Fresh Control Research Articles

Equilibration time with cryoprotectants, but not melatonin supplementation during in vitro maturation, affects viability and metaphase plate morphology of vitrified porcine mature oocytes.

The aims of this study were to investigate the effects of different equilibration times with cryoprotectants on viability and metaphase plate morphology of vitrified-warmed porcine mature oocytes (Experiment 1) and to evaluate the effects of supplementation with 10-9 M melatonin during in vitro maturation on these parameters (Experiment 2). In Experiment 1, 2,392 mature oocytes were vitrified using different equilibration times of oocytes with cryoprotectants (3, 10, 15, 20, 30, 40, 60 and 80 min). Fresh oocytes matured in vitro for 44 hr (n=509) were used as controls. In Experiment 2, a total of 573 COCs were used. COCs were matured with 10-9 M melatonin supplementation or without melatonin (control). Some oocytes from each group were vitrified with a 60-min equilibration time with cryoprotectants according to the results of Experiment 1. The remaining oocytes from each maturation group were used as fresh control groups. In both experiments, oocytes were stained with 2',7'-dichlorodihydrofuorescein diacetate and Hoechst 33342 to assess viability and metaphase plate morphology, respectively. Vitrification and warming affected (p < .01) oocyte viability compared with controls, which were all viable after 44 hr of IVM. In Experiment 1, the longer the equilibration time with cryoprotectants, the higher the viability. Oocytes equilibrated for 60 and 80 min had the highest (p < .05) viability and similar metaphase plate characteristics to the fresh control oocytes. In Experiment 2, supplementation with melatonin during in vitro maturation had no effect on oocyte viability or metaphase plate morphology of vitrified-warmed oocytes. In conclusion, under our experimental conditions, vitrified porcine mature oocytes equilibrated with cryoprotectants for 60 or 80 min exhibited the highest viability and similar metaphase plate characteristics to fresh controls. Furthermore, supplementation with 10-9 M melatonin during in vitro maturation had no effect on these parameters.

Utilization of pulsed infrared for the rapid semidrying of fresh ginseng with advanced qualities and extended shelf life

Various physiological functionalities of ginseng have been proven, however, there are little investigation on the characteristics of semidried ginseng as a foodstuff. This study aimed to investigate the effects of hot air (HA) drying temperatures and infrared (IR) pulse sizes on the drying rate of fresh ginseng and to evaluate the optimal semidrying conditions for fresh ginseng. The pulsed IR accelerated drying of ginseng compared to HA, and particularly the drying rate of IR, was rapid at the early drying stage. Dryness (moisture content) affected the rehydration properties, and ginseng dried at relatively high HA temperatures or IR treatments showed high rehydration capacity (RC) and low reconstitution (RE). The color and cutting force were affected by the dryness of ginseng rather than the drying method, and a moisture content of 0.7 kg/kg solid was regarded as the optimal semidrying condition for fresh ginseng. When applied to chicken porridge, semidried ginseng showed a lower cutting force than completely dried ginseng, and the cutting force of semidried ginseng by IR did not differ from that of the fresh control. The total viable count (TVC) indicated that semidrying by IR could extend the shelf life of ginseng by more than twice that of the fresh control. Therefore, pulsed IR has the potential to replace HA drying to produce semidried ginseng with fresh ginseng-like quality characteristics.

Fibroblast growth factor-2 promotes in vitro activation of cat primordial follicles.

This study evaluated the effect of fibroblast growth factor-2 (FGF-2) on the morphology, primordial follicle activation and growth after in vitro culture of domestic cat ovarian tissue. Ovaries (n = 12) from prepubertal domestic cats were collected and fragmented. One fragment was fixed for histological analysis (fresh control). The remaining fragments were incubated in control medium alone or with 10, 50 or 100 ng/ml FGF-2 for 7 days. After in vitro culture, the following endpoints were analyzed: morphology, activation by counting primordial and developing follicles, and growth (follicle and oocyte diameters). Treatment with 100 ng/ml FGF-2 maintained (P > 0.05) the percentage of normal follicles similar to fresh control. Follicle survival was greater (P < 0.05) after culture in 100 ng/ml FGF-2 than in 50 ng/ml FGF-2. The percentage of primordial follicles decreased (P < 0.05) and the percentage of developing follicles increased (P < 0.05) in all treatments compared with fresh tissue. The proportion of developing follicles increased (P < 0.05) in tissues incubated with 100 ng/ml FGF-2 compared with control medium and other FGF-2 concentrations. Furthermore, culture in 10 or 100 ng/ml FGF-2 resulted in increased (P < 0.05) follicle and oocyte diameters compared with fresh tissues and MEM+. In conclusion, FGF-2 at 100 ng/ml maintains follicle survival and promotes the in vitro activation and growth of cat primordial follicles.

Contents of some bioactive compounds in Norway spruce needles as affected by short-term storage at different conditions and implications for their industrial use

The main aim of the research was to evaluate changes in contents of essential oils and pigments in Norway spruce needles as affected by short-term storage under different conditions as well as to assess the potential resources of winter-harvested foliage obtainable from by-products of forest felling as a natural source of these bioactives. One- and two-year-old needles were taken for the analyses fresh (control) and after five-week storage at three different conditions: at 4 °C in fridge, at −24 °C in freezer and in the forest of their origin. Essential oils were isolated from needles by hydrodistillation and analysed by gas chromatography-mass spectrometry. Analyses of chlorophylls and carotenoids were performed spectrophotometrically. The study showed that none of the tested storage conditions of P. abies needles had significant influence on the total amounts of essential oils in them varying from 0.16% to 0.20% per dry matter (v/w) in two- and one-year-old needles, respectively. Bornyl acetate was the main compound identified in the essential oils amounting to 14.47–40.04%. The mean total contents of chlorophylls amounted to 1.10 mg/g in one-year-old needles and to 1.28 mg/g in two-year-old ones measured in fresh weight. Statistically significant differences were established in the levels of all the pigments analysed between fresh material, and stored under controlled, −24 °C and 4 °C, conditions both of 1-year-old and 2-year-old needles. The highest contents of the pigments were obtained after the storage at 4 °C as well as under the natural forest condition. Based on the above data and the national forestry statistics, potential yields of essential oils (6.45 kg), total chlorophylls (9.27 kg) and total carotenoids (1.32 kg) were assessed per hectare of a mature sprucewood stand in Lithuania.

Non-destructive Storage Time Prediction of Newhall Navel Oranges Based on the Characteristics of Rind Oil Glands.

How to non-destructively and quickly estimate the storage time of citrus fruit is necessary and urgent for freshness control in the fruit market. As a feasibility study, we present a non-destructive method for storage time prediction of Newhall navel oranges by investigating the characteristics of the rind oil glands in this paper. Through the observation using a digital microscope, the oil glands were divided into three types and the change of their proportions could indicate the rind status as well as the storage time. Images of the rind of the oranges were taken in intervals of 10 days for 40 days, and they were used to train and test the proposed prediction models based on K-Nearest Neighbors (KNN) and deep learning algorithms, respectively. The KNN-based model demonstrated explicit features for storage time prediction based on the gland characteristics and reached a high accuracy of 93.0%, and the deep learning-based model attained an even higher accuracy of 96.0% due to its strong adaptability and robustness. The workflow presented can be readily replicated to develop non-destructive methods to predict the storage time of other types of citrus fruit with similar oil gland characteristics in different storage conditions featuring high efficiency and accuracy.

Development of a porcine acellular bladder matrix for tissue-engineered bladder reconstruction

PurposeEnterocystoplasty is adopted for patients requiring bladder augmentation, but significant long-term complications highlight need for alternatives. We established a protocol for creating a natural-derived bladder extracellular matrix (BEM) for developing tissue-engineered bladder, and investigated its structural and functional characteristics.MethodsPorcine bladders were de-cellularised with a dynamic detergent–enzymatic treatment using peristaltic infusion. Samples and fresh controls were evaluated using histological staining, ultrastructure (electron microscopy), collagen, glycosaminoglycans and DNA quantification and biomechanical testing. Compliance and angiogenic properties (Chicken chorioallantoic membrane [CAM] assay) were evaluated. T test compared stiffness and glycosaminoglycans, collagen and DNA quantity. p value of < 0.05 was regarded as significant.ResultsHistological evaluation demonstrated absence of cells with preservation of tissue matrix architecture (collagen and elastin). DNA was 0.01 μg/mg, significantly reduced compared to fresh tissue 0.13 μg/mg (p < 0.01). BEM had increased tensile strength (0.259 ± 0.022 vs 0.116 ± 0.006, respectively, p < 0.0001) and stiffness (0.00075 ± 0.00016 vs 0.00726 ± 0.00216, p = 0.011). CAM assay showed significantly increased number of convergent allantoic vessels after 6 days compared to day 1 (p < 0.01). Urodynamic studies showed that BEM maintains or increases capacity and compliance.ConclusionDynamic detergent–enzymatic treatment produces a BEM which retains structural characteristics, increases strength and stiffness and is more compliant than native tissue. Furthermore, BEM shows angiogenic potential. These data suggest the use of BEM for development of tissue-engineered bladder for patients requiring bladder augmentation.

An in vitro intestinal model captures immunomodulatory properties of the microbiota in inflammation

ABSTRACT Considerable effort has been put forth to understand mechanisms by which the microbiota modulates and responds to inflammation. Here, we explored whether oxidation metabolites produced by the host during inflammation, sodium nitrate and trimethylamine oxide, impact the composition of a human stool bacterial population in a gut simulator. We then assessed whether an immune-competent in vitro intestinal model responded differently to spent medium from bacteria exposed to these cues compared to spent medium from a control bacterial population. The host-derived oxidation products were found to decrease levels of Bacteroidaceae and overall microbiota metabolic potential, while increasing levels of proinflammatory Enterobacteriaceae and lipopolysaccharide in bacterial cultures, reflecting shifts that occur in vivo in inflammation. Spent microbiota media induced elevated intracellular mucin levels and reduced intestinal monolayer integrity as reflected in transepithelial electrical resistance relative to fresh medium controls. However, multiplexed cytokine analysis revealed markedly different cytokine signatures from intestinal cultures exposed to spent medium with added oxidation products relative to spent control medium, while cytokine signatures of cultures exposed to fresh media were similar regardless of addition of host-derived cues. Further, the presence of immune cells in the intestinal model was required for this differentiation of cytokine signatures. This study indicates that simple in vitro immune-competent intestinal models can capture bacterial-mammalian cross-talk in response to host-derived oxidation products and supports utility of these systems for mechanistic studies of interactions between the gut microbiome and host in inflammation.

Cryopreservation of Testicular Tissue from Adult Red-Rumped Agoutis (Dasyprocta leporina Linnaeus, 1758).

Simple SummaryTesticular tissues are composed of many types of germ cells, including early stages which can be grown up to fully formed spermatozoa following grafting or in vitro culture. The systematic banking of testicular tissues at freezing temperatures is useful for future use in assisted reproduction and to improve the reproductive management of rare mammalian species. The present study explored testicular tissue cryopreservation in the red-rumped agouti by slow freezing or vitrification methods, using different combinations of cryoprotectants. Solid-surface vitrification using the combination of dimethyl sulfoxide and ethylene glycol was the most effective protocol to preserve testicular cell morphology and proliferative potential.This study measured the effects of different freezing techniques and permeating cryoprotectants on the preservation of testicular tissues from adult red-rumped agoutis. Tissue biopsies (3.0 mm3) from five individuals were allocated to different experimental groups: control (non-cryopreserved); slow freezing (SF), solid-surface vitrification (SSV), and conventional vitrification (CV). Each method used dimethyl sulfoxide (DMSO), ethylene glycol (EG), or a DMSO + EG combination. Morphology, viability, mitochondrial activity, and proliferative potential were assessed in fresh and frozen tissue samples. Testicular morphology was better using SSV with a combination of DMSO and EG. Across the different cryopreservation approaches, as well as cryoprotectant combinations, cell viability was comparable. Regarding mitochondrial activity, DMSO + EG/SSV or CV, and DMSO + EG/CV were similar to the EG/SF group, which was the best group that provided values similar to fresh control groups. Adequate preservation of the proliferative potential of spermatogonia, Leydig cells, and Sertoli cells was obtained using SSV with DMSO + EG. Overall, the use of SSV with DMSO + EG was the best protocol for the preservation of testicular tissues from adult red-rumped agoutis.

Impact of equilibration duration combined with temperature on the outcome of bovine oocyte vitrification

The cryopreservation of mammalian oocytes and embryos has become an integral part of assisted reproduction in both humans and veterinary species. However, the methods used to cryopreserve bovine oocytes still have significant shortcomings. A wide variety of approaches has been used to try to improve and optimize methods of cryopreservation. However, these procedures employed are not always designed to specifically take account of the osmotic tolerance response of the cells according to the temperature and time of cryoprotectant (CPA) addition. When these properties are considered, optimal procedures for the addition of CPAs can be designed proactively. Based on in silico and in vitro osmotic observations, we propose shorter dehydration-based protocols at different temperatures (25°C vs. 38.5°C) towards defining an improved cryopreservation method. In vitro matured oocytes were exposed to equilibration solution (ES) at 25°C and 38.5°C and effects of optimized exposure times for each temperature were determined prior to vitrification/warming on oocyte spindle configuration, DNA fragmentation, and further embryo development. Upon exposure to standard ES (7.5% dimethyl sulfoxide + 7.5% ethylene glycol in TCM199 medium + 20% fetal bovine serum), original oocyte volume was recovered within 2 min 30 s at 38.5°C and 5 min 30 s at 25°C. In vitro matured oocytes were then exposed to the aforementioned cryoprotectants at both temperature/duration conditions and vitrified/warmed. While similar percentages of oocytes exhibiting a normally configured spindle and DNA fragmentation were observed in the fresh control group and oocytes vitrified at 38.5°C, significantly higher apoptosis rate and lower percentages of normal spindle configuration were observed in oocytes vitrified at 25°C when compared to control fresh oocytes. Similar cleavage rates and blastocyst yields were observed in the vitrified/38.5°C and fresh controls, while these rates were lower in vitrified/25°C. These results revealed that the limitation of the exposure time of the oocytes to the ES to the point of osmotic equilibrium volume recovery could be a more efficient approach to prepare them for vitrification. Therefore, exposure time to ES to 2 min 30 s at 38.5 °C appears to improve the quality of vitrified/warmed oocytes by protecting spindle integrity and reducing DNA fragmentation thus improving blastocyst rates and embryo quality.

Isochoric supercooling cryomicroscopy

We introduce an isochoric (constant-volume) supercooling cryomicroscope (ISCM), enabling the ice-free study of biological systems and biochemical reactions at subzero temperatures at atmospheric pressure absent ice. This technology draws from thermodynamic findings on the behavior of water in isochoric systems at subfreezing temperatures. A description of the design of the ISCM and a demonstration of the stability of the supercooled solution in the ISCM is followed by an illustration of the possible use of the ISCM in the preservation of biological matter research. A comparison was made between the survival of HeLa cells in the University of Wisconsin (UW) solution in the ISCM at +4 °C under conventional atmospheric conditions and at −5 °C under isochoric supercooled conditions. Continuous real-time monitoring at cryopreservation temperature via fluorescence microscopy showed that after three days of isochoric supercooling storage, the percentage of compromised cells remained similar to fresh controls, while storage at +4 °C yielded approximately three times the mortality rate of cells preserved at −5 °C.

Effect of Ascorbic Acid and Proline Amino Acid Supplementations on Cryosurvival and Fertility Rates of Cryopreserved Honey Bee (Apis mellifera) Semen.

This study investigated the effect of ascorbic acid (vitamin C) and proline amino acid alone or together on the quality and fertility of frozen/thawed honey bee spermatozoa. The experiments were designed to compare a single ascorbic acid, a single proline amino acid, and different combinations of ascorbic acid with proline amino acid on the cryopreservation of honey bee semen based on sperm motility, viability, intact membrane (hypo-osmotic swelling test), and fertility rates. Eight cryopreserved study groups comprised Control II with no supplement, along with groups with ascorbic acid (2 mg), proline 25 mM, proline 50 mM, proline 100 mM, and combination groups of both ascorbic acid (2 mg) and proline 25 mM, proline 50 mM, and lastly proline 100 mM groups, respectively. Using 50 mM proline in the tested groups had the greatest impact on sperm motility, viability, the percentage of spermatozoa with intact membrane, and fertility. The cryopreservation process caused a gradual decrease in motility, viability, intact membrane (p < 0.05), and fertility rates (p < 0.01) in all the tested research groups as against the fresh semen control group. Successful honey bee sperm cryopreservation and fertility are achievable when using an appropriate sperm freezing protocol and antioxidant. Proline amino acid as an antioxidant in semen extender had a more beneficial influence on sperm quality parameters and fertility. The success of cryopreservation with antioxidants is related to the chosen antioxidant in a dose-dependent manner.

High survival of bovine mature oocytes after nylon mesh vitrification, as assessed by intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) is an alternative technique to in vitro fertilization (IVF) for producing transferable blastocysts, especially in combination with cryopreserved oocytes, when the IVF system does not work sufficiently. The present study was conducted to directly compare the efficacy of producing bovine blastocysts by ICSI and IVF from vitrified-warmed and fresh oocytes. Denuded oocytes with a detectable first polar body were vitrified-warmed using a nylon mesh device. In the non-vitrified control group, blastocyst yields 8 days after IVF and ICSI were 32.0 and 26.8%, respectively. Oocyte vitrification and subsequent IVF resulted in an impaired blastocyst yield (15.0%); however, such a loss of efficacy due to vitrification was not observed in the ICSI group (blastocyst yield, 25.2%). The alignment of cortical granules beneath the oolemma was comparable between the fresh control and vitrified-warmed oocytes. Here, we report the high survival of vitrified-warmed bovine oocytes, as assessed by ICSI.

Influence of storage at 5°C and vitrification on apoptotic changes in equine blastocysts

The aim of the study was to determine the extent of nuclear DNA fragmentation in equine embryos at the blastocyst stage stored at 5° C for 6 and 24 h or vitrified in straws or in the Rapid-i system. An additional aim was to determine the relationship between the age of fresh and stored embryos and the intensity of apoptosis. In this study, 42 embryos were analyzed by the TUNEL method and allocated to the following groups: fresh (control -14 embryos), stored at 5° C for 6 h (7 embryos), stored at 5° C for 24 h (7 embryos), vitrified in straws (7 embryos), and vitrified in the ultra-fast Rapid-i system (7 embryos). It was found that 36.7% of fresh equine embryos had apoptotic nuclei, and the mean dead cell index (DCI) was 0.3%. This was evident as an increase in DNA fragmentation in equine embryos after vitrification in straws (2.3%) compared to fresh embryos (0.3%) and those vitrified in the Rapid-i system (0.3%). Embryo storage at 5° C did not affect the apoptosis frequency in nuclei regardless of the time of storage. DCI for embryos stored for 6 h was 0.44%, and after 24 h of incubation it was 0.64%. The intensity of apoptotic changes observed in equine embryos depended on their age and size. The apoptosis frequency was negatively correlated with the age and diameter of fresh embryos, embryos stored at 5° C, and embryos vitrified in Rapid-I, whereas for embryos vitrified in straws the correlation was positive.

Short-term preservation of porcine zygotes at ambient temperature using a chemically defined medium.

We aimed to develop a simple method for the short-term preservation of in vitro-produced porcine zygotes at 25°C for up to 2 days. Firstly, we evaluated the efficiency of three storage solutions to preserve porcine zygotes at 25°C for 24 h. Two of these were commercially available defined media for cell storage (Cell-W and Cell-S), and the third was fetal bovine serum (FBS). Thereafter, we examined the effects of storing the zygotes in the Cell-W solution for 24-72 h at 25°C. The Cell-W solution was the most efficient for 24 h storage of porcine zygotes at 25°C, with no apparent effects on blastocyst quality. However, short-term storage of porcine zygotes for 24 h reduced the blastocyst formation rate compared with the fresh control group. As storage duration increased from 24 to 48 or 72 h, blastocyst formation rates were significantly decreased from 11.3% to 4.4% and 0%, respectively. In conclusion, during zygote storage, the developmental competence to the blastocyst stage decreased with time. Storage of zygotes in chemically defined media did not affect blastocyst quality, but the storage in 100% serum had an adverse effect on developing embryos causing apoptosis.

Issues with Tropical and Temperate Ensilage Protein and Amino Acid Feeds Utilization: A Research Note

Alfalfa protein breakdown was to soluble NPN of oligopeptide-N, AA-N, amide-N, amine-N and NH3-N. Acidity (pH) and moisture (Aw) are critical in determining extent of fermentation and changes in composition. Further changes in digestive flows and post-prandial plasma AA are indicators of protein status. Dual-purpose cropping and tree plant cropping was with ensiling management of the undergrowth. On-farm field-drying and probiotic additives are promising. It is suggested acidity with propionic acid and microbial inoculants together with field-drying and chop length are required to optimize profile qualities in silage. It is proposed use of denaturing with acid and dust cropping with a hypothetical PNA-Auxin repressor to plant protease. Further study with field-drying to follow is needed. Feeding HIS, ARG and LEU AA supplement to change GRH and GH profiles could be used to promote LBM in production. Dual-purpose cropping can expand subsistence to mixed farming with expanded livestock products and services and resources. PNA-Auxin and PNA-ARF penetrates the plant shoot tips to deliver a TF mRNA to boost proteins in residual cell tissues. Ensiled % AA-N delivery per os to per duodenum was higher; yet total AA-N flow was higher in the control. It is suggested that “bulk” flow was less but with a “tighter” conversion on TAA. FAA was 145% higher in the ensiled versus the fresh control indicating the ENU with less PFAA supplied. FAA on the ensiled diet is high inferred to be more soluble and escape lower from the rumen. WSC are less supplied in fermented forage with VFA being lower and presenting the question whether WSC should be supplied for energy and also with EFE through breaking down of polymers of lignocellulose. It was surmised, although not known, that higher dilution rate (% hr-1) was true on the fresh diet compared to the ensiled although end-products may initially detract with feed but that further digestion in the fresh feed may be higher with intake. Plasma AA before and after absorption or feeding are indicators of synthesis and breakdown. No data was available on N status; protein nutrition on neat silage was probably due to net efflux of AA with mobilization before influx with feeding and subsequent insulin action for uptake. Estuarine aquatic plant spp., water hyacinth used in the Philippines and duckweed studies in Australia, and post-harvest treatment with chemical additives and anti-microbial agents to help control potential transfer of diseases. “Greens” as supplements has yet to be established for anti-microbial properties for animal health and welfare. In conclusion, alfalfa silage fed at standard 0.6 cm particle size and wilted led to dramatic changes with AA breakdown, dramatic changes in duodenal AA flows from escape and recapture into microbial cells. Also N status of animals was compromised by lack of adequate “stores”, mobilized, resulting in a net decrease in total plasma AA with insulin-dependent uptake to tissue.

Cryoprotective effect of antifreeze protein III on the rabbit ovary.

Ovarian tissue cryopreservation is effective in preserving fertility in cancer patients who have concerns about fertility loss due to cancer treatment. However, ischemia reduces the lifespan of grafts. Microvascular transplantation of cryopreserved whole ovary may allow immediate revascularisation, but the damage incurred during the cryopreservation procedure may cause follicular depletion; hence, preventing chilling injury would help maintain ovarian function. This study was designed to investigate the beneficial effects of antifreeze protein III (AFP III) on rabbit ovary cryopreservation. Ovaries (n =25) obtained from 5-month-old female rabbits (n =13) were frozen by slow freezing and vitrification. Cryoprotectant media were supplemented with and without 1mg/mL of AFP III. The experiment was divided into five groups: fresh control group (F), slow freezing group (S), slow freezing group with AFP III (AFP III-S), vitrification group (V) and vitrification group with AFP III (AFP III-V). All groups of ovaries were examined by histological characteristics analysis, ultrastructural analysis, apoptosis detection and follicle viability test. With slow freezing, the normal rate of change in follicle morphology, density of stromal cells and the survival rate of follicles in the AFP III supplemented group were significantly higher than those in the non-supplemented group, and a lower oocyte apoptotic rate was shown in the AFP III supplemented group. In the vitrification groups, the normal rate of change in follicle morphology and density of stromal cells in the AFP III supplemented group were significantly higher than those in the non-supplemented group, and a lower oocyte apoptotic rate was found in the AFP III supplemented group. But there was no obvious difference in the survival rate of follicles between the two groups. There was also no significant difference in the normal rate of change in follicle morphology, the survival rate of follicles and the apoptotic rate of oocytes between the vitrification and slow freezing groups (P >0.05), but the density of stromal cells in the vitrification groups was statistically higher than that of the slow freezing group (P <0.05). The addition of AFP III in slow freezing and vitrification could improve the cryoprotective effect of ovaries, which was more evident in slow freezing. The findings of this study provide a foundation for further research on the effects of AFP III in human ovarian tissue.

Rosemary (Rosmarinus officinalis) as a Preservative Agent in Canned Bonito (Sarda sarda)

This study was conducted to determine the shelf life and quality of traditional canned fish made from bonito (Sarda sarda) fish. The effect of the rosemary plant on some quality parameters and storage time of bonito fish with tomato sauce stored for 510 days at room temperature was determined by sensory, physical, chemical and microbiological freshness control methods. Nutrient composition analyses were performed in bonito fish before the canning process. Crude protein, crude oil, moisture and crude ash values were found to be, 23.125±1.062%, 2.783±0.339%, 72.717±0.652% and 1.150±0.212%, respectively. The amount of carbohydrates was determined as 0.275 g/100 g, the amount of energy was determined as 157.43 kcal/100 g. During storage, the total amount of volatile basic nitrogen (TVB-N), the number of thiobarbituric acid (TBA), pH, water activity (aw), the total number of aerobic mesophilic bacteria (TAMB), the total number of yeast and mold (TYM) were calculated. When TBA, pH, aw and microbiological analyzes were evaluated, both groups did not exceed the consumption limit values during the storage period. But in the research, the antioxidant effect of rosemary was clearly seen.

Quantification of residual cryoprotectants and cytotoxicity in thawed bovine ovarian tissues after slow freezing or vitrification.

How much residual cryoprotectant remains in thawed/warmed ovarian tissues after slow freezing or vitrification? After thawing/warming, at least 60 min of diffusion washing in media was necessary to significantly reduce the residual cryoprotectants in ovarian tissues frozen by slow freezing or vitrification. Ovarian tissue cryopreservation (OTC) by slow freezing has been the conventional method; while the vitrification method has gained popularity for its practicality. The main concern about vitrification is how much potentially toxic residual cryoprotectant remains in the warmed tissues at the time of transplantation. This was an animal study using the ovarian tissues from 20 bovine ovaries. The duration of this study was from 2018 to 2020. Ovarian cortex tissues were prepared from 20 bovine ovaries and assigned randomly to groups of fresh (non-frozen) control, slow freezing with 1.5 M dimethyl sulfoxide (DMSO), 1.5 M 1,2-propanediol (PROH) and vitrification with 35% ethylene glycol (EG). The residual cryoprotectant concentrations in thawed/warmed tissues were measured by gas chromatography at the following time points: frozen (before thawing/warming), 0 min (immediately after thawing/warming), 30, 60 and 120 min after diffusion washing in media. Next, the ultrastructural changes of primordial follicles, granulosa cells, organelles and stromal cells in the ovarian tissues (1 mm × 1 mm × 1 mm) were examined in fresh (non-frozen) control, slow freezing with DMSO or PROH and vitrification with EG groups. Real-time quantitative PCR was carried out to examine the expressions of poly (ADP-ribose) polymerase-1 (PARP1), a DNA damage sensor and caspase-3 (CASP3), an apoptosis precursor, in thawed/warmed ovarian tissues that were washed for either 0 or 120 min and subsequently in tissues that were ex vivo cultured for 24 or 48 h. The same set of tissues were also used to analyze the protein expressions of gamma H2A histone family member X (γH2AX) for DNA double-strand breaks and activated caspase-3 (AC3) for apoptosis by immunohistochemistry. The residual cryoprotectant concentrations decreased with the extension of diffusion washing time. After 60 min washing, the differences of residual cryoprotectant between DMSO, PROH and EG were negligible (P > 0.05). This washing did not affect the tissue integrity or significantly elevate the percentage of AC3 and γH2AX positive cells, indicating that tissues are safe and of good quality for transplantation. N/A. Since the study was performed with ovarian tissues from bovines, generalizability to humans may be limited. Potential changes in ovarian tissue beyond 120 min were not investigated. This study addresses concerns about the cytotoxicity of EG in warmed ovarian tissues and could provide insights when devising a standard vitrification protocol for OTC. The study was funded by a Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science to N.S.

Preservation Method of Rumen Fluid Collected From Harvested Cattle Alters in vitro Digestibility of Reference Feedstuffs

The objective of our experiment was to evaluate preservation and revitalization strategies for rumen inoculum anticipating research and veterinary applications. Rumen fluid samples were collected from 12 harvested cattle. Liquid samples were divided into five 500-mL aliquots which were randomly allocated to one of five treatments in a 2 × 2 + 1 augmented factorial design. Factors included preservation method [freezing (FZN) or lyophilization (LYO)] and preservative (glycerol; + or –). A fresh control (CON) was maintained from each sample. Feedstuffs used in this experiment were alfalfa hay, Coastal bermudagrass hay, cracked maize, rice bran, and soybean meal. Reference feedstuffs were subjected to batch culture in vitro true digestibility (IVTD) and in vitro NDF digestibility (IVNDFD) assays using inoculum from each of the five treatments. There was an effect (P &amp;lt; 0.05) of preservation method, preservative, and their interaction for both IVTD and IVNDFD of each of the five references feedstuffs. Freezing or lyophilization of rumen inoculum reduced (P &amp;lt; 0.05) IVTD and IVNDFD of reference feeds relative to the CON. Despite lower degradation of feeds when frozen or lyophilized rumen fluid was used rather than fresh, differences between them in IVTD and IVNDFD suggest that, in the absence of fresh inoculum, preserved rumen fluid may be a viable option for veterinary applications, such as transfaunation, but likely will not be viable for research applications.

Aloe vera increases collagen fibres in extracellular matrix and mRNA expression of peroxiredoxin-6 in bovine ovarian cortical tissues cultured in vitro.

In vitro culture of ovarian tissue containing primordial follicles is an important tool to study the initiation of follicular populations and to develop efficient culture systems to support in vitro follicle growth. Considering that in vitro culture favours oxidative stress, it is very important to supplement culture medium with antioxidant substances such as Aloe vera extract. This study aims to evaluate the effects of different concentrations of Aloe vera on the distribution of collagen fibres in the extracellular matrix, follicular activation, development and survival in bovine ovarian cortical tissues cultured in vitro, as well as on expression of mRNAs for antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), peroxiredoxin 6 (PRDX6) and glutathione peroxidase 1 (GPX1)]. To this end, ovarian cortical tissues were cultured for 6 days in α-MEM alone or supplemented with different concentrations of Aloe vera extract (1.0, 5.0, 10.0 or 50.0%). After culture, fragments were fixed and processed histologically to evaluate follicular morphology and activation, as well as the extracellular matrix by staining with picrosirius red. The levels of mRNA for SOD, CAT, PRDX6 and GPX1 in cultured ovarian tissues were evaluated by real-time polymerase chain reaction (PCR). Ovarian tissues cultured with 10.0 or 50.0% Aloe vera had higher percentages of collagen fibres than tissues cultured in control medium. A significant increase in developing follicles was observed in ovarian tissues cultured in α-MEM alone or supplemented with 10% Aloe vera when compared with fresh control or tissues cultured with 1.0% Aloe vera. Presence of Aloe vera did not influence the percentage of morphologically normal follicles when compared with control medium. Ovarian tissues cultured with 50.0% Aloe vera had higher percentages of morphologically normal follicles than those cultured with 10.0% Aloe vera. Furthermore, 10% Aloe vera significantly increased mRNA levels for PRDX6. In conclusion, 10.0% Aloe vera improves extracellular matrix distribution in cultured tissues and increases the expression of mRNA for PRDX6 after 6 days in vitro.