Identification of key genes in bovine mammary epithelial cells challenged with Escherichia coli and Staphylococcus aureus by integrated bioinformatics analysis

This study integrated four microarray datasets by Robust Rank Aggregation (RRA) method to identify the differentially expressed genes (DEG) in bovine mammary epithelial (BME) cells in response to Escherichia coli and Staphylococcus aureus infection. Furthermore, the GO function and KEGG pathway enrichment analysis of the integrated DEG were performed. Finally, the protein-protein interaction (PPI) network was constructed. A total of 72 integrated DEG were identified from the four datasets. The most significantly enriched terms within the integrated DEG were mainly involved in the immune response. The PPI network of DEG was constructed with 53 nodes. Seventeen genes, which constitute a significant module, were identified as hub genes. Among them, CD40, CXCL6, and NFKBIZ were further screened as the key genes and have the potential to become biomarkers of E. coli and S. aureus mastitis, considering the specificity of biomarkers for diseases. The identified key genes and pathways in this study can assist in the search for biomarkers for mastitis diagnosis and disease resistance breeding.

Cytotoxicity of isolated compounds from Picrasma crenata (Vell) Engl. in animal tumor cell (HTC) / Citotoxicidade de compostos isolados de Picrasma crenata (Vell) Engl. em células tumorais animais (HTC)

The study aim was to evaluate the cytotoxic activity, using the MTT test [3-(4,5-Dimethilthiazol-2-yl)-2,5-diphenil tetrazolium bromide], from the crude extract of Picrasma crenata (Pau Tenente) and its isolated compounds, quassin and parain, in culture of rat liver tumor cells (HTC). The test was carried out exposing the cells for 24, 48 and 72 hours to concentrations of 5, 10, 50, 100, 200, 300, 400, 500 and 1000 µg of crude extract of Pau Tenente/mL of culture medium and 1, 5, 10, 15, 20, 40, 60, 80 and 100 µg of quassin or parain compounds/mL of culture medium. The absorbances averages results obtained showed that the crude extract did not present cytotoxicity for the HTC cells in all the concentrations and evaluated times. For quassin, the concentrations of 80 and 100 µg/mL were cytotoxic, after 72 hours of treatment. For parain, the concentrations of 1, 5, 20, 40, 60, 80 and 100 µg/mL, in 72 hours, were cytotoxic, revealing a new activity for this compound. Thus, the results demonstrate a first indication of the cytotoxic activity of compounds quassin and parain, adding an important social and economic value to them, and may have application in future research and in pharmaceutical industry.

O objetivo do estudo foi avaliar a atividade citotóxica, por meio do teste MTT [brometo de 3-(4,5-dimetiltiazol-2-il)-2,5-difenil tetrazólio], do extrato bruto de Picrasma crenata (Pau Tenente) e seus compostos isolados, quassina e paraína, em cultura de células tumorais de fígado de rato (HTC). O teste foi realizado expondo as células por 24, 48 e 72 horas às concentrações de 5, 10, 50, 100, 200, 300, 400, 500 e 1000 µg de extrato bruto de Pau Tenente/mL de meio de cultura e 1, 5, 10, 15, 20, 40, 60, 80 e 100 µg de compostos de quassina ou paraína/mL de meio de cultura. Os resultados das médias de absorbâncias obtidos mostraram que o extrato bruto não apresentou citotoxicidade para as células HTC em todas as concentrações e tempos avaliados. Para quassina, as concentrações de 80 e 100 µg/mL foram citotóxicas, após 72 horas de tratamento. Para a paraína, as concentrações de 1, 5, 20, 40, 60, 80 e 100 µg/mL, em 72 horas, foram citotóxicas, revelando uma nova atividade para este composto. Assim, os resultados demonstram uma primeira indicação da atividade citotóxica dos compostos quassina e paraína, agregando-lhes importante valor social e econômico, podendo ter aplicação em pesquisas futuras e na indústria farmacêutica.

Acid resistance of Salmonella Typhimurium ATCC 14028 after desiccation stress during peanut storage / Avaliação da resistência ácida de Salmonella Typhimurium ATCC 14028 após o estresse dessecativo durante o armazenamento de amendoim

ABSTRACT: Salmonella is a relevant pathogen, which causes foodborne outbreaks associated with both high and low moisture foods (LMF). This study evaluated the effect of previous desiccation stress on the acid resistance of S. Typhimurium ATCC 14028 using blanched peanut kernels as an LMF model. Salmonella was recovered from the peanut samples throughout 180 days of blanched peanut kernels storage at 28 ºC. During this period two death rates were verified, 0.04 log cfu/g/day in the first 30 days and 0.007 log cfu/g/day between 30 and180 days.Regarding acid resistance, there was no difference (P > 0.05) in the Salmonella growth/death kinetics between the undesiccated sample (TSB)and the cells recovered from peanut samples over 180 days of storage after 4 h at pHs 3.0, 3.5, 4.5 and 7.2. The average growth rate observed for pH 7.2 was 0.44 log cfu/ml/h. At pH 4.5, the Salmonella counts did not change significantly over 4 h. In contrast, Salmonella populations declined by 0.14 to 0.29 log cfu/ml/h at pH 3.5. At pH 3.0 declines were estimated to be 0.65 log cfu/ml/h for the undesiccated sample and 2.07 log cfu/ml/h for Salmonella recovered from peanuts stored for 120 days. Therefore, our data indicated that desiccation stress caused during the peanut storagedid not influence the Salmonella acid resistance.

RESUMO: Em alimentos de baixa umidade (LMF), o primeiro desafio encontrado por patógenos como a Salmonella é o estresse de dessecação. Neste estudo, o efeito prévio do estresse de dessecação sobre a resistência ácida de S. Typhimurium ATCC 14028 foi avaliado utilizando amendoim blancheado como um modelo de LMF. Salmonella foi recuperada das amostras de amendoim após 180 dias de estocagem a 28 ºC. Durante este período foram verificadas duas taxas de mortalidade, 0,04 log ufc/g/dia nos primeiros 30 dias e 0,007 log ufc/g/dia entre 30 e 180 dias. Com relação à resistência ácida, não houve diferença (P > 0,05) na cinética de crescimento/morte de Salmonella entre a amostra sem estresse dessecativo (TSB) e as amostras de amendoim após 4 h em pHs 3,0, 3,5, 4,5 e 7,2. A taxa média de crescimento observada para o pH 7,2 foi de 0,44 log cfu/ml/h. No pH 4,5, a contagem de Salmonella não mudou significativamente durante 4 h. Em contraste, a população de Salmonella diminuiu de 0,14 a 0,29 log cfu/ml/h no pH 3,5. No pH 3,0, a queda foi estimada em 0,65 log cfu/ml/h para a amostra sem estresse dessecativo e 2,07 log cfu/ml/h para a Salmonella recuperada das amostras de amendoim estocadas por 120 dias. Portanto, nossos dados indicaram que o estresse dessecativo causado durante a estocagem do amendoim não influenciou a resistência ácida de Salmonella.

Acid resistance of Salmonella Typhimurium ATCC 14028 after desiccation stress during peanut storage / Avaliação da resistência ácida de Salmonella Typhimurium ATCC 14028 após o estresse dessecativo durante o armazenamento de amendoim

Salmonella is a relevant pathogen, which causes foodborne outbreaks associated with both high and low moisture foods (LMF). This study evaluated the effect of previous desiccation stress on the acid resistance of S. Typhimurium ATCC 14028 using blanched peanut kernels as an LMF model. Salmonella was recovered from the peanut samples throughout 180 days of blanched peanut kernels storage at 28 ºC. During this period two death rates were verified, 0.04 log cfu/g/day in the first 30 days and 0.007 log cfu/g/day between 30 and180 days.Regarding acid resistance, there was no difference (P > 0.05) in the Salmonella growth/death kinetics between the undesiccated sample (TSB)and the cells recovered from peanut samples over 180 days of storage after 4 h at pHs 3.0, 3.5, 4.5 and 7.2. The average growth rate observed for pH 7.2 was 0.44 log cfu/ml/h. At pH 4.5, the Salmonella counts did not change significantly over 4 h. In contrast, Salmonella populations declined by 0.14 to 0.29 log cfu/ml/h at pH 3.5. At pH 3.0 declines were estimated to be 0.65 log cfu/ml/h for the undesiccated sample and 2.07 log cfu/ml/h for Salmonella recovered from peanuts stored for 120 days. Therefore, our data indicated that desiccation stress caused during the peanut storagedid not influence the Salmonella acid resistance.

Em alimentos de baixa umidade (LMF), o primeiro desafio encontrado por patógenos como a Salmonella é o estresse de dessecação. Neste estudo, o efeito prévio do estresse de dessecação sobre a resistência ácida de S. Typhimurium ATCC 14028 foi avaliado utilizando amendoim blancheado como um modelo de LMF. Salmonella foi recuperada das amostras de amendoim após 180 dias de estocagem a 28 ºC. Durante este período foram verificadas duas taxas de mortalidade, 0,04 log ufc/g/dia nos primeiros 30 dias e 0,007 log ufc/g/dia entre 30 e 180 dias. Com relação à resistência ácida, não houve diferença (P > 0,05) na cinética de crescimento/morte de Salmonella entre a amostra sem estresse dessecativo (TSB) e as amostras de amendoim após 4 h em pHs 3,0, 3,5, 4,5 e 7,2. A taxa média de crescimento observada para o pH 7,2 foi de 0,44 log cfu/ml/h. No pH 4,5, a contagem de Salmonella não mudou significativamente durante 4 h. Em contraste, a população de Salmonella diminuiu de 0,14 a 0,29 log cfu/ml/h no pH 3,5. No pH 3,0, a queda foi estimada em 0,65 log cfu/ml/h para a amostra sem estresse dessecativo e 2,07 log cfu/ml/h para a Salmonella recuperada das amostras de amendoim estocadas por 120 dias. Portanto, nossos dados indicaram que o estresse dessecativo causado durante a estocagem do amendoim não influenciou a resistência ácida de Salmonella.

Enhancement of Annexin V in response to combination of epigallocatechin gallate and quercetin as a potent arrest the cell cycle of colorectal cancer

Abstract Colorectal cancer (CRC) is one of the most common cancers leading to comorbidities and mortalities globally. The rational of current study was to evaluate the combined epigallocatechin gallate and quercetin as a potent antitumor agent as commentary agent for therapeutic protocol. The present study investigated the effect of epigallocatechin Gallate (EGCG) (150mg) and quercetin (200mg) at different proportions on proliferation and induction of apoptosis in human colon cancer cells (HCT-116). Cell growth, colonogenic, Annexin V in addition cell cycle were detected in response to phytomolecules. Data obtained showed that, the colony formation was inhibited significantly in CRC starting from the lowest concentration tested of 10 µg/mL resulting in no colonies as visualized by a phase-contrast microscope. Data showed a significant elevation in the annexin V at 100 µg/mL EGCG(25.85%) and 150 µg/mL quercetin (48.35%). Moreover, cell cycle analysis showed that this combination caused cell cycle arrest at the G1 phase at concentration of 100 µg/mL (72.7%) and 150 µg/mL (75.25%). The combined effect of epigallocatechin Gallate and quercetin exert antiproliferative activity against CRC, it is promising in alternative conventional chemotherapeutic agent.

Resumo O câncer colorretal (CCR) é um dos cânceres mais comuns, levando a comorbidades e mortalidade em todo o mundo. O racional do presente estudo foi avaliar a combinação de galato de epigalocatequina e quercetina como um agente antitumoral potente como agente de comentário para protocolo terapêutico. O presente estudo investigou o efeito de galato de epigalocatequina (EGCG) (150 mg) e quercetina (200 mg) em diferentes proporções na proliferação e indução de apoptose em células de câncer de cólon humano (HCT-116). O crescimento celular, colonogênico, anexina V, além do ciclo celular foram detectados em resposta a fitomoléculas. Os dados obtidos mostraram que a formação de colônias foi inibida significativamente no CRC a partir da concentração mais baixa testada de 10 µg/mL, resultando em nenhuma colônia conforme visualizado por um microscópio de contraste de fase. Os dados mostraram uma elevação significativa na anexina V a 100 µg/mL de EGCG (25,85%) e 150 µg/mL de quercetina (48,35%). Além disso, a análise do ciclo celular mostrou que essa combinação causou parada do ciclo celular na fase G1 na concentração de 100 µg/mL (72,7%) e 150 µg/mL (75,25%). O efeito combinado da epigalocatequina galato e quercetina exerce atividade antiproliferativa contra o CCR, é promissor como agente quimioterápico alternativo convencional.

Spectral analysis, in vitro cytotoxicity and antibacterial studies of bioactive principles from the leaves of Conocarpus lancifolius, a common tree of Jazan, Saudi Arabia

Abstract The objective of the present study was to analyse the bioactive compounds of the leaves of Conocarpus lancifolius (C. lancifolius). The GC-MS analysis of the hot methanolic extract of the leaves (HMEL) of C. lancifolius exhibited the bioactive compounds such as 1-(3-Methoxy-2-nitrobenzyl) iso quinoline, morphin-4-ol-6,7-dione, 1-bromo-N-methyl-, phytol, hexadecanoic acid, 2,3-dihydroxypropyl ester, 2,2':4',2-terthiophene, ethyl iso-allocholate, caryophyllene oxide, campesterol, epiglobulol, cholestan-3-ol, 2-methylene-, (3á,5à)-, dasycarpidan-1-methanol, acetate (ester) and oleic acid, eicosyl ester. The FT-IR analysis of HMEL of C. lancifolius showed a unique peak at 3184, 2413, 1657 cm-1 representing coumaric acid, chlorogenic acid and ferulic acid. The HMEL of C. lancifolius was actively inhibiting the proliferation of breast cancer cells MCF-7 ATCC at the concentration of 72.66 ± 8.21 µg/ml as IC50 value. The HMEL of C. lancifolius also revealed a good spectrum of activity against Gram-positive and Gram-negative bacterial cultures screened in this work. The activity observed has shown more or less similar effects against screened bacteria. However, the magnitude of potentiality was significantly lesser compared to standard ciprofloxacin disc at p 0.001 level (99% confidence intervals). Furthermore, the study demonstrating the bioactive compounds can be isolated from the leaves of C. lancifolius.

Resumo O objetivo do presente estudo foi analisar os compostos bioativos das folhas de Conocarpus lancifolius (C. lancifolius). A análise por GC-MS do extrato metanólico quente das folhas (HMEL) de C. lancifolius exibiu os compostos bioativos como 1- (3-Metoxi-2-nitrobenzil) isoquinolina, morfina-4-ol-6,7- diona, 1-bromo-N-metil-, fitol, ácido hexadecanoico, 2,3-di-hidroxipropil éster, 2,2 ': 4', 2 - tertiofeno, isoalocolato de etil, óxido de cariofileno, campesterol, epiglobulol, colestano -3-ol, 2-metileno-, (3á, 5à) -, dasycarpidan-1-metanol, acetato (éster) e ácido oleico, éster eicosílico. A análise FT-IR de HMEL de C. lancifolius mostrou um pico único em 3184, 2413, 1657 cm-1 representando ácido cumarico, ácido clorogênico e ácido ferúlico. O HMEL de C. lancifolius inibiu ativamente a proliferação de células de câncer de mama MCF-7 ATCC na concentração de 72,66 ± 8,21 µg / ml como valor de IC50. O HMEL de C. lancifolius também revelou bom espectro de atividade contra culturas de bactérias Gram-positivas e Gram-negativas rastreadas neste trabalho. A atividade observada mostrou efeitos mais ou menos semelhantes contra bactérias rastreadas. No entanto, a magnitude da potencialidade foi significativamente menor em comparação com o disco de ciprofloxacina padrão em nível de p 0,001 (intervalos de confiança de 99%). Além disso, o estudo demonstrando os compostos bioativos pode ser isolado das folhas de C. lancifolius.

Full confluency, serum starvation, and roscovitine for inducing arrest in the G0/G1 phase of the cell cycle in puma skin-derived fibroblast lines

The puma population is constantly decreasing, and cloning by somatic cell nuclear transfer can be used to conserve the species. One of the factors determining the success of the development of cloned embryos is the cell cycle stage of the donor cells. We evaluated the effects of full confluency (~100%), serum starvation (0.5% serum), and roscovitine (15 µM) treatments on the cell cycle synchronization in G0/G1 of puma skin-derived fibroblasts by flow cytometric analysis. Also, we assessed the effects of these synchronization methods on morphology, viability, and apoptosis levels using microscopy tools. The results showed that culturing the cells to confluence for 24 h (84.0%), 48 h (84.6%), and 72 h (84.2%) and serum starvation for 96 h (85.4%) yielded a significantly higher percentage of cells arrested in the G0/G1 (P < 0.05) phase than cells not subjected to any cell cycle synchronization method (73.9%). Nevertheless, while serum starvation reduced the percentage of viable cells, no difference was observed for the full confluence and roscovitine treatments (P > 0.05). Moreover, roscovitine for 12 h (78.6%) and 24 h (82.1%) was unable to synchronize cells in G0/G1 (P > 0.05). In summary, full confluency induces puma fibroblast cell cycle synchronization at the G0/G1 stage without affecting cell viability. These outcomes may be valuable for planning donor cells for somatic cell nuclear transfer in pumas.(AU)

Effect of zeolite-chlorella top dressing on scar metabolism and conversion of dairy cows' feed / Efeito de aperitivos de zeólita-chlorella no metabolismo cicatricial e na conversão de ração para vacas leiteiras

In scientific and economic experience, the effect of zeolite-chlorella top dressing (CCP) from 28-29% zeolite, 1.5-2% Chlorella vulgaris powder and 75-76% cake on the productive effect of feed and digestion metabolism of dairy cows has been studied. Tetra-edric frame-hollow zeolite crystals have selectively adsorbing and ion-filtering properties, and chlorella enriches with amino acids and vitamins. The introduction of CCP into the diet of cows of the experimental group increased the mineral-vitamin balance and positively affected the metabolism of cicatricial digestion. The pH shift from 6.14 to 6.17 activated the ecosystem of the rumen microflora, increased the synthesis of LVH 0.79 mmol/100ml, due to an increase in the volume of acetates from 54.1±3.0 to 57.2±2.2 mmol/100ml, increased the number of infusoria by 41.1 thousand/ml more than the control group. An increase in the amylolytic activity of the rumen chyme by 2.8 mg/starch, and cellulolytic activity by 2.8% increased scar digestion. This increased the consumption of the dry matter of the diet by cows of the experimental group by 0.48 ± 0.06 kg/head. /day. and daily milk yields by 1.06 ± 0.03 kg against the control. An increase in protein and fat in milk with a decrease in somatic cells, and in the blood of erythrocytes and hemoglobin increased biosynthesis, which increased the conversion rate of feed from 0.68 to 0.72, and protein from 17.0% to 18.9%.

A experiência científica e econômica estudou o efeito produtivo da alimentação e o metabolismo da digestão de vacas leiteiras, de Zeolite-Chlorella (CHP), de 28-29% de zeólita, 1,5-2% de pó de Chlorella vulgaris e 75-76% de bagaço. Os cristais ocos de estrutura tetraedrítica do zeólito possuem propriedades seletivas de adsorção e ionofiltração, e a Chlorella enriquece com aminoácidos e vitaminas. A introdução na dieta de vacas de um grupo experiente de CHP aumentou o equilíbrio mineral-vitamínico e afetou significativamente o metabolismo da digestão cicatricial. A mudança de pH de 6,14 para 6,17 ativou o ecossistema da microflora cicatricial, aumentou a síntese de LLA 0,79 mmol/ 100ml, devido à uveistivanie do volume de acetatos de 54,1±3,0 para 57,2±2,2 mmol/100ml, aumentou o número de infusórios em 41,1 mil/ml mais do que o grupo controle. O aumento da atividade amilolítica do quimo da cicatriz em 2,8 mg / amido e da atividade celulosolítica em 2,8% aumentou a digestão cicatricial. Isso aumentou a ingestão de matéria seca das vacas do grupo experimental em 0,48±0,06 kg/gol./dia e a produção diária de leite em 1,06±0,03 kg contra o controle. O aumento da proteína e da gordura no leite, com a diminuição das células somáticas, e no sangue de glóbulos vermelhos e hemoglobina aumentou a biossíntese, o que aumentou a taxa de conversão alimentar de 0,68 para 0,72 e proteína de 17,0% para 18,9%.

Spectral analysis, in vitro cytotoxicity and antibacterial studies of bioactive principles from the leaves of Conocarpus lancifolius, a common tree of Jazan, Saudi Arabia / Análise espectral, citotoxicidade in vitro e estudos antibacterianos de princípios bioativos das folhas de Conocarpus lancifolius, uma árvore comum de Jazan, Arábia Saudita

The objective of the present study was to analyse the bioactive compounds of the leaves of Conocarpus lancifolius (C. lancifolius). The GC-MS analysis of the hot methanolic extract of the leaves (HMEL) of C. lancifolius exhibited the bioactive compounds such as 1-(3-Methoxy-2-nitrobenzyl) iso quinoline, morphin-4-ol-6,7-dione, 1-bromo N-methyl-, phytol, hexadecanoic acid, 2,3-dihydroxypropyl ester, 2,2’:4’,2”-terthiophene, ethyl iso-allocholate, caryophyllene oxide, campesterol, epiglobulol, cholestan-3-ol, 2-methylene-, (3á,5à)-, dasycarpidan-1-methanol, acetate (ester) and oleic acid, eicosyl ester. The FT-IR analysis of HMEL of C. lancifolius showed a unique peak at 3184, 2413, 1657 cm-¹ representing coumaric acid, chlorogenic acid and ferulic acid. The HMEL of C. lancifolius was actively inhibiting the proliferation of breast cancer cells MCF-7 ATCC at the concentration of 72.66 ± 8.21 µg/ml as IC50 value. The HMEL of C. lancifolius also revealed a good spectrum of activity against Gram-positive and Gram negative bacterial cultures screened in this work. The activity observed has shown more or less similar effects against screened bacteria. However, the magnitude of potentiality was significantly lesser compared to standard ciprofloxacin disc at p< 0.001 level (99% confidence intervals). Furthermore, the study demonstrating the bioactive compounds can be isolated from the leaves of C. lancifolius.

O objetivo do presente estudo foi analisar os compostos bioativos das folhas de Conocarpus lancifolius (C. lancifolius). A análise por GC-MS do extrato metanólico quente das folhas (HMEL) de C. lancifolius exibiu os compostos bioativos como 1- (3-Metoxi-2-nitrobenzil) isoquinolina, morfina-4-ol-6,7- diona, 1-bromo-N-metil-, fitol, ácido hexadecanoico, 2,3-di-hidroxipropil éster, 2,2 ‘: 4’, 2 ” - tertiofeno, isoalocolato de etil, óxido de cariofileno, campesterol, epiglobulol, colestano -3-ol, 2-metileno-, (3á, 5à) -, dasycarpidan-1-metanol, acetato (éster) e ácido oleico, éster eicosílico. A análise FT-IR de HMEL de C. lancifolius mostrou um pico único em 3184, 2413, 1657 cm-¹ representando ácido cumarico, ácido clorogênico e ácido ferúlico. O HMEL de C. lancifolius inibiu ativamente a proliferação de células de câncer de mama MCF-7 ATCC na concentração de 72,66 ± 8,21 µg/ml como valor de IC50. O HMEL de C. lancifolius também revelou bom espectro de atividade contra culturas de bactérias Gram-positivas e Gram-negativas rastreadas neste trabalho. A atividade observada mostrou efeitos mais ou menos semelhantes contra bactérias rastreadas. No entanto, a magnitude da potencialidade foi significativamente menor em comparação com o disco de ciprofloxacina padrão em nível de p < 0,001 (intervalos de confiança de 99%). Além disso, o estudo demonstrando os compostos bioativos pode ser isolado das folhas de C. lancifolius.

Spectral analysis, in vitro cytotoxicity and antibacterial studies of bioactive principles from the leaves of Conocarpus lancifolius, a common tree of Jazan, Saudi Arabia / Análise espectral, citotoxicidade in vitro e estudos antibacterianos de princípios bioativos das folhas de Conocarpus lancifolius, uma árvore comum de Jazan, Arábia Saudita

The objective of the present study was to analyse the bioactive compounds of the leaves of Conocarpus lancifolius (C. lancifolius). The GC-MS analysis of the hot methanolic extract of the leaves (HMEL) of C. lancifolius exhibited the bioactive compounds such as 1-(3-Methoxy-2-nitrobenzyl) iso quinoline, morphin-4-ol-6,7-dione, 1-bromo N-methyl-, phytol, hexadecanoic acid, 2,3-dihydroxypropyl ester, 2,2:4,2”-terthiophene, ethyl iso-allocholate, caryophyllene oxide, campesterol, epiglobulol, cholestan-3-ol, 2-methylene-, (3á,5à)-, dasycarpidan-1-methanol, acetate (ester) and oleic acid, eicosyl ester. The FT-IR analysis of HMEL of C. lancifolius showed a unique peak at 3184, 2413, 1657 cm-¹ representing coumaric acid, chlorogenic acid and ferulic acid. The HMEL of C. lancifolius was actively inhibiting the proliferation of breast cancer cells MCF-7 ATCC at the concentration of 72.66 ± 8.21 µg/ml as IC50 value. The HMEL of C. lancifolius also revealed a good spectrum of activity against Gram-positive and Gram negative bacterial cultures screened in this work. The activity observed has shown more or less similar effects against screened bacteria. However, the magnitude of potentiality was significantly lesser compared to standard ciprofloxacin disc at p< 0.001 level (99% confidence intervals). Furthermore, the study demonstrating the bioactive compounds can be isolated from the leaves of C. lancifolius.(AU)

O objetivo do presente estudo foi analisar os compostos bioativos das folhas de Conocarpus lancifolius (C. lancifolius). A análise por GC-MS do extrato metanólico quente das folhas (HMEL) de C. lancifolius exibiu os compostos bioativos como 1- (3-Metoxi-2-nitrobenzil) isoquinolina, morfina-4-ol-6,7- diona, 1-bromo-N-metil-, fitol, ácido hexadecanoico, 2,3-di-hidroxipropil éster, 2,2 ‘: 4, 2 ” - tertiofeno, isoalocolato de etil, óxido de cariofileno, campesterol, epiglobulol, colestano -3-ol, 2-metileno-, (3á, 5à) -, dasycarpidan-1-metanol, acetato (éster) e ácido oleico, éster eicosílico. A análise FT-IR de HMEL de C. lancifolius mostrou um pico único em 3184, 2413, 1657 cm-¹ representando ácido cumarico, ácido clorogênico e ácido ferúlico. O HMEL de C. lancifolius inibiu ativamente a proliferação de células de câncer de mama MCF-7 ATCC na concentração de 72,66 ± 8,21 µg/ml como valor de IC50. O HMEL de C. lancifolius também revelou bom espectro de atividade contra culturas de bactérias Gram-positivas e Gram-negativas rastreadas neste trabalho. A atividade observada mostrou efeitos mais ou menos semelhantes contra bactérias rastreadas. No entanto, a magnitude da potencialidade foi significativamente menor em comparação com o disco de ciprofloxacina padrão em nível de p < 0,001 (intervalos de confiança de 99%). Além disso, o estudo demonstrando os compostos bioativos pode ser isolado das folhas de C. lancifolius.(AU)

Spectral analysis, in vitro cytotoxicity and antibacterial studies of bioactive principles from the leaves of Conocarpus lancifolius, a common tree of Jazan, Saudi Arabia / Análise espectral, citotoxicidade in vitro e estudos antibacterianos de princípios bioativos das folhas de Conocarpus lancifolius, uma árvore comum de Jazan, Arábia Saudita

Abstract The objective of the present study was to analyse the bioactive compounds of the leaves of Conocarpus lancifolius (C. lancifolius). The GC-MS analysis of the hot methanolic extract of the leaves (HMEL) of C. lancifolius exhibited the bioactive compounds such as 1-(3-Methoxy-2-nitrobenzyl) iso quinoline, morphin-4-ol-6,7-dione, 1-bromo-N-methyl-, phytol, hexadecanoic acid, 2,3-dihydroxypropyl ester, 2,2':4',2"-terthiophene, ethyl iso-allocholate, caryophyllene oxide, campesterol, epiglobulol, cholestan-3-ol, 2-methylene-, (3á,5à)-, dasycarpidan-1-methanol, acetate (ester) and oleic acid, eicosyl ester. The FT-IR analysis of HMEL of C. lancifolius showed a unique peak at 3184, 2413, 1657 cm-1 representing coumaric acid, chlorogenic acid and ferulic acid. The HMEL of C. lancifolius was actively inhibiting the proliferation of breast cancer cells MCF-7 ATCC at the concentration of 72.66 ± 8.21 µg/ml as IC50 value. The HMEL of C. lancifolius also revealed a good spectrum of activity against Gram-positive and Gram-negative bacterial cultures screened in this work. The activity observed has shown more or less similar effects against screened bacteria. However, the magnitude of potentiality was significantly lesser compared to standard ciprofloxacin disc at p< 0.001 level (99% confidence intervals). Furthermore, the study demonstrating the bioactive compounds can be isolated from the leaves of C. lancifolius.

Resumo O objetivo do presente estudo foi analisar os compostos bioativos das folhas de Conocarpus lancifolius (C. lancifolius). A análise por GC-MS do extrato metanólico quente das folhas (HMEL) de C. lancifolius exibiu os compostos bioativos como 1- (3-Metoxi-2-nitrobenzil) isoquinolina, morfina-4-ol-6,7- diona, 1-bromo-N-metil-, fitol, ácido hexadecanoico, 2,3-di-hidroxipropil éster, 2,2 ': 4', 2 " - tertiofeno, isoalocolato de etil, óxido de cariofileno, campesterol, epiglobulol, colestano -3-ol, 2-metileno-, (3á, 5à) -, dasycarpidan-1-metanol, acetato (éster) e ácido oleico, éster eicosílico. A análise FT-IR de HMEL de C. lancifolius mostrou um pico único em 3184, 2413, 1657 cm-1 representando ácido cumarico, ácido clorogênico e ácido ferúlico. O HMEL de C. lancifolius inibiu ativamente a proliferação de células de câncer de mama MCF-7 ATCC na concentração de 72,66 ± 8,21 µg / ml como valor de IC50. O HMEL de C. lancifolius também revelou bom espectro de atividade contra culturas de bactérias Gram-positivas e Gram-negativas rastreadas neste trabalho. A atividade observada mostrou efeitos mais ou menos semelhantes contra bactérias rastreadas. No entanto, a magnitude da potencialidade foi significativamente menor em comparação com o disco de ciprofloxacina padrão em nível de p < 0,001 (intervalos de confiança de 99%). Além disso, o estudo demonstrando os compostos bioativos pode ser isolado das folhas de C. lancifolius.

Enhancement of Annexin V in response to combination of epigallocatechin gallate and quercetin as a potent arrest the cell cycle of colorectal cancer / Aumento da anexina V em resposta à combinação de galato de epigalocatequina e quercetina como uma potente parada do ciclo celular do câncer colorretal

Colorectal cancer (CRC) is one of the most common cancers leading to comorbidities and mortalities globally. The rational of current study was to evaluate the combined epigallocatechin gallate and quercetin as a potent antitumor agent as commentary agent for therapeutic protocol. The present study investigated the effect of epigallocatechin Gallate (EGCG) (150mg) and quercetin (200mg) at different proportions on proliferation and induction of apoptosis in human colon cancer cells (HCT-116). Cell growth, colonogenic, Annexin V in addition cell cycle were detected in response to phytomolecules. Data obtained showed that, the colony formation was inhibited significantly in CRC starting from the lowest concentration tested of 10 µg/mL resulting in no colonies as visualized by a phase-contrast microscope. Data showed a significant elevation in the annexin V at 100 µg/mL EGCG(25.85%) and 150 µg/mL quercetin (48.35%). Moreover, cell cycle analysis showed that this combination caused cell cycle arrest at the G1 phase at concentration of 100 µg/mL (72.7%) and 150 µg/mL (75.25%). The combined effect of epigallocatechin Gallate and quercetin exert antiproliferative activity against CRC, it is promising in alternative conventional chemotherapeutic agent.

O câncer colorretal (CCR) é um dos cânceres mais comuns, levando a comorbidades e mortalidade em todo o mundo. O racional do presente estudo foi avaliar a combinação de galato de epigalocatequina e quercetina como um agente antitumoral potente como agente de comentário para protocolo terapêutico. O presente estudo investigou o efeito de galato de epigalocatequina (EGCG) (150 mg) e quercetina (200 mg) em diferentes proporções na proliferação e indução de apoptose em células de câncer de cólon humano (HCT-116). O crescimento celular, colonogênico, anexina V, além do ciclo celular foram detectados em resposta a fitomoléculas. Os dados obtidos mostraram que a formação de colônias foi inibida significativamente no CRC a partir da concentração mais baixa testada de 10 µg/mL, resultando em nenhuma colônia conforme visualizado por um microscópio de contraste de fase. Os dados mostraram uma elevação significativa na anexina V a 100 µg/mL de EGCG (25,85%) e 150 µg/mL de quercetina (48,35%). Além disso, a análise do ciclo celular mostrou que essa combinação causou parada do ciclo celular na fase G1 na concentração de 100 µg/mL (72,7%) e 150 µg/mL (75,25%). O efeito combinado da epigalocatequina galato e quercetina exerce atividade antiproliferativa contra o CCR, é promissor como agente quimioterápico alternativo convencional.

Enhancement of Annexin V in response to combination of epigallocatechin gallate and quercetin as a potent arrest the cell cycle of colorectal cancer / Aumento da anexina V em resposta à combinação de galato de epigalocatequina e quercetina como uma potente parada do ciclo celular do câncer colorretal

Abstract Colorectal cancer (CRC) is one of the most common cancers leading to comorbidities and mortalities globally. The rational of current study was to evaluate the combined epigallocatechin gallate and quercetin as a potent antitumor agent as commentary agent for therapeutic protocol. The present study investigated the effect of epigallocatechin Gallate (EGCG) (150mg) and quercetin (200mg) at different proportions on proliferation and induction of apoptosis in human colon cancer cells (HCT-116). Cell growth, colonogenic, Annexin V in addition cell cycle were detected in response to phytomolecules. Data obtained showed that, the colony formation was inhibited significantly in CRC starting from the lowest concentration tested of 10 µg/mL resulting in no colonies as visualized by a phase-contrast microscope. Data showed a significant elevation in the annexin V at 100 µg/mL EGCG(25.85%) and 150 µg/mL quercetin (48.35%). Moreover, cell cycle analysis showed that this combination caused cell cycle arrest at the G1 phase at concentration of 100 µg/mL (72.7%) and 150 µg/mL (75.25%). The combined effect of epigallocatechin Gallate and quercetin exert antiproliferative activity against CRC, it is promising in alternative conventional chemotherapeutic agent.

Resumo O câncer colorretal (CCR) é um dos cânceres mais comuns, levando a comorbidades e mortalidade em todo o mundo. O racional do presente estudo foi avaliar a combinação de galato de epigalocatequina e quercetina como um agente antitumoral potente como agente de comentário para protocolo terapêutico. O presente estudo investigou o efeito de galato de epigalocatequina (EGCG) (150 mg) e quercetina (200 mg) em diferentes proporções na proliferação e indução de apoptose em células de câncer de cólon humano (HCT-116). O crescimento celular, colonogênico, anexina V, além do ciclo celular foram detectados em resposta a fitomoléculas. Os dados obtidos mostraram que a formação de colônias foi inibida significativamente no CRC a partir da concentração mais baixa testada de 10 µg/mL, resultando em nenhuma colônia conforme visualizado por um microscópio de contraste de fase. Os dados mostraram uma elevação significativa na anexina V a 100 µg/mL de EGCG (25,85%) e 150 µg/mL de quercetina (48,35%). Além disso, a análise do ciclo celular mostrou que essa combinação causou parada do ciclo celular na fase G1 na concentração de 100 µg/mL (72,7%) e 150 µg/mL (75,25%). O efeito combinado da epigalocatequina galato e quercetina exerce atividade antiproliferativa contra o CCR, é promissor como agente quimioterápico alternativo convencional.

Enhancement of Annexin V in response to combination of epigallocatechin gallate and quercetin as a potent arrest the cell cycle of colorectal cancer / Aumento da anexina V em resposta à combinação de galato de epigalocatequina e quercetina como uma potente parada do ciclo celular do câncer colorretal

Colorectal cancer (CRC) is one of the most common cancers leading to comorbidities and mortalities globally. The rational of current study was to evaluate the combined epigallocatechin gallate and quercetin as a potent antitumor agent as commentary agent for therapeutic protocol. The present study investigated the effect of epigallocatechin Gallate (EGCG) (150mg) and quercetin (200mg) at different proportions on proliferation and induction of apoptosis in human colon cancer cells (HCT-116). Cell growth, colonogenic, Annexin V in addition cell cycle were detected in response to phytomolecules. Data obtained showed that, the colony formation was inhibited significantly in CRC starting from the lowest concentration tested of 10 µg/mL resulting in no colonies as visualized by a phase-contrast microscope. Data showed a significant elevation in the annexin V at 100 µg/mL EGCG(25.85%) and 150 µg/mL quercetin (48.35%). Moreover, cell cycle analysis showed that this combination caused cell cycle arrest at the G1 phase at concentration of 100 µg/mL (72.7%) and 150 µg/mL (75.25%). The combined effect of epigallocatechin Gallate and quercetin exert antiproliferative activity against CRC, it is promising in alternative conventional chemotherapeutic agent.(AU)

O câncer colorretal (CCR) é um dos cânceres mais comuns, levando a comorbidades e mortalidade em todo o mundo. O racional do presente estudo foi avaliar a combinação de galato de epigalocatequina e quercetina como um agente antitumoral potente como agente de comentário para protocolo terapêutico. O presente estudo investigou o efeito de galato de epigalocatequina (EGCG) (150 mg) e quercetina (200 mg) em diferentes proporções na proliferação e indução de apoptose em células de câncer de cólon humano (HCT-116). O crescimento celular, colonogênico, anexina V, além do ciclo celular foram detectados em resposta a fitomoléculas. Os dados obtidos mostraram que a formação de colônias foi inibida significativamente no CRC a partir da concentração mais baixa testada de 10 µg/mL, resultando em nenhuma colônia conforme visualizado por um microscópio de contraste de fase. Os dados mostraram uma elevação significativa na anexina V a 100 µg/mL de EGCG (25,85%) e 150 µg/mL de quercetina (48,35%). Além disso, a análise do ciclo celular mostrou que essa combinação causou parada do ciclo celular na fase G1 na concentração de 100 µg/mL (72,7%) e 150 µg/mL (75,25%). O efeito combinado da epigalocatequina galato e quercetina exerce atividade antiproliferativa contra o CCR, é promissor como agente quimioterápico alternativo convencional.(AU)

Cytotoxicity of extracts and compounds isolated from Croton echioides in animal tumor cell (HTC) / Citotoxicidade de extratos e compostos isolados de Croton echioides em célula tumoral animal (HTC)

The search for compounds with anticancer effects is of paramount importance today due to the high incidence of the disease. The Euphorbiaceae family is known for having compounds with therapeutic properties, one of its genera being Croton. It has several species, which contain compounds already known for their biological activities, presenting anti-inflammatory, antimicrobial and anticancer properties. Thus, the cytotoxicity/antiproliferative activity of semi-purified fractions and compounds isolated from Croton echioides in liver tumor cells of Rattus norvegicus (HTC) was evaluated by the MTT test. The semi-purified fractions showed cytotoxicity at concentrations above 200 µg/mL, at 24, 48 and 72 hours, reaching cell viability of 24.78% [400 µg/mL] at 24 hours, 12.79% [500 µg/mL] at 48 hours and 10.57% [300 µg/mL] at 72 hours. For the isolated compounds, lupeol had a cytotoxic effect in all concentrations (1, 5, 10, 15, 20, 40, 60, 80 and 100 µg/mL) and tested times (24, 48 and 72 hours), reaching minimum viability of 4.37% [100 µg/mL], within 72 hours. The clerodan diterpenes CEH-1 and CEH-4 also showed antiproliferative activity, with minimum viability of 36.19% [100 µg/mL] over 72 hours and 21.33% [100 µg/mL] over 48 hours, respectively. However, the clerodan diterpenes CEH-2 and CEH-3 did not shows a cytotoxic effect for HTC cells. Thus, there is a cytotoxic/antiproliferative potential of C. echioides against tumor cells, with targeted to mitochondrial enzymes, associated with cell proliferation, indicating that this species deserves prominence in the search for new molecules for the treatment of cancer.

A busca por compostos com efeitos anticâncer é de suma importância nos dias atuais devido à alta incidência desta doença. A família Euphorbiaceae é conhecida por possuir compostos com propriedades terapêuticas, sendo um de seus gêneros o Croton. Este possui diversas espécies, que contêm compostos já conhecidos por suas atividades biológicas, apresentando propriedades anti-inflamatórias, antimicrobianas e anticancerígenas. Assim, a citotoxicidade/atividade antiproliferativa de frações semipurificadas e compostos isolados de Croton echioides em células tumorais hepáticas de Rattus norvegicus (HTC) foi avaliada pelo teste MTT. As frações semipurificadas apresentaram citotoxicidade em concentrações acima de 200 µg/mL, em 24, 48 e 72 horas, atingindo viabilidade celular de 24,78% [400 µg/mL] em 24 horas, 12,79% [500 µg/mL] em 48 horas e 10,57% [300 µg/mL] às 72 horas. Para os compostos isolados, o lupeol teve efeito citotóxico em todas as concentrações (1, 5, 10, 15, 20, 40, 60, 80 e 100 µg/mL) e tempos testados (24, 48 e 72 horas), atingindo a viabilidade mínima de 4,37% [100 µg/mL], em 72 horas. Os diterpenos clerodan CEH-1 e CEH-4 também apresentaram atividade antiproliferativa, com viabilidade mínima de 36,19% [100 µg/mL] em 72 horas e 21,33% [100 µg/mL] em 48 horas, respectivamente. No entanto, os diterpenos clerodanos CEH-2 e CEH-3 não apresentaram efeito citotóxico para células HTC. Assim, existe um potencial citotóxico/antiproliferativo de C. echioides contra células tumorais, com enzimas direcionadas a enzimas mitocondriais, associadas à proliferação celular, indicando que esta espécie merece destaque na busca de novas moléculas para o tratamento do câncer.

Reproductive performance of single fixed-time inseminated sows with semen doses submitted to different semen storage times

This study aimed to evaluate the reproductive performance of sows submitted to single fixed-time insemination (SFTAI) using boars according to capacity for liquid in vitro semen preservation, type of extender, and storage time. Boars (n = 12) were classified into two groups based on progressive motility (PM) at 120 h of semen storage: low (PM - 64.5%) - and high-preservation (PM - 83.9%) capacity for semen storage. Weaned sows (n = 397, parity - 1 to 7) were inseminated (1.5×109 sperm cells) in a factorial design: two classes of boars (low- or high-preservation), two types of extenders (short- or long-term), and two semen storage times at insemination (24 or 72 h). An adapted triptorelin acetate protocol was used for SFTAI. Total sperm motility (TM) and PM at insemination were greater in high-preservation boars at 72 h compared with low-preservation boars at 24 or 72 h (P < 0.01). Short- or long-term extender did not affect (P ≥ 0.68) TM and PM in high-preservation boars; however, long-term extender improved these parameters in low-preservation boars (P < 0.01). Pregnancy and farrowing rates were not affected by groups (P > 0.05). Total piglets born (TPB) was reduced (P = 0.05) in low-preservation boars with 72 h of storage (13.6 ± 0.5) compared to high-preservation boars with semen stored for 24 or 72 h (15.2 ± 0.5 and 15.5 ± 0.5, respectively). The low-preservation boars reduced the TPB in sows submitted to SFTAI, and this reduction was greater using semen stored for 72 h.(AU)

Recycled pequi (Caryocar brasiliense, Camb) shell ethanolic extract induces apoptosis in canine osteosarcoma cell / Extrato etanólico de cascas recicladas de pequi (Caryocar brasiliense, Camb) induz apoptose em células de osteossarcoma canino

Osteosarcoma is a mesenchymal cancer associated with high mortality in dogs and in humans. The biodiversity-rich Cerrado, the predominant biome in the Midwest region of Brazil, is native to locally appreciated fruits such as pequi (Caryocar brasiliense, Camb). Although this plant has been frequently used in folk medicine, the pharmacological properties of pequi fruit shells have not been fully evaluated. Thus, this study aimed to determine the cytotoxic activity of ethanol extract of recycled pequi shells on canine osteosarcoma cells in vitro. Cells were cultured and treated with final extract concentrations of 0, 0.029 µg/µL, 0.29 µg/µL, and 2.91 µg/µL for 24, 48, or 72 hours. Cell viability assay using trypan blue exclusion method and tetrazolium reduction method, cell survival assay, and double labeling with annexin V and propidium iodide were performed in the treated osteosarcoma cells. These allowed the determination of IC50, survival fraction, and type of cell death, respectively. Pequi shell ethanol extract at a concentration of 2.91 µg/µL showed the greatest inhibition of osteosarcoma cell growth in vitro, resulting in a 71.80% decrease in growth compared to the control. The mean IC50 was 155.2 µg/mL at 72 hours. The calculated survival fractions showed that cell growth at 72 hours was 3.33% lower in cells treated with 2.91 µg/µL extract. Results from the double labeling experiment suggest that apoptosis was the predominant type of cell death in cells treated with 2.91 µg/µL extract. These results demonstrate that ethanol extract of recycled pequi shells promotes apoptosis in canine osteosarcoma cells.

O osteossarcoma é uma neoplasia mesenquimal associada à alta mortalidade em cães e humanos. O pequi (Caryocar brasiliense, Camb.) é nativo do Cerrado, bioma rico em biodiversidade predominante na região Centro-Oeste do Brasil. Embora esta planta seja frequentemente utilizada na medicina popular, as propriedades farmacológicas da casca do pequi ainda são pouco conhecidas. Este estudo teve como objetivo determinar a atividade citotóxica do extrato etanólico de cascas de pequi recicladas em células de osteossarcoma canino. As células foram cultivadas e tratadas com concentrações finais de extrato de zero, 0,029 µg/µL, 0,29 µg/µL e 2,91 µg/µL por 24, 48 ou 72 horas. As células foram submetidas a ensaio de viabilidade celular pelos métodos de exclusão de azul de tripano da redução de tetrazólio para determinar a IC50, ao ensaio de sobrevivência celular para calcular a fração de sobrevivência e à dupla marcação com anexina V e iodeto de propídio para determinar o tipo de morte celular. O extrato etanólico de casca de pequi na concentração de 2,91 µg/µL apresentou a maior inibição do crescimento de células de osteossarcoma, com redução de 71,80% no crescimento em relação ao controle. A IC50 média foi de 155,2 µg/mL em 72 horas. O crescimento celular em 72 horas foi 3,33% menor nas células tratadas com 2,91 µg/µL de extrato. A apoptose foi o tipo predominante de morte celular em células tratadas com 2,91 µg/µL de extrato. Esses resultados demonstram que o extrato etanólico de cascas de pequi recicladas promove apoptose em células de osteossarcoma canino.

Sperm cell capacitation status of ram semen after cooling

Background: The use of conventional artificial insemination (AI) in sheep production is usually associated with lower fertility rates when frozen semen is used. Cooled ram semen has been an alternative over frozen semen due to the higher viability, seminal quality and fertility rates following AI. The semen preservation process promotes sperm cell modifications similar to capacitation (capacitation-like) that causes cell damage affecting viability and seminal quality, but such effects are unclear for cooled semen. The aim of this study was to determine the status of sperm cell capacitation (CA) and acrosome reaction (AR) during ram semen processing and cooling under different extenders, dilution factors, and aerobiosis conditions as a function of storage time at 5o C. Materials, Methods & Results: Two consecutive ejaculates per day per male were collected from 2 adult rams by artificial vagina at 48-72 h intervals, in three replications. After macro- and microscopic evaluations, semen was segregated into groups under 3 extenders (Tris-egg yolk or TY, citrate-egg yolk or CY, skimmed milk or SM), 2 dilution factors (1 x 109 or Bi, 100 x 106 or Mi cells/mL), and 2 aerobiosis conditions (aerobic or A, semi-anaerobic or SA). Diluted semen was cooled to 5ºC and stored for up to 72 h, with evaluations every 24 h. Aliquots of fresh ejaculates and of each cooled diluted subgroup, according to extender, dilution, and aerobiosis, were collected at times T0 and T72 for determination of acrosome status and membrane integrity by the chlortetracycline (CTC) and trypan blue-Giemsa stainings, respectively. No differences were detected in sperm cell motility (M) and motility vigor (V) between fresh and diluted semen. After cooling, a significant decrease in M was observed after 48 h in CY and SM compared with fresh semen and 0 h of cooling, while V started to decrease after 24 h in CY compared with TY. Likewise, M/V from different dilutions and aerobic conditions decreased more significantly after 48 and 24 h of cooling, respectively. The sperm capacitation status did not show differences in the proportion of non-capacitated (NCA), CA and AR sperm cells between TY, CY, and SM extenders (NCA: 75.0%, 71.3%, 74.0%; CA: 15.7%, 17.2%, 15.9%; AR: 9.3%, 11.5%, 10.2%) or between Bi and Mi dilutions (NCA: 74.0%, 72.9%; CA: 15.9%, 16.6%; AR: 10.1%, 10.5%), respectively. However, differences (P < 0.05) were observed between A and SA aerobic conditions, with CA (17.0% vs. 15.5%) and AR (11.9% vs. 8.7%) rates being higher in A than SA, respectively, with no differences in NCA (71.1% vs. 75.8%), irrespective of the storage time. Sperm cell viability decreased after 48 h, especially in CY (P < 0.05). Discussion: Ram sperm cells can suffer irreversible damage due to thermal shock during cooling. Egg yolk-based extenders provide phospholipids and cholesterol to protect the sperm cell membrane during the thermal shock caused by the change in temperature. In this study, sperm cells had irreversible decreases in M/V, with increase in acrosome and plasma membrane damage after cooling to 5ºC. The largest and smallest decreases in M and V over time were observed in the CY and TY extenders, respectively. In addition to the extender type, the semen preservation method and storage time promoted changes in the capacitation status, AR and in sperm cell viability, which per se were associated with a decrease in semen fertility. In fact, the proportions of CA and/or AR sperm cells gradually increased over time after dilution and storage at 5ºC, with a negative correlation between sperm cell viability and M/V over time. In summary, extender and cooling time affected mostly M/V, while aerobiosis condition and dilution factor were more associated with acrosome status and sperm survival, with the extender having less impact on the acrosome status as a function of time.

Melanomas em cães no Sertão do Nordeste do Brasil - epidemiologia, fatores de risco e achados clinicopatológicos / Melanoma in dogs in the backlands of Northeastern Brazil - epidemiology, risk factors and clinicopathological findings

Background: Melanoma is a malignant neoplasm that arises from melanocytes and malanoblasts. It is also more frequently reported in dogs than in other species. They may arise from melanocytes in the skin, on the surfaces of the mucous membranes, and eyes. The aim of this study was to describe the epidemiological aspects, risk factors and clinicopathological findings of melanoma in dogs in the backlands, northeastern Brazil. Materials, Methods & Results: A retrospective study was carried out in all biopsy samples and necropsy examinations of dogs, from January 2003 to December 2021, at the Animal Pathology Laboratory of the Federal University of Campina Grande, Patos, Paraiba, northeastern Brazil. Epidemiological data, clinical signs, and gross lesions were reviewed from the diagnostic laboratory reports. Samples of the skin, lymph nodes, central nervous system and organs of the thoracic and abdominal cavities were fixed in 10% buffered formalin, processed routinely for histopathology, embedded in paraffin wax, cut into 4 µm sections, and stained with hematoxylin and eosin (HE). Histological sections were also submitted to immunohistochemistry with the primary antibody anti-Melan A. Of the 4717 records found, 1158 (24.5%) were diagnosed with neoplasms, of which 48 (4.14%) cases were of melanoma. Of this total, 28 (58.3%) dogs were elderly, 19 (39.6%) were adults, and 1 (2.1%) was young. Mixed breed animals were the most affected (42.6%), followed by the pinscher breed (19.1%). According to the anatomical region, the most affected site was the skin (38/53=71.7%), followed by the oral cavity (12/53=22.65%) and the eyes (3/53=5.7%). Grossly, the skin lesions were characterized by exophytic and usually blackened, sometimes irregular and firm, nodules. At cut, they had a smooth, compact and blackened surface. Lesions in the oral cavity were characterized by blackened, irregular and infiltrating nodules or masses. The ocular lesions were always unilateral and were characterized by an enlarged and diffusely blackened eyeball, with areas of ulceration and subversion of tissue architecture. In 5 animals there was more than one anatomical site affected, totaling 53 lesions. In 9 (17%) cases, metastases were identified, 8 in regional lymph nodes and 1 in the lung. Histopathology showed a densely non-encapsulated, poorly delimited, expansive and infiltrative neoplasm, composed of neoplastic cells arranged in islands or nests and supported by fibrovascular stroma, containing a variable amount of brownish pigment (melanin). Immunohistochemistry showed strong immunostaining of the neoplastic cells in brown by the anti-Melan A antibody. Discussion: The diagnosis of melanoma was established based on epidemiological, clinical, anatomopathological, and immunohistochemical findings. Gender is not a predisposing factor, and although there was no statistically significant relationship, males were more affected. Senescence is a conditioning risk factor. Elderly animals were more affected (P < 0.0001) than adult ones, with OR = 4.38; and young ones (P = 0.0051), with OR = 12.65. Some breeds, especially those with marked skin pigmentation, were more affected, however the most affected ones in this survey were pinscher and poodle. Cutaneous melanoma accounted for almost 72% of cases, contesting recent studies where oral cavity melanoma was more frequent. Therefore, it is believed that the climatic conditions of the backlands sub-region, in northeastern Brazil, associated with the individual characteristics of the dogs, are involved in the development of these neoplasms, since the climate is predominantly dry, with high temperatures throughout the year, with maximums that can reach 40ºC, favoring the exposure to high incidence of ultraviolet radiation.

Babesia bovis in large ruminants in Pakistan - molecular detection and haemato-biochemical alterations

Background: Babesiosis is endemic in Pakistan and is one of the most important bovine diseases that causes huge economic losses and high mortality in young animals. A hematobiochemical study was conducted to unveil the difference between diseased and healthy animals in selected districts i.e., Faisalabad (31° 25' 7.3740'' N and 73° 4' 44.7924'' E), Toba Tek Singh (30° 58' 9.7392'' N and 72° 27' 40.7484'' E) and Jhang (31° 16' 40.9656'' N and 72° 18' 42.3360'' E) of Punjab, Pakistan. Materials, Methods & Results: A total of 518 (Cattle = 360, Buffalo = 158) blood samples were collected. The samples were analyzed by polymerase chain reaction (PCR) targeting apocytochrome b-gene (Babesia bovis-gene) (CYTb) followed by haemato-biochemical analysis. Chi-square test for univariate analysis was used to analyze the data. In summer the PCR-based prevalence was 29.4 (53/180) and 24.05% (19/79) in cows and buffaloes, respectively. On the other hand, in winter results showed that 12.7 (23/180), 13.92 % (11/79) samples positive for Babesia genus from cows and buffaloes, respectively. The positive samples were further investigated for hematological and biochemical analysis. The results revealed that, the mean value of hematological parameters like RBCs, Hb, PCV, MCV and MCHC was significantly (P < 0.05) decreased in infected animals (cows and buffaloes) as compared to the non-infected ones. While the biochemical parameters like Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), cholesterol and Lactate dehydrogenase were significantly (P < 0.05) increased in infected animals as compared to healthy animals. This study is the first molecular and hematobiochemical evidence of Babesia bovis in dairy herds of Punjab province, Pakistan. Discussion: Bovine babesiosis is one of the important tick-borne diseases (TBD) affecting dairy industry. In bovines, among 3 Babesia species that cause the disease B. bovis is more pathogenic with high mortality and morbidity. Pakistan is situated in tropical and sub-tropical region where the humidity is high in some part of countries. This high humidity mostly favors the reproduction of the ticks thus higher prevalence of TBDs in this region. Initially the babesiosis was diagnosed by light microscopy using thin blood smear stained with Giemsa stain. Many studies verified that PCR is a more specific and sensitive tool than conventional techniques for the detection of carrier / asymptomatic ruminants. The haemato-biochemical profile is another valuable footprint to track the disease. Keeping in view the above-mentioned fact the present project has been planned to evaluate the haemato-biochemical alteration between health and Babesia infected cattle along with the molecular detection of Babesia species involved in bovine babesiosis. The mean values of haematobiochemical parameters in clinically ill and healthy animals revealed that the mean values of hematological parameters like RBCs, Hb, PCV, and HCT were significantly decreased in diseased animals as compared to the healthy ones. All these might be due the fact that the parasite is intra-erythrocytic in nature and destruction of red blood cells results in significant (P < 0.05) decrease level of all the hematological parameters. The mean value of ALT in babesiosis infected cattle was significantly higher as compared to healthy cattle. The mean values of AST and LDH in babesiosis infected cows was significantly higher as compared to that in healthy cows. The elevation in liver enzymes in babesiosis may be due to the hepatic damage and lesions induced by the parasite during its multiplication in the blood followed by disturbed liver function. These enzymes are present in high concentrations in the muscles and liver. High level of these enzymes in the blood is indicator of organ necrosis or damage.