The Use of Fullerene C60 to Preserve Testicular Tissue after Cryopreservation

Autologous transplantation of cryopreserved fragments of an immature testis is an actively developing approach to save fertility in patients facing a gonadotoxic therapy. The use of bioavailable fullerene C60 as a powerful antioxidant opens up a new potential for the prevention and correction of ischemic-reperfusion pathological processes in tissues including those associated with freezing-thawing procedure. In this work, we aimed to study the antioxidant status, apoptotic/necrotic processes, and morphological characteristics of cryopreserved fragments of the seminiferous tubules of testis (CrFSTT) of immature rats after incubation in media with different concentrations of fullerene C60 (10, 15, and 20 μg/mL). Our results indicated that the addition of C60 in a concentration of 15 μg/mL decreased ROS production, cytochrom C release, and degree of histological damage of spermatogenic epithelium as well as increased the activity of the mitochondria, antioxidant defense system, and cell density in histological sections of CrFSTT compared to the control. Fullerene C60 at investigated concentrations did not impact significantly on apoptosis in cells of CrFSTT but, after incubation with 15 μg/mL C60, a percentage of living cells was 1.2-fold higher and a value of necrotic ones in this group was 1.6-fold lower than the control samples ( p < 0.05 ). Relative amount of cells of the spermatogonia germ layer did not differ between the studied concentrations. The general analysis of obtained data showed that the C60 addition in the concentration of 15 μg/mL was the most optimal for the rehabilitation of CrFSTT. The results can be used for the development of an effective rehabilitation medium for the cryopreserved testicular tissue.

Cryopreserved embryo replacement is associated with higher birthweight compared with fresh embryo: multicentric sibling embryo cohort study

Birth weight (BW) is higher after frozen embryo transfer (FET) than after fresh embryo replacement. No study has compared the BW of siblings conceived using the same oocyte/embryo cohort. The aim of this study was to determine whether the freezing-thawing procedure is involved in such difference. Multicenter study at Montpellier University Hospital, Clinique Ovo, Canada and Grenoble-Alpes University Hospital. The first cohort (Fresh/FET) included in vitro fertilization (IVF) cycles where the older was born after fresh embryo transfer (n = 158) and the younger after transfer of frozen supernumerary embryos (n = 158). The second cohort (FET/FET) included IVF cycles where older and younger were born after FET of embryos from the same cohort. The mean adjusted BW of the FET group was higher than that of the fresh group (3508.9 ± 452.4 g vs 3237.7 ± 463.3 g; p < 0.01). In the FET/FET cohort, the mean adjusted BW was higher for the younger by 93.1 g but this difference is not significant (3430.2 ± 347.6 g vs 3337.1 ± 391.9 g; p = 0.3789). Our results strongly suggest that cryopreservation is directly involved in the BW variation. Comparing BW difference between Fresh/FET cohort and FET/FET one, it suggests that parity is not the only responsible, increasing the role of cryopreservation step in BW variation.

Development and Characterization of Novel Freeze-thawed Polyvinyl Alcohol/ Halloysite Hydrogels. An approach for drug delivery application

The influence of aluminosilicates on the structure and drug release profiles of polyvinyl alcohol (PVA) - halloysite (HNT) hydrogels containing acetylsalicylic acid (ASA) as a model drug was monitored. The hydrogels were synthesized using a three cycle freeze - thawing procedure and were characterized by FTIR, XRD and SEM. The swelling degree and cytotoxicity were also determined. All hydrogels properties were influenced by HNT concentration from the polymer matrix. The release of ASA, from PVA - HNT hydrogels was monitored in the gastrointestinal tract conditions.

35 EFFECTS OF THAWING TEMPERATURE OF FROZEN SEMEN ON VIABILITY OF REFROZEN AND THAWED CHICKSO (KOREAN BRINDLE CATTLE) AND KOREAN ALBINO CATTLE SPERMATOZOA

Germplasm cryopreservation from a desired species with agricultural and genetic importance would protect them from the risk for extinction. Semen freezing from Korean native cattle would be a good approach for protecting genetic resources due to their limited numbers. It has been known that sperm could resist cryo-damages by freeze-thaw cycles. Thus, we performed 2 refreezing experiments with different initial thawing temperatures using frozen Korean native cattle semen. A total of 5 Hanwoo, Korean Albino, and brindle cattle were used as semen donors. After thawing by using 5°C/2 min or 37°C/40 s with cooling rates, the semen was diluted with the same volume of cryo-media in the first thawing temperature and refrozen. Sperm motilities were determined and compared between animals and groups after rethawing. The mean sperm concentration and motility was 45 × 106 mL–1 (range 2.3 to 89 × 106 mL–1) and 40% (range 13 to 55%). Mean values of motility and viability of sperm that underwent second preservation were significantly higher in 5°C than in 37°C (P < 0.01). However, the activity of viable sperm thawed at 5°C was significantly decreased before refreezing. It is estimated that refreezing of frozen semen from rare Korean native cattle is possible with resistant properties of survived spermatozoa. The higher motility and viability of refrozen semen could be obtained with 5°C thawing procedure for reuse of frozen semen.

Morphological and Spectroscopic Studies of Chitin Nanowhiskers

ABSTRACTChitin nanowhiskers were obtained with the purpose to be used as astaxanthin protectors against the photo and thermal degradation. These nanostructures were generated by a freezing/thawing procedure using two stirring methods: mechanical and sonication, which were named as FTM and FTS respectively. Morphological and spectroscopic studies were carried out on chitin nanowhiskers by scanning electronic microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). Through a SEM analysis, chitin fibers were found uniformly spaced and oriented with the width ranged from of 20-40 nm. Furthermore, the nanowhiskers obtained by FTM showed long and flattened structures and bundles of homogeneous sizes, which have the capacity of being sites of stress concentration. In contrast, by FTS, the nanowhiskers showed coarse fibers exhibiting numerous peaks. By comparing the two methods is appreciated that FTS method provides more surface area, giving more sites for functionalization. Fourier transform infrared spectroscopy (FTIR) allowed the determination of free movement of functional groups on the surface of samples obtained by FTM and FTS methods. Significant differences of signals in the spectra indicate that there were more unassociated amides in the nanowhiskers obtained by FTS than by FTM.

151 COMPARATIVE ANALYSIS OF FRESH AND CRYOPRESERVED BOAR SPERMATOZOA USING RNA SEQUENCING

Fertility of cryopreserved spermatozoa is significantly reduced compared with that of their fresh counterparts, which is certainly due to the inflicted sublethal damage to spermatozoa that is observed at various molecular and cellular levels. The identification and characterisation of this damage will help us better understand sperm cryobiology and therefore develop suitable media and procedures to improve sperm cryopreservation and fertility outcomes, especially in swine. Here, we present our preliminary assessment of RNA pools of fresh and frozen‐thawed spermatozoa using RNA-sequencing technology. Semen ejaculates of 8 fertile boars were harvested and divided into 2 fractions for each ejaculate. Fraction 1 was freshly extended in commercial diluent (FD) and fraction 2 was frozen in 5-mL plastic straws (FT). Both specimens were shipped to our laboratory for analyses. The samples were purified through Percoll gradient centrifugation and resulting motile spermatozoa were washed in cold PBS. Pelleted spermatozoa were used for total RNA extraction, followed by an in-column DNase digestion. Purity and integrity of RNA samples were checked and rRNA depleted. After random priming, 40 million short cDNA reads were produced using Illumina RNA-Seq technology (Illumina Inc., San Diego, CA, USA). All reads were aligned to the pig reference genome and the produced genome-scale transcription maps consisted of both the transcript structure and the expression level of each gene mapped. Analysis of FD sperm RNA revealed a total of 18 357 sequence tags that were successfully mapped to all pig chromosomes and the mitochondrial genome. Frozen‐thawed spermatozoa showed only 16 864 sequence tags. In both FD and FT samples, chromosomes 1, 2, 6, 7, and 13 contained, in total, the highest density of mapped transcripts (>42%). Chromosome Y and mitochondrial RNAs had the lowest sequence tags mapped (<0.08%). A comparative analysis of FD and FT datasets revealed a net decrease in the total number of sequence tags (1493) with each chromosome being affected, except mitochondria. Chromosomes of FT samples showed a strong (>10%; 17, 7, 4, Y, and X) to moderate (10 to 5%) or weak (≤5%) reduction in RNA numbers. Structural annotation revealed a diverse population of sperm transcripts comprising both coding (mRNA) and noncoding (rRNA, snRNA, and mtRNA) RNAs. In both FD and FT samples, noncoding RNAs were among the most abundant sequence tags. Approximately 12 355 of sequence tags in FD v. 10 948 in FT spermatozoa were annotated with ENSEMBL and the selected genes are under investigation for comparative analyses using RT-PCR. In conclusion, mature boar spermatozoa contain a large pool of coding and non-coding RNAs that can be affected by the freezing-thawing procedure. Inflicted damage affects RNAs of all chromosomes with a great effect being seen on chromosome X. Generated datasets have the potential to lead to further study of the cryo-damage associated with reduced fertility of cryopreserved spermatozoa. Study was supported by USDA-ARS Biophotonics initiative grant # 58-6402-3-0120 and MAFES-SRI grants.