Storage Time Research Articles

Assessment of fecal bacterial viability and diversity in fresh and frozen fecal microbiota transplant (FMT) product in horses

BackgroundCurrently, lack of standardization for fecal microbiota transplantation (FMT) in equine practice has resulted in highly variable techniques, and there is no data on the bacterial metabolic activity or viability of the administered product. The objectives of this study were to compare the total and potentially metabolically active bacterial populations in equine FMT, and assess the effect of different frozen storage times, buffers, and temperatures on an equine FMT product. Fresh feces collected from three healthy adult horses was subjected to different storage methods. This included different preservation solutions (saline plus glycerol or saline only), temperature (-20 °C or -80 °C), and time (fresh, 30, 60, or 90 days). Samples underwent DNA extraction to assess total bacterial populations (both live and dead combined) and RNA extraction followed by reverse transcription to cDNA as a proxy to assess viable bacteria, then 16s rRNA gene amplicon sequencing using the V1-V2 region.ResultsThe largest difference in population indices and taxonomic composition at the genus level was seen when evaluating the results of DNA-based (total) and cDNA-based (potentially metabolically active) extraction method. At the community level, alpha diversity (observed species, Shannon diversity) was significantly decreased in frozen samples for DNA-based analysis (P < 0.05), with less difference seen for cDNA-based sequencing. Using DNA-based analysis, length of storage had a significant impact (P < 0.05) on the bacterial community profiles. For potentially metabolically active populations, storage overall had less of an effect on the bacterial community composition, with a significant effect of buffer (P < 0.05). Individual horse had the most significant effect within both DNA and cDNA bacterial communities.ConclusionsFrozen storage of equine FMT material can preserve potentially metabolically active bacteria of the equine fecal microbiome, with saline plus glycerol preservation more effective than saline alone. Larger studies are needed to determine if these findings apply to other individual horses. The ability to freeze FMT material for use in equine patients could allow for easier clinical use of fecal transplant in horses with disturbances in their intestinal microbiome.

Formaldehyde contamination in PET-bottled mineral water: impact of CO2, temperature, and storage time with analytical method validation

Formaldehyde contamination in PET-bottled mineral water: impact of CO2, temperature, and storage time with analytical method validation

Evaluation of the Quality of Guinea Fowl (Numida meleagris) Eggs from Free-Range Farming Depending on the Storage Period and Age of Laying Hens

The aim of the study was to determine the changes occurring in the eggs of helmeted guinea fowl (Numida meleagris) from free-range farming in relation to the laying season and storage time. The experimental material consisted of 360 guinea fowl eggs, collected in the first, second and third laying seasons and stored for 7, 14 and 21 days. After each period, physical and physicochemical characteristics of the eggs were determined, as well as the basic chemical composition and mineral content of the albumen and yolk and the yolk fatty acid profile. The age of the guinea fowls affected certain physical parameters of the eggs. The egg weight, shape index and shell thickness increased with the age of the laying hens; however, a decrease in the proportion of shell in the egg was demonstrated. Storage time had a significant effect on egg weight, weight loss during storage and air cell height. Significant differences were found in the chemical composition of guinea fowl eggs depending on the age of the laying hens. Eggs obtained from older laying hens were characterized by higher yolk fat content and lower ash content, while the albumen contained higher water content and lower ash content. During the three-year laying period, changes were observed in the mineral composition of the eggs. The fatty acid profile underwent significant changes; however, no important differences were observed in the total content of SFA, MUFA, PUFA and n-6 fatty acids. Conversely, significant differences were found for n-3 acids and the n-6/n-3 ratio. Eggs in the first and second laying seasons exhibited the most favorable composition. The slow dynamics of changes occurring in successive laying seasons and egg storage time indicated that the raw material studied was safe and could be used by consumers

Exploring the Evolution of Postharvest Quality and Composition in Novel Mini Plum Tomatoes with Different Fruit Pigmentations

This study evaluated the postharvest evolution in terms of quality and functional traits of recently introduced mini plum tomato cultivars, differing for fruit pigmentation. The cultivars Santy Naranja (orange fruit) and Melange (dark red, green-striped fruit), along with the well-established ‘Angelle’ (red fruit), were monitored during refrigerated storage (10 °C) for 0, 7, and 14 days (hereafter S0, S7, and S14, respectively) inside closed polylactic acid trays. ‘Melange’ displayed the highest fruit fresh weight loss and total soluble solids (TSS) (9.28 °Bx), along with the lowest firmness (4.69 N), whereas ‘Santy Naranja’ showed the strongest color deviation during the storage period. Titratable acidity (TA) was relatively stable in ‘Melange’, while ‘Angelle’ experienced the highest decrease between S7 and S14, resulting in stronger TSS/TA fluctuations. Regarding the functional traits, ‘Melange’ had the highest total polyphenols content (TPC), whereas ‘Angelle’ excelled in total carotenoid content (TCC), ascorbic acid content (AsA), and fruits’ antioxidant activity, as revealed by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays. With reference to storage time, the O2 concentration in the headspace, TPC, AsA, and DPPH decreased between S0 and S14, whereas CO2 concentration, TCC, and FRAP increased, but these variations were mostly cultivar-dependent. This study provides insights into the postharvest behavior of novel mini plum tomato typologies and their ability to maintain quality over time, providing information for developing strategies to preserve their sensory and nutraceutical characteristics during refrigerated storage.

Kinetic Insights into the Antioxidant Effect of Isatin-Thiosemicarbazone in Biodiesel Blends

Biodiesel has several drawbacks, such as being prone to oxidation, having reduced stability, and having limited storage time. Antioxidants compatible with biodiesel are being used to address its drawbacks. Utilizing antioxidants effectively improves the quality of biodiesel. Enhancing the quality of biodiesel for use as a clean energy source benefits both the global economy and ecology. Therefore, we believe that our work will contribute to the advancement of the biodiesel industry worldwide. This study used blends consisting of 20% biodiesel and 80% diesel fuel. Isatin-thiosemicarbazones were tested as additives in blends at a concentration of 3000 parts per million (ppm) using an oxifast device and were compared with the chemical antioxidant Trolox. FT-IR, DSC, and TGA were used to characterize these samples. DSC measured sample crystallization temperatures (Tc). Samples with antioxidants showed decreased values compared to the non-antioxidant diesel sample D100. Several DSC tests were conducted to determine the antioxidant strengths of various samples. The results show that the FT-IR spectrum’s antioxidant effect regions grow clearer with antioxidants. The extra antioxidant is effective. Biodiesel’s oxidative stability improves with isatin-thiosemicarbazones at varying concentrations. The kinetics of thermal decomposition of isatin-thiosemicarbazones under non-isothermal conditions were determined using the Kissinger, Ozawa, and Boswell techniques. The activation energies of compounds 1 and 2 were calculated as 137–147 kJ mol−1 and 173–183 kJ mol−1, respectively.

Antibacterial and antibiofilm potentials of vancomycin-loaded niosomal drug delivery system against methicillin-resistant Staphylococcus aureus (MRSA) infections

The threat of methicillin-resistant Staphylococcus aureus (MRSA) is increasing worldwide, making it significantly necessary to discover a novel way of dealing with related infections. The quick spread of MRSA isolates among infected individuals has heightened public health concerns and significantly limited treatment options. Vancomycin (VAN) can be applied to treat severe MRSA infections, and the indiscriminate administration of this antimicrobial agent has caused several concerns in medical settings. Owing to several advantageous characteristics, a niosomal drug delivery system may increase the potential of loaded antimicrobial agents. This work aims to examine the antibacterial and anti-biofilm properties of VAN-niosome against MRSA clinical isolates with emphasis on cytotoxicity and stability studies. Furthermore, we aim to suggest an effective approach against MRSA infections by investigating the inhibitory effect of formulated niosome on the expression of the biofilm-associated gene (icaR). The thin-film hydration approach was used to prepare the niosome (Tween 60, Span 60, and cholesterol), and field emission scanning electron microscopy (FE-SEM), an in vitro drug release, dynamic light scattering (DLS), and entrapment efficiency (EE%) were used to investigate the physicochemical properties. The physical stability of VAN-niosome, including hydrodynamic size, polydispersity index (PDI), and EE%, was analyzed for a 30-day storage time at 4 °C and 25 °C. In addition, the human foreskin fibroblast (HFF) cell line was used to evaluate the cytotoxic effect of synthesized niosome. Moreover, minimum inhibitory and bactericidal concentrations (MICs/MBCs) were applied to assess the antibacterial properties of niosomal VAN formulation. Also, the antibiofilm potential of VAN-niosome was investigated by microtiter plate (MTP) and real-time PCR methods. The FE-SEM result revealed that synthesized VAN-niosome had a spherical morphology. The hydrodynamic size and PDI of VAN-niosome reported by the DLS method were 201.2 nm and 0.301, respectively. Also, the surface zeta charge of the prepared niosome was − 35.4 mV, and the EE% ranged between 58.9 and 62.5%. Moreover, in vitro release study revealed a sustained-release profile for synthesized niosomal formulation. Our study showed that VAN-niosome had acceptable stability during a 30-day storage time. Additionally, the VAN-niosome had stronger antibacterial and anti-biofilm properties against MRSA clinical isolates compared with free VAN. In conclusion, the result of our study demonstrated that niosomal VAN could be promising as a successful drug delivery system due to sustained drug release, negligible toxicity, and high encapsulation capacity. Also, the antibacterial and anti-biofilm studies showed the high capacity of VAN-niosome against MRSA clinical isolates. Furthermore, the results of real-time PCR exhibited that VAN-niosome could be proposed as a powerful strategy against MRSA biofilm via down-regulation of icaR gene expression.

Surface mineral deposition and mechanical properties of one-step bonding agent with copper-modified niobium nanoparticles – An in vitro study

This study aimed to investigate the incorporation of copper-modified niobium (Nb/Cu) nanoparticles at concentrations of 0.1 % and 0.3 wt% in a one-step adhesive (PacBond Adhesive). Thirty sound human third molars were randomly divided into three groups: control, 0.1NbCu, and 0.3NbCu. Specimens were restored with PacBond and resin composite OptiComp, Composite LC. Stick specimens (0.64 mm2) were obtained and subjected to microtensile bond strength (MTBS) testing (n = 10) using a universal testing machine at a rate of 0.5 mm/min after 24 h and 6 months. Scanning electron microscope (SEM) analysis, microhardness after 24 h and immersion in ethanol (HK) (n = 7), degree of conversion (DC) (n = 6), and surface mineral deposition (n = 3) were also conducted. Data were analyzed using two-way ANOVA for MTBS and HK, and one-way ANOVA for %SHL and DC, all of them were followed by Tukey's test (p < 0.05). For MTBS, statistical difference in storage times was observed for 0.1NbCu after 24 h (p = 0.007). There was no statistical difference between the control and 0.3NbCu regarding time and materials after 24 h and 6 months (p = 0.065). There was no statistical difference among the groups for DC (p = 0.278), but there was a significant difference among the groups for the factor of time in HK measurements (p = 0.047). The 0.1NbCu and 0.3NbCu groups exhibited absorbance Bands indicative of surface mineral deposition after 14 and 21 days, and the 0.3NbCu interface showed better preservation in SEM analysis. The concentration of 0.3 % NbCu in a one-step adhesive appears promising and may offer enhanced longevity for the tooth/restoration interface.

Preparation and Evaluation of Nanoemulsion Formulation Containing Kojic Acid and Kojyl 3-aminopropylphosphonic Acid.

The kojyl 3-aminopropylphosphonic acid (KAP) was synthesized by kojic acid (KA) with a 3-aminopropylphosphonic acid. Which is more stable than KA and showed better skin penetration and anti-pigmentation efficacy in melanocytes. However, up till now, there have been no studies aimed at incorporating KAP into an emulsion system and evaluating its effectiveness. We develop a novel skin-lightening agent using KAP as the active ingredient and a low-cytotoxic nanoemulsion as the delivery system in this study. The sorbitan monooleate and polysorbate surfactants with polyethylene glycol (PEG) co-surfactant were used to generate a nanoemulsion system. The transparency and particle size stability over various storage times indicate that the formulated nanoemulsions are suitable for long-term storage. Besides, results demonstrate that the anti-pigmentation function of KA and KAP-containing nanoemulsions (NE-KA and NEKAP) evidently outperformed that of the non-packed KA and KAP group. Despite having the lowest concentration among other treatments, NE-KAP was able to reduce melanin content to approximately 80% of the blank. Our findings suggest that this newly developed nanoemulsion containing KAP could potentially serve as a sustainable alternative to hydroquinone for treating dermal hyperpigmentation disorders in future applications.

The effect of maltodextrin edible coating containing pyracantha extract and potassium nano-carbonate on secondary metabolites, antioxidant capacity and microbiological properties of grape during cold storage

This study aimed to investigate the postharvest application of edible coating of maltodextrin containing potassium nanoparticles (KNPs; 0–2%) and pyracantha extract (PE; 0–1.5 %) on the maintenance of phenolic compounds, antioxidant capacity and microbial properties of ‘Rishbaba’ grape during 60 days at −1 °C using response surface methodology and central composite design. The results showed that the applied coating on Rishbaba grape maintained total phenol, total flavonoids, total anthocyanin, stilbenes (resveratrol and viniferin) and catechin. That also caused higher antioxidant capacity and lower mold and yeast growth in grape during the storage time. Finally, the best cold storage conditions of ‘Rishbaba’ grape were determined by using the edible maltodextrin coating containing 2 % KNPs and 1.5 % of PE for 34 days with a desirability of 0.728 in terms of investigated Characteristics. The optimized sample has the amount of total phenol (5.79 mg/g), total flavonoid (8.95 mg/g), total anthocyanin (6.48 mg/g) and the greatest ability to inhibit DPPH free radical (42.56 %) and the lowest growth rate of mold and yeast (21 Cfu/g).

Vitrification preservation of good-quality blastocysts for more than 5years reduces implantation and live birth rates.

Does vitrification cryopreservation of embryos for more than 5 years affect the pregnancy outcomes after frozen embryo transfer (FET)? Vitrification cryopreservation of good-quality blastocysts for more than 5 years is associated with a decrease in the implantation rate (IR) and live birth rate (LBR). Previous studies have predominantly focused on embryos cryopreserved for relatively short durations (less than 5 years), yet the impact of extended cryopreservation duration on pregnancy outcomes remains a controversial issue. There is a relative scarcity of data regarding the efficacy and safety of storing embryos for 5 years or longer. This retrospective study involved 36665 eligible vitrified-thawed embryo transfer cycles from 1 January 2016 to 31 December 2022, at a single fertility center in China. Patients were divided into three groups according to embryo storage time: Group 1 consisted of 31565 cycles, with storage time of 0-2 years; Group 2 consisted of 4458 cycles, with a storage time of 2-5 years; and Group 3 included 642 cycles, with storage time exceeding 5 years. The main outcome measures were IR and LBR. Secondary outcome variables included rates of biochemical pregnancy, multiple pregnancy, ectopic pregnancy, and miscarriage, as well as neonatal outcomes. Reproductive outcomes were analyzed as binary variables. Multivariate logistic regression analysis was used to explore the effect of preservation time on pregnancy outcomes after correcting for confounding factors. In addition, we also assessed neonatal outcomes, such as large for gestational age (LGA) and small for gestational age (SGA). IRs in the three groups (0-2, 2-5, and >5 years) were 37.37%, 39.03%, and 35.78%, respectively (P = 0.017), and LBRs in the three groups were 37.29%, 39.09%, and 34.91%, respectively (P = 0.028). After adjustment for potential confounding factors, compared with the 0-2 years storage group, prolonged embryo vitrification preservation time (2-5 years or >5 years) did not affect secondary outcomes such as rates of biochemical pregnancy, multiple pregnancy, ectopic pregnancy, and miscarriage (P > 0.05). But cryopreservation of embryos for more than 5 years reduced the IR (adjusted odds ratio (aOR) 0.82, 95% CI 0.69-0.97, P = 0.020) and LBR (aOR 0.76, 95% CI 0.64-0.91, P = 0.002). Multivariate stratified analysis also showed that prolonging the cryopreservation time of blastocysts (>5 years) reduced the IR (aOR 0.78, 95% CI 0.62-0.98, P = 0.033) and LBR (aOR 0.68, 95% CI 0.53-0.87, P = 0.002). However, no effect on cleavage embryos was observed (P > 0.05). We further conducted stratified analyses based on the number and quality of frozen blastocysts transferred, and the results showed that the FET results after transfers of good-quality blastocysts in the >5 years storage group were negatively affected. However, the storage time of non-good-quality blastocysts was not significantly associated with pregnancy outcomes. Regarding the neonatal outcomes (of singletons), embryo vitrification preservation time had no effect on preterm birth rates, fetal birth weight, or neonatal sex ratios. However, as the storage time increased, rates of SGA (5.60%, 4.10%, and 1.18%) decreased, while rates of LGA (5.22%, 6.75%, and 9.47%) increased (P < 0.05). After adjusting for confounding factors, the increase in LGA and the decrease in SGA were significantly correlated with the duration of storage time. This was a retrospective study using data from a single fertility center, even though the data had been adjusted, our findings still need to be validated in further studies. With the full implementation of the two-child policy in China, there may be more patients whose embryos have been frozen for a longer time in the future. Patients should be aware that the IR and LBR of blastocysts are negatively affected when the cryopreservation time is longer than 5 years. Couples may therefore consider shortening the time until FET treatment. This study was supported by the National Nature Science Foundation of China (No. 82101672), Science and Technology Projects in Guangzhou (No. 2024A03J0180), General Guidance Program for Western Medicine of Guangzhou Municipal Health Commission (No. 20231A011096), and the Medical Key Discipline of Guangzhou (2021-2023). None of the authors have any conflicts of interest to declare. N/A.

O-298 Embryo long-term storage does not affect ART outcome: analysis of 58,001 vitrified blastocysts

Abstract Study question Does duration of storage affect clinical outcome in frozen embryo transfer (FET) cycles? Summary answer Long-term storage does not affect live birth rate (LBR) in elective and non-elective FET cycles. What is known already Despite the success rates provided by the embryo vitrification programs, some studies found an association between the duration of cryo-storage and decreased clinical outcome, raising concern about the potential negative effect that prolonged storage may have on embryos. Study design, size, duration This was a retrospective multicenter study including a total of 58001 vitrified/warmed day-5 blastocysts from two different populations according to the reason for FET: elective FET which involved patients undergoing freeze all cycles (N = 16,615 blastocysts and 16,615 patients); and non-elective FET including 41,386 embryos from 25,571 patients. FETs were performed in the period between January 2010 and December 2020. Participants/materials, setting, methods Main outcome was live birth rate (LBR) and secondary outcomes were embryo survival, miscarriage, clinical and ongoing pregnancy rates. The impact of storage time and the potential confounding variables was assessed by univariate analyses. Those covariables found to significantly modify the LBR were included in a multivariable logistic regression. GEE (generalized estimated equation) was performed to allow the analysis of repeated measurements. Odds ratios (OR) and 95% confidence intervals (CI) were provided. Main results and the role of chance The blastocysts sample was clustered into deciles which provided specific storage duration categories that ranked from ≤0.67 to ≥ 4.34 and ≤1.8 to ≥ 34.81 months in freeze-all and non-elective FET, respectively. In the freeze-all group, maximum storage time was 34.8 months (2.9 years), while it was 121.2 months (10.1 years) in the non-elective FET group. Embryo survival did not show statistical differences across the categories of storage time in freeze-all and non-elective FET groups. In the freeze-all group, statistical differences were found for LBR across some, but not all the subgroups of storage duration. Similarly, in the non-elective FET group, LBR among the deciles showed statistical differences in some comparisons but not in all. However, after adjusting for confounding variables, the multivariate analysis showed no association between storage time and LBR in both groups. Blastocyst quality, body mass index (BMI), number of retrieved oocytes, endometrial preparation, male factor and uterine factor were related to the drop in the LBR in the freeze-all group (p &amp;lt; 0.05). In the non-elective FET group, the variables that showed significant association with the LBR were age at retrieval and at FET, number of embryos transferred, number of retrieved oocytes, BMI, endometrial preparation, sperm origin and female factor. Limitations, reasons for caution The retrospective design of the study represents its main limitation. Wider implications of the findings Our results demonstrate the safety of long-term cryo-storage. This is reassuring for both IVF practitioners and patients especially considering the exponential increase in FET cycles observed in the last decades. Trial registration number Not applicable

P-006 Fertility outcomes in men diagnosed with oncohematological diseases who stored their semen for fertility preservation

Abstract Study question What is the prevalence of conceptions among patients who underwent semen cryopreservation before receiving treatment for oncohematological disease? Summary answer Natural live birth rate after oncohematological treatments is around 38%(95%CI:31-45%), but can rise to 61%(95%CI:54-67%) with the use of frozen semen during ART cycles. What is known already Due to advancements in cancer treatments, there is a growing population of male adult cancer survivors. These individuals are at risk of experiencing treatment-induced infertility, and it is generally advised for them to preserve their semen before starting therapies. However, the details regarding the long-term utilization and effectiveness of frozen sperm samples in this particular population remain unclear. Study design, size, duration The study included males with oncohematological disease who stored semen (1987-2018) at Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico’s Assisted Reproductive Technology Unit in Milan. Data, gathered from records or proactive inquiry, assessed natural and ART conceptions post-treatment. The study also examined semen analysis, parenthood attempts, and the usage rates of stored spermatozoa as primary outcomes. Participants/materials, setting, methods The study is limited to men who meet the following eligibility criteria: i) semen cryopreserved for fertility preservation between 1987 and 2018; ii) diagnosis of oncohematological disease; iii) survival of at least 5 years from cryopreservation. Main results and the role of chance A total of 424 patients who underwent semen cryopreservation for oncohematological diseases were included in the study. The median [IQR] age at the time of sperm banking was 27 [23-33]. The most common indication was lymphoma (86%), and 102 subjects (24%) underwent hematopoietic stem cell transplantation. The median sperm concentration at the time of storage was 54 [20-98] million/ml. Among the participants, 242 (57%) men underwent post-treatment sperm analysis, revealing azoospermia in 87 of them (36%, 95%CI: 30-42%). Of the remaining, only 93 (38%) exhibited normal semen parameters. A total of 188 out of the 424 men included (44%) expressed a desire for childbearing and seventy-one achieved a natural live birth (38%, 95%CI: 31-45%). Eighty-nine individuals used their cryopreserved sperm samples, constituting 21% (95%CI: 17-25%) of the entire cohort. Among these 89 men, 45 experienced at least one live birth (51%, 95%CI: 40-61%). Notably, thirty-three men, despite actively seeking pregnancy, did not utilize their frozen semen. In summary, out of the 188 men interested in fatherhood, a total of 114 achieved a live birth (61%, 95%CI: 54-67%). Limitations, reasons for caution Our study is constrained by the lack of detailed information on the illness status, comorbidities, and specific cancer therapies administered. Furthermore, the data concern a single-center population, potentially limiting the generalizability of the results. Wider implications of the findings Oncohematological disease often require systemic therapies or even hematopoietic stem cell transplantation resulting in a very high percentage of infertile patients These findings support the need to prioritize sperm preservation before therapies in these patients, even in cases who are not candidates for stem cell transplantation in the first instance Trial registration number NA

P-270 Analysis of patient response rates to automated cryomanagement platform communications by length of cryostorage and tissue type

Abstract Study question Does the length of storage time of cryopreserved samples impact patient response rate and is this influenced by the tissue type stored. Summary answer Patient response rates decline as storage time increases. The decline is faster and higher with sperm and testicular tissue, compared to oocytes and embryos What is known already Clinics are experiencing increasing inventories of cryopreserved samples. While clinics provide storage consents and contracts, an increasing issue has emerged of abandoned/unclaimed samples, even with provision of multiple disposition options. Cryomanagement is labour intensive, both in terms of physical and administrative maintenance, the latter further increasing when communication and payment from the patient inadvertently or deliberately ceases. Full cryomanagement automation including patient billing, education and engagement with disposition decisions reduces the workload for the embryologist and increases consistency. It may also provide data insights allowing clinics to identify issues and implement strategies to improve patient engagement Study design, size, duration Data were retrospectively analysed from an automated cryomanagement platform. In total, 418,895 patient accounts from 122 clinics based in the USA over the period 2015 to 2024 Participants/materials, setting, methods Data was extracted from the automated cryomanagement platform database - a live feed of clinic information – and encrypted. Logic was created to develop calculations utilising the coding language, Structured Query Language (SQL). Revenue collected was compared with collectable charges to generate the collection/response rate. Data was analysed by collection rate by tissue type to identify trends and differences Main results and the role of chance An inverse relationship was identified between storage duration and response rates. Collection rates fell from 97.44% to 68.44% in relation to oocytes, 94.87% to 45.08% for embryos, 87.01% to 29.41% for sperm, and 95.67% to 29.75% for testicular tissue. Patients with stored oocytes are least likely to abandon cryopreserved samples, with 68.44% of storage payments continuing to be received beyond 14yrs. Patients with stored sperm or testicular tissue are the most likely to abandon cryopreserved samples. Collection rates fall below 50% at 6-8yrs and 10-12yrs respectively, and beyond 14yrs storage have fallen to 29.41% and 29.75% Limitations, reasons for caution The storage duration does not necessarily reflect the duration of patient interaction with the automated cryomanagement platform, where freeze date preceeded platform implementation. Further research is necessary to identify if patients exclusively interacting with the platform from freeze date have altered response rates Wider implications of the findings Data held by automated cryomanagement systems allows accurate review and analysis of cryostorage and cryomanagement metrics. Mined data could be used to develop policies based on real-life data. Including patient treatment information and patient engagement with the system from freeze date may help identify and address reasons for abandonment Trial registration number Not Applicable

P-073 Microfluidic sperm selection yields competent spermatozoa with higher genomic integrity and cryostress tolerance

Abstract Study question Can microfluidic sperm sperm selection (MFSS) contribute to isolating spermatozoa with higher genomic integrity and cryostress resistance, while retaining embryo developmental competence? Summary answer In comparison to conventional density gradient (DG), MFSS yielded a higher proportion of motile spermatozoa coupled with enhanced genomic integrity and resistance to cryostress. What is known already The cryopreservation of human spermatozoa has long been a cornerstone in reproductive treatments, serving as a pivotal method for preserving male fertility for several decades. Despite the unlimited storage time of cryopreserved semen specimens, the thawing process inevitably diminishes the number of viable spermatozoa. The implementation of a technique capable of enhancing the motility of spermatozoa cryopreserved would yield a higher proportion of gametes with higher kinetics and genomic integrity at thawing. Study design, size, duration Since 2017, 183 men underwent cryopreservation utilizing MFSS. Post-processing semen parameters, including Sperm Chromatin Fragmentation(SCF), and post-thaw motility were compared with a DG control(n = 1979). Additionally, post-thaw parameters for 30 men who had previously employed DG and subsequently adopted MFSS for semen cryopreservation were included. In 12 specimens, a paired analysis was carried out on a same ejaculate. Moreover, we explored pregnancy outcomes with MFSS-selected specimens in some men that underwent ART. Participants/materials, setting, methods Semen parameters were analyzed, and DG was performed according to WHO 2021 guidelines. MFSS was carried out following manufacture’s protocol (ZyMōt® Multi 850µL). Cryoprotectant (Test Yolk Buffer) was added to the final sample before being immersed in liquid nitrogen. A minute aliquot of the processed sample, cryopreserved separately, was thawed for post-thaw motility. TUNEL assay was used to measure SCF (≤ 15% normal threshold). Main results and the role of chance In this study, 1979 men (40.0±7y) underwent DG prior to cryopreservation, resulting in a 44.3±25x106/mL concentration, 88.2±4% motility, and 2.9±1% normal morphology, serving as control. The remaining 183 men of a similar age underwent MFSS prior to cryopreservation. Despite a lower concentration at 26.1±15x106/mL(P &amp;lt; 0.001), MFSS resulted in a higher post-processing motility(97.8±2%, P &amp;lt; 0.001) and normal morphology(3.4±1%, P &amp;lt; 0.001). Post-thaw analysis revealed a remarkably higher post-thaw motility in MFSS (51.2±7% vs 43.6%±4%,P &amp;lt; 0.001). SCF averaged 17%(raw), 11%(DG) and 2%(MFSS,P&amp;lt;0.001). To confirm research hypothesis, a pilot study was done in 12 men (37.3±3y,3.0±2d abstinence) with their semen specimen equally aliquoted for DG or MFSS and cryopreserved. The post-thaw motility increased from 45.4±8%(DG) to 61.3±13%(MFSS,P&amp;lt;0.001). Subsequently, in men(n = 30, 40.8±6y, 3.0±2d abstinence) who underwent semen cryopreservation previously by DG and subsequent by MFSS, a similar trend was observed with a higher post-thaw motility (53.2±6% vs 43.2%±6%,P &amp;lt; 0.001). Among the 36 patients who utilized DG cryopreserved specimen for reproductive purposes, 11/19 (57.9%) patients achieved pregnancy in ICSI cycles. while 5/17 (29.4%) achieved pregnancy in an IUI cycle, Among the 18 couples who used MFSS cryopreserved specimen for ICSI, a pregnancy rate was reported (8/18,44.4%). A IUI pregnancy rate was also reported (4/18,22.2%) using MFSS cryopreserved specimen. Limitations, reasons for caution While MFSS has proven to increase post-thaw survival rate and maintain a lower SCF, it did not necessarily trend in a higher pregnancy in IUI cycles, possibly due to the borderline elevated SCF. This finding needs to be confirmed in a larger population. Wider implications of the findings In reproductive medicine, we strive to identify new techniques aimed at providing more competent gametes. MFSS was capable of isolating the most motile potion of spermatozoa with a higher genomic integrity. The utilization of MFSS for specimens to be cryopreserved appears promising in generating higher post-thaw recovery of motile spermatozoa. Trial registration number not applicable

P-472 Oocyte cryopreservation for social reasons in women ≥ 40 years who decline oocyte donation: anticipated outcomes, myth dispelling, and addressing concerns

Abstract Study question Is oocyte vitrification a reproductive viable alternative for women aged over 40 who decline oocyte donation? Under what specific conditions? Summary answer Oocyte vitrification could be considered as an acceptable alternative in this patient cohort, with patients being adequately informed about the realistic possibilities. What is known already In Argentina, legislation on Assisted Reproduction Technologies endorses assisted reproductive treatments employing women’s own oocytes up to the age of forty-four. This legislative framework has resulted in an increased number of such procedures for women over 40 who decline oocyte donation, as well as a rise in those women in this age group who wish to postpone maternity for social reasons. There are scarce published studies presenting reproductive outcomes using vitrified and thawed autologous oocytes in this specific demographic. Study design, size, duration This retrospective observational study, conducted between January 2008 and December 2023, involved 299 patients aged 40 and above who underwent 356 cycles of autologous oocyte cryopreservation (1.2 cycles per patient). Statistical analysis utilized the InStat GraphPad statistical package, including one-way ANOVA and two-tailed Student’s t-test as required. Participants/materials, setting, methods Eighty-seven patients thawed their oocytes and performed 95 ICSI procedures followed by 75 embryo transfers. The primary outcome was the live birth rate, with secondary outcomes including oocyte survival rate, fertilization rate, implantation rate, clinical pregnancy rate, and miscarriage rate. Vitrification and thawing of oocytes were performed using the Cryotop method. Main results and the role of chance Average age: 41.4± 1.8 years. Average vitrified oocytes: 4.5 ± 3.9 [1-24]. 29.1%(87/299) patients thawed their eggs. Average age at thawing:44.0± 2.7 years [41-50]. Average storage time until use was 2.6 ± 1.9 years. 465 oocytes were thawed (4.9 ± 3.0 oocytes per patient). 31.0% used a donor semen sample and 69% used their partne’s semen sample. Survival rate was 87.1%(405/465). Fertilization rate was 74.3% (301/405). Cleavage rate was 98.0 % (295/301). 78.9% (75/95) of ICSI procedures had embryo transfer (1.9 ± 0.6 embryos per patient). Implantation rate was 8.9% (13/146). Clinical pregnancy rate per transfer was 17.3% (13/75). Miscarriage rate was 23.1% (3/13). Miscarriage rate per transfer was 4.0% (3/75). Live birth rate per transfer was 13.3% (10/75), 11.5% (10/87) per patient who thawed their oocytes and 10.5% (10/95) per ICSI procedure performed. Live birth rate per cryopreserved oocyte was 2.1% (10/465) in the general population and 9.1% (10/110) in those women who achieved at least clinical pregnancy. Women who achieved a healthy live birth had a greater number of vitrified oocytes (7.1 ± 3.1) than those who did not achieve pregnancy (4.9 ± 3.0) (p = 0.02). Limitations, reasons for caution This retrospective study’s conclusions are pertinent to a well-defined group of patients, specifically women aged over 40 who decline oocyte donation and have undergone oocyte vitrification. Wider implications of the findings Despite the absence of an assurance for a live birth through oocyte vitrification in this cohort, offering clear counsel grounded in variables such as the number of retrieved oocytes and age at the moment of cryopreservation allows for informed communication of realistic possibilities for those patients who decline oocyte donation. Trial registration number na

Investigation of the qualitative and appearance characteristics of Eryngium caeruleum L. based on colorimetric and browning indices in storage conditions

AbstractThe eryngo plant is an herb related to the Apiaceae family with the greatest diversity of species, has a gorgeous taste when eaten as a vegetable, and is traditionally used in folk medicine for its health benefits. The present study was to assess the effects of different drying methods and storage times on the quality and appearance of Eryngium caeruleum. The treatments of this study were drying methods (room temperature (25°C) with proper ventilation, oven temperature 55°C, vacuum oven temperature 55°C, and a microwave with a power of 500 W) and storage times (1, 75, and 150 days). The lowest brightness index and the highest browning index were found in the shade‐dried samples kept in the refrigerator for 150 days. In these samples, the numerical values of chlorophyll were lower than others. After 75 days, in the vacuum oven‐dried samples, the maximum levels of total phenolics and flavonoids and antioxidant activity were calculated. The colorimetric test showed that the oven‐dried samples maintained their green color well. However, the storage decreased the quality of plant samples due to the degradation of chlorophyll. Overall, this study showed that the highest greenness and appearance qualities were found in the samples dried in a vacuum oven at 55°C. It seems that appearance indices can be introduced as an initial and quick step in the qualitative assessment of dried leafy products.

Synthesis of Silver Nanoparticles Using Extracts from Different Parts of the Paullinia cupana Kunth Plant: Characterization and In Vitro Antimicrobial Activity

The green synthesis of silver nanoparticles (AgNPs) can be developed using safe and environmentally friendly routes, can replace potentially toxic chemical methods, and can increase the scale of production. This study aimed to synthesize AgNPs from aqueous extracts of guarana (Paullinia cupana) leaves and flowers, collected in different seasons of the year, as a source of active biomolecules capable of reducing silver ions (Ag+) and promoting the stabilization of colloidal silver (Ag0). The plant aqueous extracts were characterized regarding their metabolic composition by liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS/MS), phenolic compound content, and antioxidant potential against free radicals. The synthesized AgNPs were characterized by UV/Vis spectrophotometry, dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and scanning electron microscopy coupled to energy-dispersive X-ray spectrometry (EDX). The results demonstrated that the chemical characterization indicated the presence of secondary metabolites of many classes of compounds in the studied aqueous extracts studied, but alkaloids and flavonoids were predominant, which are widely recognized for their antioxidant capabilities. It was possible to notice subtle changes in the properties of the nanostructures depending on parameters such as seasonality and the part of the plant used, with the AgNPs showing surface plasmon resonance bands between 410 and 420 nm using the leaf extract and between 440 and 460 nm when prepared using the flower extract. Overall, the average hydrodynamic diameters of the AgNPs were similar among the samples (61.98 to 101.6 nm). Polydispersity index remained in the range of 0.2 to 0.4, indicating that colloidal stability did not change with storage time. Zeta potential was above −30 mV after one month of analysis, which is adequate for biological applications. TEM images showed AgNPs with diameters between 40.72 to 48.85 nm and particles of different morphologies. EDX indicated silver content by weight between 24.06 and 28.81%. The synthesized AgNPs exhibited antimicrobial efficacy against various pathogenic microorganisms of clinical and environmental interest, with MIC values between 2.12 and 21.25 µg/mL, which is close to those described for MBC values. Therefore, our results revealed the potential use of a native species of plant from Brazilian biodiversity combined with nanotechnology to produce antimicrobial agents.

Self-organization of C60 fullerene molecules in xylene/ethanol mixtures

Fullerene molecules have been intensively studied over the past few decades due to their unique chemical and physical properties, which have great potential in a wide range of applications. In this article, the formation and aging of C60 colloids in the xylene/ethanol system is considered. The peculiarities of these processes are studied by the methods of refractometry, optical absorption, Raman spectroscopy and dynamic light scattering (DLS). It has been experimentally established that the formation of colloidal particles in a C60/xylene/ethanol mixture depends on the volume content of the components and the storage time of the solution. The time required to achieve stability of self-organization of solute molecules in a C60/xylene/ethanol mixture is discussed.

EVALUATION OF BACILLUS CEREUS GROWTH IN COOKED RICE

The present study aimed to evaluate the dependence of growth dynamics of toxigenic B. cereus on rice type, inoculum type, storage time, and temperature using appropriate mathematical models. Samples of cooked white (milled) and brown (husked) rice inoculated with vegetative cell/spore suspension of B. cereus were stored for 48 h at 4 °C (correct storage) and 24 °C (inappropriate storage method). No growth of B. cereus was observed during 48 hours of storage at 4 °C and was characterised by a linear model. To assess the growth of B. cereus in the cooked rice stored at 24 °C, the Baranyi-Roberts, Gompertz and Buchanan mathematical models were used. The Gompertz model characterized the growth of B. cereus best. The type of rice and the type of inoculum did not have a significant effect on the bacterial growth. After 48 h of storage at 24°C, the B. cereus counts were in the range of 8-9 log cfu.g-1, critical value of 5 log cfu.g-1 was reached in less than 10 hours of storage. The study results indicate that cooked rice stored at 24 °C quickly becomes a health risk for consumers due to the rapid multiplication of B. cereus within a few hours. In conclusion, it is crucial to cool cooked rice immediately to 4 °C if it is not to be consumed right after cooking.

Association between Storage Time of Corneal Grafts and Primary Graft Failure in Penetrating Keratoplasty

Purpose: To determine association between storage time of corneal grafts and primary graft failure in Penetrating Keratoplasty (PK). Study Design: Quasi experimental study. Place and Duration of Study: Institute of Ophthalmology, Eye Unit-III, King Edward Medical University, Mayo Hospital, Lahore from June, 2019 to December, 2019. Methods: In this study, 120 patients (between 11 and 30 years of age) diagnosed with keratoconus were enrolled and divided into two groups. Group A comprised individuals who underwent PK with donor tissues stored for 8 to 14 days, while Group B consisted of patients whose donor tissues were stored for a duration not exceeding 7 days. Patients were followed up for three weeks post-operatively. The data was analyzed using SPSS version 19 and stratified for age, gender and primary graft failure. Post stratification RR was calculated with p-value ≤ 0.05 as significant. Results: The mean age of patients in Group A was 21.38 ± 6.29 years and in Group B was19.80 ± 5.81 years. In Group A there were 32 male and 28 female while in Group B there were 29 male and 31 female. In Group A 16(26.7%) patients and in group B, 4 (6.7%) patients had primary graft failure. The risk of primary graft failure was higher in Group A, with RR = 4, p-value &lt; 0.05. Conclusion: The risk of primary graft failure in PK is higher when the storage time of corneal grafts is longer. Hence, minimization of storage time may ensure better success rate of corneal grafting.