RESUMO
BACKGROUND: The proteomic profile of cryopreserved in vitro produced bovine embryos is little known but can provide insights on the successful application of cryo procedures in support of animal breeding. OBJECTIVE: To identify embryonic proteins and biomarkers related to improved cryotolerance of vitrified in vitro produced bovine embryos. MATERIALS AND METHODS: Proteins were isolated from embryo pools (n = 25 embryos per replicate) and analyzed using the nanoLC - MS/MS system. Further, the UniProtKB database (Uniprot -http://www.uniprot.org/) was used for protein identification. Proteins were classified based on their molecular mass, isoelectric point, and enzymatic activity. Post-translational modification predictions and functional gene ontology analysis were performed as well. Finally, a protein-protein interaction network was created to shed light on the embryo interactome. RESULTS: Based on the MS/MS approach, 66 proteins were identified from vitrified Bos taurus embryos. The retrieved proteins were presumably annotated, which allowed a description of the qualitative and functional aspects of the embryo proteome after the vitrification process. CONCLUSION: These findings allowed us to conclude that in vitro-produced vitrified embryos expressed proteins that underlie biological processes related to reproduction, stress and lipid metabolic process, which are essential to maintain embryo viability. doi.org/10.54680/fr22410110512.
Assuntos
Criopreservação , Fertilização in vitro , Bovinos , Animais , Fertilização in vitro/veterinária , Criopreservação/veterinária , Criopreservação/métodos , Espectrometria de Massas em Tandem , Proteômica , Vitrificação , Blastocisto , Técnicas de Cultura Embrionária/veterinária , Técnicas de Cultura Embrionária/métodosRESUMO
In vitro embryo production methods induce DNA damage in the embryos. In response to these injuries, histone H2AX is phosphorylated (γH2AX) and forms foci at the sites of DNA breaks to recruit repair proteins. In this work, we quantified the DNA damage in bovine embryos undergoing parthenogenetic activation (PA), in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT) by measuring γH2AX accumulation at different developmental stages: 1-cell, 2-cell and blastocyst. At the 1-cell stage, IVF embryos exhibited a greater number of γH2AX foci (606.1 ± 103.2) and greater area of γH2AX staining (12923.6 ± 3214.1) than did PA and SCNT embryos. No differences at the 2-cell stage were observed among embryo types. Although PA, IVF and SCNT were associated with different blastocyst formation rates (31.1%, 19.7% and 8.3%, P < 0.05), no differences in the number of γH2AX foci or area were detected among the treatments. γH2AX is detected in bovine preimplantation embryos produced by PA, IVF and SCNT; the amount of DNA damage was comparable among those embryos developing to the blastocyst stage among different methods for in vitro embryo production. While IVF resulted in increased damage at the 1-cell embryo stage, no difference was observed between PA and SCNT embryos at any developmental stage. The decrease in the number of double-stranded breaks at the blastocyst stage seems to indicate that DNA repair mechanisms are functional during embryo development.
Assuntos
Blastocisto/fisiologia , Clonagem de Organismos , Histonas/metabolismo , Partenogênese , Fosfoproteínas/metabolismo , Animais , Bovinos , Cromatina/metabolismo , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Embrião de Mamíferos/metabolismo , Feminino , Fertilização in vitro , Masculino , Técnicas de Transferência NuclearRESUMO
The present study aimed to compare laparoscopic (LP) and ultrasound-guided (US) biopsy methods to obtain either liver or splenic tissue samples for ectopic gene expression analysis in transgenic goats. Tissue samples were collected from human granulocyte colony stimulating factor (hG-CSF)-transgenic bucks and submitted to real-time PCR for the endogenous genes (Sp1, Baff, and Gapdh) and the transgene (hG-CSF). Both LP and US biopsy methods were successful in obtaining liver and splenic samples that could be analyzed by PCR (i.e., sufficient sample sizes and RNA yield were obtained). Although the number of attempts made to obtain the tissue samples was similar (P > 0.05), LP procedures took considerably longer than the US method (P = 0.03). Finally, transgene transcripts were not detected in spleen or liver samples. Thus, for the phenotypic characterization of a transgenic goat line, investigation of ectopic gene expression can be made successfully by LP or US biopsy, avoiding the traditional approach of euthanasia.
Assuntos
Animais Geneticamente Modificados/genética , Perfilação da Expressão Gênica , Cabras/genética , Animais , Animais Geneticamente Modificados/metabolismo , Cabras/metabolismo , Fator Estimulador de Colônias de Granulócitos/genética , Humanos , Biópsia Guiada por Imagem , Laparoscopia , Fígado/diagnóstico por imagem , Fígado/metabolismo , Masculino , Reação em Cadeia da Polimerase em Tempo Real , Baço/diagnóstico por imagem , Baço/metabolismo , Transcriptoma , UltrassonografiaRESUMO
This study aims to define the best method (slow freezing or vitrification) and fragment size (1, 5, or 9 mm³) for prepubertal goat testis cryopreservation, as well as to evaluate testicular morphological integrity after cryopreservation and in vitro culture (IVC). Initially (experiment I), 1, 5, or 9 mm³ testis fragments were cryopreserved by slow freezing using a Mr. Frosty container with 20% Dimethylsulfoxide (DMSO) or vitrified using the Ovarian Tissue Cryosystem (OTC) device, (Equilibration solution - ES: 10% DMSO and 10% ethylene glycol - EG; Vitrification solution - VS: 20% DMSO and 20% EG) and then subjected to morphological analysis, type I and III collagen quantification and gene expression (Oct4, C-kit, Bax, and Bcl-2). Subsequently, (experiment II), fresh or cryopreserved by slow freezing testis fragments were cultured in vitro and submitted to morphological analysis by scanning electron microscopy. The data from the experiment I revealed fewer morphological alterations in 1 and 5 mm³ fragments after vitrification and slow freezing, respectively. The percentage of type I collagen fibers in 5 and 9 mm³ frozen was higher than in fresh or vitrified fragments. For type III collagen, fresh or frozen fragments of 1 and 5 mm3 showed a higher percentage than fragments of 9 mm3. Gene expression for Oct4 and C-kit after slow freezing or vitrification in the 5 mm3 fragments was lower than that observed in the fresh fragments. The Bax:Bcl-2 ratio in the 1 and 9 mm³ fragments was lower than in the 5 mm³ fragments for fresh fragments or after freezing. In experiment II, fragments cultured in vitro, previously frozen or not, showed more morphological alterations than fresh or frozen fragments. We concluded that slow freezing of 5 mm³ fragments was the best protocol for cryopreserving prepubertal goat testis and although the results of IVC are encouraging, it still needs improvement to restore testicular function after cryopreservation.
Assuntos
Dimetil Sulfóxido , Cabras , Animais , Masculino , Proteína X Associada a bcl-2 , Criopreservação/veterinária , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas c-kitRESUMO
The CD44 family belongs to a larger group of hyaluronic acid-binding proteins and plays important roles in oocyte maturation, fertilization and preimplantational embryo development. We analyzed the CD44 receptor in sheep oocytes and embryos. Immature oocytes (N = 66) were obtained from a local abattoir; mature oocytes (N = 35) and embryos (N = 41) were obtained by laparotomy from adult hair ewes submitted to ovarian stimulation treatment. The CD44 mRNA was detected by hemi-nested PCR, after reverse transcription, while proteins were located by indirect immunofluorescence, using anti-human CD44 monoclonal antibody. Human lymphocytes and immature bovine oocytes were used as positive and negative controls, respectively. Assessment of the oocyte nuclear stages as well as classification of the embryonic development stage were made with Hoechst 33342 staining. Indirect immunofluorescence detected CD44 expression on the surface of mature oocytes and embryos; immature oocytes did not take up the stain. These findings were supported by the RT-PCR data, which showed no mRNA templates for CD44, even after two consecutive amplifications, in material from immature oocytes and cumulus cells. The CD44 amplicons were detected after a second hemi-nested PCR in mature oocytes and embryos. The finding of CD44 in mature oocytes and preimplantational embryos could reflect the expression profile of hyaluronic acid during terminal folliculogenesis and preimplantational embryo development in sheep.
Assuntos
Blastocisto , Receptores de Hialuronatos/imunologia , Oócitos/imunologia , Animais , Anticorpos Monoclonais/imunologia , Sequência de Bases , Primers do DNA , Feminino , Receptores de Hialuronatos/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , OvinosRESUMO
Hormonal ovarian stimulation may affect the success of embryo production by regulating transcripts in recovered cumulus-oocyte complexes (COCs). Here, in parallel to morphological classification and in vitro maturation (IVM) rate analysis, we investigated the expression of epidermal growth factor (EGF) and its receptor (EGFR) in oocytes and cumulus cells from goat COCs recovered by laparoscopy after standard [multi-dose follicle-stimulating hormone (FSH)] or one-shot (single dose FSH plus eCG) treatments. No differences were observed among the number of recovered and morphologically graded COCs or the IVM rates for both gonadotropic treatments. However, the standard protocol produced COCs with higher EGFR expression in the cumulus cells than the one-shot treatment. Additionally, EGF mRNA levels were less than EGFR mRNA levels, and they did not differ among COCs from both treatments. However, during maturation, the EGF transcripts increased in oocytes derived only from the standard protocol. Interestingly, IVM strikingly increased EGFR expression in oocytes and cumulus cells but not in oocytes that fail in first polar body extrusion, irrespective of hormonal treatment. These results appear to be related to the resumption of meiosis and suggest that EGF may act through the cumulus cells or directly on the oocyte receptor.
Assuntos
Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Hormônio Foliculoestimulante/administração & dosagem , Hormônio Foliculoestimulante/farmacologia , Cabras , Oócitos/fisiologia , Animais , Gonadotropina Coriônica/administração & dosagem , Gonadotropina Coriônica/farmacologia , Fator de Crescimento Epidérmico/genética , Receptores ErbB/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hormônios/administração & dosagem , Hormônios/farmacologia , LigantesRESUMO
Ovarian stimulation with exogenous follicle stimulating hormone (FSH) has been used to increase the number of viable oocytes for laparoscopic oocyte recovery (LOR) in goats. The aim of this study was to evaluate the effect of two FSH protocols for ovarian stimulation in goats on the expression pattern of epidermal growth factor (EGF) receptor (EGFR) in cumulus-oocyte complexes (COCs) recovered by LOR. After real-time qRT-PCR analysis, expression profiles of morphologically graded COCs were compared prior to and after in vitro maturation (IVM) on a FSH protocol basis. The use of a protocol with higher number of FSH injections at a shorter interval resulted in GI/GII COCs with a higher level of EGFR expression in cumulus cells, but not in the oocyte, which was correlated with an elevated meiotic competence following IVM. Based on the maturation profile and EGFR expression patterns observed between groups, the morphological selection of COCs prior to IVM was not a good predictor of oocyte meiotic competence. Therefore, EGFR may be a good candidate marker for indirect prediction of goat oocyte quality. The IVM process of goat COCs increased the EGFR expression in oocytes and cumulus cells, which seemed to be strongly associated with the resumption of meiosis. In summary, differential EGFR expression in goat cumulus cells was associated with the in vivo prematuration process, and in turn, the upregulation in the entire COC was associated with IVM. Cause-and-effect relationships between such increased expression levels, particularly in the oocyte, and oocyte competence itself still need to be further investigated.
Assuntos
Células do Cúmulo/metabolismo , Receptores ErbB/genética , Oócitos/metabolismo , Animais , Receptores ErbB/metabolismo , Feminino , Hormônio Foliculoestimulante/metabolismo , Cabras , Laparoscopia , Recuperação de Oócitos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
The present study determined i) the presence of proteins (oviduct-specific glycoprotein, OVGP1; heat shock protein-70A, HSPA1A; heat shock protein-A8, HSPA8; annexin A1, ANXA1; annexin A5, ANXA5; and myosin-9, MYH9) known to be involved in early reproduction in the oviduct fluid (OF) of anestrous goats; and ii) the functional effect of during IVF on polyspermy modulation and embryonic development. In vitro-matured oocytes were co-cultured with spermatozoa (1.0, 2.0, or 4.0 x 106 cells/mL) for 18 h in SOF medium supplemented with 5 µg/mL of heparin, 4 µg/mL gentamicin, and 10% estrus sheep serum (CTRL1, CTRL2, and CTRL4 groups) or the same medium plus 10% OF (OF1, OF2, and OF4 groups) obtained from anestrus goats. The analysis of OF by western blotting confirmed the presence of the six proteins tested for. The increase in sperm concentration had no effect (P > 0.05) on the penetration rate in any group; however, monospermy rate decreased as sperm concentration was increased in both OF and CTRL. Regardless of the concentration used, when data were pooled, OF supplementation improved (P < 0.05) monospermy and tended (P = 0.057) to enhance IVF efficiency. Additionally, IVF efficiency was higher (P < 0.05) in OF1 than in OF4 [60 ± 13 vs 37 ± 5%). The development capacity was not affected (P > 0.05) by the sperm concentration and OF treatment, and the average values were cleavage (72 ± 2.6%), blastocyst (37 ± 3.0%), blastocyst in relation to the cleaved (51 ± 4.8%), hatched (62 ± 1.2%), and number of cells per blastocyst (174 ± 1.8%). In conclusion, the six proteins analyzed are present in the OF of anestrous goats, and the supplementation of this OF during IVF may modulate the polyspermy incidence and enhance IVF efficiency, especially when 1x106 sperm per mL is used.
Assuntos
Fertilização in vitro , Cabras , Animais , Blastocisto , Feminino , Fertilização in vitro/veterinária , Masculino , Oócitos , Oviductos , Gravidez , Estações do Ano , Ovinos , EspermatozoidesRESUMO
The objective of this study was to examine the effect of donor breed on pronuclear-stage embryo yield to be used for DNA microinjection in a transgenesis goat program. Twelve Canindé and twelve Saanen goats were heat synchronized using a progestagen-cloprostenol treatment. Forty-eight hours before the sponge removal, superovulation was induced with a total administration of 4.4 mg/kg bodyweight NIH-FSH-P1, given twice daily in decreasing doses over 3 days. In addition, goats received 100 µg of GnRH and they were hand-mated at 36 and 48 h after progestagen removal. Embryo recovery was performed by oviduct flushing at 72 h after sponge removal. Embryos were microinjected with a DNA construct and noticeable swelling of the nuclei was the criterion for successful microinjection. The total diameter, cytoplasm diameter, zona pellucida thickness and pronuclei diameter were measured for each microinjected embryo. A higher (p < 0.05) percentage of fertilized ova was observed in Canindé (89.9%) than Saanen (36.2%) goats. In addition, Canindé donors produced a higher percentage of pronuclear embryos when compared with Saanen: 72.5% vs 20.6% (p < 0.05), respectively. Successful microinjection was verified in 96.7% and 73.3% of times in Canindé and Saanen embryos, respectively (p < 0.05). Significant differences were observed for all morphometric parameters except for cytoplasm diameter. In conclusion, under our study experimental conditions, Canindé were more efficient than Saanen goats concerning the pronuclear embryo yield and manipulation. The use of Canindé goats in transgenesis programs could be increase the interest in their breeding and could be contribute to saving them from extinction.
Assuntos
DNA/genética , Cabras/embriologia , Cabras/genética , Microinjeções/veterinária , Zigoto/fisiologia , Animais , Animais Geneticamente Modificados , Sincronização do Estro , Feminino , Fármacos para a Fertilidade Feminina/administração & dosagem , Fármacos para a Fertilidade Feminina/farmacologia , Superovulação , Zigoto/efeitos dos fármacosRESUMO
Low purification efficiency and incomplete characterization of male goat (buck) spermadhesins (Bdhs) prompted us to develop an effective system to produce recombinant Bdhs (rBdhs). Bdh-2 cDNA was inserted into a prokaryotic expression plasmid, pTrcHis TOPO. The pTrcHis-Bdh-2 system was constructed to produce a His(6) fusion protein in Escherichia coli Top10 cells. Recombinant clones were selected by growth in ampicillin-enriched medium, PCR amplification and nucleotide sequencing. The inserted cDNA was completely identified and recombinant protein synthesis was monitored by SDS-PAGE, followed by immunoblotting with monoclonal anti-His antibody. Expression of insoluble rBdh-2 was achieved at 0.1 to 2.0 mM IPTG, after 2 to 6 h of induction. Significantly increased production of rBdh-2 (P < 0.01) occurred with 1.5 mM IPTG after 2 h of induction, and with 0.3 mM IPTG after 4 h in culture. Among the induction times investigated, a period of 6 h gave the lowest levels of rBdh-2 production; with a 6-h incubation, there were no significant differences in rBdh-2 production for the various concentrations of IPTG tested (P > 0.05). The apparent molecular weight of rBdh-2 was 15.85 +/- 0.09 kDa, calculated by image analysis of membranes. This is similar to the theoretical molecular weight of 15.5 kDa predicted from the nucleotide sequence. Prior to this study, expression of recombinant goat spermadhesin had never been reported. Thus, an effective prokaryotic rBdh-2 expression system was developed in order to provide an adequate tool for studying biofunctions of goat spermadhesins.
Assuntos
Perfilação da Expressão Gênica , Proteínas de Plasma Seminal/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar/metabolismo , Escherichia coli/metabolismo , Técnicas Genéticas , Cabras , Isopropiltiogalactosídeo/química , Masculino , Modelos Genéticos , Dados de Sequência Molecular , Proteínas Recombinantes/química , Fatores de TempoRESUMO
Seventeen adult and cyclic Moxoto goats were synchronized using 60 mg MPA vaginal sponge for 11 days and 50 mug cloprostenol, 48 h before sponge removal, and superovulated with 120 mg pFSH i.m. in decreasing doses at 12 h intervals for three consecutive days. In seven goats, 0.2 IU/kg BW/day of long acting insulin was subcutaneously injected at same time as pFSH, and in the other five goats, the same dose of insulin was injected for three consecutive days starting 24 h after mating. Finally, five goats were supplemented with an oral dose of 80 ml/goat/day of propylene glycol continuously during the experiment. The animals were flushed at 7 days after mating and the embryos were classified based on International Embryo Transfer Society criteria. Blood samples were collected every 3 days for insulin assay. Administration of insulin raised the insulin levels of the goats (p < 0.05), whereas in the group treated with propylene glycol, insulin rate was different only between FSH treatment and after mating (p < 0.05). Similar rates of recovery for total (80.05 +/- 9.78%) or transferable structures (61.03 +/- 15.13%) were obtained. Treatment was not influenced (p > 0.05) by responsiveness to superovulation, which averaged 64%. By contrast, insulin treatments were shown to increase the number of embryos considered excellent with respect to goats supplemented with propylene glycol (p < 0.05). When insulin was given before mating, a strong relationship (r = 0. 90) (p < 0.05) between number of transferable embryo and ovulations was observed in the animals. In conclusion, superovulated goats treated with low doses of exogenous insulin resulted in an enhancement in embryo quality, which was related to changes in circulating insulin concentrations.
Assuntos
Transferência Embrionária/veterinária , Cabras/fisiologia , Insulina/farmacologia , Propilenoglicol/farmacologia , Superovulação/efeitos dos fármacos , Administração Oral , Animais , Cruzamento , Feminino , Injeções Subcutâneas/veterinária , Insulina/administração & dosagem , Masculino , Propilenoglicol/administração & dosagemRESUMO
Spermadhesins are a family of secretory proteins expressed in the male genital tract of pig, horse and bull. Their function and structure have been widely studied, especially those isolated from boar. However, there are no data concerning spermadhesins isolated from buck. Buck seminal plasma was collected and subjected to ion exchange chromatography on DEAE-Sephacel column followed by chromatography in a C18 column coupled to a HPLC system. The purification of the protein was determined by SDS-PAGE and MALDI-TOF analysis exhibiting a molecular mass of 12.5 KDa and showed to be structurally homologous to spermadhesins from boar and stallion.
Assuntos
Cabras , Sêmen/química , Proteínas de Plasma Seminal/química , Proteínas de Plasma Seminal/isolamento & purificação , Sequência de Aminoácidos , Animais , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Proteínas de Plasma Seminal/genética , Alinhamento de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
In tropical areas, local goats are often reported as being able to reproduce throughout the year, whereas an influence of season is found to be a factor when importing different dairy breeds. In these areas, oestrus synchronisation in goats is of interest for both technical (synchronisation of kidding, adjustment to forage availability or to continuous milk supply) and genetic reasons (dissemination of improved genotypes by AI). The use of a progestagen vaginal sponge combined with equine chorionic gonadotrophin (eCG)-cloprostenol injections remains an efficient tool to achieve synchronisation in temperate and tropical zones. However, the oestrus synchronisation treatments currently used for goats in tropical regions were originally developed for goats bred in temperate regions. For this reason, several alternative possibilities for improving the efficiency of the hormonal treatment are evaluated. Oestrus synchronisation with luteolytic agents is efficient (resulting in more than 70% of goats in oestrus) and it takes into account female cyclicity. In developing regions of the tropics, the use of buck teasing appears to be a promising approach to control oestrus and ovulation. The use of this technique provides 60% of females in oestrus within 5 days of introducing the bucks. Considering the availability of nutrients as the ultimate regulator of reproduction in the tropics, the control of nutritional condition is essential before the use of hormonal treatments for oestrus synchronisation in goats bred in these regions takes place.
Assuntos
Sincronização do Estro/métodos , Estro/efeitos dos fármacos , Cabras , Hormônios/farmacologia , Animais , Estro/fisiologia , Feminino , Estações do Ano , Clima TropicalRESUMO
This pilot project was designed to determine if normal kids could be produced after microinjection in pronuclear embryos and subsequent transfer to recipients in a transgenic goat program in Brazil. Twelve donors of the Saanen breed and 17 recipients of an undefined breed were used. The estrus of both donors and recipients was synchronized by a standard progestagen treatment and superovulation obtained by six pFSH injections. Donors in estrus were mated with fertile Saanen bucks. Zygotes were recovered surgically by flushing oviducts. The recovered zygotes with visible pronuclei were microinjected with 500 to 1000 copies of the human G-CSF gene. Two or four embryos were surgically transferred into the oviducts of recipients. One recipient became pregnant and two kids were born. No transgenic goat was identified after PCR analysis. Even though transgenic goats were not obtained, this experiment establishes the basis of a synchronization and superovulation regimen for use in goats raised in Brazil, for the purpose of collecting and manipulating the pronuclear embryos. This project also showed that microinjected one-cell goat embryos can survive to produce live young following surgical transfer.
Assuntos
Animais Geneticamente Modificados/embriologia , Transferência Embrionária , Cabras/genética , Fator Estimulador de Colônias de Granulócitos/genética , Zigoto/ultraestrutura , Animais , Brasil , Feminino , Cabras/embriologia , Microinjeções , Projetos Piloto , GravidezRESUMO
The prevalence of pseudopregnancy over 44 months was investigated in 23 Saanen goats raised in Northeast Brazil during continuous oestrous cycling (cyclic group) or after synchronization of oestrus (synchronized group). The goats were monitored by ultrasonography and their plasma progesterone profile. The overall prevalence of pseudopregnancy was 30.4% (7/23). In the cyclic group, 28.6% (4/14) of goats showed pseudopregnancy, while in the synchronized group the prevalence was 33.3% (3/9). There was no significant difference between the groups (p>0.05). The mean (+/- SD) length of pseudopregnancy, as shown by the progesterone profile, was 121.6 +/- 33.5 days, ranging from 70 to 155 days. The study defined the prevalence of pseudopregnancy in Saanen goats raised in Northeast Brazil for the first time. This finding identified a major problem for this breed, as without treatment such animals remain unproductive until the spontaneous resolution of the condition. More research seems desirable to ascertain the prevalence of this condition in other breeds in this region of Brazil.
Assuntos
Estro/sangue , Doenças das Cabras/epidemiologia , Progesterona/sangue , Pseudogravidez/veterinária , Animais , Brasil/epidemiologia , Sincronização do Estro , Feminino , Doenças das Cabras/diagnóstico por imagem , Cabras , Ovulação , Prevalência , Pseudogravidez/diagnóstico por imagem , Pseudogravidez/epidemiologia , UltrassonografiaRESUMO
This study aimed to evaluate the immunolocalization and messenger RNA (mRNA) expression for transforming growth factor-beta (TGF-ß) and its receptors (TGF-ßRI and RII), as well as mRNA expression for P450 aromatase and FSH receptor in caprine preantral follicles. The effects of TGF-ß, FSH alone, or in association on the in vitro follicular development were also assessed. Immunohistochemical analyses showed the expression of TGF-ß and its receptors in oocytes of all follicle stages and granulosa cells of primary and secondary follicles. mRNA for TGF-ß receptors and for FSH receptor (FSHR) was present in preantral follicles as well as in oocytes and granulosa cells of antral follicles. Isolated secondary follicles were cultured in α-minimum essential medium (MEM) alone or supplemented with either FSH (100 ng/ml), TGF-ß (10 ng/ml), or TGF-ß + FSH for 18 d. TGF-ß increased significantly oocyte diameter when compared to FSH alone and control. After 18 d of culture, all groups showed a significant reduction in P450 aromatase and FSHR mRNA levels in comparison to fresh control. In contrast, treatment with FSH significantly increased the mRNA expression for TGF-ß in comparison to fresh control and other treatments. In conclusion, the findings showed that TGF-ß and its receptors are present in caprine ovarian follicles. Furthermore, they showed a positive effect on oocyte growth in vitro.
Assuntos
Folículo Ovariano/crescimento & desenvolvimento , RNA Mensageiro/biossíntese , Fator de Crescimento Transformador beta/biossíntese , Animais , Meios de Cultura , Feminino , Cabras , Técnicas In Vitro , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/fisiologia , Proteoglicanas/biossíntese , Proteoglicanas/fisiologia , RNA Mensageiro/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores do FSH/biossíntese , Receptores do FSH/fisiologia , Receptores de Fatores de Crescimento Transformadores beta/biossíntese , Receptores de Fatores de Crescimento Transformadores beta/fisiologia , Fator de Crescimento Transformador beta/fisiologiaRESUMO
We investigated the influence of the phase of the estrous cycle on mechanical responses elicited in sheep cervix by potassium chloride (KCl), acetylcholine chloride (ACh), prostaglandin F2 alpha (PGF2 alpha) and prostaglandin E1 (PGE1). The cervix of adult ewes (n = 48) were classified according to the presence or absence of corpora lutea (luteal or follicular phase, respectively). Muscle strips of the circular and longitudinal layers were prepared in an organ bath and coupled to an isometric force transducer. Concentration-response curves were obtained noncumulatively. KCl and ACh produced concentration-dependent contractions in all preparations in both phases of the estrous cycle. However, maximum effect, EC50 and slope values of KCl and ACh were not significantly different between muscle layers, as well as between the phases of the estrous cycle. The prostanoid, PGF2 alpha, produced a significant reduction in the amplitude of spontaneous contractions for all preparations. The depressant effect of PGF2 alpha on spontaneous contractions of circular smooth muscle was significantly greater during the follicular than the luteal phase, whilst the depressant effect of PGF2 alpha on the longitudinal layer did not differ between phases of the estrous cycle. PGE1 significantly reduced the amplitude of spontaneous contractions on circular but not on longitudinal preparations. In conclusion, we have characterized with in vitro preparations of circular and longitudinal muscle layers of ewes during the follicular and luteal phases of the estrous cycle, the parameters of the K- and ACh-induced contractions on cervix and the efficacy of PGF2 alpha and PGE1 on inhibition spontaneous contractile activity.
Assuntos
Alprostadil/farmacologia , Colo do Útero/efeitos dos fármacos , Dinoprosta/farmacologia , Estro/fisiologia , Contração Muscular/efeitos dos fármacos , Ocitócicos/farmacologia , Ovinos/fisiologia , Alprostadil/administração & dosagem , Animais , Dinoprosta/administração & dosagem , Relação Dose-Resposta a Droga , Feminino , Contração Muscular/fisiologia , Ocitócicos/administração & dosagemRESUMO
The aim of this study was to determine the period of genital tubercle (GT) migration using ultrasonography in Morada Nova sheep foetuses (n = 117) from natural mating (NM) and frozen embryo transfer (ET) to determine the window when foetal sexing can be determined. The examinations were performed using transrectal ultrasonography with a dual-frequency linear transducer (6.0 and 8.0 MHz) from day 30-54 of pregnancy at 48-h intervals. The period of GT migration of foetuses produced by NM varied from 36 to 46 days of pregnancy, resulting in an average of 39.5 +/- 2.9 days. For foetuses derived from ET, GT migration varied from 42 to 52 days of pregnancy with an average of 48.5 +/- 3.3 days, being possible the GT of foetuses from ET start to migrate 96 h later even if they are of the same gender. Migration of the GT occurred earlier (p < 0.05) in foetuses produced by NM and sexing accuracy for triplet pregnancies (77.8%) was significantly inferior (p < 0.05) to single (100%) and twin (92.9%) pregnancies for foetuses derived by NM. The results allow one to conclude that foetal sexing can be done from the 50th day onwards in foetuses produced by NM and from the 55th day onwards in foetuses derived from ET, and that multiple pregnancies compromise the sexing accuracy by ultrasonography.
Assuntos
Análise para Determinação do Sexo/veterinária , Ovinos/embriologia , Ultrassonografia Pré-Natal/veterinária , Animais , Transferência Embrionária/veterinária , Feminino , Tamanho da Ninhada de Vivíparos , Gravidez , Análise para Determinação do Sexo/métodos , Fatores de Tempo , Ultrassonografia Pré-Natal/métodosRESUMO
Milk production of transgenic does was evaluated by ultrasound measurements of the mammary gland. Two Canindé goats, which were nine months of age were used in the trial, one non-transgenic or other transgenic for hG-CSF. For hormone-induced lactation, animals were given estradiol (0.25mg/kg, IM), progesterone (0.75mg/kg, IM), and prednisolone (0.4mg/kg, IM). Ultrasonographic exams were carried out during milking, using a Falcon 100 ultrasound equipment with a 5MHz convex probe and were performed by the same operator. The results were expressed as mean±standard error. The maximum greater length and shorter length of the cistern were respectively 5.14cm and 1.36cm for the transgenic animal and 7.28cm and 2.25cm for non-transgenic, which is consistent with the maximum milk volume produced. The relationship between the average area of cisterns and milk yield was expressed as a linear correlation curve, with a correlation coefficient significantly positive for both transgenic (Y=-1.1314+10.8538*x; r=0.97) and non-transgenic (Y=-21.7551+18.3634*x; r=0.97) animals. In conclusion, the ultrasound is a practice and appropriate technique to evaluate the cisterns in ruminant udders in transgenic animal.
RESUMO
In order to evaluate embryo production in Morada Nova (white variety) ewes superovulated with porcine follicle-stimulating hormone, 20 cycling ewes were used as embryo donors and allocated into two groups according to age: group 1 (ewes aged 1-2 years; n = 9) or group 2 (ewes aged 3-4 years; n = 11). Embryo recovery was performed by laparotomy 5-6 days after oestrus. The evaluation of embryos was made under stereomicroscope according to International Embryo Transfer Society rules. The overall recovery rate was 64.6% (5.0 +/- 0.7 structures per ewe) and 86.3% of the recovered structures were fertilized. Group 1 was superior (p < 0.05) to group 2 according to recovered (6.6 +/- 0.9 vs 3.6 +/- 0.8) and fertilized structures (5.6 +/- 1.1 vs. 3.5 +/- 0.7) per ewe. In conclusion, the ovarian response and the embryo production in Morada Nova (white variety) sheep subjected to a standard superovulation treatment were considered satisfactory. In addition, the use of multiple ovulation and embryo transfer in younger ewes ( < or = 2 years old) of this sheep breed appears to be an efficient tool to accelerate the preservation of the Morada Nova (white variety) breed, since younger ewes are as responsive as older ones.