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Micromanipulation of early cleavage stage sheep embryos to produce chimeric embryos of mixed breed parentage
Theriogenology41:279,
1994
MICROMANIPULATION OF EARLY CLEAVAGE STAGE SHEEP EMBRYOS TO PRODUCE CHIMERIC EMBRYOS OF MIXED BREED PARENTAGE P.A. Pugh 1, D. Scobie 2 and H.R.Tervit 1 AgResearch, 1 Ruakura Agricultural Centre, Hamilton, New Zealand and 2 Canterbury Agricultural Centre, Lincoln, New Zealand Chimeric embryos have been produced in a number of domestic animal species including interspecific chimeras between sheep and goats (Ruffing et al., Biol Reprod, 48, 889; 1993). The production of chimeric embryos by the aggregation of pre-compaction blastomeres generally requires embedding embryos in agar chips to protect the embryo from destruction in the reproductive tract of temporary recipients (Fehilly & Willadsen, Oxford Rev Reprod Biol, 8, 379; 1986). This procedure was necessary because suitable in vitro culture systems for early sheep embryos were not available at that time. We have used a simple micromanipulation technique, that does not require agar encapsulation, followed by an in vitro culture period, to produce chimeric embryos derived from three breeds of sheep with markedly different wool characteristics. Two hundred and forty two embryos (8-16 cell) were surgically recovered 4 days after mating from 48 superovulated ewes and were held at 39°C in Hepesbuffered synthetic oviduct fluid + 3 mg/ml BSA (Hepes-SOF) until manipulation. Pairs of embryos were manipulated in Hepes-SOF containing 5ug/ml cytochalasin B at room temperature (26°C). One donor embryo was held by suction and a bevelled sharpened micropipette (45urn O.D.) was inserted through the zona pellucida and approximately half the blastomeres were aspirated into the pipette. The pipette was withdrawn and the blastomeres discharged onto the bottom of the petri dish. The same procedure was repeated for the second embryo but the blastomeres were not discharged. Blastomeres from the first embryo were again picked up into the micropipette and were inserted into the second embryo through the same zona wound. The first embryo was again secured and the remaining blastomeres in the micropipette were injected. Manipulated pairs and non -manipulated control embryos were cultured for 3 days in 50ul drops of SOF + 8mg/ml BSA + amino acids under oil and embryos that developed to late morulae or blastocysts were transferred singly or in pairs to synchronised (Day 7) recipients. Recipients were scanned on approximately day 50 to confirm pregnancy. One hundred and fifty eight chimeric embryos were produced from 165 parent embryos originating from the three different sheep breeds. The micromanipulation procedure did not affect developmental ability in vitro with 138 (87%) manipulated embryos developing in culture compared to 68/83 (82%) non-manipulated controls. Chimeric embryos that had developed in vitro were transferred to 83 recipients. Recipient pregnancy rate at scanning was 36% (30/83) and 44 conceptuses (32% embryo survival) were detected. Neither breed of the donor nor embryo stage affected the ability of chimeric embryos to develop in culture or following transfer. Eight of the 28 (28%) recipients receiving one embryo were pregnant, while 22/55 (40%) of recipients were pregnant following the transfer of two embryos. Fourteen of these latter ewes were carrying twins and 8 singletons ( overall embryo survival of 33%). Lambs are due in late August and will be examined for overt chimerism by assessing wool types and follicle density. The results demonstrate that putative chimeric sheep embryos can now be produced by using a simple micromanipulation method, followed by a short noncoculture period that overcomes the need to encapsulate the resultant chimeric embryos in agar.
Copyright © 1994 Butterworth-Heinemann
279
1994
MICROMANIPULATION OF EARLY CLEAVAGE STAGE SHEEP EMBRYOS TO PRODUCE CHIMERIC EMBRYOS OF MIXED BREED PARENTAGE P.A. Pugh 1, D. Scobie 2 and H.R.Tervit 1 AgResearch, 1 Ruakura Agricultural Centre, Hamilton, New Zealand and 2 Canterbury Agricultural Centre, Lincoln, New Zealand Chimeric embryos have been produced in a number of domestic animal species including interspecific chimeras between sheep and goats (Ruffing et al., Biol Reprod, 48, 889; 1993). The production of chimeric embryos by the aggregation of pre-compaction blastomeres generally requires embedding embryos in agar chips to protect the embryo from destruction in the reproductive tract of temporary recipients (Fehilly & Willadsen, Oxford Rev Reprod Biol, 8, 379; 1986). This procedure was necessary because suitable in vitro culture systems for early sheep embryos were not available at that time. We have used a simple micromanipulation technique, that does not require agar encapsulation, followed by an in vitro culture period, to produce chimeric embryos derived from three breeds of sheep with markedly different wool characteristics. Two hundred and forty two embryos (8-16 cell) were surgically recovered 4 days after mating from 48 superovulated ewes and were held at 39°C in Hepesbuffered synthetic oviduct fluid + 3 mg/ml BSA (Hepes-SOF) until manipulation. Pairs of embryos were manipulated in Hepes-SOF containing 5ug/ml cytochalasin B at room temperature (26°C). One donor embryo was held by suction and a bevelled sharpened micropipette (45urn O.D.) was inserted through the zona pellucida and approximately half the blastomeres were aspirated into the pipette. The pipette was withdrawn and the blastomeres discharged onto the bottom of the petri dish. The same procedure was repeated for the second embryo but the blastomeres were not discharged. Blastomeres from the first embryo were again picked up into the micropipette and were inserted into the second embryo through the same zona wound. The first embryo was again secured and the remaining blastomeres in the micropipette were injected. Manipulated pairs and non -manipulated control embryos were cultured for 3 days in 50ul drops of SOF + 8mg/ml BSA + amino acids under oil and embryos that developed to late morulae or blastocysts were transferred singly or in pairs to synchronised (Day 7) recipients. Recipients were scanned on approximately day 50 to confirm pregnancy. One hundred and fifty eight chimeric embryos were produced from 165 parent embryos originating from the three different sheep breeds. The micromanipulation procedure did not affect developmental ability in vitro with 138 (87%) manipulated embryos developing in culture compared to 68/83 (82%) non-manipulated controls. Chimeric embryos that had developed in vitro were transferred to 83 recipients. Recipient pregnancy rate at scanning was 36% (30/83) and 44 conceptuses (32% embryo survival) were detected. Neither breed of the donor nor embryo stage affected the ability of chimeric embryos to develop in culture or following transfer. Eight of the 28 (28%) recipients receiving one embryo were pregnant, while 22/55 (40%) of recipients were pregnant following the transfer of two embryos. Fourteen of these latter ewes were carrying twins and 8 singletons ( overall embryo survival of 33%). Lambs are due in late August and will be examined for overt chimerism by assessing wool types and follicle density. The results demonstrate that putative chimeric sheep embryos can now be produced by using a simple micromanipulation method, followed by a short noncoculture period that overcomes the need to encapsulate the resultant chimeric embryos in agar.
Copyright © 1994 Butterworth-Heinemann
279