Vitrification of In Vitro Produced Porcine Blastocysts: Influence of Cryoprotectants Toxicity and Embryo Age

Background: Porcine embryos are sensible to all assisted reproduction manipulations, especially the ones that involve cryopreservation. Despite the high cryoprotectant concentrations routinely applied, vitrification is the most effective technique to date. These substances toxicity can also play a negative role in embryo viability. During in vitro porcine embryo production, the speed of development is often unevenly distributed. It is possible that their development speed, affects embryo tolerance to cryoprotectants. This study aimed to evaluate the toxicity of porcine embryos of days 5 or 6 of culture to cryoprotectant agents; as well as to assess embryo survival to vitrification.Material, Methods & Results: Parthenogenetic porcine blastocysts and expanded blastocysts of days 5 and 6 of culture were exposed to toxicity tests (experiments 1 and 2) and vitrification (experiment 3) using different protocols. In the first experiment, three different cryoprotectants were used (Dimethyl sulfoxide - DMSO, Ethylene glycol – EG, and Sucrose - SUC), combined in three different associations (G1: 15% EG + 15% DMSO with 0.5M SUC; G2: 16% EG + 16% DMSO with 0.4M SUC; G3: 18% EG + 18% DMSO with 0.5M SUC). In the fresh Control, embryos of day 6 are more sensible than the ones of day 5, whom showed a lower hatching rate (39.7 vs. 60.8%). After the toxicity (Experiment 1) test, the G1 showed better expansion rates in day 6 (50.0 vs 31.0 and 3.6% for G2 and G3) and higher hatching of day 6 compared to G2 and G3 (23.2, vs. 8.6 and 0.0% for G2 and G3). The fresh non hatched embryos at day 8, derived at day 6, had a lower percentage of cells with cleaved caspase-3 (20.2%) compared with the G1 (30.5%), G2 (31.4%) and G3 (30.5%). The hatched embryos of day 5 from G2 had lower total cell number (TCN) compared with the day 6 hatched embryos, whereas in G1 the TCN was not affected. The second experiment compared EG combined to one of these three extracellular cryoprotectants: Polyvinylpyrrolidone/sucrose/trehalose (respectively groups: PVP, SUC, TRE). The group SUC has raised the best results for day 5 embryos, whereas for day 6 embryos SUC and TRE were both best. The third experiment tested four vitrification protocols, being P1: EG+DMSO+TRE/warming with SUC; P2: EG+DMSO+TRE/warming TRE; P3: EG+TRE/ warming SUC; P4: EG+TRE/warming TRE. The expansion of vitrified day 5 embryos was higher in the P1 (20.0%) in comparison with the other three groups (4.3, 4.3 and 4.4% for P2, P3 and P4, respectively), with no difference for their hatching rates, been it lower comparing to the Control. Day 6 embryos showed no difference in expansion and hatching for the vitrified groups, been them lower than the Control.Discussion: Embryos obtained on day 6 are more sensible than the ones of day 5, fact observed when the embryos were exposed to cryoprotectant solution, as well by the behavior of the no treated Control embryos. The toxicity increases as it does the concentration of intracellular cryoprotectant, where over 16% of the intracellular cryoprotectors already affected the day 6 embryos development. For the day 5 embryos however, 15 or 16% of the intracellular cryoptrotectors, had similar behavior to the embryos. For the extracellular solutions, however, it is variable according the embryos development speed. Indeed, it is necessary to adjust the cryoprotectors to be used to cryopreserve porcine in vitro produced embryos obtained at days 5 and 6 of culture.

25 Cryopreservation of horse testicular tissue as a model for rhinoceros

Cryopreservation of testicular tissue (TT) allows retention of valuable genetic material that can be used for conservation of endangered species, such as the northern white rhinoceros (NWR; Ceratotherium simum cottoni). Previously, we found that cryopreservation of NWR TT with a slow controlled cooling rate (CR) method induced morphological alterations in the seminiferous tubules (ST). However, the relative influence of CR, type of medium, and condition of TT from the aged NWR male on TT integrity was not clear. Due to the limited availability of rhinoceros TT, we used the horse as a model for optimization of TT cryopreservation. We evaluated the effect of (1) cryoprotectant solution [PBS (PBS +1.5M dimethyl sulfoxide) v. DMEM (DMEM/F12+10.0% fetal bovine serum+0.05M sucrose+1.5M dimethyl sulfoxide)] and (2) CR [CR1 (−2.0°C min−1 from 0°C to −4.0°C, −15°C min−1 to −12°C, and −0.3°C min−1 to −40°C in a programmable freezer) v. CR2 (same as CR1 but cooled to −8°C and held for 5min before cooling to −40°C) v. CR3 (−1.0°C min−1 from 0°C to −80°C in a CoolCell® freezing device; Corning, Corning, NY, USA)] on the structural integrity of ST from a 2-year-old horse (n=20 ST), cell viability, and expression of spermatogonial stem cells (SSC; GFRα1, and GRP125) and pluripotent markers (SSEA-4, SSEA-1, and OCT-4) in spermatogonial cells isolated from TT frozen with the above treatments (n=3). We found a positive interaction between CR and cryoprotectant solution on structural integrity of fixed and stained TT after freezing in PBS and CR2 that resulted in lower detachment of epithelium cells from the basement membrane (score±standard error of the mean; 0.50±0.1) than that of TT frozen in PBS and CR1 and CR3 (1.00±0.1 and 1.80±0.1, respectively; P<0.001) or in DMEM and CR1 (1.25±0.1), CR2 (1.35±0.1), and CR3 (1.40±0.1; P<0.01) and in lower incidence of basement membrane damage (0.75±0.1) than that of TT frozen in PBS and CR1 (1.17±0.07) and CR3 (1.16±0.07) or in DMEM and CR1 (1.10±0.1), CR2 (1.15±0.1), or CR3 (1.45±0.1; P<0.01). A lower rate of pyknosis was observed in TT frozen with PBS (1.15±0.06) than in TT frozen in DMEM (1.43±0.06; P<0.001). Overall, integrity of ST was improved when TT was frozen in PBS at CR2 having similar percentages of ST with intact epithelium (60%) and basement membrane (35%) as that of refrigerated TT (45 and 50%, respectively) but different from that of TT frozen with PBS at CR1 (10 and 15%, respectively; P<0.05). Flow cytometry analysis of spermatogonial cells revealed that the percentages of live cells from TT frozen in PBS (CR1: 61.5±7.4%; CR2: 59.7±4.8%; CR3: 51.5±4.1%) or DMEM (CR1: 66.2±6.0%; CR2: 59.8±6.0%; CR3: 58.9±6.9%), and expression of SSC and pluripotent markers was similar among all freezing treatments. However, the percentages of live cells from frozen-thawed TT were lower than those of cells isolated from refrigerated TT (80.6±2.2%; P<0.001). Overall, our results showed that (1) structural integrity of horse ST was better maintained when TT was frozen in PBS at CR2 and (2) SSC can be isolated from frozen-thawed TT with a similar relative frequency to that of refrigerated TT.

Tolerance of jenipapo seeds to cryoprotectants and thawing after immersion in liquid nitrogen

The objective of this study was to evaluate the effects of two cryoprotectants followed by thawing on the physiological potential of Genipa americana L. seeds. Two experiments testing 12 treatments were conducted, one for each cryoprotectant, both in a factorial scheme of 6 × 2 (cryoprotectant concentrations × thawing methods). We tested 0, 5, 10, 15, 20, and 25% for dimethyl sulfoxide, and 0, 0.25, 0.5, 0.75, 1.0, and 1.25 M for sucrose. The thawing methods were fast (38 °C for 30 min) and slow (25 °C for 4 h). The seeds were immersed in the cryoprotectant solution for 3 h, stored for 120 h, and then thawed. The seeds were then sown in substrate (sand and vermiculite, 1:1). Emergence percentage along with speed index, length, fresh and dry matter mass of seedlings were evaluated. Dimethyl sulfoxide and sucrose can be used as cryoprotectants in G. americana seeds. Thawing should be slow when treating seeds with dimethyl sulfoxide.

Post-cooling survival, growth and deformity rates in zebrafish embryos (Danio rerio)

SummaryThis study investigated and analysed survival, growth and macro- and microscopic damage during the development of zebrafish embryos up to the adult stage after undergoing cooling. The embryos at 50% epiboly stage were selected, submerged in cryoprotectant solution of methanol and sucrose, cooled gradually to 0 ± 2°C temperature, and divided into two groups with different storage times (6 and 18 h). Subsequently, the embryos were reheated, rehydrated and incubated normally. The experiment lasted 5 months and, from hatching onward, the larvae were examined, collected and processed at pre-established time intervals. The hatching rate was significantly higher for the larvae stored for 18 h compared with the 6-h group. However, embryos from this group gave rise to a larger number of malformations, and these were much more severe compared with those in the 6 h group, which led to a higher mortality in the long term. Regarding larval length, the animals of the 6 h group had higher mean total length compared with the 18 h group, but both treatments were inferior to the control. Numerous macro- and microscopic malformations were observed and, in both treatments, only the morphologically normal individuals were able to develop to the adult stage, with organ development similar to the control, except for the gonads that were still undifferentiated in treated animals.

Survival of Macrobrachium amazonicum embryos submitted to cooling

SummaryCooling techniques have several applications for reproduction in aquaculture. However, few studies have sought to create protocols for cooling and cryopreservation of Macrobrachium amazonicum embryos. Thus, the objective of this work was to verify the survival of M. amazonicum embryos and the correlation between embryonic volume and mortality of M. amazonicum embryos after cooling. Embryo pools were collected from three females and divided into two treatment groups: dimethyl sulfoxide (DMSO) 3% and ethylene glycol (EG) 0.5%, both of them associated with 2 M sucrose. Positive and negative control groups consisted of seawater 10%. Aliquots of 10 µg of embryos were placed in Falcon® tubes containing a cryoprotectant solution and submitted directly to the test temperature of 2°C for 2 and 6 h of cooling. Further analysis of survival and embryonic volume were performed under a stereoscopic microscope. Data were subjected to analysis of variance (ANOVA), and means were compared using the Tukey test at 5%. The highest embryonic survival rate was observed after the shortest storage time for both the DMSO 3% and the 0.5% EG groups, with survival rates of 84.8 ± 3.9 and 79.7 ± 2.8%, respectively. There was a reduction in survival after 24 h, with the DMSO 3% group presenting a survival rate of 71.7 ± 6.6%, and the EG 0.5% group, 66 ± 6.9%. Survival showed a statistically significant difference when compared with the positive controls after 2 h and 24 h of cooling, with 99 ± 0.5% and 95.8 ± 1.5% survival rates, respectively. There was no significant statistical difference in the embryonic volume, but it was possible to observe a change in the appearance of the embryos, from a translucent coloration to an opaque white or brownish coloration, after 24 h in incubators. Thus, it can be concluded that survival is inversely proportional to storage time and that, although there was no change in the embryonic volume after cooling, a change in the appearance of embryos could be observed.

Cryopreservation of Coffea arabica L. Zygotic Embryos by Vitrification

As a consequence of the difficulty in conventional coffee seed storage, biotechnological alternatives such as cryopreservation have been investigated. The objective of this study was to develop a protocol for the cryopreservation of Coffea arabica L. (cv. ‘Catuaí Vermelho’ - IAC 144) zygotic embryos by vitrification. For the cryopreservation study, the embryos were immersed in Plant Vitrification Solution 2 at different times (0, 10, 25, 50, 100, and 250 min) and two temperatures (0 and 25 °C). Subsequently, the best thawing time was determined in a water bath (1, 3, 5 minutes or directly in Recovery Solution). An anatomical study was conducted on non-stored and stored embryos, with or without the use of Plant Vitrification Solution 2. The immersion in cryoprotectant solution for 100 min at 0 °C allows embryo cryopreservation. Embryos can be directly thawed in Recovery Solution after storage in liquid nitrogen. It was observed that Plant Vitrification Solution 2 reduced internal water content in the cells, allowing subsequent embryo growth resumption.

CRYOPRESERVATION OF TRUE-SEED AND EMBRYO OF MAIZE AND SOYBEAN FOR LONG-TERM STORAGE

<br />Study on cryopreservation of Indonesian local cultivars and improved varieties of maize and soybean has never been done. This method may be used for long-term preservation of seeds of maize and soybean. In this study, the method was applied to maize and soybean, Arjuna and Wilis respectively, as a model for preserving germplasm of ortodox seeds. Whole seeds and excised embryos of both varieties were subjected to two methods of cryopreservation, i.e., two-stage cooling and rapid freezing with or without 15% dimethyl sulfoxide (DMSO) as cryoprotectant solution prior to immersion in liquid nitrogen (-196oC). Results indicated that there was no significant difference between the use of DMSO for both species in terms of viability, although pretreatment in DMSO was slightly reduced the percentage of viability of both species. Slow freezing to -30oC prior to immersion in the liquid nitrogen could give as high as 76.67% and 51.67% surviving whole seeds of maize and soybean, respectively. Preserving excised embryos of maize in the liquid nitrogen using either slow or rapid freezing significantly reduced the percentage of viability from 20-76.67% to 5-18.33% (four folds) depending on treatments applied. Results also showed that one day or 15 minutes of immersion of samples in the liquid nitrogen gave rise to similar values of viability of maize and soybean, i.e., 20-60% and 20-51.67%, respectively depending on treatments applied. These results implied that for long-term storage of maize and soybean seeds as they could survive at the rate of 76.67% and 51.67% respectively, the seed can be treated by prefreezing to -30oC<br />without the presence of DMSO prior to immersion in liquid nitrogen.<br /><br />