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Pueraria candollei var. mirifica produces and accumulates various phytoestrogen compounds in its tuberous roots, including daidzein and genistein. Plant cell culture methods have been established to alleviate the problems associated with producing valuable phytochemicals from natural or field-cultivated plants, and hairy root culture is one of the most promising methods for the in vitro production of plant secondary metabolites. Thus, this study aimed to produce daidzein and genistein from hairy root cultures of P. candollei var. mirifica. The influences of cultivation parameters, including the culture medium, light conditions, sugar content in the culture medium, incubation temperature, and agitation speed, on biomass and daidzein and genistein production in hairy root cultures of this medicinal plant were investigated. The results revealed that the optimal cultivation conditions for biomass and bioactive compound production were Murashige & Skoog (MS) medium, a sucrose concentration of 30 g/L, a 16/8 h light/dark photoperiod, an incubation temperature of 26 °C, and an agitation speed of 90 rpm. The highest biomass and daidzein and genistein contents achieved in this study were 17.76 g/L, 6.85 mg/g DW, and 0.96 mg/g DW, respectively. Interestingly, the daidzein and genistein contents obtained from hairy roots were approximately 45.7- and 12.0-fold greater than those obtained from normal roots, respectively, suggesting that hairy root culture is a suitable method for the sustainable production of phytoestrogen, daidzein, and genistein from this medicinal plant. [ABSTRACT FROM AUTHOR]

Ovum Pick Up (OPU) is a minimally invasive technique widely used in cattle and mares for oocyte retrieval, involving ultrasound-guided puncture of ovarian follicles. It has been demonstrated that this technique is safe for its repeated use in the same female without affecting her reproductive health, allowing for the retrieval of oocytes in individuals regardless of their reproductive status. The oocytes obtained through OPU can subsequently be used for in vitro embryo production (IVP) using assisted reproductive techniques (ARTs) or be cryopreserved in biobanks for their future use. Traditionally, the minimally invasive technique of choice performed in vivo in domestic and wild felines was LOPU (laparoscopic-guided ovum pick up). The present study was designed to explore if ultrasound-guided OPU in the domestic cat is safe and effective. In an initial series of ex vivo experiments (n = 92 ovaries, n = 434 oocytes), the effect of different aspiration pressures for oocyte collection was explored. These experiments identified 43 mmHg as the optimal aspiration pressure, resulting in the highest recovery rate and a favorable maturation and blastocyst rate. Subsequently, 16 grade I and II oocytes were retrieved by OPU and 101 oocytes were retrieved following ovariectomy and slicing. Sixteen oocytes obtained with each technique were subjected to in vitro maturation (IVM) and in vitro fertilization (IVF). A total of 14 presumptive zygotes were selected for in vitro culture (IVC) from each group (OPU and slicing), obtaining a cleavage rate of 57.1 % and 64.2 %, a morula rate of 28.5 % in both groups, and a blastocyst rate of 7.14 % and 14.2 % respectively. The hormonal stimulation protocol was well-tolerated, with no adverse effects observed. Moreover, no complications arose during the ovariectomy performed post-OPU. The use of this technique in domestic cats represents a significant step forward in terms of safety, replicability, and invasiveness, serving as a valuable model for its application in wild felids species. Additional research involving a greater number of animals is required to validate these encouraging findings. • This study presents the first successful ultrasound-guided ovum pick-up (OPU) in the domestic cat. • OPU is a minimally invasive technique that offers advantages in terms of invasiveness and safety compared to LOPU. • In vitro embryo production from OPU and slicing showed comparable cleavage, morula, and blastocyst rates. • This research provides a replicable model for potential application in wild felid species. [ABSTRACT FROM AUTHOR]

Camptothecin (CPT), an indole alkaloid popular for its anticancer property, is considered the third most promising drug after taxol and famous alkaloids from Vinca for the treatment of cancer in humans. Camptothecin was first identified in Camptotheca acuminata followed by several other plant species and endophytic fungi. Increased harvesting driven by rising global demand is depleting the availability of elite plant genotypes, such as Camptotheca acuminata and Nothapodytes nimmoniana, crucial for producing alkaloids used in treating diseases like cancer. Conservation of these genotypes for the future is imperative. Therefore, research on different plant tissue culture techniques such as cell suspension culture, hairy roots, adventitious root culture, elicitation strategies, and endophytic fungi has been adopted for the production of CPT to meet the increasing demand without affecting the source plant's existence. Currently, another strategy to increase camptothecin yield by genetic manipulation is underway. The present review discusses the plants and endophytes that are employed for camptothecin production and throws light on the plant tissue culture techniques for the regeneration of plants, callus culture, and selection of cell lines for the highest camptothecin production. The review further explains the simple, accurate, and cost-effective extraction and quantification methods. There is enormous potential for the sustainable production of CPT which could be met by culturing of suitable endophytes or plant cell or organ culture in a bioreactor scale production. Also, different gene editing tools provide opportunities for engineering the biosynthetic pathway of CPT, and the overall CPT production can be improved. Key points: • Camptothecin is a naturally occurring alkaloid with potent anticancer properties, primarily known for its ability to inhibit DNA topoisomerase I. • Plants and endophytes offer a potential approach for camptothecin production. • Biotechnology approaches like plant tissue culture techniques enhanced camptothecin production. [ABSTRACT FROM AUTHOR]

Simple Summary: Early embryonic mortality is one of the main causes of poor reproductive health and is influenced by many factors, including mycotoxins and their metabolites in feed. When several mycotoxins are present in feed, they can have different effects and cause significant animal health problems; however, their synergistic effects have not yet been explored. We found that the blastocyst rate was affected by acetonitrile as a vehicle for the mycotoxin. When the desired mycotoxin concentrations were diluted with the lowest possible amount of acetonitrile, we found no statistical difference between blastocyst rates, although there was a trend suggesting that the combination caused poorer blastocyst development. However, a less invasive vehicle should be used to see if the combination of mycotoxins can affect blastocyst rates. The quality of animal feed is increasingly affected by weather conditions, high humidity, and damage to grains, which have led to various mycotoxin-producing moulds. The aim of this study was to determine the effects of the combination of deepoxy-deoxynivalenol and beta-zearalenol on the development of preimplantation bovine embryos, the extent to which the presence of both mycotoxin metabolites affects the development of in vitro cultured bovine embryos, or whether the effect of both toxins enhances embryotoxicity. Ovaries were transported from the abattoir to the laboratory and, after maturation and fertilisation, zygotes were placed in an embryo culture medium (IVC) with different mycotoxin metabolite concentrations diluted in acetonitrile. It was found that the blastocyst rate of cleaved embryos was affected by 1 μL acetonitrile in 400 μL medium (0.25%) compared to the group without acetonitrile. For this reason, it was decided to use acetonitrile as a control group, and the desired mycotoxin metabolite concentrations were diluted in the lowest possible amount of acetonitrile (0.5 μL) that could be accurately added to the study groups. There was no statistical difference when the higher mycotoxin metabolite concentrations were added. [ABSTRACT FROM AUTHOR]

Introduction: Embryo cryopreservation is a valuable technique used for preserving genetic resources for long periods. However, the survival rate of embryos is dependent on the method used. Therefore, in this study, we evaluated the efficiency of slow freezing method but with an additional dehydration step prior to freezing to overcome the formation of ice crystals. Methods: Oocytes collected from the ovaries of native Korean cattle subjected to in vitro fertilization were cultured for 7 days until the formation of expanded blastocysts. Before freezing, the blastocysts were placed in four pre-equilibration media: a control medium with no addition of sucrose, and three experimental media with the addition of 0.1, 0.25, and 0.5 M sucrose, respectively. Then, the pre-equilibrated embryos were frozen. Embryo survival and hatching rates were evaluated morphologically at 24, 48, and 72 h after thawing. Immunofluorescence staining, terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL) assay, and gene expression analysis of the re-expanded blastocytes were examined 24 h after freeze-thawing. Results: The survival rate was significantly higher in the 0.1 M group than in the control group (p < 0.05), and the hatching rate at 72 h was significantly higher in the 0.25 and 0.5 M groups than in the control group (p < 0.05). TUNEL-positive cells were significantly lower in the 0.25 M group than in the control group (12.5 ± 0.9 vs. 8.3 ± 0.8; p < 0.05). The gene expression of BCL2 associated X, heat shock protein 70 kDa, and aquaporin 3 in the 0.25 M group was significantly lower than that in the control group (p < 0.05). Conclusion: Our study revealed that treatment with 0.25 M sucrose before slow freezing improved the viability of bovine embryos after freeze-thawing. [ABSTRACT FROM AUTHOR]

Anthraquinones (AQs) are potent secondary metabolites distributed among plants with a multitude of bioactivities and therapeutic applications. They are most commonly present in the Rubiaceae, Leguminosae, and Polygonaceae families. At present, globally many anthraquinone-formulated products are synthesized on a commercial scale as dietary supplements and their global demand in the market is significantly increasing day by day. The synthesis of AQs in plants depends on various physiological conditions. Hence, in vitro cultures are considered as an alternative source to produce the compounds with limited availability of explants, independent of environmental conditions. Moreover, the synthesis of anthraquinones from in vitro callus, cell suspension, and organ cultures using biotechnological strategies such as the feeding of precursor or elicitors could eventually enhance the anthraquinone yield. This review, highlights the different plant sources, biosynthesis, in vitro plant cells, organ cultures, elicitation strategies, metabolic engineering, and bioreactors for enhancing anthraquinone production. [ABSTRACT FROM AUTHOR]

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. • TNF-α has a detrimental effect on embryonic developmental competence. • TNF-α increased the embryonic apoptosis rate after cryopreservation. • IL-10, when combined with TNF-α, appears to attenuate the negative effect of TNF-α. • IL-10 stand-alone treatment appears to not favor competence of fresh and vitrified embryos. [ABSTRACT FROM AUTHOR]

Abstract Traditional autoclaving, slow degradation rate and preservation of biomass treated by fungi are the main factors restricting biological treatment. In our previous studies, strains with high efficiency and selective lignin degradation ability were obtained. To further solve the limiting factors of biological treatment, this paper proposed a composite treatment technology, which could replace autoclaves for fungal treatment and improve the preservation and utilization of fungal-pretreated straw. The autoclaved and expanded buckwheat straw were, respectively, degraded by Irpex lacteus for 14 days (CIL, EIL), followed by ensiling of raw materials (CK) and biodegraded straw of CIL and EIL samples with Lactobacillus plantarum for different days, respectively (CP, CIP, EIP). An expansion led to lactic acid bacteria, mold, and yeast of the samples below the detection line, and aerobic bacteria was significantly reduced, indicating a positive sterilization effect. Expansion before I. lacteus significantly enhanced lignin selective degradation by about 6%, and the absolute content of natural detergent solute was about 5% higher than that of the CIL. Moreover, EIL decreased pH by producing higher organic acids. The combination treatment created favorable conditions for ensiling. During ensiling, EIP silage produced high lactic acid about 26.83 g/kg DM and the highest acetic acid about 22.35 g/kg DM, and the pH value could be stable at 4.50. Expansion before I. lacteus optimized the microbial community for ensiling, resulting in EIP silage co-dominated by Lactobacillus, Pediococcus and Weissella, whereas only Lactobacillus was always dominant in CP and CIP silage. Clavispora gradually replaced Irpex in EIP silage, which potentially promoted lactic acid bacteria growth and acetic acid production. In vitro gas production (IVGP) in EIL was increased by 30% relative to CK and was higher than 24% in CIL. The role of expansion was more significant after ensiling, the IVGP in EIP was increased by 22% relative to CP, while that in CIP silage was only increased by 9%. Silage of fungal-treated samples reduced methane emissions by 28% to 31%. The study demonstrated that expansion provides advantages for fungal colonization and delignification, and further improves the microbial community and fermentation quality for silage, enhancing the nutrition and utilization value. This has practical application value for scaling up biological treatment and preserving the fungal-treated lignocellulose. Graphical Abstract

Traditional autoclaving, slow degradation rate and preservation of biomass treated by fungi are the main factors restricting biological treatment. In our previous studies, strains with high efficiency and selective lignin degradation ability were obtained. To further solve the limiting factors of biological treatment, this paper proposed a composite treatment technology, which could replace autoclaves for fungal treatment and improve the preservation and utilization of fungal-pretreated straw. The autoclaved and expanded buckwheat straw were, respectively, degraded by Irpex lacteus for 14 days (CIL, EIL), followed by ensiling of raw materials (CK) and biodegraded straw of CIL and EIL samples with Lactobacillus plantarum for different days, respectively (CP, CIP, EIP). An expansion led to lactic acid bacteria, mold, and yeast of the samples below the detection line, and aerobic bacteria was significantly reduced, indicating a positive sterilization effect. Expansion before I. lacteus significantly enhanced lignin selective degradation by about 6%, and the absolute content of natural detergent solute was about 5% higher than that of the CIL. Moreover, EIL decreased pH by producing higher organic acids. The combination treatment created favorable conditions for ensiling. During ensiling, EIP silage produced high lactic acid about 26.83 g/kg DM and the highest acetic acid about 22.35 g/kg DM, and the pH value could be stable at 4.50. Expansion before I. lacteus optimized the microbial community for ensiling, resulting in EIP silage co-dominated by Lactobacillus, Pediococcus and Weissella, whereas only Lactobacillus was always dominant in CP and CIP silage. Clavispora gradually replaced Irpex in EIP silage, which potentially promoted lactic acid bacteria growth and acetic acid production. In vitro gas production (IVGP) in EIL was increased by 30% relative to CK and was higher than 24% in CIL. The role of expansion was more significant after ensiling, the IVGP in EIP was increased by 22% relative to CP, while that in CIP silage was only increased by 9%. Silage of fungal-treated samples reduced methane emissions by 28% to 31%. The study demonstrated that expansion provides advantages for fungal colonization and delignification, and further improves the microbial community and fermentation quality for silage, enhancing the nutrition and utilization value. This has practical application value for scaling up biological treatment and preserving the fungal-treated lignocellulose. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Lignans are a large group of dimeric phenylpropanoids with a long and distinguished history of medicinal use in the ancient cultures of many peoples. The two main groups, -aryltetralin and arylnaphthalene lignans, are leading compounds with important pharmacological properties and a wide range of biological activities. While the first group is well studied mainly for the production of podophyllotoxin, for arylnaphthalene lignans, the data on the availability of a sustainable resource for their production is are still insufficient. The Linum genus, comprising approximately 180 species, is notable for its arylnaphthalene lignans production like justicidin B and isojusticidin B. The pharmacological potential of arylnaphthalene lignans includes cytotoxic, antiviral, anti-inflammatory and antiprotozoal effects. The review highlights the use of biotechnology by in vitro cultures for optimising lignan production. Structural elucidation of novel lignans underscores the ongoing diversity and potential discoveries in this botanical domain, providing an important additional information of arylnaphthalene lignans. [ABSTRACT FROM AUTHOR]

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. • Nine endophytic fungi isolated from two Antarctic mosses cultured in vitro for biotechnological screening. • Two of then shows a differential L -ASNase production free of glutaminase and urease. • Endophytic fungi from Antarctic moss have a high potential for the production of the antileukemic enzyme L -ASNase. [ABSTRACT FROM AUTHOR]

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. • The more manipulated IVP embryos showed increased heterogeneity in body weight at birth, with a shift toward heavier calves. • A variation in biochemical values indicate higher heterogeneity in IVP calves compared to calves produced by AI. • Biochemical and hematological values were within normal range. [ABSTRACT FROM AUTHOR]

Platelets play a role in hemostasis in vivo, and platelet transfusion is the main means to treat bleeding diseases caused by thrombocytopenia or platelet dysfunction. However, platelets are in short supply due to the increasing demand for platelet products in clinical, the limited number of blood donors and the disadvantages of platelet products such as short shelf life and bacteria contamination. Currently, induced pluripotent stem cells are considered an ideal source for producing platelets in vitro. They have the potential for self-renewal and differentiation into any cell type, and can be obtained and manipulated easily. Given the recent advances in megakaryocytic series, bioreactors, feeder-free cell production and large-scale propagation research, platelet preparations derived from induced pluripotent stem cells have gradually shown great potential for clinical applications. Considering the minimal risk of alloimmunization and tumorigenesis with these blood products, they are promising to become the standard source of future blood transfusions. This paper reviews the research progress of the methodological techniques of in vitro generation of platelets from induced pluripotent stem cells.

Pueraria candollei var. mirifica produces and accumulates various phytoestrogen compounds in its tuberous roots, including daidzein and genistein. Plant cell culture methods have been established to alleviate the problems associated with producing valuable phytochemicals from natural or field-cultivated plants, and hairy root culture is one of the most promising methods for the in vitro production of plant secondary metabolites. Thus, this study aimed to produce daidzein and genistein from hairy root cultures of P. candollei var. mirifica. The influences of cultivation parameters, including the culture medium, light conditions, sugar content in the culture medium, incubation temperature, and agitation speed, on biomass and daidzein and genistein production in hairy root cultures of this medicinal plant were investigated. The results revealed that the optimal cultivation conditions for biomass and bioactive compound production were Murashige & Skoog (MS) medium, a sucrose concentration of 30 g/L, a 16/8 h light/dark photoperiod, an incubation temperature of 26 °C, and an agitation speed of 90 rpm. The highest biomass and daidzein and genistein contents achieved in this study were 17.76 g/L, 6.85 mg/g DW, and 0.96 mg/g DW, respectively. Interestingly, the daidzein and genistein contents obtained from hairy roots were approximately 45.7- and 12.0-fold greater than those obtained from normal roots, respectively, suggesting that hairy root culture is a suitable method for the sustainable production of phytoestrogen, daidzein, and genistein from this medicinal plant.

The quality of animal feed is increasingly affected by weather conditions, high humidity, and damage to grains, which have led to various mycotoxin-producing moulds. The aim of this study was to determine the effects of the combination of deepoxy-deoxynivalenol and beta-zearalenol on the development of preimplantation bovine embryos, the extent to which the presence of both mycotoxin metabolites affects the development of in vitro cultured bovine embryos, or whether the effect of both toxins enhances embryotoxicity. Ovaries were transported from the abattoir to the laboratory and, after maturation and fertilisation, zygotes were placed in an embryo culture medium (IVC) with different mycotoxin metabolite concentrations diluted in acetonitrile. It was found that the blastocyst rate of cleaved embryos was affected by 1 μL acetonitrile in 400 μL medium (0.25%) compared to the group without acetonitrile. For this reason, it was decided to use acetonitrile as a control group, and the desired mycotoxin metabolite concentrations were diluted in the lowest possible amount of acetonitrile (0.5 μL) that could be accurately added to the study groups. There was no statistical difference when the higher mycotoxin metabolite concentrations were added.

Despite numerous advances in in-vitro embryo production (IVP), many documented factors have been shown to influence the development of mammalian preimplantation embryos and the success of IVP. In this sense, elevated levels of reactive oxygen species (ROS) correlate with poor outcomes in assisted reproductive technologies (ART) due to oxidative stress (OS), which results from an imbalance between ROS production and neutralization. Indeed, excessive production of ROS compromises the structural and functional integrity of gametes and embryos both in vivo and in vitro. In particular, OS damages proteins, lipids, and DNA and accelerates cell apoptosis. Several in-vivo and in-vitro studies report an improvement in qualityrelevant parameters after the use of various antioxidants. In this review, we focus on OS and the source of free radicals and their effects on oocytes, sperm, and the embryo during IVP. In addition, antioxidants and their important role in IVP, supplementation during oocyte in vitro maturation (IVM), in vitro culture (IVC), and semen extenders were discussed. Nevertheless, various methods for determining the level of ROS in germ cells have been briefly described. Still, it is crucial to develop standardized antioxidant supplement systems to improve overall IVP success. Further studies should explore the safety, efficacy, mechanism of action, and combination of different antioxidants to improve IVP outcomes.

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. [ABSTRACT FROM AUTHOR]

Steinernema glaseri is a potential biocontrol agent against white grubs of the Phyllophaga spp. complex; however, its suitability for in vitro multiplication has been scarcely investigated. In this study, the effects of the surface culture of NJ-43 strain with its symbiotic bacteria Xenorhabdus poinarii on egg-yolk agar (P2), chickenliver agar and nutrient meat-peptone (MP) agar on infective juvenile (IJ) productivity and their virulence against Galleria mellonella larvae were investigated. The bacteria on the surface of the agar were first incubated in darkness at 30 °C for 54-102 h, and then 100 surface-sterilised IJs were added. After two harvests, the accumulated productivity was higher on chicken-liver agar (536 × 10³ IJs/m² day) and P2 agar (534 × 10³ IJs/m² day) than on nutrient MP agar (58 × 10³ IJs/m2 day). The mean virulence of the in vitro produced IJs was 46-60% and showed no statistically significant difference among the three culture media. In conclusion, the maximum multiplication factor of S. glaseri NJ-43 on solid media was 385, and its original virulence was retained. [ABSTRACT FROM AUTHOR]

Simple Summary: Early embryo development is a dynamic and orchestrated event. Oxygen plays a fundamental role in several key biological processes necessary to sustain and complete embryonic development. Variations in the atmospheric concentrations during preimplantation development can promote several modifications in metabolic function and, consequently, biological processes affecting embryonic competence. Here, we have reviewed the impact of oxygen tension on essential embryonic traits during early development and addressed strategies to mitigate harmful effects due to unbalanced oxygen tensions. Oxygen tension, therefore, is crucial for embryonic competence acquisition and pregnancy success of embryos produced in vitro. 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. [ABSTRACT FROM AUTHOR]

Simple Summary: With conventional breeding practices, even the best dairy/beef cows will likely produce no more than half-a-dozen calves in their lifetimes; a crime, would you not agree? In contrast, with embryo transfer technologies, a cow can pass her genetics to hundreds of calves through surrogate cows. As such, embryo transfer, particularly involving embryos produced by in vitro fertilization, also known as IVF, is being widely adopted. However, pregnancy rates from the transfer of IVF embryos remain unimpressive. Prior to transfer, embryos are evaluated under a microscope, and only high-quality embryos are transferred. Unfortunately, microscopy cannot accurately distinguish between high-quality and low-quality embryos. Many embryo evaluation methodologies have been tested to date to find a replacement for microscopy-based embryo evaluation, e.g., advanced microscopic systems and the evaluation of genetics and secreted proteins/molecules. The good news: many of them can accurately separate the wheat from the chaff. The bad news: many of them are expensive, kill embryos during evaluation, are too complicated, and/or are laborious and thus useless in the frontline, i.e., thousands of embryo production/transfer facilities. This article reviews the most common (and not so common) techniques that have been tested to date and provides insights about those that have the highest potential to replace microscopy-based embryo evaluation in-field applications. 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. [ABSTRACT FROM AUTHOR]

This study examined the effect of a chemically defined media system and co-culture system on the in vitro production of porcine embryos. Cumulus-oocyte complexes (COCs) from porcine ovaries were cultured for 22 h in TCM-199 with Earle’s salts, L-glutamine, and NaHCO3 supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 μg/ml), 10 IU/ml of human chorionic gonadotropin, 25 μg/ml of follicle-stimulating hormone, 0.2 mM sodium pyruvate. But without the hormone for 22-44 h in vitro maturation (IVM) media. Matured oocytes were fertilized in basal IVF-media (modified Tris-buffered medium; mTBM). The porcine zygotes were randomly cultured in different culture conditions as follows: (1)a chemically defined media with porcine zygote medium (PZM), and (2) co-culture with the oviductal epithelial cells for embryo development. The results showed that the percentage of >4-cells, >16-cells developments and blastocysts in the PZM group (71.67%, 45.97%, and 24.33%, respectively) were higher than in the co-culture group (53.05%, 22.47%, and 8.85%, respectively) (p0.05). These results indicated that in vitro production of porcine embryos using a chemically defined medium significantly increased the percentage of >4-cells, >16-cells development, and the blastocyst stage.

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.

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.

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.

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.

Aquilaria malaccensis Lamk. is one of the most widespread agarwood-producing plants that face extinction due to overexploitation. Agarwood propagation using in vitro culture techniques is capable of producing large quantities of plants in a shorter time and free from pests and diseases. Therefore, this study was conducted to analyze the effect of auxins type and concentration on agarwood plantlet formation using a split-plot design. The main plot was the type of auxin which included IAA, IBA and NAA, while the subplot was the concentration used which consisted of 0; 5; 10; 15 and 20 µM. The variable observed was agarwood plantlet formation with parameters measured including the number of shoots and leaves, plant height, and number of roots. The results showed that the formation of agarwood plantlets was controlled by the type, concentration, and interaction between the type and concentration of auxin. Furthermore, explants cultured on Murashige Skoog (MS) medium supplemented with 10 µM IBA produced the highest number of shoots (3.39 shoots explant-1) and leaves (7.25 leaves explants-1), while the addition of 10 uM NAA resulted in the highest number of roots (2.52 roots explant-1). This is the first time a study is conducted to evaluate the effect of type and concentration of auxins on agarwood plantlet formation. The production of high-quality shoots and plantlets increased agarwood germplasm availability to prevent extinction and support sustainable production.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The ever-evolving procedures of the practice of in vitro production (IVP) of embryos has well outpaced the ability of governments and institutions to create standardized rules and regulations around the global trade of these reproductive products. There are several challenges related to identifying and quantifying disease transmission risk in IVP embryos, not the least of which is a lack of published information. The International Embryo Technology Society (IETS) Health and Safety Advisory Committee (HASAC) has identified and addressed several of the challenges related to the potential for disease transmission via IVP embryos. This review will outline the current sanitation practices recommended by the IETS for in vivo derived (IVD) embryos, as well as the current practices at most in vitro fertilization (IVF) labs and will present the case that IVP embryos are not only just as safe as IVD embryos from a disease transmission risk standpoint, but perhaps may even be safer than IVD embryos, as the oocyte, zygote, and embryo in the in vitro production system has no contact with the downstream reproductive anatomy (fimbria, isthmus, oviduct, or uterus) of the donor female., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Embryo transfer in cattle is an increasingly important technique for cattle production. Full attainment of the benefits of the technology will depend on overcoming hurdles to optimal performance using embryos produced in vitro. Given its importance, embryo technology research should become a global research priority for animal reproduction science. Among the goals of that research should be developing methods to increase the proportion of oocytes becoming embryos through optimization of in vitro oocyte maturation and in vitro fertilization, producing an embryo competent to establish and maintain pregnancy after transfer, and increasing recipient fertility through selection, management and pharmacological manipulation. The embryo produced in vitro is susceptible to epigenetic reprogramming and methods should be found to minimize deleterious epigenetic change while altering the developmental program of the resultant calf to increase its health and productivity. There are widening opportunities to rethink the technological basis for much of the current practices for production and transfer of embryos because of explosive advances in fields of bioengineering such as microfluidics, three-dimensional printing of cell culture materials, organoid culture, live-cell imaging, and cryopreservation., Competing Interests: Conflicts of interest: The authors have no conflict of interest to declare., (Copyright © The Author(s).)

Abstract 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.

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.

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.

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.

Abstract Background: Embryos are usually produced in culture systems with an oil overlay, which conveys protection against the evaporation of water and microbial contamination. The oil can also release toxic substances and absorb essential components, such as hormones, which adversely affect the quality of the oocytes and the development of embryos in vitro. Objective: The aim of this study was to validate an oil‐free bovine in vitro production (IVP) system. Method: Cumulus–oocyte complexes collected from abattoir‐derived ovaries were matured, fertilized and cultured employing a standard system. The quantity of medium in both groups (with and without an oil overlay) and throughout all stages of IVP was maintained at a volume of 100 μl. The oil group was covered with paraffin oil. The maturation stage of oocytes was assessed using fluorescence staining after 24 hr and developmental stages of embryos were evaluated on day 8. The expanded day 8 blastocysts were assessed by live–dead staining. Results: Oocytes matured in the absence of an oil overlay had significantly higher maturation rates when compared against matured oocytes in medium with an oil overlay. Steroid concentration is higher in medium after maturation without oil cover. The developmental rate was significantly higher after culture without oil overlay. The total cell number and the live–dead ratio was not significantly different. The osmolality did not differ between both groups during maturation and slightly decreased during culture without oil. Conclusion: Based on the current study, bovine oil‐free IVP systems can be suggested as an alternative to oil‐covered medium.

Aloeswood or Agarwood has long been perfumery commodity traded between Mediterranean Region and Southeast Asia since ancient times. Oil or chips are obtained by destructive harvesting of several Thymeleaceous genera includes Aquilaria and Gonystylus and are expensive and highly demanded ingredients in the global market. The recent recovery of Gyrinops walla as a potential producer of market-quality agarwood in mature damaged woods and branches, the intense illicit felling and exportation 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 G. walla through biotechnology is the only option available for sustainable exploitation and conservation of the vulnerable G. walla species. An electro-elicitation apparatuses were designed and built to stimulate calli and cell suspensions. The elicited and calli and cell suspensions were harvested at different time periods to extract sesquiterpenes. Current intensities of 10 mA and 50 mA, and 0.1 mA were the optimum values for the induction of sesquiterpenes; γ-selinene, β-caryophyllene, α-cadinol and α-guaiene, production in both calli and cell suspension after 8 weeks and 6 hours, respectively. The findings of the study led to conclude the possibility of induction of production of sesquiterpenes through electro-elicitation of G. walla calli and cell suspension in an in vitro system for sustainable utilization and conservation endeavours.

This review discusses the production and sale of fertile oocytes for in vitro fertilization technology, calf production through transplantation and delivery, and the current circulation of calves produced by in vitro production (IVP) embryos.

Abstract The quality of porcine oocytes for use in IVF is commonly graded according to the number of layers of cumulus cells (CCs) surrounding the oocyte; together these form the cumulus oocyte complex (COC). At least three compact layers of CCs is regarded as important for efficient IVP. To test this, oocytes were scored according to cumulus investment, with grade A representing COCs with three or more cumulus layers including granulosa cell‐cumulus oocyte complexes, grade B those with an intact corona radiata surrounded by another layer of cumulus cells and grades C and D representing COCs with lower CC investment. These oocytes were then monitored for in vitro maturation (IVM), as assessed by tubulin immunostaining for meiotic progression, the development of a cortical granule ring, and by glutathione levels. Results indicate that grading correlates closely with nuclear maturation and cytoplasmic maturation, suggesting that grading oocytes by cumulus investment is a reliable method to predict IVM success. Importantly, Grade A and B oocytes showed no significant differences in any measure and hence using a cut‐off of two or more CC layers may be optimal. We also determined the effect of assisted aspiration for oocyte retrieval, comparing the effect of needle size and applied pressure on the retrieval rate. These data indicated that both variables affected oocyte recovery rates and the quality of recovered oocytes. In combination, these experiments indicate that grade A and B oocytes have a similar developmental potential and that the recovery of oocytes of these grades is maximised by use of an 18‐gauge needle and 50 mmHg aspiration pressure.

The main bovine reproductive biotechnologies are recapitulated herein in five sections, and their historical development and current status are analyzed, including the results generated in Mexico. In the 1970s, estrus synchronization and ovulation induction began; thus, the reproductive cycle started to be controlled with the resources available at that time, based on the knowledge of bovine reproductive physiology. Over the years, hormone therapy evolved as new compounds were discovered, refining methods to standardize the effect and generating new methods for the release of hormones. The most widely used biotechnology in the world, artificial insemination, owes its expansion to advances in semen processing, among which the development of diluents, cryopreservation, semen sexing, and computer-assisted sperm analysis stand out. The embryonic era began with the development of multi-ovulation and methods for collecting, evaluating, transferring, and cryopreserving embryos. The second embryonic era came with the fully in vitro production of embryos from immature eggs and frozen sperm, known as in vitro embryo production. Great research and material resources have been invested in this procedure, rendering it a pillar of genetic improvement and productivity, in combination with two other tools: sexed semen and genomic evaluations. A golden age of in vitro embryo production is on the horizon, with the possibility to produce accurate modifications in the embryo genome, thanks to gene editing technology. [ABSTRACT FROM AUTHOR]

Many factors influence the final oocyte maturation, fertilisation, and early embryo development, and there are both similarities and differences between species. When comparing the advancement of assisted reproductive technologies (ARTs), the development in the bovine species is not far behind the medical front, with around one million in vitro-produced bovine embryos each year. This rate of progress is not seen in the other domestic species. This review aims to give an overview of the development and specific difficulties of in vitro embryo production in various domestic animal species, with the main focus on cows, pigs, and cats. In production animals, the aim of ARTs is commonly to increase the genetic progress, not to treat reproductive failure. The ARTs are also used for preservation of genetic diversity for the future. However, specifically for oocyte maturation, fertilisation, and early embryonic development, domestic mammals such as the cow and pig can be used as models for humans. This is particularly attractive from an animal welfare point of view since bovine and porcine oocytes are available in large numbers from discarded slaughterhouse material, thereby decreasing the need for research animals. Both for researchers on the animal and human medical fronts, we aim for the development of in vitro production systems that will produce embryos and offspring that are no different from those conceived and developed in vivo. Species-comparative research and development can provide us with crucial knowledge to achieve this aim and hopefully help us avoid unnecessary problems in the future.

The objective of this study was to determine the effect of a semi-defined culture media system developed in laboratory, named Pigs Media System (PMS) on the in vitro production of porcine embryos. In a first assay, the cummulus-oocytes complexes (COCs) were matured, fertilized and cultured for embryo development in PMS supplemented with bovine serum albumin (BSA), and in North Carolina State University-23 (NCSU-23) supplemented with follicular fluid, until blastocysts evaluation. In the assay 2, maturation and culture were performed in PMS using BSA or polyvinyl alcohol (PVA) in a 2 × 2 factorial arrangement (PMS-BSA/BSA, PMS-BSA/PVA, PMS-PVA/PVA, PMS-PVA/BSA). The PMS had a positive effect on the total cell number (58.04) and the decrease of the total lipids (49.4 %) regarding the NCSU-23 medium (37.98 and 59.2 % respectively; P

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. [ABSTRACT FROM AUTHOR]

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. • Vitrification (V) yielded a higher cryo-survival rate than slow freezing (SF) in biopsied (B) bovine IVP blastocysts. • ICM, TE, total cell numbers, and ICM:TE ratios were comparable between the BV and BSF blastocysts. • Vitrification and slow freezing produced comparable apoptotic and TUNEL-positive cell rates in biopsied IVP blastocysts. • TEM revealed extracellular space expansion and intracellular vacuoles appearance in the ICMs of the BV and BSF blastocysts. • TEMs of BV and BSF showed mitochondrial distension and cristae disorganization in ICM and TE, and weakened TE-TE junctions. [ABSTRACT FROM AUTHOR]

Background: Embryos are usually produced in culture systems with an oil overlay, which conveys protection against the evaporation of water and microbial contamination. The oil can also release toxic substances and absorb essential components, such as hormones, which adversely affect the quality of the oocytes and the development of embryos in vitro. Objective: The aim of this study was to validate an oil‐free bovine in vitro production (IVP) system. Method: Cumulus–oocyte complexes collected from abattoir‐derived ovaries were matured, fertilized and cultured employing a standard system. The quantity of medium in both groups (with and without an oil overlay) and throughout all stages of IVP was maintained at a volume of 100 μl. The oil group was covered with paraffin oil. The maturation stage of oocytes was assessed using fluorescence staining after 24 hr and developmental stages of embryos were evaluated on day 8. The expanded day 8 blastocysts were assessed by live–dead staining. Results: Oocytes matured in the absence of an oil overlay had significantly higher maturation rates when compared against matured oocytes in medium with an oil overlay. Steroid concentration is higher in medium after maturation without oil cover. The developmental rate was significantly higher after culture without oil overlay. The total cell number and the live–dead ratio was not significantly different. The osmolality did not differ between both groups during maturation and slightly decreased during culture without oil. Conclusion: Based on the current study, bovine oil‐free IVP systems can be suggested as an alternative to oil‐covered medium. [ABSTRACT FROM AUTHOR]

Some antioxidants and growth factors play an important role in promoting oocyte maturation and embryo development in many mammalian species, but there is little information about the yak (Bos grunniens). Therefore, the objective of this study was to evaluate Cys, insulin-like growth factor-1 (IGF-1) and epidermis growth factor (EGF) on yak oocyte maturation, cleavage and blastocyst rates after in vitro fertilized with Jersey sperm. A single or different combination of Cys, EGF and IGF-1 was added to in vitro maturation (IVM) and in vitro culture (IVC) media. The results showed that a single addition of Cys and IGF-1 increased oocyte maturation and blastocyst rates (p