Doi:10.1016/j.theriogenology.2006.02.040 Improved vitrification method allowing direct transfer F. Guignot , A. Bouttier , G. Baril P. Salvetti P. Pignon J.F. Beckers , J.L. Touze´ , J. Cognie´ , A.S. Traldi a INRA-CNRS-Universite´ de Tours-Haras Nationaux, Physiologie de la Reproduction et des Comportements, 37380 Nouzilly, France b Universite´ de Lie`ge, Faculte´ de Me´decine Ve´te´rinaire, Physiologie de la Reproduction, B-4000 Lie`ge, Belgium c Dep. de Reproduc¸ao Animal, FMVZ-USP, cp 23, 13630 000 Pirassununga, SP, Brazil Received 30 September 2005; received in revised form 24 February 2006; accepted 26 February 2006 The aim of this study was to design a vitrification method suited to field embryo transfer experiments in goat. In a first experiment, a standard vitrification protocol, previously designed for sheep embryos was compared to slow freezing of goatembryos. No significant difference was observed on kidding rate (48% versus 69%, respectively), nor on embryo survival rate (35%versus 45%). Second experiment: all embryos were vitrified. After warming, embryos were either transferred directly (directtransfer), or after in vitro dilution of the cryoprotectants (conventional transfer). The kidding rate was not affected by the transfermethod (38% versus 23%, respectively). However, embryo survival rate tended to be higher after direct transfer (26% versus 14%).
Third experiment: OPS vitrification was compared to standard vitrification. The kidding rate was not affected (22% versus 39%,respectively), but the embryo survival rate was lower after OPS (14% versus 28%). Fourth experiment: 0.4 M sucrose was addedwith cryoprotectants in vitrification. The kidding rate after direct transfer was significantly enhanced after addition of sucrose (56%versus 27%, respectively), whereas embryo survival rate was not significantly affected (32% versus 18%). Fifth experiment:vitrification with sucrose supplementation was compared to slow freezing. No significant difference was observed after directtransfer on kidding rate (52% versus 31%, respectively), but embryo survival rate tended to be higher after vitrification (34% versus21%). In conclusion, our results indicate that addition of 0.4 M sucrose in association with direct transfer improves significantly theviability of goat vitrified embryos.
# 2006 Elsevier Inc. All rights reserved.
Keywords: In vivo goat embryo; Vitrification; Slow freezing; Transfer; Embryo survival settings. Embryo cryopreservation has simplified themanagement of genetic resources in domestic and wild Cryopreservation and thawing under controlled species and is essential in commercial embryo transfer conditions have permitted the use of cells and tissues technology providing easier and cheaper embryo after long-term storage in both commercial and research transport, reducing health risk and avoiding loss ofanimals during transport. In domestic animals itprovides methodology to maintain genetic diversityconservation and preservation of endangered species it * Corresponding author. Tel.: +33 2 47 42 77 94; enables maintenance and storage of biodiversity via 0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved.
F. Guignot et al. / Theriogenology 66 (2006) 1004–1011 Cryopreservation of mammalian embryos was Many different vitrification protocols have been used developed during the 1970s in mouse and cow with success to cryopreserve in vivo and in vitro and has since been modified to attempt to cryopreserve produced embryos in several species. All of these embryos from most species, at different developmental protocols aim to minimize the toxicity due to high stages, as well as more sensitive embryos such as in cryoprotectant concentrations. A lower temperature of vitro produced, cloned or biopsied embryos vitrification, an accelerated rate of cooling, a combina- tion of several cryoprotectants rather than a single one, slow freezing which is designed to maintain a delicate partial replacement of cryoprotectants by macromole- balance between cryoprotectants at low concentration cules (PVP, Ficoll) and sugars to increase the viscosity (1–1.5 M) and aqueous embryo compartment, whereas of the vitrification medium have been proposed to the strategy of the vitrification method is a rapid solidification of liquid with high concentrations of cryoprotectants (6–7.5 M) avoiding ice crystal forma- vitrification solution seems to be the most critical tion. In goat, the first successful cryopreservation of factor to determine the success of vitrification. Some embryos was with the slow freezing method in 1976 .
studies have specially noticed the positive effect of Subsequently, most goat embryos were cryopreserved sugar addition in vitrification solution, as trehalose or by slow freezing until 1990 with a rate of embryo 0.1 and 0.3 M sucrose in the last step of equilibration 1990, the first successful transfer of vitrified goat procedure has significantly enhanced the hatching rate embryos was reported by Yuswiati and Holtz . They of Day 6 and Day 7 embryos as compared to 0.0 and used standard vitrification with ethylene glycol (EG) 0.5 M sucrose . To our knowledge, vitrification with and propanediol, in 0.25 mL straw. In 2001, the first addition of sugar in the last step of equilibration successful transfer of goat embryos using open pulled procedure has never been tested with goat embryos.
straw (OPS) technique was obtained by El-Gayar and In the present study, we compared in vivo goat Holtz . However, only limited results are available embryo survival rate after slow freezing and vitrifica- after transfer of goat vitrified embryos with generally tion. We tested the effect of cryoprotectant removal lower survival rates than those obtained after slow before transfer compared with direct transfer and we tried to improve the vitrification method by the addition In small ruminants, the use of embryo transfer of sucrose at the last step of equilibration procedure and technique in breeding schemes is limited as compared by using the open pulled straw (OPS) technique.
with cattle This is probably due to the excessivecost when compared to the value of the animal The more satisfactory and commonly used technique ofcryopreservation in goats (slow freezing) is unfortu- nately the more expensive since it requires an expensivebiological freezer and is labour intensive, whereas The estrus cycles of Alpine and Saanen dairy goats vitrification techniques with direct transfer offers a real were synchronized in donor and recipient females by possibility to reduce the cost of embryo transfer .
insertion of intravaginal sponges containing 45 mg FGA Efficient and economical cryopreservation techniques (Chronogest1, Intervet, Angers, France) for 12 days (day for goat embryos would be advantageous since goats 0 = sponge insertion), and 50 mg cloprostenol (Estru- produce diversified products of commercial interest. In mate1, Shering-Plough, Levallois, France) were admi- addition, emerging biotechnologies such as transgenesis nistrated in the morning of the 10th day. Two days before and somatic cell nuclear transfer allows for generation sponge removal, the recipients received 400–500 IU of and propagation of transgenic founder animals that eCG (Chronogest1, lntervet, Angers, France). Donors produce valuable recombinant proteins of pharmaceutic were superovulated with a total of 160 mg pFSH (Me´rial, or biomedical interest in goat milk Moreover, Belgium) injected intramuscularly every 12 h in six his short gestation period (5 months) small size, easy decreasing doses (40, 40, 20, 20, 20, and 20 mg) during breeding and relatively high milk production make goat the last three days of progestagen treatment (days 10–12).
competitive as compared to the cow for the application pFSH was supplemented by 66 mg LH in the last two of biotechnologies. Therefore, improvement of embryo- injections. When the onset of estrus was detected (24– based techniques is warranted in this species, especially 36 h after sponge removal), donors were mated with several bucks in experiments 2, 3, 4 and 5, while in F. Guignot et al. / Theriogenology 66 (2006) 1004–1011 experiment 1, donors were inseminated in utero under or to the two transfer methods applied in each endoscopic control with frozen/thawed semen from experiment. After embryo collection, local antibiotic several fertile bucks (200 Â 106 spermatozoa per female) (Aluspray) was spreaded on the abdominal incision, and 20–24 h after the onset of estrus.
each recipient received benzylpenicilline and dihydro-streptomycine during 3 days (i.m. 106 UI per day).
2.3. Cryopreservation and warming procedures Embryos were recovered surgically by laparotomy under general anesthesia (iv thiopental 10 mg kgÀ1, The different cryopreservation and transfer methods atropine sulphate 0.3 mg kgÀ1 and endotracheal intuba- applied in each experiment are summarized in .
tion with isoflurane and oxygen) 7–8 days after the onset Five to six embryos were cryopreserved together in of estrus. Each uterine horn was flushed with 40 mL of a same straw for conventional transfer (experiments 1 pre-warmed PBS containing 2% BSA (Euroflush, IMV, and 2) while only two embryos were loaded in each L’Aigle, France). The embryos were immediately straw for direct and pseudo-direct transfer (experiments retrieved and placed in PBS containing 4% BSA (Embryo Holding medium, IMV, L’Aigle, France). Theywere classified according to their stage of development 2.3.1. Slow freezing (experiments 1 and 5) and only morphologically normal embryos were selected Embryos were frozen according to the method of Le for cryopreservation (compact morulae, blastocysts and Gal et al. Briefly, embryos were equilibrated in expanded blastocysts). Embryos were kept for less than freezing solution consisting successively of 0.5, 1 and 30 min in Embryo Holding medium at +20 to +25 8C 1.5 M EG in Embryo Holding medium, 5 min each, at before cryopreservation. In the five experiments, the room temperature. During the last equilibration step, embryos from each donor were divided between two embryos were loaded into the centre of a 0.25 mL groups according to the two cryopreservation techniques plastic straw (IVM, L’Aigle, France) within 20–30 mL Table 1Pregnancy rate of in vivo produced goat embryos transferred after different protocols of cryopreservation The letter a and b denotes the values differ significantly in each experiment (chi-square test): (P < 0.05). The symbol a and b denotes the values tendto be significant in each experiment (chi-square test): (P < 0.10). Standard vitrify.: standard vitrification without sucrose; OPS: open pulled straw.
b Ultrasound examination and PAG assay (pregnancy-associated glycoprotein).
d Defined as the kids born/thawed embryos ratio.
e Two pregnant goats were died few days before kidding.
F. Guignot et al. / Theriogenology 66 (2006) 1004–1011 of medium. Embryos were separated from two surround- were then equilibrated in a freezing solution consisting of ing segments of freezing medium by two air bubbles 7.5% EG and 7.5% DMSO dissolved in Hepes-buffered (experiment 1) or of Embryo Holding medium (experi- TCM199–NBCS for 3 min. They were then transferred ment 5) of about 90 mL each. The straws were sealed and with approximately 1 or 2 mL of solution into a 20 mL placed in a programmable freezer (Agrogen-EF1, droplet of 18% EG, 18% DMSO and 0.4 M sucrose in Freiburg, Switzerland) and cooled from room tempera- Hepes-buffered TCM199–NBCS. Embryos were drawn ture (22 8C) to À7 8C at 4 8C/min. After 5 min, each up together in another 1–2 mL drop, loaded by capillarity straw was manually seeded. Following a further 10 min at into the narrow end of an OPS and plunged into liquid À7 8C, straws were cooled to À30 8C at a rate of 0.3 8C/ nitrogen. The time between the contact of the embryos min, and finally plunged and stored into liquid nitrogen.
with the concentrated cryoprotectant solution and the For thawing, straws were held in air for 5 s and then liquid nitrogen did not exceed 45 s. For warming, straws dipped into a 37 8C water bath for at least 15 s. In were held in air for 5 s before the narrow end was experiment 1, the content of each straw was emptied into immerged in Hepes-buffered TCM199–NBCS with a Petri dish containing 0.25 M sucrose in Embryo 0.2 M sucrose. After 1 min, embryos were transferred Holding medium. After 10 min (two baths of 5 min), the for 5 min in new Hepes-buffered TCM199–NBCS with embryos were washed twice in Embryo Holding medium 0.2 M sucrose, following by 5 min with 0.1 M sucrose.
without sucrose for 5 min each to eliminate cryoprotec- All warmed embryos were transferred.
tants before conventional transfer. In experiment 5,embryos were directly transferred after thawing.
2.3.4. Sucrose addition in standard vitrification(experiments 4 and 5) 2.3.2. Standard vitrification (experiments 1, 2, 3 The first and the second steps of equilibration procedure were the same as described in the standard The vitrification method described by Mermillod vitrification. The third step consisted of 25% glycerol et al. was applied. Embryos were kept at room and 25% EG for 30 s in PBS–NBCS supplemented with temperature for 5 min in PBS supplemented with 20% 0.4 M sucrose. Freezing and warming are identical to new-born calf serum (NBCS). They were then vitrified standard vitrification protocol. After warming, embryos in three steps at room temperature as follows: 10% glycerol for 5 min, 10% glycerol and 20% EG for 5 min,and finally 25% glycerol and 25% EG for 30 s in PBS– NBCS. During the last step, embryos were quicklyaspirated into the centre of a 0.25 mL plastic straw In all experiments, embryos were transferred into (IVM, L’Aigle, France) within 20–30 mL of vitrification synchronized Alpine and Saanen goats, 7 days after the solution. Embryos were separated by two air bubbles onset of estrus, in different locations (replicates). Three from two surrounding segments of PBS–NBCS contain- replicates were done in all experiments, except in ing 0.8 M galactose (about 90 mL each). The straws experiment 2 (only two replicates). The recipients were were sealed and immediately plunged directly into sedated with 0.3 mg kgÀ1 iv acepromazine (Calmivet1, liquid nitrogen. For warming, straws were held 5 s in air Vetoquinol, Lure, France). After endoscopic control of followed by 15 s in a 22 8C water bath. For conventional the presence of at least one functional corpus luteum, transfer in experiments 1 and 2 (half of straws), the embryos were transferred as follows.
straw content was expelled and embryos were incubatedduring 5 min in the content of the straw and washed twice for 5 min in 3 mL PBS to eliminate cryoprotec- tants before conventional transfer. In experiments 2 (the After warming and cryoprotectant removal, the other half of straws), 3 and 4, embryos were directly embryo quality was evaluated under stereomicroscope according to morphological criteria, and all degeneratedembryos, i.e. those with abnormal morphology and disrupted membrane, were eliminated. For each The OPS vitrification method was applied according recipient, one to three embryos were aspirated with to the method described by Vajta et al. . All 20 mL PBS in a glass capillary connected to a 1 mL equilibrations were performed at 34–35 8C. Briefly, syringe and were introduced surgically into the top of embryos were washed twice in Hepes-buffered TCM199 the uterine horn ipsilateral to the ovary bearing at least solution supplemented with 20% NBCS for 5 min. They one functional corpus luteum. As five to six embryos F. Guignot et al. / Theriogenology 66 (2006) 1004–1011 were cryopreserved together in a same straw for were obtained with slow freezing and standard vitrifica- conventional transfer, some good embryos were tion, respectively. In the second experiment, the kidding exceptionally not transferred in experiment 1 because rate was not affected by the transfer method used (P > 0.05): 24% versus 38% of kidding were obtainedafter conventional or direct transfer of vitrified/warmed 2.4.2. Direct transfer (experiments 2, 3, 4 and 5) embryos, respectively. However, the rate of embryo After warming, the whole content of the straw survival tended to be higher after direct transfer as (250 mL, two embryos) was deposited surgically, with a compared to conventional transfer (26% versus 14%, prototype instrument (IMV, L’Aigle, France), into the respectively). The OPS vitrification method (experiment uterine horn ipsilateral to the ovary bearing at least one 3) did not enhance the kidding rate, whereas the embryo functional corpus luteum without embryo selection or survival rate was significantly lower compared with standard vitrification (14% versus 28% for OPS andstandard vitrification, respectively). The addition of 2.4.3. Pseudo-direct transfer (experiment 3: OPS sucrose to the vitrification medium (experiment 4) significantly enhanced the kidding rate after direct After warming and partial cryoprotectant removal, transfer (56% versus 27% without sucrose, P < 0.05), all warmed embryos were transferred. For each whereas embryo survival rate was not significantly recipient, two embryos were aspirated with 20 mL affected (32% versus 18.2%). Only in one replicate true Hepes-buffered TCM199–NBCS with 0.1 M sucrose in statistically significant difference was detected: 41% a glass capillary the same way as conventional transfer.
(n = 22) versus 10% (n = 20) with and without sucrose, Embryos were then introduced surgically into the top of respectively (P < 0.05). When vitrification with addition of 0.4 M sucrose and slow freezing were compared usingdirect transfer (experiment 5), no significant difference was observed on kidding rate (52% versus 31%,respectively), but embryo survival rate tended to be Pregnancy was diagnosed at day 21 after estrus by higher after vitrification (34% versus 21%, respectively).
progesterone assay (RIA method) a female wasconsidered pregnant if progesterone concentration was higher than 1.5 ng/mL. It was confirmed by ultrasoundexaminations on day 43 and PAG assay (pregnancy- In the present study, embryo survival and kidding associated glycoprotein; RIA method) on day 43 was studied to compare slow freezing and vitrification, and 70, and finally by observation of kidding at term.
with or without addition of 0.4 M sucrose in the last step The kidding rate was defined as the pregnant/transferred of equilibration procedure, and OPS applied to in vivo recipient ratio and the embryo survival rate was defined produced goat embryos followed by either direct as the number of kids born/thawed embryos ratio.
transfer into recipients, without cryoprotectant removal,or after conventional transfer after in vitro cryoprotec- tants removal and embryo quality evaluation. Embryosurvival rates obtained in the first experiment after slow This study was designed to compare two cryopre- freezing and conventional transfer (45%) were compar- servation protocols in each experiment. Comparisons able to results reported by Baril et al. (47.3%) with among protocols were performed using the Chi-square exactly the same freezing/thawing/transfer method used test. Statistical significance was denoted as P < 0.05.
in our study, and by Holm et al. (53%) for Angoragoat and McKelvey and Bhattacharyya (53%) for Cashmere goat after slow freezing with ethylene glycol.
Similarly, embryo survival rate after vitrification The rate of pregnancy obtained in each experiment is observed here (35%) was also comparable to the results showed in . In experiment 1, 83% and 88% of reported previously by Traldi (40%) and Branca thawed embryos were transferable after slow freezing et al. (44%) with the same method. In contrast, the and standard vitrification, respectively. After conven- embryo survival rate obtained in our second experiment tional transfer, there was no significant difference (14%), using the same standard vitrification technique between cryopreservation techniques: 69% versus 48% and conventional transfer, was markedly lower than of kidding and 45% versus 35% of embryo survival rates those reported by Traldi and those obtained in our F. Guignot et al. / Theriogenology 66 (2006) 1004–1011 first experiment using slow freezing. Human error was sucrose acts in the same way as galactose contained in the ruled out because this experiment was conducted by the marginal parts of the straw during vitrification procedure, same technical team, suggesting that the difference due to its osmotic properties. These non-permeable observed may be related to the different batches and sugars prevent excess water inflow because water breeds of recipient females. Indeed, recipients were not permeates more rapidly than cryoprotectant diffuses the same between these two experiments and moreover out of the embryo, reducing membrane damage and they did not come from the same farm. Differences in increasing the embryo survival Another advantage embryo quality (ability to survive vitrification) could of sucrose, and other carbohydrates, is the preservation of also contribute to the variability observed between the structural and functional integrity of membranes at experiments. In addition, age of donors and season of embryo production may have affected embryo quality.
The low embryo survival rate obtained after slow Although the same person vitrified all embryos, the freezing in experiment 5 (21%), compared to that concentration of cryoprotectants was very high in this obtained in the first experiment (45%) and reported in method (7.5 M) and in the last step of equilibration literature (27–59%) is difficult to explain. The procedure the additional seconds that were required variability of the in vivo produced embryos quality and could induce (1) excessive dehydratation of the embryo, the different recipient batches could perhaps explain a resulting in an increased salt concentration into embryo part of this unexpected difference as suggested above.
cells, which may be detrimental to protein integrity However, as the embryos from each donor were equally , and (2) an increase of intracellular cryoprotectants distributed between both cryopreservation methods, it concentration which are known to damage the embryos, could be envisioned that embryo survival after 0.4 M even if the chosen cryoprotectants, ethylene glycol and sucrose vitrification, which had a tendency to be higher glycerol, are the least toxic compared to DMSO or than after slow freezing, could be even better with propylene glycol . The OPS technique applied in experiment 3 did not affect the rate of embryo survival The pregnancy rate decreased dramatically by about compared to standard vitrification (14% versus 28%).
20% between days 21 and 43 in experiments 1 and 4 This result differed from those obtained by El Gayar and (standard vitrification) and in experiment 3 (OPS). As pregnancy rate was diagnosed by progesterone assay on elevated rates. In our experimental conditions, OPS day 21, this variation could be attributed to a longer technique was not suited for goat embryo cryopreserva- luteal phase of the recipients, responsible of high level tion. To avoid these problems of variability, we have of progesterone at day 21, whereas embryos were no attempted to improve the vitrification technique in more viable. It could be also attributed to a pseudo- experiments 4 and 5. In these experiments, a repeatable gestation, a phenomenon which occurs often in goats embryo survival rate of 32% and 34% was obtained as a result of a growing trophoblastic vesicle after addition of 0.4 M sucrose in the last step of without a viable embryo. Because of this complication, equilibration procedure and direct transfer to a total of the most accurate test of early pregnancy was 54 recipient goats. Moreover, the addition of 0.4 M ultrasound examination or PAG on day 40–43.
sucrose in the last step increased the kidding rate as Despite the low overall survival rates obtained in compared to standard vitrification (without sucrose) and experiment 2, direct transfer of the embryos seemed to tended to enhance embryo survival rate as compared to be as effective as conventional transfer after cryopro- tectant removal. This result was previously observed in As shown by Martinez et al. in bovine embryos, goats after slow freezing and in sheep after sucrose confers an additional protective effect for the standard vitrification In cattle, several freezing embryos during vitrification. Addition of sucrose may methods have been designed to allow direct transfer of reduce the toxicity of the high level of cryoprotectants the embryos , even under field conditions applied in this technique, probably by reducing the As no embryo selection is done after thawing, intracellular amount of cryoprotectant . Indeed, the direct transfer can be performed without specific extra osmolarity created by the non-permeable carbohy- expertise in embryo quality evaluation, reducing the drate causes embryo dehydratation and rapid shrinkage cost of the transfer, avoiding embryo elimination after and thereby reduces the amount of cryoprotectants in the thawing and simplifying the embryo transfer method in cells Embryo dehydratation caused by introduc- field conditions. Moreover, in sheep, this technique tion of this non-permeable carbohydrate also reduces the allows a potential gain of 7–8% in terms of lambs born risk of ice crystal formation . During warming, which could increase the gain/cost ratio .
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