Phytohemagglutinin improves the development and ultrastructure of in vitro-cultured goat (Capra hircus) preantral follicles.

The objective this study was to determine the effect of phytohemagglutinin (PHA) on survival, growth and gene expression in caprine secondary follicles cultured in vitro. Secondary follicles (∼0.2 mm) were isolated from the cortex of caprine ovaries and cultured individually for 6 days in α-MEM+ supplemented with PHA (0, 1, 10, 50, 100, or 200 µg/mL). After 6 days of culture, follicle diameter and survival, antrum formation, ultrastructure and expression of mRNA for FSH receptors (FSH-R), proliferating cell nuclear antigen (PCNA), and neuronal nitric oxide synthase were determined. All treatments maintained follicular survival [α-MEM+ (94.59%); 1 µg/mL PHA (96.43%); 10 µg/mL PHA (84.85%); 50 µg/mL PHA (85.29%); 100 µg/mL PHA (88.57%), and 200 µg/mL PHA (87.50)], but the presence of 10 µg/mL PHA in the culture medium increased the antrum formation rate (21.21%) when compared with control (5.41%, P < 0.05) and ensured the maintenance of oocyte and granulosa cell ultrastructures after 6 days of culture. The expression of mRNA for FSH-R (2.7 ± 0.1) and PCNA (4.4 ± 0.2) was also significantly increased in follicles cultured with 10 µg/mL PHA in relation to those cultured in α-MEM+ (1.0 ± 0.1). In conclusion, supplementation of culture medium with 10 µg/mL PHA maintains the follicular viability and ultrastructure, and promotes the formation of antral cavity after 6 days of culture in vitro.

Phytohemagglutinin improves the development and ultrastructure of in vitro-cultured goat (Capra hircus) preantral follicles

The objective this study was to determine the effect of phytohemagglutinin (PHA) on survival, growth and gene expression in caprine secondary follicles cultured in vitro. Secondary follicles (∼0.2 mm) were isolated from the cortex of caprine ovaries and cultured individually for 6 days in α-MEM+ supplemented with PHA (0, 1, 10, 50, 100, or 200 µg/mL). After 6 days of culture, follicle diameter and survival, antrum formation, ultrastructure and expression of mRNA for FSH receptors (FSH-R), proliferating cell nuclear antigen (PCNA), and neuronal nitric oxide synthase were determined. All treatments maintained follicular survival [α-MEM+ (94.59%); 1 µg/mL PHA (96.43%); 10 µg/mL PHA (84.85%); 50 µg/mL PHA (85.29%); 100 µg/mL PHA (88.57%), and 200 µg/mL PHA (87.50)], but the presence of 10 µg/mL PHA in the culture medium increased the antrum formation rate (21.21%) when compared with control (5.41%, P < 0.05) and ensured the maintenance of oocyte and granulosa cell ultrastructures after 6 days of culture. The expression of mRNA for FSH-R (2.7 ± 0.1) and PCNA (4.4 ± 0.2) was also significantly increased in follicles cultured with 10 µg/mL PHA in relation to those cultured in α-MEM+ (1.0 ± 0.1). In conclusion, supplementation of culture medium with 10 µg/mL PHA maintains the follicular viability and ultrastructure, and promotes the formation of antral cavity after 6 days of culture in vitro.

Effects of growth differentiation factor-9 and FSH on in vitro development, viability and mRNA expression in bovine preantral follicles.

The present study investigated the role of growth differentiation factor (GDF)-9 and FSH, alone or in combination, on the growth, viability and mRNA expression of FSH receptor, proliferating cell nuclear antigen (PCNA) and proteoglycan-related factors (i.e., hyaluronan synthase (HAS) 1, HAS2, versican, perlecan) in bovine secondary follicles before and after in vitro culture. After 12 days culture, sequential FSH (100 ng mL⁻¹) from Days 0 to 6 and 500 ng mL⁻¹ from Days 7 to 12) increased follicular diameter and resulted in increased antrum formation (P<0.05). Alone, 200 ng mL⁻¹ GDF-9 significantly reduced HAS1 mRNA levels, but increased versican and perlecan mRNA levels in whole follicles, which included the oocyte, theca and granulosa cells. Together, FSH and GDF-9 increased HAS2 and versican (VCAN) mRNA levels, but decreased PCNA mRNA expression, compared with levels in follicles cultured in α-minimum essential medium supplemented with 3.0 mg mL⁻¹ bovine serum albumin, 10 µg mL⁻¹ insulin, 5.5 µg mL⁻¹ transferrin, 5 ng mL⁻¹ selenium, 2 mM glutamine, 2mM hypoxanthine and 50 µg mL⁻¹ ascorbic acid (α-MEM⁺). Comparisons of uncultured (0.2 mm) and α-MEM⁺ cultured follicles revealed that HAS1 mRNA expression was higher, whereas VCAN expression was lower, in cultured follicles (P<0.05). Expression of HAS1, VCAN and perlecan (HSPG2) was higher in cultured than in vivo-grown (0.3 mm) follicles. In conclusion, FSH and/or GDF-9 promote follicular growth and antrum formation. Moreover, GDF-9 stimulates expression of versican and perlecan and interacts positively with FSH to increase HAS2 expression.

Características andrológicas e do sêmen de touros do composto Red Norte (Nelore x Tabapuã x Red Angus x Sinepol) / Andrological and semen characteristics of cross-bred Red Norte (Nelore x Tabapuã x Red Angus x Sinepol) young bulls

Avaliaram-se as características andrológicas do sêmen de touros jovens do composto Red Norte (Nelore x Tabapuã x Red Angus x Sinepol), com idade média de 13,9±0,8 meses, com o objetivo de estimar o advento da puberdade e a qualidade do sêmen. Foram avaliados o perímetro escrotal (PE), o peso e as características seminais de 70 tourinhos, classificados em três grupos, de acordo com o PE: GI=27-33cm (n=24), GII=33-35cm (n=24) e GIII=35-43cm (n=22). As médias de peso e a idade de cada grupo (G) foram, respectivamente: GI=411,2±37,4kg e 13,8±1,0 meses, GII=426,9±31,5kg e 14,0±0,7 meses e GIII=438,4±38,3kg e 14,0±0,6 meses. As características seminais para cada grupo foram, volume 4,2±3,1mL, 5,3±2,6mL e 4,5±2,1mL; motilidade 31,3±24,1 por cento, 44,2±23,9 por cento e 43,9±21,5 por cento e vigor 2,8±1,6, 3,5±1,3 e 3,5±1,3, respectivamente. O espermiograma apresentou valores médios de concentração de 130,5±266,2x10(6)/mL, 289,5±390,2x10(6)/mL e 333,9±523,7x10(6)/mL, defeitos totais de 81,4±15,9 por cento, 73,8±15,4 por cento e 67,9±19,0 por cento; defeitos maiores de 87,3±26,2 por cento, 66,8±24,9 por cento e 56,7±17,1 por cento e defeitos menores de 16,6±14,9 por cento, 33,2±24,9 por cento e 43,3±17,1 por cento, respectivamente. Dos setenta animais examinados, sete (10 por cento) foram considerados aptos à reprodução. Os resultados mostraram que a patologia espermática diminuiu em razão do aumento do PE.

Reproductive traits of cross-breed Red Norte (Nelore x Tabapuã x Red Angus x Sinepol) young bulls averaging of 13.9±0.8 month-old were evaluated, in order to determine the puberty onset and semen quality in these animals. Scrotal circumference (SC), body weight (BW), and semen parameters of 70 bulls were measured. Animals were allotted in three groups (G) according to their SC: GI=27-33cm (n=24), GII=33-35cm (n=24), and GIII=35-43cm (n=22). BW and age of each group were, respectively: GI=411.2±37.4kg and 13.8±1.0 month-old, GII=426.9±31.5kg and 14.0±0.7 month-old, and GIII=438.4±38.3kg and 14.0±0.6 month-old. Seminal physical characteristics for same order of groups were: volume 4.2±3.1mL, 5.3±2.6mL, and 4.5±2.1mL; motility 31.3±24.1 percent, 44.2±23.9 percent, and 43.9±21.5 percent; and vigor 2.8±1.6, 3.5±1.3, and 3.5±1.3. The spermiogram presented concentration values of 130.5±266.2x10(6)/mL, 289.5±390.2x10(6)/mL, and 333.9±523.7x10(6)/mL; total defects of 81.4±15.9 percent, 73.8±15.4 percent, and 67.9±19.0 percent; major defects of 87.3±26.2 percent, 66.8±24.9 percent and 56.7±17.1 percent; and minor defects of 16.6±14.9 percent, 33.2±24.9 percent, and 43.3±17.1 percent, for same order of groups. Seven out of 70 bulls were considered satisfactory potential breeders. Results showed that semen pathology progressively decreased when SC increased.

Antiviral activity of bovine uterus and placenta induced by Newcastle disease virus

The antiviral activity profile of the uterus and fetal membranes from bovine placenta, induced by the Newcastle disease virus (NDV) throughout gestation, was investigated. Explants of the endometrium and caruncles were collected from the uterus, and amniochorion, allantochorion and cotyledons, from fetal placenta. Tissue cultures were induced with ~6.0 hemagglutinating units (HU) of NDV. Supernatants were concentrated 20 fold, filtered in 100kDa cut-off membranes and antiviral activity was titrated in MDBK x VSV system. Tissues of the uterus did not exhibit antiviral activity, while allantochorion and amniochorion produced antiviral factors throughout gestation. Antiviral factors were not related with IFN-alpha, gamma, tau or TNF-alpha. The antiviral activity pattern observed showed to be related with the development of fetal membranes and increased at the end of pregnancy. Such data suggest that IFN genes inducible by virus are present in fetal membranes of the cow placenta and their expression is dependent on the age of gestation.

Investigou-se a atividade antiviral do útero e da placenta bovina, ao longo da gestação, induzidos pelo vírus da doença de Newcastle (NDV). Explantes do endométrio e carúnculas foram colhidos do útero. Os tecidos corioamniótico, corioalantóide e cotilédones foram dissecados da placenta fetal. Os cultivos celulares foram induzidos com aproximadamente 6,0 unidades hemaglutinantes do NDV. Os sobrenadantes foram concentrados 20 vezes, filtrados em dispositivos com superfície de separação de 100kDa e a atividade antiviral foi titulada em células MDBK e vírus da estomatite vesicular (VSV). Endométrio, carúnculas e cotilédones não apresentaram atividade antiviral. Corioamniótico e corioalantóide produziram fatores antivirais ao longo da gestação. Estes fatores não foram relacionados aos IFN - alfa, gama ou tau e nem ao TNF - alfa. O padrão de produção de fatores antivirais acompanhou o desenvolvimento dos tecidos fetais e títulos mais altos foram observados no final da gestação. Estes dados sugerem que os genes de IFNs induzidos por vírus localizam-se nas membranas fetais da placenta e a expressão desses genes é dependente do estádio da gestação.