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Factors affecting viability of fresh and frozen-thawed sheep demi-embryos
~~er~ogenology
37:713-721,
FACTORS AFFECTING
1992
VIABILITY OF FRESH AND FROZEN-THAWED SHEEP DEMI-EMBRYOS J.N. Shelton
John Curtin School of Medical Research, Australian National University, Canberra, A.C.T. Australia Received
for publication: August 1, 2991 Accepted: December IO, 1992 ABSTRACT
The addition of 0.1 M sucrose to the medium in which sheep embryos were bisected had no effect (39.5 vs 36.4%) on the survival rate of demi-embryos transferred (one per ewe) to recipients. There was a trend to greater survival of demi-blastocysts (44.7%) compared to demi-morulae (30%), and all the surviving twins were derived from the demiblastocysts. It is suggested that the survival of demimorulae is enhanced by the transfer of two demi-morulae to one uterine horn. In three experiments demi-embryos were frozen after addition of 1.5 M glycerol in three or six steps or after the addition of 1.5 M ethylene glycol in six steps. No treatment resulted in acceptable survival rates of the demi-embryos transferred to recipients after thawing and step-wise removal of the cryoprotectant. Overall, 8 of 142 (5.6%) cryopreserved demi-embryos survived as 50-day fetuses or term lambs compared with 14 of 31 (45.2%) whole embryos. the
Key words: cryopreservation, demi-embryos, ethylene glycol, glycerol
sucrose,
INTRODUCTION The survival rate of sheep demi-embryos when transferred fresh to surrogate ewes has been demonstrated by Gatica et al. (1) and Shelton and Szell (2) and survival rates approaching 50% have been reported using a number of splitting techniques. Following the conclusion of Willadsen and Godke (3) that the zona pellucida is of no importance for the survival of bisected sheep blastocysts, the Acknowledgements This project was supported in part by the Australian Wool Research and Development Corporation. The technical assistance of Marek Bronowiecki, Gordon Hughes, Bernie Barancewiecz and Sandra Veness is gratefully acknowledged,
Copyright (B 1992 Butterworth-Heinemann
Theriogenology
714
practice of re-inserting demi-embryos into zonae pellucidae has been discontinued. The effect of medium used during the splitting procedure has not been widely studied, but Herr et al. (4) have suggested that the addition of a low concentration of sucrose (187.5 mM) to the medium could enhance demi-embryo survival. In Experiment 1 described here the survival rates of demi-embryos were compared when bisection was done in medium with and without the addition of 0.1 M sucrose. Cryopreservation of demi-embryos would confer additional flexibility on their use in both the commercial This flexibility originates from the and research context. capacity to produce genetically identical animals of predetermined disparate ages. In Experiments 2, 3 and 4, the survival of demi-embryos was studied subsequent to the use of several different cryopreservation protocols. MATERIALS AND METHODS Source of Embryos Embryos were obtained from mature Merino ewes treated to induce multiple ovulation. Intravaginal sponges (Intervet, Sydney, Australia) containing the progestogen Cronolone (5, 6) were inserted for 12 days. On the 10th day, 14 mg Follicle Stimulating Hormone (FSH-P; Schering, Kenilworth, NJ, U.S.A.) and 400 IU of pregnant mare serum gonadotrophin (PMSG; Folligon; Intervet, Boxmeer, Holland) were given as a single intramuscular (i.m.) injection. Donor ewes were laparoscopically inseminated (7) 36 to 40 hours after sponge withdrawal on Day 12. Five to six days after insemination, embryos were flushed from the uteri with TCM199, with 20 mM Hepes (Flow Laboratories, Irvine, Scotland) containing 5% fetal calf serum at laparotomy under general anesthesia. Bisection
of Embryos
The splitting of embryos was done in drops of medium (one embryo per drop) under paraffin oil in culture dishes Each dish contained 6 drops of medium (90 mm diameter). (TCM199, 20 mM Hepes), and each alternate drop also contained 0.1 M sucrose. After pipetting one embryo into each of six drops, splitting was performed under a Laborlux microscope (Leitz, Wetzlar, Germany; 100 x mag.) using a microscalpel controlled by a Leitz manipulator. The cutting edge of the microscalpel was horizontal to the bottom of the dish, and bisection of the embryo was accomplished by a vertical motion followed by horizontal movement of the blade. No holding pipette was used; when necessary the embryo was stabilized by a series of tracks
715
Theriogenology
made on the bottom of the dish with the microscalpel. In Experiment 1, the demi-embryos were not replaced in zonae for transfer. In Experiments 2, 3 and 4, the demi-embryos were placed in zonae for cryopreservation. Experiment 1 On each occasion, the was conducted over five sessions. embryos were divided between the two splitting media on the basis of stage of development and quality. Immediately after splitting, which took about 10 minutes for six embryos, the demi-embryos were washed and held for up to one hour in TCM199 containing 20 mM Hepes with 26% fetal calf serum until transfer. Transfer
of Embryos
Experiment 1. The recipient ewes were treated with intravaginal sponges for 12 days, and i.m. injection of 400 I.U. of PMSG at sponge withdrawal to synchronize their estrous cycles with those of the donor ewes. Each demiembryo was transferred (one per ewe) laparoscopically to the uterine horn ipsilateral to the ovary with a corpus luteum. Experiments 2. 3 and 4. Embryos and demi-embryos were transferred in pairs laparoscopically to the uterine horn ipsilateral to the corpus luteum in ewes treated (as in Experiment 1) to be in synchrony with the cryopreserved embryos. Embryos and demi-embryos imbedded in alginate prior to cryopreservation were transferred without removal of the alginate Pregnancy
Tests
Pregnancy was detected by real-time ultrasonography at 30 to 37 days after the transfer of embryos and demiembryos to recipient ewes. In some cases, the number of fetuses was confirmed at surgery conducted at 50 days for an ensuing experiment; in other cases, the pregnant ewes went to term. Cryopreservation
Treatments
and Procedures
Experiment 2. Group i: Demi-embryos were equilibrated with 1.5 M glycerol in three 0.5 M steps, each of ten minutes duration. After thawing, glycerol was removed in similar steps, each containing 0.5 M sucrose. Group ii: Whole embryos received the same treatment as Group i demiembryos. Group iii: Demi-embryos were equilibrated with 1.5 M glycerol in three 0.5 M steps, each containing 0.1 M sucrose and of ten minutes duration. The removal of glycerol was performed as for Group i. Experiment 3. Group i: Demi-embryos were imbedded alginate (8) and equilibrated with 1.5 M glycerol
in in six
716
Theriogenology
0.25 M steps, each of seven minutes duration. The removal of glycerol was performed in similar steps. Group ii: Demiembryos imbedded in alginate (8) were equilibrated with 1.5 M glycerol in six 0.25 M steps, each containing 0.25 M sucrose and of five minutes duration. The removal of glycerol was as for Group i, except that each step contained 0.1 M sucrose. Experiment 4. Group i: Demi-embryos imbedded in alginate (8) were equilibrated with 1.5 M ethylene glycol in six 0.25 M steps, each of seven minutes duration. The removal of ethylene glycol was performed in similar steps. Group ii: Whole embryos were treated as in Group i. Group iii: Demi-embryos without alginate imbedding were treated as in Group i. Group iv: Whole embryos were treated as in Group iii. In all three experiments, the demi-embryos were placed in zonae pellucidae prior to treatment with cryoprotectant. After equilibration with the cryoprotectant, the embryos were loaded into 0.5-ml straws which were heat sealed and placed in a programmable freezer (Planer Products Ltd, Sunbury-on-Thames, England) which had been precooled to 4oc. The cooling rate was 2OC per minute to -60C. The straws were seeded with cold forceps after 5 minutes at this temperature, and were kept at this temperature for another 5 minutes. They were then cooled at 0.3OC per minute to -35OC, followed by O.l°C per minute to -38OC, at which time they were plunged in liquid nitrogen. To thaw, the straws were held in air at room temperature for 10 seconds then in a water bath at 37OC for 20 seconds. The demi-embryos and embryos were expelled from the straws, and the cryoprotectant was removed by the procedure described for each group. RESULTS Experiment
1
There was no difference between the two splitting media in the resulting number of pregnant recipients (17143; 39.5% vs 16144; 36.4%) or in the number of pairs of twins. (Table 1). There was a suggestion (Table 2) that the survival rate of demi-embryos was higher when they were derived from embryos of later developmental stage than morulae (44.7 vs 30%). Moreover, six pairs of twins resulted from 24 later stage embryos (47 demi-embryos) compared to no twins from 20 morulae (P < 0.05). Experiments
2, 3 and 4
Table 3 shows the number of pregnancies obtained from demi-embryos and whole embryos in Experiments 2, 3 and 4.
Theriogenology Only a small number of pregnancies from cryopreserved demiOverall, embryos was obtained in each of the experiments. %/I42 (5.6%) cryopreserved demi-embryos survived as 50-dayold fetuses or term lambs, compared with 14 of 31 (45.2%) of the whole embryos subjected to the same cryopreservation treatments. Table 1.
The effect of 0.1 M sucrose in the splitting medium on the survival following transfer (one per ewe) of sheep demi-embryos at several stages of development.
Sucrose content
Number of
0.1
M
0
Table 2.
morula
demi transfers
Developmental stage blast early expanded blast blast
Total
22
12
7
2
43
ewes pregnant
7
7
3
0
17
twins (pairs)
0
2
1
0
3
18
14
10
2
44
ewes pregnant
5
5
4
2
16
twins (pairs)
0
1
1
1
3
demi transfers
The effect of stage of embryo development at the time of splitting on the number of pregnancies and the number of twins produced.
Stage of development
embryos split
Number of demi-embryos pregnancies transferred
twins (pairs)
Morulae
20
40
12
0
Early to late blastocysts
24
47
21
6
718
Theriogenology
Table 3.
Experiment
The survival in vivo of cryopreserved demiembryos and whole embryos subjected to several cryoprotectant treatments.a Type of Grp embryo transferred i ii iii
2
i ii
i ii iii i.V
Total a b c d
Number recipients
pregnant 1
1
5 I
5 2
lb 0
0
6c 5b 4b 3
2 5 3 4
13d
8 14
demi whole demi
22 13 22
12 7 12
demi demi
10
12
6 6
demi whole demi whole
37 12 39 6
19
142 31
74 16
demi whole
6 19 3
13b
lambs
0
See text
for crvoprotectant treatments returned to estrus. Four ewes returned to estrus. Six ewes returned to estrus.
One
ewe
DISCUSSION The data from Experiment 1 showed that the addition of sucrose (0.1 M) to the medium in which sheep embryos were bisected did not increase the survival rate of the demiembryos. Herr et al.(4) suggested that splitting embryos in I 187.5 mM sucrose would enhance demi-blastocyst survival. However, they compared two concentrations of sucrose used separately on consecutive days, and did not include a control in which blastocysts were split without sucrose in the medium. Even when only blastocysts were considered in the present data, there was no advantage to sucrose treatment. A salient feature of the results of Experiment 1 (Table Z), in which demi-embryos were transferred one per ewe, was the tendency for the greater survival of demiblastocysts (44.7%) than demi-morulae (30%) and the complete failure of both halves of an individual morula to survive. This is in contrast to the results obtained in this laboratory (Shelton, unpublished observations) when demi-morulae were transferred in pairs to a single uterine In this case, 38 horn ipsilateral to a corpus luteum. demi-morulae were transferred to 19 ewes; 11 of these transfers resulted in pregnancy and produced seven sets of This represents a identical twins and four singletons. demi-morula survival rate of (47.4%). Thus it appears that demi-morulae have a better chance of survival when both
Theriogenology
719
halves are transferred to one uterine horn than when each This suggestion is is transferred to a separate recipient. consistent with the observation of Maurer (9) that the survival of demi-embryos can be enhanced by placing them in one horn. It is postulated that this could be due to a single demi-morula having insufficient surviving trophoblast cells at the blastocyst stage to stimulate adequately the endometrial anti-luteolytic mechanism (10). Two demi-morulae have a greater number of developing trophoblast cells and thus a greater stimulatory effect on In commercial practice, both the luteolytic mechanism. halves of an embryo are transferred to the one recipient, but experimental protocols often require that identical cotwins each be in its own recipient. In this case, the data presented suggest that it is more productive to use Also, they indicate that the blastocysts than morulae. transfer of trophoblastic vesicles with demi-embryos may enhance the survival rate. An alternative strategy may be to administer ovine trophoblast protein or a related interferon (11). The data from Experiments 2, 3 and 4 show that the cryopreservation of sheep demi-embryos remains a problem. In preliminary experiments, demi-embryos cryopreserved by a six-step addition and removal of cryoprotectant had a greater in vitro survival rate than demi-embryos subjected to a three-step regime (15138 vs O/31; P < 0.0005). Despite this result, the in vivo survival rate of sheep demi-embryos remains too low for either commercial or There was no evidence in experimental exploitation. Experiments 3 and 4 that imbedding in alginate enhanced the survival of cryopreserved demi-embryos. The survival.of whole embryos in these experiments confirms that there is no need to remove them from the alginate prior to transfer. Vitrification has been suggested as a superior approach to the freezing of rabbit demi-embryos (12); however, this assertion was based only on the post-thaw in vitro development. The success rate obtained to date from the vitrification of sheep embryos (13) gives little cause for optimism that vitrification would enhance survival of sheep demi-embryos. The compounding of splitting damage with freezing and thawing damage to the embryonic cells renders most demi-embryos non viable. Whether the critical loss is in trophoblastic cells or those of the embryo proper is not known. Clearly less traumatic procedures for splitting and cryopreservation need to be developed before the cryopreservation of sheep demi-embryos becomes a Nuclear transfer may eventually viable proposition. present a partial solution, as resultant identical embryos would be expected to have a greater number of viable cells than demi-embryos, and thus more likely to survive cryopreservation.
720
Theriogenoiogy REFERENCES
1. Gatica, R., Boland, M.P., Crosby,T.F. and Gordon,I. Manipulation of sheep morulae to produce monozygotic twins. Theriogenology a:555-560 (1984) 2. Shelton, J.N. and Szell, A. Survival of sheep demiembryos in vivo and in vitro. Theriogenology x:855-863 (1988) 3. Willadsen, S.M. and Godke, R-A. A simple procedure the production of identical sheep twins. Vet.Rec. u:240-243 (1984)
for
4. Herr, C., Halt, N., Pietrzak, Y., Old, K. and Reed, K. Increased number of pregnancies per collected embryo by bisection of blastocyst-stage ovine embryos. Theriogenology x:244 Abstr. (1990) 5. Shelton, J.N. and Robinson, T.J. The evaluation of several progestagen treatments in the entire cyclic ewe. In: Robinson,T.J. (ed.) The Control of the Ovarian Cycle in the Sheep. Sydney University Press, Sydney, 1967, pp. 39-47. 6. Robinson, T.J., Moore, N.W., Holst, P-J. and Smith, J.F. The evaluation of several progestagens administered in intravaginal sponges for the synchronization of oestrus in the entire cyclic Merino ewe. In: Robinson, T.J. (ed.) The Control of the Ovarian Cycle in the Sheep. Sydney University Press, Sydney, 1967, pp. 76-101. 7. Killeen,I.D, and Caffrey, G.J. Uterine insemination of ewes with the aid of a laparoscope. Aust. Vet. J. =:95 (1982) 8. Kojima, T, Hashimoto, K., Ito, S. Hori, Y. Tomizuka, and Oguri, N. Protection of rabbit embryos against fracture damage from freezing and thawing by encapsulation in calcium alginate gel. J. Exp. Zool. =:186-191 (1990) 9. Maurer, R.R. Embryo splitting and transfer Theriogenology =:276 Abstr. (1988)
in sheep
10. Roberts, R.M. Minireview: Conceptus interferons and maternal recognition of pregnancy. Biol. Reprod. 40: 449-452 (1989) 11. Nephew, K.P., McClure, K.E., Day, M.L.,Xie, S, Roberts, R.M. and Pope, W.F. Effects of intramuscular administration of recombinant bovine interferon-alpha1 during the period of maternal recognition of pregnancy J. Anim. Sci. =:2766-2770 (1990)
Tberiogenology 12. Kobayashi, Y., Nagashima, H, Yamakawa, H., Kato, Y. and Ogawa, S. The survival of whole and bisected rabbit morulae after cryopreservation by the vitrification method. Theriogenology 33~777-788 (1990) 13. Szell, A., Zhang,J.and Hudson, R. Rapid cryopreservation of sheep embryos by direct transfer into liquid nitrogen vapour at -180° C* Reprod. Fertil. Dev. 2:613-6X+ (1990)
37:713-721,
FACTORS AFFECTING
1992
VIABILITY OF FRESH AND FROZEN-THAWED SHEEP DEMI-EMBRYOS J.N. Shelton
John Curtin School of Medical Research, Australian National University, Canberra, A.C.T. Australia Received
for publication: August 1, 2991 Accepted: December IO, 1992 ABSTRACT
The addition of 0.1 M sucrose to the medium in which sheep embryos were bisected had no effect (39.5 vs 36.4%) on the survival rate of demi-embryos transferred (one per ewe) to recipients. There was a trend to greater survival of demi-blastocysts (44.7%) compared to demi-morulae (30%), and all the surviving twins were derived from the demiblastocysts. It is suggested that the survival of demimorulae is enhanced by the transfer of two demi-morulae to one uterine horn. In three experiments demi-embryos were frozen after addition of 1.5 M glycerol in three or six steps or after the addition of 1.5 M ethylene glycol in six steps. No treatment resulted in acceptable survival rates of the demi-embryos transferred to recipients after thawing and step-wise removal of the cryoprotectant. Overall, 8 of 142 (5.6%) cryopreserved demi-embryos survived as 50-day fetuses or term lambs compared with 14 of 31 (45.2%) whole embryos. the
Key words: cryopreservation, demi-embryos, ethylene glycol, glycerol
sucrose,
INTRODUCTION The survival rate of sheep demi-embryos when transferred fresh to surrogate ewes has been demonstrated by Gatica et al. (1) and Shelton and Szell (2) and survival rates approaching 50% have been reported using a number of splitting techniques. Following the conclusion of Willadsen and Godke (3) that the zona pellucida is of no importance for the survival of bisected sheep blastocysts, the Acknowledgements This project was supported in part by the Australian Wool Research and Development Corporation. The technical assistance of Marek Bronowiecki, Gordon Hughes, Bernie Barancewiecz and Sandra Veness is gratefully acknowledged,
Copyright (B 1992 Butterworth-Heinemann
Theriogenology
714
practice of re-inserting demi-embryos into zonae pellucidae has been discontinued. The effect of medium used during the splitting procedure has not been widely studied, but Herr et al. (4) have suggested that the addition of a low concentration of sucrose (187.5 mM) to the medium could enhance demi-embryo survival. In Experiment 1 described here the survival rates of demi-embryos were compared when bisection was done in medium with and without the addition of 0.1 M sucrose. Cryopreservation of demi-embryos would confer additional flexibility on their use in both the commercial This flexibility originates from the and research context. capacity to produce genetically identical animals of predetermined disparate ages. In Experiments 2, 3 and 4, the survival of demi-embryos was studied subsequent to the use of several different cryopreservation protocols. MATERIALS AND METHODS Source of Embryos Embryos were obtained from mature Merino ewes treated to induce multiple ovulation. Intravaginal sponges (Intervet, Sydney, Australia) containing the progestogen Cronolone (5, 6) were inserted for 12 days. On the 10th day, 14 mg Follicle Stimulating Hormone (FSH-P; Schering, Kenilworth, NJ, U.S.A.) and 400 IU of pregnant mare serum gonadotrophin (PMSG; Folligon; Intervet, Boxmeer, Holland) were given as a single intramuscular (i.m.) injection. Donor ewes were laparoscopically inseminated (7) 36 to 40 hours after sponge withdrawal on Day 12. Five to six days after insemination, embryos were flushed from the uteri with TCM199, with 20 mM Hepes (Flow Laboratories, Irvine, Scotland) containing 5% fetal calf serum at laparotomy under general anesthesia. Bisection
of Embryos
The splitting of embryos was done in drops of medium (one embryo per drop) under paraffin oil in culture dishes Each dish contained 6 drops of medium (90 mm diameter). (TCM199, 20 mM Hepes), and each alternate drop also contained 0.1 M sucrose. After pipetting one embryo into each of six drops, splitting was performed under a Laborlux microscope (Leitz, Wetzlar, Germany; 100 x mag.) using a microscalpel controlled by a Leitz manipulator. The cutting edge of the microscalpel was horizontal to the bottom of the dish, and bisection of the embryo was accomplished by a vertical motion followed by horizontal movement of the blade. No holding pipette was used; when necessary the embryo was stabilized by a series of tracks
715
Theriogenology
made on the bottom of the dish with the microscalpel. In Experiment 1, the demi-embryos were not replaced in zonae for transfer. In Experiments 2, 3 and 4, the demi-embryos were placed in zonae for cryopreservation. Experiment 1 On each occasion, the was conducted over five sessions. embryos were divided between the two splitting media on the basis of stage of development and quality. Immediately after splitting, which took about 10 minutes for six embryos, the demi-embryos were washed and held for up to one hour in TCM199 containing 20 mM Hepes with 26% fetal calf serum until transfer. Transfer
of Embryos
Experiment 1. The recipient ewes were treated with intravaginal sponges for 12 days, and i.m. injection of 400 I.U. of PMSG at sponge withdrawal to synchronize their estrous cycles with those of the donor ewes. Each demiembryo was transferred (one per ewe) laparoscopically to the uterine horn ipsilateral to the ovary with a corpus luteum. Experiments 2. 3 and 4. Embryos and demi-embryos were transferred in pairs laparoscopically to the uterine horn ipsilateral to the corpus luteum in ewes treated (as in Experiment 1) to be in synchrony with the cryopreserved embryos. Embryos and demi-embryos imbedded in alginate prior to cryopreservation were transferred without removal of the alginate Pregnancy
Tests
Pregnancy was detected by real-time ultrasonography at 30 to 37 days after the transfer of embryos and demiembryos to recipient ewes. In some cases, the number of fetuses was confirmed at surgery conducted at 50 days for an ensuing experiment; in other cases, the pregnant ewes went to term. Cryopreservation
Treatments
and Procedures
Experiment 2. Group i: Demi-embryos were equilibrated with 1.5 M glycerol in three 0.5 M steps, each of ten minutes duration. After thawing, glycerol was removed in similar steps, each containing 0.5 M sucrose. Group ii: Whole embryos received the same treatment as Group i demiembryos. Group iii: Demi-embryos were equilibrated with 1.5 M glycerol in three 0.5 M steps, each containing 0.1 M sucrose and of ten minutes duration. The removal of glycerol was performed as for Group i. Experiment 3. Group i: Demi-embryos were imbedded alginate (8) and equilibrated with 1.5 M glycerol
in in six
716
Theriogenology
0.25 M steps, each of seven minutes duration. The removal of glycerol was performed in similar steps. Group ii: Demiembryos imbedded in alginate (8) were equilibrated with 1.5 M glycerol in six 0.25 M steps, each containing 0.25 M sucrose and of five minutes duration. The removal of glycerol was as for Group i, except that each step contained 0.1 M sucrose. Experiment 4. Group i: Demi-embryos imbedded in alginate (8) were equilibrated with 1.5 M ethylene glycol in six 0.25 M steps, each of seven minutes duration. The removal of ethylene glycol was performed in similar steps. Group ii: Whole embryos were treated as in Group i. Group iii: Demi-embryos without alginate imbedding were treated as in Group i. Group iv: Whole embryos were treated as in Group iii. In all three experiments, the demi-embryos were placed in zonae pellucidae prior to treatment with cryoprotectant. After equilibration with the cryoprotectant, the embryos were loaded into 0.5-ml straws which were heat sealed and placed in a programmable freezer (Planer Products Ltd, Sunbury-on-Thames, England) which had been precooled to 4oc. The cooling rate was 2OC per minute to -60C. The straws were seeded with cold forceps after 5 minutes at this temperature, and were kept at this temperature for another 5 minutes. They were then cooled at 0.3OC per minute to -35OC, followed by O.l°C per minute to -38OC, at which time they were plunged in liquid nitrogen. To thaw, the straws were held in air at room temperature for 10 seconds then in a water bath at 37OC for 20 seconds. The demi-embryos and embryos were expelled from the straws, and the cryoprotectant was removed by the procedure described for each group. RESULTS Experiment
1
There was no difference between the two splitting media in the resulting number of pregnant recipients (17143; 39.5% vs 16144; 36.4%) or in the number of pairs of twins. (Table 1). There was a suggestion (Table 2) that the survival rate of demi-embryos was higher when they were derived from embryos of later developmental stage than morulae (44.7 vs 30%). Moreover, six pairs of twins resulted from 24 later stage embryos (47 demi-embryos) compared to no twins from 20 morulae (P < 0.05). Experiments
2, 3 and 4
Table 3 shows the number of pregnancies obtained from demi-embryos and whole embryos in Experiments 2, 3 and 4.
Theriogenology Only a small number of pregnancies from cryopreserved demiOverall, embryos was obtained in each of the experiments. %/I42 (5.6%) cryopreserved demi-embryos survived as 50-dayold fetuses or term lambs, compared with 14 of 31 (45.2%) of the whole embryos subjected to the same cryopreservation treatments. Table 1.
The effect of 0.1 M sucrose in the splitting medium on the survival following transfer (one per ewe) of sheep demi-embryos at several stages of development.
Sucrose content
Number of
0.1
M
0
Table 2.
morula
demi transfers
Developmental stage blast early expanded blast blast
Total
22
12
7
2
43
ewes pregnant
7
7
3
0
17
twins (pairs)
0
2
1
0
3
18
14
10
2
44
ewes pregnant
5
5
4
2
16
twins (pairs)
0
1
1
1
3
demi transfers
The effect of stage of embryo development at the time of splitting on the number of pregnancies and the number of twins produced.
Stage of development
embryos split
Number of demi-embryos pregnancies transferred
twins (pairs)
Morulae
20
40
12
0
Early to late blastocysts
24
47
21
6
718
Theriogenology
Table 3.
Experiment
The survival in vivo of cryopreserved demiembryos and whole embryos subjected to several cryoprotectant treatments.a Type of Grp embryo transferred i ii iii
2
i ii
i ii iii i.V
Total a b c d
Number recipients
pregnant 1
1
5 I
5 2
lb 0
0
6c 5b 4b 3
2 5 3 4
13d
8 14
demi whole demi
22 13 22
12 7 12
demi demi
10
12
6 6
demi whole demi whole
37 12 39 6
19
142 31
74 16
demi whole
6 19 3
13b
lambs
0
See text
for crvoprotectant treatments returned to estrus. Four ewes returned to estrus. Six ewes returned to estrus.
One
ewe
DISCUSSION The data from Experiment 1 showed that the addition of sucrose (0.1 M) to the medium in which sheep embryos were bisected did not increase the survival rate of the demiembryos. Herr et al.(4) suggested that splitting embryos in I 187.5 mM sucrose would enhance demi-blastocyst survival. However, they compared two concentrations of sucrose used separately on consecutive days, and did not include a control in which blastocysts were split without sucrose in the medium. Even when only blastocysts were considered in the present data, there was no advantage to sucrose treatment. A salient feature of the results of Experiment 1 (Table Z), in which demi-embryos were transferred one per ewe, was the tendency for the greater survival of demiblastocysts (44.7%) than demi-morulae (30%) and the complete failure of both halves of an individual morula to survive. This is in contrast to the results obtained in this laboratory (Shelton, unpublished observations) when demi-morulae were transferred in pairs to a single uterine In this case, 38 horn ipsilateral to a corpus luteum. demi-morulae were transferred to 19 ewes; 11 of these transfers resulted in pregnancy and produced seven sets of This represents a identical twins and four singletons. demi-morula survival rate of (47.4%). Thus it appears that demi-morulae have a better chance of survival when both
Theriogenology
719
halves are transferred to one uterine horn than when each This suggestion is is transferred to a separate recipient. consistent with the observation of Maurer (9) that the survival of demi-embryos can be enhanced by placing them in one horn. It is postulated that this could be due to a single demi-morula having insufficient surviving trophoblast cells at the blastocyst stage to stimulate adequately the endometrial anti-luteolytic mechanism (10). Two demi-morulae have a greater number of developing trophoblast cells and thus a greater stimulatory effect on In commercial practice, both the luteolytic mechanism. halves of an embryo are transferred to the one recipient, but experimental protocols often require that identical cotwins each be in its own recipient. In this case, the data presented suggest that it is more productive to use Also, they indicate that the blastocysts than morulae. transfer of trophoblastic vesicles with demi-embryos may enhance the survival rate. An alternative strategy may be to administer ovine trophoblast protein or a related interferon (11). The data from Experiments 2, 3 and 4 show that the cryopreservation of sheep demi-embryos remains a problem. In preliminary experiments, demi-embryos cryopreserved by a six-step addition and removal of cryoprotectant had a greater in vitro survival rate than demi-embryos subjected to a three-step regime (15138 vs O/31; P < 0.0005). Despite this result, the in vivo survival rate of sheep demi-embryos remains too low for either commercial or There was no evidence in experimental exploitation. Experiments 3 and 4 that imbedding in alginate enhanced the survival of cryopreserved demi-embryos. The survival.of whole embryos in these experiments confirms that there is no need to remove them from the alginate prior to transfer. Vitrification has been suggested as a superior approach to the freezing of rabbit demi-embryos (12); however, this assertion was based only on the post-thaw in vitro development. The success rate obtained to date from the vitrification of sheep embryos (13) gives little cause for optimism that vitrification would enhance survival of sheep demi-embryos. The compounding of splitting damage with freezing and thawing damage to the embryonic cells renders most demi-embryos non viable. Whether the critical loss is in trophoblastic cells or those of the embryo proper is not known. Clearly less traumatic procedures for splitting and cryopreservation need to be developed before the cryopreservation of sheep demi-embryos becomes a Nuclear transfer may eventually viable proposition. present a partial solution, as resultant identical embryos would be expected to have a greater number of viable cells than demi-embryos, and thus more likely to survive cryopreservation.
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Theriogenoiogy REFERENCES
1. Gatica, R., Boland, M.P., Crosby,T.F. and Gordon,I. Manipulation of sheep morulae to produce monozygotic twins. Theriogenology a:555-560 (1984) 2. Shelton, J.N. and Szell, A. Survival of sheep demiembryos in vivo and in vitro. Theriogenology x:855-863 (1988) 3. Willadsen, S.M. and Godke, R-A. A simple procedure the production of identical sheep twins. Vet.Rec. u:240-243 (1984)
for
4. Herr, C., Halt, N., Pietrzak, Y., Old, K. and Reed, K. Increased number of pregnancies per collected embryo by bisection of blastocyst-stage ovine embryos. Theriogenology x:244 Abstr. (1990) 5. Shelton, J.N. and Robinson, T.J. The evaluation of several progestagen treatments in the entire cyclic ewe. In: Robinson,T.J. (ed.) The Control of the Ovarian Cycle in the Sheep. Sydney University Press, Sydney, 1967, pp. 39-47. 6. Robinson, T.J., Moore, N.W., Holst, P-J. and Smith, J.F. The evaluation of several progestagens administered in intravaginal sponges for the synchronization of oestrus in the entire cyclic Merino ewe. In: Robinson, T.J. (ed.) The Control of the Ovarian Cycle in the Sheep. Sydney University Press, Sydney, 1967, pp. 76-101. 7. Killeen,I.D, and Caffrey, G.J. Uterine insemination of ewes with the aid of a laparoscope. Aust. Vet. J. =:95 (1982) 8. Kojima, T, Hashimoto, K., Ito, S. Hori, Y. Tomizuka, and Oguri, N. Protection of rabbit embryos against fracture damage from freezing and thawing by encapsulation in calcium alginate gel. J. Exp. Zool. =:186-191 (1990) 9. Maurer, R.R. Embryo splitting and transfer Theriogenology =:276 Abstr. (1988)
in sheep
10. Roberts, R.M. Minireview: Conceptus interferons and maternal recognition of pregnancy. Biol. Reprod. 40: 449-452 (1989) 11. Nephew, K.P., McClure, K.E., Day, M.L.,Xie, S, Roberts, R.M. and Pope, W.F. Effects of intramuscular administration of recombinant bovine interferon-alpha1 during the period of maternal recognition of pregnancy J. Anim. Sci. =:2766-2770 (1990)
Tberiogenology 12. Kobayashi, Y., Nagashima, H, Yamakawa, H., Kato, Y. and Ogawa, S. The survival of whole and bisected rabbit morulae after cryopreservation by the vitrification method. Theriogenology 33~777-788 (1990) 13. Szell, A., Zhang,J.and Hudson, R. Rapid cryopreservation of sheep embryos by direct transfer into liquid nitrogen vapour at -180° C* Reprod. Fertil. Dev. 2:613-6X+ (1990)