Leptin decreases apoptosis and promotes the activation of primordial follicles through the phosphatidylinositol-3-kinase/protein kinase B pathway in cultured ovine ovarian tissue.

This study evaluated the effects of leptin on primordial follicle survival and activation after in vitro culture of ovine ovarian tissue and if leptin acts through the phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) pathway. Ovarian fragments were fixed for histology (fresh control) or cultured for 7 days in control medium (α-MEM+) alone or supplemented with leptin (1, 5, 10, 25 or 50 ng/ml). Follicle morphology, activation and apoptosis were analyzed. Next, the fragments were cultured in the medium that showed the best results in the absence or the presence of the PI3K inhibitor (LY294002), and immunohistostaining of p-Akt protein was assessed. After culture, the percentage of normal follicles decreased (P < 0.05) in all treatments compared with the fresh control. Moreover, control medium and 1 ng/ml leptin had similar (P > 0.05) percentages of normal follicles, which were significantly higher than those in other treatments. However, culture with 1 ng/ml leptin maintained apoptosis similarly (P > 0.05) to that of the fresh control and lower (P < 0.05) than that in α-MEM+. Leptin did not influence follicle activation (P > 0.05) compared with the control medium (α-MEM+). Culture in 1 ng/ml leptin with LY294002 decreased the normal follicles and increased apoptosis, inhibited follicle activation (P < 0.05), and reduced p-Akt immunostaining, compared with the medium containing 1 ng/ml leptin without PI3K inhibitor. In conclusion, leptin at 1 ng/ml reduces apoptosis and promotes the activation of primordial follicles compared with the fresh control after in vitro culture of ovine ovarian tissue possibly through the PI3K/Akt pathway.

Folliculogenesis-related genes are differently expressed in secondary and tertiary ovarian follicles.

The relative mRNA abundance of 10 genes associated with folliculogenesis was compared between late preantral (secondary) and early antral (tertiary) ovarian follicles in goats. In total, 100 follicles in each category were mechanically isolated. The relative transcript abundance of the mRNAs were determined by qPCR. Data were analyzed using unpaired Student's t-test. Of the 10 tested genes, ABLIM mRNA was not detected in either follicle category, six genes (SLIT3, TYMS, GTPBP1, AKR1C4, PIK3R6, and MAOB) were upregulated in secondary follicles compared with tertiary follicles, and three genes (ARHGEF12, CLEC6A, and CYTL1) showed similar mRNA abundances in both secondary and tertiary follicles. In conclusion, SLIT3, GTPBP1, AKR1C4, and PIK3R6 mRNA abundance was upregulated in secondary follicles (preantral phase) compared with in tertiary follicles (antral phase) in goats.

Influência da somatotropina recombinante bovina no desenvolvimento folicular e na coleta de embriões em éguas / Influence of bovine recombinant somatotropin in the follicular development and embryo colletion in mares

Dez éguas, sem raça definida, foram submetidas a avaliações ultrassonográficas durante o intervalo interovulatório, avaliando-se folículos ≥ 5mm. Cinco éguas foram tratadas com 500mg de r-bST no primeiro e no 14º dia pós-ovulação (grupo GT), e as demais com soro fisiológico (grupo GC). Quando o folículo dominante atingiu diâmetro ≥ 40mm, foram induzidas com hCG e inseminadas 24 horas após, sendo submetidas à coleta de embrião seis dias após a ovulação. Os dados foram agrupados de acordo com o diâmetro do folículo dominante nas fases de emergência, divergência, dominância, pré-ovulatória, indução, inseminação e ovulação. Todas as éguas foram usadas duas vezes, no mesmo grupo. O GT apresentou crescimento folicular precoce para as fases de emergência, divergência, dominância e pré-ovulatória, assim como para o seu maior folículo subordinado, que cresceu mais cedo. As taxas de recuperação foram de 90% (GC) e 70% (GT), em 16 estruturas coletadas, obtendo-se uma não fecundada e um blastocisto inicial para o grupo GC; os demais, no estágio de mórula, apresentaram comportamento semelhante entre os grupos. Conclui-se que a r-bST influencia a dinâmica folicular de éguas, levando a uma antecipação do desenvolvimento folicular, que pode ser utilizada para encurtar o ciclo estral.(AU)

Ten undefined mare breeds were submitted to ultrasonographic evaluations during the interovulatory interval, evaluating follicles measuring ≥ 5mm. Five mares were treated with 500mg r-bST on the first and the 14th day after ovulation (TG group), and the others with saline (CG group). When the dominant follicle reached a diameter ≥ 40mm the ovulation was induced with hCG, and the mares were inseminated 24 hours later and submitted to embryo collection six days after ovulation. The data were grouped according to the diameter of the dominant follicle in the emergence, divergence, dominance, preovulatory, induction, insemination and ovulation phases. All mares were used twice, in the same group. The GT showed early follicular growth for the emergence, divergence, dominance and pre-ovulatory phases, as well as for its greater subordinate follicle, growing earlier. The recovery rates were 90% (CG) and 70% (TG), and 16 structures were collected, obtaining an unfertilized embryo and an initial blastocyst for the CG group, the others in the morula stage behaved similarly between the groups. It can be concluded that r-bST influences the follicular dynamics of the mares, leading to an anticipation of the follicular development that can be used to shorten the estrous cycle.(AU)

Effects of leptin on the follicular development and mitochondrial activity of ovine isolated early antral follicles cultured in vitro.

This study evaluated the effect of leptin on the in vitro culture of isolated sheep early antral follicles. Early antral follicles (300-450 µm) were isolated and cultured for 12 days in tissue culture medium 199 (TCM 199) supplemented with glutamine, hypoxanthine, transferrin, insulin, selenium, ascorbic acid, bovine serum albumin (BSA) and recombinant follicle stimulating hormone (rFSH) (TCM 199+: control medium) or TCM 199+ supplemented with 2 or 10 ng/mL leptin. After culture, oocytes were subjected to in vitro maturation (IVM). The parameters analyzed were morphology, extrusion rate, follicular diameter, growth and fully-grown oocytes (oocytes ≥110 µm) rates. After IVM, reactive oxygen species (ROS) levels, mitochondrial activity, meiotic stages and meiotic resumption rates were also analyzed. After 12 days of culture, the concentration of 2 ng/mL of leptin showed a higher percentage of morphologically normal follicles, fully-grown oocytes (≥110 µm), active mitochondria and meiotic resumption compared to the control medium (TCM 199+; P < 0.05) but did not differ when compared to leptin concentration of 10 ng/mL (P > 0.05). After culturing, no significant differences existed among treatments in terms of the follicle diameter and ROS levels. In conclusion, the addition of 2 ng/mL leptin to the base culture medium is capable of improving follicular survival, oocyte growth, mitochondrial activity and meiotic resumption after the in vitro culture of isolated sheep early antral follicles.

Análise Dopplerfluxométrica e angiogênica de tumores mamários caninos / Dopplerfluxometric and angiogenic analysis of canine mammary tumors

Foi avaliado o comportamento de índices Doppler e a expressão de genes relacionados à neovascularização tumoral, visando caracterizar a vascularização das massas neoplásicas. Foram utilizadas 27 cadelas, com diagnóstico histopatológico de neoplasia mamária, sendo submetidas à avaliação Dopplerfluxométrica tumoral e à coleta de fragmentos neoplásicos para análise de expressão gênica de VEGF, FLT-1, FLK-1 e ATR1. Foram encontrados 22 tumores de origem epitelial (carcinomas) e cinco de origem mesenquimal (sarcomas). Observou-se correlação positiva entre o FLT-1 e as variáveis PS, PI e RI. O FLK-1 apresentou correlação igualmente positiva com os parâmetros PS e PI e uma tendência para RI, enquanto o VEGF retratou correlação positiva apenas com IP. O VEGF também mostrou correlação positiva com seus receptores, porém não apresentou correlação com o ATR1. O FLT-1 e o FLK-1 apresentaram ainda correlação positiva entre si e com o ATR1. Houve maior expressão média do VEGF nos tumores epiteliais do que nos mesenquimais. As variáveis PS, PI e RI, associadas com a expressão do VEGF e seus receptores, mostraram-se relevantes para caracterizar a neovascularização de tumores malignos, e a expressão diferenciada do VEGF entre os tipos tumorais pode ser um indicador auxiliar na caracterização de neoplasias mamárias malignas em cadelas.(AU)

The behavior of the tumor Doppler indexes and gene expression related to neovascularization was evaluated aiming to improve the characterization of neoplastic masses vascularization. Twenty-seven bitches with histopathological diagnosis of mammary neoplasia were submitted to tumor Dopplerfluxometric evaluation and collection of neoplastic fragments to analyze the gene expression of VEGF, FLT-1, FLK-1 and ATR1. Were found 22 epithelial (carcinomas) and five mesenchymal (sarcomas) tumors. Positive correlation was observed between FLT-1 and PS, PI and RI. FLK-1 presented a similar positive correlation with the PS and PI parameters, and a tendency for RI (r= 0.45, P= 0.07), whereas VEGF showed a positive correlation just with PI. VEGF also showed a positive correlation with its receptors, but did not present a correlation with ATR1. FLT-1 and FLK-1 also showed positive correlation with each other, and with ATR1. There was higher mean expression of VEGF in epithelial tumors than in mesenchymal ones. The PS, PI and RI associated with the expression of VEGF and its receptors have been shown to be relevant to characterize neovascularization of malignant tumors, and the differentiated expression of VEGF between the types of mammary tumors, may be an auxiliary indicator in the characterization of malignant breast cancers in bitches.(AU)

Estudo da vascularização folicular e do corpo lúteo de éguas cíclicas tratadas com extrato de pituitária equina utilizando ultrassom Doppler colorido / Study of follicular and corpus luteum vascularization in mares treated with Equine Pituitary Extract using ultrasound color Doppler

Informações sobre a vascularização da parede folicular e do corpo lúteo equino, associadas à superovulação, são escassas. Com o objetivo de avaliar o efeito superovulatório do extrato de pituitária equina (EPE) no fluxo sanguíneo folicular e luteal, foram utilizadas seis éguas Puro Sangue Árabe, em dois ciclos estrais (controle e tratamento). As éguas foram monitoradas diariamente por ultrassonografia modo B, até que os folículos atingissem diâmetro de 23mm (desvio). No ciclo tratamento, as éguas receberam 8mg de EPE, uma vez ao dia, por via IM, até que dois ou mais folículos atingissem o diâmetro entre 32 e 35mm. A ovulação foi induzida com acetato de deslorelina, quando os folículos atingiram, no mínimo, 35mm. No momento do desvio folicular, da indução da ovulação e do último exame pré-ovulatório, foi utilizada a ultrassonografia modo B para medir o diâmetro dos folículos e, no oitavo dia pós-ovulação, para a área do corpo lúteo (CL). Utilizou-se também ultrassonografia com Doppler colorido para avaliar a perfusão sanguínea da parede folicular e do parênquima luteal. No ciclo controle, foi realizado o mesmo procedimento, exceto pelo uso do EPE. Os dados foram submetidos à análise de variância, com nível de significância de 5%. Não foi observado efeito do EPE sobre o número de ovulações, o diâmetro dos folículos, a vascularização da parede folicular e a concentração sérica de estrógeno. Os animais, tratados ou não, apresentaram CLs funcionais, não havendo diferença na área do parênquima ou da vascularização luteal, nem na concentração sérica de progesterona, no oitavo dia após a ovulação. Foi observado que o EPE proporcionou um maior número de folículos subordinados no momento da indução da ovulação do folículo dominante (P ≤ 0,05). Embora esses folículos não tenham chegado a ovular, concluiu-se que o EPE atuou no crescimento de folículos, que podem ser utilizados em outras biotécnicas, como a transferência de oócitos, com maior aproveitamento da reserva folicular de ovários equinos.(AU)

Knowledge about follicle and corpus luteum vascularization associated with superovulation in mares is scarce. Aiming to evaluate the effect of equine pituitary extract (EPE) on superovulation, the experiment was conducted using six mares Purebred Arabian in two estrous cycles (control and treatment). The mares were synchronized, and monitored daily by ultrasound B mode until the follicles reached diameter ≤ 23 mm (deviation). In the treatment cycle, from the deviation, mares received 8 mg of EPE, once a day, intramuscularly, until two or more follicles reached a diameter between 32 and 35 mm. Ovulation was induced with deslorelin acetate when follicles reached at least 35 mm. At the time of follicular deviation, induction of ovulation and final preovulatory exam, it was used B-mode ultrasound to measure the diameter of follicles and on the eighth day after ovulation to measure the area of the corpus luteum (CL); color Doppler was also used to assess blood perfusion of the follicle wall and luteal parenchyma. In the control cycle was performed the same procedure except for the use of EPE. Data were subjected to analysis of variance, with 5% significance level. There was no effect of EPE on ovulation number, diameter of follicles, vascularity of the follicular wall and serum estrogen concentration. The animals treated or not, showed functional CLs, with no difference in parenchymal area or luteal vascularization, or in serum progesterone concentration on the eighth day after ovulation. It was observed that the EPE provided a greater number of subordinate follicles at the time of induction of ovulation of the dominant follicle. Although these follicles have failed to ovulate, it was concluded that EPE influenced the follicles growth, and it can be used in other biotechnologies, with greater utilization of equine ovarian follicular reserve.(AU)

Survey of mutations in prolificacy genes in Santa Ines and Morada Nova sheep / Pesquisa de mutações em genes da prolificidade em ovelhas Santa Inês e Morada Nova

Polymorphisms in the BMP-15 gene related to Galway (FecXG) and Inverdale (FecXI) and in the BMPR-1B gene known as Booroola (FecB) mutations were investigated using the Polymerase Chain Reaction - Restriction Fragment Length Polymorphism (PCR-RFLP) method, on sheep from the breeds Santa Inês (n= 574) and Morada Nova (n=282). DNA was extracted and amplified through PCR with specific primers that introduced a restriction site in association with the mutation. The PCR products were submitted to endonucleases. The experiment found no FecXG and FecXI mutations. Six samples of animals with multiple offspring/birth history presented polymorphism for FecB similar to control samples, but this pattern was not confirmed by nucleotide sequencing. Although the absence of these mutations in the studied breeds, other factors related to prolificacy should be investigated to explain the inherent prolificity mechanisms.(AU)

Polimorfismos Galway (Fec XG ) e Inverdale (Fec XI), relacionados ao gene BMP-15, e Booroola (FecB), localizado no gene BMPR-1B, foram investigados usando-se a técnica de reação em cadeia da polimerase - polimorfismo de comprimento de fragmentos de restrição (PCR-RFLP), em ovelhas Santa Inês (n= 574) e Morada Nova (n=282). O DNA foi extraído e amplificado por PCR com iniciadores específicos, que introduziram um sítio de restrição associado à mutação, em seguida os amplicons foram submetidos à ação de endonucleases. Não foram observadas as mutações Fec XG e Fec XI nas amostras estudadas. Amostras de seis animais com histórico de partos gemelares apresentaram polimorfismo para FecB semelhantes às amostras controle, mas esse padrão não foi confirmado pelo sequenciamento de nucleotídeos. Apesar da ausência dessas mutações nos animais das raças estudadas, outros fatores relacionados à prolificidade devem ser pesquisados para explicar os mecanismos da alta prolificidade desses animais.(AU)

Long-term in vitro culture of bovine preantral follicles: Effect of base medium and medium replacement methods.

Two culture media and replacement methods were compared during long-term in vitro culture of secondary follicles of cattle using α-MEM(+) or TCM-199(+) as base media. The medium replacement methods were: Conventional - removal and subsequent addition of the same amount (60µl) in a 100µl aliquot (MEM-C and TCM-C), and Small Supplementation - addition of 5µl of fresh medium to an initial small aliquot (50µl), resulting in a final volume of 125µl on the last day of culture (MEM-S and TCM-S). A total of 207 secondary follicles were cultured individually for 32 days at 38.5°C in 5% CO2 and medium replacement was performed every other day. The MEM-S treatment resulted in a larger (P<0.01) follicular diameter, greater (P<0.02) growth rate, greater (P<0.02) antrum formation, as well as greater (P<0.0001) estradiol concentrations when compared with the MEM-C treatment. The medium change methods did not affect (P>0.05) the follicular and estradiol end points for TCM-199(+). The expression of the FSHR gene was greater (P<0.03) with the TCM-C than TCM-S treatment, while the relative amounts of mRNA for IGF1 was greater (P<0.02) with MEM-S than TCM-S treatments and for VEGF was greater (P<0.02) with MEM-C than TCM-C treatment. In conclusion, the type of base medium and the effect of periodic addition of medium differentially affected follicle development, estradiol production, and gene expression. Furthermore, α-MEM(+) can be used to replace TCM-199(+) for culture of preantral follicles of cattle if progressive addition of medium is used for medium change.

In vitro development of bovine secondary follicles in two- and three-dimensional culture systems using vascular endothelial growth factor, insulin-like growth factor-1, and growth hormone.

The aim of this study was to evaluate the development and estradiol production of isolated bovine secondary follicles in two-dimensional (2D, experiment 1) and three-dimensional (3D using alginate, experiment 2) long-term culture systems in the absence (control group; only α-MEM(+)) or presence of vascular endothelial growth factor (VEGF), insulin-like growth factor-1, or GH alone, or a combination of all. A total of 363 isolated secondary follicles were cultured individually for 32 days at 38.5 °C in 5% CO2 in a humidified incubator with addition of medium (5 µL) every other day. In 2D culture system, follicular growth and antrum formation rates were higher (P < 0.05) in VEGF treatment compared with the other treatments. In 3D culture system, only estradiol concentration was greater (P < 0.05) in the GH than in the control group, whereas the other end points were similar (P > 0.05). In summary, this study demonstrated that the benefits of using a certain type of medium supplement depended on the culture system (2D vs. 3D). Vascular endothelial growth factor was an effective supplement for the in vitro culture of bovine secondary follicles when the 2D culture system was used, whereas GH only affected estradiol production using the 3D culture system. This study sheds light on advancements in methodology to facilitate subsequent studies on bovine preantral follicle development.

The expression and localization of leptin and its receptor in goat ovarian follicles.

Leptin, a hormone that was originally identified in adipocytes, has been implicated in the regulation of ovarian folliculogenesis through endocrine, autocrine and/or paracrine mechanisms. The aim of this study was to investigate the expression patterns of leptin (LEP) and its receptor (LEPRb) in different types of ovarian follicular cells from goats. In small follicles, the expression levels of LEP were higher (P<0.001) in granulosa cells than in theca cells, cumulus cells and oocytes. The expression of LEP in granulosa cells was higher (P<0.001) in small follicles than in large follicles. In large follicles, the expression of LEPRb was higher (P<0.05) in granulosa cells than in theca cells, cumulus cells and oocytes. Higher expression (P<0.05) of LEPRb was detected in granulosa cells isolated from large follicles than in granulosa cells isolated from small follicles. Immunohistochemical analyses revealed the presence of the LEP and LEPR proteins in follicles at all stages of development. The most intense staining for LEP and LEPR was observed in the cytoplasm of oocytes and the surrounding granulosa cells. In conclusion, it was demonstrated that leptin and its receptor are expressed at both the mRNA and protein levels in goat ovarian follicles. Furthermore, the presence of a leptin signaling system in the caprine ovary suggests a potential regulatory role for leptin in follicular development and the maturation of goat oocytes.

In vitro culture of equine preantral follicles obtained via the Biopsy Pick-Up method.

The objective was to conduct a preliminary evaluation of the efficacy of two media for in vitro culture of equine preantral follicles. Ovarian cortical strips were obtained from mares (N = 10) via the Biopsy Pick-Up method during the breeding season. Ovarian tissue was immediately submitted to histological analysis (noncultured control; D0) or cultured in situ for 1 day (D1) or 7 days (D7) in either α-MEM or TCM-199 and submitted to histological analysis, generating five treatment groups: noncultured control, α-MEM:D1, TCM-199:D1, α-MEM:D7, and TCM-199:D7. Preantral follicles were evaluated for follicle class (primordial, transitional, primary, and secondary) and morphology (normal vs. abnormal). A total of 142 preantral follicles were analyzed in five replicates. No follicles were observed in the TCM-199:D7 treatment group. The proportion of primordial follicles was higher (P < 0.03) in the control compared to the α-MEM:D7 treatment group. The proportion of primary follicles was higher (P < 0.04) in the α-MEM:D7 treatment group compared to the control. The proportion of developing follicles (transitional, primary, and secondary) was higher (P < 0.03) in the α-MEM:D7 treatment group compared to the control group. There was a greater (P < 0.004) percentage of morphologically normal developing follicles in the α-MEM:D1 treatment group compared to the TCM-199:D1 treatment group. Overall, the percentage of morphologically normal follicles was higher in the control group (72%; P < 0.02) and α-MEM:D1 group (84%; P < 0.0001) compared to the α-MEM:D7 (27%) treatment group. Mean follicle diameter was greater (P < 0.04) in the α-MEM:D7 treatment group (40.6 ± 1.1 µm) compared to the control group (37.3 ± 0.7 µm). Mean oocyte diameter was greater in the α-MEM:D1 (31.0 ± 0.7 µm; P < 0.006), TCM-199:D1 (30.7 ± 1.8 µm; P < 0.006), and α-MEM:D7 (33.2 ± 1.8 µm; P < 0.006) treatment groups compared to the control group (27.4 ± 0.9 µm). In conclusion, based on these preliminary data, in vitro culture of equine ovarian fragments obtained in vivo via the Biopsy Pick-Up method promoted preantral follicle development and follicle and oocyte growth in α-MEM for 7 days, with some follicles remaining morphologically normal throughout the culture period.

Equine preantral follicles obtained via the Biopsy Pick-Up method: histological evaluation and validation of a mechanical isolation technique.

The aims of this study in mares were to: (1) compare preantral follicle parameters between in vitro Biopsy Pick-Up (BPU) and scalpel blade collection methods and between histological and mechanical isolation processing (experiment 1); (2) histologically evaluate preantral follicles (experiment 2); and (3) compare histological analysis with a previously established mechanical isolation technique using a tissue chopper (experiment 3) for ovarian cortical fragments obtained in vivo using a BPU instrument. In experiment 1, preantral follicles were analyzed (N = 220; 90% primordial and 10% primary). Proportions of primordial and primary follicles did not differ (P > 0.05) between tissue collection (BPU vs. scalpel blade dissection) or processing (mechanical isolation vs. histology) methods. Follicle viability and morphology rates were similar (P > 0.05) between tissue collection methods, but mechanical isolation produced more (P < 0.05) morphologically normal follicles than histology. For experiment 2, preantral follicles (N = 332) were analyzed and primordial and transitional (combined) follicles and oocytes were 36.3 ± 0.3 and 26.1 ± 0.3 µm in diameter, respectively, and primary follicles and oocytes averaged 42.9 ± 1.8 and 31.8 ± 2.1 µm. For experiment 3 (188 preantral follicles), within the same animals, the proportion of primordial versus primary follicles was higher (P < 0.03) for histological analysis (98%) compared to tissue chopper analysis (94%), and number of follicles per mg of tissue was not affected (P > 0.05) by processing methods. In conclusion, most parameters evaluated for preantral follicles were similar between histological and tissue chopper processing techniques; hence, mechanical isolation efficiently dissociated equine preantral follicles from the ovarian cortex. Therefore, the tissue chopper could be used to isolate large numbers of morphologically normal equine preantral follicles for cryopreservation and/or in vitro culture.

Quantification, morphology, and viability of equine preantral follicles obtained via the Biopsy Pick-Up method.

A Biopsy Pick-Up (BPU) method was tested to determine the feasibility of retrieving preantral follicles from mare ovaries in vivo. A total of 33 ovarian biopsy procedures were performed on 18 mares during the breeding season. Mares were 5 to 21 years old and biopsies were performed during the estrous and/or diestrous phase, as confirmed by transrectal ultrasonography. Follicles were mechanically isolated using a tissue chopper, counted, and classified as normal or abnormal and primordial or primary. Viability of isolated follicles was determined by Trypan Blue dye. A total of 256 biopsy attempts were made resulting in 185 successful tissue sample collections (72% success rate). The mean weight of ovarian tissue collected per procedure was 25.0 ± 1.6 mg. Overall, 620 preantral follicles were collected and isolated (95% primordial and 5% primary). The mean (±SEM) number of follicles isolated per biopsy procedure was 18.8 ± 1.9. Primordial and primary follicles had an average diameter of 31.3 ± 6.2 and 41.1 ± 6.6 µm, respectively. Viability rate was higher (P < 0.001) for primordial follicles (91%) compared with primary follicles (50%). Primordial follicles tended (P < 0.06) to have a higher rate of morphological normality (96%) compared with primary follicles (80%). The total number of follicles isolated, amount of tissue harvested, and number of follicles per mg of tissue did not differ (P > 0.05) according to phase of the estrous cycle. Younger mares (5 to 7 years old) had more (P < 0.05) follicles isolated per procedure than older mares (14 to 21 years old). The length of the interovulatory interval was not affected (P > 0.05) by any biopsy procedure, and there were no adverse effects on cyclicity or general reproductive health. In conclusion, the BPU method provided large numbers of normal and viable preantral follicles for the study of early follicular development in mares. The BPU method might be used in the future to obtain preantral follicles for in vitro culture to enable the use of numerous oocytes present within the equine ovary. This could allow for the preservation of genetic material or large-scale embryo production.

Long-term in vitro culture of ovarian cortical tissue in goats: effects of FSH and IGF-I on preantral follicular development and FSH and IGF-I receptor mRNA expression.

Long-term in vitro culture (16 days) of caprine ovarian cortical tissue was performed to test the effect of FSH and IGF-I on the viability and development of preantral follicles and mRNA expression for FSH and IGF-I receptors. Fragments were cultured in α-MEM(+) alone or supplemented with different combinations of FSH and IGF-I (sequential medium). The culture period was divided into two parts. Follicles were isolated and classified as normal or abnormal and primordial, primary or secondary. Viability of isolated follicles was determined by staining with Trypan Blue dye. Expression of FSHR and IGFR-1 mRNA was evaluated by qPCR. At day 8 of culture, more (P < 0.05) follicles in treatments containing IGF-I alone or associated with FSH were normal and viable (overall mean, 81 % and 79 % respectively) than the treatments cultured with FSH or α-MEM(+) alone (68 % and 63 %). At day 16 of culture, treatments with FSH and/or IGF-I had more (P < 0.05) viable follicles (69 %) than α-MEM(+) (38 %). The percentages of follicular development observed in the IGF-I/FSH, FSH+IGF-I/FSH+IGF-I and FSH/IGF-I treatments were similar but higher (P < 0.05) than the other treatments. FSH and IGF-I during the entire culture period maximized (P < 0.05) follicular and oocyte diameters and the percentage of secondary follicles (28 %). FSHR mRNA expression in the non-cultured control was similar to the treatment supplemented with FSH and IGF-I but higher (P < 0.05) than α-MEM(+). IGFR-1 expression did not differ among treatments. Association of FSH and IGF-I in long-term in vitro culture promoted follicular development, maintaining FSHR mRNA expression.

Detection of mutations within exons 4 to 8 of the p53 tumor suppressor gene in canine mammary glands / Identificação de mutações nos exons 4 a 8 do gene p53 supressor de tumor em glândulas mamárias caninas

Fifteen female canines with mammary tumors and 6 normal females were used to study mutations in exons 4 to 8 of the p53 gene. DNA samples from the tumors, respective adjacent normal mammary tissue and mammary glands from healthy animals were sequenced and analyzed for the presence of mutations. Mutations were found in 71.8% of the samples and the most frequent were missense mutations. The most attacked exons in the mammary tumor were 5, 7 and 8, with 23.4, 31.6 and 23.4% mutations, respectively. Canine mammary tumors are related to mutations in gene p53 and mutations mostly occur in the region of the protein that is linked to the DNA in the cell nucleus, which can change the functionality of the cell and propitiate tumor growth. Despite being macroscopically normal, the mammary tissue adjacent to the tumors has mutations that can lead to recurrence if not removed together with the tumor.

Para estudar as mutações nos exos 4 a 8 do gene p53, foram utilizados 15 tumores mamários, mamas normais das mesmas cadelas e seis mamas de cadelas normais. O DNA extraído das amostras de tecido foi sequenciado e analisado para a presença de mutações. Em 71,8% das amostras obtidas foram observadas mutações, sendo as "missense" as mais frequentes. Os exons mais comprometidos foram 5, 7 e 8 com 23,4, 31,6 e 23,4% de mutações, respectivamente. O estudo conclui que tumores mamários caninos têm relação com mutações no gene p53 e que as mutações ocorrem com maior frequência nas regiões da proteína que estão ligadas ao DNA no núcleo celular. Isto pode alterar a funcionalidade da proteína e propiciar o crescimento do tumor. As mamas adjacentes aos tumores, apesar da aparência macroscópica normal, apresentaram mutações, que podem representar recidivas se a mama não for retirada juntamente com o tumor.

Viabilidade in vitro de células espermáticas ovinas submetidas a diferentes diluentes e a refrigeração / In vitro viability of ovine sperm cells submitted to different diluents and refrigeration

Avaliou-se a viabilidade in vitro de células espermáticas após a adição de três diluentes comerciais, que foram comparados com o diluente tradicional Tris-gema, utilizados no processo de refrigeração do sêmen ovino, em até 48h de armazenamento. Foram utilizados nove ejaculados diários, obtidos de três reprodutores Dorper, com vagina artificial, em três repetições com intervalo de três dias. O sêmen foi mantido a 30ºC, e foram avaliadas suas características macro e microscópicas. Após formação do pool, repetiram-se as avaliações acrescidas da concentração espermática e da integridade do DNA e do acrossoma. Dividiu-se o pool em cinco tratamento, cada um constituído de uma parte de sêmen para três partes dos respectivos diluentes: Equimix (DI), Laiciphos Green Ovine (DII), FR 4 (DIII), Equimix-Gema- Equimix com gema de ovo (DIV) e Tris-Gema (DV). O material obtido em cada tratamento foi subdividido em quadruplicata, refrigerado e mantido a 4ºC até as avaliações da motilidade individual progressiva (MIP), do vigor e da integridade do DNA e do acrossoma, correspondendo a zero, 12, 24, 36 e 48h. Nas avaliações do sêmen, com o DI ocorreu a maior queda de MIP às 12h em relação aos demais grupos (P<0,05). Às 24h, os tratamentos DII, DIV e DV apresentaram a melhor MIP (P<0,05), que não divergiram (P>0,05) entre si; às 48h, o DII e o DV foram melhores (P<0,05) que os demais. Com relação ao vigor, os tratamentos DII e DV apresentaram valores mais elevados (P<0,05) em relação ao DI e DIII, a partir das 12h, e ao DIV, a partir das 24h (P<0,05). Concluiu-se que o diluente Laiciphos Green Ovine, da mesma forma que o Tris-gema, pode ser utilizado na conservação do sêmen a 4ºC por 48h, enquanto o Equimix, acrescido de 20 por cento de gema de ovo, pode ser seja utilizado no armazenamento do sêmen (4ºC) por até 24h.

The in vitro viability of sperm cells following the addition of three commercial diluents was evaluated and compared with the traditional Tris-yolk diluent for the refrigeration of ovine sperm up to 48h of storage. Nine daily ejaculates were obtained from three Dorper breeders using an artificial vagina; three repetitions were performed in a three-day interval. The semen was kept at 30ºC and macro and microscopic characteristics were evaluated. The samples were pooled and the evaluations were repeated, along with assessments of sperm concentration, DNA integrity, and acrosome integrity. The pool was distributed into five treatments, each with one part of semen to three parts of the following diluents: Equimix (DI), Laiciphos Green Ovine (DII), FR 4 (DIII), Equimix-Yolk-Equimix with egg yolk (DIV), and Tris-Yolk (DV). The material of each treatment was aliquoted in quadruplicate, refrigerated, and maintained at 4ºC until the evaluations of the individual progressive motility (IPM), vigor, DNA integrity, and acrosome integrity, corresponding to 0, 12, 24, 36, and 48h. The highest decrease of IPM occurred with DI (at 12h) in comparison to the other diluent groups (P<0.05). At 24h, DII, DIV and DV had the best IPM (P<0.05) and did not diverge from one another (P>0.05). At 48h, DII and DV had the highest values (P<0.05). Regarding vigor, DII and DV had higher values (P<0.05) than DI and DIII at 12h and higher values than DIV at 24h (P<0.05). From the results, like Tris-Yolk, and Laiciphos Green Ovine can be used for the conservation of semen at 4ºC for 48h, whereas Equimix plus 20 percent egg yolk may be used for the storage of semen at 4ºC for up to 24h.

Pre-parturition profile of steroids and prostaglandin in cows with or without foetal membrane retention.

Retained foetal membranes in cattle is one of the most common complications associated to the reduction in milk yield and impaired fertility in dairy cattle. In order to determine some endocrine mechanisms controlling parturition and delivery of foetal membranes, plasma concentrations of steroids and prostanoids were determined in 20 healthy Holstein cows. Samples were taken within the interval of 5 days pre-parturition to 12h after calving. Progesterone (P4) levels were similar in cows with (PR) and without (NPR) placental retention. While the estradiol-17beta (E2) peak at parturition was lower in PR than in NPR cows, cortisol levels were greater in PR cows 12 and 24h pre-parturition. The Prostaglandin F2alpha metabolite (PGFM) levels were higher at parturition in NPR compared with the PR group, but 12h later, these levels in the PR group increased so that concentrations were greater as compared with NPR cows. The Prostaglandin E2 metabolite (PGEM), 24, 48 and 72 h pre-parturition, were higher in PR cows. However, the PGFM:PGEM ratio was greater in cows up NPR at all time when included, indicating the importance of higher levels of Prostaglandin F2alpha (PGF2alpha) than Prostaglandin E2 (PGE2) for normal placental delivery. In conclusion, placental retention was related to both estrogen and PGF2alpha deficiency, which may be a consequence of metabolic stress leading to PGE2 and maternal cortisol synthesis before parturition.

Plasma concentrations of estradiol 17beta and PGF2alpha metabolite and placental fatty acid composition and antioxidant enzyme activity in cows with and without retained fetal membranes.

Fetal membrane retention is one of the most common problems in Holstein cattle after parturition. To investigate mechanisms involved, the following parameters were studied in the peri-parturition period: plasmatic concentrations of estradiol-17beta (E2) and PGFM (PGF2alpha metabolite), activity of antioxidant enzymes (superoxide dismutase-SOD, catalase-CAT and glutathione peroxidase-GSH-Px), thiobarbituric acid reagent substances (TBAR) concentrations and fatty acid composition of the placentae. E2 at parturition in the NPR group (control cows, n = 10) was higher than in PR cows (placental retention, n = 10) (P < 0.05). Activity of SOD in fetal tissue of NPR animals was higher than that of the PR group. In contrast, there was no difference between the two groups in activity of GSH-Px and CAT and the TBAR content of placental tissues. PR maternal tissues had proportionally more arachidonic and linoleic acid than tissues from NPR cows. Therefore, a complex of sequential events may cause placenta retention, starting with an unbalance of antioxidant capacity of the placenta, followed by a decrease in production of estrogen, which leads to the accumulation of arachidonic and linoleic acid in placental tissues.