The aim of this study was to compare the levels of reactive oxygen species (ROS) in Bos taurus and Bos indicus in vitro embryos cryopreserved using either slow freezing or vitrification. Embryos were divided into four groups based on subspecies and freezing method: Bos indicus slow freezing (BiSF; n = 8), Bos indicus vitrification (BiVT; n = 10), Bos taurus slow freezing (BtSF; n = 9), and Bos taurus vitrification (BtVT; n = 6). After thawing, the embryos were incubated with CellRox Green and images were obtained using a confocal microscope. The fluorescence intensity of each cell was measured and expressed as arbitrary units of fluorescence (auf) and compared using a multiple regression and unpaired t-test with α = 0.05. Results showed that subspecies and the freezing method significantly affected auf (P < 0.001; R2 = 0.1213). Bos indicus embryos had higher auf than Bos taurus embryos, whether frozen by slow freezing (67.05 ± 23.18 vs 51.30 ± 16.84, P < 0.001) or vitrification (64.44 ± 23.32 vs 47.86 ± 17.53, P < 0.001). Slow freezing induced higher auf than vitrification in both Bos taurus (51.30 ± 16.84 vs 47.86 ± 17.53, P < 0.001) and Bos indicus (67.05 ± 23.18 vs 64.44 ± 23.32, P < 0.014). In conclusion, Bos taurus embryos had lower ROS levels when frozen using vitrification, while Bos indicus embryos had consistent ROS patterns regardless of the freezing method. However, Bos indicus embryos frozen by slow freezing tended to have a higher number of cells with elevated ROS levels.

Testicular biopsies (9 mm3) from domestic cats (n = 10) submitted to orchiectomy were submitted to equilibrium vitrification in the presence of ethylene glycol (EG) alone or combined with dimethylsulfoxide (DMSO) as intracellular cryoprotectants, and sucrose or trehalose as extracellular cryoprotectants. The samples were vitrified with 40% EG or 20% EG + 20% DMSO, plus 0.1 M or 0.5 M of sucrose or trehalose. The study was divided into Step 1 and Step 2. In Step 1, intratubular cells (spermatogonia, spermatids, spermatocytes, and Sertoli cells) were quantified and classified as intact or degenerated (pyknotic and/or vacuolated cells). Cryodamage of seminiferous cords was determined by spermatogonia and Sertoli cell scoring of nuclei alterations, tubular basement membrane detachment, epithelium shrinkage, and tubular measures (total area, epithelium area, larger and smaller diameter, and height of the epithelium). In Step 2, Hoechst 33342 stain and propidium iodide (PI) fluorescent stain were used to assess the cell viability of the four best experimental groups in Step 1. The effect of treatments on all analyses was accessed using analysis of variance (ANOVA), and Fisher’s post hoc test at P < 0.05 significance was considered. In Step 1, the mean percentage of spermatogonia and Sertoli cells morphological integrity did not show a difference when using both sugars at different concentrations, but their morphology was more affected when DMSO was used. EG use associated with 0.1 M of sucrose or trehalose positively affected spermatocyte and spermatid morphology, respectively. The larger diameter and epithelium height of seminiferous tubules were increased using DMSO plus 0.5 M sucrose and DMSO plus 0.1 M trehalose. The changes in spermatogonial/Sertoli nucleoli visualization were best scored in the EG groups, while the nuclei condensation was lower with sucrose. The basement membrane was satisfactorily preserved with 0.1 M sucrose. In Step 2, the percentage of cell viability was higher when EG plus 0.1 M sucrose was used. Therefore, DMSO’s negative effect on the vitrification of testicular biopsies of adult domestic cats was evident. The EG plus 0.1 M of sucrose or trehalose associations are the most suitable CPAs to preserve the testicular histology structure of adult domestic cats in vitrification.

The influence of the method of evaluating developmentally competent oocytes on their viability after cryopreservation still needs to be better understood. The objective of this study was to determine the cleavage and embryo developmental rates after parthenogenetic activation of cumulus–oocyte complexes (COCs) selected by different concentrations of brilliant cresyl blue (BCB) and cryopreservation. In the first experiment, COCs were separated into groups and incubated for 1 h in medium containing BCB (13 μM, 16 μM, or 20 μM). The control group was not exposed to BCB staining. In the second experiment, COCs were divided into four groups: 13 μM BCB(+), 13 μM BCB(−), fresh control (selected by morphologic observation and immediately in vitro matured) and vitrified control (selected by morphologic evaluation, vitrified, and in vitro matured). In the first experiment, the 13 μM BCB group displayed greater development rates at the morula stage (65.45%, 36/55) when compared with the other groups. In the second experiment, cleavage (47.05%, 72/153) and morula development (33.55%, 51/153) of the control group of fresh COCs were increased compared with the other groups. However, when comparing morula rates between vitrified COC control and BCB(+) groups, the BCB(+) group had better results (19.23%, 5/26 and 64.7%, 11/17, respectively). Our best result in rat COC selection by BCB staining was obtained using a concentration of 13 μM. This selection could be a valuable tool to improve vitrification outcomes, as observed by the BCB(+) group that demonstrated better results compared with the vitrified COC control.

Since the birth of the first baby by in vitro fertilization in 1978, more than 9 million children have been born worldwide using medically assisted reproductive treatments. Fertilization naturally takes place in the maternal oviduct where unique physiological conditions enable the early healthy development of the embryo. During this dynamic period of early development major waves of epigenetic reprogramming, crucial for the normal fate of the embryo, take place. Increasingly, over the past 20 years concerns relating to the increased incidence of epigenetic anomalies in general, and genomic-imprinting disorders in particular, have been raised following assisted reproduction technology (ART) treatments. Epigenetic reprogramming is particularly susceptible to environmental conditions during the periconceptional period and non-physiological conditions such as ovarian stimulation, in vitro fertilization and embryo culture, as well as cryopreservation procedure, might have the potential to independently or collectively contribute to epigenetic dysregulation. Therefore, this narrative review offers a critical reappraisal of the evidence relating to the association between embryo cryopreservation and potential epigenetic regulation and the consequences on gene expression together with long-term consequences for offspring health and wellbeing. Current literature suggests that epigenetic and transcriptomic profiles are sensitive to the stress induced by vitrification, in terms of osmotic shock, temperature and pH changes, and toxicity of cryoprotectants, it is therefore, critical to have a more comprehensive understanding and recognition of potential unanticipated iatrogenic-induced perturbations of epigenetic modifications that may or may not be a consequence of vitrification.

Lipid accumulation occurs in cultured embryos and is associated with reduced cryotolerance. Here we report the use of a multiple pathway lipid modulator cocktail (l-carnitine, linoleic acid and forskolin) to improve cryosurvival. First, we stained oocytes and embryos with Oil Red to examine the time course of lipid accumulation during in vitro fertilization (IVF) and embryo culture. Then we evaluated the effects of the lipid modulators cocktail on lipid content, developmental rates and survival after vitrification. In our conditions, lipid accumulation was detected (P < 0.05) at the end of in vitro maturation (IVM) and after 4 days of embryo culture (D4-D5). In experiment 1, we used lipid modulator cocktail during IVM. Reduced (P < 0.05) lipid accumulation was detected in oocytes (Control: 49.9 ± 1.6, Lip. Mod. IVM: 45.0 ± 1.8) but no changes were present at blastocyst stage (Control: 62.4 ± 2.6, Lip. Mod. IVM: 66.8 ± 2.7). Treated oocytes presented decreased (P < 0.05) blastocyst rates and lower (P < 0.05) re-expansion after vitrification. In experiment 2, lipid modulators cocktail was used during embryo culture (from D4–D7 or D6–D7). Treatment had an effect on lipid metabolism, as lipid content was increased (P < 0.05) in D7 blastocysts in treated groups (Control: 52.7 ± 3.1a, D4: 65.9 ± 2.6b, D6: 78.1 ± 2.7b). However, no effect was present for cleavage, blastocyst and cryosurvival rates. No difference was detected in mean cell number comparing the three groups (Control: 78.9 ± 9.6, D4: 82.6 ± 16.5, D6: 68.3 ± 7.8), but apoptosis rate was increased (P < 0.05) in vitrified-warmed blastocysts from treated groups (Control: 14.77*, D4: 22.28, D6: 22.22). We concluded that the combined use of lipid modulators was efficient to promote changes in lipid content of oocytes and embryos in bovine, but those changes did not reflect positively on embryo development or cryosurvival.

This study aimed to describe the viability of domestic feline spermatozoa after epididymal tail vitrification. For this, 10 pairs of testis–epididymis complexes were used. The epididymal tails were vitrified using the solid-surface vitrification (SSV) method, in which two vitrification media containing ethylene glycol (EG) 40% or glycerol (GLY) 40% were tested. Vitrification with the presence of EG resulted in better results for all sperm motility parameters (motility, vigour and CASA) compared with GLY (P < 0.05). There were no statistical differences for sperm viability and acrosome integrity, plasma membrane integrity, or overall health of morphologically normal sperm before or after vitrification among experimental groups. In conclusion, epididymal tail vitrification appears to be a suitable method for long-term storage of cat sperm, especially if the procedure is performed with EG as the cryoprotectant.

This study aimed to evaluate to what extent the different interval times between trophectoderm (TE) biopsy and vitrification influence the clinical outcomes in preimplantation genetic testing (PGT) cycles. Patients who underwent frozen embryo transfer (FET) after PGT between 2015 and 2019 were recruited. In total, 297 cycles with single day 5 euploid blastocyst transfer were included. These cycles were divided into three groups according to the interval times: <1 h group, 1–2 h group, and ≥2 h group. Blastocyst survival, clinical pregnancy, miscarriage, and ongoing pregnancy rates were compared. The results showed that, in PGT-SR cycles, survival rate in the ≥2 h group (96.72%) was significantly lower than in the <1 h group (100%, P = 0.047). The clinical pregnancy rate in the ≥2 h group was 55.93%, significantly lower than in the <1 h group (74.26%, P = 0.017). The ongoing pregnancy rates in the 1–2 h group and the ≥2 h group were 48.28% and 47.46%, respectively, significantly lower than that in the <1 h group (67.33%, P < 0.05). The miscarriage rate in the 1–2 h group was 18.42%, significantly higher than that in the <1 h group (5.33%, P = 0.027). In PGT-A cycles, the clinical pregnancy and ongoing pregnancy rates in the <1 h group were 67.44% and 53.49%, respectively, higher than that in the 1–2 h group (52.94%, 47.06%, P > 0.05) and the ≥2 h group (52.63%, 36.84%, P > 0.05). In conclusion, vitrification of blastocysts beyond 1 h after biopsy significantly influences embryo survival and clinical outcomes and is therefore not recommended.

The objective of this study was to investigate any effect of cryopreservation of donated eggs on laboratorial and clinical outcomes of intracytoplasmic sperm injection (ICSI) cycles. This retrospective cohort study included 320 oocyte recipients undergoing 307 vitrified and 119 fresh oocyte recipient ICSI cycles, participating in an egg-sharing donation programme, from 2015 to 2018, in a private university-affiliated in vitro fertilization (IVF) centre. A review of donor and recipient ICSI cycles was charted. A general mixed models fit by restricted maximum likelihood, followed by Bonferroni post hoc test was used to compare the means between fresh and warm oocyte donation groups and investigate the effect of cryopreservation on recipient ICSI outcome. The main outcome measure was blastocyst development rates. Fertilization rate, high-quality embryo rates on days 2 and 3, normal cleavage speed rates on days 2 and 3, and blastocyst development rate were significantly higher for the fresh oocyte donation cycles compared with warmed oocyte donation cycles. In the egg-sharing donation programme, fertilization and embryo developmental competence were reduced when vitrified oocytes from infertile couples were used for ICSI compared with fresh oocytes.

Culture conditions have a profound effect on the quality of in vitro-produced embryos. Co-culturing embryos with somatic cells has some beneficial effects on embryonic development. Considering the ability of stem cells to secrete a broad range of growth factors with different biological activities, we hypothesized that bovine amniotic membrane stem cells (bAMSCs) might be superior to bovine oviduct epithelial cells (BOECs) in supporting embryonic development and enhancing their cryo-survival. Bovine abattoir-derived oocytes were matured and fertilized in vitro. The resultant presumptive zygotes were then cultured up to the blastocyst stage in the following groups: (i) co-culture with bAMSCs, (ii) co-culture with BOECs, and (iii) cell-free culture (Con). Embryos that reached the blastocyst stage were vitrified and warmed, and their post-warming re-expansion, survival and hatching rates were evaluated after 72 h culture. Results showed that the cleavage, blastocyst, and 2 h post-warming re-expansion rates of embryos did not differ between groups. However, their survival rates in BOEC and bAMSC groups were significantly higher compared with the control (72.7, 75.6 and 37.5%, respectively, P < 0.05). In conclusion, our results showed that the cryo-survivability of IVF-derived bovine embryos could be improved through co-culturing with bAMSCs. Moreover, considering the possibility to provide multiple passages from bAMSCs compared with BOECs, due to their stemness properties and their ability to produce growth factors, the use of bAMSCs is a good alternative to BOECs in embryo co-culture systems.

Characterizing non-lethal damage within dry seeds may allow us to detect early signs of ageing and accurately predict longevity. We compared RNA degradation and viability loss in seeds exposed to stressful conditions to quantify relationships between degradation rates and stress intensity or duration. We subjected recently harvested (‘fresh’) ‘Williams 82’ soya bean seeds to moisture, temperature and oxidative stresses, and measured time to 50% viability (P50) and rate of RNA degradation, the former using standard germination assays and the latter using RNA Integrity Number (RIN). RIN values from fresh seeds were also compared with those from accessions of the same cultivar harvested in the 1980s and 1990s and stored in the refrigerator (5°C), freezer (−18°C) or in vapour above liquid nitrogen (−176°C). Rates of viability loss (P50−1) and RNA degradation (RIN⋅d−1) were highly correlated in soya bean seeds that were exposed to a broad range of temperatures [holding relative humidity (RH) constant at about 30%]. However, the correlation weakened when fresh seeds were maintained at high RH (holding temperature constant at 35°C) or exposed to oxidizing agents. Both P50−1 and RIN⋅d−1 parameters exhibited breaks in Arrhenius behaviour near 50°C, suggesting that constrained molecular mobility regulates degradation kinetics of dry systems. We conclude that the kinetics of ageing reactions at RH near 30% can be simulated by temperatures up to 50°C and that RNA degradation can indicate ageing prior to and independent of seed death.

Numerous factors affect vitrification success and post-thaw development of oocytes after in vitro fertilization. Therefore, elaboration of an optimal methodology ensuring higher cryotolerance of oocytes and subsequent blastocyst yield is still of great interest. This paper describes and evaluates critical factors affecting the success of oocyte vitrification. In particular, an appropriate oocyte stage such as maturation status (germinal vesicle stage, metaphase II stage), presence/absence of cumulus cells before vitrification, and the effect of follicle size, as well as different culture systems and media for in vitro production of embryos, the types and concentrations of cryoprotectants, and cooling and warming rates at vitrification are considered. Special attention is paid to various cryocarriers used for low-volume vitrification, which ensures safe storage of oocytes/embryos in liquid nitrogen and their successful post-thaw recovery. At the end, we focussed on how age of oocyte donors (heifers, cows) influences post-thaw development. This review summarizes results of recently published studies describing different methodologies of cryopreservation and post-thaw oocyte development with the main focus on vitrification of bovine oocytes.

Visualizing biological structures and cellular processes in their native state is a major goal of many scientific laboratories. In the past 20 years, the technique of preserving samples by vitrification has greatly expanded, specifically for use in cryogenic electron microscopy (cryo-EM). Here, we report on improvements in the design and use of a portable manual cryogenic plunge freezer that is intended for use in laboratories that are not equipped for the cryopreservation of samples. The construction of the instrument is economical, can be produced by a local machine shop without specialized equipment, and lowers the entry barriers for newcomers with a reliable alternative to costly commercial equipment. The improved design allows for successful freezing of isolated proteins for single particle analysis as well as bacterial cells for cryo-electron tomography. With this instrument, groups will be able to prepare vitreous samples whenever and wherever necessary, which can then be imaged at local or national cryo-EM facilities.

The objective of this study was to compare the rates of clinical pregnancy after the transfer of vitrified and thawed human embryos on days 3, 4 and 5 of embryonic development. In this retrospective study, the results of 148 embryo transfer cycles, using embryos frozen and thawed over the 3-year period between January 2016 and December 2018 at the Gülhane Training and Research Hospital Department of Gynecology and Obsterics Reproductive Medical Center of the University of Health Sciences, Ankara, Turkey were examined. Following embryo transfer – including 29 dissolved embryos frozen on day 3, 80 frozen on day 4, and 39 frozen on day 5 – results were examined in terms of clinical pregnancy rates. In this study, across all three groups, no significant differences were observed in terms of patient age, the number of oocytes collected, infertility reasons, the number of embryos dissolved, transfer day, or the number of embryos transferred. According to the transfer day, the rates of clinical pregnancy and ongoing pregnancy were significantly higher for embryos frozen on day 4 and transferred on day 5. Significantly higher rates of pregnancy and live birth were determined during in vitro fertilization (IVF) treatment with the freezing of human embryos on day 4 and the transfer of those embryos on day 5.

This study evaluated the effects of oocyte meiosis inhibitors roscovitine (ROS) and butyrolactone I (BL-I) on in vitro production of bovine embryos. Bovine oocytes were maintained in pre in vitro maturation (pre-IVM) with 25 µM ROS or 100 µM BL-I for 24 h to delay meiosis and for 24 h in in vitro maturation (IVM). Following this treatment, the nuclear maturation index was evaluated. All embryos degenerated following this procedure. In the second set of experiments, oocytes were maintained for 6 or 12 h in pre-IVM with the following three treatments: ROS (25 µM or 12.5 µM), BL-I (100 µM or 50 µM) or a combination of both drugs (6.25 µM ROS and 12.5 µM BL-I). Oocytes were cultivated for 18 or 12 h in IVM. When a meiosis-inducing agent was used during pre-IVM for 24 h, more degenerated oocytes were observed at the end of the IVM period. This effect decreased when the meiotic blocking period was reduced to 6 or 12 h. No significant differences were observed in the blastocyst production rate of oocytes in pre-IVM for 6 h with ROS, BL-I, or ROS + BL-I compared with that of the control group (P > 0.05). However, inhibition of oocytes for 12 h resulted in decreased embryo production compared with that in the controls (P < 0.05). There was no difference in the post-vitrification embryo re-expansion rate between the study groups, showing that the meiotic inhibition for 6 or 12 h did not alter the embryo cryopreservation process.