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 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 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 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: 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.

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.

Resumo O objetivo do presente estudo foi investigar o impacto sinérgico do α-tocoferol e do ácido α-linolênico (100 µM) na MIV e CIV de oócitos de búfala Nili Ravi. Os oócitos foram obtidos dos ovários de búfalos abatidos duas horas após o abate e levados ao laboratório. Complexos de oócitos cumulus de búfalo foram colocados aleatoriamente nos cinco grupos experimentais incluídos; GRUPO 1: Meio de maturação (MM) + 100 µM ALA (controle), GRUPO 2: MM + 100 µM ALA + 50 µM α-tocoferol, GRUPO 3: MM + 100 µM ALA + 100 µM α-tocoferol, GRUPO 4: MM + 100 µM ALA + 200 μM α-tocoferol e GRUPO 5: MM + 100 µM ALA + 300 μM α-tocoferol sob uma atmosfera de 5% de CO2 em ar a 38,5 °C por 22-24 h. A expansão cumulus e o estado de maturação nuclear foram determinados (Experimento 1). No experimento 2, os oócitos foram maturados como no experimento 1. Os oócitos maturados foram então fertilizados em meio de Tyrode's Albumina Lactato Piruvato (TALP) por cerca de 20 h e cultivados em meio de fluido oviductal sintético (SOF) para determinar o efeito do ácido α-linolênico (100 µM) e α-tocoferol em meio IVM em IVC de presumíveis zigotos. Para estudar o efeito do ácido α-linolênico (100 µM) em meio IVM e aumentar a concentração de α-tocoferol no meio de cultura no desenvolvimento inicial do embrião (Experimento 3), os presumíveis zigotos foram distribuídos aleatoriamente nos cinco grupos experimentais com concentração crescente de α-tocoferol em meios de cultura. Maior porcentagem de oócitos em estágio MII no experimento 1 (65,2 ± 2,0), embriões em estágio de mórula no experimento 2 (30,4 ± 1,5) e experimento 3 (22,2 ± 2,0) foram obtidos. No entanto, os resultados gerais para a expansão das células do cumulus, maturação do oócito para o estágio MII e desenvolvimento embrionário subsequente entre os tratamentos permanecem estatisticamente semelhantes (P> 0,05). A suplementação de α-tocoferol em meios de maturação com ácido α-linolênico e / ou em meios de cultura de embriões não aumentou ainda mais a maturação in vitro de oócitos ou a produção de embriões.

Abstract The objective of the current study was to investigate the synergistic impact of α-Tocopherol and α-Linolenic acid (100 µM) on IVM and IVC of Nili Ravi buffalo oocytes. Oocytes were obtained from the ovaries of slaughtered buffaloes within two hours after slaughter and brought to laboratory. Buffalo cumulus oocyte complexes were placed randomly in the five experimental groups included; GROUP 1: Maturation media (MM) + 100 µM ALA (control), GROUP 2: MM + 100 µM ALA + 50μM α-Tocopherol, GROUP 3: MM + 100 µM ALA + 100μM α-Tocopherol, GROUP 4: MM + 100 µM ALA + 200 μM α-Tocopherol and GROUP 5: MM + 100 µM ALA + 300 μM α-Tocopherol under an atmosphere of 5% CO2 in air at 38.5 °C for 22-24 h. Cumulus expansion and nuclear maturation status was determined (Experiment 1). In experiment 2, oocytes were matured as in experiment 1. The matured oocytes were then fertilized in Tyrode’s Albumin Lactate Pyruvate (TALP) medium for about 20 h and cultured in synthetic oviductal fluid (SOF) medium to determine effect of α-Linolenic acid (100 µM) and α-Tocopherol in IVM medium on IVC of presumptive zygotes. To study the effect of α-Linolenic acid (100 µM) in IVM media and increasing concentration of α-tocopherol in the culture media on early embryo development (Experiment 3), the presumptive zygotes were randomly distributed into the five experimental groups with increasing concentration of α-tocopherol in culture media. Higher percentage of MII stage oocytes in experiment 1(65.2±2.0), embryos at morula stage in experiment 2 (30.4±1.5) and experiment 3 (22.2±2.0) were obtained. However, overall results for cumulus cell expansion, maturation of oocyte to MII stage and subsequent embryo development among treatments remain statistically similar (P > 0.05). Supplementation of α-tocopherol in maturation media having α-Linolenic acid and/or in embryo culture media did not further enhance in vitro maturation of oocyte or embryo production.

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

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 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 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

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 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 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

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

The aim of this study was to assess the effect of eugenol on the in vitro production of ovine embryos. Oocytes were aspirated and taken for in vitro maturation (IVM), where they were divided into 4 groups: the CON (control), in which the oocytes were immersed in medium without an antioxidant; in the EU10, EU20, EU40 groups, the same medium as in the COM group was used, supplemented with 10, 20, and 40 µM of eugenol, respectively. The degree of expansion of cumulus cells was evaluated. Subsequently, the oocytes underwent in vitro fertilization and in vitro culture, and at the end of this process, the rate of cleaved structures was assessed. Regarding the expansion rate of cumulus cells, the CON group exhibited higher rates compared to the groups supplemented with eugenol. Evaluating oocytes with fully expanded cumulus cells, only the EU40 group showed a lower rate when compared to the rest of the treatment groups. Whereas observing oocytes with partially expanded cumulus cells, the EU40 group showed higher expansion percentages compared to the other treatments. Concerning the presence of the first polar body in the oocytes, it was observed that there was no statistical difference between the treatment groups. Lastly, concerning the cleaved structures, the CON and EU10 groups showed the highest rates compared to the EU20 and EU40 groups. Thus, it was possible to conclude that the use of 10 µM of eugenol in the IVM of ovine oocytes proved to be the optimal concentration of this substance.

El objetivo del presente estudio fue evaluar el efecto del eugenol en la producción in vitro de embriones ovinos. Los ovocitos fueron aspirados y llevados para la maduración in vitro (MIV), donde se dividieron en 4 grupos: el CON (control), en el cual los ovocitos fueron sumergidos en medio sin antioxidantes; en los grupos EU10, EU20, EU40 se utilizó el mismo medio que en el grupo CON, pero con la adición de 10, 20 y 40 µM de eugenol, respectivamente. Se evaluó el grado de expansión de las células del cúmulo. Después de esto, los ovocitos fueron sometidos a la fecundación in vitro y al cultivo in vitro, y al final de este proceso se evaluó la tasa de estructuras clivadas. En cuanto a la tasa de expansión de las células del cúmulo, el grupo CON mostró tasas más altas en comparación con los grupos suplementados con eugenol. Al evaluar los ovocitos con células del cúmulo totalmente expandidas, solo el grupo EU40 mostró una tasa más baja en comparación con el resto de los grupos de tratamiento. Mientras que al observar los ovocitos con células del cúmulo parcialmente expandidas, el grupo EU40 mostró porcentajes de expansión más altos en comparación con los otros tratamientos. Respecto a la presencia del primer corpúsculo polar en los ovocitos, se observó que no hubo diferencia estadística entre los grupos de tratamiento. Por último, en relación con las estructuras clivadas, los grupos CON y EU10 mostraron las tasas más altas en comparación con los grupos EU20 y EU40. Por lo tanto, se pudo concluir que el uso de 10 µM de eugenol en la MIV de ovocitos ovinos resultó ser la concentración óptima de esta sustância.

Objetivando avaliar o efeito do eugenol na produção in vitro de embriões ovinos. Os oócitos, foram aspirados e levados para a maturação in vitro (MIV), onde foram divididos em 4 grupos: o CON (controle), no qual os oócitos foram imersos em meio, sem antioxidante, já nos grupos EU10, EU20, EU40 foi utilizado o mesmo meio do grupo CON, acrescido de 10, 20 e 40 µM de eugenol, respectivamente. Foi avaliado o grau de expansão das células do cumulus. Após isso, os oócitos seguiram para a fecundação in vitro e cultivo in vitro, sendo, ao final desse processo, avaliada a taxa de estruturas clivadas. Em relação à taxa de expansão das células do cumulus, o grupo CON apresentou maiores taxas, quando comparado aos grupos adicionados de eugenol. Avaliando os oócitos com células do cumulus totalmente expandidas, somente o grupo EU40, apresentou-se menor, quando comparado ao restante dos grupos de tratamento. Já observando os oócitos com células do cumulus parcialmente expandidas, o grupo EU40, apresentou maiores percentuais de expansão, quando comparado aos demais tratamentos. Sobre a presença do primeiro corpúsculo polar nos oócitos, observou-se que não houve diferença estatística entre os grupos de tratamentos. E, por último, em relação às estruturas clivadas, os grupos CON e EU10 apresentaram as maiores taxas, quando comparado aos grupos EU20 e EU40. Dessa maneira, foi possível concluir que a utilização de 10 µM de eugenol na MIV de oócitos ovinos apresentou ser a melhor concentração dessa substância.

The aim of this study was to evaluate the effect of quercetin on the in vitro maturation (IVM) of oocytes and the subsequent in vitro fertilization (IVF) of ovine embryos. For this purpose, oocytes were collected and divided into the following treatment groups: control group, containing CON medium (control); and groups Q2, Q4, and Q8, with control medium supplemented with 2, 4, and 8 µM of quercetin, respectively. After IVM, the degree of cumulus cells expansion, presence of the first polar body (PB), and the other part of the oocytes proceeded to in vitro fertilization (IVF), where oocytes were incubated with spermatozoa for a period of 20 hours. Subsequently, the presumptive zygotes underwent in vitro culture (IVC) for 48 hours, and at the end of the process, the rate of cleaved structures was evaluated. The results were expressed in percentage and compared using the Chi-square test, with a significant difference considered when P&lt;0.05. Evaluating the expansion of cumulus cells, the CON and Q2 groups showed the best expansion rates compared to the Q4 and Q8 groups. Regarding the presence of the 1st PB, it was observed that only the Q8 group exhibited lower rates compared to the control group. When evaluating the rate of cleaved structures, it was observed that the Q2 group showed a higher number compared to the Q8 group. Quercetin does not influence the maturation of ovine oocytes.

El objetivo del presente estudio fue evaluar el efecto de la quercetina en la maduración in vitro (MIV) de ovocitos y la posterior fertilización in vitro (FIV) de embriones ovinos. Para ello, se recolectaron y seleccionaron ovocitos divididos en los siguientes grupos de tratamiento: grupo control, que contenía medio CON (control); y los grupos Q2, Q4 y Q8, con medio control al que se le añadieron 2, 4 y 8 µM de quercetina, respectivamente. Tras la MIV se evaluó el grado de expansión de las células del cúmulo, la presencia del primer corpúsculo polar (CP) y la otra parte de los ovocitos se utilizó para la fecundación in vitro (FIV), donde los ovocitos fueron incubados con espermatozoides durante un período de 20 horas. Posteriormente, los presuntos cigotos pasaron a cultivo in vitro (CIV) durante 48 horas y, al final del proceso, se evaluó la tasa de estructuras clivadas. Los resultados se expresaron en porcentaje y se compararon utilizando la prueba de Chi-cuadrado, considerando una diferencia significativa cuando P&lt;0,05. Al evaluar la expansión de las células del cúmulo, los grupos CON y Q2 mostraron las mejores tasas de expansión en comparación con los grupos Q4 y Q8. En cuanto a la presencia del 1er CP, se observó que solo el grupo Q8 mostró tasas más bajas en comparación con el grupo control. Al evaluar la tasa de estructuras clivadas, se observó que el grupo Q2 mostró un número mayor en comparación con el grupo Q8. La quercetina no influye en la maduración de ovocitos ovinos.

O objetivo do presente estudo foi avaliar o efeito da quercetina na MIV de oócitos e na subsequente PIV de embriões ovinos. Para isso, foram colhidos e selecionados oócitos divididos nos seguintes grupos de tratamento: grupo controle, contendo meio CON (controle); e grupos Q2, Q4 e Q8, com meio controle, acrescido de 2, 4 e 8 µM de quercetina, respectivamente. Após a MIV foi avaliado o grau de expansão das células do cumulus, presença do primeiro corpúsculo polar (CP) e a outra parte dos oócitos seguiu para a fecundação in vitro (FIV), onde os oócitos foram incubados com os espermatozoides, por um período de 20 h. Em seguida, os presumíveis zigotos seguiram para o cultivo in vitro (CIV), por um período de 48 h e, ao final do processo, foi avaliada a taxa de estruturas clivadas. Os resultados foram expressos em porcentagem e comparados, usando o Teste do Qui-quadrado e foi considerada diferença significativa quando P&lt;0,05. Avaliando a expansão das células do cumulus, os grupos CON e Q2 apresentaram as melhores taxas de expansão, quando comparadas aos grupos Q4 e Q8. Quanto à presença do 1º CP, foi possível observar que somente o grupo Q8 apresentou taxas menores, quando comparado ao grupo controle. Já avaliando a taxa de estruturas clivadas, foi possível observar que o grupo Q2 apresentou maior número quando comparado ao grupo Q8. A quercetina não influencia na maturação de oócitos ovinos.

ABSTRACT In birds, the process of sperm maturation has not been fully described. The objective of this study was to determine in vitro parameters for the decapacitating effect of oviductal proteins on rooster spermatozoa. Aliquots with spermatozoon were incubated to in vitro induce metabolic states: capacitation, decapacitation, and the acrosomal reaction to again determined percentages of live spermatozoa, motility, and concentrations of Malondialdehyde, reduced Glutathione and Adenosine triphosphate. The percentages of motility and live sperm in fresh and capacitated semen were similar (P>0.05), but higher (P<0.05) than those determined in decapacitated semen and with acrosome reaction. The determination of nmol/ml of MDA in fresh semen (1.59) was higher (P<0.005) than in capacitated semen (1.05), with acrosomal reaction (1.07) and decapacitated semen (1.05). GSH nmol/ml were similar (P>0.05) in fresh (72.6) and capacitated (62.04) semen, similarly (P<0.05) between acrosome reaction (99.09) and decapacitated (86.07) semen. The highest concentration of ATP was in capacitated semen with 69.9 µmol/ml, with similar concentrations (P<0.05) in fresh, with acrosomal reaction, and decapacitated semen. The parameters, determined, demonstrated that the protein fraction of the utero-vaginal junction, in vitro, produces decapacitation and successfully maintains sperm viability.

RESUMEN En las aves, no se ha descrito totalmente el proceso de maduración espermática. El objetivo del estudio fue determinar parámetros in vitro, del efecto no capacitante de las proteínas oviductales, en espermatozoides de gallo. Alícuotas con espermatozoides, fueron incubadas in vitro para inducir estados metabólicos de capacitación, no capacitación y reacción acrosomal para determinar porcentajes de espermatozoides vivos, su movilidad y concentraciones de Malondialdehído, Glutation reducido y Adenosin tri fosfato, como parámetros del estado metabólico de los espermatozoides. Los resultados mostraron porcentajes de movilidad y espermatozoides vivos en semen fresco y capacitado similares (P>0.05), pero mayores (P<0.05) a los determinados en espermatozoides con reacción acrosomal. Los nmol/ml de MDA en semen fresco (1.59) fue mayor (P<0.5) que en semen capacitado (1.05), con reacción acrosomal (1.07) y en semen no capacitado (1.05). Los nmol/ml de GSG fueron similares (P>0.05) en semen fresco (72.6) y capacitado (62.04), y entre semen con reacción acrosomal (99.09) y no capacitado (86.07). La mayor concentración de ATP fue en semen capacitado con 69.9 µmol/ml, con concentraciones similares (P<0.05) en semen fresco, con reacción acrosomal y no capacitados. Los parámetros determinados, demostraron que la fracción proteica de la unión útero-vaginal, in vitro produce no capacitación y mantiene la viabilidad espermática.