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1.
PLoS One ; 15(2): e0228732, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32059008

RESUMO

Adipose tissue development begins in utero and is a key target of developmental programming. Here the influence of nutritionally-mediated prenatal growth-restriction on perirenal adipose tissue (PAT) gene expression and adipocyte phenotype in late fetal life was investigated in both sexes in an ovine model. Likewise circulating leptin concentrations and non-esterified fatty acid (NEFA) and glycerol responses to glucose challenge were determined in relation to offspring adiposity at key stages from birth to mid-adult life. In both studies' singleton-bearing adolescent sheep were fed control or high nutrient intakes to induce normal or growth-restricted pregnancies, respectively. Fetal growth-restriction at day 130 of gestation (32% lighter) was characterised by greater body-weight-specific PAT mass and higher PAT expression of peroxisome proliferator-activated receptor gamma (PPARɤ), glycerol-3-phosphate dehydrogenase, hormone sensitive lipase (HSL), insulin-like growth factor 1 receptor, and uncoupling protein 1. Independent of prenatal growth, females had a greater body-weight-specific PAT mass, more multilocular adipocytes, higher leptin and lower insulin-like growth factor 1 mRNA than males. Growth-restricted offspring of both sexes (42% lighter at birth) were characterised by higher plasma NEFA concentrations across the life-course (post-fasting and after glucose challenge at 7, 32, 60, 85 and 106 weeks of age) consistent with reduced adipose tissue insulin sensitivity. Circulating plasma leptin correlated with body fat percentage (females>males) and restricted compared with normal females had more body fat and increased abundance of PPARɤ, HSL, leptin and adiponectin mRNA in PAT at necropsy (109 weeks). Therefore, prenatal nutrient supply and sex both influence adipose tissue development with consequences for lipid metabolism and body composition persisting throughout the life-course.


Assuntos
Tecido Adiposo/citologia , Adiposidade , Metabolismo dos Lipídeos , Fenótipo , Caracteres Sexuais , Ovinos/embriologia , Animais , Feminino , Leptina/sangue , Masculino , Mães
2.
Anim Reprod Sci ; 212: 106237, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31864500

RESUMO

The study aim was to estimate gestational age (GA), expected parturition date (EPD) and growth rate by determining fetal trunk diameter (TD). Effects of fetal-dam pelvis alignment and in utero fetal position at time of ultrasonography (UG) on fetal numbers and sex determination were also studied. Trans-abdominal UG (3-6.5 MHz) was conducted on 37 ewes with known breeding dates from Days 25-120 of pregnancy. Errors in GA and EPD were studied using an equation in the same ewes at their successive breeding when date of breeding was unknown. There were four equations, Y = 1.28861X+32.656 (R2 = 0.92), for Indigenous; Y = 1.2603X+38.075 (R2 = 0.85), for Indigenous × Garole; and Y = 0.8932X+45.916 (R²â€¯= 0.99), for Garole fetuses; and the equation, Y = 1.3565X + 32.604 (R2 = 0.94), independent of breed were computed to estimate GA and the relationship between GA and TD of different breeds. The error in estimated GA and EPD using these four equations was determined and there was comparison with the data collected using US and the previously described equations. Results indicate there was the greatest (P <  0.01) error for GA and EPD values using the US TD equation for all breeds. There was the least error in estimated EPD using the breed specific equations. Error in the sex determination was 4.8 % and fetal number determination was 16.7 % with singleton and 7.7 % twin fetuses. The results indicate there is a breed specific fetal TD that is useful for predicting GA in sheep.


Assuntos
Desenvolvimento Fetal/genética , Idade Gestacional , Ovinos/embriologia , Ovinos/genética , Ultrassonografia Pré-Natal/veterinária , Animais , Feminino , Gravidez
3.
J Anim Sci ; 98(1)2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31875422

RESUMO

Poor maternal nutrition during gestation can have immediate and life-long negative effects on offspring growth and health. In livestock, this leads to reduced product quality and increased costs of production. Based on previous evidence that both restricted- and overfeeding during gestation decrease offspring muscle growth and alter metabolism postnatally, we hypothesized that poor maternal nutrition during gestation would reduce the growth and development of offspring muscle prenatally, reduce the number of myogenic progenitor cells, and result in changes in the global expression of genes involved in prenatal muscle development and function. Ewes were fed a control (100% NRC)-, restricted (60% NRC)-, or overfed (140% NRC) diet beginning on day 30 of gestation until days 45, 90, and 135 of gestation or until parturition. At each time point fetuses and offspring (referred to as CON, RES, and OVER) were euthanized and longissimus dorsi (LM), semitendinosus (STN), and triceps brachii (TB) were collected at each time point for histological and RNA-Seq analysis. In fetuses and offspring, we did not observe an effect of diet on cross-sectional area (CSA), but CSA increased over time (P < 0.05). At day 90, RES and OVER had reduced secondary:primary muscle fiber ratios in LM (P < 0.05), but not in STN and TB. However, in STN and TB percent PAX7-positive cells were decreased compared with CON (P < 0.05). Maternal diet altered LM mRNA expression of 20 genes (7 genes downregulated in OVER and 2 downregulated in RES compared with CON; 5 downregulated in OVER compared with RES; false discovery rate (FDR)-adj. P < 0.05). A diet by time interaction was not observed for any genes in the RNA-Seq analysis; however, 2,205 genes were differentially expressed over time between days 90 and 135 and birth (FDR-adj. P < 0.05). Specifically, consistent with increased protein accretion, changes in muscle function, and increased metabolic activity during myogenesis, changes in genes involved in cell cycle, metabolic processes, and protein synthesis were observed during fetal myogenesis. In conclusion, poor maternal nutrition during gestation contributes to altered offspring muscle growth during early fetal development which persists throughout the fetal stage. Based on muscle-type-specific effects of maternal diet, it is important to evaluate more than one type of muscle to fully elucidate the effects of maternal diet on offspring muscle development.


Assuntos
Fenômenos Fisiológicos da Nutrição Animal , Fenômenos Fisiológicos da Nutrição Materna , Desenvolvimento Muscular , Músculo Esquelético/embriologia , Ovinos/embriologia , Ovinos/fisiologia , Fenômenos Fisiológicos da Nutrição Animal/genética , Animais , Dieta/veterinária , Regulação para Baixo/genética , Feminino , Desenvolvimento Fetal/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Imuno-Histoquímica/veterinária , Masculino , Fenômenos Fisiológicos da Nutrição Materna/genética , Desenvolvimento Muscular/genética , Gravidez , Análise de Sequência de RNA/veterinária , Ovinos/genética , Fatores de Tempo , Regulação para Cima/genética , Vitaminas/administração & dosagem
4.
Anim Reprod Sci ; 209: 106137, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31514927

RESUMO

To evaluate follicular dynamics, there was assessment of superovulatory response and in vivo embryo production in ewes treated with relatively smaller doses of exogenous pFSH than typically used in combination with a dose of eCG at the beginning of the gonadotropin treatment period. Santa Inês ewes (n = 24) were randomly divided into three groups, based on mg dose of pFSH administered: G200 (n = 8), G133 (n = 8) and G100 (n = 8) in eight decreasing doses at 12 -h intervals. All ewes were treated with 300 IU of eCG concomitantly starting with first pFSH administration. Ovulatory follicular dynamics and follicular wall vascularization (FWV) were evaluated using a B-mode and color Doppler ultrasonic machine, respectively. Superovulatory response and embryo production were evaluated 6 days after estrous detection. In the G200 group, the preovulatory follicle size (PFS) were less (P <  0.05), ovulation time later (P <  0.05), and PFS rate greater (P <  0.05); while in the G100 group ovulation rate, and number and percentage of unfertilized eggs were greater (P <  0.05) than in the G133 group (P <  0.05). Number and percentage of viable embryos were greater in the G200 and G100 compared to G133 group (P <  0.05). The dose of 100 mg of FSH was as efficacious as the traditional dose of 200 mg, in combination with a dose of eCG, for superovulatory response and viable embryo production but there was a greater percentage of unfertilized eggs with this treatment.


Assuntos
Hormônio Foliculoestimulante/administração & dosagem , Inseminação Artificial , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/fisiologia , Indução da Ovulação , Ovinos , Animais , Brasil , Tamanho Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Feminino , Inseminação Artificial/métodos , Inseminação Artificial/veterinária , Oogênese/efeitos dos fármacos , Oogênese/fisiologia , Folículo Ovariano/irrigação sanguínea , Ovulação/efeitos dos fármacos , Indução da Ovulação/métodos , Indução da Ovulação/veterinária , Gravidez , Ovinos/embriologia , Superovulação/fisiologia , Clima Tropical
5.
Int J Dev Biol ; 63(3-4-5): 143-155, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31058293

RESUMO

Monozygotic (MZ) polyembryony is a strategy to increase the output of a single zygote, thereby producing more offspring from a limited number of oocytes. However, MZ twins and multiples (multiplets) of mammals occur rarely in nature, while their generation has been more successful experimentally. In this work, we review some of the methodological, biological and field aspects of experimental MZ polyembryony in mammals. First attempts of mechanical bisection of 2-cell stage rodent embryos provided a proof-of-principle for the survival and independent development of both blastomeres. Subsequently, experiments in other species, particularly sheep and bovine, allowed 2 methods of embryo multiplication to become routine: the separation or biopsy of blastomeres from cleavage-stage embryos and the bisection of morulae and blastocysts. We discuss how the preferable stage of bisection and the success rate can be species-specific. The scope that profited most from experimental MZ polyembryony is the production of additional copies of elite livestock individuals, the reduction of interindividual variation in test groups and the possibility of investigating discordant phenotypic traits in the same genomic background, for instance, comparing an affected twin with its healthy co-twin. By contrast, the original motivation for experimental polyembryony - efficiently generating more offspring out of the same zygote - has not been fulfilled yet. Although embryo splitting leads to an increase in quantity, there is a loss of embryo quality, thus, there is no real gain from artificially generated embryos (yet) in the field of medically assisted reproduction. In conclusion, mammalian zygotes have the regulative capacity to be polyembryonic, but this is not obligate.


Assuntos
Blastômeros/citologia , Gêmeos Monozigóticos , Animais , Blastocisto/citologia , Cruzamento , Bovinos , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Desenvolvimento Embrionário , Feminino , Ovinos/embriologia , Zigoto/citologia
6.
Int J Dev Biol ; 63(3-4-5): 187-201, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31058296

RESUMO

The preimplantation development of mammals generally follows the same plan. It starts with the formation of a totipotent zygote, and through consecutive cleavage divisions and differentiation events leads to blastocyst formation. However, the intervening events may differ between species. The regulation of these processes has been extensively studied in the mouse, which displays some unique features among eutherian mammals. Farm animals such as pigs, cattle, sheep and rabbits share several similarities with one another, and with the human developmental plan. These include the timing of epigenetic reprogramming, the moment of embryonic genome activation and the developmental time-frame. Recently, efficient techniques for genetic modification have been established for large domestic animals. Genome sequences and gene manipulation tools are now available for cattle, pigs, sheep and goats, and a larger number of genetically engineered livestock is now accessible for biomedical research. Yet, these animals still make up less than 0.5% of animals in research, mainly due to our inadequate knowledge of the processes responsible for pluripotency maintenance (to date no stable naïve embryonic stem cell lines have been established) and early development. In this review, we highlight our present knowledge of the key preimplantation events in the 3 non-rodent species which present the highest potential for biomedical research related to early embryonic development: cattle, which offer an excellent model to study human in vitro embryo development, pigs which emerge as models to study the long-term effects of gene-based therapies and rabbits, which in many aspects of embryology resemble the human.


Assuntos
Blastocisto/fisiologia , Células-Tronco Pluripotentes/metabolismo , Zigoto/metabolismo , Animais , Pesquisa Biomédica , Bovinos , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Modelos Animais , Partenogênese , Coelhos , Ovinos/embriologia , Transdução de Sinais/fisiologia , Suínos/embriologia
7.
Biosci Biotechnol Biochem ; 83(6): 1045-1061, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30935300

RESUMO

MicroRNAs (miRNAs) regulate the development and growth cycle of hair follicles (HFs). The molecular mechanism by which miRNAs determine the development of HFs in the sheep foetus remains elusive. In this study, the expression profiles of miRNAs at 11 development periods (45, 55, 65, 75, 85, 95, 105, 115, 125, 135 and 145 d) in sheep foetus skin were analysed by high-throughput sequencing and bioinformatics analysis. A total of 72 conserved miRNAs, 44 novel miRNAs and 32 known miRNAs were significantly differentially expressed. qRT-PCR results for 18 miRNAs were consistent with the sequencing data. 85 d of foetal development was the starting point for secondary hair follicle (SF) development according to tissue morphology and cluster analysis. In SF development, the prolactin signalling pathway and platelet activation played important roles, and 10 miRNAs were potential candidate miRNAs in SF initiation.


Assuntos
Desenvolvimento Fetal/genética , Perfilação da Expressão Gênica , Folículo Piloso/crescimento & desenvolvimento , MicroRNAs/genética , Ovinos/embriologia , Animais , Biologia Computacional , Regulação para Baixo , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Ativação Plaquetária , Prolactina/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Análise de Sequência de RNA/métodos , Transdução de Sinais , Regulação para Cima ,
8.
Am J Physiol Endocrinol Metab ; 317(1): E1-E10, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-30964701

RESUMO

Fetal hypoxemia is associated with pregnancy conditions that cause an early activation of fetal glucose production. However, the independent role of hypoxemia to activate this pathway is not well understood. We hypothesized that fetal hypoxemia would activate fetal glucose production by decreasing umbilical glucose uptake and increasing counter-regulatory hormone concentrations. We induced hypoxemia for 9 days with maternal tracheal N2 gas insufflation to reduce maternal and fetal arterial Po2 by ~20% (HOX) compared with fetuses from ewes receiving intratracheal compressed air (CON). At 0.9 of gestation, fetal metabolic studies were performed (n = 7 CON, 11 HOX). Umbilical blood flow rates, net fetal oxygen and glucose uptake rates, and fetal arterial plasma glucose concentrations were not different between the two groups. Fetal glucose utilization rates were lower in HOX versus CON fetuses but not different from umbilical glucose uptake rates, demonstrating the absence of endogenous glucose production. In liver tissue, mRNA expression of gluconeogenic genes G6PC (P < 0.01) and PCK1 (P = 0.06) were six- and threefold greater in HOX fetuses versus CON fetuses. Increased fetal norepinephrine and cortisol concentrations and hepatic G6PC and PCK1 expression were inversely related to fetal arterial Po2. These findings support a role for fetal hypoxemia to act with counter-regulatory hormones to potentiate fetal hepatic gluconeogenic gene expression. However, in the absence of decreased net fetal glucose uptake rates and plasma glucose concentrations, hypoxemia-induced gluconeogenic gene activation is not sufficient to activate fetal glucose production.


Assuntos
Feto/metabolismo , Gluconeogênese/genética , Hipóxia/genética , Hipóxia/metabolismo , Fígado/metabolismo , Complicações na Gravidez , Ovinos , Animais , Embrião de Mamíferos , Feminino , Sangue Fetal/metabolismo , Desenvolvimento Fetal/genética , Regulação da Expressão Gênica no Desenvolvimento , Idade Gestacional , Glucose/metabolismo , Hipóxia/veterinária , Fígado/embriologia , Oxigênio/metabolismo , Gravidez , Complicações na Gravidez/genética , Complicações na Gravidez/metabolismo , Complicações na Gravidez/veterinária , Ovinos/embriologia , Ovinos/genética , Ovinos/metabolismo
9.
Domest Anim Endocrinol ; 68: 25-31, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30784945

RESUMO

The present study aimed to examine the effects of adding different concentrations of resveratrol during in vitro culture (IVC) alone and during both in vitro maturation (IVM) and IVC on ovine blastocyst yield and quality. Therefore, this study was conducted in two separate experiments. The first experiment was carried out to test the effect of different concentrations of resveratrol (0, 0.1, 0.25, 0.5, 2.0, and 5.0 µM) in the IVC medium on cleavage, morula, developmental potential of blastocyst, and total cell number (TCN) of the embryos. Addition of 0.25 and 0.5 µM of resveratrol during IVC significantly enhanced morula and blastocyst rates as compared with other groups (P < 0.05). Also, supplementation of the IVC medium with 0.5 µM of resveratrol had beneficial effects on trophectoderm cells (TE), inner cell mass (ICM), and TCN of blastocysts. In the second experiment, the same concentrations of resveratrol (0, 0.1, 0.25, 0.5, 2.0, and 5.0 µM) were applied during IVM and IVC. Therefore, oocytes were matured in vitro in the presence of different concentrations of resveratrol for 22-24 h. After in vitro fertilization, presumptive zygotes were cultured in media containing 0, 0.1, 0.25, 0.5, 2.0, and 5.0 µM of resveratrol for 8 d. No significant difference was found in the percentage of oocytes developed to MII (0, 0.1, 0.25, 0.5, and 2.0 µM of resveratrol), but the percentage of oocytes developed to MII were significantly lower in 5.0 µM of resveratrol in comparison with other groups. Addition of 0.5 µM of resveratrol to the maturation and culture media significantly increased morula and blastocyst rates compared with other groups (P < 0.05). However, a too high concentration of resveratrol (5.0 µM) during IVM and IVC decreased cleavage, morula, and blastocyst rates compared with low concentrations (P < 0.05). Treatment with 0.5/0.5 µM of resveratrol during IVM/IVC significantly improved the TE, ICM, and TCN of blastocysts. In conclusion, sequential treatment with 0.5 µM of resveratrol during IVM and IVC and during IVC alone improved the developmental competence of oocytes, which was reflected in higher blastocyst rates and TCN of blastocysts.


Assuntos
Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/efeitos dos fármacos , Resveratrol/farmacologia , Ovinos/embriologia , Animais , Meios de Cultura/química , Técnicas de Maturação in Vitro de Oócitos/veterinária , Estrutura Molecular , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Resveratrol/química
10.
Anim Reprod Sci ; 203: 25-32, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30773245

RESUMO

The effects of estradiol esters, d-cloprostenol and oxytocin on induction of cervical dilation prior to non-surgical embryo recovery in Santa Inês ewes (Days 6-7 estrous cycle) were assessed in this study. In Trial 1, transcervical embryo flushing was performed in estrous-induced ewes administered 37.5 µg of d-cloprostenol i.m. 10 h before and 50 IU of oxytocin i.v. 20 min before uterine flushing with (EB-PGF-OT; n = 13) or without (PGF-OT; n = 11) 1 mg of estradiol benzoate i.m. administered concurrently with d-cloprostenol injection. In Trial 2, the estrous-synchronized animals were treated with 1 mg of estradiol benzoate (EB-PGF-OT; n = 12) or estradiol cypionate (EC-PGF-OT; n = 12) i.m. along with 37.5 µg of d-cloprostenol i.m. 16 h before and 50 IU of oxytocin i.v. 20 min before uterine flushing. In Trial 1, uterine flushing could be accomplished in 38% of ewes in the EB-PGF-OT and 27% those in the PGF-OT (P>0.05) group. Flushing fluid recovery averaged 90% and there were 1.0 ± 1.1 embryos/ewe collected with mean duration of the flushing procedure being ˜36 min. In Trial 2, uterine flushing was accomplished in 78% of ewes in the EB-PGF-OT and 44% of those in the EC-PGF-OT group (P>0.05) with mean flushing fluid recovery rate being 88% and time elapsing to complete flushing being ˜33 min. Within the subsets of animals treated with EB, the percentages of successful transcervical penetrations were 38% compared with 78% in Trials 1 and 2, respectively (i.e., with EB administered 10 h compared with 16 h before uterine flushing: P<0.05). The interval from EB administration to the beginning of transcervical penetration can affect the efficacy of embryo recovery procedures utilizing a combined EB/d-cloprostenol/oxytocin pre-treatment.


Assuntos
Colo do Útero/fisiologia , Cloprostenol/farmacologia , Transferência Embrionária/veterinária , Estradiol/análogos & derivados , Sincronização do Estro/efeitos dos fármacos , Ocitocina/farmacologia , Ovinos/embriologia , Animais , Colo do Útero/efeitos dos fármacos , Anticoncepcionais/farmacologia , Combinação de Medicamentos , Transferência Embrionária/métodos , Embrião de Mamíferos , Estradiol/farmacologia , Feminino , Fertilização In Vitro , Luteolíticos/farmacologia , Ocitócicos/farmacologia , Ovinos/fisiologia , Coleta de Tecidos e Órgãos/veterinária
11.
Pediatr Res ; 85(7): 1032-1040, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30739124

RESUMO

BACKGROUND: Approximately 1/3 of newborns exposed antenatally to selective serotonin reuptake inhibitors (SSRIs) exhibit poor neonatal adaptation. Although several potential mechanisms have been proposed, the actual mechanism has not been elucidated. METHODS: We investigated outcomes in neonatal lambs exposed prenatally or postnatally to fluoxetine (FX). Daily FX injections (50 mg) were given intravenously (i.v.) to five pregnant ewes via implanted catheters beginning at 131-132 days gestation (term = 147 days) for 2 weeks. In another group, lambs with implanted vascular catheters had sterile water (n = 9) or FX (1 mg/kg, n = 12) injected i.v. on ~postnatal day (PND) 4. RESULTS: Prenatal FX-exposed lambs (n = 7) were hyperactive during PND 4 to 14 and their heart rate variability (HRV) was significantly lower than in control lambs (n = 7) on PND 2. In contrast, arterial pressure, heart rate, electrocardiogram, arterial blood gases, pH, glucose, lactate, cortisol, and sleep-activity cycles were not altered following postnatal FX injection. CONCLUSION: This abnormal postnatal hyperactivity with antenatal FX exposure may reflect increased maturity in terms of locomotory activity. The results suggest that altered brain development may be involved in the poor neonatal adaptation in human infants exposed to FX in utero.


Assuntos
Fluoxetina/toxicidade , Inibidores de Captação de Serotonina/toxicidade , Ovinos/embriologia , Animais , Animais Recém-Nascidos , Feminino , Fluoxetina/administração & dosagem , Frequência Cardíaca/efeitos dos fármacos , Exposição Materna , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Inibidores de Captação de Serotonina/administração & dosagem
12.
Reprod Domest Anim ; 54(3): 456-463, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30421465

RESUMO

The aim was to compare the early luteal development in ewes superovulated with different doses of pFSH. Twenty-nine Santa Inês ewes received a progesterone device (CIDR®) for 8 days. Gonadotrophic treatment started on Day 6: G200 (control, n = 9, 200 mg); G133 (n = 10, 133 mg); and G100 (n = 10, 100 mg of pFSH). On Day 6, all females received eCG (300 IU). B-mode and spectral Doppler ultrasonography were performed daily during the early luteal phase (Days 11-15) to monitor the development of corpora lutea (CLs; dimensions) and ovarian arteries indices. CLs were also classified as normal or prematurely regressed (PRCL) on Day 15 by videolaparoscopy. Ewes from G100 and G133 showed gradual increase in luteal diameter during the early luteal phase (p < 0.001), whereas G200 animals presented increase from Day 11 to Day 13, and then decrease on Days 14 and 15 (p < 0.001). The G200 females showed greater percentage of PRCL (45.20%) than those of the other groups (p < 0.001). The normal CLs number was greater in G100 than in G133 (p = 0.04), while the PRCL number was greater in G200 than in the other groups (p = 0.03). Resistive index (RI) was greater in G200 than in G100 (p = 0.02). RI was lower in Day 12 than Day 15 (p = 0.02). Pulsatility index (PI) was greater on Days 14 and 15 (p < 0.01). In conclusion, the lowest dose of pFSH (100 mg) can be considered sufficient for an efficient superovulatory response in sheep, producing better CLs development dynamic in early luteal phase and ovarian blood perfusion and smaller number of PRCL than the traditional (200 mg) pFSH dose.


Assuntos
Hormônio Foliculoestimulante/farmacologia , Progesterona/sangue , Ovinos/embriologia , Superovulação/fisiologia , Animais , Corpo Lúteo , Feminino , Luteólise , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/fisiologia , Ovário/diagnóstico por imagem , Ovário/fisiologia , Superovulação/efeitos dos fármacos , Ultrassonografia
13.
Am J Physiol Regul Integr Comp Physiol ; 317(6): R780-R792, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-29351431

RESUMO

Phase-contrast cine MRI (PC-MRI) is the gold-standard noninvasive technique for measuring vessel blood flow and has previously been applied in the human fetal circulation. We aimed to assess the feasibility of using PC-MRI to define the distribution of the fetal circulation in sheep. Fetuses were catheterized at 119-120 days of gestation (term, 150 days) and underwent MRI at ∼123 days of gestation under isoflurane anesthesia, ventilated at a FIO2 of 1.0. PC-MRI was performed using a fetal arterial blood pressure catheter signal for cardiac triggering. Blood flows were measured in the major fetal vessels, including the main pulmonary artery, ascending and descending aorta, superior vena cava, ductus arteriosus, left and right pulmonary arteries, umbilical vein, ductus venosus, and common carotid artery and were indexed to estimated fetal weight. The combined ventricular output, pulmonary blood flow, and flow across the foramen ovale were calculated from vessel flows. Intraobserver and interobserver agreement and reproducibility was assessed. Blood flow measurements were successfully obtained in 61 out of 74 vessels (82.4%) interrogated in 9 fetuses. There was good intraobserver [R = 0.998, P < 0.0001; intraclass correlation (ICC) = 0.997] and interobserver agreement (R = 0.996, P < 0.0001; ICC = 0.996). Repeated MRI measurements showed good reproducibility (R = 0.989, P = 0.0002; ICC = 0.990). We conclude that PC-MRI using fetal catheters for gating triggers is feasible in the major vessels of late gestation fetal sheep. This approach may provide a useful new tool for assessing the circulatory characteristics of fetal sheep models of human disease, including fetal growth restriction and congenital heart disease.


Assuntos
Feto/fisiologia , Imagem Cinética por Ressonância Magnética/veterinária , Ovinos/embriologia , Animais , Velocidade do Fluxo Sanguíneo , Estudos de Viabilidade , Feminino , Feto/irrigação sanguínea , Idade Gestacional , Hemodinâmica , Variações Dependentes do Observador , Gravidez , Diagnóstico Pré-Natal/métodos , Reprodutibilidade dos Testes
14.
J Pediatr Surg ; 53(12): 2502-2506, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30503249

RESUMO

BACKGROUND: The kidney develops from an intimate interaction between the ureteric bud and the metanephric mass. We attempted to differentially stain the derivatives of the ureteric bud and the metanephric mass in ovine fetuses. METHODS: After appropriate approval, 47 fetal lambs' kidneys at 50 (4), 60 (6), 70 (5), 80 (4), 100 (10), 110 (8), 145 (10) days' gestation (term is 140-145 days) were obtained. After confirming the pregnancy, the sheep were anesthetized, and the fetuses sacrificed. The fetal kidneys were prepared for histological examination, using immunostaining for ß-catenin, Laminin, CK34ßE12, CK7, E-cadherin, and EMA. RESULTS: In the nephrogenic zone, positive staining was only seen for ß-catenin and Laminin. Areas with linear ß-catenin expression increased with increasing gestational age, whereas cytoplasmic granular expression in the nephrogenic zone diminished. At 50 days, Laminin-positive cells appeared in the ureteric bud epithelial cells, but not in the proximal tubule epithelium. They were found only in the immature collecting duct at 60 days. CONCLUSION: We have shown that the distribution of ß-catenin and Laminin positive-stained cells initially appearing in the ureteric bud changes with gestational age. Further studies may help inform the optimal timing of fetal shunt insertion in obstructive uropathy.


Assuntos
Rim/embriologia , Ovinos/embriologia , Ureter/embriologia , Animais , Caderinas/metabolismo , Feminino , Feto/embriologia , Imuno-Histoquímica , Queratina-7/metabolismo , Queratinas/metabolismo , Rim/metabolismo , Laminina/metabolismo , Mucina-1/metabolismo , Gravidez , Ureter/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo
15.
Vet Immunol Immunopathol ; 204: 59-64, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30290960

RESUMO

Recent next generation sequencing studies on host-associated microbiomes generated debatable conclusions regarding the central dogma of fetal gut sterility. These observations challenge the concepts that microbial colonization of the gut begins during and after birth as well as the concept of antigen-independent prenatal maturation of mucosal-associated lymphoid tissue in ruminants and humans. The placental barrier varies markedly among mammalian species with mice and humans having haemochorial placentas (fetal tissue in direct contact with maternal blood) versus epitheliochorial placentation (maternal and fetal blood separated by six tissue layers) in ruminants. Therefore, this study re-examined the question of fetal gut sterility using the fetal lamb as a model ruminant species with the most complete placental barrier. Use of PCR and quantitative real-time PCR with three different pairs of universal bacterial primers (27 F and 1492R, HDA1 and HDA2, U2F and U2R) to amplify 16S rRNA gene did not generate detectable PCR products from samples collected from the fetal environment (placenta, amniotic fluid) and fetal intestine during the third trimester of pregnancy. Procedures to further enrich microbial DNA from total extracted DNA also resulted in no detectable genomic DNA. Moreover, use of 16S amplicon sequencing confirmed the absence of bacteria in the fetal environment during the third trimester of pregnancy. A 'No Template' control containing only PCR reagents generated sequences that could be clustered into OTUs at 97% similarity and assigned to bacterial genera, including Staphylococcus, Lactobacillus and Escherichia-Shigella. Use of multiple molecular-based approaches to profile fetal environment-associated microbiota supports the conclusion that the fetal environment and fetal intestine remain sterile during the third trimester of pregnancy. The use of appropriate controls, both positive and no template, revealed inherent contamination in reagents and that variations in the data analysis pipeline can produce artificial microbial profiles from host tissues containing low microbial biomass. Finally, these findings confirm that extensive development of gut-associated lymphoid tissue in the ruminant fetal intestine, characterized by active B cell proliferation and immunoglobulin V gene somatic mutation, is not associated with exposure to bacterial DNA and antigens.


Assuntos
Feto/microbiologia , Microbioma Gastrointestinal , Intestinos/embriologia , Líquido Amniótico/microbiologia , Animais , DNA Bacteriano/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/veterinária , Intestinos/microbiologia , Placenta/microbiologia , Gravidez , RNA Ribossômico 16S/genética , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Ovinos/embriologia , Ovinos/microbiologia
16.
Anim Reprod Sci ; 196: 205-210, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30107934

RESUMO

The aim of the present study was to evaluate the effect of serum progesterone concentrations during the superstimulatory treatment of the first follicular wave on fertilization rate and embryo development in sheep. A total of 71 Merino ewes received a superstimulatory FSH treatment during Wave 1 of ovarian follicular development (Day 0 Protocol), which was administrated under low progesterone concentrations typical of the early luteal phase (control group, n = 33) or under high progesterone concentrations induced by the administration of an intravaginal device from Day 0 to Day 3 containing 0.3 g progesterone (n = 38). Intrauterine insemination after FSH superstimulation was followed by uterine flushing 6 days later. Serum progesterone concentrations from Day 0 to 3 were greater in those ewes treated with progesterone (P < 0.05), while serum estradiol-17ß concentrations were not affected by the treatment. Although the mean number of corpora lutea per donor was not affected by the progesterone treatment, the number of collected ova and embryos was greater in progesterone treated than untreated ewes (6.6 ± 0.7 compared with 4.6 ± 0.9, respectively; P < 0.05). Furthermore, progesterone treatment increased fertilization rate (93.3% compared with 83.3%; P < 0.05) and the proportion of Grade 1 embryos (67.7% compared with 52.7%; P < 0.05) compared with the control group. In conclusion, oocyte fertilization rate and embryo quality are improved by high progesterone concentrations during FSH superstimulation, which suggests an important role of progesterone during preovulatory follicular development.


Assuntos
Hormônio Foliculoestimulante/farmacologia , Progesterona/sangue , Ovinos/embriologia , Superovulação/fisiologia , Animais , Corpo Lúteo , Estradiol , Feminino , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/fisiologia , Superovulação/efeitos dos fármacos
17.
Pediatr Res ; 84(3): 348-351, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30013152

RESUMO

BACKGROUND: Circulatory changes during gestational development of the human fetus have been considered to be similar to those noted in studies of the lamb fetus. METHODS: Blood flow measurements derived by Doppler ultrasound and magnetic resonance imaging techniques in human fetuses at various stages of gestation have been compared with those in the lamb. RESULTS: Combined ventricular output relative to fetal body weight does not change significantly with growth in the lamb or human. However, the proportion of cardiac output to the brain increases markedly in the human, but only slightly in the lamb fetus in the latter half of gestation. Cardiac output distribution to other organs also changes little in the lamb, but in the human, there is a marked decrease in the proportion distributed to the placenta and an increase in pulmonary flow. CONCLUSION: The developmental changes in the distribution of combined ventricular output in the human fetus may modify the responses to circulatory disturbances, such as congenital cardiovascular malformations, dependent on gestation.


Assuntos
Débito Cardíaco , Circulação Cerebrovascular , Feto/irrigação sanguínea , Placenta/irrigação sanguínea , Fluxo Sanguíneo Regional , Animais , Peso Corporal , Encéfalo/irrigação sanguínea , Encéfalo/diagnóstico por imagem , Feminino , Feto/fisiologia , Idade Gestacional , Ventrículos do Coração , Humanos , Placenta/diagnóstico por imagem , Gravidez , Prenhez , Ovinos/embriologia , Veias Umbilicais
18.
Int J Mol Med ; 42(1): 525-533, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29693133

RESUMO

Cartilage stem/progenitor cells (CSPCs) are a novel stem cell population and function as promising therapeutic candidates for cell­based cartilage repair. Until now, numerous existing research materials have been obtained from humans, horses, cows and other mammals, but rarely from sheep. In the present study, CSPCs with potential applications in repairing tissue damage and cell­based therapy were isolated from 45­day­old Small­tailed Han Sheep embryos, and examined at the cellular and molecular level. The expression level of characteristic surface markers of the fetal sheep CSPCs were also evaluated by immunofluorescence, reverse transcription­polymerase chain reaction analysis and flow cytometric assays. Biological growth curves were drawn in accordance with cell numbers. Additionally, karyotype analysis showed no marked differences in the in vitro cultured CSPCs and they were genetically stable among different passages. The CSPCs were also capable of adipogenic, osteogenic and chondrogenic lineage progression under the appropriate induction medium in vitro. Together, these findings provide a theoretical basis and experimental evidence for cellular transplant therapy in tissue engineering.


Assuntos
Cartilagem/citologia , Separação Celular/métodos , Embrião de Mamíferos/citologia , Ovinos/embriologia , Células-Tronco/citologia , Adipogenia , Animais , Biomarcadores/metabolismo , Proliferação de Células , Forma Celular , Células Cultivadas , Condrogênese , Feminino , Citometria de Fluxo , Cariótipo , Masculino , Células-Tronco/metabolismo
19.
Zygote ; 26(2): 149-161, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29607799

RESUMO

SummaryThe objective of the study was to investigate the effect of l-ergothioneine (l-erg) (5 mM or 10 mM) supplementation in maturation medium on the developmental potential and OCTN1-dependant l-erg-mediated (10 mM) change in mRNA abundance of apoptotic (Bcl2, Bax, Casp3 and PCNA) and antioxidant (GPx, SOD1, SOD2 and CAT) genes in sheep oocytes and developmental stages of embryos produced in vitro. Oocytes matured with l-erg (10 mM) reduced their embryo toxicity by decreasing intracellular ROS and increasing intracellular GSH in matured oocytes that in turn improved developmental potential, resulting in significantly (P < 0.05) higher percentages of cleavage (53.72% vs 38.86, 46.56%), morulae (34.36% vs 20.62, 25.84%) and blastocysts (14.83% vs 6.98, 9.26%) compared with other lower concentrations (0 mM and 5 mM) of l-erg without change in maturation rate. l-Erg (10 mM) treatment did not influence the mRNA abundance of the majority of apoptotic and antioxidant genes studied in the matured oocytes and developmental stages of embryo. A gene expression study found that the SLC22A4 gene that encodes OCTN1, an integral membrane protein and specific transporter of l-erg was not expressed in oocytes and developmental stages of embryos. Therefore it was concluded from the study that although there was improvement in the developmental potential of sheep embryos by l-erg supplementation in maturation medium, there was no change in the expression of the majority of the genes studied due to the absence of the SLC22A4 gene in oocytes and embryos that encode OCTN1, which is responsible for transportation of l-erg across the membrane to alter gene expression.


Assuntos
Blastocisto/efeitos dos fármacos , Ergotioneína/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Ovinos/embriologia , Animais , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Blastocisto/citologia , Meios de Cultura/química , Meios de Cultura/farmacologia , Técnicas de Cultura Embrionária , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Feminino , Glutationa/metabolismo , Técnicas de Maturação in Vitro de Oócitos/métodos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Masculino , Mórula , Oócitos/fisiologia , Proteínas de Transporte de Cátions Orgânicos/genética , Testículo/efeitos dos fármacos
20.
Reprod Domest Anim ; 53(4): 895-903, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29638025

RESUMO

Early embryonic mortality is one of the main sources of reproductive loss in domestic ruminants including sheep. Fibroblast growth factor-2 (FGF-2) is a member of FGFs family that mediates trophoblast activities and regulates embryonic development in various species. In this study, we have cloned, characterized sheep FGF2 cDNA (KU316368) and studied the expression in sheep embryos. Ovaries of non-pregnant sheep were collected from local abattoir and matured in culture medium at 38.5ºC, 5% CO2 , 95% humidity for 22-24 hr. The matured oocytes were inseminated with capacitated spermatozoa in Brackett and Oliphant medium and resulted embryos were cultured in CO2 incubator for 6-7 days to complete the developmental stages from two cells to blastocyst stage. Total RNA was extracted from immature oocytes (n = 100), mature oocytes (n = 100) and different stages of embryos such as 2 cell (n = 50), 4 cell (n = 25), 8 cell (n = 12), 16 cell (n = 6), morula (n = 5) and blastocyst (n = 3). The total RNA isolated from the oocytes and embryos was reverse transcribed and subjected to real-time polymerase chain reaction using sequence-specific primers and SYBR green as the DNA dye. On sequence analysis, the nucleotide sequence of sheep FGF2 exhibited highest sequence similarity with cattle (100%) and least with rat and mouse (69.2%). At the deduced amino acid level, a highest degree of similarity was noticed with cattle, buffalo, goat, pig, camel and horse (100%) and lowest degree of identity with rat, human and mouse (98.2%). The FGF2 mRNA expression was higher in immature and mature oocytes and gradually decreases from 2-cell stage of embryo to the blastocyst stage. More over a significant differences in FGF2 mRNA expression (p < .05) were observed between immature oocytes and all pre-implantation stages of embryo. It can be concluded that FGF-2 plays a significant role in pre-implantation and early development of embryos in sheep.


Assuntos
Clonagem Molecular , Embrião de Mamíferos/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Ovinos/embriologia , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar , Técnicas de Cultura Embrionária/veterinária , Fator 2 de Crescimento de Fibroblastos/genética , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
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