Effect of season on bovine seminal plasma proteins in Thailand

Although season has been shown to affect bull sperm quality and fertility in some studies, the effect of season on seminal plasma proteins has not been examined. In the present study, seminal plasma proteins were analysed by Fast Protein Liquid Chromatography (FPLC), to separate the phosphorylcholine-binding proteins and heparin-binding proteins from the other proteins. Semen samples were collected from bulls in three seasons: winter, summer and the rainy season. Sperm quality was analysed by flow cytometry and computer assisted sperm analysis, and further aliquots of semen were used to prepare the seminal plasma for FPLC. Meteorological data were available from a location close to the bull station. There were slight differences in sperm kinematics between seasons, but other parameters of sperm quality were not different. Minor differences in the phosphorylcholine-binding proteins were detected according to season, being lower in summer than in winter or in the rainy season, although there were no changes in the heparin-binding proteins. Temperature, humidity and rainfall differed between winter and the rainy season, but no differences were observed between summer and the rainy season except in the temperature humidity index (THI). However, the THI was above the threshold indicative of heat stress in all seasons, which could explain why few seasonal differences in protein composition were detected in this study. Alternatively, the bulls could have been well-adapted to heat stress. In conclusion, there were only slight differences in bull sperm quality and seminal plasma proteins between seasons during this study

Post-thaw semen quality in young bull ejaculates before being accepted for commercial semen doses

Background: Genomic selection enables bulls with desirable characteristics to be identified at a young age, but sperm quality can be poor in the ejaculates of young bulls. Few studies have been done on post-thaw sperm quality in bulls less than 10 months old. The objective of this study was to determine the age at which post-thaw sperm quality was acceptable for artificial insemination.Methods: Semen was collected by artificial vagina; samples containing 100-500 million spermatozoa/ml were frozen for this study. Post-thaw analyses of membrane integrity (MI), mitochondrial membrane potential (MMP), chromatin integrity, morphology, production of reactive oxygen species and sperm kinematics were made.Results: The age at which ejaculates exceeded the breeding company's thresholds of acceptance varied considerably among individuals, with 285 days being the earliest. Morphology (p < 0.003), MI (p = 0.0096), high MMP (p = 0.043) and superoxide production (p = 0.0084) increased between the first and last ejaculates but reached acceptable levels at different ages for individual bulls.Conclusions: It was possible to obtain acceptable post-thaw sperm quality from samples even though sperm concentration was lower than the breeding company's threshold. Therefore, it might be feasible to use ejaculates earlier than is currently considered possible, by modifying semen handling protocols

Sperm Quality in Young Bull Semen Can Be Improved by Single Layer Centrifugation

Simple Summary Genomic selection enables bulls with desirable genes to be identified early in life. Livestock producers need to use the semen from young bulls as early as possible for efficient milk and meat production with fewer greenhouse gas emissions. However, semen from young bulls is often of lower quality than needed for freezing for commercial artificial insemination. Colloid centrifugation selects spermatozoa with the desirable characteristics needed for fertilization from the rest of the ejaculate. In this study, split ejaculates from young bulls were prepared with or without colloid centrifugation. Using this technique, sperm doses of acceptable quality for artificial insemination could be produced from ejaculates that would otherwise be discarded. Thus, the semen from young bulls would be usable for artificial insemination sooner than is currently the case. Interest in using semen from young bulls is increasing due to identifying promising animals by genomic selection. However, sperm quality in these ejaculates may not reach currently accepted standards for the cattle breeding industry. The purpose of this study was to determine if centrifugation of semen from young bulls through the Bovicoll colloid could improve sperm quality sufficiently for the frozen semen to be acceptable for artificial insemination. Ejaculates from 19 young bulls were split and either processed by Single-Layer Centrifugation (SLC) or not (CON) before freezing. After thawing, sperm quality was evaluated by determination of membrane integrity, mitochondrial membrane potential, DNA integrity, production of reactive oxygen species, sperm morphology and motility. Approximately half of the CON samples reached acceptable post-thaw quality (membrane integrity >= 40%) despite being below the breeding company ' s desired sperm concentration threshold pre-freezing. In the remaining samples, sperm quality was improved by SLC such that 45% of them reached acceptable quality post-thaw. Almost 75% of the young bull sperm samples could have produced usable frozen semen doses by adjusting the breeding company ' s current processing protocols. Since lowering the generation interval has a direct effect on the genetic gain per year, SLC could aid genetic progress in cattle breeding

Season does not have a deleterious effect on proportions of stallion seminal plasma proteins

The mechanism by which the content of the major groups of seminal plasma proteins in stallion semen changes between the breeding and non-breeding seasons remains unknown. Here, we investigated the proportions of non-heparinbinding, phosphorylcholine-binding, and heparin-binding proteins in seminal plasma with the aim of relating them to sperm quality and testosterone levels in good and bad freezer stallions. Only minor variations in the major protein groups were found between the breeding and non-breeding seasons. In the non-breeding season, a higher content of a subset of nonheparin binding proteins as well as of heparin-binding proteins was found. Analysis of semen characteristics revealed a somewhat contrasting picture. While only minor variations in sperm kinematics and sperm morphology were found between seasons, the flow-cytometric measurements of mitochondrial membrane potential and also, to some extent, reactive oxygen species production indicated lower sperm quality in the breeding season. Chromatin integrity and testosterone levels were unchanged between seasons. The results suggest that stallion ejaculates could be used year-round for freezing, since only minor differences in protein composition exist between the breeding and non-breeding seasons, as well as between good and bad freezers. In addition, sperm quality is not impaired during the non-breeding season

In vitro development of primordial follicles after long-term culture of goat ovarian tissue

This study aims to investigate the effects of follicle stimulating hormone (FSH) and fibroblast growth factor-2 (FGF-2) on the survival and growth of caprine preantral follicles. Ovarian tissues were cultured for 1, 7, 14, 21 or 28 days in medium supplemented with FSH (FSH-2d or FSH-7d, i.e., with replacement of the culture medium every 2 or 7 days, respectively) or FSH + FGF-2 (replacement of the medium every 2 days). Non-cultured (control) and cultured ovarian fragments were processed for histological and ultrastructural analysis. After 28 days of culture, the media supplemented with FSH-2d was the most effective in maintaining the percentage of normal follicles and in promoting follicular growth. Furthermore, both treatments with FSH increased the percentage of the primary follicles. However, ultrastructural studies did not confirm follicular integrity from 14 days of culture onward. In conclusion, culturing tissue for up to 7 days in medium containing FSH alone or combined with FGF-2 maintains caprine preantral follicle integrity and promotes their growth in vitro

Progesterona e hormônio folículo-estimulante interagem e promovem a sobrevivência e o desenvolvimento in vitro de folículos pré-antrais caprinos

Este trabalho veri_icou os efeitos da progesterona e do hormonio foliculo-estimulante (FSH) na sobrevivencia e no crescimento de foliculos pre-antrais caprinos. Fragmentos de tecido ovariano foram cultivados por 1 ou 7 dias em Meio Essencial Minimo (MEM) sozinho ou contendo progesterona (1, 2.5, 5, 10 ou 20ng/mL), FSH (50ng/mL) ou a combinacao entre esses dois hormonios. O tecido fresco (controle nao-cultivado) e o cultivado foram processados para analise histologica e ultra-estrutural. Apos 7 dias a adicao de FSH a todas as concentracoes de progesterone manteve o percentual de foliculos normais similar ao controle fresco. No dia 7 de cultivo, um alto percentual de foliculos em desenvolvimento foi observado somente no tratamento com 2,5ng/ml de progesterona associada ao FSH ou com 10ng/ml de progesterona sozinha, em relação ao controle fresco. Do dia 1 para o dia 7 de cultivo, um aumento signi_icativo no percentual de foliculos em desenvolvimento foi observado no MEM sozinho e adicionado de 2,5ng/ml de progesterona + FSH. Alem disso, apos 7 dias, em todos os tratamentos, houve um aumento signi_icativo no diametro folicular em relacao ao controle, exceto nos tratamentos com MEM sozinho, 5ng/ml de progesterona + FSH ou 10ng/ml de progesterona sozinha. A analise ultra-estrutural con_irmou a integridade follicular apos 7 dias de cultivo no tratamento com 2,5ng/ml de progesterona + FSH. Em conclusao, este estudo demonstrou que a interação entre progesterona e FSH mantem a integridade ultra-estrutural, estimula a ativacao de foliculos primordiais e o posterior crescimento de foliculos pre-antrais caprinos cultivados in vitro.We investigated the effects of progesterone and follicle stimulating hormone (FSH) on survival and growth of caprine preantral follicles. Pieces of ovarian tissue were cultured for 1 or 7 days in minimum essential medium (MEM) alone or containing progesterone (1, 2.5, 5, 10 or 20ng/mL), FSH (50ng/mL) or the interaction between progesterone and FSH. Fresh (non-cultured control) and cultured ovarian tissues were processed for histological and ultrastructural studies. After 7 days the addition of FSH to all progesterone concentrations maintained the percentage of normal follicles similar to fresh control. At day 7 of culture, a higher percentage of developing follicles was observed only in 2.5ng/ml of progesterone associated with FSH or 10ng/ml of progesterone alone when compared with control. From day 1 to day 7 of culture, a signi_icant increase in the percentage of developing follicles was observed in MEM and 2.5ng/ml of progesterone + FSH. In addition, after 7 days, in all treatments, there was a signi_icant increase in follicular diameter when compared with control, except for MEM alone and in 5ng/ml of progesterone + FSH or 10ng/ml of progesterone alone. Ultrastructural studies con_irmed follicular integrity after 7 days of culture in 2.5ng/ml of progesterone with FSH. In conclusion, this study demonstrated that the interaction between progesterone and FSH maintains ultrastructural integrity, stimulates primordial follicles activation and further growth of cultured caprine preantral follicles

Sphingosine 1-phosphate promotes activation of aprine preantral follicle in vitro

Este estudo descreve o efeito da esfingosina 1-fosfato (S1P) no desenvolvimento de folículos pré-antrais, portanto da ativação e viabilidade de folículos caprinos cultivados in vitro. Fragmentos de ovários foram cultivados por um ou sete dias em meio essencial mínimo com diferentes concentrações de S1P (0, 1, 10, 50, 100 ou 200ng/mL). Os fragmentos de ovário foram processados para análise histológica em microscopia óptica, microscopia eletrônica e microscopia de fluorescência. O tratamento usando 1ng/mL de S1P foi capaz de manter a porcentagem de folículos normais durante o período de cultivo de sete dias. Ao final do período de cultivo, houve uma redução significativa (p<0,05) na porcentagem de folículos primordiais em todos os grupos tratados com S1P, comparados com os grupos controle (FC e CC), seguida por um aumento do número de folículos ativados (intermediários, primários e secundários). Adicionalmente, na cultura por sete dias com meio suplementado com S1P (1ng/mL), houve preservação da ultraestrutura das organelas e manteve-se a viabilidade dos folículos pré-antrais avaliados por microscopia de fluorescência. Em conclusão, após sete dias de cultura, o meio suplementado com 1ng/mL de S1P ativa o desenvolvimento de folículos pré-antrais de caprino, cultivados in situ e mantém as viabilidades oocitária e folicular.This study describes the effect of sphingosine 1-phosphate (S1P) for development of preantral follicle, therefore the activation and follicular viability of caprine follicles cultured in vitro. Ovarian fragments were cultured for 1 or 7 days in Minimum Essential Medium with different S1P concentrations (0, 1, 10, 50, 100 or 200ng/mL). All ovarian fragments were processed for histological analysis in optical microscopy, transmission electron microscopy and fluorescence analysis. The treatment using 1ng/mL of S1P was able to maintain the percentage of normal follicles with the progression of the culture from day 1 to 7. At end of the 7-day culture period there was a significant reduction (P<0.05) in the percentage of primordial follicles in all groups treated with S1P, compared with fresh control (FC) and Control Culture (CC), which was followed by an increase of activated follicles (intermediary, primary and secondary). In addition, the culture for 7 days with media supplemented with S1P with 1ng/mL preserved the ultrastructure of organelles and kept the preantral follicular viability when evaluated by fluorescence microscopy. In conclusion, after 7 days of culture, the 1ng/mL of S1P activates the development of preantral caprine follicles, cultured in situ and maintains the oocitary and follicular viability

Diferentes origens do Hormônio Folículo Estimulante (FSH) influenciam a viabilidade e o desenvolvimento de folículos pré-antrais caprinos

The aim of this study was to evaluate the effects of pituitary (pFSH) or recombinant (rFSH) FSH on the survival and growth of caprine preantral follicles. Caprine ovarian tissues were in vitro cultured for one or seven days in Minimum Essential Medium (MEM) alone or containing 10, 50, 100 and 1000 ng/ml of pFSH or rFSH. Control tissues (non-cultured) and those cultured were processed for histological and ultrastructural studies. In addition, follicular and oocyte diameter were analysed. After seven days of culture, only 50 ng/ml of rFSH maintained the percentage of normal follicles similar to control. Moreover, 10 ng/ml of pFSH and all the concentrations of rFSH promoted primordial follicles activation. In addition, the presence of 50 ng/ml of rFSH promoted the highest follicular diameter at day seven of culture. In conclusion, 50 ng/ml of rFSH maintained the ultrastructural integrity of caprine preantral follicles, promoted primordial follicles activation and further growth of cultured follicles.O objetivo deste estudo foi avaliar os efeitos do FSH pituitário (pFSH) ou recombinante (rFSH) sobre a sobrevivência e o crescimento de folículos pré-antrais caprinos. O tecido ovariano foi cultivado in vitro por um ou sete dias em Meio Essencial Mínimo (MEM) sozinho, ou contendo 10, 50, 100 e 1000 ng/ml de pFSH ou rFSH. O grupo controle (não cultivado) e aqueles cultivados foram processados para análises histológica e ultra-estrutural. Além disso, os diâmetros folicular e oocitário foram avaliados. Após sete dias de cultivo, apenas 50 ng/ml de rFSH manteve o percentual de folículos normais semelhante ao controle. Além disso, 10 ng/ml de pFSH e todas as concentrações de rFSH promoveram ativação de folículos primordiais. A presença de 50 ng/ml de rFSH promoveu o maior diâmetro folicular após sete dias de cultivo. Em conclusão, 50 ng/ml de rFSH manteve a integridade de folículos pré-antrais caprinos e promoveu a ativação e o crescimento dos folículos cultivados

Interaction between ascorbic acid and follicle-stimulating hormone maintains follicular viability after long-term in vitro culture of caprine preantral follicles

This study evaluates the effects of ascorbic acid and its interaction with follicle-stimulating hormone (FSH) on the morphology, activation, and in vitro growth of caprine preantral follicles. Ovarian fragments were cultured for 1, 7, or 14 d in minimum essential medium (MEM) containing ascorbic acid (50 or 100 μg/mL), FSH (50 ng/mL), or both of these substances. Ovarian tissue that was either fresh (control) or cultured for 1, 7, or 14 d was processed for histological and ultrastructural evaluation. The results showed that after 14 d of culture, medium supplemented with 50 μg/mL of ascorbic acid alone or combined with FSH showed higher rates of follicular survival compared with MEM. After 7 d of culture, FSH, ascorbic acid at 50 μg/mL with or without FSH, and ascorbic acid at 100 μg/mL increased the percentage of follicular activation compared to fresh control. In addition, FSH alone significantly increased the percentage of growing follicles after 14 d. The combination of 50 μg/mL of ascorbic acid and FSH promoted a significant increase in oocyte and follicular diameter after 7 d of culture. Ultrastructural and fluorescent analysis confirmed the integrity of follicles cultured with 50 μg/mL of ascorbic acid and FSH after 14 d. In conclusion, the combination of 50 μg/mL of ascorbic acid and FSH maintained follicular integrity and promoted follicular activation and growth after long-term in vitro culture of caprine preantral follicles

Histological evidence of wound healing improvement in rats treated with oral administration of hydroalcoholic extract of Vitis labrusca

Plant extracts rich in phenolic compounds have been demonstrated to accelerate wound healing, but their use by oral route has been poorly studied. The leaves of Vitis labrusca are rich in phenolic acids and flavonoids. The goal of this study was to assess the healing properties of the oral administration of hydroalcoholic extract of V. labrusca leaves (HEVL) in a murine model. HEVL was obtained by Soxhlet and dynamic maceration, and their yield and phenolic acids and flavonoid contents were determined. For the wound healing assay, 8 mm wounds were performed on the back of 48 Wistar rats, assigned into four groups (n=12): CTR (distilled water), HEVL100, HEVL200, and HEVL300 (HEVL at 100, 200, and 300 mg/kg, respectively). On days 7 and 14, wound closure rates were assessed, and the healing wounds were subjected to histological analysis. Soxhlet-obtained extract was selected for the wound healing assay because it provided a higher yield and phenolic acid and flavonoid contents. HEVL significantly reduced leukocytosis in the peripheral blood (p < 0.05), accelerated wound closure (p < 0.05), and improved collagenization (p < 0.05) on day 7, as well as enhanced the epidermal tissue thickness (p < 0.001) and elastic fiber deposition on day 14 (p < 0.01). Furthermore, HEVL promoted an increase in the histological grading of wound healing on both days 7 and 14 (p < 0.01). The doses of 200 and 300 mg/kg provided better results than 100 mg/Kg. Our data provide histological evidence that the oral administration of HEVL improves wound healing in rodents. Therefore, the extract can be a potential oral medicine for healing purposes.Brazilian Council for Research Support (CNPq) and the Foundation of research and technological innovation of Sergipe/Brazil (FAPITEC), and by the Portuguese Science and Technology Foundation (FCT/MCT), European Funds (PRODER/COMPETE)– project UIDB/04469/2020 (strategic fund), co-financed by FEDER, under the Partnership Agreement PT2020info:eu-repo/semantics/publishedVersio