ABSTRACT Macrobrachium rosenbergii is the freshwater shrimp species of greatest economic interest, and its reproduction has been studied for commercial purposes. This study aimed to evaluate the effect of different salinities (0, 6, and 12 mg L−1) on the embryogenesis of this species. The females were observed daily according to their reproductive behavior. Upon the first sign of spawning, when their eggs were attached to the abdomen, they were collected for egg collection and transferred to the incubators at the respectively tested salinities. The eggs were collected every five days for up to 23 days, separated according to their stage of development, histologically processed, and stained for visualization of structural changes during embryogenesis using an optical microscope. From the egg's histological images at different embryonic development stages, the egg hatching time was evaluated, the changes that occurred in the eggs during embryogenesis, the total area of the eggs, the vacuole area, and the ratio between the vacuole area and the total area of the eggs. Differences were observed in hatching time, egg size, and vacuole area and area ratio. The 6 g L−1 of salinity treatment was statistically different from the other in all parameters analyzed. Therefore, it was concluded that 6 g L−1 of salinity positively influenced embryogenesis, as it presented hatches from the 15th day of embryogenesis, demonstrating that there was a greater speed of migration of vitellogenin for the formation of the embryo, accelerating embryogenesis and hatching of M. rosenbergii.

Abstract Nototheniidae is the most numerous family of notothenioids and, although they are mainly Antarctic, they are also well represented in Patagonian waters. In both Antarctic and sub-Antarctic waters, they adopt a wide variety of reproductive strategies. In Antarctic waters they usually have a biennial reproductive cycle, whereas in sub-Antarctic ones, they reproduce annually or biannually. In the intertidal zone of central Patagonia the most abundant nototheniid fish is Patagonotothen cornucola and, despite this, there is little information about their biology. The aim of this work was to study its reproductive cycle in a rocky intertidal coast of Argentine Patagonia by means of histological and morphological analyses. Samples were collected monthly at two intertidal sites in the San Jorge Gulf. Condition factor, gonadosomatic and hepatosomatic indexes were calculated and thin sections of gonads were stained with one-step trichrome to determine gonadal stages. Relative and potential fecundity were also estimated. Males and females mature synchronously and reach maximum gonadosomatic index between July and August. In females the hepatosomatic index increases synchronously with gonadosomatic index, while in males it remains constant throughout the cycle. It is concluded that P. cornucola has an annual reproductive cycle and that spawning occurs in winter. Maximum potential fecundity is approximately 27,095 oocytes in a 11.7 cm female and it increases with size.

Resumen La familia Nototheniidae es la más numerosa entre los nototénidos y, a pesar de ser principalmente Antárticos, están muy bien representados en aguas patagónicas. Desde la Antártida hasta las aguas de Sudamérica, han adoptado una gran variedad de estrategias reproductivas. Los peces antárticos suelen tener un ciclo reproductivo bienal mientras que en aguas sub-antárticas los nototénidos pueden reproducirse anualmente o bianualmente. En la zona intermareal de la Patagonia central, el pez más abundante es Patagonotothen cornucola y, a pesar de esto, hay poca información sobre su biología. Por esto, el objetivo principal de este trabajo fue estudiar su ciclo reproductivo en la zona intermareal rocosa usando métodos histológicos y morfológicos. Las muestras fueron recolectadas mensualmente en dos sitios intermareales en el Golfo San Jorge, y se calculó el factor de condición, índices hepatosomáticos y gonadosomáticos y se realizaron cortes histológicos de las gónadas que fueron teñidas con tricrómico de un tiempo para determinar los estadios gonadales, además se estimó la fecundidad potencial y relativa. Se determinó que machos y hembras maduran sincrónicamente alcanzando los valores máximos de índices gonadosomáticos entre julio y agosto. En las hembras el índice hepatosomático aumenta simultáneamente con el gonadosomático mientras que en machos se mantiene constante durante todo el ciclo. Se concluye que P. cornucola tiene un ciclo reproductivo anual y que el desove ocurre en el invierno. La fecundidad potencial máxima fue de 27.095 oocitos en una hembra de 11,7 cm y se incrementa con la talla.

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 17. The study aimed to evaluate the Fertilization Rate of four independent in vitro treatments of bovine oocytes with Sexed (SX) and Conventional (Conv) semen, capacitated (B.O and Percoll) using the Swim-Up technique. Cleavage was assessed at 48 hours post-fertilization and on the seventh day of embryonic development. The results showed a cleavage percentage of 59.4% in T1 (Conv. Percoll), 60% in T2 (Conv. B.O), 75% in T3 (SX. Percoll), and 46.6% in T4 (SX. B.O), with a statistically significant difference (p>0.05) observed specifically between T3 and T4. The embryo development rate on the seventh day was 34.4% in T1, 20% in T2, 15% in T3, and 10.51% in T4. In this case, a statistically significant difference was found in the use of conventional medium with Percoll compared to SX B.O medium (p<0.05) (T1 and T4, respectively). The results also showed similar production of morula and early blastocysts and protruded embryos among the four treatments, demonstrating the feasibility of in vitro fertilization (IVF) technique for producing bovine embryos. In conclusion, the preliminary evaluation highlights the significant influence of the sexing method and treatment on semen quality and embryonic development in animal reproduction programs. Although Sexed semen with Percoll shows potential to improve cleavage rates, conventional semen, especially in T1 treatment (Conv. Percoll), exhibits superiority in individual motility and concentration. These findings provide crucial insights for optimizing reproductive programs.

Resumen 13. El objetivo del presente estudio fue evaluar la Tasa de Fecundación in vitro de ovocitos bovinos con semen sexado (SX) y convencional (Conv.) capacitados (B.O y Percoll) con la técnica Swim-Up. Se evaluó el clivaje a las 48 horas post fecundación y al séptimo día del desarrollo embrionario. Los resultados mostraron un porcentaje de clivaje del 59,4% en el T1 (Conv. Percoll), 60% en el T2 (Conv. B.O), 75% en el T3 (SX. Percoll) y 46,6% en el T4 (SX. B.O), mostrando diferencia estadística entre T3 y T4 (p>0,05). La tasa de desarrollo del embrión al séptimo día fue 34,4% T1, 20% en T2, 15% en T3 y 10,51% en T4; en este caso, se encontró diferencia estadísticamente significativa en el uso del medio convencional más Percoll en comparación con el medio de SX B.O (p<0,05) (T1 y T4, respectivamente). Los resultados también mostraron una producción similar de mórulas y blastocitos tempranos y protruidos entre los cuatro tratamientos, y demuestran la viabilidad del desarrollo e implementación de la técnica de fertilización in vitro (FIV) para producir embriones bovinos. Concluyendo la evaluación preliminar, destaca la significativa influencia del método de sexado y del tratamiento en la calidad del semen y desarrollo embrionario en programas de reproducción animal. Aunque el semen sexado con Percoll muestra potencial para mejorar tasas de clivaje, el semen convencional, especialmente en el tratamiento T1 (Conv. Percoll), exhibe una superioridad en motilidad individual y concentración. Estos resultados informan decisiones cruciales para optimizar programas reproductivos.

Abstract A total of 381 specimens of the tilapia Oreochromis mossambicus collected monthly from May 2017 to May 2018 in the Laguna de Los Patos, Cumaná, Venezuela, to evaluate reproductive parameters of this non-native species. Significant differences were found in relation to average height and weight between males and females, with the highest values in males. The sex ratio was 1:1.5 (males:females), which deviates significantly from the expected 1:1 ratio. The mean length of sexual maturity (Lm50) was 18.0 cm in females and 20.1 cm in males, reflecting that females mature at smaller sizes than males. The monthly variations of the gonadosomatic index (GSI) and the stages of sexual maturity show two reproductive peaks during the study, in October 2017 and April 2018, coinciding with the rainy and dry seasons in the region respectively. The condition factor (CF) showed significant differences between months, but not between sexes, with an average of 1.87 in females and 1.84 in males. The average absolute fecundity was 921 ± 604.6 eggs per fish, with a relative fecundity of 8.36 ± 3.09 eggs per gram of fish. Differences in oocyte size in mature females confirm that the species can spawn repeatedly over a period, which is considered an important factor for the establishment of tilapia in non-native environments. 38 201 Patos Cumaná Venezuela nonnative non native 11.5 115 1 1.5 5 1:1. malesfemales, malesfemales , (males:females) 11 1: Lm50 Lm (Lm50 180 18 0 18. 20 20. GSI (GSI study respectively CF (CF months sexes 187 87 1.8 184 84 92 6046 604 6 604. fish 836 8 36 8.3 309 3 09 3.0 period environments 11. 15 1. (males:females Lm5 (Lm5 2 9 60 83 8. 30 3. (Lm

Resumo Um total de 381 espécimes da tilápia Oreochromis mossambicus foram coletados mensalmente de maio de 2017 a maio de 2018, na Laguna de Los Patos, Cumaná, Venezuela, para avaliar parâmetros reprodutivos desta espécie não nativa. Foram encontradas diferenças significativas em relação às médias de altura e peso entre machos e fêmeas, sendo os maiores valores no sexo masculino. A razão sexual foi de 1:1,5 (machos:fêmeas), o que se desvia significativamente da proporção esperada de 1:1. O comprimento médio de maturação sexual (Lm50) foi de 18,0 cm nas fêmeas e 20,1 cm nos machos, refletindo que as fêmeas amadurecem em tamanhos menores que os machos. As variações mensais do índice gonadossomático (IGS) e dos estágios de maturidade sexual mostram dois picos reprodutivos durante o estudo, em outubro de 2017 e abril de 2018, coincidindo com as estações chuvosa e seca na região, respectivamente. O fator de condição (FC) apresentou diferenças significativas entre os meses, mas não entre os sexos, com média de 1,87 no sexo feminino e 1,84 no masculino. A fecundidade absoluta média foi de 921 ± 604,6 ovos por peixe, com fecundidade relativa de 8,36 ± 3,09 ovos por grama de peixe. Diferenças no tamanho dos ovócitos em fêmeas maduras confirmam que a espécie pode desovar repetidamente ao longo de um período, o que é considerado um fator importante para o estabelecimento de tilápias em ambientes não nativos. 38 201 2018 Patos Cumaná Venezuela nativa masculino 11,5 115 1 1,5 5 1:1, machosfêmeas, machosfêmeas , (machos:fêmeas) 11. 11 1. 1:1 Lm50 Lm (Lm50 180 18 0 18, 20 20, IGS (IGS estudo região respectivamente FC (FC meses sexos 187 87 1,8 184 84 92 6046 604 6 604, peixe 836 8 36 8,3 309 3 09 3,0 período nativos 11, 15 1, (machos:fêmeas 1: Lm5 (Lm5 2 9 60 83 8, 30 3, (Lm

Resumo O polvo com chifres Eledone cirrhosa, uma espécie de tamanho médio encontrada no Golfo Arábico ao largo da Arábia Saudita, foi coletado mensalmente no Golfo Arábico ao largo da cidade de Dammam durante o período de janeiro a dezembro de 2022. As amostras foram dissecadas e preparadas para exame por microscopia eletrônica. Durante a maturação genital, os túbulos seminíferos se estabelecem nos testículos, com espermatogônias ativas se dividindo. Espermatócitos 1 são observados no túbulo, seguidos de aumento de espermatogônias e espermatócitos em agosto. A espermiogênese começa com espermátides esféricas evoluindo para espermátides alongadas. Em setembro, as espermatogônias ativas, as divisões meióticas e o aumento da espermiogênese continuam. Os espermatozoides aparecem na Bolsa de Needham, indicando maturidade sexual. O ovário passa por vários estágios de desenvolvimento, com oócitos no estágio I em junho e julho, seguido pelo estágio II em outubro e novembro. No estágio IIIc, os cordões foliculares invadem o citoplasma do oócito, formando numerosas inclusões lipídicas e grânulos proteicos. O citoplasma contém cisternas de retículo endoplasmático e um Aparelho de Golgi pouco desenvolvido. O estágio IV ocorre em novembro, caracterizado pelo máximo desenvolvimento dos cordões foliculares e pelo início da vitelogênese. O ooplasma contém numerosas inclusões lipídicas, um sincício e células secretoras. A partir de dezembro, os oócitos do estágio V estão principalmente presentes, indicando a fase de atividade de secreção máxima. As plaquetas da gema acumulam-se no ooplasma do ovócito e o córion se forma na zona pelúcida. Em janeiro, os primeiros óvulos lisos são encontrados no ovário de alguns polvos e sua proporção aumenta continuamente. Este estudo teve como objetivo investigar a ação mitogênica da gonadotrofina bem como identificar os períodos de intensa multiplicação celular durante o ciclo sexual por meio de métodos citológicos. cirrhosa Saudita 2022 eletrônica genital testículos dividindo túbulo agosto alongadas setembro continuam Needham julho novembro IIIc oócito proteicos desenvolvido vitelogênese secretoras presentes máxima acumulamse acumulam pelúcida continuamente citológicos 202 20 2

Abstract The horned octopod Eledone cirrhosa, a medium-sized species found in Arabian Gulf off Saudi Arabia, was collected monthly from the Arabian Gulf off Dammam city during January to December 2022. Samples were dissected and prepared for examination using transmission electron microscopy. During genital maturation, seminiferous tubules are established in the testis, with active spermatogonia dividing. Spermatocytes 1 are observed in the tubule, followed by an increase in spermatogonia and spermatocytes in August. Spermiogenesis begins, with spherical spermatids evolving into elongated spermatids. In September, active spermatogonia, meiotic divisions, and increased spermiogenesis continue. Spermatozoa appear in Needham's pouch, indicating sexual maturity. The ovary undergoes various stages of development, with oocytes at stage I in June and July, followed by stage II in October and November. In stage III, follicular cords invade the oocyte's cytoplasm, forming numerous lipid inclusions and protein granules. The cytoplasm contains cisternae of endoplasmic reticulum and a poorly developed Golgi apparatus. Stage IV occurs in November, characterized by the maximum development of follicular cords and the beginning of vitellogenesis. The ooplasm contains numerous lipid inclusions, a syncytium, and secretory cells. From December, stage V oocytes are mainly present, indicating the activity phase of maximum secretion. Yolk platelets accumulate in the oocyte ooplasm, and chorion forms at the zona pellucida. In January, the first smooth eggs are found in some octopuses' ovary, with their proportion increasing steadily. This study aimed to investigate the mitogenic action of gonadotropin and identify the periods of intense cell multiplication during the sexual cycle using cytological methods. cirrhosa mediumsized medium sized Arabia 2022 microscopy maturation testis dividing tubule August begins September divisions continue Needhams Needham s pouch maturity July November III granules apparatus vitellogenesis syncytium cells present secretion pellucida octopuses steadily methods 202 20 2

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 Steroids and gonadotrophins are essential for the regulation of late stages of preantral development and antral follicular development. Although the luteinizing hormone receptor (LHCGR) has been detected in the preantral follicles of rats, rabbits, and pigs, its expression, in bovine fetal ovary, has not been demonstrated. Based on this, we aimed to investigate the expression of the LHCGR and LHCGR mRNA binding protein (LRBP), as well as, to quantify bta-miR-222 (a regulatory microRNA of the LHCGR gene) during the development of bovine fetal ovary. In summary, LHCGR expression was observed in the preantral follicle in bovine fetal ovary, from oogonias to primordial, primary and secondary stages, and the mRNA abundance was lower on day 150 than day 60. However, the mRNA abundance of LRBP followed the opposite pattern. Similar to LRBP, the abundance of bta-miR-222 was higher on day 150 than day 60 or 90 of gestation. The LHCGR protein was detected in oogonia, primordial, primary, and secondary follicles. Moreover, both oocytes and granulosa cells showed positive immunostaining for LHCGR. In conclusion, we suggest the involvement of LHCGR/LRBP/bta-mir222 with mechanisms related to the development of preantral follicles in cattle. (LHCGR rats rabbits pigs ovary demonstrated this , (LRBP) btamiR222 btamiR bta miR 222 bta-miR-22 a gene summary primordial 15 However pattern 6 9 gestation oogonia Moreover conclusion LHCGR/LRBP/btamir222 LHCGRLRBPbtamir222 LHCGRLRBPbtamir LHCGR/LRBP/bta mir222 mir LHCGR/LRBP/bta-mir22 cattle (LRBP btamiR22 22 bta-miR-2 1 btamir222 btamir LHCGR/LRBP/btamir22 LHCGRLRBPbtamir22 LHCGRLRBPbta mir22 LHCGR/LRBP/bta-mir2 btamiR2 2 bta-miR- btamir22 LHCGR/LRBP/btamir2 LHCGRLRBPbtamir2 mir2 LHCGR/LRBP/bta-mir bta-miR btamir2 LHCGR/LRBP/btamir

Abstract: Aim This study evaluates the reproduction and fecundity of Leporinus lacustris in upper Paraná River floodplain environments with the objectives to (i) recognize the germ cells of folliculogenesis and oogenesis; (ii) characterize reproductive phases; (iii) investigate oocyte diameter and evaluate if this species has group synchronous or asynchronous oocyte development and if spawning is total or batch; (iv) estimate batch fecundity; (v) establish the relationship between fecundity/ovary weight, standard length, and total weight; (vi) verify the reproductive areas of this species in the upper Paraná River floodplain. Methods Sampling was carried out using gillnets in rivers, and open and closed lagoons, in March, June, September, and December 2013, 2014, 2015, 2016, 2018, and 2019 and March and September 2017. A total of 442 females were sampled and 111 subsampled, each of which had an ovary removed and fixed in Bouin solution. A subsample of 20 ovaries in the spawning capable phase were fixed in a 4% buffered formaldehyde solution to estimate the diameter of the oocytes and fecundity. The ovaries were dehydrated, embedded in historesin, cut, stained, and evaluated as regards folliculogenesis, oogenesis, development phases, and reproduction areas. Results The germ cells recorded were differentiated oogonia, primary and secondary growth oocytes, fully-grown and mature oocytes, and post-ovulatory follicles that permitted the recognition of the reproductive phases. Oocyte diameter information revealed that the species has group synchronous oocyte development, determinate fecundity and batch spawning, and eliminates an average of 38,490 oocytes. Conclusions The fact that Leporinus lacustris is reproductively successful in the Baía River and in the Fechada, Guaraná, Patos, Garças, Ventura and Ressaco do Pau Véio lagoons strengthens the importance of the areas of environmental protection of the islands and lowlands of the Paraná River, Ilha Grande National Park and Ivinheima River State Park. Abstract i (i oogenesis ii (ii phases iii (iii iv (iv v (v fecundityovary weight length vi (vi rivers June 2013 2014 2015 2016 2018 201 2017 44 11 subsampled 2 4 dehydrated historesin cut stained oogonia fullygrown fully grown postovulatory post ovulatory 38490 38 490 38,49 Fechada Guaraná Patos Garças 1 3849 3 49 38,4 384 38,

Resumo: Objetivo Este estudo avaliou a reprodução e fecundidade de L. lacustris em ambientes da planície de inundação do alto rio Paraná, com os objetivos de: (i) reconhecer as células germinativas da foliculogênese e oogênese; (ii) caracterizar as fases reprodutivas; (iii) investigar o diâmetro dos oócitos e avaliar se esta espécie tem o desenvolvimento dos oócitos sincrônico em grupo ou assincrônico e se a desova é total ou parcelada; (iv) estimar a fecundidade do lote; (v) estabelecer a relação entre fecundidade/peso do ovário, comprimento padrão e peso total e (vi) verificar as principais áreas de reprodução desta espécie na planície de inundação do alto rio Paraná. Métodos As amostragens foram realizadas em rios, lagoas abertas e fechadas no alto rio Paraná, com redes de espera nos meses de março, junho, setembro e dezembro de 2013, 2014, 2015, 2016, 2018 e 2019 e março e setembro de 2017. Foram amostradas 442 fêmeas e subamostradas 111, das quais foi retirada uma subamostra do ovário e fixada em solução de Bouin. Uma subamostra de 20 ovários na fase apta a desova foi fixada em solução de formaldeído tamponado a 4%, para posteriormente, estimar o diâmetro dos oócitos e fecundidade. Resultados As células germinativas registradas foram: oogônias diferenciadas, oócitos de crescimento primário e secundário, oócitos de crescimento completo e maduros, e folículos pós-ovulatórios que permitiram reconhecer as fases reprodutivas. As informações do diâmetro dos oócitos revelou que a espécie tem desenvolvimento dos oócitos sincrônico em grupo, fecundidade determinada e desova parcial, e elimina no período de desova em média 38.490 oócitos. Conclusões Leporinus lacustris reprodutivamente bem sucedido no rio Baía e nas lagoas Fechada, do Guaraná, dos Patos, das Garças, do Ventura e do Ressaco do Pau Véio reforçando a importância das áreas de proteção ambiental das ilhas e várzeas do rio Paraná, Parque Nacional da Ilha Grande e Parque Estadual do rio Ivinheima. Resumo L Paraná i (i oogênese ii (ii reprodutivas iii (iii parcelada iv (iv lote v (v fecundidadepeso vi (vi rios junho 2013 2014 2015 2016 201 2017 44 111 Bouin 2 4 4% posteriormente diferenciadas secundário maduros pósovulatórios pós ovulatórios parcial 38490 38 490 38.49 Fechada Guaraná Patos Garças Ivinheima 11 3849 3 49 38.4 1 384 38.

Abstract The number of antral follicles is considered an important fertility trait because animals with a high follicle count (HFC) produce more oocytes and embryos per cycle. Identification of these animals by genetic markers such as single nucleotide polymorphisms (SNPs) can accelerate selection of future generations. The aim of this study was to perform a genome wide association study (GWAS) on Nelore and Angus heifers with HFC and low (LFC) antral follicle counts. The groups HFC and LFC for genotyping were formed based on the average of total follicles (≥ 3 mm) counted in each breed consistently ± standard deviation. A total of 72 Nelore heifers (32 HFC and 40 LFC) and 48 Angus heifers (21 HFC and 27 LFC) were selected and the DNA was extracted from blood and hair bulb. Genotyping was done using the Illumina Bovine HD 770K BeadChip. The GWAS analysis showed 181 and 201 SNPs with genotype/phenotype association (P ≤ 0.01) in Nelore and Angus heifers, respectively. Functional enrichment analysis was performed on candidate genes that were associated with SNPs. A total of 97 genes were associated to the 181 SNPs in the Nelore heifers and the functional analysis identified genes (ROBO1 and SLIT3) in the ROBO-SLIT pathway that can be involved in the control of germ cell migration in the ovary as it is involved in lutheal cell migration and fetal ovary development. In the Angus heifers, 57 genes were associated with the 201 SNPs, highlighting Fribilin 1 (FBN1) gene, involved in regulation of growth factors directly involved in follicle activation and development. In summary, GWAS for Nelore and Angus heifers showed SNPs associated with higher follicle count phenotype. Furthermore, these findings offer valuable insights for the further investigation of potential mechanism involved in follicle formation and development, important for breeding programs for both breeds. (HFC cycle (SNPs generations (GWAS (LFC counts ≥ ( mm deviation 7 32 (3 4 21 (2 2 bulb K BeadChip 18 20 genotypephenotype genotype phenotype P 0.01 001 0 01 respectively 9 ROBO1 ROBO (ROBO SLIT3 SLIT ROBOSLIT development 5 FBN1 FBN (FBN1 gene summary Furthermore breeds 0.0 00 (FBN 0.

Abstract The subcortical maternal complex, which consists of maternal-effect genes, plays a crucial role in the development of oocytes and preimplantation embryo until the activation of the zygote genome. One such gene, known as peptidyl-arginine deiminase VI (Padi6), is involved in the oocyte maturation, fertilization and embryonic development. However, the precise function of Padi6 gene in buffalo is still unclear and requires further investigation. In this study, the sequence, mRNA and protein expression patterns of Padi6 gene were analyzed in oocytes, preimplantation embryos and somatic tissues of buffalo. The coding sequence of gene was successfully cloned and characterized. Real-time quantitative PCR results indicated an absence of Padi6 transcripts in somatic tissues. Notably, the expression levels of Padi6 in oocytes showed an increased from the germinal vesicle stage to metaphase II stage, followed by a rapid decrease during the morula and blastocyst stages. Immunofluorescence analysis confirmed these findings, revealing a noticeable decline in protein expression levels. Our research provides the initial comprehensive expression profile of Padi6 in buffalo oocytes and preimplantation embryos, serving as a solid foundation for further investigations into the functionality of maternal-effect genes in buffalo. complex maternaleffect effect genome peptidylarginine peptidyl arginine Padi6, Padi , (Padi6) maturation However investigation study characterized Realtime Real time Notably stages findings (Padi6 (Padi

Abstract Reproductive control is one of the biggest challenges in tilapia production and triploidy was developed as an alternative to sterilization. In general, polyploids present chromosomal instability but for triploid Nile tilapia it has yet to be reported. This study evaluated the chromosomal instability from juveniles to adulthood, growth performance and gonadal status of tilapia hatched from eggs submitted or not to heat shock for triploid induction. Nile tilapia oocytes were fertilized (1,476 oocytes), half of the eggs were subjected to a four-minute shock in 41 °C water four minutes after fertilization and the other half were not (Control group). The eggs were incubated (at 27°C) and 160 larvae from the treated group hatched and survived after yolk sac absorption. The determination of ploidy was performed by flow cytometry at 85th (juveniles) and 301st (adults) days of age post yolk sac absorption. At the time of the first cytometry analysis there were 73 surviving juveniles from the treated group, and only 14 were confirmed triploid. However, at the analysis of adult ploidy, one out of 8 surviving adult tilapias from the 14 confirmed triploid juveniles remained triploid. Gonadal histology showed that the non-remaining triploids continued to produce gametes. The growth performance of triploid tilapia was initially superior to that of diploid tilapia during the juvenile phase, but similar in adults. Once the chromosome sets are lost and the tilapias become diploid again, at least in tissues with a high proliferation rate, such as the hematopoietic tissue that was analyzed (and possibly in gonads), all possible advantages of triploids are probably lost. Thus, our results suggest that, due to genomic instabilities, the triploid generation of tilapia has low efficiency. sterilization general reported adulthood induction 1,476 1476 1 476 (1,47 oocytes, , oocytes) fourminute minute 4 C Control group. . group) 27°C 27C 27 16 absorption th (juveniles st adults (adults 7 However nonremaining non remaining gametes phase again rate gonads, gonads gonads) Thus instabilities efficiency 1,47 147 47 (1,4 2 1,4 (1, 1, (1 (

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 Synthetic chemical insecticides are widely used in population control of pests. Aedes aegypti is the primary vector of dengue, Zika, chikungunya and yellow fever to humans, and has proven resistance to chemical insecticides. As an alternative vector control method, the ethanolic extract of the leaves of Piper peltatum L. (Piperaceae) showed larvicidal activity against Ae. aegypti. Despite the wide medicinal use of this plant, the biological activity of its isolated constituents remains unexplored. In this sense, we isolated, identified and evaluated the larvicidal activity of 4-nerolidylcatechol (4-NC) from P. peltatum against Ae. aegypti, Culex quinquefasciatus and Anopheles darlingi, focusing on the larvicidal, adulticidal and genotoxic potential of 4-NC on Ae. aegypti. Larvae were captured in the city of Manaus, Amazonas state, Brazil. 4-NC was isolated from the extract of the leaves of P. peltatum via silica gel chromatography. This was identified using nuclear magnetic resonance spectroscopy and tested in Artemia franciscana (6.25, 12.5, 25, 50, 100 and 200 µg/mL). In the toxicity bioassay, Ae. aegypti larvae were exposed to 30, 50, 70, 90, and 110 µg/mL of 4-NC, while Cx. quinquefasciatus and An. darlingi were exposed to 6.25, 12.5, 25, 50 and 100 µg/mL. Ae. aegypti larvae were also subjected to 40 and 60 µg/mL of 4-NC (genotoxicity bioassay), and adult females to 62.5 to 1,000 µg/mL (adulticidal bioassay). The results of the 4-NC toxicity assays showed that there was 100% mortality in larvae of Ar. franciscana at the concentration of 200 µg/mL, with an LC50 of 8.0 μg/mL. In the larvae of Ae. aegypti, mortality was 100%, with an LC50 of 62 μg/mL. In larvae of Cx. quinquefasciatus, 97% mortality occurred, with an LC50 of 52.3 μg/mL, and in An. darlingi larvae there was an 83% mortality rate, with an LC50 of 55.8 μg/mL. In adults of Ae. aegypti, however, there was no adulticidal activity. In the larvae of Ae. aegypti, the genotoxic effect of 4-NC (40 and 60 µg/mL) showed significant frequency (p < 0.05) of cellular abnormalities (micronuclei, budding and nuclear bridges) of interphasic nuclei of neuroblasts and oocytes in relation to the negative control. This result may be associated with a decrease in oviposition of females, which was observed in two generations. We can confirm that 4-NC has larvicidal activity against Ae. aegypti, Cx. quinquefasciatus and An. darlingi. Although it does not present adulticidal activity in Ae. aegypti, it reduced the oviposition of females. Therefore, 4-NC seems to be a strong candidate for the development of an alternative method for the control of these mosquitoes in the immature phase. pests dengue Zika humans L Piperaceae (Piperaceae Ae plant unexplored sense 4nerolidylcatechol nerolidylcatechol 4 4NC NC (4-NC P Manaus state Brazil chromatography 625 6 25 (6.25 125 12 5 12.5 10 20 µgmL . µg mL bioassay 30 70 90 11 4NC, NC, Cx An 6.25 genotoxicity , bioassay) 62. 1000 1 000 1,00 bioassay. Ar LC LC5 80 8 0 8. μgmL μg μg/mL 97 occurred 523 52 3 52. 83 rate 558 55 55. however (4 p 0.05 005 05 micronuclei, micronuclei (micronuclei bridges generations Therefore phase 2 (6.2 12. 7 9 6.2 00 1,0 ( 0.0 (6. 6. 1, 0. (6

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