RESUMO
OBJECTIVE: Livestock droppings cause some environmental problems, but they have the potential to be used as effective biomass resources. The black soldier fly (BSF), Hermetia illucens (Diptera: Stratiomyidae), is suitable for efficiently processing such resources. By using BSF larvae for the disposal of livestock droppings, we can obtain two valuable products: protein resources and organic fertilizer. However, there is insufficient research on the digestive enzymes suitable for processing this waste. Here, we aimed to construct an efficient BSF processing system using livestock droppings, and we explored the digestive enzymes involved in this process. RESULTS: First, we investigated the characteristics of transcripts expressed in the midgut of BSF larvae and found that immune response-related genes were expressed in the midgut. Then, we investigated digestive enzymes and identified a novel serine protease, HiBrachyurin, whose mRNA was highly expressed in the posterior midgut when BSF larvae fed on horse droppings. Despite the low protein content of horse droppings, larvae that fed on horse droppings accumulated more protein than those in the other groups. Therefore, HiBrachyurin may contribute to digestibility in the early stage of protein degradation in BSF larvae fed on horse droppings.
Assuntos
Dípteros , Larva , Serina Proteases , Animais , Dípteros/genética , Dípteros/metabolismo , Dípteros/enzimologia , Larva/metabolismo , Larva/genética , Cavalos , Serina Proteases/metabolismo , Serina Proteases/genética , Proteínas de Insetos/metabolismo , Proteínas de Insetos/genética , FezesRESUMO
Antioxidants are free radical scavengers that increase oocyte quality and improve female fertility by suppressing oxidative stress. However, the related mechanisms remain unclear. The present study was designed to examine whether a reduction of oxidative stress from using the antioxidant sericin led to expanded cumulus cell (CC)-oocyte communication and oocyte developmental acquisition in a bovine model. We found that cumulus-oocyte complexes (COCs) matured in the presence of sericin showed a significantly increased oocyte meiotic maturation rate (P < 0.01) and accelerated subsequent blastocyst formation, as more blastocysts were found at the hatched stage (P < 0.05) compared to that in the control group. In contrast to the control group, sericin suppressed H2O2 levels in COCs, resulting in a markedly enhanced CC-oocyte gap junction communication index and number of transzonal projections, which were preserved until 18 h of oocyte maturation. These findings indicate that sericin reduces disruption of oocyte-follicular cell communication induced by oxidative stress. Sericin consistently increased intra-oocyte glutathione (GSH) levels and reduced oocyte H2O2 levels (P < 0.05), both of which were ablated when GSH synthesis was inhibited by buthionine sulfoximide (an inhibitor of GSH synthesis). Furthermore, the inhibition of GSH synthesis counteracted the positive effects of sericin on subsequent embryo developmental competence (P < 0.01). Intra-oocyte GSH levels were positively associated with blastocyst development and quality. These outcomes demonstrate new perspectives for the improvement of oocyte quality in assisted reproductive technology and may contribute to developing treatment strategies for infertility and cancer.
Assuntos
Antioxidantes , Sericinas , Animais , Bovinos , Feminino , Antioxidantes/farmacologia , Sericinas/farmacologia , Sericinas/metabolismo , Técnicas de Maturação in Vitro de Oócitos/métodos , Peróxido de Hidrogênio/farmacologia , Oócitos/metabolismo , Estresse Oxidativo , Comunicação Celular , Glutationa/metabolismo , Blastocisto/metabolismo , Células do Cúmulo/metabolismoRESUMO
Direct cleavage, a type of abnormal cleavage in which one zygote divides into three or more blastomeres, has been reported in mammals. The incidence of direct cleavage increases in zygotes with three or more pronuclei (multi-PN) and those showing abnormal pronuclei migration. However, there are few reports on the relationship between pronuclei and direct cleavage, and the effects of these relationships on subsequent embryogenesis have not been clarified. It is difficult to observe pronuclei under visible light, especially in bovine zygotes, because of abundant dark lipid droplets in the cytoplasm. We visualized pronuclei by removing lipid droplets from bovine zygotes and analyzed the relationship between the number of pronuclei and direct cleavage using time-lapse cinematography. The direct cleavage rate of multi-PN zygotes was 78.6%, which was significantly higher than that of zygotes with one pronucleus (1 PN, 0.0%) and two pronuclei (2 PN, 8.2%). Observation of pronuclei migration in 2 PN zygotes showed that 3.1% of 2 PN zygotes had non-apposed pronuclei. The direct cleavage rate of zygotes with non-apposed pronuclei was 66.7%, which was significantly higher than that of zygotes with apposed pronuclei (6.4%). Among multi-PN zygotes, the proportions of zygotes with apposed pronuclei and non-apposed pronuclei were 37.5% and 64.3%, respectively. The direct cleavage rate of multi-PN zygotes with non-apposed pronuclei was 100.0%, which was significantly higher than that of zygotes with apposed pronuclei (40.0%). Three-dimensional live-cell imaging of bovine zygotes injected with the mRNA-encoding histone H2B-mCherry showed that the direct cleavage rates of 2 PN and multi-PN zygotes bypassing syngamy were 63.2% and 75.5%, respectively. These rates were significantly higher than that of 2 PN and multi-PN zygotes that underwent syngamy (5.6% and 20.0%, respectively). Regardless of the number of pronuclei, a high frequency of direct cleavage was observed in zygotes in which the pronuclei did not migrate inward the cytoplasm and bypassed syngamy. These results suggest that abnormal fertilization such as multi-PN and migration error of pronuclei in cattle is the primary reason for direct cleavage during the first mitosis. Assessment of direct cleavage during the first mitosis allows exclusion of embryos with abnormal fertilization and may contribute to in vitro produced embryo transfer success.
Assuntos
Desenvolvimento Embrionário , Fertilização in vitro , Animais , Bovinos , Fertilização in vitro/veterinária , Zigoto , Mitose , Núcleo Celular , Fertilização , MamíferosRESUMO
In assisted reproductive technology (ART)-derived embryos of non-rodent mammals, including humans and cattle, chromosome segregation errors are highly likely to occur during early cleavage division, resulting in aneuploidy, including mosaicism. However, the relationship between chromosomal segregation errors during early cleavage and subsequent embryonic development has not been detailed in these mammals. In the present study, we developed non-invasive live-cell imaging of chromosome segregation dynamics using a histone H2B-mCherry mRNA probe in bovine preimplantation embryos. Chromosome segregation errors in early cleavage affected blastocyst formation. Especially, embryos that underwent abnormal chromosome segregation (ACS) with multiple or large micronucleus formation rarely developed into blastocysts. Embryos with the severe ACS had prolonged cell cycle duration. After transfer of blastocysts with live-cell imaging of chromosome segregation to ten cows, six became pregnant and four of them gave full-term offspring. Interestingly, two of them were derived from blastocysts with ACS. Hence, chromosomal segregation errors with micronucleus formation during early cleavage can be a fatal hallmark of preimplantation embryogenesis in cattle. This technique has shown potential for understanding the relationship between chromosome segregation error and subsequent embryo development, and for selecting viable ART-derived embryos for medical and livestock production.
Assuntos
Aneuploidia , Blastocisto , Animais , Blastocisto/metabolismo , Bovinos , Segregação de Cromossomos , Desenvolvimento Embrionário/genética , Feminino , Mamíferos , Mosaicismo , GravidezRESUMO
Chromosome instability leading to aneuploidy during early cleavage is well known in humans and cattle. Partial compaction (PC), which occurs only in some blastomeres, is suggested as a self-correction mechanism through which human embryos avoid aneuploid mosaicism. Partially compacted embryos show abnormal cleavages more frequently during early development; however, the mechanism by which blastomeres are excluded has not been elucidated. Here, we confirmed PC in approximately half of the tested bovine embryos, similar to that in human embryos. DNA sequencing of single-cell and intact embryos revealed that the morulae that excluded some blastomeres had euploidy, but many of the excluded blastomeres had aneuploidy. Time-lapse imaging of zygotes without the zona pellucida revealed that the excluded blastomeres underwent reverse and direct cleavages, which are abnormal cleavages, more frequently than the blastomeres involved in compaction. These results suggest the potential role of abnormal cleavage in the self-correction mechanism during the development of mammalian preimplantation embryos.
Assuntos
Blastocisto/patologia , Fase de Clivagem do Zigoto/patologia , Aneuploidia , Animais , Blastômeros/metabolismo , Bovinos , Variações do Número de Cópias de DNA/genética , Mórula/metabolismo , Imagem com Lapso de TempoRESUMO
The morphokinetics of pronuclei (PN) are considered crucial factors affecting embryogenesis in mammals. Whereas, since bovine zygotes contain a large number of cytosolic lipid droplets, detailed observation of PN has not been performed. In this study, we visualized PN using time-lapse cinematography (TLC) with light microscopy for the first time in delipidated bovine zygotes. The proportions of 0 PN, 1PN, 2PN, and multi-PN in delipidated bovine zygotes were 10.1%, 6.5%, 72.7%, and 10.8%, respectively. Abnormal fertilization, including 1 PN and multi-PN, was observed in 15.6% of blastocysts. The times from IVF to PN appearance, PN fading, and first cleavage in 2 PN bovine zygotes that developed into blastocysts were 10.4, 25.5, and 27.6 h, respectively, which were similar to PN morphokinetics in humans. The 2 PN zygotes showed that the prolonged time from IVF to the appearance of PN and from the fading of PN to the first cleavage negatively affected blastocyst formation. The time from appearance to fading of PN in multi-PN zygotes that developed into blastocysts was longer than that in multi-PN zygotes that did not develop into blastocysts. Besides, among zygotes that developed into blastocysts, the time from appearance to fading of PN in multi-PN zygotes was longer than that in 2 PN and 1 PN zygotes. These results suggest that PN morphokinetic abnormalities are associated with subsequent embryonic development. Observation of PN in bovine zygotes by using non-invasive visible light TLC by delipidation could be a powerful tool to clarify the relationship between PN morphokinetics and developmental competence.
Assuntos
Fertilização in vitro , Zigoto , Animais , Blastocisto , Bovinos , Desenvolvimento Embrionário , Feminino , Fertilização in vitro/veterinária , Gravidez , Imagem com Lapso de Tempo/veterináriaRESUMO
Wild large Japanese field mice (Apodemus speciosus) responses to cyclic seasonal changes are associated with physiological and behavioral changes. However, the detailed regulation of oogenesis in the ovary during the seasonal reproductive cycle in wild large Japanese field mice has not been studied. We assessed the dynamics and changes in ovarian morphology and hormone concentrations associated with reproductive seasonality throughout the year. The stages of the ovarian morphological breeding cycle of wild large Japanese field mice were classified as breeding, transition, and non-breeding periods during the annual reproductive cycle. Measurement of blood estradiol concentrations throughout the year showed that the levels in September and October were higher than those in other months. It is presumed that follicle development starts from a blood estradiol concentration of 38.4 ± 27.1 pg/mL, which marks a shift from the transitional season to the breeding season, followed by the transition to the non-breeding season at 26.1 ± 11.6 pg/mL. These results suggest that seasonal follicle development in wild rodents is correlated with estradiol regulation. We consider this species to be an alternative animal model for studying seasonal reproductive changes and the effects of environmental changes.
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Efforts directed toward enhancing animals' productivity are focused on evaluating the effects of non-traditional feed additives that are safer than antibiotics, which have been banned because of their health hazards. Many studies used an amino acid that contributes to heme biosynthesis, known as 5-aminolevulinic acid (5-ALA), to promote the productivity of farm animals. However, these studies demonstrate inconsistent results. In order to develop a clear understanding of the effects of 5-ALA in farm animals, we comprehensively searched PubMed and Web of Science for studies evaluating 5-ALA effects on the performance, iron status, and immune response of different farm animals. The search retrieved 1369 publications, out of which 16 trials were relevant. The 5-ALA-relevant data and methodological attributes of these trials were extracted/evaluated by two independent researchers, based on a set of defined criteria. Samples were comprised of pigs, chickens, and dairy cows. The 5-ALA doses ranged from 2 mg to 1 g/kg of feed, and treatment duration ranged from 10 to 142 days. Overall, 5-ALA improved iron status in most studies and increased white blood cells count in 3 out of 10 studies, in addition to improving animals' cell-mediated immune response following immune stimulation with lipopolysaccharide. Inconsistent findings were reported for growth performance and egg production; however, a combination of 10 mg/kg of 5-ALA with 500 mg/kg of vitamin C promoted the highest egg production. In addition, 5-ALA improved milk protein concentration. In conclusion, 5-ALA can enhance farm animals' iron status and immune response; however, the heterogeneity of the reviewed studies limits the generalizability of the findings. Standard procedures and outcome measures are needed to confirm the benefits of 5-ALA. Attention should also be paid to any adverse effects.
RESUMO
The aim of this study was to investigate the proliferation and apoptosis of male germ cells during the seasonal reproductive cycle of the large Japanese field mice (Apodemus speciosus). Male mice residing in their natural habitat were captured in Niigata, Japan. Testis sections were stained with haematoxylin and eosin, and mitotic male germ cells were identified using immunofluorescence staining for proliferating cell nuclear antigen (PCNA). Apoptosis was analysed using terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labelling (TUNEL) assay. The phases of spermatogenesis during the seasonal reproductive cycle were classified as active, transitional, and inactive based on the diameter of the seminiferous tubules. The number of PCNA-positive germ cells was less during the inactive than other phases. The percentage of TUNEL-positive germ cells per seminiferous tubule was greater during the inactive than active and transitional phases. Spermatogenesis during the seasonal reproductive cycle is controlled by proliferation and apoptosis in male germ cells. This species of undomesticated mice could be used as an animal model to study spermatogenesis as a valuable indicator of the effects of ecological and anthropogenic factors on animal reproduction.
Assuntos
Apoptose/fisiologia , Proliferação de Células/fisiologia , Células Germinativas/fisiologia , Murinae/fisiologia , Estações do Ano , Espermatogênese/fisiologia , Animais , Ecossistema , Japão , MasculinoRESUMO
PURPOSE: A microwell culture system that facilitates group culture, such as well-of-the-well (WOW), improves embryonic development in an individual culture. We examined the effect of WOW on embryonic development in vitro with commercially available human single culture media. METHODS: Using four different commercial human single culture media, in vitro development and imprinted gene expression of bovine embryos cultured in WOW were compared to droplet culture (one zygote per drop). To determine the effects of microwell and group culture on embryonic development, different numbers of embryos were cultured in droplet or WOW. Diffusion simulation of accumulating metabolites was conducted using the finite volume method. RESULTS: WOW had a positive effect on bovine embryonic development, regardless of the type of single culture media. Imprinted gene expression was not different between droplet- and WOW-derived blastocysts. The microwell and group cultures in WOW showed a significant positive effect on the rate of total blastocysts and the rate of development to the expanded and hatching blastocyst stages. The assumed cumulative metabolite concentration of WOW with one embryo was 1.47 times higher than that of droplet culture with one embryo. Furthermore, the concentration of WOW with three embryos was 1.54 times higher than that of WOW with one embryo. CONCLUSIONS: In using human single culture media, a microwell culture system that allows group culture could be a powerful clinical tool for improving the success of assisted reproductive technologies.
Assuntos
Blastocisto/citologia , Meios de Cultura , Técnicas de Cultura Embrionária/métodos , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário/fisiologia , Fertilização in vitro/métodos , Zigoto/citologia , Animais , Blastocisto/fisiologia , Bovinos , Embrião de Mamíferos/fisiologia , Feminino , Perfilação da Expressão Gênica , Humanos , Zigoto/fisiologiaRESUMO
Nuclear/chromosomal integrity is an important prerequisite for the assessment of embryo quality in artificial reproductive technology. However, lipid-rich dark cytoplasm in bovine embryos prevents its observation by visible light microscopy. We performed live-cell imaging using confocal laser microscopy that allowed long-term imaging of nuclear/chromosomal dynamics in bovine in vitro fertilised (IVF) embryos. We analysed the relationship between nuclear/chromosomal aberrations and in vitro embryonic development and morphological blastocyst quality. Three-dimensional live-cell imaging of 369 embryos injected with mRNA encoding histone H2B-mCherry and enhanced green fluorescent protein (EGFP)-α-tubulin was performed from single-cell to blastocyst stage for eight days; 17.9% reached the blastocyst stage. Abnormalities in the number of pronuclei (PN), chromosomal segregation, cytokinesis, and blastomere number at first cleavage were observed at frequencies of 48.0%, 30.6%, 8.1%, and 22.2%, respectively, and 13.0%, 6.2%, 3.3%, and 13.4%, respectively, for abnormal embryos developed into blastocysts. A multivariate analysis showed that abnormal chromosome segregation (ACS) and multiple PN correlated with delayed timing and abnormal blastomere number at first cleavage, respectively. In morphologically transferrable blastocysts, 30-40% of embryos underwent ACS and had abnormal PN. Live-cell imaging may be useful for analysing the association between nuclear/chromosomal dynamics and embryonic development in bovine embryos.
Assuntos
Bovinos/embriologia , Segregação de Cromossomos , Embrião de Mamíferos/citologia , Embrião de Mamíferos/ultraestrutura , Fertilização in vitro , Animais , Bovinos/genética , Sobrevivência Celular , Aberrações Cromossômicas , Embrião de Mamíferos/anormalidades , Embrião de Mamíferos/embriologia , Feminino , Proteínas de Fluorescência Verde/análise , Proteínas Luminescentes/análise , Microscopia Confocal/métodos , Imagem Óptica/métodos , Gravidez , Proteína Vermelha FluorescenteRESUMO
The administration of follicle-stimulating hormone (FSH) prior to oocyte retrieval improves oocyte developmental competence. During bovine embryo production in vitro, however, oocytes are typically derived from FSH-unprimed animals. In the current study, we examined the effect of pre-in vitro maturation (IVM) with cAMP modulators, also known as the second messengers of FSH, on the developmental competence of oocytes derived from small antral follicles (2-4 mm) of FSH-unprimed animals. Pre-IVM with N6,2'-O-dibutyryladenosine 3',5'-cyclicmonophosphate (dbcAMP) and 3-isobutyl-1-methylxanthine (IBMX) for 2 h improved the blastocyst formation in oocytes stimulated by FSH or amphiregulin (AREG). Furthermore, pre-IVM enhanced the expression of the FSH- or AREG-stimulated extracellular matrix-related genes HAS2, TNFAIP6, and PTGS2, and epidermal growth factor (EGF)-like peptide-related genes AREG and EREG. Additionally, pre-IVM with dbcAMP and IBMX enhanced the expression of EGFR, and also increased and prolonged cumulus cell-oocyte gap junctional communication. The improved oocyte development observed using the pre-IVM protocol was ablated by an EGF receptor phosphorylation inhibitor. These results indicate that pre-IVM with cAMP modulators could contribute to the acquisition of developmental competence by bovine oocytes from small antral follicles through the modulation of EGF receptor signaling and oocyte-cumulus/cumulus-cumulus gap junctional communication.
Assuntos
1-Metil-3-Isobutilxantina/farmacologia , Bucladesina/farmacologia , Desenvolvimento Embrionário/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/efeitos dos fármacos , Oogênese/efeitos dos fármacos , Animais , Bovinos , AMP Cíclico/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Feminino , Hormônio Foliculoestimulante , Recuperação de Oócitos , Transdução de Sinais/efeitos dos fármacosRESUMO
Mitochondrial permeability transition pore (PTP) is supposed to at least in part participate in molecular mechanisms underlying the neurotoxicity seen after overactivation of N-methyl-D-aspartate (NMDA) receptor (NMDAR) in neurons. In this study, we have evaluated whether activation of GABAB receptor (GABABR), which is linked to membrane G protein-coupled inwardly-rectifying K+ ion channels (GIRKs), leads to protection of the NMDA-induced neurotoxicity in a manner relevant to mitochondrial membrane depolarization in cultured embryonic mouse cortical neurons. The cationic fluorescent dye 3,3'-dipropylthiacarbocyanine was used for determination of mitochondrial membrane potential. The PTP opener salicylic acid induced a fluorescence increase with a vitality decrease in a manner sensitive to the PTP inhibitor ciclosporin, while ciclosporin alone was effective in significantly preventing both fluorescence increase and viability decrease by NMDA as seen with an NMDAR antagonist. The NMDA-induced fluorescence increase and viability decrease were similarly prevented by pretreatment with the GABABR agonist baclofen, but not by the GABAAR agonist muscimol, in a fashion sensitive to a GABABR antagonist. Moreover, the GIRK inhibitor tertiapin canceled the inhibition by baclofen of the NMDA-induced fluorescence increase. These results suggest that GABABR rather than GABAAR is protective against the NMDA-induced neurotoxicity mediated by mitochondrial PTP through a mechanism relevant to opening of membrane GIRKs in neurons.
Assuntos
Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , N-Metilaspartato/farmacologia , Neurônios/efeitos dos fármacos , Receptores de GABA-B/efeitos dos fármacos , Animais , Cálcio/metabolismo , Células Cultivadas , Canais Iônicos/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Muscimol/farmacologia , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
The advancement of folliculogenesis is coincident with the sequential acquisition of oocyte developmental competence. In practical bovine/porcine ART, cumulus-oocyte complexes (COCs) aspirated from small antral follicles have low developmental competence relative to COCs from medium/large antral follicles, as evidenced by a poor capacity to support embryogenesis up to the blastocyst stage. This is in part because of incomplete differentiation of cumulus cells in small antral follicles, in particular under-developed functionality of EGF signalling. Gonadotrophins and oocyte-secreted paracrine factors cooperate to establish EGF receptor functionality in cumulus cells, which appears to be involved in the acquisition of oocyte developmental competence. Here we review the modification of follicular cumulus cells during antral folliculogenesis involved in oocyte developmental competence.
RESUMO
During antral folliculogenesis, developmental competence of prospective oocytes is regulated in large part by the follicular somatic component to prepare the oocyte for the final stage of maturation and subsequent embryo development. The underlying molecular mechanisms are poorly understood. Oocytes reaching the advanced stage of follicular growth by administration of exogenous follicle-stimulating hormone (FSH) possess higher developmental competence than oocytes in FSH-untreated smaller follicles. In this study, the transcriptomic profile of the cumulus cells from cows receiving FSH administration (FSH-priming) was compared, as a model of high oocyte competence, with that from untreated donor cows (control). Ingenuity Pathway Analysis showed that cumulus cells receiving FSH-priming were rich in down-regulated transcripts associated with cell movement and migration, including the extracellular matrix-related transcripts, probably preventing the disruption of cell-to-cell contacts. Interestingly, the transcriptomic profile of up-regulated genes in the control group was similar to that of granulosa cells from atretic follicles. Interferon regulatory factor 7 was activated as the key upstream regulator of FSH-priming. Thus, acquisition of developmental competence by oocytes can be ensured by the integrity of cumulus cells involved in cell-to-cell communication and cell survival, which may help achieve enhanced oocyte-somatic cell coupling.
Assuntos
Células do Cúmulo/metabolismo , Transcriptoma , Animais , Bovinos , Comunicação Celular/genética , Movimento Celular/genética , Células Cultivadas , Células do Cúmulo/citologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Oócitos/metabolismoRESUMO
Conventionally, in vitro-fertilized (IVF) bovine embryos for transfer are morphologically evaluated at day 7-8 of embryo culture. This method is, however, subjective and results in unreliable selection. We previously described a novel selection system for IVF bovine blastocysts for transfer that traces the development of individual embryos with time-lapse monitoring in our specially developed microwell culture dishes (LinKID micro25). The system can noninvasively identify prognostic factors that reflect viability after transfer. By assessing a combination of identified prognostic factors -timing of the first cleavage; number of blastomeres at the end of the first cleavage; and number of blastomeres at the onset of lag-phase, which results in temporary developmental arrest during the fourth or fifth cell cycle- the pregnancy rate was improved over using conventional morphological evaluation. Time-lapse monitoring with LinKID micro25 could facilitate objective and reliable selection of healthy IVF bovine embryos. Here, we review the novel bovine embryo selection system that allows for prediction of viability after transfer.
Assuntos
Técnicas de Cultura Embrionária/veterinária , Transferência Embrionária/veterinária , Fertilização in vitro/veterinária , Imagem com Lapso de Tempo/veterinária , Animais , Bovinos , Técnicas de Cultura Embrionária/métodos , Transferência Embrionária/métodos , Desenvolvimento Embrionário , Feminino , Fertilização in vitro/métodos , Gravidez , Taxa de Gravidez , Imagem com Lapso de Tempo/métodosRESUMO
Bovine respiratory disease complex (BRDC) is frequently found in cattle worldwide. The etiology of BRDC is complicated by infections with multiple pathogens, making identification of the causal pathogen difficult. Here, we developed a detection system by applying TaqMan real-time PCR (Dembo respiratory-PCR) to screen a broad range of microbes associated with BRDC in a single run. We selected 16 bovine respiratory pathogens (bovine viral diarrhea virus, bovine coronavirus, bovine parainfluenza virus 3, bovine respiratory syncytial virus, influenza D virus, bovine rhinitis A virus, bovine rhinitis B virus, bovine herpesvirus 1, bovine adenovirus 3, bovine adenovirus 7, Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, Trueperella pyogenes, Mycoplasma bovis and Ureaplasma diversum) as detection targets and designed novel specific primer-probe sets for nine of them. The assay performance was assessed using standard curves from synthesized DNA. In addition, the sensitivity of the assay was evaluated by spiking solutions extracted from nasal swabs that were negative by Dembo respiratory-PCR for nucleic acids of pathogens or synthesized DNA. All primer-probe sets showed high sensitivity. In this study, a total of 40 nasal swab samples from cattle on six farms were tested by Dembo respiratory-PCR. Dembo respiratory-PCR can be applied as a screening system with wide detection targets.
Assuntos
Complexo Respiratório Bovino/microbiologia , Complexo Respiratório Bovino/virologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Animais , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
OBJECTIVE: The objectives of the study were to assess the effect of cacao bean husk as bedding material in free-stall barn on the behavior, productivity, and udder health of dairy cattle, and on the ammonia concentrations in the barn. METHODS: Four different stall surfaces (no bedding, cacao bean husk, sawdust, and chopped wheat straw) were each continuously tested for a period of 1 week to determine their effects on nine lactating Holstein cows housed in the free-stall barn with rubber matting. The lying time and the milk yield were measured between d 4 and d 7. Blood samples for plasma cortisol concentration and teat swabs for bacterial counts were obtained prior to morning milking on d 7. The time-averaged gas-phase ammonia concentrations in the barn were measured between d 2 and d 7. RESULTS: The cows spent approximately 2 h more per day lying in the stalls when bedding was available than without bedding. The milk yield increased in the experimental periods when cows had access to bedding materials as compared to the period without bedding. The lying time was positively correlated with the milk yield. Bacterial counts on the teat ends recorded for cows housed on cacao bean husk were significantly lower than those recorded for cows housed without bedding. Ammonia concentration under cacao bean husk bedding decreased by 6%, 15%, and 21% as compared to no bedding, sawdust, and chopped wheat straw, respectively. The cortisol concentration was lowest in the period when cacao bean husk bedding was used. We observed a positive correlation between the ammonia concentrations in the barn and the plasma cortisol concentrations. CONCLUSION: Cacao bean husk is a potential alternative of conventional bedding material, such as sawdust or chopped wheat straw, with beneficial effects on udder health and ammonia concentrations in the barns.
RESUMO
Diarrhea in cattle is one of the most economically costly disorders, decreasing milk production and weight gain. In the present study, we established a novel simultaneous detection system using TaqMan real-time PCR designed as a system for detection of microbes from bovine diarrhea using real-time PCR (referred to as Dembo-PCR). Dembo-PCR simultaneously detects a total of 19 diarrhea-causing pathogens, including viruses, bacteria and protozoa. Specific primer-probe sets were newly designed for 7 pathogens and were synthesized on the basis of previous reports for 12 pathogens. Assays were optimized to react under the same reaction conditions. The PCR efficiency and correlation coefficient (R(2)) of standard curves for each assay were more than 80% and 0.9766, respectively. Furthermore, the sensitivity of Dembo-PCR in fecal sample analysis was measured with feces spiked with target pathogens or synthesized DNA that included specific nucleotide target regions. The resulting limits of detection (LOD) for virus-spiked samples, bacteria and DNA fragments were 0.16-1.6 TCID50 (PFU/reaction), 1.3-13 CFU/reaction and 10-100 copies/reaction, respectively. All reactions showed high sensitivity in pathogen detection. A total of 8 fecal samples, collected from 6 diarrheic cattle, 1 diarrheic calf and 1 healthy cow, were tested using Dembo-PCR to validate the assay's clinical performance. The results revealed that bovine coronavirus had infected all diarrheic adult cattle and that bovine torovirus had infected the diarrheic calf. These results suggest that Dembo-PCR may be a powerful tool for diagnosing infectious agents in cattle diarrhea.
Assuntos
Doenças dos Bovinos/microbiologia , Diarreia/microbiologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Animais , Bovinos , Doenças dos Bovinos/diagnóstico , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/veterinária , Coronavirus Bovino , Diarreia/diagnóstico , Feminino , Torovirus , Infecções por Torovirus/diagnóstico , Infecções por Torovirus/veterináriaRESUMO
Bovine toroviruses (BToVs), belong to the subfamily Toroviridae within the family Coronaviridae, and are pathogens, causing enteric disease in cattle. In Japan, BToVs are distributed throughout the country and cause gastrointestinal infection of calves and cows. In the present study, complete genome sequences of two Japanese BToVs and partial genome sequences of two Japanese BToVs and one porcine torovirus (PToV) from distant regions in Japan were determined and genetic analyses were performed. Pairwise nucleotide comparison and phylogenetic analyses revealed that Japanese BToVs shared high identity with each other and showed high similarities with BToV Breda1 strain in S, M, and HE coding regions. Japanese BToVs showed high similarities with porcine toroviruses in ORF1a, ORF1b, and N coding regions and the 5' and 3' untranslated regions, suggestive of a natural recombination event. Recombination analyses mapped the putative recombinant breakpoints to the 3' ends of the ORF1b and HE regions. These findings suggest that the interspecies recombinant nature of Japanese BToVs resulted in a closer relationship between BToV Breda1 and PToVs.