a Available online at www.sciencedirect.com Theriogenology 78 (2012) 576–582 0 h Post-thaw viability of in vivo-produced canine blastocysts cryopreserved by slow freezing C. Renato de Freitas Guaitolinia,b, M. Onghero Taffarelb, N. Soares Teixeiraa, M. José Sudanob, P. Maria Coletto Freitasc, M. Denise Lopesb, F. da Cruz Landin-Alvarengab, C. Alvarenga de Oliveirad, M. Rezende Luza,c,* a Laboratory of Animal Reproduction, Federal University of Espirito Santo, Brazil, UFES, Department of Veterinary Medicine, Alegre Campus, Alto Universitário s/n, Caixa Postal 16, Alegre, ES, Brazil, 29500-000 b São Paulo State University, Unesp, School of Veterinary Medicine and Animal Science, FMVZ, Department of Animal Reproduction and Veterinary Radiology Rubião Jr. s/n°, Botucatu, SP, Brazil, 18618-970 c Federal University of Minas Gerais, UFMG, Veterinary School, Department of Veterinary Clinics and Surgery, Sector of Animal Reproduction, Av Antonio Carlos, 6627, Caixa Postal 567, Pampulha Campus, Belo Horizonte, Minas Gerais, Brazil, 30123-970 d Laboratory of Hormonal Assays, Department of Animal Reproduction, Faculty of Veterinary Medicine and Animal Science, University of São Paulo, USP, São Paulo, SP, Brazil, 05508-270 Received 21 June 2011; received in revised form 7 March 2012; accepted 7 March 2012 Abstract The objectives were to evaluate the reexpansion blastocoele rate, post-thaw viability, and in vitro development of canine blastocysts cryopreserved by slow freezing in 1.0 M glycerol (GLY) or 1.5 M ethylene glycol (EG). Fifty-one in vivo-produced canine blastocysts were randomly allocated in two groups: GLY (n � 26) and EG (n � 25). After thawing, embryos from M0 were immediately stained with the fluorescent probes propidium iodide and Hoechst 33 342 to evaluate cellular viability. Frozen-thawed embryos from M3 and M6 were cultured in SOFaa medium � 10% FCS at 38.5°C under an atmosphere of 5% CO2 with maximum humidity, for 3 and 6 days, respectively, and similarly stained. The blastocoele reexpansion rate (24 h after in vitro culture) did not differ between GLY (76.5%) and EG (68.8%). Post-thaw viable cells rate were not significantly different between GLY and EG (66.5 � 4.8 and 57.3 � 4.8, respectively, mean � SEM), or among M0 (62.3 � 5.7%), M3 (56.9 � 6.0%), nd M6 (66.5 � 6.0%). In conclusion, canine blastocysts cryopreserved by slow freezing in 1.0 M glycerol or 1.5 M ethylene glycol, had satisfactory blastocoele reexpansion rates, similar post-thawing viability, and remained viable for up to 6 days of in vitro culture. © 2012 Elsevier Inc. All rights reserved. Keywords: Cryopreservation; Embryo viability; Blastocoele reexpansion; Glycerol; Ethylene glycol; Dog www.theriojournal.com * Corresponding author. Tel.: �55 31 3409 2229; fax: �55 31 3409 2230. E-mail address: luzmr@uol.com.br (M.R. Luz). 093-691X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. ttp://dx.doi.org/10.1016/j.theriogenology.2012.03.003 1. Introduction Assisted reproductive technologies during the 20th century have progressed much further in live- stock than in companion animals, presumably due to the relative lack of commercial interest in the latter. However, during the last two decades, there were major advances in basic and applied research involv- mailto:luzmr@uol.com.br www.theriojournal.com http://dx.doi.org/10.1016/j.theriogenology.2012.03.003 1 f D i s t p t m w c w V a w A m 1 l a z m S I s o w u t ( fl A t 0 b fi w d 577C. Renato de Freitas Guaitolini et al. / Theriogenology 78 (2012) 576–582 ing dogs, due to their importance as companion an- imals in modern society, as well as the concern with preservation of species threatened with extinction [1,2]. Cryopreservation of canine embryos has potential for preserving the gene pool of this species and of specific breeds, with potential implications for use in conserving non-domestic canids. Furthermore, the dog can be a more relevant experimental model than mice due to their size, and similar physiology in drug and radiation reactions when compared to the human [3,4]. In addition, embryo cryopreservation allows the disso- ciation of time and/or space between embryo recovery and the transfer to recipient females, as well as it facilitates international transportation of embryos. An embryo’s capacity to survive cryopreservation depends on several factors, including species, stage of development, origin (in vivo or in vitro), cryopreserva- tion methodology, cryoprotectant [5], and intracyto- plasmic lipid content [6]. Post-thaw viability can be estimated with the blastocoel reexpansion rate, embry- onic cleavage rate, in vitro hatching [7,8], as well as with fluorescent probes (e.g., Hoechst 33 342 and pro- pidium iodide), which stain living and dead cells, re- spectively, and are useful to estimate the percentage of viable blastomeres [8,9]. There are limited reports regarding cryopreservation of canine embryos. Kim, et al. [10] cryopreserved ca- nine embryos in glycerol by conventional slow freez- ing, but they did not verify post-thaw viability and did not obtain pregnancies after the transfer of the thawed embryos to recipient females. Conversely, Abe, et al. [6] obtained embryo viability rates of 50% for morulae and 40% for blastocysts and concluded that vitrification was a good method for cryopreserving canine embryos. The objectives of this study were to evaluate the blastocoel reexpansion rate, post-thaw embryo viabil- ity, as well as in vitro development of canine embryos cryopreserved by slow freezing with 1.0 M glycerol or .5 M ethylene glycol as cryoprotectants. 2. Materials and methods A 2 � 3 factorial experimental design was used to test two cryoprotectants (1.0 M glycerol and 1.5 M ethylene glycol) and three moments of post-thaw em- bryo evaluation (M0, M3, and M6). This study was approved by the “Federal University of Espirito Santo Ethical Committee on Use of Animals” (UFES, Brasil - Protocol no. 035/2010). m Fifty-one in vivo-produced embryos were recovered rom 16 healthy donors (various breeds or cross-bred). onors, which were 5 to 20 kg and 1 to 5 yrs old, were dentified as proestrus on the basis of vulvar swelling, erosanguineous vaginal discharge, or both. After ini- ial observations, every other day vaginal cytology was erformed until 80 to 90% superficial cells were de- ected [11]. Bitches sexually receptive to a male were ated every other day, for three times, whereas those hich reached a minimum of 80 to 90% of superficial ells on vaginal smears and were not sexually receptive ere vaginally inseminated (same breeding schedule). aginal AI was done with a plastic disposable syringe nd plastic catheter. After semen deposition, the bitches ere held with their hindquarters elevated for 10 min. fertile male dog (�300 � 106 morphologically nor- al, motile sperm) was used for breeding. Embryos were randomly allocated in two groups, .0 M glycerol (GLY, n � 26) and 1.5 M ethylene glycol (EG, n � 25). Cryopreserved embryos were thawed and assessed for viability using propidium iodide and Hoechst 33 342 staining immediately post thawing (M0; GLY � 8; EG � 9), 3 days (M3; GLY � 8; EG � 8) or 6 days after in vitro culture (M6; GLY � 9; EG � 8) in SOFaa � 10% FCS medium at 38.5°C under an atmosphere of 5% CO2 with maximum humidity. 2.1. Embryo recovery Embryo recoveries were performed by uterine flush- ing, using the ex vivo technique standardized in our aboratory [12], 12 days after first mating or AI. The nesthetic protocol used was 0.1 mg/kg IM aceproma- ine (Acepran 0.2%; Vetnil, Louveira, SP, Brasil), 7.5 g/kg IM tiletamine/zolazepam (zoletil 50; Virbac aúde Animal, São Paulo, SP, Brasil), and 5.0 mg/kg V propofol (Propovan; Cristália, São Paulo, SP, Bra- il) It was noteworthy that propofol was administered nly after the uterus had been removed. Briefly, bitches ere subjected to an ovariohysterectomy. After the terus was removed, each uterine horn was flushed hree times using a total of 30 mL of Dulbecco’s PBS Nutricell, Campinas, SP, Brasil) [12]. The uterine ushes were immediately recovered into petri dishes. fter identification of embryos, they were transferred o disposable 35 � 10 mm petri dishes, in PBS with .4% BSA (Nutricell) manipulation medium, for em- ryo evaluation [13] under a stereomicroscope (magni- cation, 20–40x). The global embryo recovery rate as calculated as the number of structures collected ivided by the number of CL present in the ovaries, ultiplied by 100 [12]. a v p t ( m f t w i b s 2 v a i ( e v F w m S 2 o i s s ( p o p i 9 2 r m b a m c 3 d a t 578 C. Renato de Freitas Guaitolini et al. / Theriogenology 78 (2012) 576–582 2.2. Freezing and thawing procedures Cryopreservation was performed by slow freezing, using a programmable freezer (TK 3000, TK tecnologia em Congelação, Uberaba, MG, Brasil). Grades I and II blastocysts [13] (n � 51) were washed 10 times in DPBS solution and transferred to 200 �L of 1.0 M glycerol (GLY, n � 26) or 1.5 M EG (n � 25) droplets nd equilibrated for 10 min. The embryos were indi- idually placed into the middle column of the three ermeable glycerol-containing columns (GLY), or into he middle column between the 1.0 M sucrose columns EG). The straws were then placed into the program- able freezing at �6 °C, and held at this temperature or 2 min (equilibrium period). Seeding was induced by ouching the straws in lateral columns with LN2-cooled forceps. Five min after seeding, cooling was continued at a rate of 0.6°C/min until �35 °C (for both groups). After reaching �35 °C, straws were held at this tem- perature for 5 min, the removed from the freezing apparatus and immediately submerged in LN2. For post-thaw evaluations, straws containing the embryos were held in air for 15 s and then plunged into a 37 °C water bath for another 15 s. The contents of the straws were emptied into a petri dish. The glycerol was removed in three steps. Embryos were held for 5 min in each glycerol-decreasing solution (6.6, 3.3, and 0.0%, respectively) containing 1.0 M sucrose, and then were ashed in DPBS plus 0.4% BSA. The straws contain- ng EG embryos were similarly thawed and washed in uffer solution, but without the cryoprotectant removal teps. .3. Post-thaw evaluation of embryo viability Fifty-one embryos were evaluated as to post-thaw iability. Embryos from M0 were thawed and immedi- tely stained with propidium iodide (PI; Sigma, Chem- cal Co, St. Louis, MO, USA) and Hoechst 33 342 Sigma, Chemical Co.) for cellular viability evaluation; mbryos from M3 and M6 were thawed, cultured in itro for 3 or 6 days, respectively, and similarly stained. or staining, embryos were incubated for 30 s in DPBS ith 0.4% BSA solution with 125 �g/mL PI, then transferred to a slide containing a droplet of 1 mg/mL Hoechst 33 342 and glycerol, and after 15 min, exam- ined under fluorescence microscopy (Leica DM LB, with 535 and 617 nm filters). Blastomeres with an intact plasma membrane had a blue fluorescence and were considered live, whereas those with a disrupted plasma membrane stained by PI were considered dead. The number of viable and unviable cells was counted with the aid of ImageJ 1.43u (Wayne Rasband, Na- b tional Institutes of Health, Bethesda, MD, USA). Em- bryos with �50% live blastomeres were considered viable [7,14]. Twenty-four h after in vitro culture, embryos (GLY, n � 17, e.g., n � 16) were evaluated to determine blastocoel reexpansion rate. Embryo hatching was eval- uated 3 and 6 days after in vitro culture. 2.4. In vitro culture (IVC) Thawed-embryos were in vitro cultured at 38.5 °C under an atmosphere of 5% CO2 with maximum hu- idity, for 3 or 6 days. The culture medium used was OFaa with 10% FCS. .5. Plasma progesterone concentrations Blood samples were obtained on Day 0 (first mating r AI) and on Day 12 by jugular venipuncture and mmediately centrifuged (600g for 15 min). Plasma was tored in microvials and frozen (�20 °C) pending as- ay. Commercial solid phase radioimmunoassay kits progesterone Coat-a-Count; Diagnostic Products Cor- oration, Los Angeles, CA, USA) were used as previ- usly validated for canine plasma [15]. Samples were rocessed in duplicate (following all embryo recover- es). The intra-assay CV was 3.2% and sensitivity was 4.0% (0.03 ng/mL). .6. Statistical analysis Data for blastocyst total cell number and viable cell ate were analyzed by ANOVA using the general linear model (GLM) procedure of SAS (SAS Institute, Inc., Cary, NC, USA). Re-expansion rate data were analyzed by logistic regression, using the LOGISTIC procedure of SAS. Sources of variation in the model included cryoprotectant (glycerol and ethylene glycol), post- thaw embryo evaluation moments (M0, M3 and M6), and all first-order interactions; all factors were consid- ered fixed effects. The arcsine and logarithm transfor- ation were applied to embryo percentage rate and lastocyst total cell number data, respectively. In the bsence of significant interactions, only main effect eans are presented. For all analyses, P � 0.05 was onsidered significant. . Results and discussion Seventy-two embryos recovered from 16 female ogs, 51 were classified as blastocyst Grades I and II, nd were used in the study. All other structures (unfer- ilized eggs, early embryos, morulae and Grade-III lastocysts) were discarded. A total of 125 CL were h w [ t 4 t m a o l t ( c S w Z m c t u p v l s t v g P o 5 f c a s c b m s t 579C. Renato de Freitas Guaitolini et al. / Theriogenology 78 (2012) 576–582 identified, with an embryo recovery rate of 57.6%. Interestingly, the recovery rate in 7 of 16 (43.7%) animals was 100%, consistent with a previous report in female Labrador Retrievers [16]. However, there was a 50 to 90% recovery of the embryos in three dogs (18.7%), whereas in four others (25.0%), no embryo was recovered. The average embryo recovery rate in this study (57.6%) seemed lower than our previous report (81.0%) [12], but was similar to results obtained with silver foxes (66.0%) [17]. Furthermore, there was also a greater blastocyst recovery in this study regard- ing the number of recovered embryos, as previously described [12]. Factors, such as, endometrial cystic hyperplasia [18], variations in plasma progesterone concentrations [19], or the presence of the embryos in the uterine tubes, instead of the uterine horns [12] may ave affected the recovery rate. Moreover, it was note- orthy that expanded blastocysts were not shrunken 16] after recovery or handling (Fig. 1A). Plasma progesterone concentrations on the day of he first mating or AI and at embryo recovery were .57 � 3.77 ng/mL and 28.56 � 3.21 ng/mL, respec- ively. Therefore, we inferred that the females were ated or inseminated during their fertile period [18] nd embryo recoveries were performed at the beginning f pregnancy [19]. The percentage of embryos with ruptured zona pel- ucida immediately after thawing, did not differ be- ween GLY and EG groups [3/26 (11.5%) vs. 2/25 8.0%), respectively; P � 0.6726]. These rates were onsistent with a report by Van den Abbeel and Van teirteghem [14], who obtained rates from 2.3 to 16.6% ith human embryos cryopreserved by slow freezing. ona pellucida lesions have been reported in various ammals [20–23]. Damage to the zona pellucida of ryopreserved embryos may occur due to several fac- ors, including mechanical stress produced from non- niform volume changes of the freezing medium during hase-change [22], and may negatively affect embryo iability. The possible causes of the zona pellucida esions found in this work was not the object of the tudy, but the rates obtained were considered satisfac- ory [14,21]. The embryo reexpansion rate (%) after 24 h of in itro culture did not differ between the GLY and EG roups [13/17 (76.5%) vs. 11/16 (68.8%), respectively, � 0.6196]. Shu, et al. [7] obtained reexpansion rates f 48.0 and 50.0% in frozen human blastocysts on Days and 6, respectively. These authors concluded that the ast reexpansion of the blastocoel (2–4 h of in vitro ulture) has a direct relation with greater implantation p nd pregnancy rates, when compared to the reexpan- ion that occurred more slowly. Similarly, studies with onsumption of glucose during reexpansion of bovine lastocysts demonstrated that reexpansion is a good orphologic marker for embryo selection [24]. In heep, Leoni, et al. [25] evaluated the viability of blas- ocysts produced in vitro and vitrified, obtaining reex- Fig. 1. Morphological appearance (A,C,E,G), and PI/Hoechst 33 342 staining (B,D,F,H) of fresh canine blastocysts (A,B) and frozen- thawed blastocysts (C,D immediately after thawing; E,F in vitro cultured for 3 d; G,H in vitro cultured for 6 d). The embryos were magnified (A,C,E,G �10; D � 200; B,F,H �400). ansion rates of 55.0 to 87.0%, and also considered o p o e e o u o c e 5 C r y r e a M o n t c w n b t g p m b p t 2 580 C. Renato de Freitas Guaitolini et al. / Theriogenology 78 (2012) 576–582 reexpansion as a factor determining embryo viability. Although no studies evaluating the reexpansion of cryopreserved canine embryos were found, the rates obtained in this study were considered satisfactory [7,24,25], and also reflected the percentage of embryos that remained viable in vitro for up to 6 days. Surprisingly, none of the embryos hatched. How- ever, similar results were obtained by Lindeberg, et al. [17], who did not observe any hatching in silver fox (Vulpes vulpes) embryos cultured in vitro for up to 3 wk. Since implantation of canine embryos occurs be- tween 18 and 21 days after ovulation [26,27], and based on plasma progesterone concentrations, females were in their periovulatory period (approximately 2 days after the LH peak) [27] at breeding, at the end of culture f the M6 group, some of the embryos would be ap- roximately 18 days old. However, hatching did not ccur. The hatching process demands a lot from the mbryo. Presumably, inadequate culture conditions or ven “weakness” of the embryo were the main causes f failure to hatch [28]. Although the SOF medium sed in this study is indicated for the initial culture of ocytes and embryos [29], perhaps its composition also ontributed to the lack of hatching. There was no significant difference on post-thaw mbryo viability rates between groups (66.5 � 4.8 and 7.3 � 4.8 for GLY and e.g., respectively, P � 0.2074). ocero, et al. [29] obtained better embryo viability ates after cryopreservation of ovine embryos with eth- lene glycol instead of glycerol. Similar results were eported by Pantano, et al. [30], with a superiority of thylene glycol in comparison with propylene glycol nd glycerol for freezing mouse embryos. Conversely, artinez and Matkovic [31] compared embryo devel- pment and hatching rates of ovine embryos, and did ot find statistical differences between either cryopro- ectant. In the dog, Kim, et al. [10] used glycerol as a ryoprotectant for freezing embryos by slow freezing, hich after thawing were transferred to recipients, but o pregnancies were obtained (although post-thaw em- ryo viability was not assessed). To our knowledge, his is the first work to study the in vitro effects of lycerol and ethylene glycol in canine embryos, cryo- reserved by slow freezing. Since glycerol has a higher olecular weight than ethylene glycol, it was removed y step-wise dilution method, and osmotic shock was revented. Blastocyst total cell number was similar in frozen- hawed embryos of both groups (311.2 � 55.10 and 53.8 � 55.10 for GLY and EG, respectively) (P � 0.8550). Furthermore, post-thaw embryo viability eval- uation with PI and Hoechst 33 342 facilitated assess- ment of the percentage of living and dead blastomeres (Fig. 1). Although this method allows a good estimate of living and dead cells in the embryo, since dead cells are permeable to PI and living cells are stained by the Hoechst [8], an underestimation of dead cells may occur if they are surrounded by living cells [8]. Immediately after thawing, 62.3 � 5.7% of the blas- tocyst cells in Group M0 were stained by Hoechst (viable cells), similar to that described by Abe, et al. [6], who evaluated the viability of vitrified canine em- bryos, immediately after warming, and obtained rates of 50% for morulae and 40% for blastocysts. However, unlike this study, in which the embryos were consid- ered viable if they had more than 50% living blastom- eres, those authors had a minimum threshold of 75% live cells. As previously mentioned, factors, such as freezing and warming rates may cause alterations in the plasma membranes after thawing [20,23]. Besides, large amounts intracellular lipids, which occur in ca- nine embryos [4] may have contributed to the percent- age of dead cells stained with PI immediately after thawing. Similar to swine [32], canine embryos usually have greater sensitivity to cryopreservation due to in- tracellular lipids. Although no statistical difference was observed, the M6 group, thawed embryos cultured in vitro for 6 days (144 h), had 66.5 � 6.0% of viable cells, almost 10% of more viable cells than the M3 group, with 56.9 � 6.0% of viable cells. It is known that certain plasma membrane alterations resulting from cryopreservation are reversible, since most cells recover in a few hours [33,34]. This may have contributed to the satisfactory result obtained at the end of culture in this study. The in vitro culture of the embryos in this study was performed in SOFaa medium. It is known that the maintenance of embryo viability in vitro may be af- fected by various factors, such as the culture medium, the donor’s age, and even the addition of components, such as hormones and growth factors in the medium [29]. Although Lindeberg, et al. [17] successfully used TCM199 medium for in vitro culture of silver fox embryos, according to Hewitt and England [35], there is no difference between SOF and TCM199 for culture of canine oocytes. Thus, besides oocytes, the SOFaa medium was also apparently helpful for the in vitro culture of canine embryos, although no hatching oc- curred. In conclusion, canine blastocysts cryopreserved in 1.0 M glycerol or 1.5 M ethylene glycol, by slow freez- ing, had satisfactory blastocoel reexpansion rates, sim- [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ 581C. Renato de Freitas Guaitolini et al. / Theriogenology 78 (2012) 576–582 ilar post-thaw in vitro viability and remained viable for up to 6 days in vitro. Acknowledgments The authors thank Fundação de Amparo à Pesquisa do Espírito Santo (FAPES/MCT/CNPq/CT-INFRA; grant 36600736/2007) and CAPES-REUNI for finan- cial support, and DuMilho Rações for animal food supply. References [1] Hewitt DA, England GCW. Manipulation of canine fertility using in vitro culture techniques. J Reprod Fertil Suppl 2002; 57:111–25. [2] Songsasen N, Wildt DE. Oocyte biology and challenges in developing in vitro maturation systems in the domestic dog. Anim Reprod Sci 2007;98:2–22. [3] Schneider MR, Wolf E, Kolb HJ, Adler H. Canine embryo- derived stem cells and models for human diseases. Hum Mol Genet 2008;17:42–7. [4] Chastant-Maillard S, Chebrout M, Thoumire S, Saint-Dizier M, Chodkiewicz M, Reynaud K. Embryo biotechnology in dog: a reviewEmbryo biotechnology in the dog: a review. Reprod Fertil Dev 2010;22:1049–56. [5] Fiéni F, Beckers JP, Buggin M, Bruyas JF, Perrin J, Daubié M, et al. Evaluation of cryopreservation techniques for goat em- bryos. Reprod Nutr Dev 1995;35:367–73. [6] Abe Y, Suwa Y, Asano T, Ueta YY, Kobayashi N, Ohshima N, et al. Cryopreservation of canine embryos. Biol Reprod 2011; 84:363–8. [7] Shu Y, Watt J, Gebhardt J, Dasig J, Appling J, Behr B. The value of fast blastocoele re-expansion in the selection of a viable thawed blastocyst for transfer. Fert Steril 2009;91:401–6. [8] Pryor JH, Looneya CR, Romob S, Kraemer DC, Longc CR. Cryopreservation of in vitro produced bovine embryos: effects of lipid segregation and post-thaw laser assisted hatching. The- riogenology 2001;75:24–33. [9] Latt SA, Stetten G. Spectral studies on Hoechst 33258 and related bisbenzimidazole dyes useful for fluorescent detection of deoxyribo-nucleic acid synthesis. J Histochem Cytoch 1976;24: 24–33. 10] Kim YJ, Kim BJ, You IJ. Embryo transfer with frozen embryos in the dog. J Vet Clin 2002;19:73–9. 11] Concannon PW, Digregorio GB. Canine vaginal cytology. In: Burke TJ, editor. Small animal reproduction and fertility. Phil- adelphia: Lea & Febiger; 1986, p. 96–111. 12] Luz MR, de Holanda CC, Pereira JJ, Freitas PM, Salgado AE, Giannotti JD, et al. High embryo recovery rates with in vivo and ex vivo techniques in the bitch. Reprod Domest Anim 2011;46: 724–7. 13] Stringfellow DA, Seidel SM. Manual da Sociedade Internacio- nal de Transferência de Embriões. 3 ed. Illinois [Manual of International Embryo Transfer Society.] Sociedade Brasileira de Transferência de Embriões, 1999. p. 90. 14] Van Den Abbeel E, Van Steirteghem A. Zona pellucida damage to human embryos after cryopreservation and the consequences for their blastomere survival and in-vitro viability. Hum Reprod 2000;15:373–8. 15] Srikandakumar A, Ingraham RH, Ellsworth M, Archbald LF, Liao A, Godke RA. Comparison of a solid-phase, no-extraction radio-immunoassay for progesterone with an extraction assay for monitoring luteal function in the mare, bitch, and cow. Theriogenology 1986;26:779–93. 16] Abe Y, Suwa Y, Yanagimoto-Ueta Y, Suzuki H. Preimplanta- tion development of embryos in Labrador retrievers. J Reprod Dev 2008;54:135–7. 17] Lindeberg H, Jalkanen L, Savolainen R. In vitro culture of silver fox embryos. Theriogenology 1993;40:779–88. 18] Tsutsui T, Hori T, Okazaki H, Tanaka A, Shiono M, Yokosuka M, et al. Transfer of canine embryos at various developmental stages recovered by hysterectomy or surgical uterine flushing. J Vet Med Sci 2001;63:401–5. 19] Luz MR, Bertan CM, Binelli M, Lopes MD. Plasma concentrations of 13,14-dihydro-15-keto-prostaglandin F2-alpha (PGFM), proges- terone and estradiol in pregnant and nonpregnant diestrus cross- bred bitches. Theriogenology 2006;66:1436–41. 20] Leibo SP, Mcgrath JJ, Cravalho EG. Microscopic observation of intracellular ice formation in unfertilised mouse ova as a function of cooling rate. Cryobiology 1978;15:257–71. 21] Schiewe MC, Rall WF, Stuart LD, Wildt DE. Analysis of cryoprotectant, cooling rate and in situ dilution using conven- tional freezing or vitrification for cryopreserving sheep em- bryos. Theriogenology 1991;36:279–93. 22] Rall WF, Meyer TK. Zona fracture damage and its avoidance during the cryopreservation of mammalian embryos. Theriog- enology 1989;31:683–92. 23] Cohen J, Devane GW, Elsner CW, Fehilly CB, Kort HI, Massey JB, et al. Cryopreservation of zygotes and early cleaved human embryos. Fert Steril 1988;49:283–9. 24] Gardner DK, Pawelczynski M, Trounson AO. Nutrient up- take and utilization can be used to select viable day 7 bovine blastocysts after cryopreservation. Mol Reprod Dev 1996;44: 472–5. 25] Leoni G, Bogliolo L, Berlinguer F, Rosati I, Pintus PP, Ledda S, et al. Defined media for vitrification, warming, and rehydration: effects on post-thaw protein synthesis and viability of in vitro derived ovine embryos. Cryobiology 2002;45:204–12. 26] Holst PA, Phemister RD. The prenatal development of the dog: preimplantation events. Biol Reprod 1971;5:194–206. 27] Concannon P, Tsutsui T, Shille V. Embryo development, hor- monal requirements and maternal responses during canine preg- nancy. J Reprod Fertil Suppl 2001;57:169–79. 28] Brandão DO, Maddox-Hyttel P, Løvendahl P, Rumpf R, String- fellow D, Callesen H. Post hatching development: a novel system for extended in vitro culture of bovine embryos. Biol Reprod 2004;71:2048–55. 29] Cocero MJ, Sebastian AL, Barragan ML, Picazo RA. Differ- ences on post-thawing survival between ovine morulae and blastocysts cryopreserved with ethylene glycol or glycerol. Cryobiology 1996;33:502–7. 30] Pantano T, Mello MRB, Garcia JF, Ho LL, Visintin JA. Effects of cryoprotectant and plunging temperature in liquid nitrogen on the in vitro and in vivo development of murine. Braz J Vet Res Anim Sci 2000;37:243–8. 31] Martínez AG, Matkovic M. Cryopreservation of ovine em- bryos: slow freezing and vitrification. Theriogenology 1998; 49:1039 – 49. [ [ [ 582 C. Renato de Freitas Guaitolini et al. / Theriogenology 78 (2012) 576–582 [32] Hayashi S, Kobayashi K, Mizuno J, Saitoh K, Hirano S. Birth of piglets from frozen embryos. Vet Rec 1989;125: 43– 4. 33] Vajta G, Hyttel P, Callesen H. Morphological changes of in vitro produced bovine blastocysts after vitrification, in- straw direct rehydration, and culture. Mol Reprod 1997;48: 9 –17. 34] Kaidi S, Bernard S, Lambert P, Massip A, Dessy F, Donnay I. Effect of conventional controlled-rate freezing and vitrification on morphology and metabolism of bovine blastocysts produced in vitro. Biol Reprod 2001;65:1127–34. 35] Hewitt DA, England GC. Synthetic oviductal fluid and oviduc- tal cell coculture for canine oocyte maturation in vitro. Anim Reprod Sci 1999;55:63–75. Post-thaw viability of in vivo-produced canine blastocysts cryopreserved by slow freezing 1. Introduction 2. Materials and methods 2.1. Embryo recovery 2.2. Freezing and thawing procedures 2.3. Post-thaw evaluation of embryo viability 2.4. In vitro culture (IVC) 2.5. Plasma progesterone concentrations 2.6. Statistical analysis 3. Results and discussion Acknowledgments References