An Overview of Reactive Oxygen Species Damage Occurring during In Vitro Bovine Oocyte and Embryo Development and the Efficacy of Antioxidant Use to Limit These Adverse Effects.

The in vitro production (IVP) of bovine embryos has gained popularity worldwide and in recent years and its use for producing embryos from genetically elite heifers and cows has surpassed the use of conventional superovulation-based embryo production schemes. There are, however, several issues with the IVP of embryos that remain unresolved. One limitation of special concern is the low efficiency of the IVP of embryos. Exposure to reactive oxygen species (ROS) is one reason why the production of embryos with IVP is diminished. These highly reactive molecules are generated in small amounts through normal cellular metabolism, but their abundances increase in embryo culture because of oocyte and embryo exposure to temperature fluctuations, light exposure, pH changes, atmospheric oxygen tension, suboptimal culture media formulations, and cryopreservation. When uncontrolled, ROS produce detrimental effects on the structure and function of genomic and mitochondrial DNA, alter DNA methylation, increase lipid membrane damage, and modify protein activity. Several intrinsic enzymatic pathways control ROS abundance and damage, and antioxidants react with and reduce the reactive potential of ROS. This review will focus on exploring the efficiency of supplementing several of these antioxidant molecules on oocyte maturation, sperm viability, fertilization, and embryo culture.

Use of Antifreeze Protein from Tenebrio molitor (TmAFP) in Vitrification of In Vitro-Produced Bovine Embryos: An Ultrastructural Study.

The objective of this study was to evaluate the effects of different concentrations of antifreeze protein (AFP) extracted from the larva of the beetle, Tenebrio molitor (TmAFP), on vitrification of in vitro-produced bovine embryos. In vitro-produced blastocysts were divided into three experimental groups and vitrified using a cryotop. TmAFP was added to the equilibrium solution (ES) and vitrification solution (VS) at a concentration of 0 ng/mL (control), 500 ng/mL (500TmAFP), or 1000 ng/mL (1000TmAFP). Vitrification was carried out by first placing the blastocysts in ES for 2 minutes (7.5% ethylene glycol [EG] and 7.5% dimethyl sulfoxide [DMSO]). The blastocysts were then transferred to VS (15% EG and 15% DMSO) and promptly deposited on a cryotop stem and submerged in liquid nitrogen. Warming was carried out in three steps with decreasing sucrose concentrations. After warming, the blast cells were cultured for 24 hours for subsequent survival analysis and ultrastructural evaluation. There was a significant difference in the survival rate and expansion in the 500TmAFP group compared with the other groups. The ultrastructural analysis revealed intracellular lesions in all vitrified embryos; however, the embryos of the 500TmAFP and 1000TmAFP groups showed fewer cytoplasmic lesions compared with the control group. Taken together, addition of TmAFP can mitigate cellular changes that involve organelles and cellular components essential for proper functioning and improve the viability of warmed and vitrified in vitro-produced bovine embryos.

Impact of Sire on Embryo Development and Pregnancy.

The use of in vitro embryo production (IVP) has increased globally, particularly in the United States. Although maternal factors influencing embryo development have been extensively studied, the influence of the sire is not well understood. Sperm plays a crucial role in embryo development providing DNA, triggering oocyte maturation, and aiding in mitosis. Current sire fertility measurements do not consistently align with embryo production outcomes. Low-fertility sires may perform well in IVP systems but produce fewer pregnancies. Testing sires in vitro could identify characteristics affecting embryo development and pregnancy loss risk in IVP and embryo transfer programs.

Effect of IL-10 and TNF-α on the competence and cryosurvival of in vitro produced Bos indicus embryos.

In vitro-produced embryos are constantly exposed to stressful conditions that can lead to the activation of the apoptotic pathway. The nuclear Kappa B factor (NF-κB) is an inflammatory mediator that induces the expression of tumor necrosis factor (TNF-α), a pro-inflammatory cytokine, while interleukin-10 (IL-10), an anti-inflammatory cytokine, inhibits NF-κB activity. This study aimed to investigate the effects of IL-10 and TNF-α on the competence and cryosurvival of in vitro-produced bovine embryos. Embryos were produced in vitro using standard protocols, and Grade I blastocysts were vitrified using the Cryotop method. Non-vitrified and vitrified blastocysts were subjected to the TUNEL assay. In Experiment I, on day 6.5 (156 h post-insemination), the embryos were treated with PBS (control), 50 ng/mL of IL-10, or a combination of 25 ng/mL of TNF-α and 50 ng/mL of IL-10. Embryonic development and apoptotic rates were monitored. In Experiment II, the same groups were set up, with the addition of a group treated with 25 ng/mL of TNF-α alone. Grade I blastocysts were vitrified 5 h after treatment, and cryosurvival was monitored at until 48 h post-warming. The apoptosis rate and total cell number were investigated in the vitrified-hatched blastocysts. IL-10 alone did not affect developmental competence or cryosurvival (P > 0.05). The IL-10-treated embryos, when exposed in combination with TNF-α, presented a detrimental effect (P < 0.05) in the embryonic development of non-vitrified embryos. However, vitrified blastocysts had no negative effect (P > 0.05). The TNF-α treatment reduced (P < 0.05) the re-expansion rate at 6 h post-warming and increased (P < 0.05) the apoptosis rate in vitrified hatched blastocysts, whereas no effect (P > 0.05) of the treatments was detected in the hatching rate and total cell number post-warming. In conclusion, TNF-α has a detrimental effect on embryonic developmental competence and cryosurvival by compromising the development of non-vitrified embryos and apoptotic-related events of vitrified blastocysts, whereas IL-10, when in combination with TNF-α, appears to attenuate the detrimental effects of TNF-α.

Enhancing mass production of Heterorhabditis bacteriophora: influence of different bacterial symbionts (Photorhabdus spp.) and inoculum age on dauer juvenile recovery.

The entomopathogenic nematode Heterorhabditis bacteriophora (Nematoda: Rhabditidae) is used in biological insect control. Their dauer juveniles (DJs) are free-living and developmentally arrested, invading host insects. They carry cells of their bacterial symbiont Photorhabdus spp. in the intestine. Once inside the insect´s hemolymph the DJs perceive a food signal, triggering them to exit the DJ stage and regurgitate the Photorhabdus cells into the insect's haemocoel, which kill the host and later provide essential nutrients for nematode reproduction. The exit from the DJ stage is called "recovery". For commercial pest control, nematodes are industrially produced in monoxenic liquid cultures. Artificial media are incubated with Photorhabdus before DJs are added. In absence of the insect's food signal, DJs depend on unknown bacterial food signals to trigger exit of the DJ stage. A synchronized and high DJ recovery determines the success of the industrial in vitro production and can significantly vary between nematode strains, inbred lines and mutants. In this study, fourteen bacterial strains from H. bacteriophora were isolated and identified as P. laumondii, P. kayaii and P. thracensis. Although the influence of bacterial supernatants on the DJ recovery of three inbred lines and two mutants differed significantly, the bacterial impact on recovery has a subordinate role whereas nematode factors have a superior influence. Recovery of inbred lines decreased with age of the DJs. One mutant (M31) had very high recovery in bacterial supernatant and spontaneous recovery in Ringer solution. Another mutant (M88) was recovery defective.

Screening of endophytic fungi from Antarctic mosses: Potential production for L-asparaginase free of glutaminase and urease activity.

Studies involving endophytic fungi aim to identify organisms inhabiting extreme and relatively unexplored environments, as these fungi possess unique characteristics and uncommon biochemical pathways that enable them to produce compounds with biotechnological potential. Among various enzymes, L-Asparaginase is employed in the treatment of Acute Lymphoblastic Leukemia. In this study, we identified endophytic fungi from Sanionia uncinata and Polytrichastrum alpinum collected on King George Island in Antarctica. The fungi were categorized into morphological groups based on their characteristics, molecularly identified, and assessed for L-Asparaginase (L-ASNase) enzyme production. Subsequently, production optimization was conducted. A total of 161 endophytes were isolated from 504 moss gametophytes, with 107 originating from P. alpinum and 54 from S. uncinata. These isolates were categorized into 31 morphotypes. Fungi exhibiting high enzyme production were identified molecularly. Among them, nine identified isolates belonged to the genera Aspergillus, Collariella, Diaporthe, Epicoccum, Peroneutypa, Xylaria, and Trametes. Three of these isolates were identified at the species level through multigene phylogeny, namely Epicoccum nigrum, Collariella virescens, and Peroneutypa scoparia. All 31 fungi were subjected to solid media testing for L-ASNase enzyme production, with 22 isolates demonstrating production capability, and 13 of them produced L-ASNase free from Urease and Glutaminase. The isolates displaying solid media production underwent further testing in liquid media, all of which exhibited enzyme production ranging from 0.75 to 1.29 U g-1. Notably, the three fungi identified at the species level were the highest producers of the enzyme (1.29, 1.17, and 1.13 U g-1). The production of these fungi was optimized using the Taguchi method, resulting in production values ranging from 0.687 to 2.461 U g-1. In conclusion, our findings indicate that Antarctic moss endophytic fungi exhibit significant potential for the production of the anti-leukemic enzyme L-ASNase.

Bioreactor Production Process of Spodoptera frugiperda multiple nucleopolyhedrovirus Biopesticide.

Spodoptera frugiperda (fall armyworm) is one of the most important maize pests in the world and the baculovirus Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV), a natural pathogen of this pest, has been used as a biopesticide for its control. At present, in vivo strategies at the commercial scale are employed by multiplying the virus in the host insect in biofactory facilities; however, in vitro large-scale production is an interesting alternative to overcome the limitations of baculoviruses massal production. This study aimed to develop the process of the SfMNPV in vitro production by evaluating the effects of different multiplicities of infection (MOI) and nutritional supplements, morphological and molecular analysis of the infection on the growth of Sf9 cells and virus production. The Bioreactor Stirred Tank Reactor (STR) approach with glutamine-supplemented Sf-900 III serum free culture medium, combined with the MOI of 1.0, showed the best viral production performance, with a specific productivity above 300 occlusion bodies (OBs)/cell and volumetric productivity of 9.0 × 1011 OBs/L.

Health assessment of Holstein calves born after in vitro fertilization, biopsy-based genotyping at the blastocyst stage and subsequent embryo transfer.

Establishing methods for evaluating genomic estimated breeding values of bovine embryos can potentially increase the efficiency of breeding programs by transferring only embryos with a high genomic estimated breeding value. This may be achieved by analyzing DNA from trophectoderm biopsies. However, manipulation of bovine embryos is associated with a risk of impaired conceptus health. More knowledge on the health implications of embryonic handling procedures is required. In this study, we followed pregnancies after transfer of in vitro-produced (IVP) embryos and assessed the health of the offspring during the first 2 weeks of life. Three groups of calves were studied: i) freshly transferred non-biopsied embryos (39 transfers, 17 calves; Group B-/C-); ii) biopsied and freshly transferred IVP embryos (42 transfers, 21 calves; Group B+/C-); iii) biopsied and cryopreserved IVP embryos (17 transfers, 6 calves; Group B+/C+). Blood biochemical and hematologic values were compared between groups and to a control group of 13 calves produced by conventional artificial insemination. The pregnancy rate on day 50 and the calving rate did not differ among the groups, but the average gestation length of the B+/C+ group was significantly shorter and with wider variation than the two other groups. There was a tendency toward a higher average body weight at birth in group B+/C+ (45.1 kg) and the standard deviation in body weight was larger (11.7 kg) compared to the B-/C- (39.5 kg; 3.2 kg) and B+/C- (41.8 kg; 6 kg) groups. Body weight on day 14 was higher in the B+/C+ calves compared to the other groups. There was no difference in the biochemical and hematological values at birth between the groups and these were within the normal range. However, when compared to a group of calves produced by standard artificial insemination, significantly higher concentrations were found for the hepatic-related enzymes ALAT, ASAT, ALP, and GGT in group B-/C-and B+/C-, while only higher ALP concentrations were found in B+/C+ calves. The biochemical findings indicate higher heterogeneity in IVP calves compared to calves produced by artificial insemination. The more manipulated IVP embryos also showed increased heterogeneity in body weight at birth, with a shift toward heavier calves, which calls for closer attendance at parturition to handle dystocia in a timely manner and minimize fetal losses.

Review: Some challenges and unrealized opportunities toward widespread use of the in vitro-produced embryo in cattle production.

The embryo produced by in vitro oocyte maturation, fertilization, and embryonic development is an important resource for genetic improvement and has the potential to improve female fertility and to be programmed to produce offspring with superior ability for health and production. The cultured embryo is also an important component of several realized and potential technologies such as gene editing, somatic cell nuclear cloning, stem cell technologies and gamete generation in vitro. Full realization of the opportunities afforded by the in vitro-produced embryo will require overcoming some technical obstacles to cost-effective implementation of an embryo transfer program. Among the research goals for improving the penetration of embryo transfer in the cattle industry are development of methods to increase the supply of oocytes from genetically elite females, enhance the proportion of oocytes that become transferrable embryos, improve the fraction of embryos that establish pregnancy after transfer, reduce pregnancy wastage after pregnancy diagnosis, and identify culture conditions to optimize postnatal phenotype.

Review: Large offspring syndrome in ruminants: current status and prediction during pregnancy.

Large/abnormal Offspring Syndrome (LOS/AOS) is a congenital overgrowth condition of cattle and sheep, characterized by macrosomia, abdominal wall defects, organomegaly, difficulty to stand and suckle at parturition. The condition was first described as an exclusive consequence of assisted reproductive technologies, such as in vitro production and somatic cell nuclear transfer (cloning). However, we recently reported the spontaneous occurrence of this syndrome in cattle. The etiology of LOS is unclear, although the syndrome is an epigenetic condition characterized by multi-locus loss-of-imprinting, global dysregulation of small and long RNAs, changes in DNA methylation, and altered chromosomal architecture. These molecular and epigenetic changes affect biological pathways implicated in organ size, cell proliferation, cell survival, resulting in the phenotypes which characterize LOS. The lack of accurate tools for the prediction and diagnosis of LOS and the prevention of dystocia resulting from fetal overgrowth is a major concern for the dairy and beef industries. Furthermore, death of the calf and/or dam during calving adds animal welfare issues and affects the net income of the industry. An early diagnosis of LOS/AOS during gestation is critical to facilitate the decision-making process on whether to allow the pregnancy to continue or not in order to prevent harm to the dam as well as to provide producers with the timely necessary information to prepare for a difficult birth. The present review summarizes the definition, traits, incidence, and molecular characteristics of LOS to provide information and serve as a guide for future investigations regarding the early identification of LOS during pregnancy in cattle.

Review: Use of assisted reproduction in seasonal-calving dairy herds.

A unique aspect of seasonal-calving pasture-based systems of dairy production is the intense focus placed on achieving a concentrated herd-calving period in late winter and early spring. Hence, excellent reproductive performance is required during a short breeding period. A concentrated calving period also produces a problem in the form of a large number of male dairy calves being born at the same time; as these calves have little economic value due to poor beef merit, they present a potential welfare concern. A solution exists in the form of sex-sorted semen, but this is typically associated with poorer pregnancy per artificial insemination, and hence, the use of sex-sorted semen must be carefully considered. The logical strategy to use sex-sorted semen is to target the best genetic merit dams in the herd to generate replacement heifers, thereby accelerating herd genetic gain. On the other hand, if all dairy farmers adopt such a strategy, there will be a corresponding reduction in elite genetic merit male dairy calves being born, potentially reducing availability of the next generation of future bulls to be used for artificial insemination. Use of in vitro embryo production on elite dairy donors could avoid this problem by acting as a multiplier, potentially in tandem with Y-sorted semen to skew the offspring sex ratio towards more male calves. Use of sex-sorted semen on the best genetic merit dams can also facilitate a marked increase in the usage of beef semen on any dams that are deemed unsuitable for sex-sorted semen. The use of "beef on dairy" requires selection of beef bulls that generate offspring with traits that meet the key requirements of both the dairy farmer (e.g., gestation length and calving ease) and the beef farmer that must be motivated to purchase the calves (e.g., growth rate, age at slaughter, carcass value). Beef breed dams that have elite genetic merit for these traits could also be considered for in vitro embryo production, potentially in tandem with Y-sorted semen, to facilitate genetic gain for the growing "beef-on-dairy" market. It is possible to transfer a beef embryo (75-100% beef breed genetics) into dairy dams that are not required to generate replacements, but this is likely to remain a niche practice as there are many barriers to widespread adoption. Such combinations of assisted reproduction have the potential to improve the efficiency and sustainability metrics of seasonal-calving pasture-based dairy herds.

Pre-Implantation Bovine Embryo Evaluation-From Optics to Omics and Beyond.

Approximately 80% of the ~1.5 million bovine embryos transferred in 2021 were in vitro produced. However, only ~27% of the transferred IVP embryos will result in live births. The ~73% pregnancy failures are partly due to transferring poor-quality embryos, a result of erroneous stereomicroscopy-based morphological evaluation, the current method of choice for pre-transfer embryo evaluation. Numerous microscopic (e.g., differential interference contrast, electron, fluorescent, time-lapse, and artificial-intelligence-based microscopy) and non-microscopic (e.g., genomics, transcriptomics, epigenomics, proteomics, metabolomics, and nuclear magnetic resonance) methodologies have been tested to find an embryo evaluation technique that is superior to morphologic evaluation. Many of these research tools can accurately determine embryo quality/viability; however, most are invasive, expensive, laborious, technically sophisticated, and/or time-consuming, making them futile in the context of in-field embryo evaluation. However accurate they may be, using complex methods, such as RNA sequencing, SNP chips, mass spectrometry, and multiphoton microscopy, at thousands of embryo production/collection facilities is impractical. Therefore, future research is warranted to innovate field-friendly, simple benchtop tests using findings already available, particularly from omics-based research methodologies. Time-lapse monitoring and artificial-intelligence-based automated image analysis also have the potential for accurate embryo evaluation; however, further research is warranted to innovate economically feasible options for in-field applications.

Unraveling the Consequences of Oxygen Imbalance on Early Embryo Development: Exploring Mitigation Strategies.

Although well-established and adopted by commercial laboratories, the in vitro embryo production system still requires refinements to achieve its highest efficiency. Early embryonic development is a dynamic event, demanding suitable conditions to provide a high number of embryos with quality and competence. The first step to obtaining an optimized in vitro environment is to know the embryonic metabolism and energy request throughout the different stages of development. Oxygen plays a crucial role in several key biological processes necessary to sustain and complete embryonic development. Nonetheless, there is still controversy regarding the optimal in vitro atmospheric concentrations during culture. Herein, we discuss the impact of oxygen tension on the viability of in vitro-produced embryos during early development. The importance of oxygen tension is addressed as its roles regarding essential embryonic traits, including embryo production rates, embryonic cell viability, gene expression profile, epigenetic regulation, and post-cryopreservation survival. Finally, we highlight the damage caused by in vitro unbalanced oxygen tensions and strategies to mitigate the harmful effects.

Bioactive Materials Derived from Menstrual Blood Stem Cells Enhance the Quality of In Vitro Bovine Embryos.

Backgrounds: The aim of this study was to determine whether the addition of bioactive materials derived from Menstrual Blood Stem Cells (MenSCs) to the oocyte maturation medium may improve the quality of bovine embryos in vitro. Methods: MenSCs were collected from 6 healthy women (between 26 and 36 years old) and after 3 days of culture, their bioactive materials were frozen. The bovine Cumulus-Oocyte-Complexes (COCs) were aspirated from ovarian slaughterhouse and the oocytes with more than three layers of cumulus cells were cultured in vitro in media supplemented with (treatment) and without (control) 10% MenSCs' bioactive materials. After IVM/IVF, the presumptive zygotes were cultured for 8 days. Results: The blastocyst rate on day 8 in treatment group was higher than control (40.2±1.9 vs. 23±4.2.3, p=0.001). The ratio of Trophectoderm (TE) and Inner Cell Mass (ICM) (ICM/TE) cells was also greater in treatment group compared to control (30.3±2 vs. 14.9±1; p=0.001). The re-expansion of vitrified blastocysts, 24 hours after warming, in treatment group was higher than control (93.3±2.5 vs. 66.2±8.8; p=0.01). The expression of some genes related to pluripotency and implantation (OCT4, CDX2, and IFNT) were increased in treatment group compared to control (p<0/05). Conclusion: In conclusion, the addition of MenSCs' bioactive materials during in vitro maturation of bovine oocytes could improve the quantity and quality of bovine IVP embryos. Also, the expression of some genes associated with pluripotency and implantation in the blastocyst would be increased.

Supplement of secreted recombinant low molecular weight human fibroblast growth factor 2 in culture media enhances in vitro bovine maturation.

With the annual increase in in vitro bovine embryo production, understanding oocyte maturation is becoming more important. Previous studies have shown that oocyte maturation can be improved by adding bovine additives to in vitro maturation media. Among the additives, human fibroblast growth factor 2 (hFGF2) is well known for its positive influence on the growth rate and quality of cells and oocytes. However, the effect of LMW-hFGF2, one of the isoforms of hFGF2, on bovine in vitro maturation has not yet been identified. Therefore, the goal of this study was to elucidate the effect of LMW-hFGF2 on bovine oocyte maturation. Vectors expressing LMW-hFGF2 were cloned and transfected into cells. Afterward, secretion of LMW-hFGF2 from cells was confirmed, and used to assess the effect LMW-hFGF2 on cells and bovine oocytes. LMW-hFGF2 improved bovine oocyte maturation and embryo developmental competence. Laboratories can use LMW-hFGF2 in bovine oocyte culture media to improve in vitro embryo production success rates.

Anticancer Secondary Metabolites: From Ethnopharmacology and Identification in Native Complexes to Biotechnological Studies in Species of Genus Astragalus L. and Gloriosa L.

Some of the most effective anticancer compounds are still derived from plants since the chemical synthesis of chiral molecules is not economically efficient. Rapid discovery of lead compounds with pronounced biological activity is essential for the successful development of novel drug candidates. This work aims to present the chemical diversity of antitumor bioactive compounds and biotechnological approaches as alternative production and sustainable plant biodiversity conservation. Astragalus spp., (Fabaceae) and Gloriosa spp. (Liliaceae) are selected as research objects within this review because they are known for their anticancer activity, because they represent two of the largest families respectively in dicots and monocots, and also because many of the medicinally important plants are rare and endangered. We summarized the ethnobotanical data concerning their anticancer application, highlighted the diversity of their secondary metabolites possessing anticancer properties such as saponins, flavonoids, and alkaloids, and revealed the potential of the in vitro cultures as an alternative way of their production. Since the natural supply is limited, it is important to explore the possibility of employing plant cell or organ in vitro cultures for the biotechnological production of these compounds as an alternative.

Toward in Vitro Production of Platelet from Induced Pluripotent Stem Cells.

Platelets (PLTs) are small anucleate blood cells that release from polyploidy megakaryocytes(MKs). PLT transfusion is standard therapy to prevent hemorrhage. PLT transfusion is donor-dependent way which have limitations including the inadequate donor blood supply, poor quality, and issues related to infection and immunity. Overcoming these obstacles is possible with in vitro production of human PLTs. Currently several cells have been considered as source to in vitro production of PLTs such as hematopoietic stem cells (HSCs), embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). However, HSCs are a limited source for PLT production and large-scale expansion of HSC-derived PLT remains difficult. Alternative sources can be ESCs which have unlimited expansion capacity. But ESCs have ethical issues related to destroying human embryos. iPSCs are considered as an ideal unlimited source for PLT production. They are able to differentiate into any cells and have the capacity of self-renewal. Moreover, iPSCs can be acquired from any donor and easily manipulated. Due to new advances in development of MK cell lines, bioreactors, feeder cell-free production and the ability of large scale generation, iPSC-based PLTs are moving toward clinical applicability and considering the minimal risk of alloimmunization and tumorigenesis of these products, there is great hopefulness they will become the standard source for blood transfusions in the future. This review will focus on how to progress of in vitro generation of PLT from stem cell especially iPSCs and some of the successful strategies that can be easily used in clinic will be described.

Sustainable utilization of Gyrinops walla Gaetner: in vitro production of sesquiterpenes by chemical and biological elicitation.

BACKGROUND: The recent recovery of Gyrinops walla as a potential producer of market-quality agarwood in mature damaged woods and branches has led to the intense illicit felling and exportation of G. walla leading to the verge of extinction from Sri Lankan flora. The sustainable utilization of G. walla undoubtedly enhances the foreign exchange of the country and the non-destructive utilization through tissue culture-based techniques is the only option available for sustainable exploitation and conservation of the vulnerable species. Healthy calli and cell suspensions were chemically and biologically elicited with salicylic acid (SA) and methyl jasmonate (MJ), and the sterilized fungal homogenate (carbohydrate equivalents) of Fusariym oxysporum, Phaeocremonium parasitica, Aspergillus niger, Trichoderma viride, Penicillium commune and Lasidiplodia theobromae fungal strains, respectively. The elicited calli and cell suspensions were harvested at different time periods to extract sesquiterpenes. RESULTS: Sesquiterpenes were produced in calli under chemical elicitors with media concentrations of 10 µM SA, 100 µM SA, 10 mM MJ and 1 mM MJ and cell suspensions under 0.5 µM SA and 0.1 mM MJ. Phaeocremonium parasitica, Trichoderma viride and Lasidiplodia theobromae were more effective in the production of sesquiterpenes in G. walla callus and cell suspension by biological elicitation. CONCLUSION: The findings of the study led to the conclusion of the possibility of induction of production of sesquiterpenes through elicitation of G. walla calli and cell suspension in an in vitro system for sustainable utilization and conservation endeavours.

Vitrification yields higher cryo-survival rate than slow freezing in biopsied bovine in vitro produced blastocysts.

Vitrification and slow freezing are the two commonly used embryo cryopreservation methods. In most studies, vitrification of intact embryos has proven superior in several respects, including cell and embryo survival and pregnancy rate. However, there is a lack of data for comparing these two methods in in vitro produced (IVP) bovine blastocysts, which have been subjected to the retrieval of trophectoderm (TE) biopsy. Day 7 IVP blastocysts were pooled and randomized into four groups: 1) non-biopsy (NB), 2) biopsy (B), 3) biopsy-vitrification (BV), 4) biopsy-slow freeze (BSF). The blastocysts in the B, BV, and BSF groups were subjected to TE biopsy. For the B group, this was followed by 5 hours (h) incubation and subsequent scoring of the biopsy-survival (re-expansion) rate before processing for further analyses. For the BV and BSF groups, the biopsy procedure was followed by 2 h incubation, allowing for a quick re-expansion, after which the blastocysts were subjected to vitrification and slow freezing, respectively. After warming and thawing, respectively, they were then incubated for 5 h followed by scoring the cryo-survival (re-expansion) rates before processing for further analyses. These included quantification of ICM and TE cells, cleaved caspase-3- and TUNEL-positive cells, quantitative PCR on cellular stress markers (SOD1 and PRDX1), and ultrastructural analysis. The biopsy-survival rate in the B group was 94% (307/326). The cryo-survival rate in BV (86%, 138/161) was higher than that in BSF (57%, 81/142; P < 0.001). No differences were noted between the average ICM, TE, and total cell numbers of the groups. The percentages of cleaved caspase-3-positive cells were higher in BV vs. NB (P < 0.05), in BSF vs. NB (P < 0.001), and in BSF vs. B (P < 0.001). The percentages of TUNEL-positive cells were higher in BV vs. NB (P < 0.05) and in BSF vs. NB (P < 0.001). The levels of mRNA abundance for SOD1 and PRDX1 in B, BV, and BSF were not different from that in NB. The ultrastructural analysis of blastocysts in the BV and BSF groups showed distension of extracellular spaces and appearance of intracellular vacuoles in the ICM, distension of mitochondria, and disorganization of mitochondrial cristae in both ICM and TE, and weakened tight junctions between adjacent TE cells. In summary, our findings demonstrate that vitrification yields a higher cryo-survival rate than slow freezing in biopsied bovine IVP blastocysts. However, biopsy-vitrification and biopsy-slow-freeze values are comparable in terms of ICM, TE, and total blastocyst cell numbers, as well as cleaved caspase-3- and TUNEL-positive cell rates. Moreover, biopsy and cryopreservation performed alone had no effect on ICM, TE, total blastocyst cell numbers, or TUNEL-positive cell rates. Biopsy and vitrification performed alone had no effect on the cleaved caspase-3 positive cell rates, whereas slow freezing resulted in an increased rate. Furthermore, double traumatization with a combination of biopsy and cryopreservation, either vitrification or slow freezing, resulted in increased rates of cleaved caspase-3- and TUNEL-positive cells.

Mesenchymal Stem Cells as a Cornerstone in a Galaxy of Intercellular Signals: Basis for a New Era of Medicine.

Around 40% of the population will suffer at some point in their life a disease involving tissue loss or an inflammatory or autoimmune process that cannot be satisfactorily controlled with current therapies. An alternative for these processes is represented by stem cells and, especially, mesenchymal stem cells (MSC). Numerous preclinical studies have shown MSC to have therapeutic effects in different clinical conditions, probably due to their mesodermal origin. Thereby, MSC appear to play a central role in the control of a galaxy of intercellular signals of anti-inflammatory, regenerative, angiogenic, anti-fibrotic, anti-oxidative stress effects of anti-apoptotic, anti-tumor, or anti-microbial type. This concept forces us to return to the origin of natural physiological processes as a starting point to understand the evolution of MSC therapy in the field of regenerative medicine. These biological effects, demonstrated in countless preclinical studies, justify their first clinical applications, and draw a horizon of new therapeutic strategies. However, several limitations of MSC as cell therapy are recognized, such as safety issues, handling difficulties for therapeutic purposes, and high economic cost. For these reasons, there is an ongoing tendency to consider the use of MSC-derived secretome products as a therapeutic tool, since they reproduce the effects of their parent cells. However, it will be necessary to resolve key aspects, such as the choice of the ideal type of MSC according to their origin for each therapeutic indication and the implementation of new standardized production strategies. Therefore, stem cell science based on an intelligently designed production of MSC and or their derivative products will be able to advance towards an innovative and more personalized medical biotechnology.