Equilibration Solution Research Articles

Title: Investigation of Kinetically Favorable Chromium Speciation Distribution for Iron Chromium Redox Flow Batteries

Chromium is a promising active material for redox flow battery (RFB) electrolytes due to its low cost and reduction potential very near the negative limit of the electrochemical window of water. However, many challenges exist with the performance of the Cr3+/2+, most notably sluggish electron-transfer kinetics and parasitic hydrogen evolution during the charge cycle. This presentation will focus on our recent efforts to overcome these challenges by understanding the interplay between electrolyte composition, speciation, and electron-transfer kinetics for the aqueous Cr3+/2+ redox couple.Among the earliest published work on flow batteries was a program based at NASA introducing the iron chromium redox flow battery (ICRFB) [1,2]. These studies were valuable in demonstrating both the promise and the complications of chromium for flow battery electrolytes. The NASA team went to great lengths to enhance chromium redox kinetics and suppress the hydrogen evolution via extensive modification of the carbon negative electrode with Au, Pb, and Bi additives. The present study is based on a key observation from this early work that the electron-transfer rates for Cr-chloride complexes depend strongly on the inner-sphere coordination environment, and specifically the number of chloride ions directly bound to the Cr center.Extensive prior studies from the inorganic coordination chemistry literature further demonstrate that Cr(III) chloride complexes undergo slow equilibration in aqueous solutions to form distributions of four species where the coordination environment around the metal center can be described generally as [CrClx(H2O)6-x](3-x)+ with 0 < x < 3 [3,4]. Each of these species yields a distinct optical absorption signature, enabling quantification of the composition with UV-VIS spectroscopy. Our overarching hypothesis, based on the early NASA work, was that increased chloride coordination (i.e., higher values of x in CrClx(H2O)6-x) would yield enhanced electron-transfer kinetics by reducing inner-sphere reorganization barriers associated with aquo ligands, or by enhancing catalytic mechanisms mediated by chloride surface adsorbates. Thus, we measured optical absorbance and electrochemical reversibility for Cr(III) chloride solutions with HCl, NaCl, and LiCl supporting electrolytes over a range of total chloride concentration from 0.1 to ~10 mol/L.Representative results are summarized in Figure 1 for 0.1 M CrCl3 in aqueous LiCl. Notably, at Au electrodes the reversibility of the Cr3+/2+ redox feature increases dramatically as chloride concentration increases above 5 mol/L (Figure 1a). This shift in reversibility is broadly correlated with UV-VIS results (Figure 1b) demonstrating that the primary Cr(III) species present initially remains the predominant species in the aqueous environment for high concentrations of chloride over time, but the predominant species shifts significantly at lower concentrations of LiCl. Ongoing work, which will be discussed in this presentation, is focused on full quantification of the relationship between speciation and electron-transfer kinetics as well as performance properties for ICRFBs with a range of supporting electrolyte concentrations. We anticipate that understanding the most kinetically favorable speciation distribution will allow us to develop improved electrolytes for functional ICRFBs.

P-116 Relationship between exposure in equilibration solution pre-vitrification for human collapsed blastocysts and miscarriage rate in ART treatments

Abstract Study question To explore the relationship between equilibration time pre-vitrification of human collapse blastocyst and clinical pregnancy and miscarriage rate in patients undergoing ART treatments Summary answer The miscarriage rate was significantly lower in the group with a short equilibration time of 7-8 minutes compared to the group of 9-10 minutes What is known already The introduction of vitrification represents one of the most important advancement in ART. However, protocols currently applied to cryopreserve human embryos still have some weak points that might be improved. A critical aspect is represented by the high concentration of cryoprotectants (CPAs) used during the vitrification and some of this CPAs might impact negatively cellular metabolism and function. Thus, this study investigated whether a shorter time in the equilibration solution before vitrification of artificially collapsed blastocysts might have an impact on survival and pregnancy outcomes, as well as live birth rate and risk of miscarriage in patients undergoing ART. Study design, size, duration This prospective study was performed at the Centre for Reproductive Medicine, Haikou Mary Hospital, China from March 2018 to May 2022. Informed consent for experimentation with human subjects was obtained before the patients start the ovarian stimulation. Female age &amp;lt;35 years, and causes of infertility included male factors, female infertility, and unexplained infertility. Following the fresh embryo transfer, supernumerary good quality blastocysts ≥ 2, according to Gardner’s score were vitrified and allocated as discussed below Participants/materials, setting, methods The study included a total of 831 expanded blastocysts, which were divided into two groups according to the equilibration time before vitrification: group (A) 7-8 minutes (413); and group (B) 9-10 minutes (418). Expanded blastocysts (grade 3 or more) were artificially shrunk by applying one or two laser pulses. Patients were included in the study only when at least two blastocysts were available for vitrification, in order to be allocated one in each group Main results and the role of chance Results: A total of 831 vitrified-warmed blastocysts were analysed in this study, of which 825 survived at the warming step (99.3%: 825/831). All surviving blastocysts were transferred in 585 embryo transfers. Overall, the clinical pregnancy rate per transfer was CPR: 68.5% (401/585), and live birth and miscarriage rates: 61.0% (357/585) and 11.0% (44/401). No significant differences were observed between the two groups regarding the mean age of patients, the average number of blastocysts transferred, the basal FSH, BMI, and infertility duration. Results show the same survival rate after warming for group A (99.3%) and group B (99.3%), as well as similar CPR (A: 69.1% versus B: 68.0%).The live birth (A: 63.8% versus B: 58.3%) and multiple gestation rates (A: 20.8% versus B: 23.5%) were comparable in the two groups. When analysing the overall miscarriage rate, data displayed a statistically significant difference (P &amp;lt; 0.05) in favour of group A (7.6%) compared to group B (14.2%). There were no differences between the two groups concerning the prevalence of male babies (A: 57.1% and B: 55.4%), average gestational length (A: 38.67±1.37 versus B: 38.33±1.21), preterm birth rate (A: 19.2% versus B: 20.6%), and birth weight (A: 2.95±0.58 versus B: 3.05±0.63 kg). Limitations, reasons for caution This is not a randomized controlled trial, it is a prospective observational cohort study aiming to calculate the effect of a shorted ES time on the risk of miscarriage. Also, potential confounding factors due to the heterogeneous nature of the sample investigated may impair the validity of our conclusions. Wider implications of the findings Results demonstrate that a shorter equilibration time resulted in optimal survival, clinical pregnancy, and live birth rates compared to exposure to ES for 9-10. Thus, suggesting that a longer exposure to the ES is not needed, and might negatively effect cellular metabolism and function, increasing the risk of miscarriage. Trial registration number Not applicatione

P-141 Elevating human oocyte vitrification and warming to a new level: fast, efficient, and effective

Abstract Study question Due to the rising trend for oocyte cryopreservation and increasing demand, there is an urgency to make oocyte vitrification and warming protocols more efficient. Summary answer Oocyte vitrification and warming can be done successfully in 4 minutes, reducing the time of commonly used current protocols by up to 25 minutes. What is known already Vitrification and warming of oocytes is widely used in many IVF clinics worldwide and routinely utilized as a dominant technique for fertility preservation and freezing of donor as well as autologous oocytes in many clinical settings. Outcome data using cryopreserved oocytes is comparable to fresh oocytes. However, vitrification and warming of human oocytes can be time consuming. Usually, vitrification of oocytes is done in about 11-16 minutes, and warming requires another 11 minutes. Also, it is a common practice to load not more than 2 oocytes per carrier at time of vitrification. Study design, size, duration From November 2022 to November 2023, 504 germinal vesicles (GV) and 207 metaphase I (MI) oocytes, which did not convert into mature oocytes at day of oocyte retrieval, were vitrified in Vit Kit Freeze (FujiFilm Irvine Scientific, USA), and RapidVit Oocyte (Vitrolife, Sweden). Their survival rates were compared, and as an additional viability indicator, post-warming developmental capabilities were evaluated after 24 and 48hrs of culture in Global High Protein (LifeGlobal, USA). Participants/materials, setting, methods A group of 4 GV or MI oocytes was vitrified at room temperature for 1 minute in Equilibration solution (ES) and 1 minute in Vitrification solution (VS), loaded on a S-Cryolock (Biotech, USA) and then plunged into liquid nitrogen. Warming was done at 37ºC in 0.5M sucrose for 1 minute and Wash solution (WS) for another minute. Post warming, GV and MI oocytes were cultured for developmental evaluation. Statistical significance was defined as P &amp;lt; 0.05. Main results and the role of chance There was no statistically significant difference in the survival rate of GV oocytes between Irvine (209/219; 95.4%), and Vitrolife media (270/285; 94.7%; P = 0.72). The survival of MI oocytes was also comparable and not significantly different between both media; Irvine: 103/106; 97.2%, vs Vitrolife: 96/101; 95.0%; P = 0.43). Because of the similar survival rates of oocytes in both groups, for the developmental potential and conversion rates, oocytes of both groups were combined. Of the 504 GV, 479 survived (95.0%) and after 24hrs of culture, 441 converted to MI (92.1%). After a culture of 48hrs, 56.5% (249/441) converted to metaphase II (MII) oocytes. Combining the vitrified and warmed MI oocytes from both groups, 96.1% (199/207) survived the warming, and after 24hrs of culture, 168 (84.4%) converted to MII oocytes. Vitrification and warming of oocytes at different developmental stages can be done with a short exposure time to hypertonic (ES/VS) and hypotonic (0.5M sucrose) solutions in a total of 4 minutes. Limitations, reasons for caution Immature oocytes were used as study material to evaluate a new protocol with significantly reduced vitrification and warming times. At this moment we cannot provide data on fertilization, embryo development, and blastocyst formation. Wider implications of the findings Oocytes are exposed to less suboptimal temperatures during dehydration and rehydration and help to maintain their developmental potential. Loading 4 oocytes per carrier combined with the decrease in time resulted in increased efficiency. Warming in 0.5M instead of 1M sucrose contributes positively to survival and allows earlier resumption of functionality. Trial registration number not applicable

Vitrification of feline ovarian tissue: Comparison of protocols based on equilibration time and temperature

Global contraction of biodiversity pushed most members of Felidae into threatened or endangered list except the domestic cat (Felis catus) thence preferred as the best model for conservation studies. One of the emerging conservation strategies is vitrification of ovarian tissue which is field-friendly but not yet standardized. Thus, our main goal was to establish a suitable vitrification protocol for feline ovarian tissue in field condition. Feline ovarian tissue fragments were punched with biopsy punch (1.5 mm diameter) and divided into 4 groups. Group 1 was fresh control (Fr), while the other three were exposed to 3 vitrification protocols (VIT_CT, VIT_RT1 and VIT_RT2). VIT_CT involved two step equilibrations in solutions containing dimethyl sulfoxide (DMSO) and ethylene glycol (EG) for 10 min each at 4 °C. VIT_RT1 involved three step equilibration in solutions containing DMSO, EG, polyvinylpyrrolidone and sucrose for 14 min in total at room temperature, while in VIT_RT2 all conditions remained the same as in VIT_RT1 except equilibration timing which was reduced by half. After vitrification and warming, fragments were morphologically evaluated and then cultured for six days. Subsequently, follicular morphology, cellular proliferation (expression of Ki-67, MCM-7) and apoptosis (expression of caspase-3) were evaluated, and data obtained were analysed using generalised linear mixed model and chi square tests. Proportions of intact follicles were higher in Fr (P = 0.0001) and VIT_RT2 (P = 0.0383) in comparison to the other protocols both post warming and after the six-day culture. Generally, most follicles remained at primordial state which was confirmed by the low expression of Ki-67, MCM-7 markers. In conclusion, VIT_RT2 protocol, which has lower equilibration time at room temperature has proven superior thus recommended for vitrification of feline ovarian tissue.

Vitrification of human blastocysts for couples undergoing assisted reproduction: an updated review.

Over the past 40years there has been a worldwide critical change in the field of assisted reproduction technology (ART), leading to the increased application of single blastocyst transfer, which is extremely important to avoid the risks of multiple pregnancy and associated complications for both mother and babies. Indeed, advancements in ART over the last few decades have been obtained thanks to several improvements, including ovarian stimulation, embryo culture conditions and, of course, progress in cryopreservation methods, especially with the application of vitrification. The ability to cryopreserve human embryos has improved significantly with vitrification compared to the initially adopted slow-freezing procedures. Since the introduction of vitrification, it has become the gold standard method to effectively cryopreserve human blastocysts. However, some new protocols are now being explored, such as the short warming procedure and even shorter exposure to the equilibration solution before vitrification, which seem to provide optimal results. Therefore, the main aim of the current narrative review, will be to illustrate the benefit of vitrification as an effective method to cryopreserve the human blastocyst and to illustrate new protocols and variations which in future may increase the performance of vitrification protocols.

In silico-designed vitrification protocols: an approach to improve survival of in vitro produced-bovine embryos.

In silico predictions validated in this study demonstrate the potential for designing shorter equilibration protocols that improve post-warming re-expansion and hatching rates of D7 and D8 in vitro-produced bovine embryos. Our results benefit the livestock industry by providing a refined and reproducible approach to cryopreserving bovine embryos, which, in addition, could be useful for other mammalian species. The cryopreservation of in vitro-produced (IVP) embryos is vital in the cattle industry for genetic selection and crossbreeding programs. Despite its importance, there is no standardized protocol yielding pregnancy rates comparable to fresh embryos. Current approaches often neglect the osmotic tolerance responses to cryoprotectants based on temperature and time. Hereby, we propose improved vitrification methods using shorter dehydration-based protocols. Blastocysts cultured for 7 (D7) or 8 days (D8) were exposed to standard equilibration solution (ES) at 25ºC and 38.5ºC. Optimized exposure times for each temperature and their impact on post-warming re-expansion, hatching rates, cell counts, and apoptosis rate were determined. In silico predictions aligned with in vitro observations, showing original volume recovery within 8 min 30 s at 25ºC or 3 min 40 s at 38.5ºC (D7 blastocysts) and 4 min 25 s at 25ºC and 3 min 15 s at 38.5ºC (D8 blastocysts) after exposure to ES. Vitrification at 38.5ºC resulted in D7 blastocysts re-expansion and hatching rates (93.1% and 38.1%, respectively) comparable to fresh embryos (100.0% and 32.4%, respectively), outperforming the 25ºC protocol (86.2% and 24.4%, respectively; P < 0.05). No differences were observed between D7 and D8 blastocysts using the 38.5ºC protocol. Total cell number was maintained for D7 and D8 blastocysts vitrified at 38.5ºC but decreased at 25ºC (P < 0.05). Apoptosis rates increased post-warming (P < 0.05), except for D8 blastocysts vitrified at 38.5ºC, resembling fresh controls. In conclusion, based on biophysical permeability data, new ES incubation times of 3 min 40 s for D7 blastocysts and 3 min 15 s for D8 blastocysts at 38.5ºC were validated for optimizing vitrification/warming methods for bovine IVP blastocysts.

Successful vitrification of equine embryos >300 microns without puncture or aspiration.

Equine embryos >300 μm require puncture before vitrification. Protocols that do not require pre-puncture would make vitrification easier and allow for its widespread use. To design a successful vitrification protocol for embryos >300 μm without puncture as a pre-treatment. Experimental in vivo study. Thirty-eight embryos were divided into 3 groups (G1: ≤300 μm, n = 11; G2: >300-500 μm, n = 20; G3: >500 μm, n = 7). Embryos were vitrified using a human vitrification kit. Following a 15 min exposure to equilibration solution (ES; 7.5% DMSO +7.5% ethylene glycol [EG] in a base medium [BM] of M199 HEPES-buffered medium [H199] + hydroxypropyl cellulose + gentamycin), embryos were exposed for ≤90 s to a vitrification solution (15% DMSO +15% EG + 0.5 M trelahose in BM), loaded onto a Cryolock and plunged into LN2. Warming was undertaken by plunging the Cryolock tip into 1 mL of H199 + 20% FBS + pen/strep +1 M sucrose at 42°C for 1 min. The embryos were then moved to a 0.5 M sucrose solution for 4 min, then placed in Vigro Hold for 4 min prior to transfer to a recipient. Pregnancy rates were 81.8% (9/11) for G1, 80% (16/20) for G2, and 0% (0/7) for G3. The largest embryo to survive was 480 μm. Limited numbers and only one pregnancy was followed to term. Equine embryos ≤480 μm can be successfully vitrified using a protocol with a longer exposure time to the ES. This does not appear to have a negative effect on early embryonic development.

Ultra-Fast Vitrification: Minimizing the Toxicity of Cryoprotective Agents and Osmotic Stress in Mouse Oocyte Cryopreservation.

Globally, women have been adopting oocyte cryopreservation (OC) for fertility preservation for various reasons, such as inevitable gonadotoxic treatment for specific pathologic states and social preferences. While conventional vitrification (C-VIT) has improved the success rate of OC, challenges of possible toxicities of high-concentration cryoprotective agents and osmotic stress persist. To overcome these challenges, we evaluated the ultra-fast vitrification (UF-VIT) method, which reduces the equilibration solution stage exposure time compared to C-VIT by observing mouse oocyte intracellular organelles and embryonic development. Consequently, compared to fresh mouse oocytes, UF-VIT presented significant differences only in endoplasmic reticulum (ER) intensity and mitochondrial (MT) distribution. Meanwhile, C-VIT showed substantial differences in the survival rate, key ER and MT parameters, and embryonic development rate. UF-VIT exhibited considerably fewer negative effects on key MT parameters and resulted in a notably higher blastocyst formation rate than C-VIT. Meiotic spindle (spindle and chromosomes) morphology showed no significant changes between the groups during vitrification/warming (VW), suggesting that VW did not negatively affect the meiotic spindle of the oocytes. In conclusion, UF-VIT seems more effective in OC owing to efficient cytoplasmic water molecule extraction, osmotic stress reduction, and minimization of cell contraction and expansion amplitude, thus compensating for the drawbacks of C-VIT.

Effect of pituitary adenylate cyclase-activating polypeptide supplementation, applied during or after vitrification on mouse embryo.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with widespread occurrence and diverse functions. It occurs in high levels in the gonads suggesting a potential central role in reproduction. The aim of our study was to assess the effect of PACAP treatment during embryo vitrification on the developmental rate and the expression of the heparin-binding EGF-like growth factor gene (Hbegf). Mouse embryos, obtained from superovulated females were allocated into the four treatment groups. In EM1 and EM2, the embryos were prepared for vitrification in an Equilibration Solution that was supplemented with 1 or 2 μM PACAP1-38, respectively. The embyos in groups CM1 and CM2 were not treated prior to vitrification but were cultured in a medium supplemented with 1 or 2 μM PACAP1-38 after thawing. The Vitrified Control group consisted of embryos vitrified and thawed then cultured without PACAP1-38 treatment. A non-vitrified, non-treated Fresh Control group was also used. After 24 h of culture, the developmental rate of the embryos, as well as the relative expression level of the Hbegf gene, as determined by qPCR, were compared among groups. Higher developmental rate and Hbegf gene expression level were found in the embryos treated with a higher concentration of PACAP. These results indicate that PACAP treatment has a beneficial effect on the survival and development of vitrified/thawed mouse embryos.

Definition of protocols for cryopreservation and three-dimensional in vitro culture of prepubertal goat testicular tissue after histomorphological, ultrastructural, and functional analysis

This study aims to define the best method (slow freezing or vitrification) and fragment size (1, 5, or 9 mm³) for prepubertal goat testis cryopreservation, as well as to evaluate testicular morphological integrity after cryopreservation and in vitro culture (IVC). Initially (experiment I), 1, 5, or 9 mm³ testis fragments were cryopreserved by slow freezing using a Mr. Frosty container with 20% Dimethylsulfoxide (DMSO) or vitrified using the Ovarian Tissue Cryosystem (OTC) device, (Equilibration solution – ES: 10% DMSO and 10% ethylene glycol – EG; Vitrification solution - VS: 20% DMSO and 20% EG) and then subjected to morphological analysis, type I and III collagen quantification and gene expression (Oct4, C-kit, Bax, and Bcl-2). Subsequently, (experiment II), fresh or cryopreserved by slow freezing testis fragments were cultured in vitro and submitted to morphological analysis by scanning electron microscopy. The data from the experiment I revealed fewer morphological alterations in 1 and 5 mm³ fragments after vitrification and slow freezing, respectively. The percentage of type I collagen fibers in 5 and 9 mm³ frozen was higher than in fresh or vitrified fragments. For type III collagen, fresh or frozen fragments of 1 and 5 mm3 showed a higher percentage than fragments of 9 mm3. Gene expression for Oct4 and C-kit after slow freezing or vitrification in the 5 mm3 fragments was lower than that observed in the fresh fragments. The Bax:Bcl-2 ratio in the 1 and 9 mm³ fragments was lower than in the 5 mm³ fragments for fresh fragments or after freezing. In experiment II, fragments cultured in vitro, previously frozen or not, showed more morphological alterations than fresh or frozen fragments. We concluded that slow freezing of 5 mm³ fragments was the best protocol for cryopreserving prepubertal goat testis and although the results of IVC are encouraging, it still needs improvement to restore testicular function after cryopreservation.

A recently developed minimum volume, absorbent, vitrification device, the Kitasato Vitrification System gives excellent outcomes for in vitro produced bovine blastocysts

Cryopreservation of embryos is a crucial component of current assisted reproductive technologies (ART). While the ART outcomes for many species have been greatly improved by the introduction of minimum volume vitrification devices, these devices can be difficult to handle and load. To reduce this problem, we recently developed a vitrification carrier which has a highly absorbent surface so that it simply and rapidly removes excess free vitrification solution from the specimen before the cooling step. This Kitasato Vitrification System (KVS) gives excellent results for human and mouse embryo vitrification. This study aimed to determine whether the KVS would also be effective for bovine blastocyst vitrification by comparing outcomes for the control device that was the KVS without excess vitrification solution absorber. The effect of varying the length of time spent in the first equilibration solution (0–10 min) was also evaluated. Vitrification with the KVS resulted in significantly higher survival and hatching rates than with the control device loaded with the same volume of vitrification solution (survival: 98.6% vs 87.6%, hatching at 72 h post warming: 87.3% vs 66.7%, respectively). The best outcomes were obtained with a 10 min equilibration step prior to exposure to the vitrification solution for 30 s. We also evaluated the effect of embryo quality on blastocyst viability when using the KVS. Survival rates of high- and low-quality embryos were comparable but low quality embryos had significantly lower hatching rates. Overall, the results indicate that the KVS vitrification device is effective for bovine blastocyst vitrification.

P-190 Spontaneous blastocyst collapse during pre-vitrification equilibration is related to a lower live birth rate: a prospective cohort study

Abstract Study question This report provides updated data from a study investigating the association between the spontaneous collapse patterns of blastocysts during pre-vitrification equilibration and pregnancy success. Summary answer Live birth rates were lower for completely collapsed blastocysts, suggesting the possibility for including blastocyst collapse pattern as a criterion for selecting embryos for transfer. What is known already Previous time-lapse studies have found that blastocysts exhibiting strong contraction during culture have a low probability of hatching in animals and implanting in humans, suggesting blastocyst contraction has a negative impact on reproductive outcomes. Conversely, artificial shrinkage of human blastocysts before vitrification is considered to improve embryo survival and/or pregnancy rates by promoting cryoprotectant permeation inside the blastocoel. However, shrinkage is not induced in some vitrification protocols, including ours, as the viability is high; this demonstrates the potential of existing protocols to achieve sufficient cryoprotectant permeation. Moreover, the Alpha consensus meeting on cryopreservation did not issue recommendations regarding artificial shrinkage. Study design, size, duration This study included 798 patients who were undergoing their first autologous IVF/ICSI cycle followed by a freeze-all strategy, including blastocyst vitrification, at our clinic between June 2018 and November 2021. For patients with multiple vitrified blastocysts, embryos for transfer were selected hierarchically based only on morphological scoring during culture. To reduce bias, only data from a single blastocyst (day 5, Gardner score 4, excluding CC) transfer from each patient’s first warmed cycle was analyzed. Participants/materials, setting, methods Blastocysts were vitrified-warmed in in-house-prepared solutions using Rapid-i carriers. Prior to vitrification, intact blastocysts were equilibrated in 10% ethylene glycol (15 min, 37 °C). Their spontaneous collapse patterns were assessed under an inverted microscope before they were vitrified in 15% ethylene glycol + 15% dimethylsulfoxide + 0.5 M sucrose. Blastocyst collapse was defined as the separation of the trophectoderm cells from the zona pellucida. Collapsed blastocysts with/without a blastocoel cavity were defined as partially/completely collapsed blastocysts. Main results and the role of chance Non-collapsed (Nc), partially collapsed (Pc), and completely collapsed (Cc) blastocyst transfers (embryo survival rate, 99.6%) constituted 27.1%, 54.6%, and 18.3% of the 798 cycles, respectively. Live birth rates differed significantly between groups (Nc, 54.2%; Pc, 46.6%; Cc, 34.2%; P &amp;lt; 0.001). Regarding perinatal outcomes of 366 singletons, the female rate tended to differ between groups (Nc, 35.3%; Pc, 44.3%; Cc, 53.1%; P = 0.084). Significant differences in maternal age (years: Nc, 34.6 ± 4.0; Pc, 35.4 ± 3.7; Cc, 36.4 ± 4.0; P &amp;lt; 0.001), MII oocyte number (Nc, 13.0 ± 6.2; Pc, 12.0 ± 6.6; Cc, 10.8 ± 6.9; P = 0.009), blastocyst diameter (μM: Nc, 184 ± 18; Pc, 181 ± 20; Cc, 173 ± 23; P &amp;lt; 0.001), and good-quality (4AA/4AB/4BA) blastocyst percentage (Nc, 70.4%; Pc, 61.2%; Cc, 47.9%; P &amp;lt; 0.001) were found between groups. Upon logistic regression analysis including these potential confounders, only maternal age (odds ratio (OR), 0.89; 95% confidence interval (CI), 0.86–0.93; P &amp;lt; 0.001) and collapse pattern (Cc/Nc: OR, 0.59; 95% CI, 0.37–0.93; P = 0.023) significantly impacted the live birth rate, whereas blastocyst morphology (good/not: OR, 1.37; 95% CI, 0.97–1.94; P = 0.069) tended to impact it. Limitations, reasons for caution The cryoprotectant concentration of our pre-vitrification equilibration solution is relatively low. Thus, caution is required when assessing the collapse pattern, as the solution osmolarity can influence blastocyst shrinkage. Follow-up studies with more participants are warranted to confirm these results and the health of the children born after vitrified-warmed embryo transfers. Wider implications of the findings A negative relationship was found between spontaneous blastocyst collapse during pre-vitrification equilibration and live birth rate. Studying blastocyst collapse pattern may assist selection of the most viable blastocysts after vitrification in ethylene glycol and dimethylsulfoxide—a widely used permeable cryoprotectant combination—thus, increasing IVF success rates. Trial registration number Not applicable

Embryo morphokinetics derived from fresh and vitrified bovine oocytes predict blastocyst development and nuclear abnormalities

Embryo development is a dynamic process and critical stages may go unnoticed with the use of traditional morphologic assessments, especially the timing of embryonic divisions and aberrant zygotic cleavage patterns. Bovine embryo development is impaired after oocyte vitrification, but little is known about the underlying morphokinetic behavior. Here, bovine zygotes from fresh (n = 708) and vitrified oocytes (n = 182) were monitored by time-lapse imaging and the timing and nature of early blastomere divisions were modeled to find associations with blastocyst development at day 8. The predictive potential of morphokinetic parameters was analyzed by logistic regression and receiver operating characteristic curve analysis to determine optimal cut-off values. Lag-phase was highly correlated with embryo development. Remarkably, 100% of zygotes that reached the blastocyst stage showed a lag-phase. Fast first cleavage increased the chance of blastocyst development to 30% with a cut-off of 32 h and 22 min. Aberrant zygotic cleavage events, including multipolar division, unequal blastomere sizes, and membrane ruffling resulted in decreased blastocyst development. Multipolar division leads to uneven blastomeres, which was associated with anuclear and multinuclear blastomeres, indicating genome segregation errors. Moreover, we described for the first time morphokinetics of embryos derived from vitrified bovine oocytes. Vitrification severely affected blastocyst development, although lower cryoprotectant concentration in equilibration solutions seems to be less detrimental for embryo yield. Impaired development was linked to slow cleavages, lower lag-phase incidence, and increased early embryonic arrest. Typically, less than 15% of the embryos produced from vitrified oocytes reached more than eight cells. Interestingly, the rate of abnormal first cleavage events was not affected by oocyte vitrification. In conclusion, time to first cleavage, the presence of a lag-phase, and the absence of aberrant zygotic cleavage were the best predictors of bovine blastocyst development for both fresh and vitrified oocytes.

Cryopreservation of African painted dog (Lycaon pictus) ovarian tissue.

Development of techniques for the preservation and use of gonadal tissues are increasingly needed for the genetic management of the endangered African painted dog (Lycaon pictus). Here we evaluated two cryopreservation techniques for ovarian tissue (2 × 2 × 1 mm3 fragments, n = 11 individuals): needle immersed vitrification (NIV), with equilibration in a 7.5% dimethyl sulfoxide (DMSO) and 7.5% ethylene glycol (EG) solution, and vitrification in a 15% DMSO, 15% EG, and 0.5 M sucrose solution, and slow freezing in cryovials with either the equilibration (SF-E) or vitrification (SF-V) solutions. Following warming, tissues were either fixed and embedded for evaluation of density of morphologically normal follicles, semi-quantitative scoring of stromal cell preservation, and apoptotic index (TUNEL stain), and/or flash-frozen for expression of proliferation (PCNA), apoptosis (CASP3, BCL2), or oxidative stress (GPX3, SOD1, SOD2) pathway genes (n = 4). Needle immersed vitrification maintained higher density of morphologically normal follicles compared to the slow freezing protocols applied (p < 0.05), with no significant changes in expression of select genes among treatment groups. A slight increase in apoptotic index was observed in all cryopreservation groups, but only reached significance in SF-E compared with fresh tissue controls (p < 0.05). Future research should be dedicated to developing improved methods for ovarian tissue culture in the species, both as a means to evaluate the efficacy of tissue cryopreservation techniques and for the production of viable oocytes from banked ovarian tissue in the endangered African painted dog.

Vitrification of the ovarian tissue in sturgeons

The aim of this study was to test whether vitrification of sterlet Acipenser ruthenus and Russian sturgeon Acipenser gueldenstaedtii ovarian tissue through needle-immersed vitrification (NIV) is an efficient strategy for the preservation of oogonia (OOG) in order to supplement the current conservation efforts for these endangered fish species. Histological analyses of the gonads displayed that the ovaries of both species were immature and contained predominantly OOG and primary oocytes. The germline origin of these cells was verified by localization of the vasa protein through immunocytochemistry. NIV protocol was optimized by testing different equilibration (ES) and vitrification solutions (VS) containing various concentrations of dimethyl sulfoxide (Me2SO), propylene glycol (PG) or methanol (MeOH). In sterlet, the highest average viability (55.7 ± 11.5%) was obtained by using a combination of 1.5 M PG and 1.5 M Me2SO in the ES, and 1.5 M MeOH and 5.5 M Me2SO in the VS. In Russian sturgeon, the highest average viability (49.4 ± 17.1%) was obtained by using a combination of 1.5 M MeOH and 1.5 M Me2SO in the ES, and 3 M PG and 3 M Me2SO in the VS. To test whether vitrified/warmed OOG are functional, we have conducted an intra-specific transplantation assay to verify whether transplanted sterlet OOG will colonize the gonads of recipient fish. Fluorescently labelled cells were detected within recipient gonads at 2 and 3 months post-fertilization (mpf). Colonization rates of vitrified/warmed OOG (70% at 2 mpf and 61% at 3 mpf) were similar to those of fresh OOG (80% at 2 mpf and 70% at 3 mpf). This study has demonstrated that vitrification of ovarian tissue is an effective method for the preservation of OOG, and that the vitrified/warmed cells are functional and are able to colonize recipient gonads after transplantation similarly to the fresh cells. Since the vitrification procedure displayed in this study is simple and does not require complex and expensive laboratory equipment, it can be readily applied in field conditions, and therefore it can be invaluable for the conservation efforts of the critically endangered sturgeon species. However, care needs to be taken that despite the research conducted so far, donor-derived progeny was not yet obtained in sturgeons.

Specifics of vitrification of in vitro-produced cattle embyos at various development stages

Producing embryos in vitro is an important technology used to improve the genetic potential of cattle and perfect the programs of their breeding. Regardless of the way they are produced, all embryos that had not been used for transplantation to recipients must be conserved. Because of significantly increased interest in the problem of cryoconservation of embryos, both coming from scientists and businesses, there are emerging new commercial environments that allow the facilitation of cryoconservation and the increase in the embryo survival. Oocyte-cumulus complexes obtained from the ovaries of slaughtered clinically healthy cows matured in 22–24 h in in vitro conditions. The oocytes were co-cultured with spermatozoids in Fertilization medium, and the obtained zygotes were cultured in Culture medium with Sodium-Pyruvate for 4 or 7 days up to the stage of morula or blastocyste, respectively. For the vitrification of cow embryos, we used a commercial kit for the vitrification of human embryos, having compared the duration of equilibration. According to the results of the studies, we observed high efficiency of cryoconservation of cow embryos using the commercial kit for vitrification of human embryos. The results revealed the significant effect of equilibration on survival and further development of embryos. In addition, we described the dependence of development stage of cattle embryo on the duration of the contact of embryo with equilibration solution. Therefore, optimal time of contact of cattle embryos at the morula stage with equilibration solution was 12 minutes. On the 24th h after thawing, 46.7 ± 3.3% of the embryos were observed to undergo blastulation, and on 48th h, this parameter increased to 96.7 ± 3.3%, which corresponded to the parameters in the group of embryos that had not been subjected to cryoconservation. In the conditions of further cultivation, the percentage of blastocystes that hatched in the experimental group was no different from that of the control. At the same time, the highest efficiency of vitrification of blastocystes of cows was seen after the contact with the equilibration solution for 15 min, since the percentage of hatched blastocystes was the same as in the control group. Therefore, using the commercial kit for vitrification of human embryos is beneficial, for it promotes the parameters of cow embryos after vitrification/thawing that are similar to such of intact embryos (without freezing). The data we analyzed and presented in the paper could help to increase the efficiency of cryoconservation of cattle embryos for both scientific and commercial purposes.

Solution Chemistry of Dihydroxyacetone and Synthesis of Monomeric Dihydroxyacetone.

Dihydroxyacetone (DHA) is a major byproduct of e-cigarette combustion and is the active ingredient in sunless tanning products. Mounting evidence points to its damaging effects on cellular functions. While developing a simple synthetic route to monomeric [13C3]DHA for flux metabolic studies that compared DHA and glyceraldehyde (GA) metabolism, we uncovered that solid DHA ages upon storage and differences in the relative abundance of each of its isomer occur when reconstituted in an aqueous solution. While all three of the dimeric forms of DHA ultimately resolve to the ketone and hydrated forms of monomeric DHA once in water at room temperature, these species require hours rather than minutes to reach an equilibrium favoring the monomeric species. Consequently, when used in bolus or flux experiments, the relative abundance of each isomer and its effects at the time of application is dependent on the initial DHA isomeric composition and concentration, and time of equilibration in solution before use. Here, we make recommendations for the more consistent handling of DHA as we report conditions that ensure that DHA is present in its monomeric form while in solutions, conditions used in an isotopic tracing study that specifically compared monomeric DHA and GA metabolism in cells.

Impact of equilibration duration combined with temperature on the outcome of bovine oocyte vitrification

The cryopreservation of mammalian oocytes and embryos has become an integral part of assisted reproduction in both humans and veterinary species. However, the methods used to cryopreserve bovine oocytes still have significant shortcomings. A wide variety of approaches has been used to try to improve and optimize methods of cryopreservation. However, these procedures employed are not always designed to specifically take account of the osmotic tolerance response of the cells according to the temperature and time of cryoprotectant (CPA) addition. When these properties are considered, optimal procedures for the addition of CPAs can be designed proactively. Based on in silico and in vitro osmotic observations, we propose shorter dehydration-based protocols at different temperatures (25°C vs. 38.5°C) towards defining an improved cryopreservation method. In vitro matured oocytes were exposed to equilibration solution (ES) at 25°C and 38.5°C and effects of optimized exposure times for each temperature were determined prior to vitrification/warming on oocyte spindle configuration, DNA fragmentation, and further embryo development. Upon exposure to standard ES (7.5% dimethyl sulfoxide + 7.5% ethylene glycol in TCM199 medium + 20% fetal bovine serum), original oocyte volume was recovered within 2 min 30 s at 38.5°C and 5 min 30 s at 25°C. In vitro matured oocytes were then exposed to the aforementioned cryoprotectants at both temperature/duration conditions and vitrified/warmed. While similar percentages of oocytes exhibiting a normally configured spindle and DNA fragmentation were observed in the fresh control group and oocytes vitrified at 38.5°C, significantly higher apoptosis rate and lower percentages of normal spindle configuration were observed in oocytes vitrified at 25°C when compared to control fresh oocytes. Similar cleavage rates and blastocyst yields were observed in the vitrified/38.5°C and fresh controls, while these rates were lower in vitrified/25°C. These results revealed that the limitation of the exposure time of the oocytes to the ES to the point of osmotic equilibrium volume recovery could be a more efficient approach to prepare them for vitrification. Therefore, exposure time to ES to 2 min 30 s at 38.5 °C appears to improve the quality of vitrified/warmed oocytes by protecting spindle integrity and reducing DNA fragmentation thus improving blastocyst rates and embryo quality.

PHOSPHORUS AVAILABILITY IN SOIL INCUBATED WITH BIOCHAR: ADSORPTION STUDY

ABSTRACT Considering the losses of phosphorus (P) in the soil by the adsorption process and that the use of biochar seems promising from the agricultural point of view, the aim of this research was to evaluate P adsorption by the Ultisol incubated with different biochar doses through kinetic and isothermal study. The experiment was carried out with poultry litter biochar pyrolyzed at 350 °C. Ultisol was incubated with increasing biochar doses, from 0.0 to 61.95 t ha-1. After 60 days, the phosphorus adsorption rate, kinetics and adsorption isotherms were evaluated. The results showed that the soil had the highest capacity to adsorb phosphorus (1.105 mg g-1). Biochar was not a good P adsorbent; regardless of the applied doses, it released P to the equilibration solution. Langmuir model was better at describing the adsorption of P. The pseudo-second order model fitted well to the adsorption kinetics of P, showing that the adsorption is chemically controlled. These results suggest that biochar can increase the availability of P, limiting adsorption; therefore, it could be used as a fertilizer and/or soil conditioner.

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.