LN2 Vapor Research Articles

Novel lyophilized extenders maintain stallion semen characteristics during freezing but do not need frozen shipment or storage

Stallion semen cryopreservation is an important procedure for conservation of genetic resources having a relevant roll in the horse industry. Distribution of commercial extenders is difficult due to the necessity of keeping them frozen during transport. The aim of this work was to compare after-thawing results of two commercial frozen extenders offered worldwide (C1 and C2) with two novel lyophilized freezing extenders produced in Brazil (STAR and MX3) which can be stored at room temperature up to one year. One ejaculate of each of 28 mature stallions from three breeds (Quarter Horse, Criollo and Mangalarga Marchador) was diluted with milk-based extender (Equiplus, Minitube) to 100 × 106 total sperm/ml and split into four 50ml centrifugation tubes. After centrifugation (600 g x 20’), the remaining pellet of each tube was resuspended with one of the four extenders to a concentration of 200 × 106 total sperm/ml. From each aliquot, semen was frozen with two different freezing curves named FC1 (Room temperature + 20’ at 5˚C into refrigerator + 20’ followed by LN2 vapor and plunging) and FC2 (60’ at 5˚C). After freezing, straws were thawed at 37˚C/30 sec and total motility (TM) was checked using optical microscopy in a blind test by three skilled vets. Means± standard deviation for total motility after freezing-thawing were: FC1 (C1=46.54±19.44;C2=35.30±21.32; STAR=51.15±21.18 and MX3=46.17±20.50) and FC2 (C1=51.55±21.92; C2=41.61±19.79; STAR=44.85±16.30 and MX3=49.21±15.98). Statistical analysis was performed (ANOVA followed by Tukey test) with 5% significance. No differences on average motility among extenders within or between curves was seen. Within each curve, the distribution of best motilities of each extender considering individual stallions was FC1 (C1=6, C2=2, STAR=14, MX3=6) and FC2 (C1=14, C2=4, STAR=4, MX3=6). The results show similarity on average motility for all extenders tested. Considering FC1, the lyophilized extenders got better motility in 20 of 28 stallions. In FC2, C1 performed better (14 of 28 stallions), while STAR and MX3 were comparable with the commercial extender C2. MX3 had similar performance in both curves and STAR reached better thawed motility in FC1 (14 of 28 stallions) when compared with FC2 (4 of 28 stallions). The highest motility obtained after the freezing-test revealed a balance between FC1 and FC2 with 13 and 15 stallions, respectively. Individual variation and freezing curve splitting were similar for both kinds of extenders, but further investigations are needed to clarify the better results of STAR when using faster curves. In conclusion, lyophilized extenders can be a good option to obtain similar semen quality without restrictions of frozen extender shipment and storage.

Effect of Synthetic Cholesterol (Synthechol®) Supplementation in an Egg Yolk-free Extender on Dog Sperm Cryopreservation

BACKGROUND: SyntheChol® is a new synthetic, non-animal-derived cholesterol that is easily dissolved in ethanol, ready to use, and behaves in a similar way as natural cholesterol. Therefore, it could be used as a substitute of natural cholesterol in dog sperm freezing extender. OBJECTIVE: To evaluate the effect of supplementing an egg yolk-free (EY-free) extender with synthetic cholesterol (SyntheChol®) on cryopreserved dog sperm. MATERIALS AND METHODS: Spermatozoa (1 × 108 sperm/mL) were suspended in EY-free extender supplemented with 0% (control), 0.25, 0.5, 1, 2, 4, or 6% SyntheChol® (Extender 1), cooled at 4 °C for 1 h, and diluted (1:1, v/v) with Extender 1 containing 1 M glycerol. The spermatozoa were then cooled to 4 °C for 30 min. Sperm-containing straws were frozen using LN2 vapor. Sperm motility (computer-assisted sperm analysis, CASA), sperm membrane integrity (SYBR-14 and PI staining), and acrosome integrity (FITC-PSA) were evaluated after thawing. Thereafter, optimal concentrations were determined (0.25, 0.5, 1, or 2%) and used to evaluate reactive oxygen species (ROS) generation, apoptosis, and the gene expression of motility-related sperm mitochondria-associated cysteine-rich protein, apoptosis-related B-cell lymphoma 2 (BCL2), and BCL2-associated X protein ( BAX) in cryopreserved sperm. RESULTS: Sperm progressive motility, membrane integrity, and acrosome integrity were markedly greater in the SyntheChol®-supplemented groups (0.25, 0.5, 1, or 2%) than in the control group. Only BAX expression was significantly reduced in the SyntheChol® groups (0.25, 1, or 2%) compared with the control group. However, there were no significant effects on the ROS generation or apoptosis index. CONCLUSION: SyntheChol® (0.25, 1, or 2%) proved to be effective in reducing the BAX gene expression level and improving sperm progressive motility, and membrane and acrosome integrity.

Viability of Cryogenic Cooling to Reduce Processor Power Consumption

Abstract Through the application of cryogenic cooling via liquid nitrogen (LN2), the power consumption of a CPU was substantially reduced. Using a digitally controlled solenoid valve and an additively manufactured cold plate, the manual process of LN2 cooling was automated for precise control of cold plate temperature. The power consumption and frequency relationship of the processor were established across three different thermal solutions to demonstrate the effect of temperature on this relationship. It was found that power consumption of the processor decreased at lower temperatures due to a reduction in current leakage and the core voltage necessary for stable operation. This culminated in a reduction of up to 10.7% in processor power consumption for the automated solution and 21.5% for the manual LN2 solution when compared to the air-cooled baseline. Due to the binary nature of the solenoid valve used, flow rate was tuned via an in-line needle valve to increase thermal stability. It was found that for lower flow rates, approximately 5.0 g/s, temperatures oscillated within a range of ±11.5 °C while for higher flow rates of 10–12 g/s, generated amplitudes are as small as ±3.5 °C. Additionally, several tests measured the rate of LN2 consumption and found that the automated solution used 230%–280% more coolant than the manual thermal solution, implying there is room for improvement in the cold plate geometry, LN2 vapor exhaust design, and coolant delivery optimization.

P–153 Comparison outcome of vitrified human embryos stored in vapor phase liquid nitrogen (LN2) and direct LN2

Abstract Study question Is vapor cryopreserved LN2 storage beneficial for clinical outcomes of vitrified human embryos that are frozen compared to vitrified human embryos having direct contact with LN2. Summary answer There are no significant differences compared to clinical outcomes of human embryos stored in LN2 vapor and direct store in LN2. What is known already There has been concerned about potential cross-contamination and biohazard issues of embryos for long term storage using direct LN2. This study aimed to compare clinical outcomes of human embryos transfer between vapor phase and liquid LN2. Study design, size, duration The embryo has undergone vitrification for long term storage with vapor or direct contact in LN2. After the thawing of the embryo, we checked on the survival rates. We transferred only one or two embryos per patient and kept analyzing the implantation and pregnancy rates Participants/materials, setting, methods This retrospective study was carried out from January 2018 to December 2019 with 3272cycles 4713embryos; vitrified for long term storage in vapor phase or direct contact with LN2. We compared the clinical outcomes of frozen embryo transfer cycles using vitrified for long term storage in vapor phase and direct contact with LN2. Clinical outcomes monitored were embryo survival, subsequent implantation and pregnancy after single or double embryo transfer Main results and the role of chance A total of 4713 fertilized human embryos are vitrified and then stored in LN2 vapor (n = 2520 cycles) or direct contact LN2 (n = 752 cycles). The study showed that the blastocyst stored in vapor able to retain full development. Survival was 97.8% (vapor) and 97.6% (direct contact LN2), and the vapor storage of human embryos had no significant difference in survival rates after a long term storage. For single blastocyst transfer, pregnancy and implantation rates were 51.5%, 52.4% in vapor, 54.6%, 54.9% in direct LN2; respectively (p=NS). In double blastocyst transfer, the pregnancy and implantation rates were 61.8%, 42.0% in vapor and 64.7%, 44.5% in direct LN2; respectively (p=NS). There were also no significant differences between two groups. Limitations, reasons for caution The study showed that the blastocyst stored in vapor can retain full development. A vapor storage system thus is safe and effective for long term vapor storage of vitrified human embryos.Within the limits of this study, there was no detection of an adverse effect of vapor storage. Wider implications of the findings: Vapor storage systems thus represent a useful alternative for safe and effective long-term storage of vitrified human embryos that can avoid cross contamination chances from having direct contact with LN2. Trial registration number Not applicable

Impact of Exposure Period to Liquid Nitrogen Vapor on Criteria of Human Spermatozoa Cryopreserved in New Cryopreservation Technique

Background: The emptied sheep’s ovarian follicles recently used as a container for spermatozoa during cryopreservation, it was found a proper carrier to cryopreserving spermatozoa in vapor-dependent cryopreservation. The aim of this study was to evaluate the effect of two periods of exposure to liquid nitrogen (LN2)vapor on the parameter of spermatozoa during cryopreservation in this technique. Method: The study was conducted on 30 semen samples from patients with oligozoospermia diagnosed by semen analysis according to the standard criteria of World Health Orgnization (WHO) 2010. Sheep’s ovarian follicles obtained from local slaughterhouse and prepared by slicing the ovaries and evacuating the follicular fluid and oocyte. Each semen sample diluted 1:1 with cryosolution (glycerol 10%) and injected within eight emptied sheep’s ovarian follicles. The first four follicles represent P1; exposed to LN2 vapor for 7.5 minutes and the other four follicles represent P2; exposed to LN2 vapor for 15 minutes before emerged in liquid nitrogen. Sperm progressive motility, total motility, normal morphology and DNA fragmentation index (DFI) were analyzed for all samples pre-freezing and post-thawing. Results: After two months of cryopreservation, sperm progressive motility and total motility significantly (P<0.01) increased post-thawing in P2 as compared with P1, while both of P1 and P2 significantly (P<0.01) decreased as compared with pre-freezing. Normal morphology significantly (P<0.01) decreased post thawing in both P1 and P2 as compared with pre-freezing, while no significantly difference foundbetween P1and P2. DFI significantly (P<0.01) increased post-thawing in P1 and P2 as compared with pre-freezing, while in P2 DFI was significantly lower than in P1. Conclusions: The exposure to liquid nitrogen vapor for 15 minutes in emptied ovarian follicles technique gives a better results than exposure to the vapor for 7.5 minutes regarding sperm progressive motility, total motility and DFI.

Use of Hypertonic Media to Cryopreserve Sauger Spermatozoa

AbstractThe objective of this study was to determine the effects of extender osmolality on postthaw sperm quality and fertility in Sauger Sander canadensis. Fresh milt from 10 male Saugers was diluted by using base extenders with osmolalities of 350, 500, or 750 mOsm/kg (E350, E500, and E750, respectively) containing 10% dimethyl sulfoxide, frozen in LN2 vapor, and stored. Sperm parameters (total motility, progressive motility, velocity, and viability) were assessed at different steps of the cryopreservation process (extended, equilibrated, and postthaw). Fertilization rates were compared between fresh and frozen sperm and at two sperm‐to‐egg ratios. All of the parameters that were measured, except for progressive motility, were reduced by cryopreservation. Extender 500 yielded the highest postthaw progressive motility (32.20 ± 3.86% [mean ± SD]) and velocity (84.97 ± 16.82 μm/s), whereas both E350 and E500 displayed the highest total motility (65.30 ± 4.24 and 68.70 ± 6.46%) and viability (80.60 ± 4.84 and 78.80 ± 3.91%), respectively. By contrast, E750 yielded the lowest postthaw velocity, viability, and total and progressive motility. Despite the increase in the motility parameters, fertilization in E350 (13.93%) was approximately double that in E500 (6.58%), although not statistically different. In conclusion, traditional isosmotic base extenders (E350) were found to be superior to hypertonic base extenders in the preservation of Sauger milt. These results serve as a starting point for future investigations of cryopreservation potential for Sauger spermatozoa that work toward developing a freezing protocol that is more suitable for large‐scale application.

Action of Cholesterol-Loaded Cyclodextrin on Viability of Ram Semen

Background: Studies report that cyclodextrins have the property of carrying cholesterol to the membrane, but in some cases can also remove this cholesterol from the plasma membrane. The mechanism of action of CLC is not well understood, however, it seems to involve sperm protection during the freezing and thawing process. Studies show that its use enhancing increased osmotic tolerance and reduced premature sperm capacitation reaction. In this sense, studies report that cyclodextrins have the property of carrying cholesterol to the membrane, but in some cases can also remove this cholesterol from the plasma membrane. Improvements were reported in the sperm parameters of buffaloes, bulls, stallions and sheep. Ram naturally present less lipids in their membrane, on average 27%, while bulls have 31%, rabbits 62%, and humans 50%. The aimed of the present study was to evaluate the use of cholesterol-loaded cyclodextrin (CLC), a commercial diluent, in the kinetics and viability of frozen and thawed ram spermatozoa. Materials, Methods &amp; Results: Five ejaculates, from five rams of Dorper breed were collected and divided into three groups: control, 1 mg CLC and 2 mg CLC. Semen was diluted in different concentrations of CLC (0, 1, and 2 mg/120×106 spermatozoa), and incubated at room temperature (21°C) for 10 min. Samples were conditioned in 0.5 mL straws and incubated at 5°C for 4 h, exposed to LN2 vapor for 10 min and storing a cryogenic container. The parameters as spermatic kinetics, plasma membrane, acrosomal membrane (MPAI, %), and intracellular levels of superoxide anion (O2-) were evaluated. Sperm progressive motility (PM), rapid spermatozoa percentage (RAP), linearity (LIN, %), average path velocity (VAP, μm/s) and MPAI (%) were more satisfactory with the use of 1 mg compared to 2 mg (P &lt; 0.05). In addition, 1 mg CLC showed decreased levels of superoxide anion formation (O2-), a free radical detrimental to spermatozoa (P &lt; 0.05). The use of 2 mg of CLC reduce VAP (P &lt; 0.05) and did not have any beneficial effect on the evaluated parameters. Discussion: Authors did not observe improvement in the parameters of progressive motility when using 1 mg of CLC in goat semen and 2 mg in bull semen with the slow freezing protocol. This differs from our work, as we found that 1 mg of CLC improved the PM parameters, but not at the concentration of 2 mg CLC. Additionally, authors verified that cyclodextrin at 3 mg concentration was effective in protecting the sperm against the deleterious effects of H2O2. They obtained superior plasma membrane motility, viability, and integrity of the CLC-treated samples compared to the control group. The superoxide anion (O2-) is a free radical formed from molecular oxygen by the addition of an electron. It is generated spontaneously, mainly in the membrane of the mitochondria, by the respiratory chain and by flavoenzimes, lipoxygenases, and cicloxygenases. In our study, we found a difference between the study group with 1 mg CLC and the control group. Thus, we suggest that CLC may have a beneficial effect in stabilizing the sperm plasma membrane. Use of CLC at a concentration of 1 mg was found to be effective for the improvement of parameters of sperm progressive motility, rapid sperm percentage, and plasma and acrosomal membrane integrity. In addition, the study group with 1 mg of CLC showed decreased levels of superoxide anion formation, a free radical detrimental to spermatozoa.

101 Assessment of semen traits in servals (Leptailurus serval) and Canada lynx (Lynx canadensis)

Servals and Canada lynx are managed by species survival plans in North American zoos, but current populations are not sustainable. Increased knowledge of their reproductive biology would benefit breeding management and development of assisted reproductive techniques. The aims of our study were to (1) evaluate effectiveness of urethral catheterization and electroejaculation (EEJ) for semen collection; (2) characterise basal seminal traits; and (3) compare effectiveness of semen cryopreservation methods. Semen was collected from 6 servals and 9 Canada lynx via a urinary catheter (3.5 Fr×22 cm, inserted 15cm into the urethra), followed by EEJ under dexmedetomidine-ketamine anaesthesia. To assess the effect of seasonality on lynx seminal traits, semen was collected before (late January), during (mid-February to mid-March), and after (early April) the peak breeding season. Serval and lynx semen were frozen by conventional slow freezing (i.e. in 0.25-mL straws cooled to 4°C for 2h and frozen in LN vapor) in a soy lecithin-based (SOY) or egg yolk-based (TEY) extender with 4% glycerol and by ultra-rapid freezing (URF; direct pelleting into LN at ≈104°C/min) in SOY medium with 0.2M sucrose. To evaluate post-thaw sperm function in servals, heterologous IVF of domestic cat oocytes was performed, with cleavage rate assessed at 48h post-insemination. Data were analysed by one-way or repeated-measures ANOVA. Data are mean±standard deviation. Sperm recovery by urethral catheterization was negligible in both species, but EEJ allowed sperm collection in all males. Lynx seminal traits were similar during breeding and nonbreeding seasons. Testicular volume (4.81±1.17cm3) and sperm quality (13±11×106 sperm/ejaculate; 49±14% motility; 29±12% normal morphology; 74±13% acrosome integrity) were consistent with previous findings in the lynx genus. Post-thaw sperm quality in lynx has not yet been evaluated. In servals, testes volume was 6.56±2.11cm3 with good sperm quality for most males (46±36×106 sperm/ejaculate; 75±20% motility; 56±36% normal morphology; 84±7% acrosome integrity). Post-thaw, serval sperm acrosome integrity (31±15, 21±13, 24±13% at 0h for TEY, SOY, and URF, respectively; P&amp;gt;0.05) and motility (40±21% at 0h, 20±11% at 6h for TEY; 24±19% at 0h, 6±4% at 6h for SOY; 21±16% at 0h, 3±2% at 6h for URF; treatment: P&amp;gt;0.05; time: P&amp;lt;0.05; interaction: P&amp;gt;0.05) declined substantially. However, thawed sperm could fertilize domestic cat oocytes with no difference among treatments in cleavage success (53±6, 47±4, or 49±14%; TEY, SOY, and URF, respectively; P&amp;gt;0.05), indicating that standard freezing methods are effective in servals. Our findings provide zoos with valuable information about normative reproductive traits in both species. Supported by IMLS and the Roger &amp;amp; Kathy Gross Post-doctoral Fellowship.

Optimizing sperm cryopreservation and recovery (OSCAR)

The objective of this study is to compare three common sperm freezing methods and two common thawing methods to one another to determine if there is a superior method to enhance post-thaw sperm survival. Prospective cohort study. Twelve discarded fresh semen samples were obtained with patient consent. Each fresh sample was washed with mHTF (LifeGlobal, Guilford, CT) and centrifuged for 15 minutes at 400G. The pellet was resuspended in a 3:1 ratio of mHTF to Artic™ Sperm Cryopreservation Medium (Irvine Scientific, Santa Ana, CA). Motility was evaluated manually and each participant’s sample was divided into six aliquots containing 0.5mL of specimen. Two aliquots of each specimen underwent one of 3 different freezing methods: (a) plunge into liquid nitrogen (LN2), (b) suspension in LN2 vapor for 15 minutes followed by plunge into LN2 or (c) suspension in LN2 vapor for 1 hour followed by plunge into liquid nitrogen. Each of the freezing methods was subject to two different thaw methods: (a) 37°C dry bath for 20 minutes followed by 40 minutes at room temperature or (b) room temperature (RT) for 1 hour. Following recovery, a second evaluation of motility was performed for each aliquot as a measure of sperm viability (percent motility versus initial). Analysis by two way repeated measures ANOVA was utilized with a p-value of 0.05 to determine significance. The mean motility for plunge sperm thawed at RT and 37°C were 22.9 ± 0.04% and 20.3 ± 0.04%, respectively. The mean motility for 15 minutes vapor then plunge thawed at RT and 37°C were 31.7 ± 0.06% and 31.5 ± 0.07%, respectively. For 1 hour in vapor then plunge, the mean motility at RT and 37°C were 43.8 ± 0.07% and 48.1 ± 0.06%, respectively. Survival rates between all freezing methods varied significantly, with greater recovery noted in the 1 hour vapor phase when compared both plunge (p<0.001) and 15 minutes in vapor (p=0.005). The shorter vapor phase of 15 minutes also demonstrated statistically greater viability compared to plunge (p = 0.027). Thawing methods did not significantly affect sperm recovery. The recovery of motile sperm varied directly with the length of cooling prior to plunge in LN2 with an hour suspension in vapor resulting in greatest motility compared to the initial sample. Recovery was not dependent on the thaw method used, with either of the two methods producing similar results for a given cryopreservation method. Interestingly, it was also noted that samples plunged directly from room temperature into LN2 retained appreciable, if diminished viability

59 EFFECT OF PELLET VOLUME AND THAWING TEMPERATURE ON VITRIFICATION EFFICACY WITH DOMESTIC CAT SEMEN COLLECTED VIA URETHRAL CATHETERIZATION

Historically, semen banking in felids has required sample collection via electroejaculation followed by sperm freezing in straws over LN2 vapor. Recent modifications include urethral catheterization of males treated with α-2 agonists for semen recovery and vitrification of cat sperm by suspension in a sucrose-based cryomedium and direct pelleting into LN2. In combination, these latter methods greatly simplify semen cryopreservation in cats but protocols need to be optimized for applied usage. In the present study, our goal was to assess the effect of 2 variables—pellet volume and thawing temperature—on post-thaw sperm motility, acrosome status, and in vitro fertility. Semen was collected from 3 males (3 ejaculates/male) via urethral catheterization under dexmedetomidine-ketamine anaesthesia. Sperm were diluted in Feline Optimized Culture Medium (FOCM), centrifuged (8 min; 300 × g), and resuspended in a soy-lecithin-based vitrification medium (with 0.2 M sucrose). After a 5-min equilibration, sperm was vitrified in 2 volumes (20 or 30 µL) by direct pipetting into LN2. Sperm pellets were thawed in FOCM at 1 of 2 temperatures (37 or 55°C) and the 4 treatment groups (20 µL-37°C, 20–55, 30–37, 30–55) assessed for percentage of progressively motile and acrosome intact sperm. To assess sperm function, additional 30-µL pellets were thawed at 37 or 55°C, and recovered sperm were used to inseminate in vitro-matured domestic cat oocytes (n = 10–25/ejaculate). At 48 h post-insemination, oocytes and embryos were fixed (1% NBF). Hoechst fluorescent stain (#33342) was used to evaluate embryo cleavage and maturation status of unfertilized ova. Sperm motility and acrosomal integrity percentages were analysed by ANOVA, and oocyte cleavage proportions were analysed by chi-squared. Mean (± SEM) progressive sperm motility post-thaw did not differ (P &gt; 0.05) among treatments (38 ± 8, 34 ± 7, 41 ± 7, 32 ± 7% for 20 µL-37°C; 20–55, 30–37, and 30–55, respectively). Similarly, acrosomal integrity did not differ (P &gt; 0.05) among treatments (26 ± 4, 25 ± 4, 17 ± 3, 17 ± 2% for 20 µL-37°C, 20–55, 30–37, and 30–55, respectively). Oocyte cleavage proportions did not differ (P &gt; 0.05) between thawing temperatures for total inseminated oocytes but, after correcting for oocyte maturation status, was higher (P &lt; 0.01) for samples thawed at 55°C (60%, 67/112) compared with 37°C (39%, 52/133). In summary, although variations in pellet volume and thawing temperature had minimal effect on sperm motility or acrosome status immediately post-thaw, sperm function appeared to be enhanced when vitrified pellets were thawed at a higher temperature. In vitro fertility success (~60% embryo cleavage) is comparable to values reported by our laboratory with conventionally collected and frozen cat semen, suggesting these newer methods may be suitable for applied usage in felids. This study was funded by the Institute of Museums and Library Services.

Abstract 1377: Impact of biospecimen pre-analytical factors on molecular analysis

Abstract The Biospecimen Pre-analytical Variables (BPV) Program of the Biorepositories and Biospecimen Research Branch (BBRB) at the National Cancer Institute (NCI) is designed to systematically investigate the effects of preanalytical factors on the molecular integrity of biospecimens. Specific parameters related to formalin fixation and paraffin embedding (FFPE) of cancer tissues were examined, including the effects of cold ischemic time (delay to fixation (DTF)) and time in fixative (TIF). Additional preanalytical studies focus on snap freezing method for tissue specimens (dry Ice vs. LN2 vapor), storage temperature (-80°C vs. LN2 vapor), and duration of storage for frozen specimens. Biospecimens for the BPV program were collected at four medical centers, within a tightly regulated infrastructure and strict adherence to SOPs, to enable consistent collection and handling across all sites. Biospecimens were collected from research participants undergoing surgical treatment for renal cell carcinoma; ovarian, fallopian tube, and peritoneal carcinoma; lung adenocarcinoma and squamous cell carcinoma; and colorectal adenocarcinoma. Biospecimens were subjected to specific experimental protocols to systematically vary the preanalytical factors of interest. Extensive annotation was performed with 300+ data elements that include steps in the collection, handling, and processing of the biospecimens, pathological evaluation of the tumor, and clinical information collected from donors. Biospecimen collections concluded in April 2015 with a total of 364 tumor tissue cases collected. The BPV program has conducted multiple, simultaneous molecular analyses to understand the impact of FFPE preanalytical factors on the expression and detection of various molecular analytes. Initial efforts were focused on evaluating the impact of DTF and TIF on the quality of DNA and RNA from FFPE samples using multiple methods and approaches such as NanoDrop 8000 UV-Vis spectrophotometer, Qubit 2.0 Fluorometer, Agilent Bioanalyzer and KAPA Human Genomic DNA Quantification. The QC data from both RIN and Kappa assays showed that FFPE samples have a significant drop in RNA and DNA quality compared with matched frozen samples. 72 hr TIF samples showed a significant drop in both RNA and DNA quality compared to shorter time points (6, 12, or 23 hr TIF), as measured by DV200 and Kappa assays. No significant differences were observed in RNA/DNA quality between the shorter TIF time points or between the DTF time points (1, 2, 3, 12 hr DTF). Further studies are now underway to evaluate additional preanalytical factors. The data from BPV studies will be widely shared with the research community through publication and deposition at a public data repository. The results from these studies will be used to develop evidence-based protocols and best practices for fit-for-purpose collection, processing, and storage of biospecimens. This project is funded by NCI Contract No. HHSN261200800001E. Citation Format: Rachana Agarwal, Ping Guan, Mary Barcus, Jasmin Bavarva, Robin Burges, Philip Branton, Latarsha Carithers, Corinne Camalier, Biswajit Das, Jason Lih, Hana Odeh, Nancy Roche, Dan Rohrer, Michael Sachs, Leslie Sobin, Jewell Scott, Anna Smith, Conrado Soria, Kimberly Valentino, Dana Valley, Mickey Williams, Helen Moore. Impact of biospecimen pre-analytical factors on molecular analysis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1377.

Validation-verification of a highly effective, practical human testicular tissue in vitro culture-cryopreservation procedure aimed to optimize pre-freeze and post-thaw motility.

Purpose The aim of our paper was to validate a testicular biopsy procedure that simplifies handling, processing, and cryopreservation, while at the same time optimizes sperm motility before freezing and after thawing.MethodsTwo prospective studies were conducted to verify, optimize, and understand the virtues of pre-freeze testicular tissue IVC at different temperatures (21, 30, or 37 °C). Testicular tissue was obtained from clinical specimens designated for whole tissue cryopreservation (i.e., intact mass of tubules) and/or for fresh use in IVF-ICSI cycles. Whole testicular biopsy pieces (1–3 mm3) were diluted in glycerol containing freeze solutions, slow cooled to 4 °C and then rapidly frozen in LN2 vapor. Fresh and post-thaw testicular biopsy tissue were evaluated for changes in the quantity (%) and pattern of motility (I–IV: twitching to rapid progression, respectively) over a 1 week duration. The clinical effectiveness of IVC-cryopreserved whole testicular biopsy tissue was also validated analyzing fresh embryo transfers.ResultsMore reliable recovery of motile testicular sperm was achieved using whole tissue freeze preservation combined with IVC (24–96 h) post-acquisition at an incubation temperature of 30 °C compared to ambient temperature (21 °C) or 37 °C. Up to 85 % of the pre-freeze motility was conserved post-thaw (+3 h) for easy ICSI selection. Sperm longevity was optimized to fresh tissue levels by implementing testicular biopsy sucrose dilution post-thaw. Favorable clinical outcomes were proven using frozen-thawed testicular biopsy sperm for ICSI.ConclusionsBy employing minimal tissue manipulation, integrating pre-freeze IVC processing at 30 °C and the freezing of whole testicular biopsy tissue, we have reduced the labor and improved the efficacy of processing testicular tissue for freeze-preservation and subsequent ICSI use.

49 EFFECT OF ADDING Trolox C AND ASCORBIC ACID TO RAM SPERM BEFORE CRYOPRESERVATION ON THE MOTILITY AND BINDING CAPABILITY

Different antioxidants have been tested to improve sperm quality, but distinct and consistent beneficial effects are lacking. The objective of this study was to evaluate whether the addition of different concentrations of the antioxidant Trolox C and ascorbic acid before cryopreservation could improve the binding of sperm to chicken egg perivitelline membrane (PM) after cryopreservation. Three ejaculates of ram were split and diluted with Tris egg yolk diluent to a final concentration of 200 × 106 cells mL–1, and after, the ejaculates were divided into 5 tubes. Each tube received one of the following antioxidants: control, no antioxidant; 200 μM of Trolox C; 300 μM of Trolox C; 0.05% of ascorbic acid; and 0.25% of ascorbic acid. The samples were cooled to 5°C/2 h, packaged into 0.5-mL straws, and frozen in static LN vapor for 15 min before being plunged into LN. Straws were thawed (37°C/30 s). The motility was determined using computer-assisted semen analysis. For PM binding test, PM was put in tubes with 1 mL of TALP and inseminated with 50 000 sperm. The PM and sperm were incubated for 90 min at 37°C in an atmosphere of 5% CO2 in air, and 20 min before the end of the incubation time, 10 μL of Hoechst 33342 was added in each treatment. After each PM was washed 5 times in TALP, placed under the coverslip on a slide, and evaluated by fluorescence microscopy at 400×. Spermatozoa were counted in 6 random fields of each piece of PM. Percentage data were transformed using arcsine prior analysis. Treatment differences were determine by analysis of variance and Tukey test. The total and progressive motility of sperm treated with 0.25% ascorbic acid Trolox C was higher (64.5 and 45%) than 100 μM of Trolox C (61.9 and 42.6%) and 200 μM of Trolox C (64.3 and 46.7%) and control (59.8 and 39.6%; P &lt; 0.05), respectively. The binding test was higher when using 0.25% of ascorbic acid (155.73 cells; P &lt; 0.05) compared with other treatments. Addition of 0.25% ascorbic acid to ram sperm before cryopreservation improved cell cryosurvival rates.

Liquid nitrogen vapor is comparable to liquid nitrogen for storage of cryopreserved human sperm: evidence from the characteristics of post-thaw human sperm

To compare the differences in the characteristics of post-thaw human sperm after storage in either liquid nitrogen (LN2; -196 °C) or LN2 vapor (-167 °C). Experimental study. University hospital. Thirty healthy volunteers who agreed to donate their normal semen samples for infertility or research were included in the study. Semen samples (n = 30) were divided into eight aliquots and frozen. Four aliquots of each human semen sample were stored in LN2 (-196 °C), and the other four aliquots were stored in LN2 vapor (-167 °C). After 1, 3, 6, or 12 months, samples were thawed and analyzed. The motility was evaluated by the manual counting method. The viability was estimated by eosin staining. The morphology was analyzed by Diff-Quik staining. The sperm DNA integrity was determined with acridine orange fluorescent staining, and acrosin activity was assayed by the modified Kennedy method. The characteristics of post-thaw human sperm, including motility, viability, morphology, DNA integrity, and acrosin activity, showed no significant difference between LN2 and LN2 vapor storage for the different time periods. LN2 vapor was comparable to LN2 in post-thaw sperm characteristics, suggesting that LN2 vapor may be substituted for LN2 for the long-term storage of human sperm.

A novel filtration device for point of care preparation of cellular therapies

Many biological samples are stored at cryogenic temperatures (i.e. temperatures below -1500C) to preserve their viability. Typically, these samples are stored in liquid nitrogen vapor-phase freezers. The underlying assumption is that biological samples show highly reduced degradation and metabolic activity while below Tg (the glass transition temperature). On the other hand, every time a sample is manipulated or temporarily removed from an LN2 freezer, the sample will experience thermal excursions marked by warm-up rates of several degrees per second. In these cases, the risk of harming a sample by inadvertently crossing the Tg threshold is very likely. This paper’s objective is to provide supporting data to fully characterize the thermal excursions of cryogenically frozen single vials filled with H2O during typical transient temperature events. Specifically, we focus our attention on the cold-chain steps that involve transferring a single vial from an LN2 vapor environment to either an ambient temperature or transportable dry ice (-800C) environment. In general, the warm-up rates experienced by the biological sample correlate to the thermal energy exchanged with the warmer environment via convective and conductive heat transfer. The magnitude of the heat transfer is driven by multiple factors: the warmer environment temperature and the overall time of exposure, the size and shape of the vial, its placement in a cryobox, the type of handling (manual or automated), the handling speed, etc. In this paper, we rationalize all the above factors and present experimental measurements supported by calibrated finite elements simulations showing typical expected warm-up rates. As a result, best-practice time constants for handling vials of biological samples without risking excessive thermal excursions above Tg are suggested.

Influence of Egg Yolk on the Quality of Cryopreserved Spermatozoa of Common Carp, Cyprinus carpio

Cryopreservation of fish gametes can help in producing quality fish seeds. Success of cryopreservation is evaluated by the post-thaw motility of the spermatozoa. The changes in the seminal plasma during cryopreservation would alter the energy supply for the motility of the spermatozoa, and thus energy supplementation is found to be useful during cryopreservation. Cyprinus carpio spermatozoa were cryopreserved along with egg yolk as a co-cryoprotectant after 1:100 dilution with 0.85% physiological saline as extender and DMSO as cryoprotectant (85:15). The diluents contained egg yolk at three different concentrations, viz., T1 (5%), T2 (10%), and T3 (15%). The diluted milt was equilibrated for 10 min at 5°C and loaded into 0.25 ml straws. The loaded straws were then frozen with LN2 vapor for 5 min and immersed in liquid nitrogen. Observations were made once in 7 days for 42 days on motility parameters based on which the duration, score, pattern, and percentage were determined. There were significant differences in the motility duration between treatments, and egg yolk at 5% (T1) concentration was found to support the cryopreserved spermatozoa better than the other concentrations; the difference in motility duration was statistically significant (P > 0.005).

67 EFFECTS OF ANTIOXIDANTS LACTOFERRIN AND CATALASE ON STALLION FROZEN SEMEN

During cryopreservation, the sperm were submitted to an increased generation of reactive oxygen species. Furthermore, because of the large portion of seminal plasma removal, there is a decrease of sperm antioxidant protection. Addition of antioxidants proteins found in seminal plasma, such as lactoferrin (Lf) and catalase (Cat), to the freezing semen extenders could protect the sperm during cryopreservation. Lactoferrin is a transferrin, which prevents the hydroxyl radicals generation, and Cat plays an antioxidant role. The aim of this study was to determine the effects of Lf and Cat supplementation to the INRA 82 freezing extender (Battelier et al. 1997) on sperm motility parameters and membrane functionality of stallion frozen semen. Semen from 6 stallions was collected with an artificial vagina, diluted with Kenney extender (1 : 1), and centrifuged (500 × g, 10 min). The supernatant was discarded, and sperm number per milliliter was calculated. Semen was resuspended with 3 extenders to 100 × 106 sperm mL–1. The treatments were distributed in (F1) control, INRA 82 freezing extender (Battelier et al. 1997), (F2) F1+ 500 μg mL–1 lactoferrin, and F3) F1 + 200 IU mL–1 catalase. Semen samples were packaged in 0.5-mL straws and cooled to 5°C (0.27°C min–1). For semen freezing, the straws were laid over the LN vapor for 20 min and plunged into the LN. The straws were thawed at 37°C for 30 s. Motility parameters of frozen semen were determined using a computer sperm cell analysis, and sperm membrane functionality was assessed by the hyposmotic swelling test (Lagares et al. 1998). The data were analysed using Friedman test using stallion as a block. A probability of P &lt; 0.05 was considered significant. There was no significant difference between the percentage of total sperm motility (median, minimum-maximum value; F1: 29.9, 11.0–82.7; F2: 49.8, 7.7–55.2; F3: 39.8, 5.7–92) and progressive sperm motility (F1: 7.1, 3.2–23.3; F2: 13.4, 2.6-22.4; F3: 15.6, 1.1–29.6), and functional sperm membrane (F1: 26.7, 14.7–56.2; F2: 50.5, 15.7–61.7; F3: 46.6, 13.8–50.9) with regard to the treatment. However, the velocity parameters: velocity average path (F1: 29.3, 22.1–33.80; F2: 34.6, 24.8–44; F3: 35.7, 18.2–42.6), velocity curvilinear (F1: 36.9, 30.5–45.1; F2: 42.5, 34.7–51; F3: 44.6, 25.5–50.9), and velocity straight line (F1: 23.4, 17–3.60; F2: 28.9, 18.8–38.2; F3: 26.6, 13.6–37.2) in the treatment with Lf (F2) were higher compared with the control (F1; P &lt; 0.05). These results corroborate with studies reporting the lack of positive effect on equine sperm motility when antioxidants were added to skim milk-based extenders. Although the addition of Lf or Cat to skim milk-based extenders did not improve the motility sperm characteristics and sperm membrane functionality, more studies about the positive effect of Lf on the velocity parameters are necessary. Lactoferrin could then play an important role on the oxidative metabolism, which provides energy to the sperm movement.Acknowledgments to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil, for the financial support.

49 PRACTICAL APPLICATION OF THE HOLLOW FIBER VITRIFICATION METHOD FOR CRYOPRESERVATION OF MAMMALIAN EMBRYOS

We recently developed the hollow fibro vitrification (HFV) method, which is a novel, high-performance embryo cryopreservation method (Matsunari et al., 2012). In this study, we aimed to verify the applicability of the HFV method for cryopreserving various types of embryos; BDF1 mouse embryos at the 2-cell stage, porcine parthenogenetic morulae derived from in vitro-matured oocytes, bovine morulae produced by in vitro maturation/fertilization (LIAJ Animal Biotechnology Center, Tokyo, Japan), and in vivo-derived blastocysts of common marmosets were vitrified, and their survival was assessed by culture or transfer. The embryos were vitrified using 20 mM HEPES-buffered TCM-199 containing 20% calf serum as a base medium. Cellulose acetate hollow fibres (25 mm) containing 1 to 20 embryos were placed in an equilibration solution containing 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 5 to 7 min, followed by incubation for 1 min in vitrification solution containing 15% EG, 15% DMSO, and 0.5 M sucrose. The embryos were then vitrified by immersion in LN. The embryos were devitrified by immersing the hollow fibre in a 1 M sucrose solution at 38.5°C, which was followed by stepwise dilution of the cryoprotectants and washing. For a subset of the vitrified mouse embryos, rewarming in a non-ultra-rapid manner by melting a hollow fibre in air at room temperature for 5 s was tested. Embryo transfer was performed to assess the viability of the vitrified mouse embryos. For porcine embryos, vitrification in LN vapor (–150°C) was tested. Development of the vitrified mouse embryos to blastocysts was equal to that of the non-vitrified embryos [105/110 (95.5%) v. 109/110 (99.1%)]. Post-transfer development to fetuses was also equal between the vitrified and non-vitrified embryos [pregnancy rates: 4/4 v. 2/2; developmental rates: 55/80 (68.8%) v. 35/40 (87.5%)]. Non-ultra-rapid rewarming did not decrease the survival of the vitrified mouse embryos [blastocysts: 94/100 (94.0%); pregnancy: 4/4; fetuses: 55/80 (68.8%)]. Blastocyst formation was equivalent for vitrification of porcine embryos in LN vapor [27/34 (79.4%)], direct immersion into LN [28/35 (80.0%)], and the non-vitrified control [31/32 (96.9%)]. Vitrification of 191 bovine morulae resulted in 153 (80.1%) blastocysts. In preliminary experiments, survival of marmoset blastocysts was 100% (n = 6). These data demonstrate that the HFV method is (1) effective for embryos of various species and production methods; (2) effective even for porcine in vitro-derived morulae, which are highly cryosensitive; and (3) amenable to modifications such as non-ultra-rapid warming and cooling in LN vapor, increasing the potential applicability of the HFV method. For instance, vitrification in LN vapor may allow embryo cryopreservation with high hygienic standards. This study was supported by JST, ERATO, Nakauchi Stem Cell and Organ Regeneration Project.

Cryobiology of cephalopod (Illex coindetii) spermatophores

Cephalopod culture is expected to increase in the near future and sperm cryopreservation would be a valuable tool to guarantee sperm availability throughout the year and to improve artificial insemination programs. We have studied the tolerance of spermatophores from the oceanic squid Illex coindetii to several cryoprotectants, in two toxicity experiments and a cryopreservation test. Five permeating cryoprotectants were tested: Dimethyl sulfoxide (Me2SO), methanol, glycerol, propylene glycol and ethylene glycol. In the first experiment, spermatophores were exposed to the five cryoprotectants at 5% (v/v) and 15% (v/v) at 4°C for 5min. In the second experiment, spermatophores were exposed to the cryoprotectants at 15% using different exposure times: 5, 15 and 30min. In a third experiment, we tested two cryopreservation protocols: LN2 vapor or −80°C freezer, using a 15% cryoprotectant and 15 or 30min of exposure. Viability and mitochondrial activity were assessed using Mitotracker deep red, YOPRO1 and Hoechst 33342, by flow cytometry. Spermatozoa in this species remain viable after cryoprotectant exposure but their quality decreased considerably after cryopreservation, only 5–10% of spermatozoa being motile. Flow cytometry demonstrated that Me2SO may be the most appropriate cryoprotectant for I. coindetii spermatozoa, and shows a first approach on cephalopod sperm cryopreservation, opening new possibilities for the research and culture of this group of molluscs.

Morphological and functional preservation of pre-antral follicles after vitrification of macaque ovarian tissue in a closed system

What are the appropriate conditions to vitrify the macaque ovarian cortex in a large-volume, closed system that will preserve functional pre-antral follicles? The combination of glycerol, ethylene glycol (EG) and polymers with cooling in liquid nitrogen (LN2) vapor and a two-step warming procedure was able to preserve tissue and follicle morphology as well as function of a small population of secondary follicles in the macaque ovarian cortex following vitrification in a closed system. For prepubertal cancer patients or those who require immediate cancer therapy, ovarian tissue cryopreservation offers the only hope for future fertility. However, the efficacy of live birth from the transplantation of cryopreserved ovarian tissue is still unclear. In addition, live birth from cryopreserved ovarian tissue has only been demonstrated after tissue autotransplantation, which poses the risk of transmitting metastatic cancer cells back to the cancer survivor in certain cancers. Non-human primate model, n = 4, randomized, control versus treatment. End-points were collected from tissue histology, tissue culture (48 h) and isolated secondary follicle culture (6 weeks). Two vitrification solutions (VSs) containing EG + glycerol (VEG) and EG + dimethylsulfoxide (VED) were examined for vitrification, devitrification and thermodynamic properties. Once the optimal VS was determined, macaque ovarian cortical pieces (3 × 3 × 0.5 mm(3)) were divided into fresh and two vitrified groups (VEG and VED). For the vitrification groups, tissues were exposed to 1/4, 1/2 and 1× VS for 5 min/step as well as 1× VS + polymers for 1 min at 37°C, loaded into high-security straws with 1 ml of VS + polymers, heat sealed and cooled in LN2 vapor. Samples were warmed in a 40°C water bath and cryoprotective agents were diluted with 1, 0.5, 0.25 and 0 M sucrose. Tissues were fixed for histological analysis and cultured with bromodeoxyuridine (BrdU). Secondary follicles from VEG tissues were encapsulated and cultured (n = 24/treatment/animal). Follicle health, diameter and steroid [progesterone, androstenedione (A4), estradiol (E2)] production were analyzed weekly. Dense stroma and intact pre-antral follicles were observed using VS containing 27% glycerol, 27% EG and 0.8% polymers with cooling in LN2 vapor and a two-step warming. Higher cooling and warming rates led to fracturing. BrdU uptake was evident in granulosa cells of growing follicles in fresh and vitrified tissues. Secondary follicles from fresh tissues (70 ± 12%) and tissues vitrified with VEG (52 ± 2%) showed similar survival rates (all data: mean ± SEM; P > 0.05). For both groups, the initial follicle diameter was similar and increased (P < 0.05) by Week 3, but diameters in vitrified follicles were smaller (P < 0.05) by Week 6 (566 ± 27 µm) than those of the fresh follicles (757 ± 26 µm). Antrum formation rates were lower (P < 0.05) for vitrified (37 ± 6%) relative to fresh (64 ± 8%) follicles. There was no significant change in levels in culture media of E2, P4 and A4 between fresh and VEG groups at any time point during culture. Only in vitro studies are reported. Future in vivo tissue transplantation studies will be needed to confirm long-term function and fertility potential of vitrified ovarian tissues. This is the first demonstration of antral follicle development during 3D culture following ovarian tissue vitrification in a closed system using primate ovarian tissue. While diminished antrum formation and slower growth in vitro reflect residual cryodamage, continued development of ovarian tissue vitrification based on cryobiology principles using a non-human primate model will identify safe, practical and efficient protocols for eventual clinical use. Tissue function following heterotopic transplantation is currently being examined. National Institutes of Health (NIH) Oncofertility Consortium UL1 RR024926 (1RL1-HD058293, HD058295, PL1 EB008542), the Eunice Kennedy Shriver NICHD/NIH (U54 HD018185) and ONPRC 8P51OD011092-53. G.M.F. works for the company that makes the polymers used in the current study.