Comparison of the developmental competence of in vitro-produced mouse embryos cultured under 5 versus 2% O2 with in vivo-derived blastocysts.

PURPOSE: The prevalence of infertility in Canada has substantially increased over 30 years, and plateaued success rates of culture systems warrant further optimization for transfer outcomes. In clinical programs, embryos commonly undergo extended culture under 5% O2 until the blastocyst stage. The aim of this study is to characterize the developmental competence and stress-related responses of embryos cultured under 5 versus 2% O2 in comparison to in vivo-derived blastocysts. We hypothesized 2% O2 compromises developmental competence through altered embryonic stress responses and induction of apoptosis-related genes relative to those cultured under 5% O2 and in vivo-derived blastocysts. METHODS: Quantitative measures of development and relative expressions of a cohort of stress-related genes in CD1 mouse zygotes cultured to blastocysts under 5 or 2% O2 were compared to in vivo-derived embryos. Apoptotic responses were evaluated using an immunofluorescence assay for Caspase-3. RESULTS: The mean percentage of blastocysts developed, and total cell number of embryos derived in vivo or cultured under 5% O2 was significantly higher than those cultured under 2% O2. Blastocyst expansion was greatest in embryos cultured under 5% O2. Stress response genes were significantly upregulated in embryos cultured under 2% O2, and expression of antioxidant-related genes was significantly lower in cultured versus in vivo-derived embryos. Caspase-3 immunofluorescence was significantly higher in cultured embryos versus in vivo-derived embryos. CONCLUSION: We inferred that 5% O2 systems better approximate physiologic oxygen availability for culture of mouse embryos, warranting re-evaluation of culturing embryos under threshold or sub-physiologic oxygen concentrations during clinical IVF programs.

New method to induce neurotrophin gene expression in human adipose-derived stem cells in vitro.

Rosemary leaf extract, a well-known medicinal plant, can induce neurotrophin gene expression and proliferation in stem cells. Human adipose-derived stem cells (hASCs) with high proliferation and differentiation capacity are easily accessible and can be extracted with the least damage. This study evaluated the effect of rosemary extract (RE) on neurotrophin gene expression at 48 h postinduction in hASCs. hASCs were isolated from healthy female donors, aged 28-35 years, who had undergone abdominal liposuction. Passage-4 stem cells were cultured and treated with different doses of RE (from 30 to 70 µg/ml) containing 40% carnosic acid for 48 h. Reverse transcription-polymerase chain reaction was used to check the expression of neurotrophin genes. The expression of NTF3, NTF4, and nerve growth factor genes in cells treated with 40-60 µg/ml and the expression of GDNF in cells treated with 50-70 µg/ml of RE for 48 h showed a significant increase compared to cells cultured in serum-containing medium. However, different doses of RE showed no effect on brain-derived neurotrophic factor gene expression in the treated cells. RE (50, 60 µg/ml) leads to an increase of neurotrophin gene expression in hASCs as compared to routine cell culture. Hence, this protocol can be used to prepare ideal cell sources for cell therapy.

Establishment and characterization of fibroblast lines from the northern tiger cat (Leopardus tigrinus, Schreber, 1775) during extended passage and cryopreservation.

The establishment of fibroblast lines enables several applications from the formation of biobanks for the conservation of biodiversity to the use of these cells in physiological and toxicological assays. Considered a species vulnerable to extinction, the characterization of fibroblastic lines of northern tiger cat would contribute to its conservation. Therefore, we established and characterized fibroblasts derived from northern tiger cat during extended passage (third, seventh, and eleventh passages) and cryopreservation with regard to the morphology, viability, apoptotic classification, metabolism, proliferative activity, and oxidative stress by reactive oxygen species (ROS) levels and mitochondrial membrane potential (ΔΨm). Initially, we identified four dermal fibroblast lines by morphology, immunophenotyping, and karyotyping assays. In vitro culture after the third, seventh, and eleventh passages did not affect the viability, apoptotic classification, and ROS levels. Nevertheless, cells at seventh and eleventh passages featured a reduction in metabolism and an alteration in ΔΨm when compared to third passage cells. Additionally, cells at eleventh passage showed changes in the proliferative activity and morphology when compared to other passages. Regarding cryopreservation, no effect was observed on cryopreserved cells for morphology, viability, apoptotic classification, metabolism, and proliferative activity. Nevertheless, cryopreserved cells had alteration for ROS levels and ΔΨm. In summary, fibroblasts from northern tiger cat were affected by extended passage (seventh and eleventh passages) and cryopreservation. Adjustments to the in vitro culture and cryopreservation are necessary to reduce cellular oxidative stress caused by in vitro conditions.

In vitro production of viable eggs from undeveloped oocytes in mouse preantral follicles by reconstructing granulosa cell-oocyte complexes†.

In vitro culture of ungrown oocytes in preantral follicles is one of the intriguing subjects being pursued to produce viable eggs in assisted reproductive technology. Previous studies have succeeded in obtaining mature eggs after in vitro culture of preantral follicles, while denuded undeveloped oocytes, which are obtained occasionally when collecting preantral follicles, seem to be almost useless. Moreover, methods to culture them efficiently to produce viable eggs have not been established yet. The present study was conducted to demonstrate in vitro culture of mouse denuded undeveloped oocytes by reconstructing granulosa cell-oocyte complexes, and to analyze cellular communication in reconstructed granulosa cell-oocyte complexes. Single denuded undeveloped oocytes were aggregated with 1 × 104 granulosa cells in wells with U-shaped bottoms in a low-binding cell culture plate for 8 days under either 20% or 5% O2, and then the reconstructed granulosa cell-oocyte complexes formed were cultured on a collagen-coated culture membrane insert for 4 days under 5% O2. At day 8 of culture, the rates of reconstructed granulosa cell-oocyte complexes formation were significantly higher in the culture group under 5% O2 (64.9%) than that under 20% O2 (42.3%; P < 0.001); furthermore, the formation of transzonal projections was observed. After maturation and fertilization, we produced matured eggs and blastocysts at higher rates (>90% and 61.9%, respectively) in the group cultured under 5% O2. After transferring 126 two- to four-cell stage embryos, six live pups were obtained. This is the first report that demonstrates production of viable eggs after in vitro culture of denuded undeveloped oocytes from preantral follicles by reconstruction of granulosa cell-oocyte complexes.

ThymoSpheres culture: A model to study human polyclonal unconventional T cells.

In vitro cultures remain crucial for studying the fundamental mechanisms of human T-cell development. Here, we introduce a novel in vitro cultivation system based on ThymoSpheres (TS): dense spheroids consisting of DLL4-expressing stromal cells and human hematopoietic precursor cells, in the absence of thymic epithelial cells. These spheroids are subsequently cultured at the air-liquid interphase. TS generate large numbers of mature T cells, are easy to manipulate, scalable, and can be repeatably sampled to monitor T-cell differentiation. The mature T cells generated from primary human hematopoietic precursor cells were extensively characterized using single-cell RNA and combined T-cell receptor (TCR) sequencing. These predominantly CD8α T cells exhibit transcriptional and TCR CDR3 characteristics similar to the recently described human polyclonal αß unconventional T cell (UTC) lineage. This includes the expression of hallmark genes associated with agonist selection, such as IKZF2 (Helios), and the expression of various natural killer receptors. The TCR repertoire of these UTCs is polyclonal and enriched for CDR3-associated autoreactive features and early rearrangements of the TCR-α chain. In conclusion, TS cultures offer an intriguing platform to study the development of this human polyclonal UTC lineage and its inducing selection mechanisms.

Investigating cryopreservation techniques for maintaining morphology and in vitro viability of cartilage and skin from Spix's yellow-toothed cavies (Galea spixii Wagler, 1831) for conservation through biobanks.

Conservation of the genetic diversity through skin and cartilage biobanks represents an essential strategy for maintaining biodiversity. Biobanks for the wild species of the order Rodentia have been little studied. Considering that the cryopreservation technique has specific relationships with the tissue and species of interest, we propose investigating different techniques for preserving tissue integrity and cell viability after cartilage and skin culture from Spix's yellow-toothed cavies. Subsequently, two techniques [solid-surface vitrification (SSV) vs. slow freezing (SF)] were used for cartilage and skin cryopreservation. Tissues not subjected to cryopreservation were used as controls. All tissues were evaluated for morphology and proliferation by histological techniques. Moreover, fragments were cultured, and cells were evaluated for viability, proliferation, metabolism, and apoptosis. Regardless of the cryopreservation technique, no differences were observed for the thickness of the epidermis, dermis, skin, spinous and basal layers, fibroblasts, and proliferative activity regarding the number of nucleolar organizer regions (NOR). SSV ensured better maintenance of epidermal cells, normal chondrocytes, filled gaps, collagen fibers, proliferative activity by NOR area/cell, and reduced perinuclear halos and empty gaps compared to SF. SF ensured the conservation of corneum thickness compared to the control. Although both techniques promoted cell recovery after culture, cells from SF resulted in better subconfluence time and day with cell growth around fragments compared to SSV. In conclusion, both cryopreservation techniques resulted in viable cells after culture. However, SSV promoted better maintenance of tissue morphological integrity, and SF ensured the preservation of all cell quality parameters in Spix's yellow-toothed cavies.

Tapping the potential of Calotropis procera hairy roots for cardiac glycosides production and their identification using UHPLC/QTOF-MS.

The present work deals with the establishment of hairy root cultures from different explants of C. procera using Agrobacterium rhizogenes strain A4. A high transformation frequency (95%) was obtained from leaves followed by cotyledons (81.6%) and hypocotyls (38.3%). Genetic transformation of hairy roots was confirmed through PCR by amplifying a 400 bp fragment of the rolB gene. Hairy roots were highly branched, possessed plagiotropic and rapid growth on hormone-free ½ B5 medium. Ten cardiac glycosides, including calotropagenin, calotoxin, frugoside, coroglaucigenin, calotropin, calactin, uzarigenin, asclepin, uscharidin, and uscharin, based on their specific masses and fragmentation properties were identified in ethanolic extracts of hairy roots by ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry UHPLC/QTOF-MS. This protocol could be used as a powerful tool for large-scale in vitro production of highly valued cardiac glycosides and for further transcriptomics or metabolomics studies.

Generation of mouse testicular organoids with highly compartmentalized tubular lumen structure and their cryopreservation.

Testicular organoids have great potential for maintaining male fertility and even restoring male infertility. However, existing studies on generating organoids with testis-specific structure and function are scarce and come with many limitations. Research on cryopreservation of testicular organoids is even more limited, and inappropriate cryopreservation methods may result in the loss of properties in resuscitated or regenerated organoids, rendering them unsuitable for clinical or research needs. In this paper, we investigated the effects of mouse age and cell number on the self-aggregation of testicular cells into spheres in low-adsorption plates. Various media compositions, culture systems, and cell numbers were used to culture cell spheres for 14 days to form testicular organoids, and the self-organization of the organoids was assessed by histological and immunofluorescence staining. We determined the appropriate cryopreservation conditions for testicular cells, cell spheres, and tissues. Subsequently, organoids derived from cryopreserved testicular tissues, testicular cells, and testicular cell spheres were compared and evaluated by histological and immunofluorescence staining. The results indicate that testicular cell spheres consisting of 30 × 104 testicular cells from 2-week-old mice were able to form organoids highly similar to the luminal structure and cell distribution of natural mouse testicular tissues. This transformation occurred over 14 days of incubation in α-MEM medium containing 10 % knockout serum replacer (KSR) using an agarose hydrogel culture system. Additionally, the Sertoli cells were tightly connected to form a blood-testis barrier. The relative rates of tubular area, germ cells, Sertoli cells, and peritubular myoid cells were 36.985 % ± 0.695, 13.347 % ± 3.102, 47.570 % ± 0.379, and 27.406 % ± 1.832, respectively. The optimal cryopreservation protocol for primary testicular cells involved slow freezing with a cryoprotectant consisting of α-MEM with 10 % dimethyl sulfoxide (DMSO). Slow freezing with cryoprotectants containing 5 % DMSO and 5 % ethylene glycol (EG) was optimal for all different volumes of testicular cell spheres. Compared to testicular organoids generated from frozen testicular tissue and cell spheres, freezing testicular cells proved most effective in maintaining organoid differentiation characteristics and cell-cell interactions. The findings of this study contribute to a "universal" testicular organoid in vitro culture protocol with promising applications for fertility preservation and restoration in prepubertal cancer patients and adult infertile patients.

Survival and growth of M. perstans larvae in a human colon carcinoma cell line-based in vitro culture.

Mansonella perstans infections are widespread in Sub-Saharan Africa and Central and South America and thus can be considered as the most prevalent parasite of man in tropical Africa. In contrast to the high prevalence, knowledge about the biology of this filarial nematode is restricted and no effective treatment regimens of this ivermectin-resistant parasite is lacking. An obstacle for the research is that M. perstans resides in body cavities and thus have been only rarely recovered during surgery or autopsy. Therefore, alternative methods like in vitro culture systems need to be implemented to decipher the nature of mansonellosis and effective drugs. Previously, we have established a monkey kidney epithelial cell-based in vitro culture for the maintenance of M. perstans infective larvae (L3) up to 77 days. However, no alternative for this culture system have been postulated to allow longer survival rates and development of adult worms in vitro. Thus, we aim to establish an alternative in vitro culture system for M. perstans L3. M. perstans L3 were isolated from engorged and laboratory reared Culicoides midges. The larvae were then cultured in Dulbecco's Modified Eagle Medium supplemented with either 10% foetal bovine serum (FBS), 10% newborn calf serum (NCS) or 1% bovine serum albumin (BSA) together with human colon carcinoma cells (HCT-8) as feeder cells. Survival and growth were recorded. We obtained that the 10% NCS culture condition was superior allowing long-term maintenance of M. perstans L3 for up to 100 days and boosted growth of the parasites for up to 5-folds compared to the initial size at culture inception. Although no moulting of the L3 into L4 or adult worms could be overserved, the human colon carcinoma cell-based in vitro culture provides an alternative platform to analyse M. perstans biology and screen for novel drugs against M. perstans.

Advancing fertility preservation in prepubertal mice: Efficacy of ovarian tissue culture and in vitro growth in mature oocyte development.

AIM: This study aimed to evaluate the ovarian tissue culture and in vitro follicle growth as safer alternatives to cryopreservation for generating in vitro fertilization (IVF)-ready mature oocytes from prepubertal mice without the risk of cancer cell contamination. METHODS: Ovaries from prepubertal B6D2F1 mice were cultured in α-minimum essential medium supplemented with an estrogen receptor antagonist, ICI 182780. Culture duration was investigated to identify the optimal timeframe for follicle growth and oocyte maturation. Follicles were isolated mechanically or using 1 mg/mL collagenase and cultured in Matrigel matrix or polyvinylpyrrolidone. Oocyte development at metaphase II was induced by in vitro maturation, followed by IVF. RESULTS: The optimal culture duration was 2-4 days, and tissues cultured beyond this period showed significant follicular degeneration. ICI 182780 supplementation resulted in the recovery of 20.5 follicles per ovary compared with 9.5 follicles in non-supplemented cultures (p < 0.05). Of the 452 isolated follicles, 237 (52.4%) showed growth, 150 (33.2%) underwent germinal vesicle breakdown, and 18 (4.0%) reached metaphase II. However, none of the metaphase II oocytes were successfully fertilized after IVF. Matrigel demonstrated a significantly higher in vitro maturation rate compared with polyvinylpyrrolidone in a comparative analysis of culture matrices (p < 0.001). CONCLUSIONS: This study highlighted ovarian tissue culture and in vitro growth as effective strategies for producing mature oocytes from prepubertal mice. Further studies are required to overcome fertilization hurdles and understand the mechanisms that improve post-IVF embryo viability.

In vitro extracellular replication of Wolbachia endobacteria.

Obligate intracellular endobacteria of the genus Wolbachia are widespread in arthropods and several filarial nematodes. Control programs for vector-borne diseases (dengue, Zika, malaria) and anti-filarial therapy with antibiotics are based on this important endosymbiont. Investigating Wolbachia, however, is impeded by the need for host cells. In this study, the requirements for Wolbachia wAlbB growth in a host cell-free in vitro culture system were characterized via qPCRs. A cell lysate fraction from Aedes albopictus C6/36 insect cells containing cell membranes and medium with fetal bovine serum were identified as requisite for cell-free replication of Wolbachia. Supplementation with the membrane fraction of insect cell lysate increased extracellular Wolbachia replication by 4.2-fold. Replication rates in the insect cell-free culture were lower compared to Wolbachia grown inside insect cells. However, the endobacteria were able to replicate for up to 12 days and to infect uninfected C6/36 cells. Cell-free Wolbachia treated with the lipid II biosynthesis inhibitor fosfomycin had an enlarged phenotype, seen previously for intracellular Wolbachia in C6/36 cells, indicating that the bacteria were unable to divide. In conclusion, we have developed a cell-free culture system in which Wolbachia replicate for up to 12 days, providing an in vitro tool to elucidate the biology of these endobacteria, e.g., cell division by using compounds that may not enter the C6/36 cells. A better understanding of Wolbachia biology, and in particular host-symbiont interactions, is key to the use of Wolbachia in vector control programs and to future drug development against filarial diseases.

Kidney-tonifying formula facilitates the development and maturation of mouse preantral follicle in vitro.

OBJECTIVE: Kidney-tonifying formulas are frequently used in clinical practices to enhance follicular development and maturation. This research explored the impacts of the Bushen Tiaojing formula (BSTJF) on the development of mouse preantral follicles in vitro and its relationship with granulosa cells and gonadotropins. METHODS: Preantral follicles were extracted from mice and cultured with or without serum from rats that were previously treated with or without BSTJF. During cultivation, the follicles were monitored for morphological changes and developmental maturation. Exhausted medium was collected every other day for the measurement of progesterone and estradiol (E2) levels by ELISA. Granulosa cells in in-vitro medium were collected on days 8, 10, and 12 and analyzed for determining the expressions of apoptosis-associated genes (Bax, Bcl-2, and Caspase-3). Propagation and apoptosis rates of collected granulosa cells were measured by CCK-8 assay and flow cytometry. RESULTS: Compared with control follicles, follicles cultured with serum from BSTJF-treated rats had a higher survival rate, larger follicle diameter, higher Bcl-2 expression, and lower Bax and Caspase-3 expressions (all P ≤ 0.05). In addition, their granulosa cells presented substantially elevated proliferation (P ≤ 0.05) and a lower rate of apoptosis (P ≤ 0.05) compared with granulosa cells from control follicles. The level of E2 in the culture media of all groups increased slowly in the first 6 days. Subsequently, after formation of the antrum, the levels of E2 and progesterone were enhanced in the medium of follicles cultured with serum from BSTJF-treated rats compared with those in the media of control follicles (all P ≤ 0.05). CONCLUSION: Serum from BSTJF-treated rats facilitated the in vitro development and maturation of mouse follicles by increasing the expression of anti-apoptotic gene Bcl-2, reducing the expressions of pro-apoptotic genes Bax and Caspase-3 as well as the apoptosis of granulosa cells, promoting the proliferation of granulosa cells and increasing the secretion of E2 and progesterone in the cells.

Developing an Optimized Protocol for Regeneration and Transformation in Pepper.

Capsicum annuum L. is extensively cultivated in subtropical and temperate regions globally, respectively, when grown in a medium with 8 holding significant economic importance. Despite the availability of genome sequences and editing tools, gene editing in peppers is limited by the lack of a stable regeneration and transformation method. This study assessed regeneration and transformation protocols in seven chili pepper varieties, including CM334, Zunla-1, Zhongjiao6 (ZJ6), 0818, 0819, 297, and 348, in order to enhance genetic improvement efforts. Several explants, media compositions, and hormonal combinations were systematically evaluated to optimize the in vitro regeneration process across different chili pepper varieties. The optimal concentrations for shoot formation, shoot elongation, and rooting in regeneration experiments were determined as 5 mg/L of 6-Benzylaminopurine (BAP) with 5 mg/L of silver nitrate (AgNO3), 0.5 mg/L of Gibberellic acid (GA3), and 1 mg/L of Indole-3-butyric acid (IBA), respectively. The highest regeneration rate of 41% was observed from CM334 cotyledon explants. Transformation optimization established 300 mg/L of cefotaxime for bacterial control, with a 72-h co-cultivation period at OD600 = 0.1. This study optimizes the protocols for chili pepper regeneration and transformation, thereby contributing to genetic improvement efforts.

Undisturbed culture: a clinical examination of this culture strategy on embryo in vitro development and clinical outcomes.

OBJECTIVE: To compare the effect of a fully undisturbed culture strategy over a sequential one on embryo in vitro development and clinical outcomes in intracytoplasmic sperm injection (ICSI) cycles. DESIGN: Retrospective cohort study. SETTING: University-affiliated private IVF center. PATIENT(S): This study included 4,564 ICSI cycles performed over 5 years, including autologous and oocyte donation treatments with extended embryo culture until blastocyst in one of the two defined culture strategies. INTERVENTION(S): Embryo cohorts were cultured in one of two culture systems: a fully undisturbed culture, including an incubator with integrated time-lapse technology, a one-step culture medium and embryo selection assisted by semi-automatic tools on the basis of embryo morphokinetics, or a sequential culture, using a conventional benchtop incubator, sequential media and traditional morphological evaluation under optical microscope. The effect of the culture strategies on embryo development and clinical outcomes was quantified by generalized estimated equations, controlling for possible confounders through the inverse probability of the treatment weighting method. MAIN OUTCOME MEASURE(S): Weighted odds ratios (ORs) and 95% confidence intervals (CIs) for live birth rate after fresh single embryo transfer and the cumulative live birth rate. In addition, blastocyst development and morphology and other intermediate outcomes were also assessed. RESULT(S): A significant positive association was found between the employment of undisturbed embryo culture and higher live birth rate in the first embryo transfer in both autologous (OR, 1.617; 95% CI, 1.074-2.435) and oocyte donation cycles (OR, 1.316; 95% CI, 1.036-1.672). Cumulative live birth rate after 1-year follow-up was also positively associated with the undisturbed culture strategy in oocyte donation cycles (OR, 1.5; 95% CI, 1.179-1.909), but not in autologous cycles (OR, 1.051; 95% CI, 0.777-1.423). Similarly, blastocyst rate, good morphology blastocyst rate, and utilization rate were positively associated with the employment of undisturbed culture in oocyte donation cycles, but not in autologous cycles. CONCLUSION(S): These findings imply that a culture system combining integrated time-lapse incubators with a one-step culture medium may enhance the success rates of patients undergoing ICSI treatment by increasing the production of higher quality blastocysts and improving embryo selection while streamlining laboratory procedures and workflow.

Cryopreservation and passaging optimization for Galea spixii (Wagler, 1831) adult skin fibroblast lines: A step forward in species management and genetic studies.

BACKGROUND: In vitro culture of fibroblasts is a technique based on cell isolation, physiological characterization, and cryopreservation. This technique has not been described for Galea spixii, therefore, it can be used to learn about its cellular biology and genetic diversity. OBJECTIVE: We established fibroblast lines of six G. spixii individuals from several passages (second, fifth, eighth, and tenth) and cryopreserved them. METHODS: Fibroblasts recovered from skin biopsies were identified based on morphology, immunocytochemistry, and karyotyping. The cells were analyzed for morphology, ultrastructure, viability, proliferation, metabolism, oxidative stress, bioenergetic potential, and apoptosis before and after cryopreservation. RESULTS: After the eighth passage, the fibroblasts showed morphological and karyotypic changes, although their viability, metabolism, and proliferation did not change. An increase in oxidative stress and bioenergetic potential from the fifth to the eighth passages were also observed. Post cryopreservation, cell damage with respect to the ultrastructure, viability, proliferative rate, apoptotic levels, oxidative stress, and bioenergetic potential were verified. CONCLUSION: Fibroblasts up to the tenth passage could be cultured in vitro. However, cells at the fifth passage were of better quality to be used for reproductive techniques. Additionally, optimization of the cryopreservation protocol is essential to improve the physiological parameters of these cells.

In vitro production of meiotically competent oocytes from early antral follicles in sheep.

The potential of using long in vitro culture (LIVC) of cumulus-oocyte complexes (COCs) from early antral follicles (EAFs) as an assisted reproductive technology in cattle has shown promising results. This study explored the feasibility of applying this technology to sheep as seasonal breeding animals. Ovaries from sheep were collected during both the breeding and non-breeding seasons. COCs were isolated from EAFs (350-450 µm) and cultured in TCM199 medium supplemented with 0.15 µg/mL Zn sulfate, 10-4IU/mL FSH, 10 ng/mL estradiol, 50 ng/mL testosterone, 50 ng/mL progesterone, and 5 µM Cilostamide. After five days of LIVC, the COCs were submitted to an in vitro maturation procedure. The results indicate successful in vitro development of COCs, evidenced by a significant increase in oocyte diameter (p < 0.000) and the preservation of gap junction communication between oocyte and cumulus cells. The gradual uncoupling was accompanied by a progressive chromatin transition from the non-surrounded nucleolus (NSN) to the surrounded nucleolus (SN) (p < 0.000), coupled with a gradual decrease in global transcriptional activity and an increase in oocyte meiotic competence (p < 0.000). Maintenance of oocyte-cumulus investment architecture, viability, and metaphase II capability was significantly higher in COCs collected during the breeding season (p < 0.000), suggesting higher quality than those obtained during the non-breeding season. In conclusion, our study confirms LIVC feasibility in sheep, emphasizing increased effectiveness during the breeding season in isolating higher-quality COCs from EAFs. These findings can influence improving the LIVC system in mammals with seasonal reproduction.

Tissue explants as tools for studying the epigenetic modulation of the GH-IGF-I axis in farmed fish.

Somatic growth in vertebrates is mainly controlled by the growth hormone (GH)/insulin-like growth factor I (IGF-I) axis. The role of epigenetic mechanisms in regulating this axis in fish is far from being understood. This work aimed to optimize and evaluate the use of short-term culture of pituitary and liver explants from a farmed fish, the gilthead seabream Sparus aurata, for studying epigenetic mechanisms involved in GH/IGF-I axis regulation. Our results on viability, structure, proliferation, and functionality of explants support their use in short-term assays. Pituitary explants showed no variation in gh expression after exposure to the DNA methylation inhibitor decitabine (5-Aza-2'-deoxycytidine; DAC), despite responding to DAC by changing dnmt3bb and tet1 expression, and TET activity, producing an increase in overall DNA hydroxymethylation. Conversely, in liver explants, DAC had no effects on dnmt s and tet s expression or activity, but modified the expression of genes from the GH-IGF-I axis. In particular, the expression of igfbp2a was increased and that of igfbp4, ghri and ghrii was decreased by DAC as well as by genistein, which is suggestive of impaired growth. While incubation of liver explants with S-adenosylmethionine (SAM) produced no clear effects, it is proposed that nutrients must ensure the methylation milieu within the liver in the fish to sustain proper growth, which need further in vivo verification. Pituitary and liver explants from S. aurata can be further used as described herein for the screening of inhibitors or activators of epigenetic regulators, as well as for assessing epigenetic mechanisms behind GH-IGF-I variation in farmed fish.

Protocol for Generating Arabidopsis thaliana Cell Suspension Cultures Under Different Light Conditions.

Plant cell suspension cultures (PCSCs) are in vitro-cultured cells that can divide indefinitely in a sterile growth medium. These PCSCs can be derived from various plant tissues, such as the root, stem, leaves, or seeds, and are maintained in a suitable culture medium containing nutrients, vitamins, hormones, and other essential components necessary for their growth. PCSCs have extensive applications in biotechnology, particularly in producing pharmaceutical and chemical compounds. This chapter presents a protocol for generating cell lines from Arabidopsis thaliana root callus under different light conditions, which can be used to investigate the effects of light on plant cell growth and development. The protocol described in this chapter is a valuable tool for researchers interested in utilizing PCSCs in their studies.

Chloroplast Isolation in Agavaceas.

Chloroplast isolation protocols have been extensively developed for various species of plants, particularly model organisms with easily manipulable physical characteristics. However, succulent plants, such as Agave angustifolia Haw., which possess adaptations for arid environments like the Crassulacean acid metabolism (CAM) and a thicker cuticle, have received less attention, resulting in a potential knowledge gap. This chapter presents a specialized protocol focusing on isolating chloroplast from A. angustifolia, a species exhibiting adaptations to arid conditions and holding ecological and economic significance due to its role in producing bacanora and mezcal beverages. By successfully isolating chloroplast from A. angustifolia plant growth in ex vitro and in vitro conditions, this protocol enables comprehensive future analyses to elucidate metabolic processes and explore potential applications in related species. Consequently, this research aims to bridge this knowledge gap in chloroplast isolation for succulent plants, providing new insights for future investigations in the field.

Optimal isolation, culture, and in vitro propagation of spermatogonial stem cells in Huaixiang chicken.

Spermatogonial stem cells (SSCs) comprise the foundation of spermatogenesis and hence have great potential for fertility preservation of rare or endangered species and the development of transgenic animals and birds. Yet, developing optimal conditions for the isolation, culture, and maintenance of SSCs in vitro remains challenging, especially for chicken. The objectives of this study were to (1) find the optimal age for SSC isolation in Huaixiang chicken, (2) develop efficient protocols for the isolation, (3) enrichment, and (4) culture of isolated SSCs. In the present study, we first compared the efficiency of SSC isolation using 11 different age groups (8-79 days of age) of Huaixiang chicken. We found that the testes of 21-day-old chicken yielded the highest cell viability. Next, we compared two different enzymatic combinations for isolating SSCs and found that 0.125% trypsin and 0.02 g/L EDTA supported the highest number and viability of SSCs. This was followed by investigating optimal conditions for the enrichment of SSCs, where we observed that differential plating had the highest enrichment efficiency compared to the Percoll gradient and magnetic-activated cell sorting methods. Lastly, to find the optimal culture conditions of SSCs, we compared adding different concentrations of foetal bovine serum (FBS; 2%, 5%, 7%, and 10%) and different concentrations of GDNF, bFGF, or LIF (5, 10, 20, or 30 ng/mL). We found that a combination of 2% FBS and individual growth factors, including GDNF (20 ng/mL), bFGF (30 ng/mL), or LIF (5 ng/mL), best supported the proliferation and colony formation of SSCs. In conclusion, SSCs can be optimally isolated through enzymatic digestion from testes of 21-day-old chicken, followed by enrichment using differential plating. Furthermore, adding 2% FBS and optimized concentrations of GFNF, bFGF, or LIF in the culture promotes the proliferation of chicken SSCs.