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Successful direct transfer of vitrified sheep embryos
I~~i ~' ~~.. i ,'j ELSEVIER
SUCCESSFUL DIRECT TRANSFER OF VITRIFIED SHEEP EMBRYOS G. Baril, j A-L.Traldi, ta Y. Cogni6, j B. Leboeu f~2 J.F. Beckers 3 and P. Mermillod tb JlNRA-PRC 37380 Nouzilly, France 2INRA-SEIA 86480 Rouill6, France 3Universit6 de Li6ge, Facult6 de M6decine V6t6rinaire, Physiologie de la Reproduction B4000 Sart-Tilman, Belgique Received for publication: August ] , 2000 Accepted: February 8, 2001
ABSTRACT The use of a simple cryopreservation method, adapted to direct transfer of thawed embryos may help to reduce the costs of embryo transfer in sheep and increase the use of this technique genetic improvement of this species. Two experiments were made to test a vitrification method that is easy to apply in field conditions. All embryos were collected at Day 7 of the estrous cycle of FSH-stimulated donor ewes and were assessed morphologically, washed in modified PBS and incubated for 5 rain in 10% glycerol, for 5 rain in 10% glycerol and 20% ethylene glycol and were transferred into the vitrification solution (25% glycerol and 25% ethylene glycol). All solutions were based on mPBS. Embryos were loaded in straws (1 cm central part, the remaining parts being filled with 0.8 M galactose in mPBS) and plunged into liquid N2 within 30 sec of contact with the vitrification solution. The straws were thawed (10 sec at 20°C) and the embryos were either transferred directly or after 5 min of incubation in the content of the straw (followed by washing in PBS) into the uterus of a recipient ewe. In Trial 1, the pregnancy rates at term (72 vs. 72%) as well as the embryo survival rates (60 vs 50% respectively) were not different between fresh (n = 48 embryos) and vitrified (n = 50) embryos. In a second trial no difference was observed between vitrified embryos transferred after in vitro removal of the cryoprotectant (n = 86 embryos) or directly after thawing (n = 72) both in terms of lambing rate (67 vs. 75%, respectively) and embryo survival rate (lambs born/embryos transferred; 49 vs. 53%). This method of sheep embryo cryopreservation provided high pregnancy and embryo survival, even after direct transfer of the embryos. © 2001 by Elsevier Science inc.
Key words : sheep, embryo, transfer, vitrification, cryopreservation Acknowledgments The authors thank the Lacaune sheep-breeding associations (Conffidfration des Eleveurs de Roquefort and OVITEST) as well as F. Dupont and collaborators (INRA, Nouzilly) for care and treatment of donor and recipient ewes. The authors also thank Instruments de M6decine V6t6rinaire (IMV, L'Aigle, France) for designing the direct transfer device. Part of this work was supported by a grant from the "Bureau des Ressources G6n6tiques" (France). Dr A-L. Traldi was supported by an FAPESP (Brazil) post doctoral fellowship. ~' Present adress: Universidade de S~.o Paulo, Faculdade de Medicina Veterin~iria e Zootecnia, Departamento de Reproduq~.o Animal, c.p. 23, Pirassununga, SP, Brasil. b Correspondence and reprints requests.
Yheriogenology56:299-305, 2001 ©2001 ElsevierScience Inc.
0093-691X/O1/$-seefront matter PII: S0093-691X(01)00564-7
Theriogenology
300 INTRODUCTION
Although embryo transfer techniques are as efficient in sheep (2) as in cattle (10), embryo transfer activity is dramatically lower in sheep than in cattle. In 1999, more than 520,000 cattle embryos and only 13,700 sheep embryos were transferred worldwide (16). This low use of embryo transfer in sheep may be related to the relatively high cost of the technique when compared to the value of animals. Reduction of costs at any stage of embryo production or transfer is likely to increase the use of ovine Multiple Ovulation and Embryo Transfer (MOET) techniques and may improve genetic selection. Efficient cryopreservation techniques are required to allow the delayed transfer of embryos and to use MOET schemes more widely to increase the genetic gain resulting from selection. The slow-freezing protocols that are widely used to cryopreserve embryos from different species require an expensive biological freezer and are time consuming. The use of ultra rapid techniques such as vitrification may help to reduce a part of the costs in ovine MOET. Vitrification does not require any special equipment and, therefore, may be very well adapted to routine field use. Several vitrification methods have been described for embryos from different species (7, 11, 18). However, most of these methods involved complex media with high protein concentrations and/or precise embryo handling techniques that are difficult to adapt to field conditions (17, 19). Furthermore, few of these vitrification studies dealt with ovine embryos. Ultra rapid techniques may be more adapted to embryos with a higher cryosensitivity such as in vitro produced or biopsied embryos (8). The following experiments were designed to evaluate the possibility of using a simple vitrification method to cryopreserve and transfer sheep embryos in field conditions. In a first trial the viability of vitrified/thawed ovine embryos was compared to results obtained after transfer of fresh embryos. In a second trial the possibility to transfer the vitrified embryos directly after thawing (without washing or evaluation of the morphological status after thawing) was tested by comparison with the standard technique of transfer of vitrified/thawed embryos (removal of cryoprotectant and morphological evaluation). MATERIALS AND METHODS Animals, Stimulation and Insemination These experiments were carried out in Lacaune and Ile de France ewes between April 1998 and November 1999. Synchronization of estrus in donor and recipient ewes was induced by progestagen treatment with a vaginal sponge containing 40mg FGA (Chronogest ND, Intervet, Angers, France) inserted for 14 days. At the time of sponge removal, the recipients received eCG (500 IU im) (Chronogest ND, lntervet, Angers, France). Donors ewes were induced to superovulate according to the method previously described by Cogni6 et al. (3), involving pretreatment with the GnRH antagonist Antarelix ND (0.5 mg sc per day) (Europeptides, Argenteuil, France) during the first ten days of progestagen treatment, followed by FSH/LH (40 Armour units im) (Stimufol ND-University of Liege and M6rial-Belgium) superovulatory treatment in eight decreasing doses during the last four days of progestagen treatment.
Theriogenology
301
Insemination of donor ewes was performed in utero under laparoscopic control 54 h after the end of progestagen treatment with fresh semen (80× l06 spermatozoa/female). Several males were used randomly in each breed. Embryo Recovery Embryos were recovered surgically by laparotomy 7 days afier the onset of estrus. Each uterine horn was flushed with 40 mL PBS containing 2%0 bovine serum albumin (BSA) (IMV-l'Aigle-France). Only morphologically normal embryos were selected for transfer or vitrification. Embryos were kept less than 30 min in PBS containing 20% newborn calf serum (NBCS-Sigma) at +20 to +25°C before transfer (Experiment 1, Group 1: fresh embryos) or vitrification (Experiment 1, Group 2 and Experiment 2). Vitrification and Thawing Procedures The methods previously described (4, 9) were used for vitrification and thawing. Before vitrification, embryos were kept at room temperature for 5 min in PBS-NBCS containing 10% glycerol, they were placed for 5 min in PBS-NBCS solution containing 10% glycerol and 20% ethylene glycol, and finally plunged for 30 set: into the vitrification solution (PBS-NBCS with glycerol 25% and ethylene glycol 25%). During the last step, embryos were quickly aspirated in the central part of a 0.25 mL straw, and the remaining parts of the straw were filled with galactose 0.8 M in PBS-NBCS. Groups of 5 to 6 embryos were treated together for conventional transfer (Experiments 1 and 2) and groups of 2 embryos were vitrified for direct transfer (Experiment 2). Straws were thawed 5 sec in air followed by 10 sec in a 20°C water bath. Embryos were transferred surgically to recipients either directly (Experiment 2) or after removal of the cryoprotectant by incubation during 5 min in the content of the straw and two washes of 5 min in 3 mL PBS-NBCS (Experiments 1 and 2). Transfer Methods Experiment 1 This experiment used 50 multiparous Lacaune ewes to compare the rate of success after transfer of vitrified embryos (after removal of cryoprotectant; 25 recipients) with that obtained after transfer of fresh embryos (without cryopreservation; 25 recipients). Thawed or fresh embryos were transferred into synchronized females (+/- 24 h) within 30 min after removal of the cryoprotectant or after recovery. For each recipient, 1 to 3 embryos were aspirated with 20 pL PBS in a glass capillary connected to a 1 mL syringe and were introduced surgically into the top of the uterine horn ipsilateral to the ovary showing at least one functional corpus luteum (CL) (fresh embryos :1 embryo n = 4; 2 embryos n = 19 ; 3 embryos: n = 2; vitrified embryos: 1 embryo: n = 3; 2 embryos: n = 19 ; 3 embryos n = 3).
Thefiogenology
302 Experiment 2
This experiment used two flocks [A: n=39 multiparous Lacaune ewes (Lacaune ewes used in Experiments 1 and 2 were issued from the same flock); B: n= 40 nulliparous Ile de France ewes] to compare two transfer procedures for vitrified embryos. Embryos were transferred either after cryoprotectant removal and evaluation of embryo quality after thawing, followed by transfer in 20 ~aL PBS in a glass capillary connected to 1 mL syringe (conventional transfer technique) or (direct transfer) without cryoprotectant removal nor embryo evaluation after thawing and transfer of the whole content of the straw (250 lal) with direct transfer instrument prototype (IMV-L'Aigle-France). Regardless of the procedure used for embryo transfer, two embryos were transferred per female using the same surgical method previously described for the first experiment. Each ewe received embryos from her own breed. Determination of Treatment Success Rate The fertility (number of pregnant ewes/number of recipients ewes) was assessed by ultrasonography at D45 after estrus and at lambing. The embryo survival rate (number of lambs born/number of embryos transferred) was also recorded. Pregnancy and embryo survival rates were compared by Chi-square test. RESULTS Experiment 1 No difference was observed for pregnancy rates at Day 45 and at lambing according to origin of the embryos (fresh or vitrified) (Table l). Embryo survival rates tended to be better for fresh embryos than for vitrified embryos although the results were not statistically different (P = 0.30). No effect of the number of embryos transferred per recipient was observed on the pregnancy rate and embryo survival rate (although the numbers were too low in recipients with I or 3 embryos to allow statistical analysis). Table 1. Pregnancy rate and embryo survival after transfer of ti'esh or vitrified sheep embryos Embryos
Recipients
Embryos transferred
Pregnancy Day 45 Lambing (%) (%) Fresh 25 48 76 72 Vitrified 25 50 72 72 a Embryo survival rate = Lambs born/embryos transferred (%) No significant differences (Chi-square)
Embryo survival" (%)
60 50
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Experiment 2 For traditional transfer method, 84% (86/102) of the thawed embryos were evaluated as transferable according to morphological criteria. No effect of the breed was observed on this percentage [Lacaune 87% (46/53) ; Ile de France 82% (40/49)]. Pregnancy rate, lambing rate and embryo survival rate were not affected by the embryo transfer method (Table 2). However, the embryo survival rate was significantly higher in multiparous Lacaune ewes than for the nulliparous Ile de France ewes (59 vs. 42% respectively; P < 0.05) (Table2). Experiments 1 and 2 In order to verify a possible effect of embryo developmental stage on embryo survival after transfer, the data of vitrified embryos were pooled from Experiments 1 and 2. For the two groups considered (morulae to early blastocyst : Group 1 and blastocyst to expanded blastocyst : Group 2), the survival rate was not significantly different according to embryo developmental stage (Table3). However, for nulliparous lie de France ewes, the survival rate tended to be lower in Group 1 than in Group 2 (P < 0.15). Table 2. Success rate after transfer of vitrified/thawed sheep embryos according to the embryo transfer method and parity/breed of recipients Transfer method
Parity/Breed
Recipients
Pregnancy D 45 (%)
Lambing (%)
Embryo survival (%)
Traditional
Multiparous/Lacaune Nulliparous/Ile de France Total
23 20 43
87 60 74
74 60 67 ~
59 ~ 37 f 49 ~
Direct
Multiparous/Lacau ne Nulliparous/Ile de France Total
16 20 36
81 70 75
8l 70 75 b
59 g 47 h 53 d
a vs. b ; c vs. d : no significant difference e + g vs. f + h: P < 0.05
Table 3. Embryo survival rate after transfer of vitrified/thawed sheep embryos according to the developmental stage of the embryos.
Development stage Morulae to early blastocyst. Blastocyst to expanded blastocyst ( ) : embryos transferred a vs. b : P <0.15
Embryo survival rate (%) Multiparous Nulliparous Lacaune Ile de France 61 (46) 33 a (30) 58 (50) 52 b (44)
Total 50 (76) 55 (94)
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304
DISCUSSION In the first experiment, the pregnancy rate and embryo survival rate observed after transfer of vitrified embryos was similar to those obtained with fresh embryos. The results with vitrified embryos (percentage of surviving embryos afte, thawing, pregnancy and embryo survival rates after transfer) are similar to those reported previously for embryos preserved by slow freezing (5, 12, 14, 15) or with other vitrification methods ( 1, 13). Embryo survival after vitrification and thawing was not affected by the developmental stage reached by the embryo at the time of collection. However, in the nulliparous Ile de France ewes the survival rate tended to be lower lot embryos at an early stage of development (morula to early blastocyst, 33%) than for more advanced embryos (blastocyst to expanded blastocyst, 52%). This lower survival rate of early embryos was already reported for frozen embryos (6, 12) as well as for embryos vitrified by other methods (1, 13, 15). This difference may be explained by a less efficient evaluation of embryo quality for early stages of development leading to the cryopreservation of embryos of lower quality or by a higher sensitivity of these embryos to the different cryoprcservation methods. On the other hand, a link between developmental kinetics and viability of the embryos was also reported for in vitro produced embryos (8) and this may explain the lower cryoresistance of less advanced in vivo produced embryos. These results show for the first time that the embryos vitrified with this simple technique can be transferred directly with the content of the straw without dilution of the cryoprotectant. Because 15 to 20% of the thawed embryos are discarded at morphological examination after thawing, the direct transfer of vitrified embryos represents a potential gain of 7 to 8% in terms of lambs born which could increase the gain/cost ratio in sheep MOET. This also shows that morphological evaluation of frozen/thawed embryos is not accurate and leads to the elimination of viable embryos or that the methods used for cryoprotectant removal are less efficient than in straw dilution of the cryoprotectant and direct transfer. During intermttional commercial exchange of embryos, the evaluation of post thawing morphology of embryos is always a problem and a source of conflicts. The use of direct trausfer in this case will avoid these problems. Additionally, direct embryo transfer may be pertbrmed by a technician without experience in embryo evaluation. In the case of direct transfer, 250 gL of medium are transferred into the uterine horn of the recipient. This represents a 10-told increase of liquid volume transferred into the uterus when compared with the traditional transfer method. However, this increased volume does not seem to negatively affect embryo survival. In conclusion, this simple method of sheep embryo cryopreservation provided very promising pregnancy and embryo survival rates, even alter direct transfer of thawed embryos. The use of this technique in sheep MOET schemes may decrease the cost of the procedure and may increase the reliability of the results, leading to a larger use of embryo transfer in sheep. REFERENCES I. All J, Shelton JN. Successful vitrification of day-6 sheep embryos. J Reprod Fertil 1993; 99: 65-70.
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2. Baril G, Brebion P, Chesn6 P. Training manual on embryo transfer in sheep and goats. Animal Production and Health, paper n°115, Food and Agriculture Organization of the United Nations, Rome, Italy, pp183. 3. Cogni6 Y. State of the art in sheep-goat embryo transfer. Theriogenology 1999; 51: 105116. 4. Donnay I, Auquier P, Kaidi S, Carolan C, Lonergan P M P, Massip A. Vitrification of in vitro produced bovine blastocysts: methodological studies and developmental capacity. Anim Reprod Sci 1998; 52: 93-104. 5. Heyman Y, Vincent C, Garnier V, Cogni6 Y. Transfer of frozen-thawed embryos in sheep. Vet Rec 1987; 120: 83-85. 6. Martinez AL, Nibart M, Humblot P, Thibier M. Production et cong61ation d'embryons chez la brebis. E levage - Ins6mination 1985; 208: 7-16. 7. Massip A. La vitrification: une m6thode de cryopr6servation int6ressante pour I'embryon de mammif~re. Contracept Fertil Sex 1996; 24: 665-673. 8. Massip A, Mermillod P and Dinnyes A. Morphology and biochemistry of in-vitro produced bovine embryos: implications for their cryopreservation. Hum Reprod 1995; 10:103-108. 9. Mermillod P, Traldi AS, GuerinY, Poulin N, Massip A, Cognie Y. Successful vitrification of in vitro produced ovine embryos. Proc 13th Scientific Meeting of European Embryo Transfer Association, Lyon, France, 1997; 182 abstr. 10. Nibart M. Embryo transfer and biotechnologies associated to it: bissection and sexing. Recueil de Mddecinne V6t6rinaire- Sp6cial Rcproduction des Ruminants 1991; 261-290. 11. Palasz AT and Mapletoft RJ Cryopreservation of mammalian embryos and oocytes : recent advances. Biotech Adv 1996;14: 127-149. 12. Sakull H, Bradford GE, BonDurant RH, Anderson GB, Donahue SE. Cryopreservation of embryos as a means of germ plasm conservation in sheep. Theriogenology 1993; 39: 401-409. 13. Sz611A, Windsor DP. Survival of vitrified embryos in vitro and in vivo. Theriogenology 1994; 42: 881-889. 14. Sz611A, Zhang J, Hudson R. Rapid cryopreservation of sheep embryos by direct transfer into liquid nitrogen vapour at -180°C. Reprod Fertil Dev 1990; 2: 613-618. 15. Tervit HR, Goold PG. Deep freezing of sheep embryos. Theriogenology 1984; 21:268 abstr. 16. Thibier M. The IETS statistics of embryo transfers in livestock in the world for the year 1999: A new record for bovine in vivo-derived embryos transferred. Embryo Transfer Newsletter 2000; 18: 24-28. 17. Vajta G, Holm P, Kuwayama M, Booth P J, Jacobsen H, Greve T, Callesen H. Open Pulled Straw (OPS) vitrification: a new wa3' to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev 1998; 51: 53-58. 18. Vajta G. Vitrification of the oocytes and embryos of domestic animals. Anim Reprod Sci. 2000 Jul 2; 60-61:357-64. 19. Wagtendonk-de Leeuw A M v, Daas J H G d, Rall W F. Field trial to compare pregnancy rates of bovine embryo cryopreservation methods: vitrification and one-step dilution versus slow freezing and three-step dilution. Theriogenology 1997; 48: 10711084.
SUCCESSFUL DIRECT TRANSFER OF VITRIFIED SHEEP EMBRYOS G. Baril, j A-L.Traldi, ta Y. Cogni6, j B. Leboeu f~2 J.F. Beckers 3 and P. Mermillod tb JlNRA-PRC 37380 Nouzilly, France 2INRA-SEIA 86480 Rouill6, France 3Universit6 de Li6ge, Facult6 de M6decine V6t6rinaire, Physiologie de la Reproduction B4000 Sart-Tilman, Belgique Received for publication: August ] , 2000 Accepted: February 8, 2001
ABSTRACT The use of a simple cryopreservation method, adapted to direct transfer of thawed embryos may help to reduce the costs of embryo transfer in sheep and increase the use of this technique genetic improvement of this species. Two experiments were made to test a vitrification method that is easy to apply in field conditions. All embryos were collected at Day 7 of the estrous cycle of FSH-stimulated donor ewes and were assessed morphologically, washed in modified PBS and incubated for 5 rain in 10% glycerol, for 5 rain in 10% glycerol and 20% ethylene glycol and were transferred into the vitrification solution (25% glycerol and 25% ethylene glycol). All solutions were based on mPBS. Embryos were loaded in straws (1 cm central part, the remaining parts being filled with 0.8 M galactose in mPBS) and plunged into liquid N2 within 30 sec of contact with the vitrification solution. The straws were thawed (10 sec at 20°C) and the embryos were either transferred directly or after 5 min of incubation in the content of the straw (followed by washing in PBS) into the uterus of a recipient ewe. In Trial 1, the pregnancy rates at term (72 vs. 72%) as well as the embryo survival rates (60 vs 50% respectively) were not different between fresh (n = 48 embryos) and vitrified (n = 50) embryos. In a second trial no difference was observed between vitrified embryos transferred after in vitro removal of the cryoprotectant (n = 86 embryos) or directly after thawing (n = 72) both in terms of lambing rate (67 vs. 75%, respectively) and embryo survival rate (lambs born/embryos transferred; 49 vs. 53%). This method of sheep embryo cryopreservation provided high pregnancy and embryo survival, even after direct transfer of the embryos. © 2001 by Elsevier Science inc.
Key words : sheep, embryo, transfer, vitrification, cryopreservation Acknowledgments The authors thank the Lacaune sheep-breeding associations (Conffidfration des Eleveurs de Roquefort and OVITEST) as well as F. Dupont and collaborators (INRA, Nouzilly) for care and treatment of donor and recipient ewes. The authors also thank Instruments de M6decine V6t6rinaire (IMV, L'Aigle, France) for designing the direct transfer device. Part of this work was supported by a grant from the "Bureau des Ressources G6n6tiques" (France). Dr A-L. Traldi was supported by an FAPESP (Brazil) post doctoral fellowship. ~' Present adress: Universidade de S~.o Paulo, Faculdade de Medicina Veterin~iria e Zootecnia, Departamento de Reproduq~.o Animal, c.p. 23, Pirassununga, SP, Brasil. b Correspondence and reprints requests.
Yheriogenology56:299-305, 2001 ©2001 ElsevierScience Inc.
0093-691X/O1/$-seefront matter PII: S0093-691X(01)00564-7
Theriogenology
300 INTRODUCTION
Although embryo transfer techniques are as efficient in sheep (2) as in cattle (10), embryo transfer activity is dramatically lower in sheep than in cattle. In 1999, more than 520,000 cattle embryos and only 13,700 sheep embryos were transferred worldwide (16). This low use of embryo transfer in sheep may be related to the relatively high cost of the technique when compared to the value of animals. Reduction of costs at any stage of embryo production or transfer is likely to increase the use of ovine Multiple Ovulation and Embryo Transfer (MOET) techniques and may improve genetic selection. Efficient cryopreservation techniques are required to allow the delayed transfer of embryos and to use MOET schemes more widely to increase the genetic gain resulting from selection. The slow-freezing protocols that are widely used to cryopreserve embryos from different species require an expensive biological freezer and are time consuming. The use of ultra rapid techniques such as vitrification may help to reduce a part of the costs in ovine MOET. Vitrification does not require any special equipment and, therefore, may be very well adapted to routine field use. Several vitrification methods have been described for embryos from different species (7, 11, 18). However, most of these methods involved complex media with high protein concentrations and/or precise embryo handling techniques that are difficult to adapt to field conditions (17, 19). Furthermore, few of these vitrification studies dealt with ovine embryos. Ultra rapid techniques may be more adapted to embryos with a higher cryosensitivity such as in vitro produced or biopsied embryos (8). The following experiments were designed to evaluate the possibility of using a simple vitrification method to cryopreserve and transfer sheep embryos in field conditions. In a first trial the viability of vitrified/thawed ovine embryos was compared to results obtained after transfer of fresh embryos. In a second trial the possibility to transfer the vitrified embryos directly after thawing (without washing or evaluation of the morphological status after thawing) was tested by comparison with the standard technique of transfer of vitrified/thawed embryos (removal of cryoprotectant and morphological evaluation). MATERIALS AND METHODS Animals, Stimulation and Insemination These experiments were carried out in Lacaune and Ile de France ewes between April 1998 and November 1999. Synchronization of estrus in donor and recipient ewes was induced by progestagen treatment with a vaginal sponge containing 40mg FGA (Chronogest ND, Intervet, Angers, France) inserted for 14 days. At the time of sponge removal, the recipients received eCG (500 IU im) (Chronogest ND, lntervet, Angers, France). Donors ewes were induced to superovulate according to the method previously described by Cogni6 et al. (3), involving pretreatment with the GnRH antagonist Antarelix ND (0.5 mg sc per day) (Europeptides, Argenteuil, France) during the first ten days of progestagen treatment, followed by FSH/LH (40 Armour units im) (Stimufol ND-University of Liege and M6rial-Belgium) superovulatory treatment in eight decreasing doses during the last four days of progestagen treatment.
Theriogenology
301
Insemination of donor ewes was performed in utero under laparoscopic control 54 h after the end of progestagen treatment with fresh semen (80× l06 spermatozoa/female). Several males were used randomly in each breed. Embryo Recovery Embryos were recovered surgically by laparotomy 7 days afier the onset of estrus. Each uterine horn was flushed with 40 mL PBS containing 2%0 bovine serum albumin (BSA) (IMV-l'Aigle-France). Only morphologically normal embryos were selected for transfer or vitrification. Embryos were kept less than 30 min in PBS containing 20% newborn calf serum (NBCS-Sigma) at +20 to +25°C before transfer (Experiment 1, Group 1: fresh embryos) or vitrification (Experiment 1, Group 2 and Experiment 2). Vitrification and Thawing Procedures The methods previously described (4, 9) were used for vitrification and thawing. Before vitrification, embryos were kept at room temperature for 5 min in PBS-NBCS containing 10% glycerol, they were placed for 5 min in PBS-NBCS solution containing 10% glycerol and 20% ethylene glycol, and finally plunged for 30 set: into the vitrification solution (PBS-NBCS with glycerol 25% and ethylene glycol 25%). During the last step, embryos were quickly aspirated in the central part of a 0.25 mL straw, and the remaining parts of the straw were filled with galactose 0.8 M in PBS-NBCS. Groups of 5 to 6 embryos were treated together for conventional transfer (Experiments 1 and 2) and groups of 2 embryos were vitrified for direct transfer (Experiment 2). Straws were thawed 5 sec in air followed by 10 sec in a 20°C water bath. Embryos were transferred surgically to recipients either directly (Experiment 2) or after removal of the cryoprotectant by incubation during 5 min in the content of the straw and two washes of 5 min in 3 mL PBS-NBCS (Experiments 1 and 2). Transfer Methods Experiment 1 This experiment used 50 multiparous Lacaune ewes to compare the rate of success after transfer of vitrified embryos (after removal of cryoprotectant; 25 recipients) with that obtained after transfer of fresh embryos (without cryopreservation; 25 recipients). Thawed or fresh embryos were transferred into synchronized females (+/- 24 h) within 30 min after removal of the cryoprotectant or after recovery. For each recipient, 1 to 3 embryos were aspirated with 20 pL PBS in a glass capillary connected to a 1 mL syringe and were introduced surgically into the top of the uterine horn ipsilateral to the ovary showing at least one functional corpus luteum (CL) (fresh embryos :1 embryo n = 4; 2 embryos n = 19 ; 3 embryos: n = 2; vitrified embryos: 1 embryo: n = 3; 2 embryos: n = 19 ; 3 embryos n = 3).
Thefiogenology
302 Experiment 2
This experiment used two flocks [A: n=39 multiparous Lacaune ewes (Lacaune ewes used in Experiments 1 and 2 were issued from the same flock); B: n= 40 nulliparous Ile de France ewes] to compare two transfer procedures for vitrified embryos. Embryos were transferred either after cryoprotectant removal and evaluation of embryo quality after thawing, followed by transfer in 20 ~aL PBS in a glass capillary connected to 1 mL syringe (conventional transfer technique) or (direct transfer) without cryoprotectant removal nor embryo evaluation after thawing and transfer of the whole content of the straw (250 lal) with direct transfer instrument prototype (IMV-L'Aigle-France). Regardless of the procedure used for embryo transfer, two embryos were transferred per female using the same surgical method previously described for the first experiment. Each ewe received embryos from her own breed. Determination of Treatment Success Rate The fertility (number of pregnant ewes/number of recipients ewes) was assessed by ultrasonography at D45 after estrus and at lambing. The embryo survival rate (number of lambs born/number of embryos transferred) was also recorded. Pregnancy and embryo survival rates were compared by Chi-square test. RESULTS Experiment 1 No difference was observed for pregnancy rates at Day 45 and at lambing according to origin of the embryos (fresh or vitrified) (Table l). Embryo survival rates tended to be better for fresh embryos than for vitrified embryos although the results were not statistically different (P = 0.30). No effect of the number of embryos transferred per recipient was observed on the pregnancy rate and embryo survival rate (although the numbers were too low in recipients with I or 3 embryos to allow statistical analysis). Table 1. Pregnancy rate and embryo survival after transfer of ti'esh or vitrified sheep embryos Embryos
Recipients
Embryos transferred
Pregnancy Day 45 Lambing (%) (%) Fresh 25 48 76 72 Vitrified 25 50 72 72 a Embryo survival rate = Lambs born/embryos transferred (%) No significant differences (Chi-square)
Embryo survival" (%)
60 50
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Experiment 2 For traditional transfer method, 84% (86/102) of the thawed embryos were evaluated as transferable according to morphological criteria. No effect of the breed was observed on this percentage [Lacaune 87% (46/53) ; Ile de France 82% (40/49)]. Pregnancy rate, lambing rate and embryo survival rate were not affected by the embryo transfer method (Table 2). However, the embryo survival rate was significantly higher in multiparous Lacaune ewes than for the nulliparous Ile de France ewes (59 vs. 42% respectively; P < 0.05) (Table2). Experiments 1 and 2 In order to verify a possible effect of embryo developmental stage on embryo survival after transfer, the data of vitrified embryos were pooled from Experiments 1 and 2. For the two groups considered (morulae to early blastocyst : Group 1 and blastocyst to expanded blastocyst : Group 2), the survival rate was not significantly different according to embryo developmental stage (Table3). However, for nulliparous lie de France ewes, the survival rate tended to be lower in Group 1 than in Group 2 (P < 0.15). Table 2. Success rate after transfer of vitrified/thawed sheep embryos according to the embryo transfer method and parity/breed of recipients Transfer method
Parity/Breed
Recipients
Pregnancy D 45 (%)
Lambing (%)
Embryo survival (%)
Traditional
Multiparous/Lacaune Nulliparous/Ile de France Total
23 20 43
87 60 74
74 60 67 ~
59 ~ 37 f 49 ~
Direct
Multiparous/Lacau ne Nulliparous/Ile de France Total
16 20 36
81 70 75
8l 70 75 b
59 g 47 h 53 d
a vs. b ; c vs. d : no significant difference e + g vs. f + h: P < 0.05
Table 3. Embryo survival rate after transfer of vitrified/thawed sheep embryos according to the developmental stage of the embryos.
Development stage Morulae to early blastocyst. Blastocyst to expanded blastocyst ( ) : embryos transferred a vs. b : P <0.15
Embryo survival rate (%) Multiparous Nulliparous Lacaune Ile de France 61 (46) 33 a (30) 58 (50) 52 b (44)
Total 50 (76) 55 (94)
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DISCUSSION In the first experiment, the pregnancy rate and embryo survival rate observed after transfer of vitrified embryos was similar to those obtained with fresh embryos. The results with vitrified embryos (percentage of surviving embryos afte, thawing, pregnancy and embryo survival rates after transfer) are similar to those reported previously for embryos preserved by slow freezing (5, 12, 14, 15) or with other vitrification methods ( 1, 13). Embryo survival after vitrification and thawing was not affected by the developmental stage reached by the embryo at the time of collection. However, in the nulliparous Ile de France ewes the survival rate tended to be lower lot embryos at an early stage of development (morula to early blastocyst, 33%) than for more advanced embryos (blastocyst to expanded blastocyst, 52%). This lower survival rate of early embryos was already reported for frozen embryos (6, 12) as well as for embryos vitrified by other methods (1, 13, 15). This difference may be explained by a less efficient evaluation of embryo quality for early stages of development leading to the cryopreservation of embryos of lower quality or by a higher sensitivity of these embryos to the different cryoprcservation methods. On the other hand, a link between developmental kinetics and viability of the embryos was also reported for in vitro produced embryos (8) and this may explain the lower cryoresistance of less advanced in vivo produced embryos. These results show for the first time that the embryos vitrified with this simple technique can be transferred directly with the content of the straw without dilution of the cryoprotectant. Because 15 to 20% of the thawed embryos are discarded at morphological examination after thawing, the direct transfer of vitrified embryos represents a potential gain of 7 to 8% in terms of lambs born which could increase the gain/cost ratio in sheep MOET. This also shows that morphological evaluation of frozen/thawed embryos is not accurate and leads to the elimination of viable embryos or that the methods used for cryoprotectant removal are less efficient than in straw dilution of the cryoprotectant and direct transfer. During intermttional commercial exchange of embryos, the evaluation of post thawing morphology of embryos is always a problem and a source of conflicts. The use of direct trausfer in this case will avoid these problems. Additionally, direct embryo transfer may be pertbrmed by a technician without experience in embryo evaluation. In the case of direct transfer, 250 gL of medium are transferred into the uterine horn of the recipient. This represents a 10-told increase of liquid volume transferred into the uterus when compared with the traditional transfer method. However, this increased volume does not seem to negatively affect embryo survival. In conclusion, this simple method of sheep embryo cryopreservation provided very promising pregnancy and embryo survival rates, even alter direct transfer of thawed embryos. The use of this technique in sheep MOET schemes may decrease the cost of the procedure and may increase the reliability of the results, leading to a larger use of embryo transfer in sheep. REFERENCES I. All J, Shelton JN. Successful vitrification of day-6 sheep embryos. J Reprod Fertil 1993; 99: 65-70.
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2. Baril G, Brebion P, Chesn6 P. Training manual on embryo transfer in sheep and goats. Animal Production and Health, paper n°115, Food and Agriculture Organization of the United Nations, Rome, Italy, pp183. 3. Cogni6 Y. State of the art in sheep-goat embryo transfer. Theriogenology 1999; 51: 105116. 4. Donnay I, Auquier P, Kaidi S, Carolan C, Lonergan P M P, Massip A. Vitrification of in vitro produced bovine blastocysts: methodological studies and developmental capacity. Anim Reprod Sci 1998; 52: 93-104. 5. Heyman Y, Vincent C, Garnier V, Cogni6 Y. Transfer of frozen-thawed embryos in sheep. Vet Rec 1987; 120: 83-85. 6. Martinez AL, Nibart M, Humblot P, Thibier M. Production et cong61ation d'embryons chez la brebis. E levage - Ins6mination 1985; 208: 7-16. 7. Massip A. La vitrification: une m6thode de cryopr6servation int6ressante pour I'embryon de mammif~re. Contracept Fertil Sex 1996; 24: 665-673. 8. Massip A, Mermillod P and Dinnyes A. Morphology and biochemistry of in-vitro produced bovine embryos: implications for their cryopreservation. Hum Reprod 1995; 10:103-108. 9. Mermillod P, Traldi AS, GuerinY, Poulin N, Massip A, Cognie Y. Successful vitrification of in vitro produced ovine embryos. Proc 13th Scientific Meeting of European Embryo Transfer Association, Lyon, France, 1997; 182 abstr. 10. Nibart M. Embryo transfer and biotechnologies associated to it: bissection and sexing. Recueil de Mddecinne V6t6rinaire- Sp6cial Rcproduction des Ruminants 1991; 261-290. 11. Palasz AT and Mapletoft RJ Cryopreservation of mammalian embryos and oocytes : recent advances. Biotech Adv 1996;14: 127-149. 12. Sakull H, Bradford GE, BonDurant RH, Anderson GB, Donahue SE. Cryopreservation of embryos as a means of germ plasm conservation in sheep. Theriogenology 1993; 39: 401-409. 13. Sz611A, Windsor DP. Survival of vitrified embryos in vitro and in vivo. Theriogenology 1994; 42: 881-889. 14. Sz611A, Zhang J, Hudson R. Rapid cryopreservation of sheep embryos by direct transfer into liquid nitrogen vapour at -180°C. Reprod Fertil Dev 1990; 2: 613-618. 15. Tervit HR, Goold PG. Deep freezing of sheep embryos. Theriogenology 1984; 21:268 abstr. 16. Thibier M. The IETS statistics of embryo transfers in livestock in the world for the year 1999: A new record for bovine in vivo-derived embryos transferred. Embryo Transfer Newsletter 2000; 18: 24-28. 17. Vajta G, Holm P, Kuwayama M, Booth P J, Jacobsen H, Greve T, Callesen H. Open Pulled Straw (OPS) vitrification: a new wa3' to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev 1998; 51: 53-58. 18. Vajta G. Vitrification of the oocytes and embryos of domestic animals. Anim Reprod Sci. 2000 Jul 2; 60-61:357-64. 19. Wagtendonk-de Leeuw A M v, Daas J H G d, Rall W F. Field trial to compare pregnancy rates of bovine embryo cryopreservation methods: vitrification and one-step dilution versus slow freezing and three-step dilution. Theriogenology 1997; 48: 10711084.