Equilibration Solution Research Articles

Equilibration solution composition and extended exposure to equilibration phase affect embryo development and lipid profile of mouse oocytes

Research questionCan exposure time to equilibration solutions during oocyte vitrification affect the lipid profile of oocytes and embryonic development? Could vitrification media supplemented with oleic, linoleic acids and L-carnitine effectively minimize damage induced by vitrification on embryo development and oocyte membrane lipid profile? DesignExperimental study including 936 oocytes from C57BL/6J mice, randomly divided into fresh IVF (control) and equilibration solution groups. Oocytes were exposed to equilibration solution from Irvine Scientific, Tvitri-4 or Tvitri-4 supplemented with L-carnitine and fatty acids for 7 or 10 min, vitrified–warmed, and submitted to IVF. The lipid profile of oocytes immediately after equilibration solution exposure was also asessed using the same equilibration times and solution compositions. ResultsLonger equilibration time resulted in lower oocyte survival and blastocyst rates, and reduced relative abundance of structural lipids, i.e. phosphatidylcholines and sphingomyelins, varying according to equilibration solution composition. It also induced membrane disruptions resembling bubbles in the oocyte surface predominantly in equilibration solution from Irvine Scientific, rarely in Tvitri-4 and absent in Tvitri-4 supplemented with L-carnitine and fatty acids. To reveal the metabolic pathways associated with the equilibration phase of vitrification, lipid pathway analysis was conducted; both P-values and pathway impact values showed that the linoleic acid metabolism (P = 0.00223; impact =1) and alpha-Linolenic acid metabolism (P = 0.00084; impact = 0.33) were the most pathway perturbed, followed by glycerophospholipid metabolism (P = 0.0167; impact = 0.25) ConclusionA longer equilibration phase pre-vitrification can influence embryo development and induce changes in oocyte lipid composition related to membrane integrity. The results suggest internalization of oleic and linoleic acids added to equilibration solution by the oocyte, which, to some extent, contributed to membrane phospholipids preservation, regardless of the equilibration times assessed.

The vitrification system may affect preterm and cesarean delivery rates after single vitrified blastocyst transfer

ABSTRACT The purpose of this study was to investigate the possible effects of different vitrification systems on single vitrified blastocyst transfer cycles. The clinical and birth outcomes of 412 patients who underwent single vitrified blastocyst transfer between January 2018 and June 2020 were retrospectively analyzed and compared between patients who underwent blastocyst vitrification with kit A (group A, 196 patients) and those who underwent blastocyst vitrification with kit B (group B, 216 patients). Clinical outcomes, including the clinical pregnancy rate, ongoing pregnancy rate, early miscarriage rate, late miscarriage rate, ectopic pregnancy rate, twin pregnancy rate, and induced labor rate due to fetal malformation, were not significantly different between the two groups (P > 0.05). The preterm delivery rate among singleton newborns (11.57% vs. 3.23%, P < 0.05) and the cesarean delivery rate were significantly higher in group B than in group A (70.25% vs. 57.26%, P < 0.05). Birth outcomes, including the male-to-female ratio, low-birth-weight rate, macrosomia rate, birth defect rate, newborn gestational age, neonatal body weight, and singleton neonatal body length, were not significantly different (P > 0.05). Our findings suggest that different vitrification systems might differentially affect birth outcomes. Such disparity could reflect differences in kit composition and/or protocol. ABBREVIATIONS: DMSO: dimethyl sulfoxide; ES: equilibration solution; VS: vitrification solution; BMI: body mass index; ICSI: intracytoplasmic sperm injection; OR: odds ratio; CI: confidence interval

Effect of Different Cryoprotectant Agents on Mitochondrial Distribution and Oocyte Developmental Competence in the Buffalo (Bubalus Bubalis )

Oocytes cryopreservation in mammalian species has gained a rapid pace during the past couple of decades emphasizing its importance in various assisted reproductive technologies .Aim of this work was to study effects of different cryoprprotectant agents: Ethylene Glycol (EG), Dimethyl sulfoxide (DMSO) and combination of them (EG+DMSO) on the viability, mitochondrial distribution and intensity of in vitro matured vitrified/ thawed buffalo oocytes by determine: 1) the effect of different cryoprotectants on the viability and developmental competence of vitrified/thawed in vitro matured buffalo oocytes. 2) The effect of different cryoprotectants on the mitochondria distribution and intensity of vitrified/thawed in vitro matured buffalo oocytes. Ovaries were collected from EL-Warak slaughter house, Cairo, Egypt. Excellent and good oocytes were in vitro matured in TCM-199 +IGF for 22 h in incubator at 38.5 ₒC in 5% CO2 and humidified atmosphere. Morphologically normal matured oocytes with first polar were placed in equilibration solution (VS1) for 1 min then oocytes were transferred to (VS2) for 30 sec. VS1 is half concentration of VS2 which contain EG 40% (EG group) or DMSO 40% (DMSO group) or EG 20%+DMSO 20% (Combination group). Oocytes were loaded in sterile 0.25 ml straws and stores in liquid nitrogen for 7-10 days. Morphological changes (normal and abnormal) of vitrified thawed in vitro mature buffalo oocyte were detected under invertd microscope. Mitotracker red stain and confocal microscope (Zeiss 710) used to study the mitochondrial distribution and Hochest dye to study the viability of in vitro vitrified/ thawed matured buffalo oocytes. The obtained results revealed that using a combination of EG+DMSO for vitrification resulted in the best quality of in vitro vitrified thawed buffalo oocytes which was demonstrated by significantly higher percent of morphologically normal recovered oocytes and transferable embryos when compared with EG and DMSO. More mitochondria were diffusely distributed in the fresh oocytes (93.33%) than in all vitrified groups oocytes: DMSO group (63.33%), EG group (79.99%) and DMSO+ EG group (79.99%). The Mean No. of mitochondrial intensity of recovered mature oocytes vitrified in EG+DMSO group (186.22) was significantly higher (P < 0.05) than those vitrified in EG group (153.33) and DMSO group (146.98). In conclusion: Combination of EG+DMSO cryoprotectants improve the viability and developmental competence of in vitro matured vitrified/thawed buffalo oocytes.

Successful vitrification of manually punctured equine embryos.

Successful vitrification of equine expanded blastocysts requires collapse of the blastocoele cavity using a micromanipulator-mounted biopsy pipette on an inverted microscope. Such equipment is expensive and requires user skill. To develop a manual method of blastocoele collapse prior to vitrification using commercial products. In vivo experiment. Seventy-nine Day 7 or 8 embryos were measured and graded. Twenty were vitrified following micromanipulator-assisted puncture and aspiration before being used to validate commercial human vitrification and warming kits containing, respectively, 2-step concentrations of DMSO and ethylene glycol (7.5%-15% v:v) and decreasing concentrations of sucrose. After warming, embryos were transferred to recipient mares. Once validated, the commercial kits were used to vitrify and warm a further 39 embryos which were punctured manually using a microneedle, 2 (5%) were damaged during puncture and excluded; 20 more embryos were vitrified without puncture. Embryos were grouped as follows: non-punctured≤300µm (n=10) and>300 to≤560µm (n=10), punctured small (>300 to≤560µm; n=17) and large (>560µm; n=10) and exposed to the equilibration solution (ES) in the kit for 6min. An additional group of punctured large embryos was exposed to ES for 8min (n=10). For the initial warming step, embryos were exposed for 1min to the thawing solution at 42°C, before being moved to a dilution solution at room temperature. Vitrified, manually punctured embryos≤560µm exposed to ES for 6min resulted in a pregnancy rate of 82% (14/17). Unpunctured embryos≤300µm gave an 80% (8/10) pregnancy rate. Larger unpunctured embryos, punctured embryos>560µm and embryos exposed to ES for 8min gave significantly reduced pregnancy rates. Limited group sizes. High pregnancy rates can be achieved by manually puncturing≤560µm equine embryos prior to their vitrification and subsequent warming in commercial media.

25 Effect of taxol and epothilone B on meiotic spindle stabilization in vitrified bovine oocytes

Vitrification has the potential to be a valuable technique for preservation of bovine oocytes; however, this method often results in abnormal microtubule and chromosome arrangement. The aim of this experiment was to evaluate taxol and epothilone B as meiotic spindle stabilising pretreatments in a vitrification protocol. Bovine oocytes were purchased and matured invitro during shipment. At 18h of maturation, oocytes were divided randomly into control, taxol, and epothilone B treatments (Table 1). All treatments were prepared in invitro maturation (IVM) medium (IVF Biosciences). Partially denuded oocytes were incubated in either control or treatment medium for 15min at 38.5°C before vitrification. Oocytes were incubated in an equilibration solution (10% dimethyl sulfoxide, 10% ethylene glycol) for 5min, transferred to a vitrification solution (20% dimethyl sulfoxide, 20% ethylene glycol, 0.5M sucrose), loaded onto a Cryolock, and plunged into liquid nitrogen within 45s. For warming, a Cryolock was placed directly into a 0.5M sucrose solution and incubated for 3min. Oocytes were transferred to a 0.25M solution for 3min and washed in the basal solution used for vitrification and warming media (Dulbecco’s phosphate-buffered saline, 20% fetal bovine serum). Once warmed, oocytes were transferred to IVM medium for a 4-h recovery period and completely denuded before staining. Staining to evaluate spindle morphology was performed with anti α-tubulin primary antibody and secondary antibody Alexa Fluor 488. Oocytes were also stained with Hoechst to evaluate chromosome arrangement. Both spindle morphology and chromosome arrangement data were analysed using a logistic regression with a binomial response variable (normal/abnormal). Both 0.5μM and 1.0μM Taxol treatments had no effect on either meiotic spindle or chromosome arrangement compared with the control group (P&amp;gt;0.05). The 2.0μM taxol treatment improved chromosome configuration (P&amp;lt;0.05) with no effect on microtubule distribution compared with the control group (P&amp;gt;0.05). All epothilone B treatments resulted in disruption of microtubule distribution and chromosome arrangement compared with control (P&amp;lt;0.001) and resulted in a consistent abnormality hypothesised to be tubulin polymerization. These results indicate that taxol is capable of increasing the occurrence of normal chromosome arrangement in vitrified bovine oocytes and that epothilone B may cause additional harm to the oocyte that is not associated with the metaphase plate. Table 1. Effect of stabilisation agents on meiotic spindle of invitro-matured bovine oocytes Treatment n Normal microtubule distribution (%) Normal chromosome arrangement (%) Control 100 44 47 0.5μM Taxol 104 44 37 1.0μM Taxol 98 43 56 2.0μM Taxol 102 49 62a 0.5μM Epothilone B 103 11b 11b 1.0μM Epothilone B 97 6b 8b 2.0μM Epothilone B 100 2b 1b aP&amp;lt;0.05;. bP&amp;lt;0.001: Different superscripts within a column indicate a significant difference.

28 Evaluation invitro of two protocols of vitrification from alpaca (Vicugna pacos) embryos

The reproductive efficiency of South American camelids as the alpaca is low, with a few number of animals having a good genetic characteristic. The transfer of cryopreserved embryos has great potential to disseminate valuable genetic, but the suitable protocol for such cryopreservation still needs to be developed. In this study, two protocols of vitrification of alpaca embryos were tested. Day 6.5 post-mating, embryos (n=66) were recovered from 14 female alpacas through a non-surgical technique and classified according to the characteristics of old world camelids reported by Skidmore et al. 2004 (Reprod. Fertil. Dev. 16, 605–609). Only quality 1 and 2 embryos were used for the study. They were placed together in 50-µL drops of holding medium for 30min and transferred to a 100-µL drop of equilibration solution 1, consisting of 7.5% (v/v) ethylene glycol (EG) + 0.25M sucrose. After 1min, embryos were transferred to equilibration solution 2, consisting of 15% (v/v) EG + 0.5M sucrose. After 2min, embryos were transferred into 2 consecutive drops of vitrification solutions A [SA: 30% (v/v) EG + 1M sucrose] for 20s each, then in 2 other drops of vitrification solution B [SB: 30% (v/v) EG + 3% glycerol + 1M sucrose] for 20s each. Thereafter, embryos were quickly loaded into open pulled straws (OPS) in a volume of 10µL and then plunged into liquid nitrogen. For warming, the OPS were held in air for 5s and subsequently thawed at 37°C for 50s. Straws were emptied into 1mL of prewarmed holding medium solution (HMS1) containing 1M sucrose for wash and the thawed blastocysts were transferred into a second 1mL of prewarmed HMS1. After 5min incubation at 37°C, the blastocysts were transferred into 1mL of warmed Holding medium solution 2 (HMS2) containing 0.5M sucrose maintained at room temperature (∼24°C) for evaluation. Data were analysed by the Chi-squared test. Post-thaw embryo expansion results were 81.3% and 58.8% for SA and SB (P&amp;lt;0.05), respectively. Post-thaw embryo quality (1 and 2) were found at 62.5% and 29.1% with SA and SB, respectively (P&amp;lt;0.05). In conclusion, the vitrification of alpaca embryos with the ethylene glycol:sucrose solution results in better post-thaw outcomes than the ethylene glycol:sucrose:glycerol. Further experiments with embryo transfer are needed. This research was funded by FONDECYT project no. 149-2017.

Uterine receptor activation in response to uterotonic agents in late-pregnancy rat myometrium following pretreatment with oxytocin: an in vitro study

Background: Long term exposure to oxytocin reduces the ability of myometrium to respond to oxytocin, leading to oxytocin receptor (OXTR) desensitization. In this study we analyzed the response to other uterotonics such as prostaglandin, as well as investigating prostaglandin E2 receptors (EP3) and prostaglandin F2α receptors (FP). We hypothesized that compensatory mechanisms would increase the expression and activation of FP and EP3 following OXTR desensitization. Methods: Myometrium from late-pregnancy rats was collected in order to assess mRNA expression levels for OXTR, FP, and EP3 using RT-PCR. This was done after 2 hours of pretreatment with 10-6 M oxytocin to induce OXTR desensitization, or equilibration in physiological salt solution (PSS). Myometrium was exposed to increasing concentrations of uterotonic agents (10-10 to 10-5 M) following 2 hours of pretreatment with 10-6 M oxytocin (experimental group) or with PSS (control group). Myometrium from the experimental group was washed with PSS and OXTR expression was assessed using Western blot and RT-PCR. Results: mRNA expression levels for EP3, FP and OXTR were not statistically different between the experimental (OXTR desensitization) and control groups. Compared to the control group, the (mean ± SD) contractile potency of carboprost (pEC50: 7.74 ± 0.56 vs 6.81 ± 0.25, P = 0.03) and maximal contractility of misoprostol (Emax(ratio): 4.44 ± 3.60 vs 1.32 ± 0.22, P = 0.02) were significantly increased in the OXTR desensitization group, while the contractility of oxytocin was significantly reduced (Emax(ratio): 1.62 ± 0.27 vs 2.82 ± 0.98, P = 0.015). No significant differences in myometrial OXTR expression were observed between the PSS, carboprost and misoprostol groups following OXTR desensitization. Discussion: Following OXTR desensitization of myometrium, FP and EP3 activation increased in a compensatory manner, but not FP and EP3 receptor expression.

Biotechnological approaches to the preservation and use of bovine ovarian cumulus-oocyte complexes in the system of reproductive technologies

Aim. To study the effect of different concentrations of ethylene glycol and glycerin in equilibration and vitrifi cation solutions on 1) the viability and further development of frozen-thawed bovine ovarian cumulus-oocyte complexes (COCs), cryopreserved by vitrifi cation method, 2) on the effectiveness of inseminating mature oocytes, obtained from them, and 3) on the formation of embryos. Methods. Biotechnological, cryobiological, morphological, cytogenetic, and statistical methods, as well as methods of statistical data processing were used in the research. Results. The results of experimental studies on the effect of different concentrations of ethylene glycol and glycerin in the equilibration solution during cryopreservation of bovine ovarian COCs (n = 502) on their viability and further development after freezing-thawing are presented. We also show the results of the comparative analysis of cryoresistant properties of bovine ovarian COCs (n = 560) using different concentrations of ethylene glycol and glycerin, as cryoprotectants, in the vitrifi cation solution in terms of the viability and maturation of the oocytes, which originated from these COCs, up to metaphase II of meiosis. A comparative analysis of the application of ethylene glycol and glycerol in differ- ent concentrations for the equilibration and vitrifi cation solutions in cryopreserving bovine ovarian COCs (n = 220) demonstrated a relationship between the level of concentration of these cryoprotectants and the number of embryos after in vitro insemination of mature gametes, obtained from these frozen-thawed COCs. Conclusions. It was found that the use of 25 % ethylene glycol and 5 % glycerin in the equilibration solution and 10 % ethylene glycol and 40 % glycerin in the vitrifi cation solution during cryopreservation of bovine ovarian COCs ensures lower toxicity of these solutions and promotes more effi cient (up to 14.3 %) formation and development of embryos after in vitro insemina- tion of mature gametes, obtained from these COCs

Development and ultrastructure of bovine matured oocytes vitrified using electron microscopy grids

The aim of this study was to establish a methodology of cryopreservation of cattle oocytes and the quality assessment of oocytes and subsequent embryos produced in vitro under our laboratory conditions. Previously in vitro matured (IVM) oocytes were vitrified in minimum volume by ultra-rapid cooling technique. The oocytes were put into the equilibration solution (3% ethylene glycol in M199-HEPES + 10% foetal bovine serum) for 12 min, transferred to vitrification solution (30% ethylene glycol + 1 M sucrose in M199-HEPES + 10% foetal bovine serum) at room temperature for 25 s, then placed onto nickel electron microscopy grids and plunged into liquid nitrogen. After warming 75% of the oocytes were assessed as viable. Part of viable oocytes was taken for electron microscopy, the remaining oocytes were fertilized in vitro, and the presumptive zygotes were cultured until the blastocyst stage. Embryo cleavage and blastocyst rates in vitrified group after warming were 64.98% and 17.3%, resp. versus 70.72% and 25.54% in the control group (P < 0.05). No significant differences were found in the blastocyst total cell number, TUNEL and dead cell indexes between both groups. Ultrastructure of vitrified oocytes showed damages in smooth endoplasmic reticulum (SER) vesicles and lipid droplets as well as irregular arrangement of solitary cortical granules. Several mitochondria were damaged and the microtubules around the chromosomes were less occurred compared to the control group. However, the extent of injuries was lower than reported by other authors studying the ultrastructure of vitrified bovine oocytes, what is also supported by the better development of our oocytes after IVF. In conclusion, the designed oocyte vitrification technique ensures obtaining the blastocysts of the quality comparable to the fresh oocytes.

RELATIONSHIP BETWEEN THE DURATION OF EQUILIBRATION STEP OF THE VITRIFICATION AND CHANGES ON ABUNDANCE AND COMPOSITION OF OOCYTE MEMBRANE LIPIDS

Volumetric damages and cytotoxicity due to cryoprotectants exposure are time-sensitive in the vitrification process and can trigger the lipid remodeling of the plasma membrane. Here, we investigate the effects of exposure time to equilibration solution (ES) on membrane lipid profile of C57BL/6J mice oocytes using MRM-profiling, a sensitive exploratory method for lipidomics of oocytes and embryos1. We also assessed the effects of supplementing ES with antioxidants and unsaturated fatty acids. Experimental groups included oocytes equilibrated with Irvine Scientific (IRV), a commercial standard vitrification medium; Tvitri-4 (T4) produced in small scale for research by INVITRA®, and Tvitri-4 supplemented with L-carnitine (LC) and oleic and linoleic fatty acids (FA) (T4-LC/FA; Patent n. BR102019013697-9). Experimental study. Oocytes were randomly divided in 7 groups: non-exposed (NE) and exposed to either 10-minute equilibration step (ES10) according to the manufacturer’s procedure or 7-minute equilibration step (ES7), using IRV, T4, or T4-LC/FA media. After equilibration, oocytes were washed in methanol: H2O and lipids extracted with methanol. Diluted lipid extracts were flow injected into the triple quadrupole spectrometer with electrospray ionization (ESI). Relative ion intensities were used for univariate (ANOVA, fold-change, t-test, volcano plot) and multivariate analysis (PCA, PLS-DA, cluster analysis). Most informative lipids were sorted out using statistical significance of p<0.05 or partial least square discriminant analysis (PLS-DA) variables of importance (VIP) scores>1. One-way ANOVA showed PC (38:8) differently represented among NE, ES7 and ES10 groups, upregulated in T4-LC/FA oocytes. In ES7 groups, two by two comparisons between IRV, T4 and T4-LC/FA using volcano plot (p-value≤0.05; fold-change≥2.0) detected 4, 5, and 4 significant lipids between IRV vs. T4, IRV vs. T4-LC/FA, and T4 vs. T4-LC/FA, respectively, with the PEo(36:1) overrepresented in IRV (fold-change=12.5). In ES10 groups, were detected 11, 19 and 4 lipids differentially represented among IRV vs. T4, IRV vs. T4-LC/FA, and T4 vs. T4-LC/FA, respectively. PLS-DAVIP scores identified in IRV oocytes 8 free- fatty acids, phosphatidyletanolamine PEo(36:1), phosphatidylcholines PCo(32:0) and PC(36:3), phosphatidylserines PS(14:1), PS(16:0), PS(30:2), PSo(40:5), and phosphatidylinositols PI(22:4) and PI(40:8) among the top features, whereas unsaturated PC and SM predominated in T4 and T4-LC/FA oocytes. Significant lipids observed between T4 and T4-LC/FA pointed oleic (18:1) and linoleic (18:2) acids upregulated in T4-LC/FA oocytes exposed for 10 min to ES. The duration of the equilibration phase changed the abundance and composition of membrane lipids. Phospholipids were upregulated at 10-minute exposure to ES, except in IRV oocytes. The oleic and linoleic acids used as supplements in T4-LC/FA medium were promptly detected in the oocytes and seem contribute to better preserving membrane phospholipids during cell equilibration.

Effect of equilibration time on clinical and neonatal outcomes in human blastocysts vitrification.

PurposeProlonged exposure to equilibration solutions may be detrimental to an embryo's developmental potential, whereas a shorter exposure may affect the penetration of cryoprotectants into blastomeres. The purpose of this study was to evaluate the effects of different equilibration times on the clinical and neonatal outcomes of human blastocyst vitrification.MethodsThis is a retrospective study based on data collected between November 2008 and November 2015. A total of 192 blastocysts (80 non‐expanded and 112 expanded) obtained from 167 patients were analyzed. The blastocysts were divided into two groups according to their equilibration time: 8‐11 minutes or 12‐15 minutes. The clinical and neonatal outcomes of warmed blastocysts were evaluated.ResultsThe survival, implantation, and live birth rates of non‐expanded blastocysts were not different between the two groups, but they significantly improved for the expanded blastocysts in the 12‐15 minutes group compared to the 8‐11 minutes group. The results were similar for the neonatal outcomes after vitrified embryo transfer, when partitioned by equilibration time and blastocyst stage at vitrification.ConclusionsFor the non‐expanded blastocysts, a shortened equilibration time (8‐11 minutes) is sufficient for effective vitrification.

NMR Spectroscopic and Theoretical Studies on Tautomerism and Isomerism of Perfluoroalkyl‐Substituted 1,5‐Benzodiazepines

AbstractThe tautomerism and isomerism of several ethyl 2‐(4‐(perfluoroalkyl)dihydro‐2H‐benzo(naphtho)[b][1,4]diazepin‐2‐ylidene)acetates were investigated by 1H‐, 13C‐, 15N‐ and 19F‐NMR spectroscopy and DFT computations. Depending on the polarity and basicity of the solvent a different behaviour was observed. In CDCl3, (D6)acetone and CD3CN solution two chelate hydrogen bonded tautomers, the enaminoimine and the diaminodiene, were found to exist. In the polar and more basic solvents (D6)DMSO and (D7)DMF two further species, the chelate ring‐opened isomers, were detected. The ratio of the isomers is influenced by the solvents and the substituents at the chelate ring. The chelate enaminoimine is favoured in the less polar chloroform, whereas the chelate diaminodiene predominates with increasing polarity of solvents. The length of the perfluoroalkyl chain and the substituents of the phenyl ring do not influence the isomeric equilibrium as much as the chlorine atom in 4‐position. The structures of the isomers were unambiguously confirmed by one‐ and two‐dimensional techniques (1H,1H‐COSY, 1H,1H‐NOESY (EXSY), 1H,13C‐ and 1H,15N‐chemical shift correlation spectra (HSQC and HMBC)). The observed 15N NMR data are typical for enamines, amides and diazepines and additionally confirm the proposed structures of the four isomers. The time‐dependent course of the equilibration in DMSO solution was followed by recording 1H and 19F spectra. Calculated Gibbs free energies (ΔG) correlate essentially with our experimental findings.

Assessment of DNA Repair Gene Expressions in Vitrified Mouse Preantral Follicles.

Objective Vitrification of the ovarian tissue is one of the techniques recommended for preserving the fertility of women who are dealing with infertility. Despite its benefits, our information about the molecular aspects of ovarian follicles vitrification is somehow ambiguous. Therefore, the aim of this study was to evaluate the expression pattern of DNA repair genes in vitrified preantral follicles. Materials and Methods In this experimental study, the isolated preantral follicles (n=906) from 14-16 days old mice (n=12) were divided into three groups: fresh, toxic and vitrified which were cultured in vitro for 12 days. Preantral follicles were vitrified using cryotop followed by exposure to equilibration solution for five minutes and vitrification solution (VS) for 30 seconds. In the toxic group, preantral follicles were only placed in equilibration and vitrification media and they were then placed in the warming solutions without exposure to liquid nitrogen. On the second and sixth days of the culture period, real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was carried out to evaluate expression of the selected genes involved in DNA repair, including Msh6 (MutS homolog 6), Mre11 (Meiotic recombination 11), Brca1 (Breast cancer type 1), Rad51 (RAD51 recombinase), Pcna (Proliferating cell nuclear antigen) and Atm (ATM serine/threonine kinase). In addition, developmental parameters including growth, survival rate, antrum cavity formation and ovulation were analyzed. Results The relative mRNA expression of Msh6, Mre11, Brca1, Rad51, Pcna and Atm on the second and sixth days of the culture period in vitrified group was significantly higher than those of the control and toxic groups, but there was no significant difference between the toxic and control groups. In addition, developmental parameters of follicles were similar in both toxic and control groups, while both were significantly higher than that of vitrified group. ConclusionVitrification changes the expression pattern of DNA repair genes of the mouse preantral follicles.

28 Short equilibration improves vitrification of invitro-produced bovine embryos using VitTrans device

For the successful application of vitrification technology to field conditions, the procedures for the warming and transfer of the cryopreserved bovine embryos should be as simple as possible. The device VitTrans, designed by our group, enables warming/dilution of embryos and their transfer directly to recipient females in field conditions (Morato and Mogas 2014 Cryobiology 68, 288). VitTrans vitrification protocol consists of an incubation in equilibration solution during 12min followed by an exposure of 40s to vitrification solution. However, there are other reports using similar vitrification devices where equilibration length is shorter than ours. This study aimed to improve VitTrans methodology by comparing two vitrification protocols: short equilibration (SE) and long equilibration (LE). A total of 63 invitro-produced Day 7 blastocysts (IETS stage code 7) were randomly placed in an equilibration solution with 7.5% ethylene glycol + 7.5% dimethyl sulfoxide in holding medium (tissue culture medium-199 HEPES + 20% fetal calf serum) for either 3min (SE) or 12min (LE). Then, blastocysts were transferred to vitrification solution (15% ethylene glycol + 15% dimethyl sulfoxide + 0.5M sucrose in holding medium) for 40s, loaded onto the VitTrans device, plunged into liquid nitrogen, and covered with a 0.5mL straw. Fresh nonvitrified blastocysts (n=30) were set as control. For warming, the VitTrans was quickly submerged into a water bath at 45°C, while a syringe containing 0.3mL of diluting solution (0.5M sucrose in holding medium) at 45°C was injected through the hollow of the device. Blastocysts were then transferred to synthetic oviductal fluid medium and cultured for 24h at 38.5% in a 5% CO2 and 5% O2 environment in a humidified atmosphere. Re-expansion rates were recorded 3 and 24h after warming. Blastocysts were fixed and stained with SOX2 (Invitrogen) for inner cell mass (ICM) count, TUNEL (Roche) for apoptosis index assessment, and DAPI (Vector Laboratories) for total cell count (TCC). Images were captured using a Leica TCS SP5 confocal microscope (Leica Microsystems) and examined with Imaris 9.2 software (Oxford Instruments). Blastocyst survival rates were compared between groups using chi-squared test. Blastocyst TCC, ICM count, and apoptosis indices were analysed using analysis of variance. Significance was set at P ≤ 0.05. No differences were observed in re-expansion rate at 3h postwarming (61.3 and 59.4% for SE and LE, respectively). However, significantly higher re-expansion rates were found after 24h of culture for the blastocysts of the SE group (74.2%) when compared with the blastocysts of the LE group (65.7%). Blastocysts vitrified using the LE protocol produced the lowest TCC (115±5.9; P ≤ 0.05), whereas TCC of the SE (152±9.7) and fresh control (138±8.6) treatments were similar. No differences were found in ICM count among groups. Nevertheless, apoptosis index was higher (P ≤ 0.05) in both vitrification groups when compared with fresh control. These results indicate that short equilibration vitrification not only improves VitTrans outcomes but adds efficiency by taking less time to perform. Supported by MCIU, Spain (Project AGL2016-79802-P and Grant BES-2017-081962).

36 Extended culture after vitrification-warming helps in spindle recovery of bovine oocytes

The meiotic spindle is one of the most vulnerable cytoplasmic organelles when performing oocyte vitrification. It has been proposed that submitting oocytes to a post-warming incubation period in maturation medium helps in the reorganization of microtubules and chromosomes. Our previous experiments found no differences in spindle morphology after submitting vitrified oocytes to a 2-h incubation period. The aim of this experiment was to determine the effect of extended culture on the reorganization of the meiotic spindle of vitrified-warmed bovine oocytes. Oocytes were purchased from a commercial vendor (n=86) and matured during shipment. In this experiment, three treatments were evaluated: fresh oocytes (F) (n=30), vitrified-warmed (VW; n=26), and extended culture (EC; n=30). Cumulus-oocyte complexes were removed at 18h of maturation. Fresh oocytes were denuded by vortexing in hyaluronidase (1.5mgmL−1) and immediately fixed using 4% paraformaldehyde. Oocytes undergoing vitrification were partially denuded by pipetting in hyaluronidase (1.5mgmL−1). The vitrification protocol consisted of incubation in equilibration solution (7.5% dimethyl sulfoxide + 7.5% ethylene glycol) for 9min and then in vitrification solution (15% dimethyl sulfoxide + 15% ethylene glycol + 0.5M sucrose). While in vitrification solution, oocytes were mounted onto a Cryolock and plunged into liquid nitrogen in less than 1min. Warming was performed by placing a Cryolock into 0.5M sucrose for 3min and then into 0.25M sucrose for 3min. Finally, oocytes were washed in base medium. The base medium used for cryoprotectant and warming solutions was Dulbecco's phosphate-buffered saline supplemented with 20% fetal bovine serum. Both, vitrification and warming, were performed at 38.5°C. After warming, half of the oocytes were completely denuded and fixed and the other half underwent a 6-h incubation period in maturation medium (IVF-Bioscience). To examine microtubule distribution and chromosome arrangement, fixed oocytes were submitted to an immunostaining protocol using α tubulin antibody (1:100) and anti IgG-Alexa Fluor 488 (1:1000; Thermo Fisher Scientific) and counterstained with Hoechst. The effect of extended culture on the incidence of abnormal microtubule distribution and chromosome arrangement was analysed using logistic regression with a binomial response variable (normal/abnormal). There was no difference in maturation rates among groups (F=73.3%, VW=77%, EC=86.6%; P=0.43). For microtubule distribution, oocytes fixed immediately after warming had a higher incidence of abnormal spindles (57.7%) when compared with oocytes submitted to extended culture (26.6%; P=0.02). The most common abnormality seen in oocytes fixed after warming was small and faintly stained spindles. Microtubule distribution in fresh oocytes did not differ from oocytes in the other groups. There were no differences in chromosome arrangement among groups (P=0.11). Future research will focus on evaluating the benefits that this technique offers to improve development following IVF using vitrified-warmed oocytes.

Cryopreservation and transplantation of common carp spermatogonia.

Common carp (Cyprinus carpio) is one of the most cultured fish species over the world with many different breeds and plenty of published protocols for sperm cryopreservation. However, data regarding preservation of gonadal tissue and surrogate production is still missing. A protocol for freezing common carp spermatogonia was developed through varying different factors along a set of serial subsequent experiments. Among the six cryoprotectants tested, the best survival was achieved with dimethyl sulfoxide (Me2SO). In the next experiment, a wide range of cooling rates (0.5–10°C/min) and different concentrations of Me2SO were tested resulting in the highest survival achieved using 2 M Me2SO and cooling rate of -1°C/min. When testing different tissue sizes and incubation times in the cryomedia, the highest viability was observed when incubating 100 mg tissue fragments for 30 min. Finally, sugar supplementation did not yield significant differences. When testing different equilibration (ES) and vitrification solutions (VS) used for needle-immersed vitrification, no significant differences were observed between the tested groups. Additionally, varied exposure time to VS did not improve the vitrification outcome where the viability was 4-fold lower than that of freezing. The functionality of cryopreserved cells was tested by interspecific transplantation into sterilized goldfish recipients. The exogenous origin of the germ cells in gonads of goldfish recipient was confirmed by molecular markers and incorporation rate was over 40% at 3 months post-transplantation. Results of this study can serve for long-term preservation of germplasm in carp which can be recovered in a surrogate recipient.

22 Vitrification of bovine embryo using antifreeze polyamino acid

Carboxylated poly-l-lysine (CPLL) is an ampholytic polymer compound and a polyamino acid with a known functional resemblance to antifreeze proteins. We previously reported that CPLL is an effective cryoprotectant for bovine cells, sperm, and slow-frozen embryos. In this study, we investigated CPLL as a cryoprotectant for vitrified bovine embryos. We developed bovine embryos in vitro and vitrified them at the blastocyst stage. Embryos were equilibrated (3min) and vitrified (1min). Vitrified embryos were cryopreserved in LN (Cryotop® device; Kitazato Corp., Tokyo, Japan) for at least 1 week, thawed with a 0.3M sucrose warming solution, and then cultured in a basal medium (Gibco® medium 199, Grand Island, NY, USA; supplemented with 100µM 2-mercaptoethanol, 10% fetal bovine serum, and antibiotics) at 38.5°C in a humidified atmosphere (5% CO2, 5% O2, 90% N2). We evaluated the embryos morphologically for survival and hatched rate at 0, 24, 48, and 72h post-thawing. In control, the equilibration solution (ES) consisted of 7.5% (vol/vol) dimethyl sulfoxide (DMSO) and 7.5% (vol/vol) ethylene glycol, and the vitrification solution (VS) consisted of 16.5% (vol/vol) DMSO and 16.5% (vol/vol) ethylene glycol and 0.5M sucrose. In this study, CPLL was added to ES and VS at various concentrations instead of DMSO. The CPLL was added at 16.5, 11.0, 5.5, and 2.2% (wt/vol) to VS; respectively, these solutions were named P16.5, P11.0, P5.5, and P2.2. The ES was used 45% CPLL of VS each. Embryos underwent the above procedure concurrently, with testing replicated at least 3 times. We evaluated 88, 34, 38, 44, and 28 embryos with each solution (control, P16.5, P11.0, P5.5, and P2.2, respectively). Results were analysed statistically with a chi-square test and residual analysis, regarding P&amp;lt;0.05 as significant. Survival rates were significantly greater in P11.0 at 24h post-thawing (55.7% v. 89.5%; P&amp;lt;0.05) and in P11.0 and P5.5 at 48h post-thawing (47.7% v. 78.9% and 47.7% v. 79.5%, respectively; P&amp;lt;0.05) relative to controls but showed no significant differences at 0h post-thawing. Hatched rates were significantly greater in P11.0 and P5.5 through 72h post-thawing relative to controls (44.7% v. 22.7% and 52.3% v. 22.7%, respectively; P&amp;lt;0.05). The CPLL improved post-thawing embryo survival and hatched rates when applied during vitrification, thus demonstrating cryoprotective effectiveness. We conclude that CPLL acts as a low-toxicity cryoprotectant for vitrified bovine embryos, and our results are consistent with previous reports of protective CPLL effects for cells and cell membranes.

Effects of cryoprotectants and cryoprotectant combinations on viability and maturation rates of Camelus dromedarius oocytes vitrified at germinal vesicle stage.

Camel fertility faces many problems, which could be solved by assisted reproductive technologies (ARTs). We designed the experiment to explore the effect of different cryoprotectant concentrations and combinations on viability and maturation rates of vitrified/warmed camel oocytes. We collected ovaries directly after slaughtering from local abattoir and transported them to laboratory in a thermo-flask containing normal physiological saline. We aspirated the oocytes from follicles, which is 2-8mm in diameter, washed three times in TCM-199 and then examined under stereo-microscope for selection. We selected morphologically normal oocytes with an evenly granulated cytoplasm and a compact cumulus cell layer. We equilibrated morphologically normal oocytes in equilibration solution (ES), which is half concentration of vitrification one. After equilibration, We transported oocytes to vitrification solution using ethylene glycol (EG, 40%), dimethyl sulphoxide (DMSO, 40%) and EG 40%+DMSO 40%. The obtained results revealed that addition of EG 40%+DMSO 40% resulted in the best quality of vitrified/warmed oocytes, which is demonstrated by higher per cent survival rate (90.16%) and maturation rate (58.95%). While DMSO 40% resulted in 62.79% and 29.54%, respectively, EG 40% reported 86.11% and 53.47%, respectively. We could conclude that vitrification of immature camel oocytes by using 40% EG+40% DMSO is suitable methods to limit drawbacks of vitrification methods, and we need further studies to assess the ability of in vitro-produced blastocyst to develop in vivo and establish pregnancy after embryo transfer.

CRYOPRESERVATION OF OOCYTE-CUMULUS COMPLEXES OF COWS WITH DIFFERENT BIOACTIVE SUBSTANCES

Implementation of biotechnological process in livestock should be considered not only in terms of selection process intensification (obtain of embryos in vitro, their transplantation) and to a greater extent as development of effective methods of freezing and long storage of mammalian cells, including ova and embryos. Application of biotechnology in livestock breeding increases rate of genetic progress, preservation of gene pool of breeds via banks of sperm, embryos and cryobank of oocytes, obtaining and regulation of progeny of the desired sex, providing genetic evaluation of gametes and embryos, and it will enable to use genetic potential of animals after culling by age, replicate and create new genotypes with desired properties repeatedly. Solution to this problem is to improve medium and conditions of gametes and embryos freezing. Although the overall development of cryopreservation method is through simplification of the equilibration and vitrification solutions which would be able to ensure the full development frozen-thawed gametes. Addition of biologically active substances to the solution for cryopreservation contributes to protection of gametes during freezing and thawing, and determination of the consistent patterns of these substances will improve procedures of frozen-thawed oocyte cultivation outside the body. So it is necessary to deepen the fundamental research on the mechanisms of formation of a mature ovum of cows obtained from frozen-thawed oocytes to obtain embryos. The aim of the research is to conduct comparative analysis of different biologically active substances in equilibration and vitrification solutions at cryopreservation of oocyte-cumulus complexes of cows. Material and methods of the research. The objects of experimental studies were oocyte-cumulus complexes of black-and-white cows. The oocytes with homogeneous fine-grained ooplasm, undamaged pellucid zone, thick or partially loosened cumulus were used for freezing. The gametes of cows were treated by equilibration solution before freezing and then were transferred into vitrification solution. All the equilibration (10% glycerol + 20% propanediol) and vitrification (25% glycerol + 25% propanediol) solutions for cryopreservation of cows’ oocyte-cumulus complexes were prepared in Dulbecco phosphatebuffered saline with addition of 20% fetal serum of cows, 1х10-4 M unithiol, 1х10-6 M acetylcholine and without addition of bioactive substances. The research on adding some biologically active substances (fetal serum of cows – version A, unithiol – version B, acetylcholine – version C, without addition of biologically active substances – version D and not frozen cells of the control group (K)) in equilibration and vitrification solutions at freezing the oocyte-cumulus complexes of cows was carried out. It was found by the results of experimental studies that introduction of the above-mentioned components into the equilibration solution for freezing cows’ oocyte-cumulus complexes increased their cryoresistance, as evidenced by increasing indicator of maturation of frozen-thawed gametes outside the body to metaphase-2 of meiosis after 27-hour cultivation by 5,4-23,0% and decreasing indicator of number of oocytes with chromosomal abnormalities by 2,9-15,3%. The introduction of biologically active substances into the vitrification solution and subsequent cultivation during 27 hours after freezing and thawing cows’ oocyte-cumulus complexes showed that the indicator of maturation of frozen-thawed gametes outside the body to metaphase-2 of meiosis increased by 3,9-16,4% and the indicator of number of oocytes with chromosomal abnormalities decreased by 2,9-8,4%. Comparative analysis of the results of in vitro fertilization of cows’ frozen-thawed ova which were frozen using fetal serum of cows (version A) and without it (version - B) has shown a positive effect of adding it to equilibration and vitrification medium at freezing gametes of cows; it led to increase of obtaining bovine embryos in vitro by 11,5%. Thus, the analysis of experimental results showed different efficiency of using fetal serum of cows, unithiol, acetylcholine in the equilibration solution for freezing cows’ oocyte-cumulus complexes. The advantage of using these biologically active substances in the vitrification solution for cryopreservation of cow’s gametes by the indicator of maturation of frozen-thawed oocyte-cumulus complexes outside the body to metaphase-2 of meiosis wasn’t established. Conclusions. Introduction of fetal serum into cryopreservation solution increases cryoresistance of cow’s oocytes to cooling leading to increasing the indicator of the matured frozen-thawed gametes outside the body to metaphase-2 of meiosis by 23,0% and the obtained bovine embryos in vitro by 11,5%.

Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling.

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods for safe storage of genetic resources beyond the common practices of keeping breeding colonies. The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Cryopreservation of early-stage germ cells by whole testes NIV offers possibilities for the storage of zebrafish genetic resources, especially since after transplantation they can mature into both male and female gametes. Testes were excised, pinned on an acupuncture needle, equilibrated in two cryoprotective media (equilibration solution containing 1.5 M methanol and 1.5 M propylene glycol; and vitrification solution containing 3 M dimethyl sulfoxide and 3 M propylene glycol) and plunged into liquid nitrogen. Samples were warmed in a series of three consequent warming solutions. The main advantages of this technique are (1) the lack of spermatozoa after digestion of warmed testes thus facilitating downstream manipulations; (2) ultra-rapid cooling enabling the optimal exposure of tissues to liquid nitrogen therefore maximizing the cooling and reducing the required concentration of cryoprotectants, thereby reducing their toxicity; (3) synchronous exposure of several testes to cryoprotectants and liquid nitrogen; and (4) repeatability demonstrated by obtaining viability of above 50% in five different zebrafish strains.