Decreasing Concentrations Of Sucrose Research Articles

Use of Antifreeze Protein from Tenebrio molitor (TmAFP) in Vitrification of In Vitro-Produced Bovine Embryos: An Ultrastructural Study.

The objective of this study was to evaluate the effects of different concentrations of antifreeze protein (AFP) extracted from the larva of the beetle, Tenebrio molitor (TmAFP), on vitrification of in vitro-produced bovine embryos. In vitro-produced blastocysts were divided into three experimental groups and vitrified using a cryotop. TmAFP was added to the equilibrium solution (ES) and vitrification solution (VS) at a concentration of 0 ng/mL (control), 500 ng/mL (500TmAFP), or 1000 ng/mL (1000TmAFP). Vitrification was carried out by first placing the blastocysts in ES for 2 minutes (7.5% ethylene glycol [EG] and 7.5% dimethyl sulfoxide [DMSO]). The blastocysts were then transferred to VS (15% EG and 15% DMSO) and promptly deposited on a cryotop stem and submerged in liquid nitrogen. Warming was carried out in three steps with decreasing sucrose concentrations. After warming, the blast cells were cultured for 24 hours for subsequent survival analysis and ultrastructural evaluation. There was a significant difference in the survival rate and expansion in the 500TmAFP group compared with the other groups. The ultrastructural analysis revealed intracellular lesions in all vitrified embryos; however, the embryos of the 500TmAFP and 1000TmAFP groups showed fewer cytoplasmic lesions compared with the control group. Taken together, addition of TmAFP can mitigate cellular changes that involve organelles and cellular components essential for proper functioning and improve the viability of warmed and vitrified in vitro-produced bovine embryos.

Just noticeable difference in sweetness perception of cola: Small changes in sugar are noticeable.

These data could help researchers and industry product developers know how much sugar can be reduced from a cola-flavored beverage before the change is apparent to consumers.

Simulated nitrogen deposition promotes the carbon assimilation of shrubs rather than tree species in an evergreen broad-leaved forest

Although understory addition of nitrogen (UAN) is commonly used to simulate nitrogen deposition in field studies in forest ecosystems, it ignores the effects of atmospheric nitrogen deposition on the canopy. We studied the effects of nitrogen deposition simulated by UAN and by canopy addition of nitrogen (CAN) on leaf structure, chemical properties, Calvin cycle, and photosynthate distribution strategy of representative woody plant species in a subtropical evergreen broadleaved forest in South China. The results showed that maximum photosynthetic rate (Amax) of shrub species Blastus cochinchinensis and Ardisia quinquegona under CAN treatments was significantly higher than that of UAN treatments at the same N addition concentration. The concentrations of intermediates (PGK, DPGA and G3P) in Calvin cycle of B. cochinchinensis and A. quinquegona, and Castanea henryi were significantly increased with CAN treatments, but the opposite was true with UAN treatments. CAN25 significantly increased starch concentrations of shrub species Lasianthus chinensis and B. cochinchinensis, and significantly decreased sucrose concentrations of shrub species A. quinquegona and tree species C. henryi. Correlation analyses showed that nitrogen application amount under different modes helped explain the changes in Amax and Calvin cycle intermediates. In summary, nitrogen deposition may promote the Amax and Calvin cycle of shrub species, and the adaptability of shrub species to nitrogen deposition is higher than that of tree species, which may help to explain the degradation of subtropical evergreen broad-leaved forest.

Optimization of physicochemical, textural, and rheological properties of sour cherry jam containing stevioside by using response surface methodology.

The response surface method (RSM) was used to produce sour cherry jam containing stevia sweetener with favorable physicochemical, textural, and rheological properties. The experiments were designed based on RSM using a central composite design (CCD) with three independent variables: sugar, pectin, and stevia. Sample processing was performed at five levels of sucrose (10%–50%), pectin (0.1%–0.5%), and stevia (0.2%–0.6%) at a constant fruit weight of 300 g. To evaluate the jam, the physicochemical, textural, and rheological properties of the jam were determined and optimized. The concentrations of sucrose, pectin, and stevia had a significant effect on the textural and rheological properties of the jam. The results showed that increasing the concentration of sucrose is the main factor in increasing the soluble solids and the total sugar content of the jam. Pectin had a positive effect on textural characteristics such as hardness and adhesiveness of the jam, while the cohesiveness of the jam decreased with increasing pectin. Herschel–Bulkley model was appropriate for describing the rheological behavior of the stable state of sour cherry jam containing stevia. Decreasing sucrose concentration is accompanied by a decrease in yield stress and consistency index of jam samples due to the TSS reduction. Pectin also had a positive effect on the amount of yield stress and consistency index. Dynamic rheological tests indicated that the sour cherry jam is a weak gel. The predominant viscose behavior and the formation of a dilute solution were evident in the samples with 10% and 20% sucrose concentrations. The most favorable condition for the production of the jam was obtained at a 36.5% sugar, 0.277% pectin, and stevia 0.30%.

A COMPREHENSIVE DESCRIPTION OF EX-VITRO ROOTING OF PISTACHIO ROOTSTOCKS GROWN IN PHOTOMIXOTROPHIC AND PHOTOAUTOTROPHIC CONDITION

As a basic principle, ex-vitro rhizogenesis increases the micropropagation efficiency of Micro propagation in any plant from both biological and economic viewpoints. In the current study, we surveyed the effects of number of air exchanges along with sucrose concentration on in-vitro rooting of two pistachio rootstocks consisting of UCB1 and Qazvini versus ex-vitro rooting. Based on our findings for the UCB1 rootstock, microshoot ex-vitro rooting reached the highest percentage (63.70 %) after six weeks' treatment with indole butyric acid (IBA) (5000 ppm) and free naphthalene acetic acid (NAA), while for Qazvini rootstock treated with NAA (6000 ppm) along with IBA (5000 ppm), rooting achieved 35.06%. Photomixotrophic resulted from decreasing sucrose concentration from 30 to 15 (g L-1) in corporation with ventilation condition increased UCB1 rooting (67.89%) as well as plant survival (58.34%). For Qazvini rootstock, maximum sucrose concentration (30 g L-1) improved rooting parameters. For in-vitro rooting experiment, rooting percentage of UCB1 plantlets as well as the main and lateral produced roots were higher in media supplemented with (1 mg l-1) IBA, free NAA, and BA. Regarding Qazvini rootstock, the highest in-vitro rooting percentage (43.75%) and root length were associated with the media supplemented with BA (0.5 mg l-1), IBA (2 mg l-1), and NAA (2 mg l-1). As a result, for both the studied rootstocks, better rooting parameters were observed in the ex-vitro rooted microshoots than in-vitro rooted.

FTIR Spectroscopy Detects Intermolecular \u03b2-Sheet Formation Above the High Temperature Tm for Two Monoclonal Antibodies

The temperature-dependent secondary structure of two monoclonal IgG antibodies, anti-IGF1R and anti-TSLP, were examined by transmission mode Fourier Transform Infrared (FTIR) spectroscopy. Anti-IGF1R and anti-TSLP are IgG monoclonal antibodies (mAbs) directed against human Insulin-like Growth Factor 1 Receptor for anti-tumor activity and Thymic Stromal Lymphopoietin cytokine for anti-asthma activity, respectively. Differential scanning calorimetry (DSC) clearly indicates both antibodies in their base formulations have a lower temperature protein conformational change near 70 °C (Tm1) and a higher temperature protein conformational change near 85 °C (Tm2). Thermal scanning dynamic light scatting (TS-DLS) indicates a significant particle size increase for both antibodies near Tm2 suggesting a high level of protein aggregation. The nature of these protein conformational changes associated with increasing the formulation temperature and decreasing sucrose concentration were identified by transmission mode FTIR and second derivative FTIR spectroscopy of temperature controlled aqueous solutions of both monoclonal antibodies. The transition from intra-molecular β sheets to inter-molecular β sheets was clearly captured for both monoclonal antibodies using FTIR spectroscopy. Finally, FTIR Spectroscopy was able to show the impact of a common excipient such as sucrose on the stability of each monoclonal antibody, further demonstrating the usefulness of FTIR spectroscopy for studying protein aggregation and formulation effects.

NMR-based fruit metabonomic analysis of watermelon grafted onto different rootstocks under two potassium levels

Grafting watermelon onto nonself-rootstock is an effective way to solve the soilborne disease, and potassium plays an important role in fruit quality; however, a comprehensive metabonomic analysis of rootstock on the fruit quality of watermelon under different potassium levels is lacking. In this study, the NMR-based metabonomic approaches was used to reveal the effects of nonself-rootstocks (wild watermelon, bottle gourd, pumpkin) on the fruit metabolites accumulation of watermelon under two potassium levels (normal potassium: 6.0 mM K, low potassium: 0.1 mM K) at ripening stage. Thirty eight metabolites were identified, including nineteen amino acids and ammonium compounds, four organic acids, six sugars, four nucleic acid and five other compounds. Compared with self-grafted plants, nonself-rootstock grafting significantly increased the concentrations of some health-related compounds in watermelon fruit under normal potassium, such as citrulline, methionine, isoleucine, phenylalanine, leucine and valine. Low potassium decreased sucrose concentration, but increased the concentrations of some important fruit metabolites, including citrulline and glucose in watermelon fruit. Nonself-rootstock grafting increased the sucrose and citrulline concentrations under low potassium. Our results suggested that the accumulation of some fruit quality-related compounds in watermelon can be enhanced by grafting onto nonself-rootstocks under both normal and low potassium conditions.

The Effect of Improvised Media and Gelling Agents on In Vitro Germination of Cotton (Gossypium hirsutum. L.)

The significance of cotton can hardly be over emphasized in Turkish economy as it contributes momentously to agricultural value added products. The success of propagating cotton in in vitro conditions or to modify it genetically largely depends on an efficient, reproducible and cost effective protocol for in vitro germination of seeds, seedlings development and different explant sources. The present study was conducted to investigate the effect of different media (various sucrose concentrations) and gelling agents (Agar and Phytagel) on in vitro germination and seedling growth of different cotton cultivars. Results showed that MS medium devoid of sucrose contents induced maximum germination rates. The decreased sucrose concentration resulted in increased germination rates. Furthermore, experiments with different concentrations of gelling agents exhibited that MS media supplemented with 0.4 (w/v) % Phytagel results in induction of maximum in vitro germination rates; leading to robust seedling development suitable for further in vitro propagation experiments. According to our knowledge, this is the first report of increased germination rates of cotton cultivars incubated on MS media without the presence of sucrose; hence MS medium devoid of sucrose and solidified with 0.4% of Phytagel is most suitable for in vitro seed germination of cotton cultivars to obtained healthy seedlings in a shorter period.

Effects of Different Kinds of Potassium and Chloride Salts on Sugarcane Quality and Photosynthesis

Appropriate fertilizer management practices are important to improve sugarcane quality. We have ever reported that KCl and K2SO4 had different effects on sugarcane quality and that only excessive KCl application decreased sucrose concentration in juice, indicating that Cl− is the key factor for the quality improvement. In this study, to see the individual effects of Cl− independent of K+, we established treatments using different kinds of K+ and Cl− salts (50 mM KCl, 25 mM K2SO4, mixture of 12.5 mM MgCl2 and 12.5 mM CaCl2, and 50 mM NaCl). The treatments did not greatly affect quantitative parameters. Plants treated with KCl and K2SO4 had significantly higher juice K+ concentrations. On the other hand, Cl− concentration was remarkably high only in the KCl treatment and the other Cl− salt treatments hardly enhanced Cl− accumulation, suggesting that Cl− needs K+ to be efficiently absorbed. Juice sucrose concentration was lowest in the KCl treatment, and the other treatments had no adverse effects. We observed neither declines of stomatal conductance nor F v/F m; however, the KCl treatment showed a slightly lower CO2 assimilation rate, which may have accounted for the sucrose reduction. From this study, we could not evaluate the single effects of Cl− but revealed that Cl− could have a negative impact on sugarcane quality only when K+ is supplied above sufficiency levels, so that we need to reduce K+ as well as Cl− concentrations to improve sugarcane quality.

Differences in Sucrose Metabolism in Peach Fruit Stored at Chilling Stress versus Nonchilling Stress Temperatures

To explore differences in sucrose metabolism between peach fruit subjected to chilling stress (5 °C) and nonchilling stress (10 °C), sucrose concentration as well as the activities and gene expression levels for enzymes associated with sucrose metabolism were compared. Fruits stored at 5 °C accumulated higher concentrations of H2O2 and developed severe chilling injury (CI) compared with fruit kept at 10 °C. Activities and gene expression levels for enzymes related to sucrose metabolism, such as acid invertase (AI), neutral invertase (NI), sucrose synthase (SS), and sucrose phosphate synthase (SPS) were higher in fruit stored at 5 °C than at 10 °C throughout or late in storage. A sharp increase in net sucrose cleavage activity dramatically decreased sucrose concentration and increased reducing sugars at 5 °C. The sucrose concentration at 10 °C increased over the first 21 days and then declined slightly, and was higher than in fruit at 5 °C throughout storage. The increase in net sucrose cleavage activity at 5 °C is contrary to the expectation that biochemical reactions ordinarily proceed more rapidly with increasing temperature. We conclude that chilling stress stimulates the activities and transcription levels of enzymes involved in sucrose metabolism, resulting in increased sucrose cleavage.

64 EX VIVO CULTURE OF FRESH AND FROZEN - THAWED SHEEP WHOLE OVARIES

Cryopreservation and retransplantation of ovarian tissue is a real option to preserve fertility in young cancer patients. However, a high risk of retransmission of malignancy exists in several tumours. In these patients, cryopreserved whole ovaries could provide an appealing source of oocytes to be grown and matured in vitro. The aim of this study was to develop a perfusion system for ex vivo culture of fresh and cryopreserved whole ovaries. Upon arrival to the laboratory, all ovaries were perfused via the ovarian artery with Ringer's solution and 10 UI L–1 of heparin for 10 min. Ovaries to be frozen were subsequently perfused with cryoprotectant solution [L-15 medium, 10% FBS, and 1.5 M dimethyl sulfoxide (DMSO)] and then frozen using Multi Thermal Gradient freezing technology (Core Dynamics Ltd., Ness Ziona, Israel), pushing the samples along the thermal gradient (4 to –70°C) at 0.01 mm s–1, resulting in a cooling rate of 0.3°C min–1. Samples were thawed at 37°C and immediately perfused with L-15 medium supplemented with decreasing sucrose concentrations (0.25, 0.125, and 0 M). In a closed-circuit perfusion system, 100 mL of recirculating medium (M199, 25 mM HEPES, 1% BSA, 2 mM glutamine, and antibiotic/antimycotic) was pumped into the ovarian artery using a peristaltic pump. The flow rate through the ovary was maintained between 1 and 1.5 mL min–1. Whole sheep ovaries were cultured at 38.5°C for 1 or 3 days. After culture, ovaries were fixed with 10% formaldehyde. Statistical analysis was performed using Student's t-test (SPSS 20, IBM Corp., Armonk, NY, USA). Morphological analysis showed that the rate of intact follicles was inversely related to the days of culture but was not affected by cryopreservation. In fact, the percentage of morphologically normal follicles in fresh and frozen ovaries cultured for 1 day (87 ± 3.4 and 83 ± 3.2%, respectively; P = 0.058) was higher (P = 0.048) than in ovaries cultured for 3 days (75 ± 2.9 and 71 ± 2.8%, respectively; P = 0.053). Cell proliferation, measured as Ki67-positive stromal cells, decreased during culture (P = 0.028) and was affected by cryopreservation both on Day 1 (13 ± 7 v. 15 ± 4%; P = 0.047) and Day 3 (10 ± 4 v. 12 ± 6%; P = 0.039). Similar results were observed for the apoptotic index that increased during culture both in fresh and cryopreserved ovaries (P = 0.028). The number of apoptotic cells per millimeter squared was lower (P = 0.031) in fresh (23 ± 10%) than in frozen ovaries (27 ± 15%) both on Day 1 and on Day 3 (30 ± 14 v. 33 ± 20%, respectively; P = 0.03). Cell viability and active endocrine function during culture is confirmed by steroid secretion, which is conserved in both fresh and cryopreserved ovaries for up to 3 days. Our results show that it is possible to culture both fresh and cryopreserved whole ovaries for up to 3 days. Although fresh ovaries, on average, did better than cryopreserved ones, we observed large individual variations, with positive and negative results overlapping between fresh and frozen samples. Further studies are in progress to explain the reason of such variations. Supported by AIRC IG 10376, Carraresi Foundation, and by Legge 7 (R.A.S).

Night Temperature and Source-sink Effects on Growth, Leaf Carbon Exchange Rate, and Carbohydrate Accumulation in Bell Pepper Ovaries

Low night temperatures and/or high source-sink ratios increase ovary swelling and subsequent fruit malformation in many sweet peppers (Capsicum annuum), including bell pepper. Although this response has been correlated with increased ovary carbohydrate accumulation, evidence for this is limited. Furthermore, it is unknown how the combined effects of night temperature and source-sink ratio affect ovary carbohydrate accumulation and ovary swelling. The objectives of the present work were to determine night temperature and source-sink effects on ovary swelling, net carbon exchange rate (CER), and soluble sugar and starch concentrations in bell pepper ovaries at anthesis. Source-sink and temperature effects were tested by comparing fruiting (low source-sink ratio or high sink demand) with non-fruiting (high source-sink ratio or low sink demand) ‘Legionnaire’ bell pepper plants grown at 22/20 °C [high night temperature (HNT)] or 22/12 °C [low night temperature (LNT)] day:night temperatures. Flowers that opened after imposition of the temperature and fruiting treatments were harvested at anthesis. Ovaries from harvested flowers were weighed and analyzed for non-structural carbohydrates. Leaf gas exchange measurements were performed every 3 days. Ovary fresh weight of flowers harvested at anthesis was highest in non-fruiting plants under LNT and lowest in plants grown under HNT regardless of fruiting status. Mean CER averaged over the experimental period was significantly higher in fruiting plants under HNT compared with all other treatments. There were no significant interactions between night temperature and fruiting status on ovary soluble sugar or starch concentrations. Low night temperature increased glucose, fructose, and starch concentration and decreased sucrose concentration in the ovary wall compared with HNT. There were no differences in soluble sugar or starch concentrations in the ovary wall between fruiting and non-fruiting plants. Thus, although both low temperature and high source-sink ratio (i.e., non-fruiting plants) resulted in ovary swelling, the mechanisms appear to differ. Whereas LNT effects on ovary swelling were associated with increased ovary carbohydrate accumulation, this association was not apparent when ovary swelling occurred in response to high source-sink ratios.

58 MULTI-THERMAL GRADIENT FREEZING ALLOWS THE CRYOPRESERVATION OF SHEEP WHOLE OVARIES WITH THE SAME EFFICIENCY OF OVARIAN FRAGMENTS

Ovarian tissue cryobanking is proposed as an effective option for preserving female fertility in cancer patients. At present 2 options are available: cryopreservation of ovarian cortical fragments or of the whole ovary. The use of whole ovary reduces ischemic insult. However, the larger the sample volume, the more difficult it is to introduce the cryoprotective agents and to ensure an adequate cooling rate that minimizes tissue damage. For this reason, we used the multi-thermal gradient method, based on running the sample through a temperature gradient. This allows a homogeneous cooling rate through the whole sample independently from its volume. The aim of the study was to determine whether multi-thermal gradient freezing allows a substantial reduction of the damages induced by cryopreservation of large samples by comparing the viability of cortical fragments versus whole ovaries after thawing and grafting in nude mice. Sheep ovaries were collected at the local abattoir and randomly divided into 3 groups: A) ovaries frozen as cortical fragments, B) ovaries frozen as whole organs, and C) fresh ovaries immediately processed for further analysis (control). Ovarian fragments (10 × 5 × 1 mm) were sliced from the cortical region and immersed into cryoprotectant solution (Leibovitz L-15 medium, 10% FCS, and 1.5 M dimethyl sulfoxide), while whole ovaries were perfused with the same solution. Samples were placed into glass freezing tubes 16 mm in diameter filled with cryoprotectant solution. Samples were frozen with the multi-thermal gradient freezing apparatus (Core Dynamics, Ness Ziona, Israel) progressing along the thermal gradient at a rate of 0.01 mm s–1, resulting in a cooling rate of 0.3°C min–1. Two weeks later, samples were thawed by plunging the tubes into a 37°C water bath with gentle shaking. Whole ovaries were perfused with 10 mL of HEPES-Talp medium, 0.5 M sucrose, and 10 IU mL–1 of heparin and their cortical region was cut into fragments. These fragments and those derived from group A were rehydrated in L-15 medium with decreasing sucrose concentrations. Fragments (2 × 2 × 1 mm) were xenografted in the dorsal region of 6 nude mice for each group. Mice were killed after 8 weeks and grafts were collected for analysis. Cryopreserved samples were compared with each other and fresh controls (group C). Morphologically normal follicles at primordial, primary, and secondary stages were visible in all samples. Cell proliferation was assessed measuring Ki-67 mRNA and counting immunohistochemically positive cells. The FSH receptor and GDF9 gene expression were used to evaluate tissue viability. No significant differences for any of these parameters were measured amongst the groups. We conclude that directional freezing is an effective method for ovarian tissue cryopreservation independently from the sample volume, thus overriding the limitations usually associated with whole-organ banking. Supported by AIRC IG 10376 and by Carraresi Foundation.

Sodium Chloride Stress Induced Changes in Leaf Osmotic Adjustment of Trifoliate Orange (Poncirus trifoliata) Seedlings Inoculated with Mycorrhizal Fungi

Citrus plants are sensitive to salinity, and thus employing new approaches to alleviate salt damage are necessary. The present study evaluated the effect of two arbuscular mycorrhizal fungi (AMF), Glomus mosseae and G. versiforme, on leaf osmotic adjustment of trifoliate orange (Poncirus trifoliata) seedings exposed to 100 mM NaCl. Salinity significantly inhibited mycorrhizal colonization, plant biomass and leaf relative water content, whereas the reduce of plant biomass was notably alleviated by the mycorrhizal colonization. Mycorrhizal seedlings exhibited significantly lower Na+ and Ca2+ concentrations, whilst also recorded higher K+ concentration and K+/Na+, Ca2+/Na+ and Mg2+/Na+ ratios at both salinity levels. Under salinity stress, mycorrhizal symbiosis markedly decreased sucrose concentrations of leaves and also increased glucose, fructose and proline concentrations of leaves. The results suggest that arbuscular mycorrhizas improved leaf osmotic adjustment responses of the seedlings to salt stress, thus enhancing salt tolerance of mycorrhizal plants.

Storage and conversion of Eclipta alba synseeds and RAPD analysis of the converted plantlets

The encapsulated shoot tips and nodal segments of Eclipta alba were stored at 4, 12 and 20 °C under irradiance of 1.5 gmmol m-2 s-1 and high conversion was observed in synseeds stored at 4 °C for 8 weeks. Duration of storage was extended up to 12 weeks by decreasing sucrose concentration in the alginate matrix from 3 to 1 or 2 % and conversion frequency was 71.2-76.1 %. Synseed-derived plantlets survived by 100 % in ex vitro conditions. RAPD analysis revealed uniform amplification profile in donor and synseed derived plantlets.

101 EFFECT OF NITROCOOLER NEGATIVE PRESSURE AND RECOVERY INTERVAL ON CRYOTOLERANCE OF BOVINE IN VITRO-PRODUCED EMBRYOS

Exposing bovine embryos and porcine oocytes to hydrostatic pressure has been shown to increase cryosurvival, possibly by a resulting expression of stress tolerance proteins. This study aimed to evaluate the effect of the negative pressure stress condition (a 5-min-long embryo exposure to a negative pressure) and the interval between vacuum exposure and vitrification (40 min or 2 h) on survival of bovine in vitro-produced (IVP) embryos. The negative pressure was achieved with the same apparatus used previously for the cryopreservation of embryos (Nitrocooler; Mezzalira et al. 2009 Reprod. Fert. Dev. (1) 134), in which a negative pressure (vacuum) is applied to liquid nitrogen to increase the cooling rate through the slush phenomenon, except that in this study, the vacuum was applied to the chamber without liquid nitrogen, at room temperature. Grades 1 and 2 bovine IVP expanded blastocysts were allocated to 1 of 5 experimental groups: embryos in vitro-cultured as fresh (control) or after vitrification (Vitri); and embryos subjected to the negative pressure for 5 min and then in vitro-cultured as fresh (NP-fresh) or after vitrification performed 40 min (NP-Vitri-40 min) or 2 h (NP-Vitri-2h) following the vacuum exposure. Embryos were vitrified in pulled glass micropipettes in a solution with 20% ethylene glycol + 20% dimethylsulfoxide + 20% fetal bovine serum and rewarmed in decreasing sucrose concentrations (Mezzalira et al. 1999 Acta Scientiae Veterinariae 27 262-262). In vitro culture was carried out in all treatments for 72 h for the assessment of re-expansion and hatching rates (Table 1), which were analyzed by the chi-square test, for P < 0.05. No differences in re-expansion rates were observed between groups. However, the vitrification of embryos after 2 h of exposure to a 5-min-long negative pressure (NP-Vitri-2h) improved embryo survival expressed by a higher hatching rate than for embryos vitrified without vacuum exposure (Vitri) or after 40 min following the 5-min-long exposure to vacuum. In addition, hatching rates in group NP-Vitri-2h were similar to those for fresh embryos (control and NP-fresh). Our results indicated that a short exposure of embryos to a negative pressure can improve cryotolerance following vitrification, which is dependent on the time interval between NP exposure and cryopreservation. Bovine IVP embryos should be allowed to recover for at least 2 h after NP exposure before the increase in cryotolerance is achieved. Table 1.Effect of negative pressure on re-expansion and hatching rates of fresh and vitrified

Vitrification of Ovarian Cortex from Prepubertal and Adult Cats Induces Less Damage to the Primordial Follicles than Slow Freezing.

Due to certain similarities in reproductive mechanisms, the domestic cat has become a valuable model for exploring fertility preservation strategies for young girls and women. The objective of this study was to assess the survival of primordial follicles in prepubertal and adult ovarian tissues during slow freezing or vitrification. Freshly-excised ovaries from prepubertal (4-6 mo; n = 12 total ovaries; 6 replicates) and adult (>2 yr; n = 12 total ovaries; 6 replicates) females were transported at 4°C before processing. In each replicate, the cortex of each ovary (n = 2 from prepubertal and n = 2 from adult cats) was dissected and cut into 18 pieces (1-2 mm3) that were allocated to different treatments. Three pieces of each cortex were directly fixed (4% paraformaldehyde) to determine primordial follicle density in histological sections (i.e., number of follicles with visible oocyte nuclei/mm2 of section) as well as the proportion of intact primordial follicles (i.e., number of oocytes and surrounding granulosa cells without pycnotic nuclei, shrunken cytoplasm or vacuoles relative to a total of 60 follicles examined per ovary). Three other cortical pieces were dissociated (1 mg/ml collagenase) and stained (2 μM calcein-AM and 5 μM ethidium) to evaluate the proportion of viable primordial follicles (i.e., number of oocytes with surrounding granulosa cells stained only with calcein-AM relative to a total of 60 follicles examined per ovary). Six cortical pieces were exposed to 1.5 M ethylene glycol (EG; 15 min) and frozen using a slow cooling device (−1°C/min to −80°C ) before plunging into liquid nitrogen. The remaining six pieces were exposed to 1.5 M EG + 1.5 M DMSO + 0.5 M sucrose (5 min) before vitrification in liquid nitrogen. After 1 week of storage, all samples were rapidly warmed to 38°C and diluted stepwise in decreasing sucrose concentrations before fixation for histological assessment or dissociation for viability staining (as above). Primordial follicle density was higher (P<0.05; ANOVA) in the ovarian cortex of prepubertal (mean ±; SD; 93.0 ± 15.8 follicles/mm2) compared to adult (43.0 ± 13.6) females. Proportions of intact and viable follicles were similar (P>0.05) in fresh controls from prepubertal and adult ovarian tissues (intact follicles, 90.0 ± 5.8 and 92.5 ± 8.7%, respectively; viable follicles, 86.0 ± 2.7 and 82.5 ± 3.5%, respectively). Although cryopreservation decreased (P<0.05) the proportions of intact follicles compared to fresh controls, vitrification was superior (P<0.05) to slow freezing for preserving follicular structure in prepubertal (vitrification, 70.0 ± 3.4% intact follicles; slow freezing, 57.5 ± 8.7%) and adult ovarian tissues (vitrification, 73.5 ± 3.3%; slow freezing, 56.3 ± 4.8%). A similar pattern was observed for proportions of viable follicles, with vitrification exceeding (P<0.05) slow freezing in prepubertal (vitrification, 59.8 ± 4.6%; slow freezing, 37.8 ± 2.8%) and adult (vitrification, 62.5 ± 3.5%; slow freezing, 36.3 ± 4.5%) tissues. Interestingly, higher follicle densities in prepubertal versus adult ovaries had no influence (P>0.05) on follicle structure and viability post-thawing. Results demonstrate, for the first time, that vitrification permits retaining ≈80% of the original structure and ≈70% of the original viability of cat primordial follicles regardless of their density in the ovarian cortex. (Supported by the National Institutes of Health K01 RR020045). (platform)

Better ongoing pregnancy rates from frozen embryo transfers when vitrified blastocysts are used

Better ongoing pregnancy rates from frozen embryo transfers when vitrified blastocysts are used

83 VITRIFICATION OF IMMATURE AND IN VITRO-MATURED HORSE OOCYTES

Few attempts have been carried out to cryopreserve equine oocytes, and an effective cryopreservation protocol is not defined yet. Studies were conducted to compare the viability of immature and in vitro-matured horse oocytes vitrified by the minimal volume cooling (MVC) cryotop vitrification method (Kuwayama et al. 2005 Reprod. BioMed. Online 11, 300–308). Oocytes were recovered from slaughterhouse ovaries and divided, on the basis of the morphology of cumulus cells, into cumulus-expanded (CE) and cumulus-compacted (CC) oocytes. Groups of CC and CE oocytes were vitrified immediately after recovery [germinal vesicle (GV) stage] or matured in vitro (IVM) and cryopreserved at the MII stage as follows: oocytes were incubated 30 min in TCM-199 + 20% FCS + 10% ethylene glycol (EG) + 10% DMSO, followed by 20 min in TCM-199 + 20% FCS + 20% EG + 20% DMSO + 0.25 M sucrose, loaded in cryotops (2 µL), and plunged into liquid nitrogen. Warming was performed at 38.5°C by washing the oocytes in TCM-199 + 20% FCS with decreasing sucrose concentrations (1.25 M, 0.62 M, 0.31 M). After warming oocytes cryopreserved at the GV stage were matured in vitro for 24 h (CE) or 36 h (CC) in TCM-199 + 10% FCS + FSH, LH each at (0.1 UI/mL) + cysteamine, fixed, and stained with glycerol-Hoechst 33342 to assess nuclear maturation. Oocytes vitrified at the MII stage were in vitro cultured for 2 h to evaluate their morphological survival on the basis of the presence of an intact zona pellucida and membrane. Nonvitrified oocytes undergoing the same maturation protocol were used as controls. Results (Table 1) indicated that the survival rate of oocytes vitrified at the GV stage, after IVM, was similar between CE and CC oocytes (43.6% vs 42.6%). Significantly (P &lt; 0.01) higher numbers of vitrified CE MII oocytes (52.9%) survived, compared to CC (34.8%), after 2-h culture. The percentages of viable MII oocytes from CE and CC GV vitrified oocytes were 43.6% and 40.9% respectively and were comparable to those from vitrified MII oocytes (CE, 52.9%; CC, 34.8%) and control oocytes (CE, 56.4%; CC, 53.3%). In conclusion, the results of this study showed that vitrification by the MCV Cryotop method of horse oocytes at either the GV or the MII stage allows a similar number of viable mature oocytes to be recovered. Table 1. Maturation and survival rates of immature and mature equine oocytes vitrified by the MCV Cryotop method

METODOLOGIAS DE DESNUDAMENTO PARCIAL DE OÓCITOS BOVINOS MATURADOS E SUBMETIDOS À VITRIFICAÇÃO

Diferentes metodologias de desnudamento parcial de oócitos bovinos maturados foram avaliadas na produção in vitro de embriões e na vitrificação, em três experimentos. No primeiro avaliou-se o efeito do desnudamento parcial realizado por sucessivas pipetagens. No segundo, comparou-se o desnudamento através de sucessivas pipetagens, com o realizado pela enzima hialuronidase e no terceiro, avaliou-se a influência das duas metodologias na vitrificação de oócitos. Os oócitos foram submetidos aos tratamentos após 22 horas de maturação em meio TCM 199. Para a vitrificação, foram expostos por 30 segundos a uma solução de equilíbrio e 20 segundos a uma solução de vitrificação (20%EG + 20% Dimitilsufoxido (DMSO) + 0,3MSAC), envasados em palhetas abertas e estiradas (OPS) e mergulhados em N2 líquido. O reaquecimento foi realizado com exposição a concentrações decrescentes de sacarose. Os oócitos de todos os tratamentos foram submetidos a duas horas adicionais de maturação, seguida da fecundação e os possíveis zigotos cultivados em meio SOFaaci. No experimento I foi observado um decréscimo de 28,7% para 20,5% na taxa de blastocistos com o emprego do desnudamento (P