Small Ruminant Research 82 (2009) 112–116 Fertility rates following the transfer of ovine embryos cryopreservedusing three protocols Elisa Maria Bettencourt , Carlos Manuel Bettencourt , João Chagas e Silva ,Paulo Ferreira , Claudino Pereira Matos , Ricardo Jorge Romão , António Rocha a Évora University, Veterinary Medicine Department, ICAM, 7000-092 Évora, Portugalb Herdade da Abóbada, V.N.S. Bento 7830-908, Portugalc Ex-Divisão de Selecc¸ão e Reproduc¸ão Animal (DGV), R. Elias Garcia, 30, Venda Nova, 2704-507 Amadora, Portugald ICBAS University of Porto and UNIB, Campus Agrário de Vairão, Vairão, Portugal The aim of this study was to compare the efficiency of three cryopreservation techniques namely, controlled slow freezing, conventional vitrification and open pulled straw (OPS) Received in revised form 18 February 2009 vitrification, for the cryopreservation of in vivo produced Portuguese Black Merino ovine embryos. Fresh (control group) and cryopreserved embryos (3 methods) were transferred by laparoscopy to recipient ewes (2 embryos/recipient). Embryo survival rates were assessedand pregnancy diagnosis was performed by serum progesterone concentration on day 18 and by ultrasonography on day 45, and at lambing. Successful pregnancy and embryo sur- vival rates did not differ between the fresh (73.3 and 50.0%, respectively) and cryopreserved (58.3 and 38.5%, respectively) embryos. No differences in lambing and embryo survival rates were observed between the three cryopreservation methods used (68.4 and 44.7%; 50.0 and 33.3%; 54.5 and 36.3% for controlled slow freezing, vitrification and OPS, respectively).
Pregnancy rate based on serum progesterone concentration on day 18 was higher than thepregnancy diagnosed at 45 days following transfer for the cryopreserved embryos. It couldbe concluded that all three cryopreservation techniques were equally efficient in preservingand propagating genetic material in Portuguese Black Merino sheep.
2009 Elsevier B.V. All rights reserved.
1. Introduction
the use of the ovine Multiple Ovulation and Embryo Trans-fer (MOET) technique and may improve genetic progress Embryo cryopreservation technology can be used for the long-term storage of selected genetic material and com- Slow freezing is the most widely used technique mercial lines from the industry and research flocks, as well for ovine embryo cryopreservation, but it requires an as for maintaining rare genotypes worthy of conservation expensive biological programmable freezer and is time use of embryo transfer in sheep may be related to the rel- techniques of cryopreservation such as vitrification, may atively high cost of the technique, when compared to the be an alternative option—to reduce costs. This technique value of the animal (Reduction of costs at any does not require any special equipment and may be easily stage of embryo production or transfer, is likely to increase adapted for routine field use The open pulled straw (OPS) method on theother hand is a modification of conventional vitrification ∗ Corresponding author. Tel.: +351266760809; fax: +351266760944.
which reduces the volume of the vitrification solutions and E-mail address: (E.M. Bettencourt).
0921-4488/$ – see front matter 2009 Elsevier B.V. All rights reserved.
doi: E.M. Bettencourt et al. / Small Ruminant Research 82 (2009) 112–116 It thus remains important to identify the least costly 2.2. Cryopreservation and thawing protocols and most efficient technique of ovine embryo preservation.
A genetic conservation program has been set up for the Only embryos in the morulae and blastocyst stages, classified as excel- lent or good (Grades 1 and 2, respectively) were considered freezable.
Black Merino sheep, a Portuguese native breed which is on Embryos were pooled and randomly allocated to one of the three cryop- the FAO’s endangered breed list. Currently no studies have been performed on the efficiency of embryo cryopreserva-tion of this particular breed. The aim of the present study was to compare the efficiency of controlled slow freezing, The protocol for embryo cryopreservation as described by as used, with certain modifications. Embryos were immersed conventional vitrification and OPS vitrification as cryop- for 5 min in a cryopreservation solution of 0.4% BSA in PBS, with 1.5 M reservation techniques of embryos in the Portuguese Black ethylene glycol (EG) added in one step. Two embryos were loaded into the middle portion of each straw (PBS, air bubble, PBS, air bubble, cry-opreservation solution with embryos, air bubble, 0.3 M sucrose). Straws 2. Materials and methods
were sealed with plastic plugs (plastic plugs for 0.25 ml French palettes,IMV®) and placed vertically in the cooling chamber of the programmable All experiments were conducted during spring at the Baixo Alentejo freezer (Bio-Cool III, FTS Systems Inc., Stone Ridge, NY) at −6 ◦C for 3 min Experiment Station - Herdade da Abóbada, located in southern Portugal and then subjected to manual seeding. After seeding, straws were kept (latitude 37◦57 N, longitude 7◦26 W) at 242 m above sea level.
for 5 additional min at −6 ◦C. Embryos were then cooled at 0.5 ◦C/min Ewes were maintained on natural pastures all year round. During the until reaching −35 ◦C, where they were maintained at this temperature experimental period ewes were group-housed in straw-bedded pens, with for 15 min, and then rapidly immersed into liquid nitrogen. Thawing was ad libitum access to ray grass hay, and supplemented daily with 400 g carried out by exposing the straws for 10 s at room temperature, followed by the immersion in a 35 ◦C water bath, for 10 s. After thawing, embryos Except otherwise indicated, all chemicals used were obtained from were left for 5 min in the content of the straw and then washed in holding Sigma Chemicals Company (Barcelona, Spain).
2.2.2. Conventional vitrification The vitrification procedure was performed according to Adult (3–6 years old) multiparous Portuguese Black Merino ewes media were prepared with a PBS solution containing 20% new (n = 32), with a mean BCS of 3 (0-extremely thin, 5-obese), were used as born calf serum (NBCS). Embryos were exposed to the vitrification solution embryo donors. None of the ewes utilized in the experiment had been at room temperature, according to the following procedure: incubation in previously subjected to a MOET program. Estrous synchronization was a 10% glycerol solution for 5 min, followed by incubation in a mixture of carried out with the aid of intravaginal sponges containing 40 mg fluroge- 10% glycerol + 20% ethylene glycol for an additional 5 min, before being stone acetate (FGA; Chronogest®, Intervet Laboratories, Boxmeer, Holland) transferred to the vitrification medium (25% glycerol + 25% ethylene gly- for a period of 12 days. On day 9 of progestagen treatment, and during the col) for 30 s. Embryos were then aspirated into a 0.25cc straw (2 embryos 4 consecutive days, ewes were treated (im) twice daily with 1.25 ml oFSH per straw) in a central column. In the straws, the vitrification solution con- (Ovagen®, Immunological Products, Ldt. Aukland, New Zealand). Intrav- taining the embryos was kept separated by two air bubble columns from aginal sponges were removed at the 7th oFSH administration. Estrous two columns of a 0.8 M sucrose solution. After sealing, the straws were detection was performed at 12, 24, 30, 36 and 48 h following sponge immediately plunged into liquid nitrogen. Thawing was done for 5 s in air followed by 10 s in a water bath at 20 ◦C. For cryoprotectant removal, the Ewes were sedated with acepromazine (0.1–0.5 mg/kg; Calmivet®, embryos were kept for 5 min in the content of the straw and subsequently Vetoquinol, Portugal) and laparoscopic intrauterine inseminations were subjected to two 5-min washes in 3 ml of a PBS solution containing 20% performed with fresh diluted semen 48 h after sponge removal A minimum of 50 × 106 motile sperm was inseminated ineach uterine horn. Additionally, ewes were hand mated at 36 and 48 h after intravaginal sponge removal, and left with rams for an additional Vitrification by the open pulled straw (OPS) method was conducted 4–6 h period (at an ewe:ram ratio of 3 to 1). All rams used in this study as described by yos were equilibrated for 5 min had passed a breeding soundness evaluation test.
in a holding medium (HEPES-buffered TCM-199 medium supplemented Feed and water were withheld from the ewes for at least 24 h prior with 20% of fetal calf serum), transferred for 3 min to the dilution medium to laparotomy. The abdominal area anterior to the udder was shaved and (holding medium with 10% ethylene glycol and 10% dimethyl sulfoxide), sprayed with an iodine solution and 70% alcohol. Animals were sedated followed by 30 s in the vitrification medium (holding medium with 20% EG with 0.4 mg/kg acepromazine (im) (Calmivet® Vetoquinol, Portugal) and and 20% DMSO). Embryos were loaded in the OPS via capillarity, by placing 0.1–1 mg/kg atropine (sc) (Sultropin® Sorologic Laboratory, Amadora Por- the narrow end of the pulled straw into the holding medium. The straws tugal) and anesthetized (iv) with thiopental (22 mg/kg; Thiopental® 0.5 g were then immediately submerged into liquid nitrogen. For thawing, the Braun). A mid-ventral laparotomy was performed after local anesthesia end of the straw was directly immersed in a holding medium contain- by infiltration with 6–8 ml of 2% lidocaine hydrochloride at the incision ing 0.25 M sucrose. Embryos were left for 1 min in this medium and then area (Anestesin® Sorologic Laboratory, Amadora, Portugal). On the day of double washed for 5 min in a holding medium containing 0.15 M sucrose.
surgery and for 4 consecutive days after surgery all ewes were treated with2.2 mg/kg flunixine-meglumine im (Finadyne®, Schering-Plough Portu- gal) and kept in an enclosed pen, in groups of 4 and observed twice dailyfor signs of discomfort and/or inflammation in the incision area.
Sixty-three recipient ewes (Portuguese Black Merino, 3–6 years of Embryos were recovered as described in detail by age with a BCS of 3), were synchronized in spring with intravagi- , each uterine horn was flushed with 40 ml flushing media nal sponges containing 40 mg FGA (Chronogest®, Intervet Laboratories, (PBS containing 2% of bovine serum albumin - BSA), through the inser- Boxmeer, Holand) for 12 days, followed by the administration of eCG tion of a needle attached to a syringe, near the utero-tubal junction. A (450IU, im; Intergonan®, Intervet Laboratories, Boxmeer, Holland) at the 10 gauge Foley catheter was inserted in the base of the uterine horns time of sponge removal. Estrous was detected using vasectomized rams for recovery of the embryos, and the media collected and examined for and a synchrony of ±12 h with the donor ewes was permitted. On day 6 the presence of oocytes and/or embryos under a stereomicroscope. After after estrous detection, recipient ewes were sedated with acepromazine location, embryos were immediately placed in a holding medium (PBS (0.1–0.5 mg/kg; Calmivet®, Vetoquinol, Portugal) and local anesthetic supplemented with 10% FCS—or with 4% BSA). Embryos were classified (2–3 ml of 2% lidocaine hydrochloride - Anestesin® Sorologic Labora- based on the morphological criteria and according to their stage of devel- tory, Amadora, Portugal) was infiltrated at the laparocopic puncture sites.
Embryo transfer was then carried out using a modified method of the of the International Embryo Transfer Society ( laparoscopic technique Before transfer, the pres- ence of at least one newly formed CL was confirmed in the recipient ewe E.M. Bettencourt et al. / Small Ruminant Research 82 (2009) 112–116 Table 1
Pregnancy, lambing and embryo survival rates following the transfer of fresh or cryopreserved embryos using three methods.
by laparoscopy. Morulae or blastocysts that had been cryopreserved using were recorded between the cryopreservation methods either controlled slow freezing (n = 38), conventional vitrification (n = 36) or OPS (n = 22) were then transferred to recipient ewes (2 embryos per The overall pregnancy rate obtained with cryopreserved recipient ewe). Embryos were transferred independently of their classi- embryos at day 18 (68.3%) was higher (p < 0.05) than the fication at thawing. Fresh Grade 1 or 2 embryos, obtained as previouslydescribed, were transferred as a control group (n = 30). All embryos were pregnancy rate at day 45 (61.9%; All ewes pregnant transferred within 30 min of cryoprotectant removal (frozen embryos) or at 45 days following embryo transfer subsequently lambed.
There was no difference between pregnancy rates on days Blood samples were collected from the recipient ewes at the following 18 and 45, when fresh embryos (controls) were used.
times: sponge insertion, estrous (day 0), day of embryo transfer (day 6)and at days 12, 15, 18 and 21 following transfer. The blood samples were Differences (p < 0.05) in the circulating progesterone centrifuged and the serum stored at −20 ◦C until assayed. Plasma pro- concentration between pregnant and non-pregnant ewes gesterone (P4) was assayed by RIA using commercial kits (Coat-A-Count, were recorded on days 15, 18 and 21 following trans- diagnostic Product Corporation, Los Angeles, CA, USA). The intra-assay fer (Serum progesterone concentration on day 6 coefficient of variation was 2.77% and inter-assay coefficients of variation was positively correlated with the number of CL’s (r = 0.29, p < 0.0001; Y = 2.1 + 2.2X), but the embryo survival was not affected either by serum progesterone concentration onday 6 or the number of CL’s.
An ewe was considered pregnant on day 18 after estrous when the serum progesterone concentration was greater than 1.0 ng/ml maintenance of pregnancy was confirmed at day 45 by abdom-inal ultrasonography (Pie Medical 200 Scanner with a 7.5-MHz sectorial 4. Discussion and conclusions
probe) and at lambing. The pregnancy rate (%) was defined as the num-ber of pregnant females divided by the number of recipients (×100) and The lambing and embryo survival rates observed in this lambing rate as the number of ewes lambing divided by the number of study with fresh (73.3 and 50.0%, respectively) and over- recipients (×100). Embryo survival rate, defined as number of lambs borndivided by the number of embryos transferred (×100), was also calculated.
all cryopreserved (58.3 and 38.5%, respectively) embryoswere comparable to those reported for other breeds of Pregnancy and lambing rates were analyzed using the chi-square test.
Embryo survival rate was analyzed using the categorical data modeling ing pregnancy, lambing and embryo survival rates after (CATMOD) and a one-way ANOVA was used to assess the effect of the the transfer of the fresh or cryopreserved embryos, as well number of CL’s on the serum progesterone concentration on day 6 and the between the 3 cryopreservation methods used. However, effect of serum progesterone concentration at days 6 and 18 on embryosurvival rate. All data was analyzed using the statistical software package when all cryopreserved embryos were treated as a group and compared to the fresh embryos, the fresh embryostended to record a higher pregnancy rate at 45 days fol- 3. Results
lowing embryo transfer, partially due to their absence ofembryo mortalities between days 18 and 45.
Only 10 embryos were classified as Grade 3 (poor qual- The pregnancy and lambing rates obtained after the ity) following morphological embryo evaluation at thawing transfer of cryopreserved embryos by controlled slow and thus embryo score was not included in the statisti- freezing, compared favorably with those reported else- cal model. There were no significant interactions between embryonic stage of development and pregnancy, lamb- The results of the vitrified embryos, using ing or embryo survival rates. No significant differences either OPS or conventional vitrification, were also simi- in pregnancy and lambing rates or in embryo survival lar to those obtained by other researchers Table 2
Mean (±SE) serum progesterone concentration at days 6, 12, 15, 18 and 21 following transfer in pregnant and non-pregnant ewes.
Means in the same column with different superscripts differ significantly (*p < 0.05; **p < 0.001).
E.M. Bettencourt et al. / Small Ruminant Research 82 (2009) 112–116 efficiency was detected between the three protocols uti- and embryo survival rates have been reported in a few lized. Therefore, the choice of method to cryopreserve embryos of this sheep breed may be dictated by the fact that in those trials only blastocysts that re-expanded availability of equipment, qualified staff and management after thawing, were transferred. However, re-selection of embryos at thawing may decrease the potential number oftotal lambs born. It has been shown that the transfer of all Acknowledgements
embryos at thawing, with no further selection, allow for apotential gain of 7–8% in terms of lambs born—which could This work was supported by funds from the Portuguese AGRO projects (AGRO 438 Portuguese Animal Germplasm The use of the laparoscopic technique utilized in this Bank); FEDER and FCT (SFRH/BD/30575/2006).
trial, allows for the visualization of the ovaries, thus permit- The authors thank Prof. Luisa Mateus, Reproduction Lab- ting the culling of recipient ewes without luteal structures oratory, FMV, for help in the RIA analyses.
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Témoignage de Carmen J’ai un trouble obsessionnel-compulsif (TOC) depuis à peu près l’âge de six ans. A cette époque je ne me rendais pas compte de ce qui m’arrivait. Maintenant je me rappelle trois événements qui m'ont fait réaliser que le TOC est apparu dès cette période. Un jour, mon père m’a grondée car il avait trouvé dans ma corbeille à papiers beaucoup de feuilles pres

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