The aim of this study was to determine the effect of dimethylformamide (DF) associated with ethylene glycol (EG) or 1-2 propanediol (PROH) during vitrification, on the in vitro development of mouse blastocysts. Cryoprotectant toxicity was evaluated exposing embryos into three different equilibrium solutions (ES) composed by DF, EG or PROH mixtures (10% v/v of each) in mPBS + 0.5% PVA at different interval times (1, 3 and 10min). In a second experiment, embryos were exposed to the same ES (either 1 or 3min), following for the three respectively vitrification solutions (VS) (20% v/v of each) for 30s. After 72 hours of in vitro culture, embryo hatching and expansion rates were similar for the ES1 and ES2 equilibration solutions during the time interval of 1 or 3min. However embryos exposed for 10 min to the DF equilibration solutions, had lower survival rates than EG-PROH solution (P<0.01). Furthermore, survival rates for embryos exposed to DF-PROH (ES+VS) were lower than embryos exposed to the other solutions (P<0.01). Blastocyst vitrification was performed with the three ES+VS (for 1min and 30s, respectively), using glass micropipettes (GMP). Survival rates were lower for blastocysts vitrified with DF solutions (3%-3/108 and 17.1%-19/111) (P<0.01) than with PROH+EG vitrification solutions (69%-73/105). In conclusion, DF as a cryoprotectant into vitrification solutions have deleterious effects on the in vitro developmental competence of vitrified mouse blastocysts.

Although slow programmable freezing is currently the standard method for bovine embryo cryopreservation, vitrification has become an alternative for in vitro-produced embryos. A study was designed to compare the in vitro survival rates of in vivo- and in vitro-produced bovine embryos with 1 of 2 commercially available methods of cryopreservation: slow freezing and solid surface vitrification. In vivo-produced Grade 1 blastocysts (n = 210) collected from superovulated donor cows 7 days post-insemination and in vitro-produced Grade 1 blastocysts (n = 122) from slaughterhouse oocytes, produced with the procedure described by Chaubal et al. (2007 Theriogenology 67, 719–728) were randomly allocated in 2 groups. Group 1 (slow freezing) embryos were exposed to 1.5 M ethylene glycol (ViGro EG; Bioniche Animal Health USA Inc., Pullman, WA, USA) for 5 min and loaded in 0.25-mL plastic straws. The straws were placed in a Freeze Control CL 5500 freezer (CryoLogic, Victoria, Australia) at –6.5°C, seeded and after 10 min of equilibration, cooled at –0.6°C min–1 until –CE°C, before plunging into liquid nitrogen. Group B (vitrification) embryos were exposed to a AE% EG+0.BEM trehalose solution for A min and then into C0% EG+AM trehalose solution for C0 sec at room temperature to be vitrified using the CVM system (CryoLogic). The CVM used a cryohook and the solutions with the embryos are exposed to a metal solid surface cooled at –AIF°C. The vitrification solution was chosen after a toxicity test in which several EG and trehalose combinations were tested (Rodriguez Villamil et al. Ith IRAC Symposium, Argentina B0AA). After at least 1 wk of storage, embryos in the slow freezing groups were thawed in water bath at C0°C for AB s, placed in holding medium for E min and then cultured in SOF. Vitrified embryos were placed directly in a 0.BE M sucrose solution for E min (at CG°C) and then cultured in SOF medium. Re-expansion and hatching rates were evaluated at BD and GB h, respectively. Data was analyzed by nonparametric tests with type of embryo and cryopreservation procedure as main effects, using the software Infostat (UNC, Argentina, B0A0). In vivo-produced embryos had higher (P < 0.0A) re-expansion (AGI/BB0, HA% vs FI/ABB, EF%) and hatching rates (AEI/BB0, GB% vs EC/ABB, DC%) than in vitro-produced embryos, regardless of cryopreservation method. However, re-expansion (DE/FC, GA%) and hatching (CI/FC, FB%) rates were higher (P < 0.0A) with in vitro-produced vitrified embryos than in vitro-produced embryos in the slow freezing group (re-expansion: BD/EI, D0% and hatching: AD/EI, BD%). Although similar re-expansion rates (IC/AA0, HE% vs HF/A00, HF%) were obtained with in vivo- produced embryos cryopreserved by the 2 systems, hatching rates tended to be lower (P = 0.0I) with in vivo-produced embryos that were vitrified compared with slow freezing (GH/AA0, GA% vs HA/AA0, HA%). In conclusion, solid surface vitrification improved the cryosurvival rates of in vitro-produced embryos compared with the conventional slow, controlled freezing procedure.

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The placenta plays a key role in normal conceptus development and in mediating effects of the maternal system on the fetus. Changes in the environment or in placental function may affect fetal developmental and lead to what is known as “developmental origins of health and disease.” Thus, the aim of this study was to analyse the protein profile of bovine placentomes of different sizes at distinct gestation ages to determine spatial and temporal differences or similarities in the protein pool of biological significance to the conceptus and the newborn. Samples of placentomes (n = 36) representing small, medium, and large sizes among placentomes at 60, 90, 120, and 160 days of gestation were collected at a local slaughterhouse and freeze-dried for protein analysis. For that, 5 mg of each tissue sample was used for protein extraction with 1% Triton X and sonication for 10 min at 4°C, with protein concentration per sample determined by the Bradford method. A total of 300 μg of protein was subjected to 2D-SDS PAGE electrophoresis (GE Healthcare®), and gels were stained with Coomassie Blue G-250. Spots were analysed using the PDQuest, with data compared by ANOVA or the Kruskall-Wallis test (SAS). In silico protein identification was performed using ExPASy. On average, 74 proteins were detected in the gels, regardless of placentome sizes and gestational ages. No interactions were detected between placentome sizes and gestational periods. However, differences were observed in the mean number of proteins between small (n = 69), medium (n = 90), and large (n = 64) placentomes (P = 0.002), and between Days 60 (n = 113), 90 (n = 65), 120 (n = 44), and 160 (n = 74) of gestation (P = 0.001). One specific spot (32.45 kDa, 4.6 pI) represented 1.0, 10.8, 12.1, and 2.1% of the intensity of all valid spots for Day 60, 90, 120, and 160 placentomes, respectively, indicating a bell-like expression. For small placentomes, all the spots had similar intensities, whereas for medium placentomes, the 32.45 kDa (4.6 pI) spot represented 3.0% of the intensity of all valid spots. For large placentomes, the 32.45 kDa (4.6 pI) spot and a 46.19 kDa (5.4 pI) spot represented 5.8 and 5.0% of the total intensity for all spots, respectively. Six and 10 spots were differentially identified between placentome sizes and between gestational periods, respectively, from which male-enhanced antigen-1 proteins and angio-associated migratory cell protein were identified by in silico analysis, with the former only present on Day 90 placentomes, and the latter for 120 days of gestation. Further analyzes on the identities of such placental proteins and their profiles is underway, which will be crucial to the comprehension of placentome function and growth during pregnancy, and for the understanding of physiological mechanisms and processes associated with normal conceptus development and life ex utero.

An experiment was designed to evaluate the effect of brilliant cresyl blue (BCB) selection of immature oocytes and the addition of sodium hyaluronate (HA) to the vitrification solution on survival rates of bovine oocytes vitrified using solid-phase vitrification. Bovine cumulus–oocyte complexes (COC; n = 716) obtained from slaughterhouse ovaries were used in 6 replicates. Cumulus–oocyte complexes were washed in tissue culture medium 199 (TCM-199) and randomly allocated to 2 groups to be exposed to BCB stain (Sigma Chemical Company, St. Louis, MO, USA) for 90 min as described by Alm et al. (2005 Theriogenology 63, 2194–2205) or (control) maintained in Vigro holding medium (Bioniche Animal Health, Belleville, Canada) for 90 min (n = 220). Cumulus–oocyte complexes in the BCB group were selected based on their response to BCB as BCB+ (colored, n = 248) or BCB– (colorless, n = 248), whereas those in the control group were selected morphologically as described by Rodríguez-González et al. (2002 Theriogenology 57, 1397–1409). Oocytes from both BCB groups and 100 oocytes in the control group were vitrified by solid-phase vitrification as previously described by Rodriguez et al. (2012 Reprod. Fertil. Dev. 24, 132). The remaining 120 oocytes in the control group were not vitrified and were matured, fertilized, and cultured in vitro (in SOFaa in a controlled atmosphere) for 7 days. Vitrified oocytes were exposed to 10% ethylene glycol for 10 min, and 20% ethylene glycol + 0.2-M trehalose for 30 s, and then were subdivided to be exposed to 30% ethylene glycol + 0.5-M trehalose with or without 0.1 mg mL–1 HA (MAP 5, Bioniche Animal Health). Vitrified oocytes were stored in liquid nitrogen for at least one week and then placed directly into a 0.5-M sucrose solution (in TCM 199) at 37°C for 5 min, 0.25 M of sucrose for another 5 min, and finally TCM-199 and matured, fertilized, and cultured. Development rates (i.e. proportion of blastocysts) were examined on Day 7 after fertilization. Proportional data were first transformed by square root and then analyzed by ANOVA to detect the effect of replicate, type of oocyte (BCB+, BCB–, controls), and vitrified with or without HA or not vitrified as main effects, using the software Infostat (UNC, Argentina, 2010). There was a significant effect of oocyte type on blastocyst rate (P < 0.01) following vitrification (BCB+, 6.4 ± 0.4%. v. BCB–, 1.6 ± 0.6%). Control oocytes (not exposed to BCB) resulted in 3.0 ± 2.0% blastocysts following vitrification, which was lower to that obtained with the BCB+ oocytes. Vitrification also influenced development rates (3.0 ± 2.0 v. 32.0 ± 1.3%) for blastocysts produced from vitrified v. nonvitrified oocytes, respectively (P < 0.01). Furthermore, the use of HA in the vitrification solutions did not have a significant effect on development rates (4.7 ± 0.9 v. 3.3 ± 0.9%, for blastocysts obtained from vitrified oocytes with or without HA, respectively). In conclusion, the selection of oocytes by BCB increased the in vitro development rates of vitrified immature oocytes, whereas the use of HA in the vitrification solution did not improve the survival rates of vitrified oocytes.