This study evaluated the effects of three maturation systems, namely invitro (MatV) and invivo (MatS) systems, as well as intrafollicular transfer of immature oocytes (IFIOT; MatT), on the accumulation of lipid droplets in bovine oocytes. Lipids were evaluated using confocal microscopy and transmission electron microscopy. The expression of genes related to lipid metabolism, namely acyl-CoA synthetase short chain family member 2 (ACSS2), ELOVL fatty acid elongase 1 (ELOVL1) and fatty acid binding protein 3 (FABP3), was quantified by quantitative polymerase chain reaction. The mean (±s.d.) area occupied by lipids in immature oocytes (13±2%) was similar to those matured invivo (MatS, 16±2%; MatT, 12±2%). However, there was a significant increase in lipids in oocytes in the MatV group (24±2%) compared with all other groups (P<0.001). In the ultrastructural evaluations, MatV oocytes also showed the highest lipid content. The expression of ELOVL1 and FABP3 was similar in the MatS and IFIOT groups. However, transcript levels of ACSS2 were lower in IFIOT than MatV oocytes. These results indicate, for the first time, that oocytes matured by IFIOT are similar to those matured invivo with regard to lipid accumulation, which indicates better quality than those matured invitro.

In vitro produced embryos are still sensitive to the freezing process which can be explained, in part, by the high‐lipid accumulation that characterizes these embryos. Therefore, we aimed to evaluate the effect of delipidating agents, L‐carnitine and the trans‐10 cis‐12 conjugated linoleic acid (CLA) isomer, on blastocyst development, lipid content, gene expression and cryotolerance when added to embryo culture media. Embryos were cultured in four different media: T1: control (n = 616), synthetic oviduct fluid (SOF) media with 5% foetal bovine serum (FBS); T2: L‐carnitine (n = 648), SOF medium with 5% FBS and 0.6 mg/ml of L‐carnitine; T3: CLA (n = 627), SOF medium with 5% FBS and 100 μM trans‐10 cis‐12 CLA; and T4: L‐carnitine + CLA: (n = 597), SOF medium with 5% FBS plus 0.6 mg/ml L‐carnitine and 100 μM trans‐10 cis‐12 CLA. Supplementation of culture medium with either or both delipidating agents reduced (p < .05) blastocyst rate on D7 (T1 = 49 ± 3.5; T2 = 39 ± 3.0; T3 = 42 ± 3.9 and T4 = 39 ± 3.9), but did not affected gene expression (p > .05). Although embryos cultured in the presence of L‐carnitine contained fewer (p < .05) lipid droplets than the control embryos, they showed a lower re‐expansion rate 24 hr post‐thaw than those (p < .05). In conclusion, although L‐carnitine reduced the amount of lipids in cultured embryos, the use of L‐carnitine and CLA during in vitro culture was not able to improve the embryo production and the response to cryopreservation.

Cows intensively used as oocyte donors for in vitro embryo production (IVEP) are usually kept nonpregnant for prolonged intervals, exposed to successive hormonal treatments, and frequently become overweight. These are all risk factors for the development of endocrine unbalance and, consequently, cystic ovarian disease (COD). The aim of this study was to evaluate the effect of active immunization against gonadotropin-releasing hormone (GnRH) on (1) ovarian follicular population, and (2) development potential of oocytes used for IVEP. Nelore (Bos indicus) cows (n = 14), previously diagnosed with chronic COD (Faria et al. 2017 Anim. Reprod., in press), weighing 620.0 ± 12.8 kg and with body condition score of 4.1 ± 0.2, were assigned to control (n = 6) or treatment (n = 8) group. Cows in the treatment group received 2 SC injections of 1.0 mL of anti-GnRH vaccine (Bopriva, Zoetis, Brazil), 28 days apart (weeks 0 and 4), whereas cows in the control group received placebo on the same schedule. Transrectal ultrasonography was performed weekly from week 0 to evaluate the number and distribution of follicles among size classes, endometrial thickness, and clinical presence of mucometra. Immunization was considered effective (E-IM) when no follicles ≥5.0 mm were observed on the ovaries during a given examination. Cows having E-IM were then used as oocyte donors for IVEP. Cumulus-oocyte complexes (COC) were collected in 5 consecutive ovum pick-up weekly sessions. As a control for IVEP, oocytes from a slaughterhouse were used, with similar procedures performed on the same days and using the same semen batch. The MIXED procedure of SAS (SAS Institute Inc., Cary, NC, USA) with repeated-measures statement was used to evaluate the effects of treatment, time, and interactions on ovarian endpoints; and the GLM procedure was used to analyse embryo production data. Results are shown as mean ± SEM. There were time and time × treatment effects on ovarian parameters. Treated cows had a decrease (P < 0.05) in the average diameter of the largest follicle and in the number of follicles ≥8 mm, and an increase (P < 0.05) in follicular population after week 6. Nonetheless, individual response to treatment was variable: only 50% of the cows (4 of 8) were E-IM at week 8, whereas 25% (2 of 8) still had COD (largest follicle ≥18.0 mm) at this timepoint. Overall, a negative correlation was detected between follicular population and the diameter of the largest follicle (r = –0.60, P < 0.0001) or the number of follicles ≥8 mm (r = –0.47, P < 0.0001). There was no effect (P > 0.05) of treatment on endometrial thickness or mucometra score. Cows with E-IM produced 22.2 ± 3.6 total and 12.9 ± 2.3 viable COC. Cleavage rate did not differ between E-IM and control (slaughterhouse) oocytes (70.8 ± 7.0 v. 75.1 ± 3.0%, respectively; P > 0.05); however, blastocyst rate was greater in the E-IM group compared with controls (39.7 ± 5.5 v. 20.5 ± 4.7%, respectively; P < 0.02). In summary, our results suggest that active immunization against GnRH leads to variable results in the distribution of the follicular population in cows with COD, but it does not negatively affect IVEP efficiency. This research was supported by Zoetis, CNPq, and CAPES.

ResumoA neosporose bovina é uma doença infecciosa de expressão global, de origem parasitária, causada pelo protozoário Neospora caninum, que gera grande impacto econômico por causar o aborto em bovinos de corte e de leite em vários países. Apesar de altas prevalências de anticorpos nos animais, a frequência de neosporose clínica tem sido pouco registrada. Para controle efetivo da doença, deve-se coletar e eliminar fetos abortados, membranas fetais, placentas e bezerros natimortos, impedindo assim a ingestão por cães de material contaminado. Esta revisão de literatura propõe-se a abordar aspectos importantes da neosporose bovina, incluindo a etiologia, epidemiologia, patogenia, sintomas, diagnóstico, prognóstico, terapêutica e controle.

For the development of in vitro produced (IVP) as well as in vivo produced bovine embryos, it is extremely important that their energy metabolism works properly because the embryo must be able to metabolize energy substrates that are necessary for producing energy. Lipids play an important role in early embryonic development, acting as source of energy for oocytes and embryos. However, it is known that oocytes and embryos, mainly IVP, accumulate large amounts of lipids in the cytoplasm. Although they are extremely important in embryonic development, lipids have been associated with the reduced survival of bovine embryos following cryopreservation. There is evidence that at least four different categories of lipids affect embryo survival after cryopreservation, including triglycerides (TAG), free fatty acids, cholesterol and phospholipids. Thus, many studies are being conducted to improve the resistance of IVP embryos to the cryopreservation process by reducing the concentration or removing the source of serum from the medium or by reducing oocyte/embryo lipids using mechanical or chemical means. Regarding the use of delipidating agents that reduce the uptake and synthesis of fatty acids (FA) by cells, substances such as phenazine ethosulfate (PES), forskolin, L-carnitine and isomers of conjugated linoleic acid (CLA) have been utilized. This review aims to address important issues related to embryonic energy metabolism, the importance of lipid metabolism and its relation to the cryopreservation of IVP bovine embryos by summarizing the latest research in this field. Key words: Bovine embryos. Cryopreservation. Energy metabolism. Lipids. ResumoPara o desenvolvimento de embriões bovinos produzidos in vitro (PIV) e in vivo, é extremamente importante que o metabolismo energético funcione de maneira adequada, pois o embrião precisa ser capaz de metabolizar os substratos energéticos necessários para a produção de energia. Os lipídios desempenham papel importante no desenvolvimento embrionário inicial, atuando como fonte de energia para oócitos e embriões. Entretanto, sabe-se que oócitos e embriões bovinos, principalmente os PIV, acumulam grande quantidade de lipídios no citoplasma. Apesar de serem extremamente importantes no desenvolvimento embrionário, os lipídios têm sido associados com a reduzida sobrevivência após a criopreservação de embriões bovinos. Existem evidências de que pelo menos quatro classes de lipídios afetam a sobrevivência embrionária pós-criopreservação, sendo os triglicerídeos (TAG), ácidos graxos livres, colesterol e os fosfolipídios. Sendo assim, vários estudos estão sendo realizados com o intuito de melhorar a resistência dos embriões PIV ao processo de criopreservação, realizando a redução ou retirada do soro do meio ou a redução mecânica ou química de lipídios. Com relação ao uso de agentes delipidantes que diminuam a captação e síntese de ácidos graxos (AG) pelas células, substâncias como fenazina etossulfato (PES), forskolin, L-carnitina e isômeros do ácido linoleico conjugado (CLA) t...

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ResumoA ecografia é um método de obtenção de imagens através de pulsos sonoros, operados em frequências de um a vinte MHz, transmitidos para dentro do organismo que são refletidos em forma de eco. Além de ser uma técnica com ausência de radiação ionizante permite a avaliação em tempo real. Uma onda de ultrassom é uma forma de energia acústica, gerada quando vários cristais piezoelétricos no interior de um transdutor vibram a uma frequência elevada.O exame tridimensional fundamenta-se na reconstrução digital de uma imagem bidimensional adquirida por meio de transdutores multifrequenciais comuns. A otimização de imagens em escala de cinza pode ser obtida através de ajustes na vazão de potência, ganho, zona focal, campo de visão, densidade de linhas, curvas da escala de cinza, alcance dinâmico e

Dedico esse trabalho aos meus pais, Henrique e Dinair, à minha irmã Heloísa, às minhas sobrinhas Geovanna e Maria Clara. Tudo que faço é por vocês e para vocês! vi AGRADECIMENTOSAgradeço a Deus por todas as oportunidades que me concede e principalmente por ser minha força maior para superar todas as dificuldades.Agradeço aos meus pais, Henrique e Dinair, pelo apoio incondicional, amor, carinho, confiança e compreensão. Em especial à minha mãe, por ser meu "esteio", principal motivo para não desistir nos momentos mais difíceis. À minha irmã Heloísa, por acreditar na minha capacidade e por me dar mais dois motivos para sempre melhorar e crescer cada vez mais: Gevonna e Maria Clara (sobrinhas).Ao Dr. Ivo Pivato, pela oportunidade por ter aceitado me orientar e pela paciência.À Dra Margot, pela oportunidade de fazer parte da sua equipe, por todo conhecimento transmitido, pelas cobranças, por toda seriedade e conduta profissional, as quais admiro.À colega e parceira de laboratório Ligi (Ligiane Leme), por toda experiência compartilhada, paciência, por todas as palavras de apoio e por toda ajuda disponibilizada. Sem sua ajuda não teria dado certo! Ao Regivaldo, por todos os cálculos e ensinamentos para utilização de equipamentos sofisticados.Ao Dr. Luciano Paulino, pela disponibilidade para realizar as análises de lipidoma e pelas explicações direcionadas à técnica.Aos amigos Lucas Prado, João Ricardo (amigo-irmão), Dany Helena, Isabella Dambros, Nessinha, Aline Vasques, Noemi e Nathália Arioza, por acreditarem no meu esforço para alcançar minhas metas, pelos desabafos, pela palavra amiga e pelo incentivo.