RESUMO
Mastitis is the most important disease in the global dairy industry, and causes large economic losses. Staphylococcus aureus is one of most common pathogens that cause bovine mastitis. CXCR1 has been implicated as a prospective genetic marker for mastitis resistance in dairy cows; CXCR1 expression significantly increases when cows have mastitis. To investigate the mechanisms involved in its increased expression, bisulfite sequencing polymerase chain reaction (PCR) was used to detect the methylation status of CXCR1 CpG island, and quantitative fluorescence PCR was used to detect CXCR1 expression in bovine mammary tissue induced with S. aureus in three Chinese Holstein cows. No CpG island was found for bovine CXCR1 in the upstream 2-kb region, whereas one CpG island that contained 13 CpG sites was found in exon 1 of CXCR1. All of the CpG sites were under hypermethylation from 90 to 100% in the mammary tissues. When the mammary gland mRNA expression of CXCR1 was 12.10-fold higher in infected cow quarters than in uninfected quarters, the methylation levels of the CpG site at position 519 were significantly lower in the infected quarters than in the uninfected quarters. Pearson correlation analysis showed that the methylation level at position 519 was significantly negatively correlated with the CXCR1 mRNA expression level (P < 0.05). These results indicate that the methylation of the CpG site at position 519 may regulate CXCR1 expression in cows with mastitis induced by S. aureus, but further studies are needed to elucidate the mechanisms involved.
Assuntos
Metilação de DNA , Glândulas Mamárias Animais/metabolismo , Mastite Bovina/genética , Receptores de Interleucina-8A/genética , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/isolamento & purificação , Animais , Bovinos , Ilhas de CpG , Feminino , Mastite Bovina/metabolismo , Mastite Bovina/microbiologia , Reação em Cadeia da Polimerase , Estudos Prospectivos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Interleucina-8A/metabolismo , Infecções Estafilocócicas/metabolismoRESUMO
Spermatogenesis, is a complex process of precisely regulated intracellular events, where it is affected by many factors. Long-distance transport of animals is one of the stressors that may influence spermatogenesis and sperm quality. The present study chose luteinizing hormone receptor (LHR), androgen receptor (AR), and heat-shock protein 70 (HSP70) as our target genes to investigate their mRNA and protein expression in the testes of long-distance transported (about 1000 km) mice. Histological analysis showed that there was a reduction in the thickness of the seminiferous epithelium in the transported mice, and a significant decrease in body weight and sperm count in the epididymis was also observed. mRNA expression was determined by QPCR in the testis of transported and control mice. The levels for AR decreased significantly in transported mice. LHR and HSP70 expression in the testes of the transported mice was slightly higher than that of control mice but did not reach a significant level. A similar tendency of protein expression was also observed by Western blot analysis. The levels of LHR and HSP70 increased slightly after transportation. However, none of the changes were statistically significant compared with the control mice. In conclusion, long-distance transport has an adverse effect on reproductive organs and spermatozoa in adult mice.
Assuntos
Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/genética , Receptores Androgênicos/genética , Receptores do LH/genética , Testículo/metabolismo , Animais , Peso Corporal , Masculino , Camundongos , Tamanho do Órgão , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Contagem de Espermatozoides , Espermatogênese/genética , Estresse Fisiológico/genética , Testículo/anatomia & histologiaRESUMO
DNA vaccination has been studied intensively as a potential vaccine technology. We evaluated the effect of an attenuated Salmonella choleraesuis-mediated inhibin DNA vaccine in rats. First, 15 rats were treated with different doses of an inhibin vaccine to evaluate vaccine safety. Next, 30 rats were divided into 3 groups and injected intramuscularly with the inhibin vaccine two (T1) or three times (T2) or with control bacteria (Con) at 4-week intervals. The inhibin antibody levels increased [positive/negative well (P/N) value: T1 vs Con = 2.39 ± 0.01 vs 1.08 ± 0.1; T2 vs Con = 2.36 ± 0.1 vs 1.08 ± 0.1, P < 0.05] at week 2 and were maintained at a high level in T1 and T2 until week 8, although a small decrease in T2 was observed at week 10. Rats in the T1 group showed more corpora lutea compared with the Con group (10.50 ± 0.87 vs 7.4 ± 0.51, P < 0.05). Estradiol (0.439 ± 0.052 vs 0.719 ± 0.063 ng/mL, P < 0.05) and progesterone (1.315 ± 0.2 vs 0.737 ± 0.11 ng/mL, P < 0.05) levels differed significantly at metestrus after week 10 between rats in the T1 and Con groups. However, there were no significant differences in body, ovary, uterus weights, or pathological signs in the ovaries after immunization, indicating that this vaccine is safe. In conclusion, the attenuated S. choleraesuis-mediated inhibin vaccine may be an alternative to naked inhibin plasmids for stimulating ovarian follicular development to increase the ovulation rate in rats.
Assuntos
Inibinas/genética , Inibinas/imunologia , Salmonella/genética , Vacinas Atenuadas/imunologia , Vacinas de DNA/imunologia , Animais , Anticorpos/sangue , Anticorpos/imunologia , Estradiol/sangue , Feminino , Imunização , Folículo Ovariano/imunologia , Folículo Ovariano/patologia , Ovulação , Progesterona/sangue , Ratos , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/efeitos adversos , Vacinas de DNA/administração & dosagem , Vacinas de DNA/efeitos adversosRESUMO
Follicle-stimulating hormone receptor (FSHR), which mediates the functioning of FSH, plays a central role in reproduction. We investigated bovine FSHR gene polymorphisms and analyzed their relationships with pregnancy rates after embryo transfer and with hormone concentrations on the day of embryo transfer. One reported SNP of FSHR, G-278A, located in the 5'-upstream region, was analyzed and three genotypes (GG, GA and AA) were detected in 132 Luxi cattle recipients. Statistical analysis revealed that recipients with the GG genotype had significantly higher estrogen levels on the day of embryo transfer than did GA and AA genotypes. There were no significant differences in pregnancy rates among genotypes, after embryo transfer. We conclude that variation at these loci of the FSHR gene has no significant effect on pregnancy rates in Luxi cattle.
Assuntos
Transferência Embrionária , Estrogênios/sangue , Receptores do FSH/genética , Reprodução/genética , Animais , Bovinos , Feminino , Genótipo , Polimorfismo de Nucleotídeo Único , Gravidez , Taxa de Gravidez , Receptores do FSH/sangueRESUMO
Bone morphogenetic protein 15 (BMP-15) expression has been detected in the testis, but its roles in this organ has not been well elucidated. We evaluated polymorphisms of the BMP-15 gene by PCR-SSCP and PCR-RFLP in 212 Chinese Holstein bulls, and investigated possible associations with sperm quality traits, including semen volume per ejaculate, sperm density, fresh sperm motility, thawed sperm motility, acrosome integrity rate, and abnormal sperm rate. A single nucleotide polymorphism (C5697T) in intron 1 of the BMP-15 gene was identified in these bulls. Age was found to have significant effects on both fresh sperm motility and abnormal sperm rate. A significant effect of genotype on fresh sperm motility was also observed. Least square analysis showed that CT genotype bulls had significantly lower fresh sperm motility than CC or TT genotype bulls. In conclusion, BMP-15 should be considered as a potential genetic marker for sperm quality, based on its association with fresh sperm motility.
Assuntos
Proteína Morfogenética Óssea 15/genética , Motilidade dos Espermatozoides/genética , Espermatozoides , Animais , Bovinos , Marcadores Genéticos , Genótipo , Masculino , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Inhibin is a major regulator of secretion of follicle-stimulating hormone, which is involved in follicular development and regulation of steroidogenesis in females. The objectives of this study were to detect polymorphisms of the bovine inhibin beta-A subunit (INHßA) gene and to evaluate its associations with superovulatory responses in 171 Chinese Holstein cows treated for superovulation. Polymerase chain reaction-restricted fragment length polymorphism revealed a C>T transition determining the StyI polymorphism at position 7639 in intron I of the bovine INHßA gene, and three genotypes (CC, CT, and TT) were detected. The frequencies of the three genotypes showed a tendency for CT > TT > CC, and this polymorphism was in Hardy-Weinberg equilibrium. Statistical analysis revealed no significant differences of least square means for superovulation traits among the three genotypes (P > 0.05). These results demonstrate, for the first time, that the detected loci of the INHßA gene have no significant effects on superovulation performance in Chinese Holstein cows.
Assuntos
Bovinos/genética , Subunidades beta de Inibinas/genética , Polimorfismo Genético , Superovulação/genética , Animais , Estudos de Casos e Controles , Bovinos/fisiologia , FemininoRESUMO
Members of the transforming growth factor-ß (TGFß) superfamily are critical regulators of germ cell development that act as extracellular ligands of the signal transduction pathways regulating proliferation, differentiation, apoptosis, and other aspects of cell behavior. Growth differentiation factor 9 (GDF9) is a member of the TGFß superfamily that plays a critical role in ovarian follicular development and ovulation rate in females; however, its role in the testis has not been well elucidated. Therefore, in this study we investigated the effects of GDF9 mutations on the quality of fresh and frozen semen of Holstein bulls. Two reported single nucleotide polymorphisms of GDF9, A485TA and A625C, were analyzed in 129 Holstein bulls. Analysis of variance revealed that the A485T polymorphism had significant effects on the acrosome integrity rate (P < 0.05), whereas the A625T polymorphism was significantly associated with sperm concentration (P < 0.05). In addition, a significant additive effect on sperm concentration was detected for the A485T polymorphism (P < 0.05), whereas the polymorphisms A485TA and A625C had significant dominant effects on acrosome integrity rate and sperm motility in frozen semen, respectively (P < 0.05). This study is the first to show a significant association of GDF9 with sperm quality traits, and the results implied that GDF9 is involved in the initiation or maintenance of spermatogenesis; however, further verification is needed.
Assuntos
Acrossomo/fisiologia , Fator 9 de Diferenciação de Crescimento/genética , Polimorfismo de Nucleotídeo Único , Motilidade dos Espermatozoides/genética , Animais , Animais Endogâmicos , Bovinos , Masculino , Contagem de EspermatozoidesRESUMO
Growth differentiation factor 9 (GDF9) belongs to the transforming growth factor ß superfamily and plays a critical role in ovarian follicular development and ovulation rate. We examined the bovine GDF9 gene polymorphism and analyzed its association with superovulation performance. Based on the sequence of the bovine GDF9 gene, six pairs of primers were designed to detect single nucleotide polymorphisms of two exons and intron 1 of GDF9 using polymerase chain reaction-single-strand conformation polymorphism. Only the products amplified by primer 3-1 displayed polymorphisms. Sequencing revealed two mutations of A485T and A625T in intron 1 of the GDF9 gene in 171 Chinese Holstein cows treated for superovulation. Association analysis showed that these two single nucleotide polymorphisms of A485T and A625T had significant effects on the number of transferable embryos (P < 0.05), and the A625T polymorphism was significantly associated with the total number of ova (P < 0.05). In addition, a significant additive effect on the number of transferable embryos was detected in polymorphisms of A485T (P < 0.05). This study is the first to identify two polymorphisms in bovine GDF9 and describe their correlation with superovulation traits in Chinese Holstein cows.
Assuntos
Bovinos/genética , Fator 9 de Diferenciação de Crescimento/genética , Superovulação/genética , Animais , Feminino , Frequência do Gene , Estudos de Associação Genética , Genótipo , Íntrons , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Chinese native buffaloes have faced the threat of extinction, along with an increase in crossbreeding with domesticated river buffaloes; consequently, conservation of local buffalo genetic resources has become a priority. A Chinese native breed, Jianghan, is often crossed intentionally and unintentionally with imported breeds from India and Pakistan, Murrah, and Nili-Ravi. A total of 128 buffaloes of the breeds Jianghan, Murrah, and Nili-Ravi and their presumed hybrid offspring were genotyped for 10 microsatellite markers. Heterozygosity and Wright's F-statistics were calculated to determine the genetic variation in those populations. The observed average heterozygosities ranged from 0.836 (Murrah) to 0.986 (Jianghan), higher than the expected heterozygosities and all the inbreeding values within the populations were negative. The genetic distances between the presumed hybrid buffaloes and the two imported river type dairy buffalo breeds (Murrah and Nili-Ravi) were lower than with the native Jianghan, indicating strong contributions of the imported breeds to this presumed hybrid buffalo population. This information will be useful for the development of rational breeding for the dairy buffalo industry and for conservation strategies for the Jianghan buffalo.
Assuntos
Cruzamento , Búfalos/genética , Bases de Dados Genéticas , Variação Genética , Hibridização Genética , Repetições de Microssatélites/genética , Alelos , Animais , China , Frequência do Gene/genética , Genética Populacional , Heterozigoto , Filogenia , Rios , Tamanho da Amostra , Estatística como AssuntoRESUMO
Complex vertebral malformation (CVM) is a recently described monogenic autosomal recessive hereditary defect of Holstein dairy cattle that causes premature birth, aborted fetuses and stillborn calves. Guanine is substituted by thymine (G>T) in the solute carrier family 35 member A3 gene (SLC35A3). A valine is changed to a phenylalanine at position 180 of uridine 5'-diphosphate-N-acetyl-glucosamine transporter protein. CVM is expected to occur in many countries due to the widespread use of sire semen. We developed a created restriction site PCR (CRS-PCR) method to diagnose CVM in dairy cows. This was tested on 217 cows and 125 bulls selected randomly from a Holstein cattle population in south China. Five Holstein cows and five Holstein bulls were identified to be CVM carriers; the percentages of CVM carriers were estimated to be 2.3, 4.0 and 2.9% in the cows, bulls and entire Holstein cattle sample, respectively.