipment was being serviced.) incubated for 30 h, they were prepared for freezing by transferring them through a series of solutions of modified PBS with increasing molarities The final glycerol concentration was of glycerol as the cryoprotectant. 1.5 M and was added in six steps (0.25, 0.5, 0.75, 1.0, 1.25, and 1.5 M) at lo-min intervals. Individual embryos were placed in 0.5-ml French straws and frozen at a rate of loC/min from room temperature to -60C. They were then ice-nucleated and held at that temperature for 10 min. Next, they were cooled at 0.3oClmin to -350C and at O.loC/min to -380C before being plunged into liquid nitrogen. Following storage in were thawed by placing the the straw were placed in a The glycerol evaluated.
DECEMBER
liquid nitrogen for 1 wk, the frozen embryos straw in a 220C water bath. The contents of petri dish and the embryos were located and was removed by passing the embryos through
1986 VOL. 26 NO. 6
805
I’HERIOGENOLOGY
seven PBS solutions of decreasing glycerol concentrations (in reverse The seventh solution order of its addition) at IO-min intervals. Five embryos were contained PBS with 15% BTFCS and no cryoprotectant. incubated, frozen, and thawed, but only four were judgel to be of sufficiently high quality for transfer. Pregnancy examination was made on all recipients 35 to 40 d after transfer and confirmation of pregnancy was done at 90 d of gestation.
RESULTS
The five embryos transferred immediately after collection 1) were evaluated as being of very good quality and resulted pregnancies (60%) which were confirmed at 90 d of gestation.
(Figure in three
The cultured embryos (Figures 2 and 3) devefoped from the morula to These were cultured for 30 h prior to freezing. the blastocyst stage. After storage in liquid nitrogen, thawing, and deglycerolization, the embryos were again evaluated. One embryo was judged to be of poor quality and was not transferred. The four ramaining embryos (Figure 4) and transferred to four recipients. were deglycerolated, photographed, Two of the four frozen-thawed embryos transferred resulted in confirmed 90-d pregnancies (50%).
DISCUSSIOR
Earlier reports indicate that the pregnancy rates obtained after transfer of cultured bovine embryos are lower than those rates obtained after transfer of freshly collected bovine embryos. Wright (23) reported pregnancy rates of less than 10% after keeping the embryos for 14 h from the time of collection to onset of freezing. The pregnancy rates of embryos freshly callected and immediately transferred surgically has reached 60 to 65% (20). With nonsurgical techniques, the pregnancy rates obtained are very similar (Fahning and Garcia, unpublished data). The techniques for freezing embryos as well as those for handling frozen-thawed embryos have been improved in the last years; however, additional work is needed to improve embryo survival rates of postthawed embryos and their pregnancy rates. The latter range from 30 to 56% (24-26). Embryos should be frozen as soon as possible after collection to achieve higher pregnancy rates. These results illustrate that it is possible to culture bovine embryos prior to freezing and achieve an acceptable pregnancy rate. Tbe sample size was very small and at this point we do not suggest that embryos routinely be cultured prior to freezing. However, cul tur ing may be desirable or necessary if freezing equipment is not available at the time or place of embryo collection. Culturing also maybe desirable for embryos of questionable quality to evaluate their progressive development to assess which embryos are to be frozen.
DECEMBER
1986 VOL. 26 NO. 6
THERIOGENOLOGY
A
Figure 1,
Nonfrozen embryos transferred to recipients within 3 h after collection.
DECEMBER 1986 VOL. 26 NO. 6
807
I’HERIOGENOLOGY
Figure
808
2.
Embryos cultured in vitro for 30 h in Dulbecco's phosphate buffered saline with 15% heat-inactivated fetal calf serum at 37OC. prior to freezing. Embryos A and B resulted in pregnancies. Embryo E was not transferred to a recipient after thawing.
DECEMBER
1986 VOL. 26 NO. 6
THERIOGENOLOGY
Figure
3.
DECEMBER
Embryos cultured in vitro for 30 h in Dulbecco’s phosphate buffered saline with 15% heat-inactivated fetal calf serum at 37oC, prior to freezing. Embryos C and D did not result in pregnancies after their transfer to recipients.
1986 VOL.
26 NO. 6
809
THERIOGENOLOGY
Figure 4.
810
Post-thaw embryos that were cultured in vitro for 30 h prior to freezing. At 90 d after transfer recipients of EanbryosA. B, C. and D were pregnant. pregnant, nonpregnant, and nonpregnant, respectively.
DECEMBER 1986 VOL. 26 NO. 6
THERIOGENOLOGY
REFERENCES
1.
Das Saugethieri Kunstlick befruchtet ausserhalb des Schenk.S.L. Mutter-tieres. In: Rothblat. G.H. and Cristofalo. V.J. (eds.). New York and London Academic Metabolism. Growth, Nutritionand Press, 1972, pp. 251-286.
2.
Brinster. R.L. Cultivation of the mammalian embryo. In: Rothblatt. G.H. and Cristofalo. V.J. (eds.). Growth. Nutrition zd Metabolism. New York and London Academic Press, 1972. pp. 251-286.
3.
Brinster, R.L. A method for in vitro cultivation of mouse ova from two-cell to blatocyst. Exp. Cell. Res. z:205-208 (1963).
4.
Wright, R.W.. Anderson. G.B.. Cupps. P.T. and Drost. M. Blasto-cyst J. Anim. expansion and hatching of bwine ova cultured in vitro. Sci. 42:170-174 (1976).
5.
Wright. R.W.. Anderson, G.B. and Drost. M. Culture of bovine wa in various media. Proc. 8th Ann. Meeting, Sot. Study of Reprod.. Fort Collins. co. p.73 (1975).
6.
Thibault. C. Le culture in vitro de I'oeuf de vache. Anim. Biochim. Biophys. -lo:141 abstr. (1970).
7.
Whittingham. D.G. Fertilization. early development and storage of mammalian wa in vitro. In: Balls, M. and Wild, A.E. (eds.). The Cambridge Univ. Press. 1975. pp. Early Development of Mammals. l-24.
a.
Whittingham, D.G. General aspects of egg culture and preservation. In: Rowson. L.E.A. ted.). Egg Transfer in Cattle. Commission of fT;eEuropean Committies. Luxembourg, Europe. 1976, pp. 101-113.
9.
Tervit. H.R.. Whittingham. D.G. and Rowson. L.E.A. Successful culture in vitro of sheep and cattle ova. J. Reprod. Fertil. 30:493-497 (1972). -
10.
Elsden. R.P. Conventional techniques of bovine embryo transfer. Paper presented at February 28-29. USDA Vet. Serv. Embryo Transfer Seminar, Washington. D.C., 1984. pp. l-9.
11.
Trounson. A.O.. Willadsen. S.M. and Rowson. L.E.A. The influence of in vitro culture and cooling on the survival and development of 47:367-370 (1976). cow embryos. J. Reprod. Fertil. -
Annal. Biol.
12. Trounson. A.O., Willadsen. S.M.. Rowson. L.E.A. and Newcomb. R. The storane of cow eaes at room temperature and at low -temperatures. J. Reprod,-Fertil. c:173-178 (1976). 13.
Peters, D.F., Anderson, G.B.. BonDurant. R.. Cupps. P.T. and Drost, M. Transfer of cultured bovine embryos. Theriogenology -10:337-342 (1978).
DECEMBER
1986 VOL. 26 NO. 6
811
THERIOGENOLOGY
14. Heyman. Y. Embryonic loss in cattle after transfer of fresh. frozen or cultured embryos. 10th Internat. Congress on Anim. Reprod. and Artif. Insem.. Urbana-Champaign. IL.1984. p. 228. H. Bovine egg transplantation. 15. Lehn-Jensen. embryos. Nord. Vet. Med. 3: 523-532 (1980). 16.
Preservation
of
Schneider, J. and Mazur. P. Osmotic consequences of cryoprotectant permeability and its relation to the survival of frozen thawed embryos. Theriogenology z:68-71 (1984).
17. Chupin. D. and Procureur. R. Glycerol equilibration for deep freezing of cattle blastocysts: effect of number of steps and of total duration. Theriogenology -21:230 abstr. (1984). 18. Willadsen, S.M.. Polge. C. and Rowson. L.E.A. The viability of deep frozen cow embryos. J. Reprod. Fertil. -52:391-393 (1978). 19. Lehn-Jensen. H., Grave. T. and Perez, A. Two step freezing of cow embryos in 1.4 M glycerol. Theriogenology -15:427-432 (1981). 20.
Fahning. M.L. Ova transfer in cattle. Proc. 77th Ann. Meeting North Dakota Vet. Med. Assoc., 1982. pp. 1-15.
21.
G. and Wright, R.W. Lindner. Bovine embryo morphology evaluation. Theriogenology %:407-416 (1982).
and
22. Whittingham. D.G. and Wales, R.G. Survival of mouse embryo after (1971). freezing and thawing. Nature (Land.) =:125-126 23. Wright, J.M. Commercial freezing of bovine Theriogenology 2~17-30 (1985). 24.
embryos in straws.
Leibo. S.P. Commercial production of pregnancies from one-step diluted frozen-thawed bovine embryos. Theriogenology -25:166 abstr. (1986).
25. Lehn-Jensen. H. and Rail. W.F. Cryomicroscopic observations of cattle embryos during freezing and thawing. Theriogenology 2:263-277 (1983). 26. Rail. W.F. Embryo cryopreservation. Current application. Prospects for the future. Proc. Ann. Conf. Artifical Insemination and Embryo Transfer in Beef Cattle. Denver, CO. 1986. pp. 48-52.
812
DECEMBER
1986 VOL. 26 NO. 6