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Micromanipulation of sheep morulae to produce monozygotic twins
THERIOGENOLOGY MICROMANIPULATION OF SHEEP MORULAE TO PRODUCE MONOZYGOTIC TWINS R. Gatica, (a) M.P. Boland, T.F. Crosby and I. Gordon Department of Agriculture, University College Lyons Estate, Newcastle, Co. Dublin, Ireland Received for publication: Accepted:
July 29, 1983 February 10, 1984
ABSTRACT Donor ewes were treated with an intravaginal sponge containing 30 mg flrrorogestoneacetate (FGA) which was removed 12 days later. On the mornings of days 10, 11, and 12, each animal received 30 mg horse anterior pituitary (HAP) extract. Donors were mated twice daily during oestrus. Egg recovery was attempted seven days after progestagen withdrawal, when late morula/early blastocysts could be expected. The zona pellucida was opened with the aid of a micromanipulator, and the embryo was removed by gentle positive pressure from an evacuating pipette. After removal from the zona pellucida, the embryo was bisected using a fine glass needle; each demi-embryo was immediately placed either into the original zona pellucida or into one from an evacuated oocyte. The original opening was closed and both halves were transferred bilaterally into previously synchronized recipients. From 17 embryos split and transferred to 17 ewes, nine became pregnant and produced seven sets of monozygotic twins and two singletons. INTRODUCTION The incidence of monozygotic twinning in sheep is thought to be extremly low (l), although embryological evidence suggests that it does exist (2) There are no data in the literature on the birth of supernumerary lambs following transfer (l), although it has been reported that nonozygotic twins in cattle can occur after the transfer of a single day-8 blastocyst (3). A unique and original means of producing monozygotic twins in sheep and cattle has been described (4,5). This technique has proved to be useful for experimental purposes, but it requires the presence of an intermediate host. More recently, techniques to produce monozygotic twinsin cattleby bisecting embryos and transferring these into suitable recipients without the use of an intermediate host have been described (6, 7). Earlier attempts at the production of monozygotic twins in sheep were generally unsuccessful (8). Although 24 out of 82 half-sections of day-6 and day-7 embryos developed into apparently normal blastocysts, no monozygotic twins were produced following transfer after a period of in vitro culture (8). The present experiments, therefore, sought G ane the development of sheep demi-embryos without the use of an intermediate host.
(a)
Present address:
APRIL
Animal Reproduction Institute, Austral University,P.O. Box 567, Valdivia, Chile.
1984 VOL. 21 NO. 4
555
THERIOGENOLOGY MATERIALS AND METHODS Supply of embryos. Eggs for micromanipulation were provided following the hormonal induction of superovulation (9). Ewes were treated with an intravaginal sponge containing 30 mg fluorogestone acetate (Intervet Laboratories Ltd., Milton Road, Cambridge, England); the sponge was removed 12 days later. On the mornings of days 10, 11 and 12, ewes were subcutaneously injected with 30 mg HAP in an aqueous suspension. Animals were bred using rams in natural service twice daily until the end of heat. Eggs were recovered seven days after progestagen withdrawal, when late morulae or very early blastocysts could be expected. Manipulation of embryos. A Leitz micromanipulator (Ernst Leitz, GMBH, Wetzlar, W. Germany) was used to bisect each embryo. Four types of pipette were used to bisect the embryo (Fig. 1). The embryo was held under suction in the holding pipette and the dissecting needle was used to pierce a hole in the zona pellucida; care was taken not to damage the embryonic mass. The needle was then used to make a "slit" in the zona pellucida. Next, the evacuating pipette was used to expel the embryo from the zona pellucida with gentle pressure by injecting a small volume of medium. After removal, the embryo was bisected by horizontally sawing with the opening needle (Fig. 2). Once the embryo had been separated into approximately two halves (Fig. 3), one demiembryo was replaced into its own zona pellucida and the other was placed into a previously evacuated zona pellucida of an unfertilised sheep egg (Fig. 4). This was accomplished with a transfer pipette; the opening in the zona pellucida was then closed by pressing both sides of the zona pellucida. Transfer of embryos. Embryos were recovered in Dulbecco's phosphate buffered saline supplemented with 5p!sheep serum. During the manipulation procedure, embryos were held in 15X sheep serum and transferred in similar medium. Recipients were prepared by similar intravaginal treatment as the donors, but pessaries were withdrawn 12 hours ahead of the donors, when the recipients received 500 i.u. PMSG to increase the degree of synchronization (10). Embryos were transferred into recipients within 30 to 40 minutes of bisection and generally withinthree hours of recovery from the donors. RESULTS AND DISCUSSION All recipients were detected in oestrus at either the same time or within 12 hours after the onset of oestrus in the donors. We were able to divide and place the demi-embryos into new zonae pellucidae within 20 minutes. Data in Table 1 contain results of transfers of demi-embryos.
556
APRIL
1984 VOL. 21 NO. 4
THERIOGENOLOGY
Fig. 1. Pipettes used for manipulation of embryos: (a) opening and cutting needle, (b) holding ';ifette, (c) replacing pipette, separating pipette for early-stage embryos.
Fig. 2. Morula outside the zona pellucida with the needle above the cell mass and being brought down vertically.
APRIL
1984 VOL. 21 NO. 4
557
THERIOGENOLOGY
Fig. 3. An embryo divided into demi-embryos the position of the needle.
Fig. 4.
558
showing
Two demi-embryos replaced inside the zona pellucida.
APRIL
1984 VOL. 21 NO. 4
THERIOGENOLOGY Table 1.
Results of bisection and transfer of sheep embryos
No. of embryos bisected
17
No. of halves transferred
34
No. of recipient ewes
17
No. (%) of ewes lambing
9(52.?)
No. (Z) of ewes producing twins
7(77.8)
No. (Z) of ewes producing singles
2(22.2)
The pregnancy rate in the present experiment is somewhat lower than but the embryo survival rate is almost identical to an earlier report (4). This represents an embryo survival rate of 10 to 15% below that reported following the transfer of nonmicromanipulated day-6 sheep embryos (11). The survival rate of the transferred embryos is also similar to results reported for cattle, where the embryos had been bisected either within or outside the zona pellucida and transferred immediately (6, 7). Results of the present experiment are much more encouraging than those of Trounson and Moore (8): in their study, no pregnancies followed bisection of day-7 embryos and only ;! of 14 ewes became pregnant after transfer of demi-embryos on day 6 following a period of -in vitro culture. There was no attempt made in the work of Trounson and Moore (8) to place the embryo in a new zona pellucida, and this may have affected the survival rate of the embryos. However, it has been demonstrated recently that enzymatic and mechanical procedures can be used to remove zonae pellucidae from hamster and cow eggs without adversely affecting their subsequen survival (13). The present experiment clearly demonstrates that sheep embryos can be bisected at the morula stage and implanted in recipients immediately without a period in an intermediate host. Therefore,the zona pellucida does not need to be sealed at the time of transfer in order to achieve pregnancy. The gestation lengthof the ewes was normal and ranged from 146 to 150 days. One twin lamb was dead at birth and appeared to have been dead for at least one week. All six sets of twins that were alive at birth survived to weaning. There was a high level of twinning in those animals which became pregnant. Most of the recipients had bilateral ovulations and therefore both transfers were regarded as ipsilateral. It has been clearly shown that ipsilateral as opposed to contralateral transfers in cattle can result in higher embryo survival rates (12). Our results indicate that if the recipient ewe becomes pregnant, she is likely to carry both embryos to term (although Williams -et al. (7) have not recommended twinning of demiembryos in cattle because of the possibility of increased rate of resorption and abortion). The present results show that it is possible to bisect sheep embryos with the simultaneous use of three instruments, which is a simplification of the method described by Ozil -et al. (6). APRIL
1984 VOL. 21 NO. 4
559
THERIOGENOLOGY REFERENCES 1.
Willadsen, S.M. The viability of early cleavage stages containing half the normal number of blastomeres in sheep. J. Reprod. Fert. -59 : 357-362 (1980).
2.
Rowson, L.E.A. and Moor, R. Occurrence and development of identical twins in sheep. Nature, London 201 : 521-522 (1964).
3.
McGeady, T.A., Roland, M.P. and Roche, J.F. Conjoined bovine embryos from a single transplanted blastocyst. Ir. Vet. J. -33 : 136-141 (1979).
4.
A method for culture of micromanipulated sheep Willadsen, S.M. embryos and its use to produce monozygotic twins. Nature, London -277 : 298-300 (1979).
5.
Willadsen, S.M. and Polge, C. Attempts to produce monozygotic quadruplets in cattle by blastomere separation. Vet. Rec. 108 : 211-213 (1981).
6.
Ozil, J.P., Heyman, Y. and Renard, J.P. Production of monozygotic twins by micromanipulation and cervical transfer in the cow. Vet. Rec. -110 : 126-127 (1982).
7.
Bisecting Williams, T.J., Elsden, R.P. and Seidel, G.E. Jr. bovine embryos: Methods, applications and success rates. Proc. Ann. Conf. on A.I. and Embryo Transfer in Beef Cattle. Denver,CO. Jan. 1983, pp.45-51.
a.
Attempts to produce identical Trounson, A.O. and Moore, N.W. offspring in the sheep by mechanical division of the ovum. Aust. J. Biol. Sci. -27 : 505-510 (1974).
9.
Superovulation in the anoestrous Boland, M.P. and Gordon, I. Galway ewe following PMSG and HAP. J. Ir. Dept. Agric. -74 : al-83 (1978).
10.
Effect of mating Boland, M.P., Crosby, T.F. and Gordon, I. management and PMSG dose level on lambing outcome in ewes bred in late anoestrus. J. Agric. Sci. Camb. -97 : 445-447 (1981).
11.
Moor, R.M. and Trounson, A.O. Hormonal and follicular factors affecting maturation of sheep oocytes -in vitro and their subsequent developmental capacity. J. Reprod. Fert. -49 : 101-109 (1977).
12.
Egg transfer in the cow: Effect of site of Sreenan, J.M. transfer. Proc. 8th Int. Congr. Anim. Reprod. A.I. Cracow, (1976). -3 : 269-272
13.
Effect of removing the zona Hoppe, R.W. and Bavister, B.D. pellucida on development of hamster and bovine embryos in vitro and -in vivo. Theriogenology -19 : 391-404 (1983).-
560
APRIL
1984 VOL. 21 NO. 4
July 29, 1983 February 10, 1984
ABSTRACT Donor ewes were treated with an intravaginal sponge containing 30 mg flrrorogestoneacetate (FGA) which was removed 12 days later. On the mornings of days 10, 11, and 12, each animal received 30 mg horse anterior pituitary (HAP) extract. Donors were mated twice daily during oestrus. Egg recovery was attempted seven days after progestagen withdrawal, when late morula/early blastocysts could be expected. The zona pellucida was opened with the aid of a micromanipulator, and the embryo was removed by gentle positive pressure from an evacuating pipette. After removal from the zona pellucida, the embryo was bisected using a fine glass needle; each demi-embryo was immediately placed either into the original zona pellucida or into one from an evacuated oocyte. The original opening was closed and both halves were transferred bilaterally into previously synchronized recipients. From 17 embryos split and transferred to 17 ewes, nine became pregnant and produced seven sets of monozygotic twins and two singletons. INTRODUCTION The incidence of monozygotic twinning in sheep is thought to be extremly low (l), although embryological evidence suggests that it does exist (2) There are no data in the literature on the birth of supernumerary lambs following transfer (l), although it has been reported that nonozygotic twins in cattle can occur after the transfer of a single day-8 blastocyst (3). A unique and original means of producing monozygotic twins in sheep and cattle has been described (4,5). This technique has proved to be useful for experimental purposes, but it requires the presence of an intermediate host. More recently, techniques to produce monozygotic twinsin cattleby bisecting embryos and transferring these into suitable recipients without the use of an intermediate host have been described (6, 7). Earlier attempts at the production of monozygotic twins in sheep were generally unsuccessful (8). Although 24 out of 82 half-sections of day-6 and day-7 embryos developed into apparently normal blastocysts, no monozygotic twins were produced following transfer after a period of in vitro culture (8). The present experiments, therefore, sought G ane the development of sheep demi-embryos without the use of an intermediate host.
(a)
Present address:
APRIL
Animal Reproduction Institute, Austral University,P.O. Box 567, Valdivia, Chile.
1984 VOL. 21 NO. 4
555
THERIOGENOLOGY MATERIALS AND METHODS Supply of embryos. Eggs for micromanipulation were provided following the hormonal induction of superovulation (9). Ewes were treated with an intravaginal sponge containing 30 mg fluorogestone acetate (Intervet Laboratories Ltd., Milton Road, Cambridge, England); the sponge was removed 12 days later. On the mornings of days 10, 11 and 12, ewes were subcutaneously injected with 30 mg HAP in an aqueous suspension. Animals were bred using rams in natural service twice daily until the end of heat. Eggs were recovered seven days after progestagen withdrawal, when late morulae or very early blastocysts could be expected. Manipulation of embryos. A Leitz micromanipulator (Ernst Leitz, GMBH, Wetzlar, W. Germany) was used to bisect each embryo. Four types of pipette were used to bisect the embryo (Fig. 1). The embryo was held under suction in the holding pipette and the dissecting needle was used to pierce a hole in the zona pellucida; care was taken not to damage the embryonic mass. The needle was then used to make a "slit" in the zona pellucida. Next, the evacuating pipette was used to expel the embryo from the zona pellucida with gentle pressure by injecting a small volume of medium. After removal, the embryo was bisected by horizontally sawing with the opening needle (Fig. 2). Once the embryo had been separated into approximately two halves (Fig. 3), one demiembryo was replaced into its own zona pellucida and the other was placed into a previously evacuated zona pellucida of an unfertilised sheep egg (Fig. 4). This was accomplished with a transfer pipette; the opening in the zona pellucida was then closed by pressing both sides of the zona pellucida. Transfer of embryos. Embryos were recovered in Dulbecco's phosphate buffered saline supplemented with 5p!sheep serum. During the manipulation procedure, embryos were held in 15X sheep serum and transferred in similar medium. Recipients were prepared by similar intravaginal treatment as the donors, but pessaries were withdrawn 12 hours ahead of the donors, when the recipients received 500 i.u. PMSG to increase the degree of synchronization (10). Embryos were transferred into recipients within 30 to 40 minutes of bisection and generally withinthree hours of recovery from the donors. RESULTS AND DISCUSSION All recipients were detected in oestrus at either the same time or within 12 hours after the onset of oestrus in the donors. We were able to divide and place the demi-embryos into new zonae pellucidae within 20 minutes. Data in Table 1 contain results of transfers of demi-embryos.
556
APRIL
1984 VOL. 21 NO. 4
THERIOGENOLOGY
Fig. 1. Pipettes used for manipulation of embryos: (a) opening and cutting needle, (b) holding ';ifette, (c) replacing pipette, separating pipette for early-stage embryos.
Fig. 2. Morula outside the zona pellucida with the needle above the cell mass and being brought down vertically.
APRIL
1984 VOL. 21 NO. 4
557
THERIOGENOLOGY
Fig. 3. An embryo divided into demi-embryos the position of the needle.
Fig. 4.
558
showing
Two demi-embryos replaced inside the zona pellucida.
APRIL
1984 VOL. 21 NO. 4
THERIOGENOLOGY Table 1.
Results of bisection and transfer of sheep embryos
No. of embryos bisected
17
No. of halves transferred
34
No. of recipient ewes
17
No. (%) of ewes lambing
9(52.?)
No. (Z) of ewes producing twins
7(77.8)
No. (Z) of ewes producing singles
2(22.2)
The pregnancy rate in the present experiment is somewhat lower than but the embryo survival rate is almost identical to an earlier report (4). This represents an embryo survival rate of 10 to 15% below that reported following the transfer of nonmicromanipulated day-6 sheep embryos (11). The survival rate of the transferred embryos is also similar to results reported for cattle, where the embryos had been bisected either within or outside the zona pellucida and transferred immediately (6, 7). Results of the present experiment are much more encouraging than those of Trounson and Moore (8): in their study, no pregnancies followed bisection of day-7 embryos and only ;! of 14 ewes became pregnant after transfer of demi-embryos on day 6 following a period of -in vitro culture. There was no attempt made in the work of Trounson and Moore (8) to place the embryo in a new zona pellucida, and this may have affected the survival rate of the embryos. However, it has been demonstrated recently that enzymatic and mechanical procedures can be used to remove zonae pellucidae from hamster and cow eggs without adversely affecting their subsequen survival (13). The present experiment clearly demonstrates that sheep embryos can be bisected at the morula stage and implanted in recipients immediately without a period in an intermediate host. Therefore,the zona pellucida does not need to be sealed at the time of transfer in order to achieve pregnancy. The gestation lengthof the ewes was normal and ranged from 146 to 150 days. One twin lamb was dead at birth and appeared to have been dead for at least one week. All six sets of twins that were alive at birth survived to weaning. There was a high level of twinning in those animals which became pregnant. Most of the recipients had bilateral ovulations and therefore both transfers were regarded as ipsilateral. It has been clearly shown that ipsilateral as opposed to contralateral transfers in cattle can result in higher embryo survival rates (12). Our results indicate that if the recipient ewe becomes pregnant, she is likely to carry both embryos to term (although Williams -et al. (7) have not recommended twinning of demiembryos in cattle because of the possibility of increased rate of resorption and abortion). The present results show that it is possible to bisect sheep embryos with the simultaneous use of three instruments, which is a simplification of the method described by Ozil -et al. (6). APRIL
1984 VOL. 21 NO. 4
559
THERIOGENOLOGY REFERENCES 1.
Willadsen, S.M. The viability of early cleavage stages containing half the normal number of blastomeres in sheep. J. Reprod. Fert. -59 : 357-362 (1980).
2.
Rowson, L.E.A. and Moor, R. Occurrence and development of identical twins in sheep. Nature, London 201 : 521-522 (1964).
3.
McGeady, T.A., Roland, M.P. and Roche, J.F. Conjoined bovine embryos from a single transplanted blastocyst. Ir. Vet. J. -33 : 136-141 (1979).
4.
A method for culture of micromanipulated sheep Willadsen, S.M. embryos and its use to produce monozygotic twins. Nature, London -277 : 298-300 (1979).
5.
Willadsen, S.M. and Polge, C. Attempts to produce monozygotic quadruplets in cattle by blastomere separation. Vet. Rec. 108 : 211-213 (1981).
6.
Ozil, J.P., Heyman, Y. and Renard, J.P. Production of monozygotic twins by micromanipulation and cervical transfer in the cow. Vet. Rec. -110 : 126-127 (1982).
7.
Bisecting Williams, T.J., Elsden, R.P. and Seidel, G.E. Jr. bovine embryos: Methods, applications and success rates. Proc. Ann. Conf. on A.I. and Embryo Transfer in Beef Cattle. Denver,CO. Jan. 1983, pp.45-51.
a.
Attempts to produce identical Trounson, A.O. and Moore, N.W. offspring in the sheep by mechanical division of the ovum. Aust. J. Biol. Sci. -27 : 505-510 (1974).
9.
Superovulation in the anoestrous Boland, M.P. and Gordon, I. Galway ewe following PMSG and HAP. J. Ir. Dept. Agric. -74 : al-83 (1978).
10.
Effect of mating Boland, M.P., Crosby, T.F. and Gordon, I. management and PMSG dose level on lambing outcome in ewes bred in late anoestrus. J. Agric. Sci. Camb. -97 : 445-447 (1981).
11.
Moor, R.M. and Trounson, A.O. Hormonal and follicular factors affecting maturation of sheep oocytes -in vitro and their subsequent developmental capacity. J. Reprod. Fert. -49 : 101-109 (1977).
12.
Egg transfer in the cow: Effect of site of Sreenan, J.M. transfer. Proc. 8th Int. Congr. Anim. Reprod. A.I. Cracow, (1976). -3 : 269-272
13.
Effect of removing the zona Hoppe, R.W. and Bavister, B.D. pellucida on development of hamster and bovine embryos in vitro and -in vivo. Theriogenology -19 : 391-404 (1983).-
560
APRIL
1984 VOL. 21 NO. 4