Post-thaw Survival Rate Research Articles

P-031 Viral crossroads: revisiting sperm processing in the context of Human Immunodeficiency Virus (HIV) infection

Abstract Study question Is extensive sperm washing, followed by viral load testing, necessary to ensure safety of assisted reproductive techniques in HIV-positive men? Summary answer In context of viral suppression, there is no added value of extensive sperm washing followed by viral load testing for HIV-positive men seeking fertility treatment. What is known already Sperm washing was initially introduced as a safe reproductive strategy for serodiscordant couples in which the male partner is seropositive. This technique uses a combination of density gradient centrifugation (DGC) and swim-up (SU) to separate the HIV-free spermatozoa from the seminal plasma containing most of the viral particles. Nowadays, due to the increased availability and efficacy of antiretroviral drugs, more patients are adequately suppressed and have no risk of transmitting HIV, which is reinforced by the ‘Undetectable Equals Untransmittable’ principle. This questions the need for extensive sperm washing followed by quantitative polymerase chain reaction (qPCR) testing in the IVF laboratory. Study design, size, duration This single center retrospective study investigates the effectiveness of our existing sperm washing procedures by evaluating the HIV viral load (VL) both in the raw and processed semen fractions, while considering the patient’s viral suppression status. All sperm wash procedures performed on fresh ejaculates between 2012 and 2023 were included. Additionally, clinical outcomes of the subsequent intracytoplasmic sperm injection (ICSI)-cycles were collected. Participants/materials, setting, methods Type 1 (wash-DGC-SU), type 2 (wash-DGC) or type 3 (double wash) sperm processing was executed based on the concentration and motility of the raw sample. The processed fraction was cryopreserved, and the VL was determined in both the raw and processed semen fraction using qPCR. A semen VL below 20 copies/mL was considered negative. Only processed fractions with a negative VL were suitable for further therapeutic use. Threshold for viral suppression was 50 copies/mL. Main results and the role of chance A number of 82 sperm processing procedures were performed for 68 seropositive men, with a mean age at donation of 41.9 ± 7.0 years. All patients were confirmed to be HIV-1 positive, with the exception of one individual presenting a co-infection with both HIV-1 and HIV-2. Incidences for type 1, type 2 and type 3 processing were 23.2%, 62.2% and 14.6%, with mean initial concentration of 91.3(±63.6), 42.1(±47.9) and 0.12(±0.15) million/mL, and mean progressive motility of 54(±12.7)%, 33(±16.8)% and 4(±3.6)% in the respective groups. In 91.5% (n = 75/82) of cases, the patient was suppressed at the time of sperm processing. In case of viral suppression, only one raw sample (n = 1/75, 1.3%) tested positive for HIV (94 copies/mL), likely due to the phenomenon of “viral shedding”. After processing, none of the fractions contained a residual VL. Post-thaw progressive motile sperm survival rate was on average 0.40 ± 0.26. Overall, 61 couples underwent a total of 120 ICSI-cycles, resulting in cumulative clinical pregnancy and cumulative live birth rates per couple of 57.4% (n = 35/61) and 42.6% (n = 26/61), respectively. Limitations, reasons for caution Given that HIV-positive individuals represent a minority within our patient population, the limited prevalence of sperm wash procedures performed over the past decade has led to a relatively small sample size for this study. Wider implications of the findings These findings have the potential to shape future guidelines concerning the use of sperm samples from HIV-positive men. This may optimize the efficiency of sperm processing protocols, while maintaining both the safety and efficacy of assisted reproductive techniques for this specific patient population. 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 < 0.001), MFSS resulted in a higher post-processing motility(97.8±2%, P < 0.001) and normal morphology(3.4±1%, P < 0.001). Post-thaw analysis revealed a remarkably higher post-thaw motility in MFSS (51.2±7% vs 43.6%±4%,P < 0.001). SCF averaged 17%(raw), 11%(DG) and 2%(MFSS,P<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<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 < 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

#83 : Human Sperm Cryo-Survival Rate

Background and Aims: Sperm cryopreservation plays an important role in the suite of services available to fertility patients. The ability to preserve the motility and viability of human spermatozoa is a key component in delivering successful assisted reproductive technologies (ART) outcomes. Cryopreservation methods expose sperm to stress, resulting in cellular damage which consequently leads to a reduction in their survival rate. The aim of this study was to evaluate the average cryo-survival rate of semen samples frozen in a clinical setting, to help improve and manage patient expectations prior to accessing this service. Method: Pre and post sperm freeze data from patients who presented at the Genea Sydney clinic for sperm cryopreservation between 2017 and 2022 was analysed. The post-thaw survival rate for 1467 specimens was assessed by averaging the total post-thaw motility with total pre-freeze motility. Only ejaculated samples, frozen on-site, who had a raw motility calculated as well as a post-thaw motility analysis able to be performed were included in the study. Results: The range of post-thaw survival was 57% to 67% per annum over the six years of examined data, with an overall cryo-survival rate of 62%. Conclusions: The data shows a consistent recovery rate of post-thaw sperm motility over the time period analysed. Literature indicates average sperm cryo-survival rates range from 30% to 50%, and previous advice to patients has been to expect around 50% sperm survival. With this new data, Genea can revise its guidance to match the current average figure of around 60%. In addition, future internal freeze optimisation studies can use this figure as a benchmark to improve upon techniques and processes.

Successful cryopreservation of common carp (Cyprinus carpio) embryos: efficiency of methanol-based vitrification solutions with honey on post-thaw survival rate

Successful cryopreservation of common carp (Cyprinus carpio) embryos: efficiency of methanol-based vitrification solutions with honey on post-thaw survival rate

Evaluating the Effects of Cryopreservation on the Viability and Gene Expression of Porcine-Ear-Skin Fibroblasts.

Owing to the inherent heterogeneity and plasticity of fibroblasts, they are considered as the conventional biological resources for basic and clinical medical research. Thus, it is essential to generate knowledge about the establishment of fibroblast cultures and the effects of cryopreservation processes on their biological characteristics. Since the pig (Sus scrofa) possesses numerous genetic, physiological, and anatomical similarities with humans, porcine fibroblasts are naturally regarded as useful analogues of human fibroblasts. Nonetheless, less attention has been given to the alterations in viability and gene expression of cryopreserved porcine fibroblasts. In this study, we aimed to obtain fibroblasts from porcine ear skin and evaluate the effects of cryopreservation on the cell survival, proliferation, and gene expression profiles of the fibroblasts by trypan-blue-staining assay, Cell Counting kit-8 (CCK-8) assay, and RNA-sequencing analysis, respectively. Our results suggested that morphologically stable fibroblast cultures can be constructed from pig-ear skin. The post-thaw survival rate of the cryopreserved fibroblasts at 0 h and 24 h was over 90%. The proliferative activity of the cryopreserved fibroblasts was similar to that of the non-cryopreserved fibroblasts after 7 days of in vitro culture, which suggested that cryopreservation did not influence the viability. The RNA-sequencing analysis indicated that this should be attributed to the 867 differentially expressed genes (DGEs) identified, which are involved in molecular process related to cell recovery and survival after cryo-stimulation. In addition, eight important DEGs BMP2, GDF15, EREG, AREG, HBEGF, LIF, IL-6, and HOX-7 could potentially be applied to improve the efficiency of fibroblast cryopreservation, but comprehensive and systematic studies on understanding the underlying mechanisms responsible for their modulatory roles are urgently needed.

Micro-straw: An efficient cryopreservation carrier for rare human spermatozoa.

Many cryopreservation carriers have been introduced to freeze rare human spermatozoa, however, these carriers relative attributes and comparative effectivenesses have not yet been systematically studied. Is the micro-straw cryopreservation carrier more effective for cryopreserving rare human spermatozoa compared with the Cryoplus and a new micro-straw (LSL straw) carriers? This study involves 93 samples from healthy sperm donors and 40 samples from patients diagnosed with oligospermia, asthenospermia, oligoasthenospermia, or obstructive azoospermia. We determined the optimal freeze-thaw protocol for the Micro-straw carrier. The post-thaw survival rate, normal sperm morphology, acrosome integrity, and DNA fragmentation for Micro-straw, Cryoplus, and LSL carriers were then determined. Finally, we verified the effects of freezing using these carriers by comparing the qualities of post-thaw spermatozoa from patients. The highest total motility (TM) and progressive motility (PR) survival rates were obtained by placing the Micro-straw at 1cm above the LN2 surface for 70 s during freezing and in a 42°C water bath for 40 s during thawing. No differences were observed in the PR survival rate, acrosome integrity, and DNA fragmentation of the post-thaw spermatozoa from the three carriers. However, the normal morphology rate of spermatozoa frozen using the Micro-straw carrier was higher than for the Cryoplus carrier (p<0.05), and the TM survival rate of spermatozoa frozen with the Micro-straw was higher than that for the LSL carrier (p<0.01). In verification tests, there were no significant differences in the quality of post-thaw spermatozoa cryopreserved using these carriers for both rare spermatozoa or epididymal sperm. Micro-straw, Cryoplus, and LSL carriers are all efficient means of freezing rare human spermatozoa. However, the Micro-straw carrier is more economical, safe, and user-friendly.

PSXI-10 How to use ultrasonic cutter on frozen semen for multiple embryo production

Abstract The objective of the present study was to establish conditions for using technology that can potentially enhance the efficiency of bovine embryos derived from in vitro fertilization (IVF) with frozen semen. Frozen semen from selected bulls can be stored indefinitely in liquid nitrogen as genetic resources; however, these resources are considered consumable because they cannot be regenerated. Therefore, to optimize the utilization of frozen semen, as many oocytes as possible should be fertilized with one straw. However, a sufficient number of prepared oocytes might not be available for one experiment, which can limit the use of the total spermatozoa population. Thus, an economical method for producing embryos needs to be established by optimizing technology for transplantable embryos. In this study, the utilization of frozen semen was increased by dividing the straw with an ultrasonic cutter. The post-thaw survival rate of uncut straws from Korean Proven Bulls did not differ from that of half cuttings. When ultrasonic cutting was applied to frozen semen, spermatozoa could be prepared for IVF trials at least four times, and blastocysts were produced. Therefore, cutting frozen semen with an ultrasonic cutter represents a potentially useful tool to expand genetic resources from excellent breeding stocks. This approach could also be valuable in the field of IVF of endangered species or rare breeds for their preservation, as well as in ovum pick-up (OPU) techniques.

Ultrasonic Cutting of Frozen Semen Straws to Optimize the Use of Spermatozoa for In Vitro Fertilization.

Simple SummaryThe use of frozen semen is essential for the success of in vitro fertilization in the bovine embryo transfer industry. Therefore, we aimed to maximize the use of a single frozen semen straw by employing a cutting protocol. To the best of our knowledge, the current study is the first to apply ultrasonic cutting to frozen semen. The mechanical damage of spermatozoa was studied in frozen bovine semen by assaying sperm motility, acrosome abnormalities, and developmental competence. These findings may help to optimize the utilization of frozen semen for in vitro fertilization in the bovine industry.The objective of the present study was to establish conditions for using technology that can potentially enhance the efficiency of bovine embryos derived from in vitro fertilization (IVF) with frozen semen. Frozen semen from selected bulls can be stored indefinitely in liquid nitrogen as genetic resources; however, these resources are considered consumable because they cannot be regenerated. Therefore, to optimize the utilization of frozen semen, as many oocytes as possible should be fertilized with one straw. However, a sufficient number of prepared oocytes might not be available for one experiment, which can limit the use of the total spermatozoa population. Thus, an economical method for producing embryos needs to be established by optimizing technology for transplantable embryos. In this study, the utilization of frozen semen was increased by dividing the straw with an ultrasonic cutter. The post-thaw survival rate of uncut straws from Korean Proven Bulls did not differ from that of half cuttings. When ultrasonic cutting was applied to frozen semen, spermatozoa could be prepared for IVF trials at least four times, and blastocysts were produced. Therefore, cutting frozen semen with an ultrasonic cutter represents a potentially useful tool to expand genetic resources from excellent breeding stocks. This approach could also be valuable in the field of IVF of endangered species or rare breeds for their preservation, as well as in ovum pick-up (OPU) techniques.

Effects of cryoprotective agents and treatment methods on sperm cryopreservation of stone flounder, Kareius bicoloratus

This study was performed as part of the effort to develop artificial seed production techniques for stone flounder Kareius bicoloratus, which has a great potential for farming in northeast Asian countries. To develop a sperm cryopreservation protocol that can be applied for artificial fertilization, we carried out experiments on the key factors for successful fish sperm cryopreservation, including cryoprotective agents (CPAs), diluents, equilibration times, and dilution ratios. The CPAs tested in this study were dimethyl sulfoxide (DMSO), glycerol, methanol, and ethylene glycol, and the diluents were glucose and sucrose. The equilibration times tested in the experiments ranged from 0 to 210 s and the dilution ratios ranged from 1:1 to 1:1000. According to the results of our experiments, the optimal CPA for stone flounder sperm cryopreservation was DMSO in concentrations ranging from 7.5% to 10% when 300 mM glucose was used as diluent and DMSO in concentrations ranging from 15% to 17.5% when 300 mM sucrose was used as diluent. Post-thaw sperm motility was high, with equilibration times up to 150 s, and dilution ratios up to 1:10 (semen:CPA + diluent) showed no significant differences in the effect on post-thaw sperm motility. The cryopreserved sperm showed more significant DNA damage than the fresh sperm with the severity of DNA damage being in inverse proportions to post-thaw sperm motility and survival rate. It is expected that these results will be useful for applications of artificial fertilization at stone flounder hatchery farms.

Developmental potential of surplus morulas with delayed and/or incomplete compaction after freezing-thawing procedures

BackgroundMorulas with delayed growth sometimes coexist with blastocysts. There is still limited evidence regarding the optimal disposal of surplus morulas. With the advancement of vitrification, the freezing-thawing technique has been widely applied to zygotes with 2 pronuclei, as well as embryos at the cleavage and blastocyst stages. The freezing of morulas, however, has rarely been discussed. The purpose of this study was to investigate whether these poor-quality and slow-growing morulas are worthy of cryopreservation.MethodsThis is a retrospective, observational, proof-of-concept study. A total of 1033 day 5/6 surplus morulas were cryopreserved from January 2015 to December 2018. The study included 167 women undergoing 180 frozen embryo transfer cycles. After the morulas underwent freezing-thawing procedures, their development was monitored for an additional day. The primary outcome was the blastocyst formation rate. Secondary outcomes were clinical pregnancy rate, live birth rate and abortion rate.ResultsA total of 347 surplus morulas were thawed. All studied morulas showed delayed compaction (day 5, n = 329; day 6, n = 18) and were graded as having low (M1, n = 54), medium (M2, n = 138) or high (M3, n = 155) fragmentation. The post-thaw survival rate was 79.3%. After 1 day in extended culture, the blastocyst formation rate was 66.6%, and the top-quality blastocyst formation rate was 23.6%. The day 5 morulas graded as M1, M2, and M3 had blastocyst formation rates of 88.9, 74.0, and 52.8% (p < 0.001), respectively, and the top-quality blastocyst formation rates were 64.8, 25.2, and 9.0% (p < 0.001), respectively. The clinical pregnancy rate was 33.6%.ConclusionsThe post-thaw blastocyst formation rate was satisfactory, with approximately one-half of heavily fragmented morulas (M3) developing into blastocysts. Most of the poor-quality morulas were worth to freeze, with the reasonable goal of obtaining pregnancy and live birth. This alternative strategy may be a feasible approach for coping with poor-quality surplus morulas in non-PGS (preimplantation genetic screening) cycles.

Effects of different diluents, cryoprotective agents, and freezing rates on sperm cryopreservation in Epinephelus akaara

We investigated various factors, including cryoprotective agents (CPAs), diluents, and freezing rates, to develop an optimal cryopreservation protocol for Epinephelus akaara sperm. In experiments using 10% dimethyl sulfoxide (DMSO), glycerol, and methanol with various diluents, 10% DMSO and 300 mM glucose yielded the highest post-thaw sperm motility. The combination of 10% glycerol and 300 mM sucrose yielded significantly higher post-thaw sperm motility than did combinations using other diluents. Glycerol and DMSO at a concentration of 10% as CPAs with 300 mM glucose as the diluent resulted in the highest MSR and sperm activity index (SAI). An investigation to determine the effects of glycerol and DMSO concentrations on post-thaw sperm survival rate revealed no significant differences among 5, 10, 15, and 20% concentrations of either CPA. In assessing the effects of CPA concentration on the fertilization rate, the 10% concentration yielded the highest fertilization rate (81.4 ± 4.3%) in DMSO, whereas 15% was the optimal concentration for glycerol (fertilization rate = 66.7 ± 6.1%). The hatching rate was also highest in 10% DMSO (40.1 ± 0.4%) and in 15% glycerol (27.8 ± 2.3%). In conclusion, the optimal rates of post-thaw sperm motility, fertilization, and hatching were achieved when E. akaara sperm were cryopreserved in a diluent of 300 mM glucose with 10% DMSO as the CPA at a freezing rate of −5 °C/min. We therefore recommend this protocol for the cryopreservation of E. akaara sperm.

208 Cryotolerance of In Vitro-Produced Cattle Embryos Produced with Different Maturation and Culture Media

In vitro-produced (IVP) bovine embryos suffer from damage due to suboptimal culture conditions involving altered metabolism, reactive oxygen species generation and high lipid accumulation, which could be impacted by oocyte maturation × embryo culture interaction. We hypothesised that optimizing oocyte and embryo culture conditions would lead to a higher post-thaw embryo survival rate. The experiment was replicated 9 times in a factorial design with 3 oocyte maturation media: TCM-199 plus 10% serum and gonadotropins (control), BO-HEPES-IVM (BO, IVF Bioscience UK), and HMM (HEPES-buffered maturation medium) and 3 embryo culture media: BO-IVC (BOC), BBH7 (Cooley Biotech, Gainesville, FL, USA), and SCF1 (SOF for Conventional Freezing 1; Owen et al. 2017 Reprod Fertil Dev. 29, 129-130). Bovine oocytes (n = 2907) were aspirated from 2- to 8-mm follicles of abattoir ovaries, matured at 38.5°C for 22 to 24 h in 5% CO2 (control) or in BO and HMM in air, fertilized with semen from 1 of 3 bulls, and cultured in BOC, BBH7, and SCF1 at 38.5°C in 5.5% CO2, 5% O2, and 90% N2. Cleavage and blastocyst rates were evaluated at Day 3 and 7, respectively, after IVF. Selected stage 7 blastocysts were slow frozen using 1.5 M ethylene glycol supplemented with 1 mm l-ascorbic acid for 20 min (equilibration). The embryos were cooled to and seeded at –6°C, and then cooled to −32°C at 0.5°C/min. Embryos were thawed and re-expansion was assessed at 24 and 48 h. Data (Table 1) was arcsin transformed and analysed by ANOVA, and means separated by l.s.d. Results indicate that oocytes matured in BO and embryos cultured in SCF1 had a higher blastocyst rate than all oocytes matured in control medium and all embryos cultured in BBH7 (P &lt; 0.05). However, oocytes matured in control medium and embryos cultured in BOC had lower post-thaw re-expansion rates than other treatment groups (P &lt; 0.05). These results suggest that a combination of oocyte maturation and embryo culture media plays an important role in post-thaw embryo survival, al though interactions of present treatments need to be further evaluated. Table 1.Effect of maturation and culture media on blastocyst rate and cryotolerance ± SEM)

75 Improvement of Developmental Competence of Bovine In Vitro-Produced Embryos by Using Charcoal:Dextran-Stripped Fetal Bovine Serum on In Vitro Culture Media

Serum has widely been used as a main supplement to embryo in vitro culture media as it contains embryotrophic factors. Charcoal:dextran treatment of fetal bovine serum (FBS) removes lipophilic chemicals and certain steroid hormones and growth factors. The objective of this study was to investigate the effects of charcoal:dextran-stripped fetal bovine serum (CDS FBS) and heat-inactivated FBS (HI FBS) in embryo culture medium (SOF-BE1 medium supplemented with 10% of serum) on their ability to support in vitro development of bovine embryos. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, mitochondrial activity, gene expression, and cryo-tolerance. The experiment was conducted in 6 replicates (350 oocytes per group). The differences in embryo development, integrated optical intensity, and expression levels of the various genes between experimental groups were analysed by one-way ANOVA. Duncan’s multiple range tests were used to test the differences between the treatments. The level of statistical significance was set at P &lt; 0.05. The percentages of embryos that underwent cleavage and formed a blastocyst were significantly (P &lt; 0.05) higher in medium containing CDS FBS than in medium containing HI FBS (42.84 ± 0.78% v. 36.85 ± 0.89%, respectively). The total number of cells per Day 8 blastocyst was not significantly different (P &gt; 0.05) between the CDS FBS group (208.40 ± 14.77) and the HI FBS group (195.11 ± 19.15). Furthermore, the beneficial effects of CDS FBS on embryos were associated with a significantly increased mitochondrial activity, as identified by MitoTracker Green, and reduced intracellular lipid content, as identified by Nile red staining, which increased their cryo-tolerance. The post-thaw survival rate of blastocysts was significantly (P &lt; 0.05) higher after 24 h in the CDS FBS than in the HI FBS group (85.33 ± 4.84% v. 68.67 ± 1.20%). Quantitative reverse transcription PCR showed that the mRNA levels of lipid metabolism-related genes, acyl-CoA synthetase long-chain family member 3, acyl-coenzyme A dehydrogenase long-chain, and the cholesterol metabolism related gene hydroxymethylglutaryl-CoA reductase were significantly increased upon culture with CDS FBS. Moreover, the mRNA levels of survival gene sirtuin 1, antioxidant gene superoxide dismutase 2, and anti-apoptotic associated gene B-cell lymphoma 2 in frozen–thawed blastocysts were significantly (P &lt; 0.05) higher in the CDS FBS group than in the HI FBS group; however, the mRNA level of the pro-apoptotic gene BCL2-associated X protein was significantly reduced. In conclusion, these data suggest that supplementation of in vitro culture medium with CDS FBS improves in vitro bovine embryo developmental competence and the quality of blastocysts in terms of their crytolerance and gene expression. This research was supported by grant from the Next-Generation BiogGeen21 (No. PJ01107703), IPET (No. 315017-5 and 117029-3), Allergy free cat (Co.. Felix Pets), BK21plus, and KGSP.

52 CRYOPRESERVATION OF BOVINE SOMATIC CELL USING ANTIFREEZE POLYAMINO-ACID (CARBOXYLATED POLY-L-LYSINE)

Cryopreservation is used for long-term preservation of biological materials containing cell and tissue, somatic cells are important to commercial application in gene bank and medical engineer. For cell cryopreservation, dimethyl sulfoxide (DMSO) have been used widely as cryoprotective material for long time. Carboxylated poly-l-lysine (CPLL) is an ampholytic polymer compound obtained by converting 65% amino groups to carboxyl groups after synthesising ε-poly-l-lysine aqueous solution and succinic anhydride. It is reported that the addition of CPLL into cryopreservation medium improves the post-thaw survival rate of murine L929 cells, human induced pluripotent stem cells and murine embryo. In this research, the effectiveness of CPLL as bovine somatic cell cryoprotective material was examined. In experiment 1, using bovine fibroblast cells from skin, the cryopreservation media were prepared as follows: 10% (vol/vol) DMSO, 5% (vol/vol) DMSO + 5% (wt/vol) CPLL, 5% (wt/vol) CPLL, 7.5% (wt/vol) CPLL, 10% (wt/vol) CPLL and 20% (wt/vol) CPLL were dissolved in DMEM and pH was adjusted to 7.4. Cells were resuspended in 1 mL of cryopreservation medium at a cell density of 5 × 105 and stored in a −80°C freezer. After storage for at least 1 wk, cells were thawed. The 5% CPLL showed significantly higher post-thaw survival rate than 10% DMSO (96.5% v. 86.7%; P &lt; 0.05), and 7.5% CPLL and 20% CPLL showed significantly higher proliferation rates than 10% DMSO at 72 h after thawing (10% DMSO v. 7.5% CPLL v. 20% CPLL ; 99.4% v. 173.6% v. 207.4%; P &lt; 0.05). Although it is necessary to remove the cryoprotective material after thawing, the proliferation rate at 72 h after thawing of 5% CPLL and 10% CPLL without removal of cryoprotective material were significantly higher than that when removing cryoprotective material (111.2% v. 269.6%, 113.0% v. 210.0%). In addition, there was no difference between the removal treatment of CPLL and the NOT removal treatment of CPLL at the thawing. In experiment 2, using bovine cumulus cells, the cryopreservation media were prepared as follows: 10% (vol/vol) DMSO, 5% (vol/vol) DMSO + 5% (wt/vol) CPLL, 5% (wt/vol) CPLL, 10% (wt/vol) CPLL, 20% (wt/vol) CPLL, 25% (wt/vol) CPLL, and 30% (wt/vol) CPLL were dissolved in DMEM and other conditions were the same as experiment 1. There was no significant difference for post-thaw survival rate (93.7, 91.8, 89.2, 89.2, 95.3, 91.6, and 90.2%). At 72 h after thawing, 10% DMSO, 5% DMSO + 5% CPLL and 25% CPLL showed higher proliferation rate (10% DMSO v. 5% DMSO + 5% CPLL v. 25% CPLL; 258.4% v. 281.4% v. 257.4%). The significant differences in survival rate were determined by chi-squared test, and in proliferation were determined by ANOVA. In bovine somatic cells, it was possible to cryopreserve cells using CPLL without DMSO. The cryoprotective effect of CPLL is equal or superior to that of DMSO, the conventional cryoprotective material. Further, when using CPLL, it was possible to culture without removing the cryopreservation medium after thawing. These results suggest that CPLL has promise as a new cryoprotective material for bovine somatic cells.

47 CRYOPRESERVATION OF BOVINE GERM CELL USING ANTIFREEZE POLYAMINO-ACID (CARBOXYLATED POLY-L-LYSINE)

Carboxylated poly-l-lysine (CPLL) is an ampholytic polymer compound, and it is obtained by converting 65% amino groups to carboxyl groups after synthesising ε-poly-l-lysine aqueous solution and succinic anhydride. CPLL has cryoprotective property similar to antifreeze protein, and addition of CPLL into cryopreservation medium improves the post-thaw survival rate of cells and embryos. In this research, we examined the effectiveness of CPLL as a bovine germ cell cryoprotective material. In experiment 1 (in sperm), the conventional cryopreservation medium used for control group was consisted of 6.5% (vol/vol) glycerin, and the cryopreservation medium used for CPLL group was consisted of 3.25% (vol/vol) glycerin and 0.5% CPLL (wt/vol). The post-thaw survival and motility were assessed by using Sperm Motility Analysis System (DITECT Corp., Tokyo, Japan). There was no significant difference for post-thaw survival rate and motility (control v. CPLL; 98.8% v. 96.6% and 69.7% v. 62.2%, respectively). Artificial insemination was carried out in 65 cows (control v. CPLL; 34 v. 31), and the conception rate of the CPLL group was higher than that of the control group (80.6% v. 67.6%; P = 0.23). In experiment 2 (embryos), the conventional cryopreservation medium used for control group was consisted of 5% (vol/vol) ethylene glycol and 6% (vol/vol) propylene glycol in PBS. In the CPLL group, 7% (wt/vol) CPLL was added to the conventional medium. In vitro fertilization embryos were cryopreserved at Day 7 and Day 8. There was no significant difference in survival rate at 0, 24, and 48 h and hatched rate until 72 h after thawing (control v. CPLL: 93.6% v. 93.2%, 69.0% v. 64.7%, 56.1% v. 56.3%, 12.9% v. 10.2%, respectively). Embryos obtained by superovulation treatment and in vivo fertilization at Day 7 were cryopreserved using above 2 media, and transferred non-surgically into synchronized recipient cows (1 embryo per animal). Embryo transfer (ET) was carried out in 81 cows (control v. CPLL: 31 v. 50), and recipients were diagnosed for pregnancy ultrasonically 50 days after embryo transfer. Conception rate of CPLL group was higher than control group (50.0% v. 29.0%; P = 0.063). In both experiments, the significant differences between control group and CPLL group were determined by chi-squared test. The effectiveness of CPLL in cells and embryos has been reported; however, there is no report using CPLL in bovine germ cells. In this research, CPLL improved the conception rate of AI and ET, probably due to its low toxicity and protection of the cell membrane. These results suggest that CPLL is available as a new cryoprotective material for bovine sperm and embryo in slow freezing methods.

Glutathione improves survival of cryopreserved embryogenic calli of Agapanthus praecox subsp. orientalis

The effect of supplementation of reduced glutathione (GSH) to cryoprotectant solution on the generation of reactive oxygen species (ROS) (e.g., H2O2, OH·, and O2·−) and antioxidants (e.g., SOD, POD, CAT, AsA, and GSH), as well as membrane lipid peroxidation (i.e., MDA content) mitigation in cryopreserving of embryogenic calli (EC) of Agapanthus praecox subsp. orientalis was investigated. The vitrification-based cryopreservation method was used in this study. The addition of GSH at a final concentration of 0.08 mM to the cryoprotectant solution has significantly improved cryotolerance of A. praecox EC. The EC post-thaw survival rate increased by 68.34 % using the cryoprotectant solution containing 0.08 mM GSH as compared to the control (GSH-free). EC treated with GSH displayed the reduction in OH· generation activity and the contents of H2O2 and MDA, as well as enhancement in the inhibition of O2·− generation and the antioxidant activity. Treatment with exogenous GSH also increased endogenous AsA and GSH contents after dehydration step. Expression of stress-responsive genes, e.g., peroxidase (POD), peroxiredoxin, ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), and glutathione peroxidase (GPX), was also increased during cryopreservation processes. The expression of DAD1 (Defender against apoptotic cell death) was elevated, while cell death-related protease SBT was suppressed. These results demonstrated that the addition of GSH to cryoprotectant solution affects the ROS level and could effectively improve survival of A. praecox EC through enhancing antioxidant enzyme activities and decreasing cell death.

52 EFFECT OF CARBOXYLATED POLY-L-LYSINE ON THE POST-THAW VIABILITY OF IN VITRO-PRODUCED BOVINE BLASTOCYST

Recently, there has been development of an antifreeze polyamino acid (carboxylated poly-l-lysine: PLL) as new cryoprotectants (CPA). This compound counts as amphoteric macromolecular cationic and anionic substituents (polyampholyte) by chemically modifying poly-lysine. In addition, PLL is highly safe and frequently used as a food additive in substitution for other CPA. Other common CPA have high toxicity and caused physiological damage. In vitro-produced blastocysts were randomly assigned into 3 groups: (1) vitrified embryos with PLL vitrification solution [PLL-vit-1: 15% PLL + 15% ethylene glycol (EG); PLL-vit-2: 30% PLL + 30% EG + 0.5 M sucrose], (2) vitrified embryos with Vajta et al. solution (Conv-vit-1: 7.5% dimethyl sulfoxide + 7.5% EG; Conv-vit-2: 16.5% dimethyl sulfoxide + 16.5% EG + 0.5 M sucrose), and (3) nonvitrified blastocysts (control). All embryos were frozen by droplet vitrification method. First, the PLL-vitrified embryos were exposed to 5, 10, and 15 min in the PLL-vit-1 and then putted for 30 to about 60 s in the PLL-vit-2. Then, we compared with each group regarding exposure time of Vit-1. Post-thaw survival rate of each exposure time did not significantly differ among the 3 groups (100 v. 100 v. 100%). However, hatching rate of the 10-min exposure group was higher than that of 5- and 15-min groups (75.0 v. 25.0 v. 66.7; P &lt; 0.05). Therefore, we confirmed that exposure time of Vit-1 was exposed for a minimum of 10 min. The post-thawed survival rate of each vitrification method was not significantly different between PLL-vit and Conv-vit groups (97.7 v. 86.4%). The total cell numbers of blastocyst did not significantly differ among groups. However, the apoptotic cell numbers of blastocyst was significantly different between the control and Conv-vit groups (0.4 ± 0.6 v. 4.4 ± 3.9; P &lt; 0.05) but was not different in control v. PLL-vit (0.4 ± 0.6 v. 2.1 ± 2.4) and Conv-vit v. PLL-vit (4.4 ± 3.9 v. 2.1 ± 2.4). In conclusion, PLL-vit for bovine blastocyst could reduce toxicity and osmotic shock and showed high efficiency on the quality of post-thawed bovine blastocysts compared with that of Conv-vit.This work was partly supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ009587022014), IPET (Grant No. 112020-3), and a scholarship from the BK21 plus program. A-Na Ha, Kyeong-Lim Lee, and Md. Fakruzzaman were supported by BK21 plus fellowships at Gyeongsang National University, Republic of Korea.

Development of a modified straw method for vitrification of in vitro-produced bovine blastocysts and various genes expression in between the methods

This study evaluated a modified plastic straw loading method for vitrification of in vitro-produced bovine blastocysts. A modified straw was used with a depressed area on its inner surface to which embryos attach. In vitro-produced blastocysts were randomly assigned into three groups: (i) blastocysts attached to the inner surface of a plastic straw (aV), (ii) blastocysts attached to the inner surface of a modified plastic straw (maV), and (iii) non-vitrified blastocysts (control). The recovery rates were not significantly different between aV and maV groups (95.8% vs. 94.3%). The post-thaw survival rate did not significantly differ between aV and maV groups (86.4% vs. 88.2%). The total cell numbers of blastocyst was higher in control than in aV and maV groups (142±21.8 vs. 117±29.7 and 120±25.2; P<0.05), but not significantly differ between aV and maV groups. The mRNA levels of pro-apoptosis related genes Bax and Caspase-3 were higher in aV and maV than in control (P<0.05). By contrast, the mRNA levels of anti-apoptotic genes Bcl-2 and Mcl-1 and of antioxidant-related genes MnSOD and Prdx5 were lower in aV and maV than in control (P<0.05). Confocal microscopy analysis of Golgi apparatus and mitochondria showed that the fluorescence intensity of Golgi apparatus and mitochondria was higher in control than in aV and maV groups. In conclusion, both aV and maV methods can be used to successfully vitrify IVP blastocysts, with maV method to be preferable because of its easiness in embryo loading.

Neonatal outcomes after the transfer of vitrified blastocysts: closed versus open vitrification system

BackgroundIncreasing evidence indicates that closed vitrification has been successfully used in the cryopreservation of human oocytes and embryos. Little information is available regarding the neonatal outcome of closed blastocysts vitrification. The aim of this study was to evaluate the effectiveness and safety of blastocyst vitrification using a high-security closed vitrification system compared with an open vitrification system.MethodsA total of 332 vitrified-warmed blastocyst transfer cycles between April 2010 and May 2012 were analyzed retrospectively. The post-thaw survival rate, implantation rate, clinical pregnancy rate, live birth rate, and neonatal outcome were recorded.ResultsThere were no significant differences between the open vitrification group and the close vitrification group regarding the post-thaw survival rate (98% versus 95.8%), clinical pregnancy rate (47.6% versus 42.2%), implantation rate (42.9% versus 35.6%), and live birth rate (39.8% versus 32.1%). In total, 332 warming cycles produced 131 healthy babies. There were no significant differences in the mean gestational age, the birth weight, and the birth length between the two groups. No adverse neonatal outcomes were observed in the children born after the transfer of closed vitrified blastocysts compared with the transfer of open vitrified blastocysts.ConclusionsThese data suggest that blastocyst vitrification using a closed vitrification device seems safe and effective with results comparable to those obtained through open vitrification.

Postthaw survival of in vitro-produced bovine blastocysts loaded onto the inner surface of a plastic vitrification straw

In this study, we investigated whether vitrification of an embryo by attachment to the inner surface of a plastic straw, which requires a small volume of vitrification solution, improves the survival of thawed embryos. In vitro-produced Korean native cattle blastocysts were randomly assigned into four groups: (1) blastocysts attached to the inner surface of a plastic straw (aV); (2) blastocysts loaded into the column of a plastic straw (cV); (3) blastocysts directly dropped into liquid nitrogen (dV); and (4) nonvitrified blastocysts (control). The postthaw recovery rate did not significantly differ among the aV, dV, and cV groups (98.3% vs. 81.5% vs. 91.4%). The postthaw survival rate was greater in the control, aV, and dV groups than in the cV group (100%, 87.7%, and 81.8% vs. 26.4%, P < 0.05), but did not significantly differ among the control, aV, and dV groups. The total number of cells per blastocyst did not significantly differ among the groups (134.4 ± 38.9 in control vs. 114 ± 48.1 in aV, 105.6 ± 33.9 in dV, and 102 ± 35.1 in cV group). However, the number of apoptotic cells per blastocyst was higher in the dV and cV groups than in the control group (10.9 ± 9.6 and 14.5 ± 9.5 vs. 0.4 ± 1.4; P < 0.05), but did not significantly differ between the control and aV groups (0.4 ± 1.4 vs. 6.6 ± 9.5). In addition, the blastocoel of each blastocyst was left intact or was mechanically punctured to reduce its volume, and the blastocysts were then vitrified using the aV method. At 12 hours after thawing, the re-expansion rate did not significantly differ among the control, punctured aV, and nonpunctured aV groups (93.3% vs. 85.2% vs. 82.8%). However, at 24 hours after thawing, the hatching rate was greater in the control and punctured aV groups than in the nonpunctured aV group (75% and 62.9% vs. 37.1%; P < 0.05). The total number of cells per blastocyst was greater in the control group than in the nonpunctured aV group (143 ± 37.2 vs. 94.5 ± 18.6; P < 0.05), but did not significantly differ between the control and punctured aV groups (143 ± 37.2 vs. 119.4 ± 19.7). The number of apoptotic cells per blastocyst was greater in the nonpunctured aV group than in the control and punctured aV groups (11.3 ± 6.1 vs. 5.9 ± 5.8 and 6.3 ± 4.4; P < 0.05). Taken together, the data show that the aV method improved the survival and quality of vitrified-thawed blastocysts. Furthermore, puncture of the blastocoel increased the hatching rate and reduced the number of apoptotic cells in vitrified-thawed blastocysts generated using the aV method.