Vitrification of goat, sheep, and cattle skin samples from whole ear extirpated after death and maintained at different storage times and temperatures

Proper tissue preservation from a wide range of animals of different species is of paramount importance, as these tissue samples could be used to reintroduce lost genes back into the breeding pool by somatic cloning. We aim to study the temporal and thermal post-mortem limits, tested in rabbits and pigs, within which there will be guarantees of obtaining living skin cells in goat, sheep, and cattle. We also intend to study the effect of vitrification on the ability of ear skin cells, stored at different times and temperatures, to attach to the substratum and grow in vitro after warming. Ears were stored either at 4 degrees C for 12, 252, and 348 h post-mortem (hpm), or at room temperature (22-25 degrees C) for 60 and 96 hpm. In all cases, skin samples from these ears were sorted into two groups: one group was in vitro cultured immediately after storage, and the other group was vitrified after storage and further in vitro cultured. In goat and sheep, no differences in attachment (100%: goat; 90-100%: sheep) or subconfluence (75-100%: goat; 70-100%: sheep) rates were observed between experimental groups. However, in days of culture to reach subconfluence, significant differences between non-vitrified and vitrified groups were observed when ears were stored at 4 degrees C for 12 and 252 hpm. In cattle, with respect to attachment rate, vitrified samples from ears stored at 22-25 degrees C for 60 hpm were different from non-vitrified control group (60 vs. 100%, respectively; P < 0.05). Also, days of culture to reach subconfluence were analysed by a non-parametric Cox Survival Analysis. In general, results from ANOVA and Survival Analysis were similar, because the proportion of censored data was quite low (9%), so the bias when using ANOVA is not too high. In spite of all the above, the lowest survival rates (75%: goat; 70%: sheep; and 40%: cattle) were sufficiently high to enable collection of skin samples from the majority of dead animals and their cryopreservation.     

Survival of oocytes recovered from vitrified sheep ovarian tissues

The objective of this work was to develop an effective vitrification technique for cryopreserving oocytes in sheep ovarian tissues. Ovaries were surgically recovered from 15 pubertal ewes and the ovarian cortex was cut into sections. Ovarian tissues were placed in equilibration medium consisting of 4% (v/v) ethylene glycol (EG) and 20% (v/v) FBS in TCM-199 on ice for 30 min and transferred to vitrification solution (35% EG, 5% polyvinylpyrrolidone, 0.4M trehalose and 20% FBS in TCM-199) for 5 min. Ovarian tissues were vitrified by dropping the tissue on the surface of a steel cube cooled by liquid nitrogen. Cumulus-enclosed oocyte complexes (COC) were also collected and vitrified following the procedure used for ovarian tissues. After 2-3 weeks of storage in liquid nitrogen, ovarian tissues and COC were thawed at 37 degrees C in 0.3M trehalose and COC in ovarian tissues were mechanically and enzymatically isolated. Vitrified COC and freshly collected COC were washed twice in maturation medium (TCM-199 supplemented with 0.255 mM pyruvate and 10% heat-treated estrus cow serum) and cultured in 50 microl drops of maturation medium under paraffin oil for 23-25h at 39 degrees C in a humidified atmosphere of 5% CO(2) in air. After culture, cumulus cells were removed by hyaluronidase treatment and vortexing and oocytes were fixed and stained. No significant differences were observed between vitrified oocytes, oocytes recovered from vitrified ovarian tissues and non-vitrified control oocytes in the percentage of oocytes with acceptable staining per total number of oocytes fixed or with visible chromatin per total number of oocytes with acceptable staining. However, fewer (P<0.05) oocytes obtained from vitrified ovarian tissues (70%) reached metaphase II compared to vitrified oocytes (88%) and non-vitrified control oocytes (90%). In contrast, when oocytes with at least 3-5 layers of cumulus cells were considered from each of the three groups, no differences (P>0.05) were observed due to treatment in the percentages of oocytes developing to metaphase II. These results demonstrate that sheep oocytes can be successfully cryopreserved by vitrification of ovarian tissues and exhibit in vitro maturation rates similar to that of vitrified and non-vitrified oocytes.     

Rapid cooling of the amniotic membrane as a model system for the vitrification of posterior corneal lamellae

To vitrify human amniotic membrane specimens so that the maximum of epithelial cells survives in order to develop a procedure for the eventual vitrification of posterior corneal lamellae without using cryoprotective agents. To assess different methods of tissue sample preparation preceding vitrification. In group 1, the amniotic membrane specimens were stretched on nitrocellulose support. In group 2, mechanical pressure was used to remove the excess culture medium between the support and the membrane. The samples were frozen in liquid ethane (?183 °C) and stored in liquid nitrogen. The specimens in the control group were not vitrified. Re-warming was performed at 40 °C. The epithelial cell survival rate was assessed after 1, 3 and 7 days of storage following re-warming using calcein and ethidium homodimer-1 fluorescence. A wide range of values was observed among the different groups and among individual specimens within the groups. Resulting average survival rate was 41 % for group 1 and 53 % for group 2; in several samples the cell survival rate exceeded 70 %. The storage period did not significantly affect the survival rates. The results of the rapid cooling of amniotic membranes in liquid ethane indicate that significant percentage of epithelial cells remain viable after the re-warming.     

Factors affecting nuclear maturation, cleavage and embryo development of vitrified bovine cumulus-oocyte complexes

The objective was to investigate the effects of cryodevice, vitrification solutions, and equilibration time on in vitro maturation, cleavage, and embryo development of vitrified bovine oocytes. In Experiment 1, the nuclear maturation (MII) rate of immature bovine COCs vitrified was compared between two equilibration times (0 vs 10 min) in vitrification solution 1 (VS1) and two cryodevices (cryotop vs 0.25 mL straw). The MII rate was higher in the non-vitrified control group than in vitrified groups (61 vs 16%, P < 0.0001). Equilibration time did not affect MII rate (P = 0.964); however, the MII rate was higher for COCs vitrified on cryotops than in straws (23 vs 9%, P = 0.007). In Experiment 2, bovine COCs were vitrified on cryotops using two equilibration times (0 vs 5 min) in VS1 and two kinds of vitrification solutions (freshly prepared vs frozen). Cleavage and blastocyst rates were higher (P < 0.0001) in the non-vitrified control group than vitrified groups (cleavage rate 93 vs 42% and blastocysts rate 31 vs 0.4%). Cleavage rate of COCs vitrified using frozen solutions with 5 min equilibration was higher (P = 0.05) than other treatment groups. However, blastocyst rate did not differ (P = 0.993) among treatment groups. In conclusion, cryotop was a better cryodevice than 0.25 mL straw for vitrification of bovine COCs. Furthermore, 5 min equilibration in VS1 improved cleavage. Compared with control, the vitrification procedure per se damaged bovine COCs, resulting in poor nuclear maturation and embryo development. However, vitrification did not immediately kill oocytes, as the cleavage rate was acceptable.     

Ultrastructural changes of sheep cumulus-oocyte complexes following different methods of vitrification

To determine the ultrastructural changes of sheep cumulus-oocyte complexes (COCs) following different methods of vitrification, good quality isolated COCs (GV stage) were randomly divided into the non-vitrified control, conventional straw, cryotop and solid surface vitrification groups. In both conventional and cryotop methods, vitrified COCs were respectively loaded by conventional straws and cryotops, and then plunged directly into liquid nitrogen (LN2); whereas in the solid surface group, vitrified COCs were first loaded by cryotops and then cooled before plunging into LN2. Post-warming survivability and ultrastructural changes of healthy COCs in the cryotop group especially in comparison with the conventional group revealed better viability rate and good preservation of the ooplasm organization. However in all vitrification groups except the cryotop group, mitochondria were clumped. Solely in the conventional straw group, the mitochondria showed different densities and were extremely distended. Moreover in the latter group, plenty of large irregular connected vesicles in the ooplasm were observed and in some parts their membrane ruptured. Also, in the conventional and solid surface vitrification groups, cumulus cells projections became retracted from the zona pellucida in some parts. In conclusion, the cryotop vitrification method as compared with other methods seems to have a good post-warming survivability and shows less deleterious effects on the ultrastructure of healthy vitrified-warmed sheep COCs.     

Vitrification of in vitro produced ovine embryos at various developmental stages using two methods

This study was conducted to evaluate the effects of developmental stage of in vitro produced (IVP) ovine embryos and the type of vitrification procedure used on embryo cryotolerance.The IVP embryos were vitrified at five different developmental stages: 4-, 8- and 16-cell, morula, and blastocyst. For each stage, half of the embryos were vitrified in either 30 μl 3.4 M glycerol + 4.6 M ethylene glycol in straw (method 1) or in <0.1 μl 2.7 M ethylene glycol + 2.1 M Me₂SO + 0.5 M sucrose placed on the inner surface of a straw (method 2) of vitrification solution, based on two different procedures. After warming embryo viability was determined by assessing the rates of re-expansion, survival, and blastocyst formation. The quality of surviving embryos was evaluated by their hatching rate and blastocyst cell numbers. In both vitrification methods, embryo survival progressively increased as the developmental stage progressed. In method 1 few of the early cleavage stage embryos (4-, 8- and 16-cell) could reach to the blastocyst stage following warming. There was no significant difference in blastocyst cell numbers (total, ICM, and trophectoderm cells) or hatching rate of blastocysts derived from vitrified embryos at different developmental stages. The number of dead cells in vitrified blastocysts in method 1 was higher than for non-vitrified blastocysts (P < 0.05). The number of apoptotic cells in vitrified blastocysts was higher than for non-vitrified counterparts (P < 0.05). In conclusion, both the developmental stage of IVP ovine embryos and the method of vitrification have a significant effect on the viability and developmental competence of sheep embryos.     

A new vitrification device that absorbs excess vitrification solution adaptable to a closed system for the cryopreservation of mouse embryos

Several closed vitrification devices that avoid contact with liquid nitrogen have been reported. Recently, based on the Kitasato Vitrification System (KVS), we developed the Closed-KVS, which is a closed vitrification device. The KVS is an open vitrification device that can absorb excess vitrification solution. In this study, we performed two experiments to evaluate the efficacy of the Closed-KVS as a vitrification device for the cryopreservation of mouse embryos at the blastocyst and two-cell stage. In the first experiment, the blastocysts were vitrified using either the Closed-KVS or the KVS (control device). The survival, re-expansion, and hatching rates were not significantly different between embryos vitrified using the Closed-KVS and those vitrified using the KVS. In the second experiment, we evaluated the embryonic development of the two-cell stage embryos vitrified using the Closed-KVS. There were no significant differences in the survival, blastocyst formation, or hatching rates between vitrified or non-vitrified embryos. Additionally, we evaluated the cooling and warming rates of these devices using a numerical simulation method. The cooling rates of the Closed-KVS were similar regardless of whether the outer cap was pre-cooled and were lower than those of the KVS. However, the warming rates of the Closed-KVS (irrespective of cap pre-cooling) were the same as those of the KVS (612,000 °C/min). In summary, the Closed-KVS is a novel closed vitrification device for the cryopreservation of mouse embryos at the blastocyst and two-cell stage.     

Fresh and vitrified bovine preantral follicles have different nutritional requirements during in vitro culture

The aim of this study was to compare the efficiency of different media for the in vitro culturing of fresh and vitrified bovine ovarian tissues. Fragments of the ovarian cortex were subjected to vitrification and histological and viability analyses or were immediately cultured in vitro using the alfa minimum essential medium, McCoy’s 5A medium (McCoy), or medium 199 (M199). Samples of different culture media were collected on days 1 (D1) and 5 (D5) for quantification of reactive oxygen species and for hormonal assays. In non-vitrified (i.e., fresh) ovarian tissue cultures, the percentage of morphologically normal follicles was significantly greater than that recorded for the other media (e.g., M199). In the case of previously vitrified tissues, the McCoy medium was significantly superior to the other media in preserving follicular morphology up until the last culture day (i.e., D5), thus maintaining a similar percentage from D1 to D5. Reactive oxygen species levels were higher in D1 vitrified cultured tissues, but there were no differences in the levels among the three media after 5 days. The hormonal assays showed that in the case of previously vitrified tissues, at D5, progesterone levels increased on culture in the M199 medium and estradiol levels increased on culture in the McCoy medium. In conclusion, our results indicate that the use of M199 would be recommended for fresh tissue cultures and of McCoy for vitrified tissue cultures.     

Successful pregnancy following transfer of feline embryos derived from vitrified immature cat oocytes using 'stepwise' cryoprotectant exposure technique

Oocyte cryopreservation is the desired tool for the ‘long-term’ storage of female genetic potential especially for endangered/valuable species. This study aims at examining the ability of different cryoprotectant (CPA) and CPA exposure techniques to protect immature feline oocytes against cryoinjury during vitrification. Immature oocytes were submitted to different CPA exposure techniques: 1) 2-step DMSO, 2) 4-step DMSO, 3) 2-step EG, 4) 4-step EG, 5) 2-step EG plus DMSO and 6) 4-step EG plus DMSO. Non-CPA treated, non-vitrified oocytes served as controls. The oocytes were then submitted either to in vitro maturation (Experiment 1, n = 334) or to vitrification/warming (Experiment 2, n = 440). The stage of nuclear maturation was subsequently determined. In Experiment 3, the vitrified immature oocytes (n = 254) were matured and fertilized in vitro, and their developmental competence was assessed. A total of 424 embryos derived from vitrified immature oocytes were transferred into the oviduct of 6 recipient queens (Experiment 4).Vitrification reduced significantly the meiotic and developmental competence of immature cat oocytes compared with the non-vitrified controls. The EG alone or a combination of EG and DMSO yielded higher maturation rates than DMSO, irrespective of the CPA equilibration techniques used. The 4-step EG vitrification resulted in the highest maturation rate (37.6%) but cleavage and blastocyst rates were significantly lower than the non-vitrified controls (24.8% and 30.2% vs 62.5% and 49.3%, respectively). Pregnancy was established in recipients receiving embryos derived from non-vitrified and vitrified/warmed immature oocytes. It is concluded that the stepwise CPA exposure technique can be successfully applied for vitrification of immature cat oocytes, in terms of in vitro development but it is likely to affect in utero development.     

Cryopreservation of cartilage cell and tissue for biobanking

Preservation of cell and tissue samples from endangered species is a part of biodiversity conservation strategy. Therefore, setting up proper cell and tissue cryopreservation methods is very important as these tissue samples and cells could be used to reintroduce the lost genes into the breeding pool by nuclear transfer. In this study, we investigated the effect of vitrification and slow freezing on cartilage cell and tissue viability for biobanking. Firstly, primary adult cartilage cells (ACCs) and fetal cartilage cells (FCC) were cryopreserved by vitrification and slow freezing. Cells were vitrified after a two-step equilibration in a solution composed of ethylene glycol (EG), Ficoll and sucrose. For slow freezing three different cooling rates (0.5, 1 and 2 °C/min) were tested in straws. Secondly, the tissues taken from articular cartilage were cryopreserved by vitrification and slow freezing (1 °C/min). The results revealed no significant difference between the viability ratios, proliferative activity and GAG synthesis of cartilage cells which were cryopreserved by using vitrification or slow freezing methods. Despite the significant decrease in the viability ratio of freeze–thawed cartilage tissues, cryopreservation did not prevent the establishment of primary cell cultures from cartilage tissues. The results revealed that the vitrification method could be recommended to cryopreserve cartilage tissue and cells from bovine to be used as alternative cell donor sources in nuclear transfer studies for biobanking as a part of biodiversity conservation strategy. Moreover, cartilage cell suspensions were successfully cryopreserved in straws by using a controlled-rate freezing machine in the present study.     

Vitrification of porcine articular cartilage

The limited availability of fresh osteochondral allograft tissues necessitates the use of banking for long-term storage. A vitrification solution containing a 55% cryoprotectant formulation, VS55, previously studied using rabbit articular cartilage, was evaluated using porcine articular cartilage. Specimens ranging from 2 to 6 mm in thickness were obtained from 6 mm distal femoral cartilage cores and cryopreserved by vitrification or freezing. The results of post-rewarming viability assessments employing alamarBlue demonstrated a large decrease (p < 0.001) in viability in all three sizes of cartilage specimen vitrified with VS55. This is in marked contrast with prior experience with full thickness, 0.6 mm rabbit cartilage. Microscopic examination following cryosubstitution confirmed ice formation in the chondrocytes of porcine cartilage vitrified using VS55. Experiments using a more concentrated vitrification formulation (83%), VS83, showed a significant treatment benefit for larger segments of articular cartilage. Differences between the VS55 and the VS83 treatment groups were significant at p < 0.001 for 2 mm and 4 mm plugs, and at p < 0.01 for full thickness, 6 mm plugs. The percentage viability in fresh controls, compared to VS55 and VS83, was 24.7% and 80.7% in the 2 mm size group, 18.2% and 55.5% in the 4 mm size group, and 5.2% and 43.6% in the 6 mm group, respectively. The results of this study continue to indicate that vitrification is superior to conventional cryopreservation with low concentrations of dimethyl sulfoxide by freezing for cartilage. The vitrification technology presented here may, with further process development, enable the long-term storage and transportation of living cartilage for repair of human articular surfaces.     

In-straw cryoprotectant dilution of IVP bovine blastocysts vitrified in hand-pulled glass micropipettes

The aim of this study was to determine the influence of two ethylene glycol-based vitrification solutions on in vitro and in vivo survival after in-straw cryoprotectant dilution of vitrified in vitro-produced bovine embryos. Day-7 expanded blastocysts were selected according to diameter (> or = 180 microm) and osmotic characteristics and randomly assigned to one of three groups (i) VSa: vitrification in 40% EG+17.1% SUC+0.1% PVA; (ii) VSb: vitrification in 20% EG+20% DMSO; (iii) control: non-vitrified embryos. Vitrification was performed in hand-pulled glass micropipettes (GMP) and cryoprotectant dilution in 0.25 ml straws after warming in a plastic tube. Embryo viability was assessed by re-expansion and hatching rates after 72 h of IVC and by pregnancy rates after direct transfer of vitrified embryos. No differences in re-expansion rates were observed between vitrified groups after 24 h in culture (VSa=84.5%; VSb=94.8%). However, fewer VSa embryos (55.2%, P<0.05) hatched after 72 h than the VSb (75.8%) and control embryos (80.0%). To evaluate in vivo viability, vitrified embryos (VSa=20; VSb=21) were warmed under field conditions and individually transferred to synchronous recipients. Pregnancy rates (day 60) were similar between groups (VSa=20%; VSb=19%). Greater hatching rates occurred after 72 h of IVC for EG+DMSO than EG+SUC+PVA vitrification solutions. However, using a GMP vitrification container and in-tube warming, both solutions provided similar pregnancy rates after the in-straw cryoprotectant dilution and direct embryo transfer.     

Production of transgenic mice from vitrified pronuclear-stage embryos.

Cryopreservation of pronuclear-stage embryos would be useful for transgenic technology and genome preservation purposes. We compared a novel vitrification technique (solid surface vitrification, SSV) with another vitrification method in straws for cryosurvival and to generate transgenic progeny from cryopreserved mouse zygotes following microinjection. The SSV solution consisted of 35% ethylene glycol (EG), 5% polyvinyl-pyrrolidone (PVP), and 0.4 M trehalose in M2 supplemented with 4 mg/ml BSA; the in straw vitrification solution was 7 M EG in M2 plus BSA. In experiment I, we compared the effect of the vitrification solutions alone, without cooling. No reduction was detected in survival and cleavage rates. In experiment II, SSV yielded a significantly higher percentage of morphologically normal zygotes (96%) that also cleaved at significantly higher rates (80%) when compared to that following "in straw" vitrification (68 and 66%, respectively). Cleavage rate in the non-vitrified control group (93%) was significantly higher than that of both vitrified groups. Following embryo transfer, there was no difference in the rate of pups obtained from the SSV, "in straw" vitrified, and control groups (97/457, 21%; 15/75, 20% and 56/209, 27%, respectively). In experiment III, SSV vitrified and fresh embryos were used for pronuclear DNA injection. Survival rate of vitrified embryos after microinjection was reduced compared to nonvitrified ones (64 vs. 72%, respectively; P < 0.05); however, development to two-cell stage was not different (76 vs. 72%, respectively). Following embryo transfer of vitrified vs. fresh microinjected embryos, in both cases 10% live pups were generated, including transgenic pups. The results demonstrated that the efficiency of generating transgenic pups from SSV vitrified pronuclear zygotes is comparable to that from fresh embryos.     

Tsmu solution improves rabbit osteochondral allograft preservation and transplantation outcome

To compare the effects of Tsmu solution with vitrification on chondrocyte viability and examine histological and biomechanical properties of osteochondral allografts (OCAs) after storage, OCAs from femoral condyles of New Zealand rabbits were harvested, stored for 35 days in Tsmu solution or by in vitro vitrification, and subjected to in vivo and in vitro assays. Stored OCAs were transplanted into knee femoral condyle cartilage defects in recipient rabbits. Chondrocyte viability and histological changes of cartilage grafts were assessed in vitro. Gross assessment, chondrocyte viability, histological assessment, OCA biomechanics, and immunological markers were evaluated in vivo 6 months after transplantation. Fresh OCAs served as in vitro and in vivo controls. Chondrocyte viability and scores for cartilage surface and histological quantitative assessment were superior for Tsmu solution compared with vitrification, but inferior compared with fresh OCAs in vitro and in vivo. With the exception of interleukin 6 content, biomechanical features of samples stored in Tsmu solution were superior to vitrification, and inferior to fresh OCAs in vivo. Thus, Tsmu solution provided suitable storage that improved chondrocyte viability, intact OCA cartilage matrix architecture, and transplantation outcomes.     

Glutamate dehydrogenase activity in normal and vitrified plants of Agave tequilana Weber propagated in vitro

Agave tequilana stem explants were used to produce adventitious shoots under a set of different water potentials induced by different concentrations of gelrite in the medium. At high water potentials all shoots were vitrified; as the medium water potential became more negative the degree of vitrification decreased but the number of shoots per explant also diminished. The enzymes NADH and NAD-GDH (EC. 1.4.1.2) were measured along the water potential gradient. GDH activity was high in the non-vitrified tissues and decreased significantly in the vitrified ones.Abbreviations GDH glutamate dehydrogenase - MS Murashige and Skoog medium - MSO methionine sulfoximine - PVP polyvinylpolypyrrolidone - GS glutamine synthetase - GOGAT glutamine: oxoglutarate amino transferase     

Rabbit and pig ear skin sample cryobanking: effects of storage time and temperature of the whole ear extirpated immediately after death

The post-mortem temporal and thermal limits within which there will be ample guarantees of rescuing living skin cells from dead specimens of two species, rabbit and pig, were studied. Post-mortem extirpated whole ears were stored (in non-aseptic conditions) either at 4 degrees C or at room temperature (from 22 to 25 degrees C) or at 35 degrees C for different time lapses after animal death. In both species, the post-mortem maximum time lapses where cell viability was not significantly reduced were 240, 72, and 24 h post-mortem (hpm) for 4, 22-25 and 35 degrees C, respectively. Once the post-mortem temporal limits for each tested thermal level at which cells from skin samples are able to grow in culture were defined, the survival ability of skin samples submitted to these temporal limits and cryopreserved were tested. In the pig, skin samples stored at the three tested thermal levels survived after vitrification-warming, reaching confluence in culture. In rabbit, only tissue samples from ears stored at 35 degrees C for 24 hpm did not survive after vitrification-warming. In conclusion, we should remark that cell survival rates obtained according to the assayed post-mortem time lapses and thermal levels are sufficient to collect and to cryopreserve skin samples from the majority of dead specimens.     

Influence of stage of maturation of bovine oocytes at time of vitrification on the incidence of diploid metaphase II at completion of maturation

The present study was to verify the incidence of bovine diploid oocytes when vitrified at various maturation stages. Bovine cumulus-oocyte complexes were recovered from ovaries at a slaughterhouse and then divided into five groups: control group (unvitrified oocytes), 0 h group (composed of oocytes vitrified before the onset of maturation) and 8, 12, and 22 h groups (vitrified respectively at 8, 12 and 22 h after the onset of maturation). The oocytes remained vitrified for 24 h, and then were thawed. In all groups, the oocytes completed 24 h of maturation. Subsequently, the cumulus cells were removed, and the denuded oocytes fixed on slides and stained with aceto-orcein. No differences (P>0.05) in the incidence of diploid metaphase II oocytes were observed between the control, non-vitrified group (2.4%, 1/41) and oocytes vitrified at 12 h (6.9%, 3/43) or 22 h (2%, 1/50). However, significantly (P<0.05) more diploid oocytes were detected after vitrification at 0 h (28.5%, 10/35) or 8 h (35.4%, 11/31) of maturation. These results suggest that the nuclear stage at which bovine oocytes are vitrified may affect the incidence of diploid oocytes, especially in oocytes vitrified before maturation or 8 h after the onset of maturation.     

Effects of times and storage conditions of Duddingtonia flagrans chlamydospores in sodium alginate pellets on its nematode predatory ability

This study aimed to determine the effects of Duddingtonia flagrans contained in sodium alginate pellets on trichostrongylide larvae under different storage conditions and durations. The in vitro predatory activity of D. flagrans in pellets against trichostrongylide larvae in sheep faeces were assessed at 0, 1, 3, 6, 9, and 12 months after the pellet was stored under four different conditions (i.e. ?20, 4°C, outdoors, and indoors). These results revealed that the numbers of larvae in faeces of sheep treated with pellets containing chlamydospores (treatment groups) were significantly lower than those in the control groups (without chlamydospores) for all trial months under four storage conditions for different durations (p?<?.05). The obtained reduction rates of the infective larvae (L3) in the four treatment groups ranged from 45.62% to 96.73% throughout the entire experiment. The overall mean L3 reduction percentages were 89.22%?±?3.74%, 88.97%?±?1.33%, 68.60%?±?14.31%, and 75.45%?±?13.18% for 4°C refrigeration, ?20°C refrigeration, indoor, and outdoor conditions, respectively. The pellets stored under these storage conditions for a year were provided to sheep for ingestion (in vivo test), and the results showed that the number of recovered larvae in sheep faeces at 24?h after ingestion were significantly lower than that before ingestion. For in vivo test, the L3 reduction percentage in the faeces was 90.99% (?20°C), 74.81% (outdoor), 83.53% (4°C), and 65.60% (indoor). Under the four storage conditions, D. flagrans spores contained in the pellets can maintain their survival ability to a varying degree in a year.     

Resveratrol promotes the embryonic development of vitrified mouse oocytes after <Emphasis Type="Italic">in vitro</Emphasis> fertilization

Vitrification of oocytes is closely associated with the lower embryonic developmental potential, which involves the cryopreservation injury occurred during vitrification. It indicates that vitrification may need to be further optimized. Therefore, we studied the effects of resveratrol, an antioxidant, on the developmental potential of vitrified mouse oocytes after in vitro fertilization. After adding a series of concentrations of resveratrol (0, 1, 10, 25, and 50 μM) into vitrification, warming, and post-warming mediums, we found that 25 and 50 μM resveratrol increased the blastocyst formation rate of vitrified oocytes. We further showed that 25 μM resveratrol increased the mean cell numbers of blastocyst from vitrified oocytes. 25 μM resveratrol reduced oxidative stress of vitrified oocytes through decreasing the levels of reactive oxygen species (ROS) and increasing the levels of glutathione (GSH), and 25 μM resveratrol alleviated the abnormal mitochondrial distribution pattern of oocytes after vitrification. In conclusion, our study implied that resveratrol could diminish the cryopreservation injuries during the vitrification of mouse oocytes and further confirmed that resveratrol may be an effective antioxidant to optimize vitrification.