Insulin-like growth factor-1 improves in vitro meiotic resumption of dromedary camel (Camelus Dromedarius) oocytes

Despite relatively high maturation rate of in vitro matured oocytes in the dromedary camel, however, blastocyst production is very low after in vitro fertilization (IVF). Herein, the influences of oocyte collection method (follicular aspiration vs slicing; Experiment I), the addition of Insulin-like growth factor I (IGF-I) to the maturation medium (Experiment II) on in vitro maturation (IVM) of oocyte were investigated. Although the nuclear maturation did not differ regardless of collecting method, follicular aspiration led to lower degeneration rates than those in controls (P < 0.05). The percentages of oocytes at MII were greater in the presence of IGF-1 than in its absence (71.9% vs 48.4%, respectively, P<0.05). Additionally, the percentages of degenerated oocytes were higher in the control group compared to oocytes cultured in the presence of IGF-I (23.6% vs 10.4%, respectively, P<0.05). IGF-I treatment improved the quality of MII matured oocytes as evidenced by the decrease of cathepsin B (CTSB) activity, a marker of poor quality oocytes, when compared to control ones (P < 0.05). In conclusion, follicular aspiration decreased the degeneration rate; however, it had no effect on completion of maturation. IGF-I enhanced the IVM of oocyte and decreased degeneration rate.(AU)

Maternal contributions to pregnancy success: from gamete quality to uterine environment

The establishment and maintenance of a pregnancy that goes to term is sine qua non for the long-term sustainability of dairy and beef cattle operations. The oocyte plays a critical role in providing the factors necessary for preimplantation embryonic development. Furthermore, the female, or maternal, environment where oocytes and embryos develop is crucial for the establishment and maintenance of a pregnancy to term. During folliculogenesis, the oocyte must sequentially acquire meiotic and developmental competence, which are the results of a series of molecular events preparing the highly specialized gamete to return to totipotency after fertilization. Given that folliculogenesis is a lengthy process in the cow, the occurrence of disease, metabolic imbalances, heat stress, or other adverse events can make it challenging to maintain oocyte quality. Following fertilization, the newly formed embryo must execute a tightly planned program that includes global DNA remodeling, activation of the embryonic genome, and cell fate decisions to form a blastocyst within a few days and cell divisions. The increasing use of assisted reproductive technologies creates an additional layer of complexity to ensure the highest oocyte and embryo quality given that in vitro systems do not faithfully recreate the physiological maternal environment. In this review, we discuss cellular and molecular factors and events known to be crucial for proper oocyte development and maturation, as well as adverse events that may negatively affect the oocyte; and the importance of the uterine environment, including signaling proteins in the maternal-embryonic interactions that ensure proper embryo development. We also discuss the impact of assisted reproductive technologies in oocyte and embryo quality and developmental potential, and considerations when looking into the prospects for developing systems that allow for in vitro gametogenesis as a tool for assisted reproduction in cattle.(AU)

Dynamics of nuclear and cytoplasm maturation of bovine oocytes cultivated in vitro in medium supplemented with fulerol / Dinâmica da maturação nuclear e citoplasmática de oócitos bovinos cultivados in vitro em meio suplementado com fulerol

The aim of this work was to evaluate, in vitro, the dynamics of nuclear and cytoplasmic maturation of bovine oocytes in traditional IVM medium (CT) and supplemented with fullerol (MF50), for 36 hours. The nuclear maturation of CT (n=300) and MF50 (n=270) every 6 hours, stained with Hoechst33342 and cytoplasmic, the mitochondrial distribution of CT (n=197) and MF50 (n=159) at every 12 hours, stained with Mitotracker Orange. At 6 hours, CT oocytes (19%) were in MI (metaphase I), while in MF50 they were in GV (germ vesicle) or GVB (GV breakeage), repeating at 12 hours. At 18 hours, 46.3% were matured in CT, and 20% in MF50. At 24 hours, 43.9% of maturation was observed in the MF50 group, and 63.8% in the CT. At 30 and 36 hours, the maturation pattern was stable, but with the onset of oocyte degeneration. There was a delay in cytoplasmic maturation with 36 hours (P < 0.05) in MF50 (53.9% of mature gametes), compared to CT (69.8%). With immature cytoplasm, they were 10.4% and 31.7% for CT and MF50 (P< 0.05), respectively. It was concluded that fullerol possibly interfered in the expansion of cumulus oophorus cells, as well as delayed the meiotic progression and cytoplasmic maturation.

O objetivo deste estudo foi avaliar, in vitro, a dinâmica da maturação nuclear e citoplasmática de oócitos bovinos em meio MIV tradicional (TC) e suplementado com fulerol (MF50), durante 36 horas. Na maturação nuclear do TC (n=300) e do MF50 (n=270) a cada seis horas, corados com Hoechst 33342, e na citoplasmática, avaliou-se a distribuição mitocondrial do TC (n=197) e do MF50 (n=159) a cada 12 horas, corados com Mitotracker Orange (Life® Technologies). Às seis horas, oócitos do TC (19%) se encontravam em MI (metáfase I), enquanto no MF50 estavam em VG (vesícula germinativa) ou QVG (quebra VG), repetindo com 12 horas. Às 18 horas, 46,3% estavam maturados no TC, e 20% no MF50. Com 24 horas, verificaram-se 43,9% de maturação no grupo MF50, e 63,8% no TC. Às 30 e 36 horas, o padrão de maturação foi estável, mas com início de degeneração oócitária. Houve retardo na maturação citoplasmática com 36 horas (P<0,05) no MF50 (53,9% de gametas maduros), comparado ao TC (69,8%). Com citoplasma imaturo, foram 10,4% e 31,7% para TC e MF50 (P<0,05), respectivamente. Conclui-se que o fulerol possivelmente interferiu na expansão das células do cumulus oophorus, bem como retardou a progressão meiótica e a maturação citoplasmática dos oócitos.

Effect of different gonadotropins on in vitro maturation of sheep oocytes / Efeito de diferentes gonadotrofinas na maturação in vitro de oócitos ovinos

The aim of this study was to examine the effect of replacing the use of follicle-stimulating hormone (FSH) with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) on the in vitro maturation (IVM) of sheep oocytes. After sheep ovaries were collected (n=300), the cumulus-oocyte complexes were aspirated, selected, and divided into four groups according to the IVM medium: CON group, in which the basic IVM medium was used; and eCG, hCG, and FSH groups, in which the oocytes were immersed in basic IVM medium with 10 IU/mL eCG, 10 IU/mL hCG, and 10 µg/mL FSH-p, respectively. In vitro maturation of the oocytes was performed at 38.5 °C, in a humidified atmosphere of 5% CO2 in air, for 24 h. Subsequently, the oocytes were evaluated for the degree of cumulus-cell expansion, chromatin configuration, GSH levels, and active mitochondria. There were no significant differences for the rate of cumulus cell expansion. The percentage of oocytes in MII was higher in the eCG group than in the CON and hCG groups (P<0.05) and similar to that of the FSH group. In conclusion, eCG can be used as a substitute for FSH in IVM of sheep oocytes.

O objetivo deste estudo foi avaliar o efeito da gonadotrofina coriônica equina (eCG) e da gonadotrofina coriônica humana (hCG), em substituição ao uso de hormônio folículo estimulante (FSH) na maturação in vitro (MIV) de oócitos ovinos. Após a coleta de ovários (n=300) ovinos, os complexos cúmulus-oócitos (CCOs) foram aspirados, selecionados e divididos em quatro grupos de acordo com o meio de MIV: grupo CON, em que foi utilizado o meio MIV base; e grupos ECG, HCG e FSH, em que os oócitos foram imersos em meio MIV base adicionado de 10 UI/mL de eCG, 10 UI/mL de hCG e 10 µg/mL de FSH-p, respectivamente. A MIV dos oócitos foi realizada a 38,5°C, em atmosfera umidificada de 5% de CO2 em ar, durante 24 horas. Posteriormente, os oócitos foram avaliados, quanto grau de expansão das células do cumulus, configuração da cromatina, níveis de GSH e mitocôndrias ativas. Não foram observadas diferenças significativas com relação à taxa de expansão de células do cumulus. A percentagem de oócitos em MII foi maior no grupo ECG do que no grupo CON e HCG (P<0,05) e semelhante ao grupo FSH. Em conclusão, a eCG pode ser utilizada em substituição ao FSH na MIV de oócitos ovinos.

Avances en el manejo de gametos caninos in vitro / Advance in handling canine gametes in vitro

Las características fisiológicas de los gametos caninos que los hace especiales respecto a los de otras especies han dificultado los estudios tendientes a incrementar los porcentajes de éxito en diversas biotecnologías reproductivas. Entre estas características está el inicio de la meiosis de los ovocitos posterior al nacimiento y la ovulación de un gameto inmaduro como ovocito primario, lo que ha complejizado la maduración in vitro de estos ovocitos y, por parte de los espermatozoides, una gran diferencia en la sobrevivencia de los espermatozoides eyaculados versus los congelados-descongelados, que hace imprescindible evaluar la biología reproductiva de estos gametos tendiente a optimizar nuevos protocolos haciendo énfasis previamente en una adecuada evaluación del crecimiento del ovocito in vivo y de la viabilidad espermática en aquellos sometidos a criopreservación(AU)

The physiological characteristics of canine gametes that make them unique compared to those of other species have hindered studies aimed at increasing the success rates in various reproductive biotechnologies. Among these characteristics is the meiosis of the oocytes after birth and the ovulation of an immature gamete as a primary oocyte, which has made the in vitro maturation of these oocytes more complex and, on the part of the spermatozoa, a significant difference in the viability of ejaculated versus frozen-thawed spermatozoa, which makes it essential to evaluate the reproductive biology of these gametes in order to optimize new protocols, previously emphasizing an adequate evaluation of in vivo oocyte growth and sperm viability in those subjected to cryopreservation.(AU)

Retrospectiva dos principais biomarcadores envolvidos no desenvolvimento folicular e oocitário em mamíferos / Retrospective of the main biomarkers involved in follicular and oocyte development in mammals

A análise da expressão das diversas proteínas relacionadas a foliculogênese e oogênese in vivo e in vitro, através da proteômica, tem demonstrado possíveis biomarcadores celulares. A descoberta desses biomarcadores pode ajudar na elucidação de mecanismos complexos como a foliculogênese (entendimento sobre o crescimento folicular e maturação oocitária), no tratamento de doenças relacionadas a infertilidade feminina, bem como, no aprimoramento de biotécnicas reprodutivas como a manipulação de oócitos inclusos em folículos pré-antrais. Desta forma, a presente revisão abordou as principais proteínas relatadas nos mais novos trabalhos referentes aos estudos proteômicos visando melhores esclarecimentos referentes ao desenvolvimento folicular e oocitário in vivo e in vitro. A escolha dos artigos foi realizada com base nas descobertas mais atuais e do impacto que determinadas proteínas poderiam trazer para melhorar o entendimento da foliculogênese e oogênese nas diferentes espécies de animais mamíferos.(AU)

The analysis of the expression of several proteins related to folliculogenesis and oogenesis in vivo and in vitro, through proteomics, has demonstrated possible cellular biomarkers. The discovery of these biomarkers may help in the elucidation of complex mechanisms such as folliculogenesis (understanding of follicular growth and oocyte maturation), in the treatment of diseases related to female infertility, as well as in the improvement of reproductive biotechniques such as the manipulation of oocytes enclosed in preantral follicles. Thus, the present review addressed the main proteins reported in the newest works related to proteomic studies aiming at better clarifications regarding follicular and oocyte development in vivo and in vitro. The choice of articles was based on the most current discoveries and the impact that certain proteins could bring to improve the understanding of folliculogenesis and oogenesis in different species of mammalian animals.(AU)

Relationships of morphological and phototextural attributes of presumptive ovine zygotes and early embryos to their developmental competence in vitro: a preliminary assessment using timelapse imaging

The assessment of morphology and digital image opacity may provide valuable information on the present embryo quality. Time-lapse imaging has been employed in research to establish a means of monitoring the dynamic nature of preimplantation embryo development. The aim of present study was to use time-lapse imaging for assessing various prospective morphometric and phototextural markers of the developmental potential of in vitro-derived ovine embryos. Oocytes were obtained by scarification of ovaries from nine Polish Longwool ewes. After in vitro maturation (IVM) and fertilization (IVF) of oocytes with fresh ram semen, the development of embryos to the blastocyst stage was monitored and evaluated using Primo Vision time-lapse imaging technology. Commercially available Image-Pro® Plus software was used to measure zona pellucida thickness, embryo diameter, total area of the perivitelline space, cellular grey-scale pixel intensity and cellular pixel heterogeneity. Statistical assessment of all attributes was done at various time points during embryo development (i.e., presumptive zygote stage: t(0); first cleavage detected at t(2) or t(3); and second cleavage detected at t(4) or t(6)). Out of thirty-seven zygotes analyzed in this study, five did not divide, 26 arrested before and six developed to the blastocyst stage. Our present results indicate that most parameters analyzed did not differ among embryos varying in their developmental fate except for the perivitelline space area that was greater (P<0.05) for non-dividing zygotes than future blastocysts at the presumptive zygote stage (4040±1850 vs. 857±262 µm2, respectively; means±SEM). Consequently, the measurement of perivitelline space at t(0) can potentially be used to prognosticate developmental potential of in vitro-produced ovine embryos albeit further confirmational studies are needed.(AU)

Effect of the presence and location of corpus luteum on competence of bovine cumulus-oocyte complexes

This study aimed to determine the effect of presence of the corpus luteum (CL) and its influence on cumulus­oocyte complexes (COCs) obtained from the ipsilateral or contralateral ovary in bovine on the recovery and capacity of the oocytes to sustain mono-spermic fertilization, undergo preimplantation development, and develop to the blastocyst stage. Ovaries were collected at a local slaughterhouse and kept in pairs corresponding to the same animal. In the first experiment the variables evaluated were compared between cows with (CCL+) and without (CCL- ) CL, and for the second experiment, comparisons were made between ovaries with an ipsilateral (CL+), contralateral (CL−), and no (NCL). The recovery rate of COCs was higher in ovaries from CCL− cows, and a higher proportion of grade 1 COCs were recovered from this group. A higher proportion of metaphase I oocytes at 7 h of maturation, and a higher rate of cleavage were observed in the CCL+ group; however, a higher proportion of embryos were obtained from the CCL− group. Besides, COCs from the CL+ group had a lower proportion of grades 1 and 2 morphological qualities, lower rate of metaphase II oocytes at 22 h of maturation, and lower rate of formation of two pronuclei, whereas a higher proportion of unfertilized oocytes after in vitro fertilization. On the other hand, the COCs from the CL− group displayed a lower proportion of oocytes with more than two pronuclei, higher cleavage rate, and higher final blastocyst production were obtained when compared to CL+. Thus, the effects of CL on the competence of bovine COCs are different depending on the anatomical proximity of their location in the animal, negatively affecting the quality of COCs located in the same ovary, but not having negative effects on the competence of COCs in the ovaries contralateral to their location.(AU)

The effect of adding L-ergothioneine in culture medium on blastocyst development rates

Background: Oocytes and embryos produce energy through mitochondrial oxidative phosphorylation by using oxygen. The membrane structure of the embryo is mostly composed of unsaturated fatty acids, for this reason DNA fragmentation, apoptosis, and abnormal gene expression are shaped as a result of the lipid peroxidation during culture. Oxidative stress (OS) is one of the most important problems affecting the in vitro embryo development. Antioxidant supplementation to the culture medium has been an alternative way to reduce cell damage caused by oxidative stress in in vitro embryo production systems. In this study, it was aimed to determine the effect of L-ergothioneine on blastocyst development when added to the culture medium. Materials, Methods & Results: The material of the study consisted of oocytes aspirated from the ovaries of Holstein cows which were collected from the local slaughterhouse. The ovaries were delivered to the laboratory within 2-3 h in a thermos which provided a constant temperature of 25-30o C with physiological saline (0.9%) containing antibiotics. All follicles in the 3-8 mm range on the ovaries were aspirated using 20 G needle. The collected follicle fluid was filtered through filters with a pore diameter of 70 micrometers. Cells remaining in the filter were washed with OPU medium and transferred to the petri dishes. Fluids were examined under a stereomicroscope. The cumulus-oocyte complexes were classified, and A and B quality oocytes were included to the study (A, B, C, and D quality COC). Oocytes aspirated from the ovaries and collected later on were incubated in IVM medium for 22 h. After maturation, it was taken into IVF medium, semen was added and incubated for 20-22 h. Possible zygotes to be taken to the culture stage were transferred to culture (IVC) drops with (L-ergothioneine 100 µL/mL (n:121) added and without antioxidant (control (n:124)), and kept in the incubator for 6-7 days. Evaluated on the 7th day differences in in vitro embryo production stages were evaluated with the Chi-square test. The study was run in 5 replicates each time, with at least 20 possible zygotes for per group being cultured. It was determined that 262 (87.33%) of a total of 300 oocytes undergoing in vitro maturation were matured. It was determined that 245 of the mature oocytes were fertilized (93.51%). The cleavage rates of the groups were determined as 87.60% and 86.29%, respectively. Eighty-two (33.47%) blastocysts were obtained from 245 zygotes taken into the culture stage, and the blastocyst rates in the groups were found to be 40.50% and 26.61%, respectively. After the study, it was determined that the statistical difference between L-ergothioneine and control in cleavage rates was insignificant (P > 0.05) and blastocyst rates was significant (P < 0.05) Discussion: Oxygen content above normal ratios can increase the formation of reactive oxygen species (ROS), particularly hydrogen peroxide (H2 O2 ), hydroxyl radical (HO·), and peroxyl radicals (ROO·). The increased rate of ROS negatively affects the success of IVP in mammalian embryos. It was observed that L-ergothioneine, which has high antioxidant activity, improved blastocyst development rates, and higher blastocyst rates could be achieved compared to the control group. By investigating the use of L-ergothioneine in different doses, it was thought that the dose with the highest antioxidant activity could be added to the culture medium in in vitro embryo production and more blastocysts could be produced.

Chemical composition of forage watermelon fruit at different maturity stage or storage length

This study aimed to assess the chemical responses of forage watermelon fruit at different maturity stages or storage lengths, performing two experimental tests. In the first test, four maturity stages were assessed: 30, 45, 60, and 75 days after anthesis, with four replicates. In the second test, fruits were maintained under three storage lengths: T1D (harvest day), T3M (3 months after harvest), and T6M (6 months after harvest), with eight replicates. Experimental design was completely randomized in both experimental tests. Fruit maturity stage did not affect crude protein, total carbohydrate, neutral detergent fiber, in vitro dry matter digestibility (IVDMD), pulp firmness, soluble solids content and total pectin content, but increased acid detergent fiber content from 45 days after anthesis. Storage length up to six months after harvest increased ash, crude protein and IVDMD, and reduced the content of soluble solids. Forage watermelon fruit can be harvested from 30 to 75 days after anthesis equivalent to 75 - 120 days after planting, and they can be stored under tree shade up to 6 months after harvest.(AU)

Growth performance, reproductive parameters and fertility measures in young Nellore bulls with divergent feed efficiency

The growth, sexual maturity and fertility-related parameters related of young Nellore bulls with divergent residual feed intake (RFI) raised on pasture were evaluated. After classification of 48 young males as low and high RFI (more and less efficient, respectively), the animals were evaluated for growth and reproductive parameters at 28-day intervals from 14.3 to 24.6 months of age. The semen was cryopreserved in the last sampling and fresh and post-thaw semen samples were evaluated. Low RFI bulls exhibited higher initial and final body weight (P < 0.05), but feed intake, body condition score and growth measures evaluated by carcass ultrasound were unaffected by RFI (P > 0.05). The scrotal circumference, sperm concentration, defects, and quality of fresh semen, and ultrasonographic testicular characteristics were unaffected by RFI (P > 0.05). However, velocity parameters such as average path and curvilinear velocities determined by computer-assisted sperm analysis of thawed semen submitted to the rapid thermoresistance test were improved (P < 0.05) in low RFI bulls, but this improvement in quality did not enhance in vitro sperm fertilizing ability. Our results demonstrated significant differences in metabolism and growth performance between bulls of divergent RFI. In addition, there was slight improvement in the semen quality of bulls with low RFI bulls, but this did not enhance in vitro fertilizing ability. Selection of beef bulls for RFI can be performed, which will result in economic benefits by improving the growth performance of the animals without affecting reproductive parameters.(AU)

Effectiveness of near-infrared spectroscopy as a non-invasive tool to discriminate spectral profiles of in vitro cultured oocytes from goats

Here, we aimed to discriminate between the spectral profiles of spent culture media after oocyte in vitro maturation (IVM) and culture (IVC) from goats of different ages subjected to repeated hormonal treatments. The profiles were discriminated using near infrared (NIR) spectroscopy combined with multivariate methods. A total of 19 goats (young = 10; old = 9) were subjected to serial hormonal stimulation (HS) with gonadotropins. Cumulus oophorus complexes (COCs) were collected using laparoscopic ovum pick-up (LOPU) and subjected to IVM and parthenogenetic activation. The initial embryos were subjected to IVC. Spent culture media were collected after oocyte IVM and on day 2 of IVC and analyzed using NIR spectroscopy. NIR spectral data were interpreted through chemometric methods, such as principle component analysis (PCA) and partial least square discriminant analysis (PLS-DA). The results of PCA analysis clearly showed a separation in the spectral profiles between the experimental groups (HS sessions; young and old animals) both after IVM and IVC. Overall, the main absorption bands were attributed to the C-H group second overtone, first overtone of O-H and N-H, and C-H combinations and may serve as molecular markers. On the other hand, the spectral data obtained using PLS-DA models provided a better classification of the groups. The results showed the possibility of discriminating young and old groups as well as the three HS sessions with high specificity, sensitivity, and accuracy using NIR spectra. Thus, the culture medium analysis using NIR spectroscopy combined with multivariate methods indicated the dissimilarities between the groups and provided an insight into the in vitro development of goat oocytes. This technique serves as an efficient, objective, rapid, and non-invasive method to discriminate spectral profiles.(AU)

Effects of different stimulators of cGMP synthesis on lipid content in bovine oocytes matured in vitro

Bovine oocytes and blastocysts produced in vitro are frequently of lower quality and less cryotolerant than those produced in vivo, and greater accumulation of lipids in the cytoplasm has been pointed out as one of the reasons. In human adipocytes cGMP signaling through the activation of PKG appears to be involved in lipid metabolism, and components of this pathway have been detected in bovine cumulus-oocyte complexes (COCs). The aim of this study was to investigate the influence of this pathway on the lipid content in oocytes and expression of PLIN2 (a lipid metabolism-related gene) in cumulus cells. COCs were matured in vitro for 24 h with different stimulators of cGMP synthesis. The activation of soluble guanylyl cyclase (sGC) by Protoporphyrin IX reduced lipid content (22.7 FI) compared to control oocytes (36.45 FI; P <0.05). Stimulation of membrane guanylyl cyclase (mGC) with natriuretic peptides precursors A and C (NPPA and NPPC) had no effect (36.5 FI; P>0.05). When the PKG inhibitor KT5823 was associated with Protoporphyrin IX, its effect was reversed and lipid contents increased (52.71 FI; P<0.05). None of the stimulators of cGMP synthesis affected the expression of PLIN2 in cumulus cells. In conclusion, stimulation of sGC for cGMP synthesis promotes lipolytic activities in bovine oocytes matured in vitro and such effect is mediated by PKG. However, such effect may vary depending on the stimulus received and/or which synthesis enzyme was activated, as stimulation of mGC had no effects.(AU)

O que já sabemos sobre a reprodução de fêmeas de grandes felídeos? / What do we already know about the reproduction of big cats' females?

Os grandes felídeos são predadores de topo de cadeia com um papel essencial nos ecossistemas globais. O conceito de Conservação Única propõe a reprodução artificial como uma das ferramentas para reduzir a vulnerabilidade dessas espécies. Este manuscrito teve como objetivo avaliar o que há de novo na reprodução de grandes felídeos na última década. O conhecimento da fisiologia e do comportamento reprodutivo é o primeiro passo para o desenvolvimento de tecnologias reprodutivas em animais selvagens. Nos grandes felídeos, o comportamento copulatório é de fundamental importância, pois necessitam de mecanismos de indução da ovulação, que podem ser mecânicos, sensoriais ou via administração de hormônio luteinizante. O sucesso no cuidado neonatal representa o sucesso da tecnologia reprodutiva em fêmeas. Na última década, o sucesso da inseminação artificial foi relatado apenas em tigres-siberianos e leopardos da Anatólia, e a inseminação de onças-pintadas é foco de pesquisa do Instituto Reprocon, trocando material genético entre ambientes in situ e ex situ por meio de inseminação artificial. Para obter oócitos viáveis de alta qualidade, a técnica de escolha é a colheita de oócitos por laparoscopia. A produção de embriões in vitro enfrenta desafios para a maturação eficiente de oócitos e sua vitrificação eficiente. As técnicas reprodutivas precisam de estudos aprofundados em grandes felídeos para atingir a repetibilidade necessária para uma aplicação eficiente na conservação.

Big cats are apex predators with an essential role in global ecosystems. The One Conservation concept proposes artificial reproduction as one of the tools to reduce the vulnerability of these species. This manuscript aimed to assess what is new in big cat reproduction in the last decade. Knowledge of reproductive physiology and behavior is the first step towards developing reproductive technologies in wild animals. In big cats, copulatory behavior is of fundamental importance because they need ovulation induction mechanisms, which can be mechanical, sensory, or via the administration of the luteinizing hormone. The success in neonatal care represents the success of reproductive technology in females. In the last decade, successful artificial insemination was only reported in Siberian tigers and Anatolian leopards. Jaguar artificial insemination focuses on research at the Reprocon Institute, exchanging genetic material between in situ and ex situ environments thru artificial insemination. The technique of choice is laparoscopic ovum pick-up to obtain high-quality viable oocytes. The production of in vitro embryos faces challenges for the efficient maturation of oocytes and their efficient vitrification. Reproductive technologies need in-depth studies in big cats to achieve the repeatability necessary for efficient application in conservation.

A L-arginina na produção in vitro de embriões bovinos: perspectivas para o futuro / L-arginine in the in vitro production of bovine embryos: perspectives for the future

A L-arginina (L-arg) é o principal precursor da síntese do NO, contudo, é precursora também da síntese de creatina, agmatina, ureia, síntese proteica, L-ornitina, poliaminas, L-prolina e L-glutamato. Nesta breve revisão, vamos falar de alguns resultados que estão sendo obtidos sobre o papel da L-arg na capacitação de espermatozoides bovinos e seu impacto na produção in vitro de embriões. Estudos in vitro mostraram que a adição de L-arg ao meio de capacitação espermática está associada a um aumento na produção de NO, que se correlaciona com aumento da motilidade e vigor, integridade da membrana plasmática e acrossomal, atividade mitocondrial, capacitação espermática, peroxidação lipídica, bem como com a produção de blastocistos. Além disso, a adição da L-arg ao meio de capacitação in vitro, altera o perfil de proteínas importantes ligadas ao processo de capacitação, fertilização e desenvolvimento embrionário inicial. Estes efeitos da L-arg são GMPc dependentes e independentes. Na maturação in vitro, entretanto, embora já tenham sido encontrados bons resultados com o uso do L-arg, mais estudos são necessários para determinar a concentração ideal a ser adicionada ao meio de maturação in vitro e seu impacto na produção de blastocistos. Visto que a pré-capacitação de espermatozoides induzida pela heparina em presença de L-arg foi o método mais eficiente na produção in vitro de embriões, sugerimos sua utilização. Mais pesquisas sobre o metabolismo da L-arg no espermatozoide e CCOs de bovinos durante eventos ligados à fertilização são necessários para se identificar novas vias que atuem nestas etapas in vitro visando o aumento da percentagem e qualidade de embriões bovinos produzidos in vitro.

L-arginine (L-arg) is the main source of NO synthesis; however, it is also a precursor of the synthesis of creatine, agmatine, urea, protein synthesis, L-ornithine, polyamines, L-proline, and Lglutamate. In this brief review, we will discuss some results obtained previously about the role of L-arg in the capacitation of bovine sperm and its impact on in vitro embryo production. In vitro studies have shown that the addition of L-arg to the sperm capacitation medium is associated with an increase in NO production, which in controlled levels is related to an increased motility and vigor, plasma and acrosomal membrane integrity, mitochondrial activity, sperm capacitation, peroxidation lipids, as well as with the blastocyst production. Furthermore, the addition of L-arg to the in vitro capacitation medium alters the profile of important proteins linked to the capacitation process, fertilization, and early embryonic development. These effects of L-arg are cGMP dependent and independent. In in vitro maturation, however, although good results have already been found with the use of L-arg, further studies are needed to determine the ideal concentration to be added to the in vitro maturation medium and its impact on the production of blastocysts. Since heparin-induced pre-capacitation of spermatozoa in the presence of L-arg was the most efficient method for in vitro embryo production, we suggest its use. More research on L-arg metabolism in bovine sperm and OCCs during events related to fertilization is needed to identify new pathways that act in these in vitro steps aiming to increase the percentage and quality of bovine embryos produced in vitro.

Morphometry of bovine blastocysts produced in vitro in culture media with antioxidants cysteamine or oily extract of Lippia origanoides / [Morfometria de blastocistos bovinos produzidos in vitro em meio de cultura com antioxidantes cisteamina ou extrato oleoso de Lippia origanoides]

This study aimed to evaluate the ultrastructural morphometry of bovine embryos produced in vitro grown at different concentrations of antioxidants. After in vitro maturation and fertilization, the presumptive zygotes were assigned into five treatments. T1) without the addition of any antioxidants (negative control); T2) addition of 50µM/mL cysteamine; and T3, T4 and T5) adding 2.5µg/mL, 5.0µg/mL or 10.0µg/mL of the antioxidants derived from the oily extract from Lippia origanoides, respectively. On D7 of culture, the embryos in the blastocyst stage were fixed and prepared for electron transmission microscopy. These were evaluated for the proportion of cytoplasm-to-nucleus, cytoplasm-to-mitochondria, cytoplasm-to-vacuoles, cytoplasm-to-autophagic vacuoles and cytoplasm-to-lipid droplets. Blastocysts cultured in media containing oily extract of Lippia origanoides presented morphological characteristics such as high cell:mitochondria ratio and low cell:vacuoles and cell:autophagic vacuole ratio, possibly been morphological indicators of embryonic quality. Inner cell mass (ICM) from blastocysts cultured in media without any antioxidants had the highest cell:vacuole ratio. Similar results were found in the trophectoderm (TE) cells of blastocysts from treatment 2. Embryo culture media supplemented with antioxidants derived from Lippia origanoides oil produced embryos with a higher cytoplasmic proportion of organelles, such as mitochondria. Also, treatments without any antioxidants or with the addition of cysteamine presented cytoplasmic vacuolization, a characteristic related to production of poor-quality embryos.(AU)

Este estudo teve como objetivo avaliar a morfometria ultraestrutural de embriões bovinos produzidos in vitro e cultivados em diferentes concentrações de antioxidantes. Após a maturação e a fertilização in vitro, os possíveis zigotos foram divididos em cinco tratamentos: T1) sem adição de antioxidantes (controle negativo); T2) adição de 50µM/mL de cisteamina; e T3, T4 e T5) adição de 2,5µg/mL, 5,0µg/mL ou 10,0µg/mL dos antioxidantes derivados do extrato oleoso de Lippia origanoides, respectivamente. No D7 de cultivo, os embriões em estágio de blastocisto foram fixados e preparados para microscopia eletrônica de transmissão. Estes foram avaliados para a proporção entre citoplasma e núcleo, citoplasma e mitocôndria, citoplasma e vacúolos, citoplasma e vacúolos autofágicos e citoplasma e gotículas lipídicas. Blastocistos cultivados em meio contendo extrato oleoso de Lippia origanoides apresentaram características morfológicas como alta relação célula:mitocôndria e baixa relação célula:vacúolos e célula:vacúolo autofágico, possíveis indicadores morfológicos de qualidade embrionária. A massa celular interna (MCI) de blastocistos cultivados em meio sem quaisquer antioxidantes teve a maior razão célula:vacúolo. Resultados semelhantes foram encontrados nas células do trofectoderma (TE) de blastocistos do tratamento 2. Portanto, o meio de cultivo embrionário suplementado com antioxidantes derivados do óleo de Lippia origanoides produziu embriões com maior proporção citoplasmática de organelas, como mitocôndrias. Além disso, tratamentos sem antioxidantes ou com adição de cisteamina apresentaram vacuolização citoplasmática, característica relacionada à produção de embriões de baixa qualidade.(AU)

Effectiveness of near-infrared spectroscopy as a non-invasive tool to discriminate spectral profiles of in vitro cultured oocytes from goats

Abstract Here, we aimed to discriminate between the spectral profiles of spent culture media after oocyte in vitro maturation (IVM) and culture (IVC) from goats of different ages subjected to repeated hormonal treatments. The profiles were discriminated using near infrared (NIR) spectroscopy combined with multivariate methods. A total of 19 goats (young = 10; old = 9) were subjected to serial hormonal stimulation (HS) with gonadotropins. Cumulus oophorus complexes (COCs) were collected using laparoscopic ovum pick-up (LOPU) and subjected to IVM and parthenogenetic activation. The initial embryos were subjected to IVC. Spent culture media were collected after oocyte IVM and on day 2 of IVC and analyzed using NIR spectroscopy. NIR spectral data were interpreted through chemometric methods, such as principle component analysis (PCA) and partial least square discriminant analysis (PLS-DA). The results of PCA analysis clearly showed a separation in the spectral profiles between the experimental groups (HS sessions; young and old animals) both after IVM and IVC. Overall, the main absorption bands were attributed to the C-H group second overtone, first overtone of O-H and N-H, and C-H combinations and may serve as molecular markers. On the other hand, the spectral data obtained using PLS-DA models provided a better classification of the groups. The results showed the possibility of discriminating young and old groups as well as the three HS sessions with high specificity, sensitivity, and accuracy using NIR spectra. Thus, the culture medium analysis using NIR spectroscopy combined with multivariate methods indicated the dissimilarities between the groups and provided an insight into the in vitro development of goat oocytes. This technique serves as an efficient, objective, rapid, and non-invasive method to discriminate spectral profiles.

Techniques of fragile renal organoids transplantation in mice

Purpose: This study aimed to develop a microsurgical technique to transplant extremely fragile renal organoids in vivo, created by in-vitro reaggregation of metanephros from fetal mice. These organoids in reaggregation and development were examined histologically after transplantation under the renal capsule. Methods: Initially, metanephros from fetal mice were enzymatically treated to form single cells, and spheroids were generated in vitro. Under a microscope, the renal capsule was detached to avoid bleeding, and the outer cylinder of the indwelling needle was inserted to detach the renal parenchyma from the renal capsule using water pressure. The reaggregated spheroid was aspirated from the culture plate using a syringe with an indwelling needle outer cylinder and carefully extruded under the capsule. Pathological analysis was performed to evaluate changes in reaggregated spheroids over time and the effects of co-culture of spinal cord and subcapsular implantation on maturation. Results: In vitro, the formation of luminal structures became clearer on day 5. These fragile organoids were successfully implanted without tissue crapes under the renal capsule and formed glomerular. The effect of spinal cord co-transplant was not obvious histrionically. Conclusions: A simple and easy method to transplant fragile spheroids and renal under the renal capsule without damage was developed.

Resposta oocitária ao estresse térmico: efeitos moleculares e na cinética do desenvolvimento embrionário / Oocytes responses to heat stress: molecular effects and on the kinetics of embryonic development

A exposição de bovinos a temperaturas elevadas pode causar hipertermia e alterações fisiológicas denominadas de estresse térmico. Este fenômeno tem sido caracterizado em várias espécies, sendo exacerbado em vacas leiteiras de alta produção. Apesar de estas alterações serem observadas em diversos órgãos e tipos celulares, oócitos e embriões pré-implantacionais são mais susceptíveis ao estresse térmico, resultando em reduções na fertilidade das vacas lactantes durante os meses mais quentes do ano. Os oócitos bovinos expostos ao estresse térmico apresentam alterações celulares e moleculares que reduzem a competência oocitária sendo carreados ao embrião pré-implantacional. A maturação in vitro de oócitos bovinos em condições de temperatura elevada (choque térmico) recapitula as principais alterações observadas in vivo, permitindo investigar em mais detalhes a susceptibilidade e os mecanismos de sobrevivência. O entendimento dos processos mediados pelo estresse térmico na fisiologia do oócito permitirá o desenvolvimento de estratégias para mitigar os efeitos negativos mantendo a competência oocitária

The exposure of cattle to high environmental temperatures may lead to hyperthermia and physiological changes known as heat stress. This phenomenon has been characterized in several species, albeit is a more profound problem in high-yielding dairy cows. Although these alterations are observed in several organs and cell types, oocytes and preimplantation embryos are more susceptible to heat stress, resulting in reductions in the fertility of lactating cows during the hottest months of the year. Bovine oocytes exposed to heat stress undergo cellular and molecular changes reducing oocyte competence which is carried out to the preimplantation embryo. In vitro maturation of bovine oocytes under conditions of high temperature (heat shock) recapitulates the main changes observed in vivo, allowing us to investigate the susceptibilities and survival mechanisms. Understanding the processes mediated by heat stress in oocyte physiology will allow the development of strategies to mitigate the negative effects while maintaining oocyte competence.

Ultra-diluted Folliculinum 6 cH impairs ovine oocyte viability and maturation after in vitro culture

This study investigated the effect of Folliculinum 6 cH on the oocyte meiosis resumption and viability rates, progesterone production and mitochondrial activity after in vitro maturation of cumulus-oocyte complexes (COCs) in sheep. Sheep ovaries were collected at a local slaughterhouse and COCs were recovered by slicing technique. The selected COCs were maturated in TCM199 (Control treatment), or control medium supplemented with 0.05% ethanol (v/v) (the vehicle of the homeopathic preparation – Ethanol treatment) or with Folliculinum 6 cH. After 24 h of in vitro maturation (IVM), oocytes were mechanically denuded and incubated with Hoechst 33342 and MitoTracker (0.5 μM) Orange CMTMRos for analysis of viability and chromatin configuration, and mitochondrial activity, respectively. The results showed that Folliculinum 6 cH addition increased oocyte degeneration and reduced meiotic resumption compared to the control (P < 0.05). Interestingly, the percentages meiotic resumption and oocyte maturation were lower in the Folliculinum 6 cH treatment compared to its vehicle (Ethanol treatment) (P < 0.05). On the other hand, when the treatments were compared, higher mitochondrial activity was observed in the Ethanol treatment (P < 0.05). In conclusion, contrary to its vehicle, the addition of Folliculinum 6 cH to the IVM medium promoted oocyte degeneration and affected negatively the mitochondrial distribution, impairing meiosis resumption.