Cryopreservation and assessment of genetic fidelity Acorus calamus Linn., an endangered medicinal plant.

BACKGROUND: Acorus calamus Linn. is a medicinally valuable monocot plant belonging to the family Acoraceae. Over-exploitation and unscientific approach towards harvesting to fulfill an ever-increasing demand have placed it in the endangered list of species. OBJECTIVE: To develop vitrification-based cryopreservation protocols for A. calamus shoot tips, using conventional vitrification and V cryo-plate. MATERIALS AND METHODS: Shoot tips (2 mm in size) were cryopreserved with the above techniques by optimizing various parameters such as preculture duration, sucrose concentration in the preculture medium, and PVS2 dehydration time. Regenerated plantlets obtained post-cryopreservation were evaluated by random amplified polymorphic DNA (RAPD) to test their genetic fidelity. RESULTS: The highest regrowth of 88.3% after PVS2 exposure of 60 min was achieved with V cryo-plate as compared to 75% after 90 min of PVS2 exposure using conventional vitrification. After cryopreservation, shoot tips developed into complete plantlets in 28 days on regrowth medium (0.5 mg/L BAP, 0.3 mg/L GA3, and 0.3 mg/L ascorbic acid). RAPD analysis revealed 100% monomorphism in all cryo-storage derived regenerants and in vitro donor (120-days-old) plants. CONCLUSION: Shoot tips of A. calamus that were cryopreserved had 88.3% regrowth using V cryo-plate technique and the regerants retained genetic fidelity. https://doi.org/10.54680/fr24210110412.

Effect of melatonin on developmental competence, mitochondrial distribution, and intensity of fresh and vitrified/thawed in vitro matured buffalo oocytes.

BACKGROUND: In livestock breeding, oocyte cryopreservation is crucial for preserving and transferring superior genetic traits. This study was conducted to examine the additional effect of melatonin to maturation and vitrification media on the in vitro developmental capacity, mitochondrial distribution, and intensity of buffalo oocytes. The study involved obtaining ovaries from a slaughterhouse and conducting two phases. In the first phase, high-quality oocytes were incubated in a maturation medium with or without 10-9M melatonin for 22 h (at 38.5°C in 5% CO2). Matured oocytes were fertilized in vitro and cultured in SOF media for seven days. In the second phase, vitrified in vitro matured oocytes were stored in vitrified media (basic media (BM) containing a combination of cryoprotectants (20% Ethyl Glycol and 20% Dimethyl sulfoxide), with or without melatonin, and then stored in liquid nitrogen. Normal vitrified/thawed oocytes were fertilized in vitro and cultured as described. Finally, the matured oocytes from the fresh and vitrified/thawed groups, both with and without melatonin, were stained using DAPI and Mitotracker red to detect their viability (nuclear maturation), mitochondrial intensity, and distribution using a confocal microscope. The study found that adding 10-9M melatonin to the maturation media significantly increased maturation (85.47%), fertilization rate (84.21%)cleavage (89.58%), and transferable embryo (48.83%) rates compared to the group without melatonin (69.85%,79.88%, 75.55%, and 37.25% respectively). Besides that, the addition of melatonin to the vitrification media improved the recovery rate of normal oocytes (83.75%), as well as the cleavage (61.80%) and transferable embryo (27.00%) rates when compared to the vitrified TCM group (67.46%, 51.40%, and 17.00%, respectively). The diffuse mitochondrial distribution was higher in fresh with melatonin (TCM + Mel) (80%) and vitrified with melatonin (VS2 + Mel groups) (76.70%), Furthermore, within the same group, while the mitochondrial intensity was higher in the TCM + Mel group (1698.60) than other group. In conclusion, Melatonin supplementation improves the developmental competence and mitochondrial distribution in buffalo oocytes in both cases(in vitro maturation and vitrification).

Fatty acid supplementation during warming improves pregnancy outcomes after frozen blastocyst transfers: a propensity score-matched study.

This study aimed to examine the viability of human blastocysts after warming with fatty acids (FAs) using an in vitro outgrowth model and to assess pregnancy outcomes after a single vitrified-warmed blastocyst transfer (SVBT). For the experimental study, we used 446 discarded vitrified human blastocysts donated for research purposes by consenting couples. The blastocysts were warmed using FA-supplemented (FA group) or non-FA-supplemented (control group) solutions. The outgrowth area was significantly larger in the FA group (P = 0.0428), despite comparable blastocyst adhesion rates between the groups. Furthermore, the incidence of outgrowth degeneration was significantly lower in the FA group than in the control group (P = 0.0158). For the clinical study, we retrospectively analyzed the treatment records of women who underwent SVBT in natural cycles between January and August 2022. Multiple covariates that affected the outcomes were used for propensity score matching as follows: 1342 patients in the FA group were matched to 2316 patients in the control group. Pregnancy outcomes were compared between the groups. The rates of implantation, clinical pregnancy, and ongoing pregnancy significantly increased in the FA group after SVBTs (P = 0.0091-0.0266). These results indicate that warming solutions supplemented with FAs improve blastocyst outgrowth and pregnancy outcomes after SVBTs.

Oocyte vitrification for fertility preservation is an evolving practice requiring a new mindset: societal, technical, clinical, and basic science-driven evolutions.

Infertility is a condition with profound social implications. Indeed, it is not surprising that evolutions in both medicine and society affect the way in vitro fertilization is practiced. The keywords in modern medicine are the four principles, which implicitly involve a constant update of our knowledge and our technologies to fulfill the "prediction" and "personalization" tasks, and a continuous reshaping of our mindset in view of all relevant societal changes to fulfill the "prevention" and "participation" tasks. A worldwide aging population whose life priorities are changing requires that we invest in fertility education, spreading actionable information to allow women and men to make meaningful reproductive choices. Fertility preservation for both medical and nonmedical reasons is still very much overlooked in many countries worldwide, demanding a comprehensive update of our approach, starting from academia and in vitro fertilization laboratories, passing through medical offices, and reaching out to social media. Reproduction medicine should evolve from being a clinical practice to treat a condition to being a holistic approach to guarantee patients' reproductive health and well-being. Oocyte vitrification for fertility preservation is the perfect use case for this transition. This tool is acquiring a new identity to comply with novel indications and social needs, persisting technical challenges, brand-new clinical technologies, and novel revolutions coming from academia. This "views and reviews" piece aims at outlining the advancement of oocyte vitrification from all these tightly connected perspectives.

Views and reviews: fertility preservation from different perspectives.

Fertility preservation for different conditions provides women the chance to buy time that can be invested in improving their well-being, by curing their condition from a holistic perspective, in line with the precepts of modern medicine.

Comparative effects of a calcium chelator (BAPTA-AM) and melatonin on cryopreservation-induced oxidative stress and damage in ovarian tissue.

Oncology treatments cause infertility, and ovarian tissue cryopreservation and transplantation (OTCT) is the only option for fertility preservation in prepubertal girls with cancer. However, OTCT is associated with massive follicle loss. Here, we aimed to determine the effect of supplementation of slow freezing and vitrification media with BAPTA-AM and melatonin alone and in combination on ovarian tissue viability, reactive oxygen species (ROS) levels, total antioxidant capacity (TAC), and follicular morphology and viability. Our results indicated that BAPTA-AM and melatonin can significantly improve ovarian tissue viability and the TAC/ROS ratio and reduce ROS generation in frozen-thawed ovarian tissues in slow freezing and vitrification procedures. BAPTA-AM was also found to be less effective on TAC compared to melatonin in vitrified ovarian tissue. While supplementation of slow freezing and vitrification media with BAPTA-AM and/or melatonin could increase the percentage of morphologically intact follicles in cryopreserved ovarian tissues, the differences were not significant. In conclusion, supplementation of cryopreservation media with BAPTA-AM or melatonin improved the outcome of ovarian tissue cryopreservation in both vitrification and slow freezing methods. Our data provide some insight into the importance of modulating redox balance and intracellular Ca2+ levels during ovarian tissue cryopreservation to optimize the current cryopreservation methods.

Cryopreservation of zygotic embryos of Podophyllum hexandrum Royle, an endangered medicinal plant, by vitrification and v cryo-plate techniques.

BACKGROUND: Podophyllum hexandrum is a highly endangered valuable medicinal plant of the Himalayas belonging to family Berberidaceae. This plant needs conservation efforts due to the over-exploitation and unscrupulous harvesting from the wild because of its ever-increasing demand. OBJECTIVE: To establish a long-term cryopreservation method for Podophyllum hexandrum using two techniques: Vitrification and V Cryo-plate. MATERIALS AND METHODS: Zygotic embryos were cryopreserved using vitrification and V cryo-plate by optimization of parameters including preculture time, loading time and PVS2 dehydration time. Recovery of zygotic embryos was performed on different regrowth media for plantlet formation. RESULTS: With V cryo-plate, 90% regrowth was obtained as compared to 73.3% with vitrification. V Cryo-plate conditions were pre-culture of zygotic embryos in 0.3 M sucrose for 4 days, treatment in loading solution with 0.8 M sucrose for 20 min, dehydration in PVS2 for 50 min, LN exposure, unloading in 1.2 M sucrose for 20 min and transfer of zygotic embryos to regrowth medium for recovery. During recovery, the maximum number of shoots (4.2) and highest shoot length (5.1 cm) were observed on regrowth medium with 1.5 mg per liter BAP and 0.1 mg per liter IAA (R7). CONCLUSION: Zygotic embryos of Podophyllum hexandrum were cryopreserved with 90% regrowth using a V cryoplate technique and plantlets were produced directly after cryopreservation. Doi: 10.54680/fr23410110712.

Effect of antioxidant procyanidin b2 (pcb2) on ovine oocyte developmental potential in response to in vitro maturation (ivm) and vitrification stress.

BACKGROUND: It was demonstrated that external stress, such as in vitro maturation (IVM) and vitrification process can induce significantly reduced development capacity in oocytes. Previous studies indicated that antioxidants play a pivotal part in the acquisition of adaptation in changed conditions. At present, the role of the natural potent antioxidant PCB2 in response to IVM and vitrification during ovine oocyte manipulation has not been explored. OBJECTIVE: To investigate whether PCB2 treatment could improve the developmental potential of ovine oocytes under IVM and vitrification stimuli. MATERIALS AND METHODS: The experiment was divided into two parts. Firstly, the effect of PCB2 on the development of oocytes during IVM was evaluated. Un-supplemented and 5 ug per mL PCB2-supplemented in the IVM solution were considered as control and experimental groups (C + 5 ug per mL PCB2). The polar body extrusion (PBE) rate, mitochondrial membrane potential (MMP), ATP, reactive oxygen species (ROS) levels and early apoptosis of oocytes were measured after IVM. Secondly, we further determine whether PCB2 could improve oocyte quality under vitrification stress. The survival rate, PBE rate and early apoptosis of oocytes were compared between fresh group, vitrified group and 5 ug per mL PCB2-supplemented in the IVM solution after vitrification (V + 5 ug per mL PCB2). RESULTS: Compared to the control group, adding PCB2 significantly increased PBE rate (79.4% vs. 62.8%, P < 0.01) and MMP level (1.9 +/- 0.08 vs. 1.3 +/- 0.04, P < 0.01), and decreased ROS level (47.1 +/- 6.3 vs. 145.3 +/- 8.9, P < 0.01). However, there was no significant difference in ATP content and early apoptosis. Compared to the fresh group, vitrification significantly reduced oocytes viability (43.0% vs. 90.8%, P < 0.01) as well as PBE rate (24.2% vs. 60.6%, P < 0.05). However, 5 ug per mL PCB2-supplemention during maturation had no effect on survival, PBE or early apoptosis in vitrified oocytes. CONCLUSION: PCB2 could effectively antagonise the oxidative stress during IVM and promote oocyte development. DOI: 10.54680/fr23210110412.

Effect of α-tocopherol in the vitrification medium on the viability, lipid peroxidation, expression of key developmental, apoptotic and stress-related genes in ovine secondary follicles.

The present study aimed to evaluate the effect of α-tocopherol on viability, lipid peroxidation and the expression of apoptosis, stress and development-related genes in the vitrified sheep secondary follicles. Ovarian secondary follicles (200-300 µm) were isolated and distributed separately to the vitrification treatment and supplemented with 5 mM, 10 mM, 20 mM and 30 mM of α-tocopherol (while the control fresh group was without vitrification and supplementation of α-tocopherol). After a week, the follicles were thawed and evaluated for follicular viability by trypan blue dye exclusion method, lipid peroxidation and gene expression studies. The results showed that the vitrification with 10 and 20 mM of α-tocopherol positively affected (p < .05) the viability of vitrified follicles in comparison with vitrified ones without α-tocopherol but the higher concentration of α-tocopherol, i.e., 30 mM negatively affected the viability (p < .05) in comparison with the 10 and 20 mM of α-tocopherol groups. The malondialdehyde (MDA) levels were significantly (p < .05) higher in the vitrified without α-tocopherol group in comparison to the vitrified with 20 mM of α-tocopherol group. The expression of apoptotic-related gene, BCL2L1 was significantly higher in 10 mM α-tocopherol group compared to the control fresh and CASPASE 3, 9 expressions were significantly higher in the vitrified group when compared to the vitrified with 10 mM α-tocopherol group. Expressions of BAX, BAD, BAK, BMP-15 and GDF-9 showed no significant difference among the groups. The mRNA expression of SOD1 was significantly higher in the vitrified without α-tocopherol group when compared to other groups. We conclude that the supplementation of 10 and 20 mM α-tocopherol in vitrification solution was the efficient vitrification procedure for the vitrification of ovine secondary follicles.

Membrane lipid changes in mouse blastocysts induced by ovarian stimulation, IVF and oocyte vitrification.

RESEARCH QUESTION: Is the membrane lipid profile of mice blastocysts affected by ovarian stimulation, IVF and oocyte vitrification? Could supplementation of vitrification media with L-carnitine and fatty acids prevent membrane phospholipid changes in blastocysts from vitrified oocytes? DESIGN: Experimental study comparing the lipid profile of murine blastocysts produced from natural mating, superovulated cycles or after IVF submitted or not to vitrification. For in-vitro experiments, 562 oocytes from superovulated females were randomly divided into four groups: fresh oocytes fertilized in vitro and vitrified groups: Irvine Scientific (IRV); Tvitri-4 (T4) or T4 supplemented with L-carnitine and fatty acids (T4-LC/FA). Fresh or vitrified-warmed oocytes were inseminated and cultured for 96 h or 120 h. The lipid profile of nine of the best quality blastocysts from each experimental group was assessed by multiple reaction monitoring profiling method. Significantly different lipids or transitions between groups were found using univariate statistics (P < 0.05; fold change = 1.5) and multivariate statistical methods. RESULTS: A total of 125 lipids in blastocysts were profiled. Statistical analysis revealed several classes of phospholipids affected in the blastocysts by ovarian stimulation, IVF, oocyte vitrification, or all. L-carnitine and fatty acid supplements prevented, to a certain extent, changes in phospholipid and sphingolipid contents in the blastocysts. CONCLUSION: Ovarian stimulation alone, or in association with IVF, promoted changes in phospholipid profile and abundance of blastocysts. A short exposure time to the lipid-based solutions during oocyte vitrification was sufficient to induce changes in the lipid profile that were sustained until the blastocyst stage.

Influences of glycine supplementation during vitrification on the developmental potential of vitrified/warmed immature dromedary camel oocytes.

Oocytes experience detrimental osmotic stress during vitrification and warming procedures because of the osmolality imbalance between the vitrification-warming fluids and the intracellular environment. Cellular osmotic homeostasis can be preserved by glycine, a powerful osmolyte with antioxidant properties. We aimed to examine the influences of supplementing glycine to the vitrification solutions (VS) on the developmental potential of vitrified/warmed immature dromedary camel oocytes following IVM/IVF and in vitro embryo culture (IVC). Cumulus oocyte complexes (COCs) were collected from dromedary camel ovaries and randomly allocated into two groups namely control (oocytes subjected directly to IVM) and vitrified (COCs were vitrified into VS supplemented with 0.0, 0.5, 1.0 or 2.0 mM glycine). For vitrification, COCs were equilibrated for 3 min in 12.5% ethylene glycol; EG plus 12.5% dimethyl sulfoxide; DMS and then they were vitrified for 60 s in VS composed of 25% EG + 25% DMSO using solid surface vitrification (SSV). Warming of vitrified oocytes was conducted in decreasing concentrations of trehalose solution. Following vitrification and warming, the morphologically viable oocytes were subjected to IVM for 36 h. Matured oocytes were then fertilized in vitro by epididymal spermatozoa and cultured for seven days. The results showed that the percentage of viable oocytes assessed by trypan blue stain was significantly higher (p ≤ .05) in the 1.0 mM glycine-supplemented group than 0.0- and 2.0-mM glycine-supplemented ones (90.0 % vs. 80.0% and 76.6%, respectively). However, no significant difference was observed between 0.5 mM glycine and other vitrified groups. Nuclear maturation rates, cleavage (48-h post-insemination; pi) and blastocyst rate (7-days pi) were significantly lower in vitrified groups than control ones (p ≤ .05). Among vitrified groups, these parameters were the highest in the 1.0 mM glycine-supplemented group. Taken together, supplementation of vitrification solutions with 1.0 mM glycine could enhance the developmental potential of vitrified/warmed immature dromedary camel oocytes.

Effect of supplementation of cryoprotectant solution with hydroxypropyl cellulose for vitrification of bovine oocytes.

BACKGROUND: Successful cryopreservation of bovine oocytes is very important for research and commercial applications. However, the survival and development rate of vitrified-thawed (VT) oocytes are lower than those of non-vitrified-thawed (non-VT) oocytes. OBJECTIVE: To investigate the effect of adding hydroxypropyl cellulose (HPC) to the vitrification solution for bovine oocytes. MATERIALS AND METHODS: For vitrification, bovine metaphase II oocytes were pretreated with a solution containing 10% ethylene glycol supplemented with 0, 10, 50, or 100 ug/mL HPC for 5 min, exposed to a solution containing 30% ethylene glycol supplemented with 0, 10, 50, or 100 ug/mL HPC for 30 s, and then directly plunged into liquid nitrogen. RESULTS: The survival rate of oocytes was significantly higher in the 50 HPC group than in the 0, 10, and 100 HPC groups. The reactive oxygen species level was lower in the non-VT and 50 HPC groups than in the other groups. The mRNA levels of proapoptotic genes (Bax) were lower in the non-VT, 0, and 50 HPC groups than in the other groups. The mRNA levels of antiapoptotic genes (BCl2) were higher in the non-VT than in the other groups. The development rates of embryos (day 8) obtained via parthenogenetic activation (PA) were determined in the non-VT, 0 HPC, and 50 HPC groups. The cleavage rate was significantly higher in the non-VT group. CONCLUSION: Supplementation of vitrification solution with HPC improves the survival of VT bovine oocytes and the development capacity of embryos derived from these oocytes via PA. doi.org/10.54680/fr23110110212.

Successful production of offspring derived from mouse zygotes vitrified with carboxylated ε-poly-L-lysine and polyvinyl alcohol without serum.

The vitrification of zygotes is important for their use as donors for generating genome-edited mice. We previously reported the successful vitrification of mouse zygotes using carboxylated ε-poly-L-lysine (COOH-PLL). However, this vitrification solution contains fetal calf serum (FCS), which contains unknown factors and presents risks of pathogenic viral and microbial contamination. In this study, we examined whether polyvinyl alcohol (PVA) can be used as an alternative to FCS in vitrification solutions for mouse zygotes. When COOH-PLL was added to the vitrification solutions, zygotes vitrified with solutions containing 0.01% PVA (PV0.01) and those vitrified in a control solution containing FCS (75.6%) developed into blastocysts (78.4%). In addition, there were no significant differences in the ability to develop to term between the control solution (46.6%) and PV0.01 (44.1%) groups. In conclusion, we clearly demonstrated that PVA can replace FCS in our vitrification solution supplemented with COOH-PLL for mouse zygotes.

Feline ovarian tissue vitrification: The effect of fragment size and base medium on follicular viability and morphology.

To achieve optimal vitrification, tissue structure and fragment size represent a challenge for obtaining sufficient cooling velocity. Theoretically, thin ovarian tissue fragments lead to higher surface contact, hence higher solute penetration. Another critical factor is the concentration of cryoprotectants (CPA): CPA toxicity may occur with high concentrations, and as such, this may induce local apoptosis. Therefore two experiments were conducted: In experiment I, we compared the effect of sucrose supplementation in vitrification solution along with ovarian fragments of different sizes on post-warming tissue viability and follicle architecture. Fragments of two different sizes, with a thickness and radius of 1.5 × 0.75 mm and 3 × 1.5 mm respectively were vitrified in vitrification solution without sucrose and with 0.5 M sucrose supplementation. Post-warming, fragments of ovarian tissue (fresh and vitrified) were evaluated for viability (Calcein AM/Propidium Iodide) and for morphology (hematoxylin-eosin). In experiment II, we aimed to reduce cryoprotectant toxicity by using lower CPA concentrations in combination with an optimized carrier medium (HypThermosol®; HTS). Ovarian tissue fragments were randomly allocated to five groups (A: fresh controls; B: vitrified in GLOBAL® TOTAL® LP w/HEPES with 15% ethylene glycol (EG) and 15% DMSO; C: vitrified in HTS with 5% EG and 5% DMSO; D: vitrified in HTS with 10% EG and 10% DMSO; E: vitrified in HTS with 15% EG and 15% DMSO). Fragments (fresh and vitrified) were evaluated for morphology (hematoxylin-eosin) and for apoptosis through the activity of caspase-3. Results showed that follicular morphology was affected by the size of the fragment; smaller sized fragments contained a greater proportion of intact follicles (53.8 ± 2.0%) compared to the larger fragments (40.3 ± 2.0%). Our results demonstrated that 1.5 × 0.75 mm sized pieces vitrified in a vitrification solution supplemented with 0.5 M sucrose had more intact follicles (54.8 ± 1.3%; P = 0.0002) after vitrification. In addition, HTS presented no additional protective effect as a base medium, neither for follicular morphology nor apoptotic rate.

The Beneficial Effects of Static Magnetic Field and Iron Oxide Nanoparticles on the Vitrification of Mature Mice Oocytes.

This study was conducted to evaluate the effects of static magnetic field (SMF) and nanoparticles (NPs) on the vitrification of cumulus-oocyte-complex (COC). To this end, the non-vitrified (nVit) and vitrified groups (Vit) that contain NPs, with or without SMF were labeled nVit_NPs, nVit_NPs_SMF, Vit_NPs, and Vit_NPs_SMF, respectively. The non-toxic dosages of NPs were first determined to be 0.008% w/v. The survival, apoptosis, and necrosis, mitochondrial activity, fertilization rate, subsequent-derived embryo development, and gene expressions were examined. The viability rates obtained by trypan blue and Anx-PI staining were meaningfully smaller in the Vit groups, compared to the nVit groups. The JC1 red/green signal ratios were reduced considerably in the Vit group, compared to the nVit. Transmission electron microscopy (TEM) was performed to assess the entry of the NPs into the oocytes. TEM images showed that NPs were present in nVit_NPs, and Vit_NPs. Thereafter, the effects of NPs and SMF on in vitro fertilization (IVF) were examined. The difference in blastocyst rates between nVit and Vit_NPs_SMF groups was significant. Finally, Nanog, Cdx2, Oct4, and Sox2 genes were evaluated. There were substantial differences in Cdx2 gene expressions between the Vit_NPs and nVit groups. The expression of Nanog in Vit was significantly higher than those of the Vit_NPs, Vit_NPs_SMF, and nVit groups. The data presented here provide deeper insight into the application of iron oxide nanoparticles in COC vitrification. It appears that using SMF and supplemented CPA by NPs inhibits cryoinjury and promote the embryo development capacity of vitrified-warmed COCs.

Alpha-lipoic acid improves bovine preimplantation blastocyst quality and cryotolerance.

In vitro embryo production has grown in recent decades due to its great potential for cattle production. However, the quality of in vitro-produced embryos is lower compared with those produced in vivo. The postfertilization culture environment has a major influence on bovine embryo quality. We hypothesize that the inclusion of the inclusion of alpha-lipoic acid (ALA) in the in vitro culture (IVC) medium during the first 24 h would have positive effects on embryo development in vitro and cryotolerance. The aims of this study were to evaluate the antioxidant effect of ALA in IVC medium for 24 h on bovine zygotes (21 h post in vitro fertilization, IVF), day 2 cleaved embryos (46 h post-IVF), and to assess embryo quality, developmental competence, and cryotolerance after vitrification. In all experiments, IVC medium was the Control, and 2.5 µM ALA was the treatment implemented. Viability and reactive oxygen species (ROS) levels in zygotes and day 2 embryos did not differ from the Control (P > 0.05). Supplementation with ALA increased total blastocyst and hatching rates (P < 0.05). It also improved embryo quality, evidenced by the increased blastocyst total cell number and the percentage of excellent-quality embryos observed (P < 0.05). In embryos cultured with ALA and then vitrified, ALA reduced intracellular ROS levels in warmed blastocysts (P < 0.05). In conclusion, ALA supplementation to IVC medium during 24 h is a new advantage in improving embryo quality for assisted bovine reproduction.

Apoptosis and glucocorticoid-related genes mRNA expression is modulated by coenzyme Q10 supplementation during in vitro maturation and vitrification of bovine oocytes and cumulus cells.

Oocyte in vitro maturation (IVM) and vitrification procedures lead to detrimental effects on the overall oocyte quality. The addition of antioxidants during IVM, such as the coenzyme Q10 (Q10), has been demonstrated to positively impact on the cumulus-oocyte complexes due to its role in protection from oxidative damage and modulating gene transcription. Furthermore, glucocorticoids (GC) regulate gene transcription, energy metabolism and apoptosis during the early steps of reproduction. In this sense, most GC actions are mediated by the glucocorticoid receptor (NR3C1), a transcription factor. However, the specific roles of GC in ovarian physiology and oocyte maturation are still unknown. In this regard, a better knowledge on the expression of GC-related and apoptosis-related genes during IVM and cryopreservation procedures could potentially benefit the refinement of assisted reproductive techniques in the bovine species. The present study aims to explore the expression of NR3C1 mRNA in fresh and vitrified bovine oocytes and cumulus cells in response to Q10 (50 µM), and the effect of cortisol addition (0.25 µM, 0.5 µM) on the expression of NR3C1. We also studied the mRNA expression of NR3C1-related genes belonging to the GC regulation pathway, such as hydroxysteroid dehydrogenases (HSD11B1; HSD11B2), immunophilins (FKBP4; FKBP5), signal transducers and activators of transcription (STAT3; STAT5A), the mineralocorticoid receptor (NR3C2), and to the apoptosis pathway, such as the anti- (BCL2) and pro-apoptotic (BAX) mRNA transcripts in oocytes and cumulus cells 1) after IVM, and 2) after vitrification, both in presence or absence of Q10 supplementation during IVM. Our results show that there is an increase in the NR3C1 receptor expression after vitrification of oocytes, but not after exogenous cortisol supplementation during IVM. In addition, Q10 reduces the mRNA expression of HSD11B1 and FKBP5 in oocytes at levels of immature oocytes (HSD11B1 mRNA expression also in cumulus cells), and the BAX:BCL2 ratio mRNA expression. After vitrification in the presence of Q10, HSD11B2 mRNA expression increases in cumulus cells, while HSD11B1 and BAX:BCL2 mRNA expression decreases significantly both in oocytes and cumulus cells. In conclusion, our results show for the first time the effect of IVM, vitrification and Q10 supplementation on the mRNA relative expression of GC-related and apoptosis genes, and the effect of vitrification in the protein expression of NR3C1.

The effect of sucrose supplementation on chondrocyte viability in porcine articular cartilage following vitrification.

Vitrification can extend the banking life of articular cartilage (AC) and improve osteochondral transplantation success. Current vitrification protocols require optimization to enable them to be implemented in clinical practice. Sucrose as a non-permeating cryoprotective agent (CPA) and clinical grade chondroitin sulfate (CS) and ascorbic acid (AA) as antioxidants were investigated for their ability to improve a current vitrification protocol for AC. The aim of this study was to assess the impact of sucrose and CS/AA supplementation on post-warming chondrocyte viability in vitrified AC. Porcine osteochondral dowels were randomly vitrified and warmed with one established protocol (Protocol 1) and seven modified protocols (Protocols 2-8) followed by chondrocyte viability assessment. Sucrose supplementation in both vitrification and warming media (Protocol 4) resulted in significantly higher (p = 0.018) post-warming chondrocyte viability compared to the protocol without sucrose (Protocol 1). There was no significant difference (p = 0.298) in terms of post-warming chondrocyte viability between sucrose-supplemented DMEM + CS solution (Protocol 4) and Unisol-CV (UCV) + CS (Protocol 6) solution. Clinical grade CS and AA contributed to similar post-warming chondrocyte viability to previous studies using research grade CS and AA, indicating their suitability for clinical use. The addition of an initial step (step 0) to reduce the initial concentration of CPAs to minimize osmotic effects did not enhance chondrocyte viability in the superficial layer of AC. In conclusion, sucrose-supplemented DMEM + clinical grade CS (Protocol 4) could be an ideal protocol to be investigated for future use in clinical applications involving vitrified AC.

Equilibration time with cryoprotectants, but not melatonin supplementation during in vitro maturation, affects viability and metaphase plate morphology of vitrified porcine mature oocytes.

The aims of this study were to investigate the effects of different equilibration times with cryoprotectants on viability and metaphase plate morphology of vitrified-warmed porcine mature oocytes (Experiment 1) and to evaluate the effects of supplementation with 10-9 M melatonin during in vitro maturation on these parameters (Experiment 2). In Experiment 1, 2,392 mature oocytes were vitrified using different equilibration times of oocytes with cryoprotectants (3, 10, 15, 20, 30, 40, 60 and 80 min). Fresh oocytes matured in vitro for 44 hr (n = 509) were used as controls. In Experiment 2, a total of 573 COCs were used. COCs were matured with 10-9 M melatonin supplementation or without melatonin (control). Some oocytes from each group were vitrified with a 60-min equilibration time with cryoprotectants according to the results of Experiment 1. The remaining oocytes from each maturation group were used as fresh control groups. In both experiments, oocytes were stained with 2',7'-dichlorodihydrofuorescein diacetate and Hoechst 33342 to assess viability and metaphase plate morphology, respectively. Vitrification and warming affected (p < .01) oocyte viability compared with controls, which were all viable after 44 hr of IVM. In Experiment 1, the longer the equilibration time with cryoprotectants, the higher the viability. Oocytes equilibrated for 60 and 80 min had the highest (p < .05) viability and similar metaphase plate characteristics to the fresh control oocytes. In Experiment 2, supplementation with melatonin during in vitro maturation had no effect on oocyte viability or metaphase plate morphology of vitrified-warmed oocytes. In conclusion, under our experimental conditions, vitrified porcine mature oocytes equilibrated with cryoprotectants for 60 or 80 min exhibited the highest viability and similar metaphase plate characteristics to fresh controls. Furthermore, supplementation with 10-9 M melatonin during in vitro maturation had no effect on these parameters.

Supplementation of EGF, IGF-1, and Connexin 37 in IVM Medium Significantly Improved the Maturation of Bovine Oocytes and Vitrification of Their IVF Blastocysts.

The quality and developmental capacity of oocytes derived from in vitro maturation (IVM) remain unsatisfactory, which greatly impairs the efficiency and application of embryo technologies. The present experiment was designed to investigate the effect of the supplementation of EGF, IGF-1, and Cx37 in an IVM medium on the maturation quality and development ability of bovine oocytes. The cytoplasmic maturation events of oocytes and the quality of in vitro fertilization (IVF) blastocysts were examined to investigate the relative mechanisms. Our results showed that the nuclear maturation and blastocyst development after the IVF of oocytes treated with 25 µg/mL Cx37 or the combination of 50 ng/mL EGF and 100 ng/mL IGF-1 were significantly increased compared to those of the control group (p < 0.05). Furthermore, the blastocyst rate, and blastocyst total cell number and survival rate after vitrification of the EGF+IGF-1+Cx37 group, were significantly higher than those of the control group (p < 0.05), but lower than those of the FSH+LH+EGF+IGF-1+Cx37 group (p < 0.05). The transzonal projection (TZP) intensity, glutathione (GSH) level, and mitochondrial function of the EGF+IGF-1+Cx37 group were significantly higher than that of the control group, and lower than those of the FSH+LH+EGF+IGF-1+Cx37 group, in contrast to the results of the reactive oxygen species (ROS) levels. In conclusion, our results showed that the supplementation of 50 ng/mL EGF, 100 ng/mL IGF-1, and 25 µg/mL Cx37 in the IVM of bovine oocytes significantly improved their quality and developmental ability by increasing the TZP, mitochondrial function, and GSH level.