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Cryopreservation of porcine embryos: state of the art
Livestock Production Science 83 (2003) 73–83 www.elsevier.com / locate / livprodsci
Cryopreservation of porcine embryos: state of the art q ´ Franc¸oise Berthelot a , *, Franc¸oise Martinat-Botte´ a , Gabor Vajta b , Michel Terqui a a
Physiologie de la Reproduction et des Comportements, UMR INRA-CNRS-Universite´ Franc¸ois Rabelais, 37380 Nouzilly, France b Section for Reproductive Biology, Danish Institute of Agricultural Sciences, DK-8830 Tjele, Denmark Received 19 February 2002; received in revised form 9 January 2003; accepted 14 January 2003
Abstract Long term storage of porcine embryos would be worthwhile both for the preservation of genetic resources and for exportation of genotypes around the world. Three methods are available: freezing, classical vitrification and Open Pulled Straw vitrification (OPS). The efficiency of these methods were mainly evaluated in vitro by the development of morulae and blastocysts and in vivo by the birth rate after transfer into recipients. Freezing involved addition of glycerol as the cryoprotectant, the temperature is slowly decreased with, at least one step. Despite some development in vitro, the in vivo survival after transfer of embryos up to the peri-hatching stage is lower than 5%. Ice crystal formation was considered to be responsible for the main damages to embryos. Vitrification that allows direct transition between liquid and vitreous phase was the second method developed. When combined with pre and post-cryopreservation treatments of the embryos, the in vivo survival rate rises up to 18.5%. The OPS method is characterised by a very high speed of vitrification and without any treatment of embryos, the in vivo survival of embryos was close to 30%. In conclusion, methods are now available for cryopreservation of porcine embryos and further improvements are expected for a better efficiency and practicability. 2003 Elsevier B.V. All rights reserved. Keywords: Cryopreservation; Pig-reproduction
1. Introduction There are two major reasons to cryopreserve porcine embryos. Firstly, as a consequence of the major effort to improve pig productivity and selections for lean meat, many breeds of domestic pigs have not been q
This review was presented in the Session Reproduction Technology in the Pig at the EAAP 2001 Meeting in Budapest *Corresponding author. Tel.: 1 33-247-427-7937; fax: 1 33247-427-743. E-mail address: [email protected] (F. Berthelot).
appropriately maintained and are now endangered in Europe and in many other countries (Simon and Buchenauer, 1993) Methods of semen preservation by freezing have been established (Labroue et al., 2000), however by using artificial insemination, the recovery of a breed will be long and expensive. Porcine embryo banking using cryopreservation will be a more efficient and economical way to preserve biodiversity. Secondly due to the worldwide competition, pig breeding companies are obliged to transport live pigs. This transportation is very expensive, and not without risk to animal and human health and also not
0301-6226 / 03 / $ – see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016 / S0301-6226(03)00038-1
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F. Berthelot et al. / Livestock Production Science 83 (2003) 73–83
always in the interest of the animal’s welfare. Until recently, only fresh embryos could be transported, however the storage of fresh embryos is limited to 24 h. Porcine embryos are extremely sensitive to chilling, this sensitivity has been attributed to lipids droplets contents (Dobrinsky, 1997). Cryopreservation of porcine embryos remained, until the end of the last century, a dream for the pig industry and a nightmare for researchers, despite a lot of efforts to freeze embryos in cryoprotectant medium with slow cooling rate. However rapid progress has been made using vitrification which involves a very rapid cooling rate. According to the recommendation of the International Embryo Transfer Society (IETS) regarding international transportation, embryos must be collected, frozen and maintained with an intact zona pellucida (Stringfellow and Seidel, 1998). The zona pellucida is known to protect embryos from contamination with most pathogenic agents. This constraint limits the stage of development to morulae and blastocysts at the time of collection and cryopreservation. This paper gives an overview of the methods described for porcine embryo collection, cryopreservation and viability assessment. The three methods currently used for cryopreservation of porcine embryo (slow freezing, vitrification and OPS vitrification) are compared.
2. Porcine embryo production The main stages of porcine embryo development prior to implantation are similar to that of bovine or caprine species. However the timing of embryonic development, composition and requirements for in vitro development are different for the porcine embryo.
2.1. Embryo development Ovulation occurs at a mean time of 40 h after onset of oestrus in non-gonadotrophin stimulated females (Terqui et al., 2000). The first cleavage of the eggs occurs 36–48 h after first insemination (i.e. 36–48 h of pregnancy; day 0: first insemination) and, by day 3 of pregnancy, the embryo reaches the 4–6
cell stage, leaves the oviduct and enters the uterine horn. Hatching takes place between days 6 and 7 of pregnancy. After which the blastocyts are still spherical or slightly ovoid. Between days 11 and 12, they undergo an important elongation stage (Martinat-Botte´ et al., 2000). This chronology of events may be different according to genotype (Terqui et al., 1992). To avoid the risk of collecting hatching blastocysts, it is recommended to collect embryos on days 5 and 6 of pregnancy. At this stage morulae and blastocysts are often collected together.
2.2. Assessment of embryo quality after thawing 2.2.1. Morphology The microscopic evaluation of the embryo after thawing might not be a valid criterion of quality or viability as the morphology may change significantly due to recovery from cold, osmotic and toxic shocks. Cryopreservation-related damage of the embryonic cytoskeleton (microtubules, microfilaments) has been observed by confocal microscopy (Dobrinsky, 1996; Hyttel et al., 2000; Vajta et al., 1997). Chromosome aberration has also been assessed using fluorescent in situ hybridisation (Viuff et al., 2000). 2.2.2. In vitro development The best method to evaluate the quality and viability of the embryos after cryopreservation is to culture them in vitro for 24–48 h. As far as morulae and non-hatched blastocysts are concerned, the most informatived criterion is the hatching rate. For hatched blastocysts, diameter, number of normal and dead cells found after staining with propidium iodide have been used to determine their quality (Berthelot and Terqui, 1996; Cameron et al., 1992; Dobrinsky, 1997; Dobrinsky and Johnson, 1994; Nagashima et al., 1999). 2.2.3. In vivo development Transfer of embryos to recipient and the determination of the number of piglets born is the most precise method to evaluate the efficiency of cryopreservation. Most embryo transfers in pigs have required surgical procedures (Cameron et al., 1989; Polge, 1982). Recipients should be oestrous asynchronous
F. Berthelot et al. / Livestock Production Science 83 (2003) 73–83
(224 h) compared to the donor females. The number of embryos ( , 10) transferred into one recipient is critical for survival rate (Martinat-Botte´ et al., 1992; Polge, 1982). Intrauterine crowding has a negative effect on foetal survival due to the limitation of available uterine space for foetal development (Dziuk, 1991). All the embryos transferred into one recipient should be at the same stage of development. However in many studies, the total number of transferred embryos is not constant between transfers and not always indicated in publication and for computing survival rates reported in Tables 2 and 4, some values were only an estimation. The breed of the recipient is also an important factor of success. In two studies (Berthelot et al., 2000, 2001a) Meishan recipients were used since they resulted in a better embryonic survival (Martinat-Botte´ et al., 1992). Results obtained for non-surgical transfer (Hazeleger and Kemp, 1999; Martinez et al., 2001, 2002) indicate that this method may soon be suitable for transferring porcine embryos.
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3. Embryo freezing
3.1. Principle Programmed cooling systems using a slow-rate of freezing are designed to maintain equilibrium between the various sources of injury together with low cryoprotectant concentrations (minimizing osmotic and toxic damage) and controlled ice formation. As a consequence, concentration and partial ice-free solidification of solutions occur in and close to the embryo. During thawing, the cryoprotectant is eliminated stepwise with sucrose, protecting the embryos against osmotic shock (Nagashima et al., 1992, 1994).
3.2. In vitro results As indicated in Table 1, the survival of frozen embryos after 24–48 h of in vitro culture is highly variable (0–50%), (Cameron et al., 1992; Fujino et
Table 1 Freezing with slow cooling: in vitro development of porcine embryos after freezing to 2 196 8C and thawing according to the stage of embryos, cryoprotectant and treatment. Bold characters emphasise the stages of development when embryos were still in uterus and within intact zona pellucida Stage of embryos
Final concentration of cryoprotectant
Embryo treatment
Number of frozen embryos
% of survival of frozen embryos
Refs.
2–4 cells
1.5 M P
Centrifuged 1 delipated
64
13 . 8cells
Nagashima et al. (1994)
Morula
1.5 M P 1 0.1 M S
Delipated
27
63
Nagashima et al. (1999)
Blastocyst
1.5 M G 1.5 M P
No Centrifuged
30 31
0 61
Nagashima et al. (1992) Nagashima et al. (1999)
Expanded Blastocyst
1.5 M G 1.5 M G 1.5 M G 1.5 M G 1 Egg yolk 1.5 M G
No No No No No
57 66 21 79 36
2 24 10 3 22
Kashiwasaki et al. (1991) Nagashima et al. (1992) Cameron et al. (1992) Fujino et al. (1993) ¨ et al. (1996) Modl
Peri-hatching
1.5 M G 1.5 M G
No No
38 19
0 26
Kashiwasaki et al. (1991) ¨ et al. (1996) Modl
Hatched
1.5 1.5 1.5 1.5 1.5
No No No No No
82 85 26 46 34
11 19 12 22 50
Kashiwasaki et al. (1991) Nagashima et al. (1992) Cameron et al. (1992) Fujino et al. (1993) ¨ et al. (1996) Modl
M M M M M
G G G G 1 Egg yolk G
P, Propanediol; S, Sucrose; G, Glycerol.
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¨ et al., al., 1993; Kashiwasaki et al., 1991; Modl 1996; Nagashima et al., 1992, 1994, 1999). However frozen / thawed hatched blastocysts have a higher and more reproducible survival rate than blastocysts and expanded blastocysts. Thus, some authors culture in vitro blastocysts to the hatched stage and then freeze them. It has been shown that centrifugation at 13 000– 15 000 g and removal of the lipid phase by aspiration before freezing allows in vitro survival of 2–8 cells embryos (Nagashima et al., 1994) (Table 1). These authors defined micro-manipulated to aspirate the lipids as ‘delipated’.
3.3. In vivo results Reports of results of in vivo survival is very limited as indicated in Table 2. Indeed only 18 transfers have been performed after freezing with slow cooling (Fujino et al., 1993; Kashiwasaki et al., ¨ 1991; Kosarcic et al., 1995; Modl et al., 1996; Nagashima et al., 1995). Furthermore, only two piglets were born after transfer of zona pellucida intact embryos into recipients (Kosarcic et al., 1995). This is much lower than might be expected from reported in vitro results (Table 1).
3.4. Conclusions The various freezing methods using slow cooling demonstrate that porcine embryos can be cryopreserved but none of the methods is efficient enough for practical application.
4. Vitrification
4.1. Principle Vitrification avoids ice crystal formation. By using high cryoprotectant concentration with high cooling and warming rates direct transition between the liquid phase to the amorphous vitreous phase, and vice versa is achieved. One critical point of vitrification is the high cryoprotectant concentrations which may be deleterious for the embryos as a result of increasing osmotic and toxic damage. On the other hand, the high speed of cooling and warming results in a quick passage through the dangerous temperatures above and around 0 8C, minimizing the chilling injury (Dobrinsky and Johnson, 1993a,b; Rall, 1987; Weber et al., 1992). Warming after vitrification is not fundamentally different from thawing after slow
Table 2 Slow cooling: in vivo development of frozen / thawed porcine embryos after transfer into recipients according to the stage of embryos, cryoprotectant and treatment. Bold characters emphasise the stages of development when embryos were still in uterus and within intact zona pellucida Stage of embryos
Concentration of cryoprotectant
Embryo treatment
Number of pregnancy / Number of transfers
Piglets born / Number of embryos transferred
Refs.
2–4 cells
1.5 M P 1 0.1 M S
Centrifuged 1 delipated
1/1
3 / 39
Nagashima et al. (1995)
Morula
1.5 M G
No
1/3
2 / 42
Kosarcic et al. (1995)
Expanded blastocyst
1.5 M G 1 Egg yolk
No
0/2
0 / 30
Fujino et al. (1993)
Peri-hatching
1.5 M G 1 Egg yolk 1.5 M G
No No
1/4 2/7
1 / 27 8 / 141
Fujino et al. (1993) ¨ et al. (1996) Modl
Hatched
1.5 M G
No
1/1
4 / 32
Kashiwasaki et al. (1991)
Blastocyst
P, Propanediol; S, Sucrose; G, Glycerol.
F. Berthelot et al. / Livestock Production Science 83 (2003) 73–83
freezing and includes also the dilution of cryoprotectant with sucrose solution. Two types of vitrification have been studied for pig embryos. The classical one, performed in standard insemination straws, with a cooling rate around 2500 8C per min (Mazur et al., 1992; Rall and Fahy, 1985) and the Open Pulled Straw (OPS) method (Vajta et al., 1997). In the OPS method the temperature drops down faster, at approximately 18 000 8C per min.
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cytochalasin B improves the in vitro survival of vitrified / thawed embryos. This treatment de-polymerised the microfilaments and reduces the damages to the cytoskeleton caused by cryoprotectant and vitrification. The in vitro survival of expanded and hatched blastocysts has been shown to be higher than that of blastocysts (Dobrinsky and Johnson, 1994; Dobrinsky et al., 1997; Kobayashi et al., 1998b; Nagashima et al., 1999).
4.2. Classical vitrification This technique recently published by (Dobrinsky et al., 2001) for blastocyst included pre- and posttreatments of the blastocysts with cytochalasin B and pronase, respectively. The cryoprotectant used was glycerol at 6.5 M. The different steps before plunging into LN 2 took 66 min and warming 199 min.
4.2.1. In vitro results As indicated in Table 3 a pre-treatment with
4.2.2. In vivo results From the results presented in Table 4, a total of 1278 vitrified embryos were transferred to 46 recipients, resulting in the birth of 125 piglets (Dobrinsky et al., 1998, 2000, 2001; Kobayashi et al., 1998a,b). The highest survival was obtained with a technique that included a pre-treatment with cytochalasin B and a post-thawing treatment to remove the zona pellucida (Dobrinsky et al., 2001). Digestion of the
Table 3 Vitrification: in vitro development of vitrified / thawed embryos according to the stage of embryo development, cryoprotectant and treatment. Bold characters emphasise the stages of development when embryos were still in uterus and within intact zona pellucida Stage of embryos
Concentration of cryoprotectant
Embryo treatment
2–4 cells
7 M EG 1 1 M S 7 M EG 1 1 M S
Centrifuged 1 delipated Centrifuged
Blastocyst
6.5 M G 1 BSA 6.5 M G 1 BSA 8 M EG 1 7 % PVP
Expanded blastocyst
Number of frozen embryos evaluated
% of survival of frozen embryos
Refs.
17
50
Nagashima et al. (1999)
17
9
Nagashima et al. (1999)
No Cytochalasin No
59 17 128
0 6 23
Dobrinsky and Johnson (1994) Dobrinsky et al. (1997) Kobayashi et al. (1998b)
6.5 M G 1 BSA 6.5 M G 1 BSA 8 M EG 1 7% PVP
No Cytochalasin No
64 25 106
27 60 54
Dobrinsky and Johnson (1994) Dobrinsky et al. (1997) Kobayashi et al. (1998b)
6.5 M G 1 BSA 6.5 M G 1 BSA 8 M EG 1 7% PVP
No Cytochalasin No
84 70 131
39 74 35
Dobrinsky and Johnson (1994) Dobrinsky et al. (1997) Kobayashi et al. (1998b)
Morula
Peri-hatching Hatched
EG, Ethylene glycol; G, Glycerol; S, Sucrose; BSA, Bovine serum albumin (g / l); PVP, Polyvinyl pyrrolidone (g / l).
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Table 4 Vitrification: in vivo development of vitrified / thawed porcine embryos after transfer into recipients according to the stage of embryo development, cryoprotectant and treatment. Bold characters emphasise the stages of development when embryos were still in uterus and within intact zona pellucida Stage of embryos
Concentration of cryoprotectant
Embryo treatment
Number of pregnancy / Number of transfer
Piglets born / Number of embryos transferred
Refs.
Blastocyst
6.5 M G 1 BSA
Cytochalasin 1 centrifugation 1 pronase
9 / 11
61 / 330
Dobrinsky et al. (2001)
Expanded blastocyst
8 M EG 1 7% PVP
No
1/1
4 / 20
Kobayashi et al. (1998b)
Peri-hatching
8 M EG 1 7% PVP 8 M EG 1 7% PVP
No No
0/2 3 / 15
0 / 44 11 / 338
Kobayashi et al. (1998b) Kobayashi et al. (1998a)
Hatched
6.5 M G 1 BSA 6.5 M G 1 BSA
Cytochalasin Cytochalasin
2/5 6 / 12
10 / 165 39 / 381
Dobrinsky et al. (1998) Dobrinsky et al. (2000)
2–4 cells Morula
EG, Ethylene glycol; G, Glycerol; BSA, Bovine serum albumin (g / l); PVP, Polyvinyl pyrrolidone (g / l).
zona pellucida by pronase is assumed to enhance the hatching rate.
4.2.3. Conclusions These results indicate that classical vitrification is much more efficient than freezing with slow cooling rates. The most efficient technique for blastocysts gives a high pregnancy rate ( | 80%) but a low rate of birth of piglets after transfer of selected embryos (Dobrinsky et al., 2001). However this method includes several steps, mainly after warming, that would need to be done in a laboratory and by a qualified technician. 4.3. High speed vitrification with open pulled straws 4.3.1. Principle The cooling rate when a straw is plunged into liquid nitrogen depends upon several factors including volume of medium around the embryos, internal diameter of the straw, thickness of the wall of the straw, direct contact between liquid nitrogen and the embryo-containing medium.
The Open Pulled Straw technique reduces these parameters. A narrow plastic tube is prepared by heating a 0.25-ml insemination straw and pulling it to half its original diameter. Embryos in 1–2 ml drops are loaded using the capillary effect. The cooling rate by plunging the straw directly into liquid nitrogen is approximately 18 000 8C / min (Vajta, 2000; Vajta et al., 1998a). The time required for equilibration-cooling and warming-rehydration is 7 and 15 min, respectively. Moreover, Vajta made some suggestions about sterile application of OPS vitrification technique (Vajta et al., 1998b) the embryo-containing OPS will be placed into a 0.5-ml plastic straw pre-cooled in LN 2 .
4.3.2. In vitro results Table 5 shows that using the OPS method morulae can also be successfully cryopreserved (Berthelot et al., 2000; Vajta et al., 1997). The rate of in vitro survival of OPS vitrified blastocysts appears to be similar or slightly higher than that obtained with the classical vitrification method (Beebe et al., 2000; Berthelot et al., 2000; Holm et al., 1999; Vajta et al., 1997).
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Table 5 OPS vitrification: in vitro development of OPS vitrified / thawed porcine embryos according to the stage of embryo development, cryoprotectant and treatment. Bold characters emphasise the stages of development when embryos were still in uterus and within intact zona pellucida Stage of embryos
Concentration of cryoprotectant
Embryo treatment
3.2 M EG 1 2.5 M DMSO 1 0.6 M S 3.6 M EG 1 2.8 M DMSO 1 0.6 M S 8 M EG 1 7% PVP
No No
Number of vitrified embryos evaluated
% of survival of frozen embryos
Refs.
10
70
Vajta et al. (1998a)
118
12
Berthelot et al. (2000)
Cytochalasin 1 centrifugation 1 dezoning
77
71
Beebe et al. (2002)
No
29
67
Vajta et al. (1998a)
Cytochalasin Cytochalasin 1 centrifugation No
25 30
0 77
Beebe et al. (2000) Beebe et al. (2000)
94
42
Berthelot et al. (2000)
No
59
71
Holm et al. (1999)
2–4 cells Morula
Blastocyst
3.2 M EG 1 2.5 M DMSO 1 0.6 M S 8 M EG 1 7% PVP 8 M EG 1 7% PVP 3.2 M EG 1 2.5 M DMSO 1 0.6 M S
Expanded blastocyst
3.2 M EG 1 2.5 M DMSO 1 0.6 M S
Peri-hatching Hatched EG, Ethylene glycol; G, Glycerol; DMSO, Dimethyl sulfoxide; S, Sucrose; PVP, Polyvinyl pyrrolidone (g / l).
4.3.3. In vivo results The data presented in Table 6 indicates that a total of 28 recipients became pregnant after transfer of 1237 embryos into 56 recipients and 128 piglets were born. As for classical vitrification all piglets were at normal appearance and the sex ratio was not altered. Live piglets were also obtained after OPS vitrification / warming of morulae (Berthelot et al., 2001a). A high pregnancy rate (90%) and survival rate (30%) after transfer of vitrified / warmed blastocysts were obtained (Berthelot et al., 2002) using TCM hepes buffer for cryoprotectant dilution instead of the phosphate saline buffer previously used (Berthelot et al., 2000). This suggests that the dilution medium is one of the factors important to maintain blastocyst integrity. Treatment of embryos (centrifugation and cytochalasin B) did not seem to improve in vivo survival
rate (Cameron et al., 2000). But, too many variables involved can have interfere on these results (different genotypes, different pre and post freezing treatment). Indeed, the highest survival rate of 30% for vitrified blastocysts was obtained without treatment and without selection of blastocysts after warming (Berthelot et al., 2002). The efficiency of OPS method can be evaluated by the number of offspring obtained after collection, cryopreservation and transfer into recipient. Tables 7 and 8 summarised these data (Berthelot et al., unpublished results). As indicated in Table 7, the collection rate of embryos as a percentage of the number of corpora lutea is less than 100% even after slaughter. Morulae and blastocysts represents 85% of the total collected. As the embryo losses due to manipulations before and after vitrification are very low (3.25%) and since
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Table 6 OPS vitrification: in vivo development of OPS vitrified / thawed porcine embryos after transfer into recipients according to the stage of embryo development, cryoprotectant, medium and treatment. Bold characters emphasise the stages of development when embryos were still in uterus and within intact zona pellucida Stage of embryos
Concentration of cryoprotectant
Embryo treatment
Number of pregnancy / Number of transfer
Piglets born / Number of embryos transferred
Refs.
3.6 M EG 1 2.8 M DMSO 1 0.6 M S 8 M EG 1 7% PVP
No
8 / 10
26 / 200
Berthelot et al. (2001a)
Cytochalasin 1 centrifugation 1 dezoning
3/4
16 / 115
Beebe et al. (2002)
8 M EG 1 7% PVP
Cytochalasin 1 centrifugation No
1/5
5 / 180
Cameron et al. (2000)
11 / 20
38 / 400
Berthelot et al. (2000)
No
8 / 10
59 / 200
Berthelot et al. (2001b)
No
0 / 11
0 / 257
Holm et al. (1999)
2–4 cells Morula
Blastocyst
3.2 M EG 1 2.5 DMSO 1 0.6 M 3.2 M EG 1 2.5 DMSO 1 0.6 M Expanded blastocyst
M S M S
3.2 M EG 1 2.5 M DMSO 1 0.6 M S
Peri-hatching Hatched EG, Ethylene glycol; DMSO, Dimethyl sulfoxide; S, Sucrose; PVP, Polyvinyl pyrrolidone (g / l).
Table 7 Number of Corpora Lutea (CL) and rates of embryo at different stage of development after embryo collection at Day 5–6 of hyperprolific Large White for vitrification by the OPS technique (Berthelot F., Martinat-Botte´ F. and Terqui M., unpublished results) Number of CL per female
Rate of collection in % of CL
Non-fertilised eggs in % of total collected
Morulae in % of total collected
Blastocysts in % of total collected
Hatched blastocysts in % of total collected
19
80
9
14
71
5.6
Losses before vitrification in %
3
3
Losses after vitrification in %
0.25
0.25
Collection
Survival rates from Table 6
no selection of vitrified and thawed embryos was made; more than 96% of collected morulae and blastocysts were transferred (Table 7). Based on these rates, the Table 8 presents the
13
29
results of collection from 20 donors, the number of morulae, blastocysts and piglets that were obtained with the OPS vitrification method (Berthelot et al., 2001b). Twelve recipients were required for transfers
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Table 8 Estimated numbers of embryos, recipients and piglets born after collection of 20 Large White hyperprolific donors based upon rates in Table 7 (Berthelot F., Martinat-Botte´ F. and Terqui M., unpublished results) 43 Morulae Collected 41 Vitrified Morulae transferred 2 recipients for Morula 5 piglets
of morulae and blastocysts and a total of 67 piglets were born representing a little more than three offspring per donor (Table 8).
4.3.4. Conclusions The OPS vitrification method appears to be an efficient technique for cryopreservation of morulae and especially blastocysts. The cooling rate may be a key factor for survival of porcine embryos by vitrification.
5. Improvements in future At present, both vitrification procedures are suitable for the in vitro survival and birth of piglets. These methods could be used at least for gene banking. However improvements are required to reduce the cost and increase the possibility for practical application. One difficulty to improve cryopreservation methods is narrow relationship between in vitro and in vivo survival (see Tables 1–6). As indicated above the medium of dilution of cryoprotectant and the concentration of cryoprotectants are key parameters for the success of vitrification. Increased embryo survival rates might be obtained if medium and cryoprotectant concentration are at optimum. Also, as indicated previously, the ¨ cooling rate is very critical. Minitube (Germany) distributed a system (VitMaster) that supercools liquid nitrogen, avoids vapours and allows cooling rates of up to 100 000 8C / min. Such system might be also beneficial for cryopreservation porcine embryos. A team (Beebe et al., 2002) had obtained 16 piglets but after dezoning 115 morulae before transfer. So far, the thawing procedures of the OPS method has also to be carried out in a laboratory. Direct transfer from the straws after thawing is easy to
216 Blastocysts collected 209 Vitrified blastocysts transferred 10 recipients for blastocysts 62 piglets
perform at farm level especially when it is associated with non-surgical transfer via the cervix (Cuello et al., 2002).
6. General conclusions Although techniques still need further improvement, cryopreservation of porcine embryos has been achieved. Two methods give acceptable results, 29 and 18.5% of in vivo survival, respectively (Berthelot et al., 2001b; Dobrinsky et al., 2001). Cost and practicability of the use of this method to export selected lines and to preserve some endangered breeds (Labroue et al., 2000) with vitrified embryos have to be determined. Attention has to be paid to the physiology of the breed to be cryopreserved. For example, timing of ovulation, stage of embryo development have to be characterized in order to obtain morula or blastocyst production. In the future, pig embryo cryopreservation and transfer will be a practical tool for genetic preservation and exportation.
Acknowledgements The authors thank Professor B. Kemp and EAAP board for the organisation of the Session on Reproduction Technology of the Pig. The authors are grateful to Dr P. Mermillod for reading this manuscript and his suggestions.
References Beebe, L.F.S., Cameron, R.D.A., Blackshaw, A.W., Higgins, A., Nottle, M.B., 2000. Piglets born from vitrified zona-intact blastocysts. Theriogenology 53, 249.
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Beebe, L.F.S., Cameron, R.D.A., Blackshaw, A.W., Verrall, R.G., 2002. Vitrification of zona pellucida intact embryos and birth of piglets using the Vit-Master. In: 17th International Pig Veterinary Society Congress. Ames, Iowa, USA, pp. 64. ´ F., Perreau, C., Locatelli, A., Berthelot, F., Martinat-Botte, Manceau, P., Venturi, E., Terqui, M., 2002. The use of an appropriate vitrification medium allows development of 30% of cryopreserved blastocysts and their birth as live piglets. Pig New Informations 23 (4), 103N–108N. ´ F., Locatelli, A., Perreau, C., Terqui, Berthelot, F., Martinat-Botte, M., 2000. Piglets born after vitrification of embryos using the open pulled straw method. Cryobiology 41, 116–124. ´ F., Perreau, C., Terqui, M., 2001a. Berthelot, F., Martinat Botte, Birth of piglets after OPS vitrification and transfer of compacted morula stage embryos with intact zona pellucida. Reprod. Nutr. Dev. 41, 267–272. ´ F., Perreau, C., Terqui, M., 2001b. Berthelot, F., Martinat-Botte, Birth of piglets after open pulled straw (OPS) vitrification and transfer of intact zona pellucida compacted morulae and unhatched blastocysts. In: 6th International Conference on Pig Reproduction, Columbia, USA, pp. Abstr. 136. Berthelot, F., Terqui, M., 1996. Effects of oxygen,CO2 / pH and medium on in vitro development of individually cultured porcine one- and two cell embryos. Reprod. Nutr. Dev. 36, 241–251. Cameron, R.D.A., Beebe, L.F.S., Blackshaw, A.W., Higgins, A., Nottle, M.B., 2000. Piglets born from vitrified early blastocysts using a simple technique. Aust. Vet. J. 78, 195–196. Cameron, R.D.A., Durack, M., Fogarty, R., Putra, D.K.H., McVeigh, J., 1989. Practical experience with commercial embryo transfer in pigs. Aust. Vet. J. 66, 314–318. Cameron, R.D.A., Lising, R., Nagashima, H., Blackshaw A.W., 1992. Cryopreservation of cultured and uncultured porcine embryos with glycerol and trehalose. In: 12th International Pig Veterinary Society Congress, The Hague, pp. 476. ´ F., Venturi, E., Vazquez, ´ Cuello, C., Berthelot, F., Martinat-Botte, ´ J.M., Roca, J., Martınez, E.A., 2002. Pregnancy rate after non-surgical transfer of vitrified pig embryos. Reproduction Abstract Series 29, Abstr. 52. Dobrinsky, J.R., 1996. Cellular approach to cryopreservation of embryos. Theriogenology 45, 17–26. Dobrinsky, J.R., 1997. Cryopreservation of pig embryos. J. Reprod. Fertil. Suppl. 52, 301–312. Dobrinsky, J.R., Johnson, L.A., 1993a. Development of porcine embryos cryopreserved by vitrification. In: 4th International Conference on Pig Reproduction. Columbia University, Missouri, USA, p. 35. Dobrinsky, J.R., Johnson, L.A., 1993b. Effect of vitrification media on the in vitro development of porcine embryos. Theriogenology 39, 209. Dobrinsky, J.R., Johnson, L.A., 1994. Cryopreservation of porcine embryos by vitrification: a study of in vitro development. Theriogenology 42, 25–35. Dobrinsky, J.R., Long, C.R., Johnson, L.A., 1997. Stability of microfilaments during swine embryo cryopreservation. Theriogenology 47, 343. Dobrinsky, J.R., Nagashima, H., Pursel, V.G., Schreier, L.L., Johnson, L.A., 2001. Cryoconservation of morula and early
blastocyst stage swine embryos: Birth of litters after transfers. Theriogenology 55, 303. Dobrinsky, J.R., Pursel, V.G., Long, C.R., Johnson, L.A., 1998. Birth of normal piglets after cytoskeletal stabilisation of embryos and cryoconservation by vitrification. Theriogenology 49, 166. Dobrinsky, J.R., Pursel, V.G., Long, C.R., Johnson, L.A., 2000. Birth of piglets after transfer of embryos cryopreserved by cytoskeletal stabilization and vitrification. Biol. Reprod. 62, 564–570. Dziuk, P., 1991. Reproduction in the pig. In: Reproduction in Domestic Animals, 4th ed. Academic Press, Inc, pp. 471–489. Fujino, Y., Ujisato, Y., Endo, K., Tomizukz, T., Kojima, T., Oguri, N., 1993. Cryoprotective effect of egg yolk in cryopreservation of porcine embryos. Cryobiology 30, 299–305. Hazeleger, W., Kemp, B., 1999. State of the art in pig embryo transfer. Theriogenology 51, 81–90. Holm, P., Vajta, G., Machaty, Z., Schmidt, M., Prather, R.S., Greve, T., Callesen, H., 1999. Open Pulled Straw (OPS) vitrification of porcine blastocysts: Simple procedure yielding excellent in vitro survival, but so far no piglets following transfer. Cryo Letters 20, 307–310. Hyttel, P., Vajta, G., Callesen, H., 2000. Vitrification of bovine oocytes with the open pulled straw method: ultrastructural consequences. Mol. Reprod. Dev. 56, 80–88. Kashiwasaki, N., Ohtani, S., Nagashima, H., Yamakawa, H., Cheng, W.T.K., Lin, A.-C., Ma, R.C.S., Ogawa, S., 1991. Production of normal piglets from hatched blastocysts frozen at 2 196 8C. Theriogenology 35, 221. Kobayashi, S., Goto, M., Kano, M., Takei, M., Minato, K., Leibo, S.P., 1998a. Farrows or pregnancies by transfer of porcine embryos vitrified at two institutions. Cryobiology 37, 436. Kobayashi, S., Takei, M., Kano, M., Tomita, M., Leibo, S.P., 1998b. Piglets produced by transfer of vitrified porcine embryos after stepwise dilution of cryoprotectants. Cryobiology 36, 20–31. Kosarcic, D., Veselinovic, S., Veselinovic, S., Ivkov, V., Medic, D., Micic, R., Ivkov, O., Kosarcic, S., 1995. Transplantation of thawed deeply frozen embryos in Swine. Macedonian J. Reprod. 1, 23–27. Labroue, F., Luquet, M., Guillouet, P., Bussiere, J.F., Glodek, P., Wemheuer, W., Gandini, G., Pizzi, F., Delgado, J.V., Poto, A., Ollivier, L., 2000. La cryoconservation des races porcines ´ de disparition. La situation en France, en Allemagne, menacees ´ de la Recherche porcine en Italie et en Espagne. In: Journees en France, Paris, pp. 419–427. ´ F., Plat, M., Procureur, R., Terqui, M., 1992. Martinat-Botte, Meishan (MS) as recipient in embryo transfer programs. In: International Symposium on Chinese Pig Breeds, Harbin, China, pp. 561–564. ´ F., Renaud, G., Madec, F., Costiou, P., Terqui, M., Martinat-Botte, 2000. Ultrasonography and reproduction in swine. Principles and practical applications. In: INRA Editions and Hoechst Roussel Vet, Paris, France, pp. 104. Martinez, E.A., Gil, M.A., Rieke, A., Roca, J., Vazquez, J.M., Didion, B.A., Day, B.N., 2002. Non-surgical deep intrauterine embryo transfer in sows. Theriogenology 57, 549. Martinez, E.A., Vazquez, J.M., Roca, J., Lucas, X., Gil, M.A.,
F. Berthelot et al. / Livestock Production Science 83 (2003) 73–83 Vazquez, J.L., 2001. Deep intrauterine insemination and embryo transfer in pigs. Reproduction Supplement 58, 301–311. Mazur, P., Schneider, U., Mahowald, A.P., 1992. Characteristics and kinetics of subzero chilling injury in Drosophila embryos. Cryobiology 29, 39–68. ¨ J., Reichenbach, H.-D., Wolf, E., Brem, G., 1996. DevelopModl, ment of frozen–thawed porcine blastocysts in vitro and in vivo. Vet. Rec. 139, 208–210. Nagashima, H., Cameron, R.D.A., Kuwayama, M., Young, M., Beebe, L.F.S., Blackshaw, A.W., Nottle, M.B., 1999. Survival of porcine delipated oocytes and embryos after cryopreservation by freezing or vitrification. J. Reprod. Dev. 45, 167–176. Nagashima, H., Kashiwasaki, N., Ashman, R., Grupen, C., Seamark, R.F., Nottle, M.B., 1994. Recent advances in cryopreservation of porcine embryos. Theriogenology 41, 113–118. Nagashima, H., Kashiwazaki, N., Ashman, R.J., Grupen, C.G., Nottle, M.B., 1995. Cryopreservation of porcine embryos. Nature 374, 416. Nagashima, H., Yamakawa, H., Nieman, H., 1992. Freezability of porcine blastocysts at different peri-hatching stages. Theriogenology 37, 839–850. Polge, C., 1982. Embryo transplantation and preservation. In: Cole, D.J.A., Foxcroft, G.R. (Eds.), Control of Pig Reproduction. Butterworth Scientific, Nottingham, London, UK, pp. 277–291. Rall, W.F., 1987. Factors affecting the survival of mouse embryos cryopreserved by vitrification. Cryobiology 24, 387–402. Rall, W.F., Fahy, G.M., 1985. Ice-free cryopreservation of mouse embryos at 2 196 8C by vitrification. Nature 313, 573–575. Simon, D.L., Buchenauer, D., 1993. Genetic diversity of European Livestock breeds. In: EAAP publication no. 66. Wageningen, pp. 581.
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Stringfellow, D.A., Seidel, G.E., 1998. Manual of the International Embryo Transfer Society (IETS): A procedural guide and general information for the use of embryo transfer technology emphasizing sanitary precautions. 2nd Ed. Stringfellow D., Seidel, S.M. USA, pp. 67. ´ F., 1992. Mechanisms of Terqui, M., Bazer, F.W., Martinat-Botte, high embryo survival in Meishan gilts. International Symposium on Chinese Pig Breeds, Harbin, China, pp. 52–58. ´ F., 2000. Terqui, M., Guillouet, P., Maurel, M.-C., Martinat-Botte, Relation between peri-oestrus progesterone levels and time of ovulation by echography in pigs and influence of the interval between ovulation and artificial insemination (AI) on litter size. Reprod. Nutr. Dev. 40, 393–404. Vajta, G., 2000. Oocyte and embryo vitrification. Reprod. Domestic Animals Suppl. 6, 45–48. Vajta, G., Holm, P., Booth, P.J., Jacobson, H., Greve, T., Callesen, H., 1998a. Open Pulled Straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol. Reprod. Dev. 51, 53–58. Vajta, G., Holm, P., Greve, T., Callesen, H., 1997. Vitrification of porcine embryos using the open pulled straw (OPS) method. Acta Vet. Scand. 38, 349–352. Vajta, G., Lewis, I.M., Kuwayama, M., Greve, T., Callesen, H., 1998b. Sterile application of the open pulled straw (OPS) vitrification method. Cryo Letters 19, 389–392. Viuff, D., Greve, T., Avery, B., Hyttel, P., Brockhoff, P.B., Thomsen, P.D., 2000. Chromosome aberrations in in vitroproduced bovine embryos at days 2–5 post insemination. Biol. Reprod. 63, 1143–1148. Weber, P.K., McGinnis, L.K., Youngs, C.R., 1992. An evaluation of potential vitrification solutions for cryopreservation of porcine embryos. Theriogenology 37, 321.
Cryopreservation of porcine embryos: state of the art q ´ Franc¸oise Berthelot a , *, Franc¸oise Martinat-Botte´ a , Gabor Vajta b , Michel Terqui a a
Physiologie de la Reproduction et des Comportements, UMR INRA-CNRS-Universite´ Franc¸ois Rabelais, 37380 Nouzilly, France b Section for Reproductive Biology, Danish Institute of Agricultural Sciences, DK-8830 Tjele, Denmark Received 19 February 2002; received in revised form 9 January 2003; accepted 14 January 2003
Abstract Long term storage of porcine embryos would be worthwhile both for the preservation of genetic resources and for exportation of genotypes around the world. Three methods are available: freezing, classical vitrification and Open Pulled Straw vitrification (OPS). The efficiency of these methods were mainly evaluated in vitro by the development of morulae and blastocysts and in vivo by the birth rate after transfer into recipients. Freezing involved addition of glycerol as the cryoprotectant, the temperature is slowly decreased with, at least one step. Despite some development in vitro, the in vivo survival after transfer of embryos up to the peri-hatching stage is lower than 5%. Ice crystal formation was considered to be responsible for the main damages to embryos. Vitrification that allows direct transition between liquid and vitreous phase was the second method developed. When combined with pre and post-cryopreservation treatments of the embryos, the in vivo survival rate rises up to 18.5%. The OPS method is characterised by a very high speed of vitrification and without any treatment of embryos, the in vivo survival of embryos was close to 30%. In conclusion, methods are now available for cryopreservation of porcine embryos and further improvements are expected for a better efficiency and practicability. 2003 Elsevier B.V. All rights reserved. Keywords: Cryopreservation; Pig-reproduction
1. Introduction There are two major reasons to cryopreserve porcine embryos. Firstly, as a consequence of the major effort to improve pig productivity and selections for lean meat, many breeds of domestic pigs have not been q
This review was presented in the Session Reproduction Technology in the Pig at the EAAP 2001 Meeting in Budapest *Corresponding author. Tel.: 1 33-247-427-7937; fax: 1 33247-427-743. E-mail address: [email protected] (F. Berthelot).
appropriately maintained and are now endangered in Europe and in many other countries (Simon and Buchenauer, 1993) Methods of semen preservation by freezing have been established (Labroue et al., 2000), however by using artificial insemination, the recovery of a breed will be long and expensive. Porcine embryo banking using cryopreservation will be a more efficient and economical way to preserve biodiversity. Secondly due to the worldwide competition, pig breeding companies are obliged to transport live pigs. This transportation is very expensive, and not without risk to animal and human health and also not
0301-6226 / 03 / $ – see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016 / S0301-6226(03)00038-1
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F. Berthelot et al. / Livestock Production Science 83 (2003) 73–83
always in the interest of the animal’s welfare. Until recently, only fresh embryos could be transported, however the storage of fresh embryos is limited to 24 h. Porcine embryos are extremely sensitive to chilling, this sensitivity has been attributed to lipids droplets contents (Dobrinsky, 1997). Cryopreservation of porcine embryos remained, until the end of the last century, a dream for the pig industry and a nightmare for researchers, despite a lot of efforts to freeze embryos in cryoprotectant medium with slow cooling rate. However rapid progress has been made using vitrification which involves a very rapid cooling rate. According to the recommendation of the International Embryo Transfer Society (IETS) regarding international transportation, embryos must be collected, frozen and maintained with an intact zona pellucida (Stringfellow and Seidel, 1998). The zona pellucida is known to protect embryos from contamination with most pathogenic agents. This constraint limits the stage of development to morulae and blastocysts at the time of collection and cryopreservation. This paper gives an overview of the methods described for porcine embryo collection, cryopreservation and viability assessment. The three methods currently used for cryopreservation of porcine embryo (slow freezing, vitrification and OPS vitrification) are compared.
2. Porcine embryo production The main stages of porcine embryo development prior to implantation are similar to that of bovine or caprine species. However the timing of embryonic development, composition and requirements for in vitro development are different for the porcine embryo.
2.1. Embryo development Ovulation occurs at a mean time of 40 h after onset of oestrus in non-gonadotrophin stimulated females (Terqui et al., 2000). The first cleavage of the eggs occurs 36–48 h after first insemination (i.e. 36–48 h of pregnancy; day 0: first insemination) and, by day 3 of pregnancy, the embryo reaches the 4–6
cell stage, leaves the oviduct and enters the uterine horn. Hatching takes place between days 6 and 7 of pregnancy. After which the blastocyts are still spherical or slightly ovoid. Between days 11 and 12, they undergo an important elongation stage (Martinat-Botte´ et al., 2000). This chronology of events may be different according to genotype (Terqui et al., 1992). To avoid the risk of collecting hatching blastocysts, it is recommended to collect embryos on days 5 and 6 of pregnancy. At this stage morulae and blastocysts are often collected together.
2.2. Assessment of embryo quality after thawing 2.2.1. Morphology The microscopic evaluation of the embryo after thawing might not be a valid criterion of quality or viability as the morphology may change significantly due to recovery from cold, osmotic and toxic shocks. Cryopreservation-related damage of the embryonic cytoskeleton (microtubules, microfilaments) has been observed by confocal microscopy (Dobrinsky, 1996; Hyttel et al., 2000; Vajta et al., 1997). Chromosome aberration has also been assessed using fluorescent in situ hybridisation (Viuff et al., 2000). 2.2.2. In vitro development The best method to evaluate the quality and viability of the embryos after cryopreservation is to culture them in vitro for 24–48 h. As far as morulae and non-hatched blastocysts are concerned, the most informatived criterion is the hatching rate. For hatched blastocysts, diameter, number of normal and dead cells found after staining with propidium iodide have been used to determine their quality (Berthelot and Terqui, 1996; Cameron et al., 1992; Dobrinsky, 1997; Dobrinsky and Johnson, 1994; Nagashima et al., 1999). 2.2.3. In vivo development Transfer of embryos to recipient and the determination of the number of piglets born is the most precise method to evaluate the efficiency of cryopreservation. Most embryo transfers in pigs have required surgical procedures (Cameron et al., 1989; Polge, 1982). Recipients should be oestrous asynchronous
F. Berthelot et al. / Livestock Production Science 83 (2003) 73–83
(224 h) compared to the donor females. The number of embryos ( , 10) transferred into one recipient is critical for survival rate (Martinat-Botte´ et al., 1992; Polge, 1982). Intrauterine crowding has a negative effect on foetal survival due to the limitation of available uterine space for foetal development (Dziuk, 1991). All the embryos transferred into one recipient should be at the same stage of development. However in many studies, the total number of transferred embryos is not constant between transfers and not always indicated in publication and for computing survival rates reported in Tables 2 and 4, some values were only an estimation. The breed of the recipient is also an important factor of success. In two studies (Berthelot et al., 2000, 2001a) Meishan recipients were used since they resulted in a better embryonic survival (Martinat-Botte´ et al., 1992). Results obtained for non-surgical transfer (Hazeleger and Kemp, 1999; Martinez et al., 2001, 2002) indicate that this method may soon be suitable for transferring porcine embryos.
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3. Embryo freezing
3.1. Principle Programmed cooling systems using a slow-rate of freezing are designed to maintain equilibrium between the various sources of injury together with low cryoprotectant concentrations (minimizing osmotic and toxic damage) and controlled ice formation. As a consequence, concentration and partial ice-free solidification of solutions occur in and close to the embryo. During thawing, the cryoprotectant is eliminated stepwise with sucrose, protecting the embryos against osmotic shock (Nagashima et al., 1992, 1994).
3.2. In vitro results As indicated in Table 1, the survival of frozen embryos after 24–48 h of in vitro culture is highly variable (0–50%), (Cameron et al., 1992; Fujino et
Table 1 Freezing with slow cooling: in vitro development of porcine embryos after freezing to 2 196 8C and thawing according to the stage of embryos, cryoprotectant and treatment. Bold characters emphasise the stages of development when embryos were still in uterus and within intact zona pellucida Stage of embryos
Final concentration of cryoprotectant
Embryo treatment
Number of frozen embryos
% of survival of frozen embryos
Refs.
2–4 cells
1.5 M P
Centrifuged 1 delipated
64
13 . 8cells
Nagashima et al. (1994)
Morula
1.5 M P 1 0.1 M S
Delipated
27
63
Nagashima et al. (1999)
Blastocyst
1.5 M G 1.5 M P
No Centrifuged
30 31
0 61
Nagashima et al. (1992) Nagashima et al. (1999)
Expanded Blastocyst
1.5 M G 1.5 M G 1.5 M G 1.5 M G 1 Egg yolk 1.5 M G
No No No No No
57 66 21 79 36
2 24 10 3 22
Kashiwasaki et al. (1991) Nagashima et al. (1992) Cameron et al. (1992) Fujino et al. (1993) ¨ et al. (1996) Modl
Peri-hatching
1.5 M G 1.5 M G
No No
38 19
0 26
Kashiwasaki et al. (1991) ¨ et al. (1996) Modl
Hatched
1.5 1.5 1.5 1.5 1.5
No No No No No
82 85 26 46 34
11 19 12 22 50
Kashiwasaki et al. (1991) Nagashima et al. (1992) Cameron et al. (1992) Fujino et al. (1993) ¨ et al. (1996) Modl
M M M M M
G G G G 1 Egg yolk G
P, Propanediol; S, Sucrose; G, Glycerol.
F. Berthelot et al. / Livestock Production Science 83 (2003) 73–83
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¨ et al., al., 1993; Kashiwasaki et al., 1991; Modl 1996; Nagashima et al., 1992, 1994, 1999). However frozen / thawed hatched blastocysts have a higher and more reproducible survival rate than blastocysts and expanded blastocysts. Thus, some authors culture in vitro blastocysts to the hatched stage and then freeze them. It has been shown that centrifugation at 13 000– 15 000 g and removal of the lipid phase by aspiration before freezing allows in vitro survival of 2–8 cells embryos (Nagashima et al., 1994) (Table 1). These authors defined micro-manipulated to aspirate the lipids as ‘delipated’.
3.3. In vivo results Reports of results of in vivo survival is very limited as indicated in Table 2. Indeed only 18 transfers have been performed after freezing with slow cooling (Fujino et al., 1993; Kashiwasaki et al., ¨ 1991; Kosarcic et al., 1995; Modl et al., 1996; Nagashima et al., 1995). Furthermore, only two piglets were born after transfer of zona pellucida intact embryos into recipients (Kosarcic et al., 1995). This is much lower than might be expected from reported in vitro results (Table 1).
3.4. Conclusions The various freezing methods using slow cooling demonstrate that porcine embryos can be cryopreserved but none of the methods is efficient enough for practical application.
4. Vitrification
4.1. Principle Vitrification avoids ice crystal formation. By using high cryoprotectant concentration with high cooling and warming rates direct transition between the liquid phase to the amorphous vitreous phase, and vice versa is achieved. One critical point of vitrification is the high cryoprotectant concentrations which may be deleterious for the embryos as a result of increasing osmotic and toxic damage. On the other hand, the high speed of cooling and warming results in a quick passage through the dangerous temperatures above and around 0 8C, minimizing the chilling injury (Dobrinsky and Johnson, 1993a,b; Rall, 1987; Weber et al., 1992). Warming after vitrification is not fundamentally different from thawing after slow
Table 2 Slow cooling: in vivo development of frozen / thawed porcine embryos after transfer into recipients according to the stage of embryos, cryoprotectant and treatment. Bold characters emphasise the stages of development when embryos were still in uterus and within intact zona pellucida Stage of embryos
Concentration of cryoprotectant
Embryo treatment
Number of pregnancy / Number of transfers
Piglets born / Number of embryos transferred
Refs.
2–4 cells
1.5 M P 1 0.1 M S
Centrifuged 1 delipated
1/1
3 / 39
Nagashima et al. (1995)
Morula
1.5 M G
No
1/3
2 / 42
Kosarcic et al. (1995)
Expanded blastocyst
1.5 M G 1 Egg yolk
No
0/2
0 / 30
Fujino et al. (1993)
Peri-hatching
1.5 M G 1 Egg yolk 1.5 M G
No No
1/4 2/7
1 / 27 8 / 141
Fujino et al. (1993) ¨ et al. (1996) Modl
Hatched
1.5 M G
No
1/1
4 / 32
Kashiwasaki et al. (1991)
Blastocyst
P, Propanediol; S, Sucrose; G, Glycerol.
F. Berthelot et al. / Livestock Production Science 83 (2003) 73–83
freezing and includes also the dilution of cryoprotectant with sucrose solution. Two types of vitrification have been studied for pig embryos. The classical one, performed in standard insemination straws, with a cooling rate around 2500 8C per min (Mazur et al., 1992; Rall and Fahy, 1985) and the Open Pulled Straw (OPS) method (Vajta et al., 1997). In the OPS method the temperature drops down faster, at approximately 18 000 8C per min.
77
cytochalasin B improves the in vitro survival of vitrified / thawed embryos. This treatment de-polymerised the microfilaments and reduces the damages to the cytoskeleton caused by cryoprotectant and vitrification. The in vitro survival of expanded and hatched blastocysts has been shown to be higher than that of blastocysts (Dobrinsky and Johnson, 1994; Dobrinsky et al., 1997; Kobayashi et al., 1998b; Nagashima et al., 1999).
4.2. Classical vitrification This technique recently published by (Dobrinsky et al., 2001) for blastocyst included pre- and posttreatments of the blastocysts with cytochalasin B and pronase, respectively. The cryoprotectant used was glycerol at 6.5 M. The different steps before plunging into LN 2 took 66 min and warming 199 min.
4.2.1. In vitro results As indicated in Table 3 a pre-treatment with
4.2.2. In vivo results From the results presented in Table 4, a total of 1278 vitrified embryos were transferred to 46 recipients, resulting in the birth of 125 piglets (Dobrinsky et al., 1998, 2000, 2001; Kobayashi et al., 1998a,b). The highest survival was obtained with a technique that included a pre-treatment with cytochalasin B and a post-thawing treatment to remove the zona pellucida (Dobrinsky et al., 2001). Digestion of the
Table 3 Vitrification: in vitro development of vitrified / thawed embryos according to the stage of embryo development, cryoprotectant and treatment. Bold characters emphasise the stages of development when embryos were still in uterus and within intact zona pellucida Stage of embryos
Concentration of cryoprotectant
Embryo treatment
2–4 cells
7 M EG 1 1 M S 7 M EG 1 1 M S
Centrifuged 1 delipated Centrifuged
Blastocyst
6.5 M G 1 BSA 6.5 M G 1 BSA 8 M EG 1 7 % PVP
Expanded blastocyst
Number of frozen embryos evaluated
% of survival of frozen embryos
Refs.
17
50
Nagashima et al. (1999)
17
9
Nagashima et al. (1999)
No Cytochalasin No
59 17 128
0 6 23
Dobrinsky and Johnson (1994) Dobrinsky et al. (1997) Kobayashi et al. (1998b)
6.5 M G 1 BSA 6.5 M G 1 BSA 8 M EG 1 7% PVP
No Cytochalasin No
64 25 106
27 60 54
Dobrinsky and Johnson (1994) Dobrinsky et al. (1997) Kobayashi et al. (1998b)
6.5 M G 1 BSA 6.5 M G 1 BSA 8 M EG 1 7% PVP
No Cytochalasin No
84 70 131
39 74 35
Dobrinsky and Johnson (1994) Dobrinsky et al. (1997) Kobayashi et al. (1998b)
Morula
Peri-hatching Hatched
EG, Ethylene glycol; G, Glycerol; S, Sucrose; BSA, Bovine serum albumin (g / l); PVP, Polyvinyl pyrrolidone (g / l).
F. Berthelot et al. / Livestock Production Science 83 (2003) 73–83
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Table 4 Vitrification: in vivo development of vitrified / thawed porcine embryos after transfer into recipients according to the stage of embryo development, cryoprotectant and treatment. Bold characters emphasise the stages of development when embryos were still in uterus and within intact zona pellucida Stage of embryos
Concentration of cryoprotectant
Embryo treatment
Number of pregnancy / Number of transfer
Piglets born / Number of embryos transferred
Refs.
Blastocyst
6.5 M G 1 BSA
Cytochalasin 1 centrifugation 1 pronase
9 / 11
61 / 330
Dobrinsky et al. (2001)
Expanded blastocyst
8 M EG 1 7% PVP
No
1/1
4 / 20
Kobayashi et al. (1998b)
Peri-hatching
8 M EG 1 7% PVP 8 M EG 1 7% PVP
No No
0/2 3 / 15
0 / 44 11 / 338
Kobayashi et al. (1998b) Kobayashi et al. (1998a)
Hatched
6.5 M G 1 BSA 6.5 M G 1 BSA
Cytochalasin Cytochalasin
2/5 6 / 12
10 / 165 39 / 381
Dobrinsky et al. (1998) Dobrinsky et al. (2000)
2–4 cells Morula
EG, Ethylene glycol; G, Glycerol; BSA, Bovine serum albumin (g / l); PVP, Polyvinyl pyrrolidone (g / l).
zona pellucida by pronase is assumed to enhance the hatching rate.
4.2.3. Conclusions These results indicate that classical vitrification is much more efficient than freezing with slow cooling rates. The most efficient technique for blastocysts gives a high pregnancy rate ( | 80%) but a low rate of birth of piglets after transfer of selected embryos (Dobrinsky et al., 2001). However this method includes several steps, mainly after warming, that would need to be done in a laboratory and by a qualified technician. 4.3. High speed vitrification with open pulled straws 4.3.1. Principle The cooling rate when a straw is plunged into liquid nitrogen depends upon several factors including volume of medium around the embryos, internal diameter of the straw, thickness of the wall of the straw, direct contact between liquid nitrogen and the embryo-containing medium.
The Open Pulled Straw technique reduces these parameters. A narrow plastic tube is prepared by heating a 0.25-ml insemination straw and pulling it to half its original diameter. Embryos in 1–2 ml drops are loaded using the capillary effect. The cooling rate by plunging the straw directly into liquid nitrogen is approximately 18 000 8C / min (Vajta, 2000; Vajta et al., 1998a). The time required for equilibration-cooling and warming-rehydration is 7 and 15 min, respectively. Moreover, Vajta made some suggestions about sterile application of OPS vitrification technique (Vajta et al., 1998b) the embryo-containing OPS will be placed into a 0.5-ml plastic straw pre-cooled in LN 2 .
4.3.2. In vitro results Table 5 shows that using the OPS method morulae can also be successfully cryopreserved (Berthelot et al., 2000; Vajta et al., 1997). The rate of in vitro survival of OPS vitrified blastocysts appears to be similar or slightly higher than that obtained with the classical vitrification method (Beebe et al., 2000; Berthelot et al., 2000; Holm et al., 1999; Vajta et al., 1997).
F. Berthelot et al. / Livestock Production Science 83 (2003) 73–83
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Table 5 OPS vitrification: in vitro development of OPS vitrified / thawed porcine embryos according to the stage of embryo development, cryoprotectant and treatment. Bold characters emphasise the stages of development when embryos were still in uterus and within intact zona pellucida Stage of embryos
Concentration of cryoprotectant
Embryo treatment
3.2 M EG 1 2.5 M DMSO 1 0.6 M S 3.6 M EG 1 2.8 M DMSO 1 0.6 M S 8 M EG 1 7% PVP
No No
Number of vitrified embryos evaluated
% of survival of frozen embryos
Refs.
10
70
Vajta et al. (1998a)
118
12
Berthelot et al. (2000)
Cytochalasin 1 centrifugation 1 dezoning
77
71
Beebe et al. (2002)
No
29
67
Vajta et al. (1998a)
Cytochalasin Cytochalasin 1 centrifugation No
25 30
0 77
Beebe et al. (2000) Beebe et al. (2000)
94
42
Berthelot et al. (2000)
No
59
71
Holm et al. (1999)
2–4 cells Morula
Blastocyst
3.2 M EG 1 2.5 M DMSO 1 0.6 M S 8 M EG 1 7% PVP 8 M EG 1 7% PVP 3.2 M EG 1 2.5 M DMSO 1 0.6 M S
Expanded blastocyst
3.2 M EG 1 2.5 M DMSO 1 0.6 M S
Peri-hatching Hatched EG, Ethylene glycol; G, Glycerol; DMSO, Dimethyl sulfoxide; S, Sucrose; PVP, Polyvinyl pyrrolidone (g / l).
4.3.3. In vivo results The data presented in Table 6 indicates that a total of 28 recipients became pregnant after transfer of 1237 embryos into 56 recipients and 128 piglets were born. As for classical vitrification all piglets were at normal appearance and the sex ratio was not altered. Live piglets were also obtained after OPS vitrification / warming of morulae (Berthelot et al., 2001a). A high pregnancy rate (90%) and survival rate (30%) after transfer of vitrified / warmed blastocysts were obtained (Berthelot et al., 2002) using TCM hepes buffer for cryoprotectant dilution instead of the phosphate saline buffer previously used (Berthelot et al., 2000). This suggests that the dilution medium is one of the factors important to maintain blastocyst integrity. Treatment of embryos (centrifugation and cytochalasin B) did not seem to improve in vivo survival
rate (Cameron et al., 2000). But, too many variables involved can have interfere on these results (different genotypes, different pre and post freezing treatment). Indeed, the highest survival rate of 30% for vitrified blastocysts was obtained without treatment and without selection of blastocysts after warming (Berthelot et al., 2002). The efficiency of OPS method can be evaluated by the number of offspring obtained after collection, cryopreservation and transfer into recipient. Tables 7 and 8 summarised these data (Berthelot et al., unpublished results). As indicated in Table 7, the collection rate of embryos as a percentage of the number of corpora lutea is less than 100% even after slaughter. Morulae and blastocysts represents 85% of the total collected. As the embryo losses due to manipulations before and after vitrification are very low (3.25%) and since
F. Berthelot et al. / Livestock Production Science 83 (2003) 73–83
80
Table 6 OPS vitrification: in vivo development of OPS vitrified / thawed porcine embryos after transfer into recipients according to the stage of embryo development, cryoprotectant, medium and treatment. Bold characters emphasise the stages of development when embryos were still in uterus and within intact zona pellucida Stage of embryos
Concentration of cryoprotectant
Embryo treatment
Number of pregnancy / Number of transfer
Piglets born / Number of embryos transferred
Refs.
3.6 M EG 1 2.8 M DMSO 1 0.6 M S 8 M EG 1 7% PVP
No
8 / 10
26 / 200
Berthelot et al. (2001a)
Cytochalasin 1 centrifugation 1 dezoning
3/4
16 / 115
Beebe et al. (2002)
8 M EG 1 7% PVP
Cytochalasin 1 centrifugation No
1/5
5 / 180
Cameron et al. (2000)
11 / 20
38 / 400
Berthelot et al. (2000)
No
8 / 10
59 / 200
Berthelot et al. (2001b)
No
0 / 11
0 / 257
Holm et al. (1999)
2–4 cells Morula
Blastocyst
3.2 M EG 1 2.5 DMSO 1 0.6 M 3.2 M EG 1 2.5 DMSO 1 0.6 M Expanded blastocyst
M S M S
3.2 M EG 1 2.5 M DMSO 1 0.6 M S
Peri-hatching Hatched EG, Ethylene glycol; DMSO, Dimethyl sulfoxide; S, Sucrose; PVP, Polyvinyl pyrrolidone (g / l).
Table 7 Number of Corpora Lutea (CL) and rates of embryo at different stage of development after embryo collection at Day 5–6 of hyperprolific Large White for vitrification by the OPS technique (Berthelot F., Martinat-Botte´ F. and Terqui M., unpublished results) Number of CL per female
Rate of collection in % of CL
Non-fertilised eggs in % of total collected
Morulae in % of total collected
Blastocysts in % of total collected
Hatched blastocysts in % of total collected
19
80
9
14
71
5.6
Losses before vitrification in %
3
3
Losses after vitrification in %
0.25
0.25
Collection
Survival rates from Table 6
no selection of vitrified and thawed embryos was made; more than 96% of collected morulae and blastocysts were transferred (Table 7). Based on these rates, the Table 8 presents the
13
29
results of collection from 20 donors, the number of morulae, blastocysts and piglets that were obtained with the OPS vitrification method (Berthelot et al., 2001b). Twelve recipients were required for transfers
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Table 8 Estimated numbers of embryos, recipients and piglets born after collection of 20 Large White hyperprolific donors based upon rates in Table 7 (Berthelot F., Martinat-Botte´ F. and Terqui M., unpublished results) 43 Morulae Collected 41 Vitrified Morulae transferred 2 recipients for Morula 5 piglets
of morulae and blastocysts and a total of 67 piglets were born representing a little more than three offspring per donor (Table 8).
4.3.4. Conclusions The OPS vitrification method appears to be an efficient technique for cryopreservation of morulae and especially blastocysts. The cooling rate may be a key factor for survival of porcine embryos by vitrification.
5. Improvements in future At present, both vitrification procedures are suitable for the in vitro survival and birth of piglets. These methods could be used at least for gene banking. However improvements are required to reduce the cost and increase the possibility for practical application. One difficulty to improve cryopreservation methods is narrow relationship between in vitro and in vivo survival (see Tables 1–6). As indicated above the medium of dilution of cryoprotectant and the concentration of cryoprotectants are key parameters for the success of vitrification. Increased embryo survival rates might be obtained if medium and cryoprotectant concentration are at optimum. Also, as indicated previously, the ¨ cooling rate is very critical. Minitube (Germany) distributed a system (VitMaster) that supercools liquid nitrogen, avoids vapours and allows cooling rates of up to 100 000 8C / min. Such system might be also beneficial for cryopreservation porcine embryos. A team (Beebe et al., 2002) had obtained 16 piglets but after dezoning 115 morulae before transfer. So far, the thawing procedures of the OPS method has also to be carried out in a laboratory. Direct transfer from the straws after thawing is easy to
216 Blastocysts collected 209 Vitrified blastocysts transferred 10 recipients for blastocysts 62 piglets
perform at farm level especially when it is associated with non-surgical transfer via the cervix (Cuello et al., 2002).
6. General conclusions Although techniques still need further improvement, cryopreservation of porcine embryos has been achieved. Two methods give acceptable results, 29 and 18.5% of in vivo survival, respectively (Berthelot et al., 2001b; Dobrinsky et al., 2001). Cost and practicability of the use of this method to export selected lines and to preserve some endangered breeds (Labroue et al., 2000) with vitrified embryos have to be determined. Attention has to be paid to the physiology of the breed to be cryopreserved. For example, timing of ovulation, stage of embryo development have to be characterized in order to obtain morula or blastocyst production. In the future, pig embryo cryopreservation and transfer will be a practical tool for genetic preservation and exportation.
Acknowledgements The authors thank Professor B. Kemp and EAAP board for the organisation of the Session on Reproduction Technology of the Pig. The authors are grateful to Dr P. Mermillod for reading this manuscript and his suggestions.
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