RESUMEN Introducción. Las células dendríticas (CD) desempeñan un papel clave en la presentación antigénica y la activación de linfocitos T, pero su función puede ser modulada por el microambiente tumoral, lo que afecta la respuesta inmunitaria antitumoral. Este estudio se centra en la interacción entre las CD y el hepatocarcinoma (HCC), explorando cómo el entorno tumoral influye en la actividad de las CD. Objetivo. Evaluar la variación en la actividad de las CD en respuesta a la expresión de citoquinas proinflamatorias, IL-10 y receptores CXCR4 y CCR7 en un modelo murino de hepatocarcinoma (PM299L). Métodos. Se realizaron ensayos in vitro cocultivando CD murinas y una línea tumoral murina de HCC. Se evaluó la expresión de citoquinas proinflamatorias (IL-12, IL-6, IL-1β, TNF-α), inmunosupresora (IL-10) y receptores asociados a migración y maduración (CXCR4 y CCR7) mediante Qpcr a las 24, 48 y 72 horas. Los ensayos se repitieron tres veces. Resultados. Las CD expuestas al entorno tumoral de HCC mostraron una mayor expresión de citoquinas proinflamatorias y IL-10 en comparación al grupo control. Además, se observó una expresión elevada de receptores CXCR4 y CCR7 en las CD expuestas al HCC. Estos cambios en la expresión de genes ocurrieron en un período de 72 horas de cocultivo. Conclusión. La actividad de las CD se ve influenciada por el entorno tumoral de HCC y el tiempo de interacción, lo que modula su función proinflamatoria y de presentación antigénica. Estos hallazgos destacan la importancia de comprender la dinámica de la respuesta inmunitaria en el hepatocarcinoma.

ABSTRACT Introduction. Dendritic cells (DCs) play a key role in antigen presentation and T cell activation, but their function can be modulated by the tumor microenvironment, affecting the antitumor immune response. This study focuses on the interaction between DCs and hepatocellular carcinoma (HCC), exploring how the tumor environment influences DC activity. Objective. To evaluate the variation in DC activity in response to the expression of proinflammatory cytokines, IL-10 and CXCR4 and CCR7 receptors in a murine model of hepatocellular carcinoma (PM299L). Methods. In vitro assays were performed co-culturing murine DCs and HCC tumor line. The expression of proinflammatory cytokines (IL-12, IL-6, IL-1β, TNF-α), immunosuppressive (IL-10) and receptors associated with migration and maturation (CXCR4 and CCR7) was evaluated by Qpcr at 24, 48 and 72 hours. The tests were repeated three times. Results. DCs exposed to the HCC tumor environment showed increased expression of proinflammatory cytokines and IL-10 compared to the control group. Furthermore, elevated expression of CXCR4 and CCR7 receptors will be observed in DCs exposed to HCC. These changes in gene expression occurred within a 72-h period of coculture. Conclusion. DC activity is influenced by HCC tumor environment and interaction time, which modulates their proinflammatory and antigen presentation function. These findings highlight the importance of understanding the dynamics of the immune response in hepatocarcinoma.

RESUMEN El estudio tuvo como objetivo evaluar el efecto del suero fetal bovino (FCS) agregado al medio de cultivo los días 5 a 7 después de la fertilización in vitro sobre la producción y posterior criotolerancia de blastocistos de vaquillonas criollas ecuatorianas. Se recolectaron complejos cúmulo-ovocitos (COCs) inmaduros mediante aspiración intravaginal guiada por ultrasonido (OPU) de 10 vaquillonas criollas y de ovarios de matadero en ocho sesiones. Los COCs se sometieron a maduración in vitro (MIV), fecundación (FIV) y cultivo (IVC). El día 1 después de la FIV, los presuntos embriones de cada fuente de ovocitos (OPU [O] y Matadero [A]) se asignaron aleatoriamente en dos grupos según si se añadió FCS al 2.5% (v/v) al medio de cultivo el día 5 después de la FIV: 1) O-FCS+, 2) O-FCS-, 3) A-FCS+ y 4) A-FCS-. El día 7 después de la FIV se vitrificaron embriones de alta calidad. La criotolerancia de los embriones vitrificados-calentados se evaluó según la reexpansión del blastocele a las 2 h y la posterior reexpansión y eclosión a las 24 y 48 h de incubación. La tasa de blastocistos el día 7 no difirió entre la fuente de ovocitos y el grupo FCS. Después del proceso de vitrificación/ calentamiento, la adición de FCS solo afectó la tasa de reexpansión a las 2 h, independientemente de la fuente de ovocitos (p<0.05). Asimismo, la tasa de eclosión de los blastocistos a las 48 h de incubación se vio drásticamente afectada únicamente en los ovocitos derivados de OPU (p<0.01). En conclusión, la suplementación de FCS el día 5 después de la FIV no mejoró la producción de blastocistos y afectó negativamente la criotolerancia de embriones bovinos in vitro derivados de vaquillonas criollas criadas en los Andes ecuatorianos.

ABSTRACT This experiment aimed to assess the effect of fetal calf serum (FCS) added to culture medium on day 5 to 7 after in vitro fertilization on production and subsequent cryotolerance of blastocysts from Ecuadorian Creole heifers. Immature cumulus-oocyte complexes (COCs) were collected by ovum pick-up (OPU) from 10 Creole heifers and from ovaries collected at abattoir in eight collection sessions. COCs were subjected to in vitro maturation (IVM), fertilization (IVF), and culture (IVC). On day 1 after IVF, presumptive embryos from each oocyte source (OPU [O] and Abattoir [A]) were allocated randomly in two groups according to whether 2.5% (v/v) FCS was added to culture medium on day 5 after IVF: 1) O-FCS+, 2) O-FCS-, 3) A-FCS+, and 4) A-FCS-. On day 7 after IVF, high quality embryos were vitrified. Cryotolerance of vitrified-warmed embryos was assessed according to blastocele re-expansion at 2 h and subsequent re-expansion and hatching at 24 and 48 h of re-incubation. Blastocysts rate on day 7 did not differ between oocyte source and FCS group. After vitrification/warming process, addition of FCS affected the re-expansion rate only at 2 h, irrespective of the oocyte source (p<0.05). Likewise, blastocysts hatching rate at 48 h of incubation was drastically affected only in OPU derived oocyte (p<0.01). Supplementation of FCS on day 5 after IVF did not improve blastocyst production and adversely affected the cryotolerance of in vitro bovine embryos derived from creole heifers raised on Ecuadorian Andean highlands.

RESUMEN El estudio tuvo como objetivo evaluar el efecto del suplemento Insulina-Transferrina-Selenio (ITS) en el cultivo embrionario sobre la tasa de blastocistos y calidad embrionaria. Los cigotos de bovino obtenidos por maduración (MIV) y fertilización in vitro (FIV) fueron transferidos a medio Evolve en 4 grupos (T1: Sin suplemento ITS; T2: ITS las primeras 48 h de cultivo (CIV1): T3: ITS después del día 2 de cultivo (CIV2), T4: ITS todo el tiempo de cultivo (CIV1 + CIV2). Todos los procedimientos se realizaron a 38.5 °C, 5% de CO2 y 90% de humedad relativa. Las tasas de clivaje y de blastocistos se determinaron a las 48 h y día 8 de cultivo, respectivamente. El número de células totales y la producción de especies reactivas de oxígeno (EROs) en los blastocistos expandidos fueron determinados por tinción del núcleo con Hoechst y la oxidación de la sonda fluorescente diacetato diclorodihidrofluoresceína (H2DCFDA). Los embriones cultivados de T4 presentaron la mayor tasa de blastocistos (32.6%) y número de células totales (145.6) comparada al grupo control sin ITS (26.2% y 98.6, respectivamente; p<0.05). Asimismo, la producción de EROs en los blastocistos de T4 fue menor (p<0.05) que la obtenida en los demás grupos de estudio. Los resultados demuestran que la adición del suplemento ITS durante todo el tiempo de cultivo embrionario tiene efectos benéficos sobre el porcentaje de blastocistos y calidad embrionaria, valorada por el número de células totales y la producción de EROs.

ABSTRACT The aim of this study was to evaluate the effect of Insulin-Transferrin-Selenium (ITS) supplementation in embryo culture on the blastocyst rate and embryo quality. The bovine zygotes obtained by in vitro maturation (MIV) and fertilization (IVF) were transferred to Evolve medium in 4 groups (T1: Without ITS supplement; T2: ITS the first 48 h of culture (CIV1): T3: ITS after day 2 of culture (CIV2), T4: ITS during all culture time (CIV1+CIV2). All procedures were performed at 38.5 °C,5%CO2and90%relativehumidity. The cleavage and blastocyst rates were determined at 48 h and day 8 of culture. The total cell number and the production of reactive oxygen species (ROS) in the expanded blastocysts were determined by staining the nucleus with Hoechst and the oxidation of the fluorescent probe dichlorodihydrofluorescein diacetate (H2DCFDA). The T4 cultured embryos presented the highest rate of blastocysts (32.6%) and total cell number (145.6) compared to the control group without STI (26.2% and 98.6, respectively; p<0.05). Likewise, the production of ROS in T4 blastocysts was lower (p<0.05) than that obtained in the other study groups. The results show that the addition of the ITS supplement throughout the embryo culture time has beneficial effects on the percentage of blastocysts and embryo quality, assessed by the number of total cells and the production of ROS.

RESUMEN: Piura es la región con mayor producción de banano orgánico en el Perú; sin embargo, la enfermedad post cosecha de la pudrición de la corona ha generado grandes pérdidas económicas. Por lo cual, se planteó los siguientes objetivos: evaluar la eficacia de biofungicidas in vitro e in vivo para el control de esta enfermedad. Los productos usados fueron: extracto de orégano, ácido cítrico; ácido ascórbico, bioflavonoides; biofertilizante y aceite del árbol de té. In vitro se utilizó el método del “medio envenenado”. In vivo, se inocularon a frutos con suspensiones de conidias a 1 x 104 UFC mL-1 de los fitopatógenos Thielaviopsis paradoxa y Colletotrichum musae. Se prepararon los biofungicidas según la dosis comercial. Se aplicaron los productos y se inocularon los hongos con asperjadores manuales. Los clústeres de banano con tratamiento se embalaron en cajas de cartón de 18,4 kg y fueron embaladas incubándose a 13 °C durante 18 días y 4 días en cámara de maduración con gas etileno. En in vitro los biofungicidas: extracto de orégano (1450 ppm) y ácido cítrico (2825 ppm) obtuvieron 100% inhibición en el crecimiento del micelio de T. paradoxa y C. musae. En in vivo los frutos el menor índice de severidad lo fue para el biofungicida extracto de cítricos (4275 ppm) registrándose 0,0% frente a T. paradoxa y C. musae.

ABSTRACT: Piura is the region with the largest organic banana production in Peru; however, the post-harvest disease crown rot has generated large economic losses. Therefore, the following objectives were raised: to evaluate the efficacy of biofungicides in vitro and in vivo for the control of this disease. The products used were: oregano extract, citric acid; ascorbic acid, bioflavonoids; biofertilizer and tea tree oil. In vitro, the "poisoned medium" method was used. In vivo, fruits were inoculated with conidial suspensions at 1 x 104 CFU ml-1 of the phytopathogens Thielaviopsis paradoxa and Colletotrichum musae. The biofungicides were prepared according to the commercial dose. The products were applied, and the fungi were inoculated with manual sprayers. The banana clusters with treatment were placed in 18,4 kg cardboard boxes and packed, incubating at 13 °C for 18 days and 4 days in a maturation chamber with ethylene gas. In vitro, the biofungicides: oregano extract (1450 ppm) and citric acid (2825 ppm) obtained 100% inhibition in the growth of the mycelium of T. paradoxa and C. musae. In fruits, the lowest severity index was for the citrus extract biofungicide (4275 ppm), registering 0,0% against T. paradoxa and C. musae.

BACKGROUND: Rhinoceros are currently one of the most threatened mammal species globally. Slow population growth, increased poaching and habitat destruction have led to increased conservation efforts for each species. Assisted reproductive technologies (ART) have been implemented in an attempt to aid reproductive outputs for the conservation of these endangered species. Developing species-specific ART programmes for wildlife have been challenging. Temperature control during oocyte recovery is essential for ensuring in vitro success OBJECTIVE: This study is the first to investigate the effect of enema warming prior to trans-rectal ovum pick-up (OPU) on in vitro oocyte maturation in Southern white rhinoceros (Ceratotherium simum simum). METHODS: OPUs were performed on 20 rhinoceros cows from three different game farms in South Africa; oocytes were transported to one of two in vitro fertilisation laboratories for culture. The enema fluid was either warmed to 32 °C or not warmed prior to the OPU. Location of the farm, the different laboratories, ambient temperature, season, aspiration probe temperature, media type and enema temperature were investigated as predictor variables for oocyte maturation success RESULTS: After considering all other potential covariates, warming of the enema fluid was the only independent predictor of in vitro oocyte maturation success during this study CONCLUSION: Oocytes retrieved from rhinoceros cows that received an enema warmed to 32 °C were 2.3 times more likely to mature in vitro compared to oocytes from cows that received an unwarmed enema; the findings can be implemented in other rhinoceros ART programmes and in conservation efforts of other endangered mammalian species

BACKGROUND: The implementation of assisted reproductive techniques and use of in vitro embryo production has highlighted their potential in attempts to prevent the extinction of the northern white rhinoceros (Ceratotherium simum cottoni). However, in vitro embryo production has proved challenging. Transrectal ovum pick-up (OPU), while done successfully with custom-made aspiration probes (AP), may expose oocytes to detrimental temperature shock OBJECTIVE: This study compares the temperature regulation properties between two AP, made from different materials and used during OPU in southern white rhinoceros (C. s. simum METHODS: The initial PVC plastic AP, produced using a computer numerical control machine, was compared to a modified aluminium AP. A rhinoceros model was constructed using a 210 L water container fitted with heating elements and a water pump. Water was warmed to 37.5-38.5 °C to simulate rhinoceros body temperature. The AP was equilibrated to room temperature (22 simulating summer) or in a walk-in fridge (4 simulating winter) overnight. Temperature loggers were placed inside and on the outside of the AP RESULTS: The aluminium AP equilibrated to the model water temperature within 15 minutes of submersion in the model, while the temperature of the PVC AP remained significantly lower during the three-hour period. The inside of the PVC AP remained significantly colder compared to outside during both winter and summer (33.23 ± 4.28°C vs. 36.51 ± 2.13 °C and 36.04 ± 1.69 °C vs. 37.22 ± 0.92 °C, respectively). There was no significant difference between the inside temperature of the aluminium AP compared to the outside temperature during both the winter and summer simulations (36.75 ± 0.49 °C vs. 37.83 ± 0.40 °C and 37.86 ± 0.16 °C vs. 38.04 ± 0.11 °C, respectively CONCLUSION: This research highlights the importance of using material with good thermal properties during assisted reproductive procedures. It also contributes to further advancement during the establishment of a successful in vitro embryo production protocol for rhinoceros in South Africa

Abstract During oocyte meiosis resumption, a coordinated program of transcript translation and decay machinery promotes a remodeling of mRNA stores, which determines the success of the acquisition of competence and early embryo development. We investigated levels of two genes related to mRNA translation (CPEB1 and CPEB4) and two related to mRNA degradation (CNOT7 and ZFP36L2) machinery and found ZFP36L2 downregulated in in vitro-matured bovine oocytes compared to in vivo counterparts. Thereafter, we tested the effects of a pre-IVM step with NPPC and a modified IVM with AREG on the modulation of members of mRNA translation and degradation pathways in cumulus cells and oocytes. Our data showed a massive upregulation of genes associated with translational and decay processes in cumulus cells, promoted by NPPC and AREG supplementation, up to 9h of IVM. The oocytes were less affected by NPPC and AREG, and even though ZFP36L2 transcript and protein levels were downregulated at 9 and 19h of IVM, only one (KDM4C) from the ten target genes evaluated was differently expressed in these treatments. These data suggest that cumulus cells are more prone to respond to NPPC and AREG supplementation in vitro, regarding translational and mRNA decay programs. Given the important nursing role of these cells, further studies could contribute to a better understanding of the impact of these modulators in maternal mRNA modulation and improve IVM outcomes. resumption stores development CPEB1 CPEB (CPEB CPEB4 CNOT7 CNOT (CNOT ZFPL ZFP L ZFP36L vitromatured vitro matured counterparts Thereafter preIVM pre h KDM4C KDMC KDM C (KDM4C treatments programs outcomes

Abstract In reproductive technologies, uncovering the molecular aspects of oocyte and embryo competence under different conditions is crucial for refining protocols and enhancing efficiency. RNA-seq generates high-throughput data and provides transcriptomes that can undergo additional computational analyses. This study presented the transcriptomic profiles of in vitro matured oocytes and blastocysts produced in vitro from buffalo crossbred (Bubalus bubalis), coupled with gene co-expression and module preservation analysis. Cumulus Oophorus Complexes, obtained from slaughterhouse-derived ovaries, were subjected to in vitro maturation to yield metaphase II oocytes (616) or followed in vitro fertilization and culture to yield blastocysts for sequencing (526). Oocyte maturation (72%, ±3.34 sd) and embryo development (21.3%, ±4.18 sd) rates were obtained from three in vitro embryo production routines following standard protocols. Sequencing of 410 metaphase II oocytes and 70 hatched blastocysts (grade 1 and 2) identified a total of 13,976 genes, with 62% being ubiquitously expressed (8,649). Among them, the differentially expressed genes (4,153) and the strongly variable genes with the higher expression (fold-change above 11) were highlighted in oocytes (BMP15, UCHL1, WEE1, NLRPs, KPNA7, ZP2, and ZP4) and blastocysts (APOA1, KRT18, ANXA2, S100A14, SLC34A2, PRSS8 and ANXA2) as representative indicators of molecular quality. Additionally, genes exclusively found in oocytes (224) and blastocysts (2,200) with specific biological functions were identified. Gene co-expression network and module preservation analysis revealed strong preservation of functional modules related to exosome components, steroid metabolism, cell proliferation, and morphogenesis. However, cell cycle and amino acid transport modules exhibited weak preservation, which may reflect differences in embryo development kinetics and the activation of cell signaling pathways between buffalo and bovine. This comprehensive transcriptomic profile serves as a valuable resource for assessing the molecular quality of buffalo oocytes and embryos in future in vitro embryo production assays. technologies efficiency RNAseq RNA seq highthroughput high throughput analyses Bubalus bubalis, bubalis , bubalis) coexpression co Complexes slaughterhousederived slaughterhouse derived ovaries 616 (616 526. 526 . (526) 72%, 72 (72% 334 3 34 ±3.3 sd 21.3%, 213 21 (21.3% 418 4 18 ±4.1 41 7 grade 2 13976 13 976 13,97 62 8,649. 8649 8,649 8 649 (8,649) them 4,153 4153 153 (4,153 foldchange fold change 11 BMP15, BMP15 BMP (BMP15 UCHL1 UCHL WEE1 WEE NLRPs KPNA7 KPNA ZP2 ZP ZP4 APOA1, APOA1 APOA (APOA1 KRT18 KRT ANXA2 ANXA S100A14 SA S A SLC34A2 SLCA SLC PRSS Additionally 224 (224 2,200 2200 200 (2,200 components metabolism proliferation morphogenesis However bovine assays 61 (61 52 (526 72% (72 33 ±3. 21.3% (21.3 ±4. 1397 97 13,9 6 864 8,64 64 (8,649 4,15 415 15 (4,15 BMP1 (BMP1 (APOA KRT1 S100A1 SLC34A 22 (22 2,20 220 20 (2,20 (6 5 (52 (7 ±3 21.3 (21. ±4 139 9 13, 86 8,6 (8,64 4,1 (4,1 (BMP S100A (2 2,2 (2,2 ( (5 ± 21. (21 8, (8,6 4, (4, 2, (2, (8, (4 (8

Abstract Assisted reproductive technologies (ART) are fundamental for cattle breeding and sustainable food production. Together with genomic selection, these technologies contribute to reducing the generation interval and accelerating genetic progress. In this paper, we discuss advancements in technologies used in the fertility evaluation of breeding animals, and the collection, processing, and preservation of the gametes. It is of utmost importance for the breeding industry to select dams and sires of the next generation as young as possible, as is the efficient and timely collection of gametes. There is a need for reliable and easily applicable methods to evaluate sexual maturity and fertility. Although gametes processing and preservation have been improved in recent decades, challenges are still encountered. The targeted use of sexed semen and beef semen has obliterated the production of surplus replacement heifers and bull calves from dairy breeds, markedly improving animal welfare and ethical considerations in production practices. Parallel with new technologies, many well-established technologies remain relevant, although with evolving applications. In vitro production (IVP) has become the predominant method of embryo production. Although fundamental improvements in IVP procedures have been established, the quality of IVP embryos remains inferior to their in vivo counterparts. Improvements to facilitate oocyte maturation and development of new culture systems, e.g. microfluidics, are presented in this paper. New non-invasive and objective tools are needed to select embryos for transfer. Cryopreservation of semen and embryos plays a pivotal role in the distribution of genetics, and we discuss the challenges and opportunities in this field. Finally, machine learning (ML) is gaining ground in agriculture and ART. This paper delves into the utilization of emerging technologies in ART, along with the current status, key challenges, and future prospects of ML in both research and practical applications within ART. ART (ART selection progress animals possible decades encountered breeds practices wellestablished well established relevant (IVP counterparts systems eg e g e.g microfluidics noninvasive non invasive transfer genetics field Finally (ML status

Abstract 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. importance science fertilization selection manipulation productivity microfluidics threedimensional three dimensional materials livecell live imaging cryopreservation

ABSTRACT Purpose: This study aimed to compare the degree of maturation and development of fetal pig segmental intestinal tissue with that of spheroids created by in-vitro reaggregation of dissociated fetal intestinal cells after transplantation into immunodeficient mice. Methods: Fetal pig small intestines were transplanted as segmental grafts into the omentum and subrenal capsules of immunodeficient mice or enzymatically treated to generate single cells. Spheroids made by in-vitro reaggregation of these cells were transplanted into the subrenal capsules of immunodeficient mice. The segmental grafts and spheroids were harvested four and eight weeks after transplantation, and the structural maturity and in-vivo development of these specimens were histologically evaluated. Results: The spheroids were engrafted and supplied blood vessels from the host mice, but an intestinal layered structure was not clearly observed, and there was almost no change in size. On the other hand, the segmental grafts formed deep crypts in the mucus membrane, the inner circular layer, and outer longitudinal muscles. The crypts of the transplanted grafts harvested at eight weeks were much deeper, and the smooth muscle layer and the enteric nervous system were more mature than those of grafts harvested at the fourth week, although the intestinal peristaltic wave was not observed. Conclusions: Spheroids created from fetal small intestinal cells could not form layered structures or mature sufficiently. Conversely, segmental tissues structurally matured and developed after in-vivo transplantation and are therefore potential grafts for transplantation. Purpose invitro vitro Methods invivo vivo evaluated Results observed size hand membrane muscles deeper week Conclusions sufficiently Conversely

Abstract One of the crucial aspects to be considered for successful in vitro production (IVP) of embryos is the composition of the various media used throughout the stages of this reproductive biotechnology. The cell culture media employed should fulfill the metabolic requirements of both gametes during oocyte maturation and sperm development, as well as the embryo during its initial cell divisions. Most IVP protocols incorporate blood serum into the media composition as a source of hormones, proteins, growth factors, and nutrients. Numerous studies have suggested Platelet-Rich Plasma (PRP) as a substitute for fetal sera in cell culture, particularly for stem cells. Therefore, the objective of this study is to assess the potential use of PRP as a replacement for fetal bovine serum (FBS) during oocyte maturation for in vitro production of bovine embryos. During in vitro maturation (IVM), cumulus-oocyte complexes (COCs) were allocated into the following experimental groups: Group G1 (IVM medium with 5% PRP); Group G2 (MIV medium with 5% PRP and 5% SFB); Group G3 (MIV medium with 5% SFB); and Group G4 (MIV medium without either PRP or SFB). Subsequently, the cumulus-oocyte complexes were fertilized with semen from a single bull, and the resulting zygotes were cultured for seven days. Cleavage and blastocyst formation rates were assessed on days 2 and 7 of embryonic development, respectively. The quality of matured COCs was also evaluated by analyzing the gene expression of HSP70, an important protein associated with cellular stress. The results demonstrated that there were no significant differences among the experimental groups in terms of embryo production rates, both in the initial cleavage stages and blastocyst formation (except for the G4 group, which exhibited a lower blastocyst formation rate on D7, as expected). This indicates that PRP could be a cost-effective alternative to SFB in the IVP of embryos. (IVP biotechnology development divisions hormones proteins factors nutrients PlateletRich Platelet Rich (PRP cells Therefore FBS (FBS IVM, IVM , (IVM) cumulusoocyte cumulus (COCs G 5 PRP) MIV SFB) SFB. . Subsequently bull respectively HSP70 HSP stress except group D7 D expected. expected expected) costeffective cost effective HSP7

Cystic echinococcosis (CE) is a zoonotic disease caused by the infection of Echinococcus granulosus (E. granulosus) larva. Currently, blocking the pathogenic cycle chain through immunoprophylaxis has become the main research direction. EgG1Y162 protein has good antigenicity and immunogenicity and is therefore a good candidate molecule for E. granulosus vaccine. Mature T cells express CTLA-4 on their surface, and its extracellular IgV region binds efficiently to the B7 molecules on antigen-presenting cells to deliver negative signals. We designed and prepared a recombinant vaccine by fusing CTLA-4IgV to the EgG1Y162 protein to exploit its binding properties. Bioinformatic methods were used to analyze the structure and epitopes of the proposed recombinant vaccine. The placement of 16 amino acids (GTDDDDKAMADIGSEF) between the CTLA-4IgV and EgG1Y162 using the skeleton structure of pET30a plasmid did not affect the correct folding of the proteins. When the recombinant proteins were co-cultured with bone marrow-induced dendritic cells (DC), the protein CTLA-4IgV-EgG1Y162 promoted its binding to DC and increased the percentage of DC maturation compared with protein EgG1Y162 in vitro and in vivo. Compared to EgG1Y162, CTLA-4IgV-EgG1Y162 promoted the proliferation of lymphocytes in spleen and the release of interferon (IFN)-γ and interleukin (IL)-4 by those lymphocytes in vitro, while it also promoted the release of protective antibodies in the serum of immunized mice in vivo. These findings indicated that the designed recombinant vaccine, CTLA-4IgV-EgG1Y162, can provide new ideas for the optimization and improvement of vaccines against E. granulosus. CE (CE E (E larva Currently direction EgGY EgG Y EgG1Y16 CTLA4 CTLA 4 CTLA- surface B antigenpresenting antigen presenting signals CTLA4IgV CTLAIgV 4IgV properties 1 GTDDDDKAMADIGSEF (GTDDDDKAMADIGSEF pETa pET cocultured co cultured marrowinduced marrow induced DC, , (DC) CTLA4IgVEgG1Y162 CTLAIgVEgGY CTLA-4IgV-EgG1Y16 vivo IFNγ IFN γ IL4 IL (IL)- CTLA4IgVEgG1Y162, EgG1Y1 (DC IgVEgG CTLA4IgVEgG1Y16 CTLA-4IgV-EgG1Y1 (IL) EgG1Y CTLA4IgVEgG1Y1 CTLA-4IgV-EgG1Y (IL CTLA4IgVEgG1Y

Abstract This brief review delves into the topic of in vitro follicle culture for in vitro embryo production, with a particular emphasis on goat models. Specifically, we examine the main findings from LAMOFOPA-Brazil over the last 20 years, highlighting the challenges posed by oxidative stress and epigenetic changes. Our focus is on strategies to improve follicular development and oocyte maturation. Furthermore, we underscore the valuable role of the antioxidant anethole in optimizing the efficacy of in vitro follicle culture and improving outcomes in in vitro embryo production. production models Specifically LAMOFOPABrazil LAMOFOPA Brazil 2 years changes maturation Furthermore

Resumo: A pitangueira é fruteira nativa do Brasil, podendo ser encontrada em países subtropicais da América Latina. O fruto apresenta diferentes tonalidades, variando pelo estágio de maturação e pelo genótipo. Porém, um genótipo pode ter maior potencial de mercado, principalmente se destinado à industrialização, pois produz frutos sem sementes. O objetivo deste estudo foi caracterizar a biologia floral e reprodutiva do genótipo apirênico de pitangueira, comparando com o pirênico. Os trabalhos foram realizados na UTFPR – Câmpus Dois Vizinhos, Brasil, e na EEAD (CSIC), Espanha. Foram avaliados o período entre o início da floração e da maturação dos frutos, horário de antese, quantificação do número de anteras por flor, pólen por antera, desenvolvimento dos grãos de pólen e germinação in vitro de pólen. O genótipo pirênico da pitangueiraapresenta maior número de anteras por flor, pólen por antera e pólen por flor. A viabilidade in vitro dos grãos de pólen foi maior com flores após antese e quando foram utilizados períodos de incubação mais longos. Em condições naturais, a germinação dos grãos de pólen e ocrescimento do tubo polínico ocorrem normalmente, sem autoincompatibilidade. Resumo Brasil Latina tonalidades Porém mercado industrialização sementes Vizinhos CSIC, CSIC , (CSIC) Espanha flor longos naturais normalmente autoincompatibilidade (CSIC

Abstract: Surinam cherry tree (Eugenia uniflora) is native to Brazil, but it is found in several subtropical countries of Latin America. Its fruit has different berries according to the maturation stage and variety. However, a genotype may have greater market potential, especially if intended for processing, as it produces seedless fruits. The aim of this study was to characterize the floral and reproductive biology of a E. uniflora apyrenic genotype, comparing possible differences with a pyrenic genotype. Works were carried at UTFPR – Câmpus of Dois Vizinhos, Brazil and EEAD (CSIC), Zaragoza, Spain. The period between the beginning of flowering and fruit maturation, anthesis time, quantification of the number of anthers per flower, pollen per anther, pollen grain development, and in vitropollen germination were evaluated. The pyrenic Surinam cherry genotype has higher number of anthers per flower, pollen per anther and pollen per flower. The in vitro viability of pollen grains was greater with flowers after anthesis and when longer incubation periods were used. Under natural conditions, pollen grain germination and pollen tube growth occur normally, with no self-incompatibility. Abstract Eugenia America variety However potential processing fruits E Vizinhos CSIC, CSIC , (CSIC) Zaragoza Spain time flower development evaluated used conditions normally selfincompatibility. selfincompatibility self incompatibility. incompatibility self-incompatibility (CSIC