Impact of water deficit in the relationship among alfalfa traits / Impacto do déficit hídrico na relação entre as características da alfafa

This study aimed to evaluate the relationship among traits related to yield and nutritive value of alfalfa genotypes grown under deficit and full irrigation conditions. Seventy-seven alfalfa genotypes were evaluated in two different cuts, the first one with full irrigation, and the second, with water deficit. A randomized block design with three replications was used. The evaluated traits were vigor, plant height, dry matter biomass, stem-to-leaf ratio, dry matter percentage, leaf and stem protein contents, in vitro dry matter digestibility, neutral detergent fiber, acid detergent fiber, and lignin. Significant interaction between genotypes and environments was reported for vigor, plant height, and lignin. The correlation between traits and path analysis of dry matter biomass was performed for each cut, aiming to identify auxiliary traits for indirect selection. Water availability did not alter the phenotypic and genotypic correlations, only their magnitudes. Regardless of the environment, plant height is one of the most promising traits for the selection of alfalfa genotypes with higher dry matter biomass since it showed a high direct effect in the same sense of its phenotypic correlations. However, the coefficient of determination obtained by the model applied to full irrigation was higher than that of the water-deficit environment, indicating the importance of variables not included in this study in the determination of alfalfa dry matter biomass under dry conditions.

Este estudo objetivou avaliar a relação entre características relacionadas à produção e o valor nutritivo de genótipos de alfafa cultivados em condições de déficit e irrigação total. Setenta e sete genótipos de alfafa foram avaliados em dois cortes diferentes, o primeiro com irrigação total e o segundo com déficit hídrico. O delineamento experimental foi em blocos casualizados, com três repetições. As características avaliadas foram: vigor, altura de planta, biomassa de matéria seca, razão colmo-folha, porcentagem de matéria seca, teores foliar e foliar de proteína, digestibilidade in vitro da matéria seca, fibra em detergente neutro, fibra em detergente ácido e lignina. Houve interação significativa entre genótipos e ambientes para vigor, altura de planta e lignina. A correlação entre características e análise de trilha da biomassa da matéria seca foi realizada para cada corte, visando identificar características auxiliares para a seleção indireta. A disponibilidade de água não alterou as correlações fenotípicas e genotípicas, apenas suas magnitudes. Independentemente do ambiente, a altura das plantas é um dos caracteres mais promissores para a seleção de genótipos de alfafa com maior biomassa de matéria seca, uma vez que apresentou alto efeito direto no mesmo sentido de suas correlações fenotípicas. Entretanto, o coeficiente de determinação obtido pelo modelo aplicado à irrigação total foi superior àquele do ambiente com déficit hídrico, indicando a importância de variáveis não incluídas neste estudo na determinação da biomassa de matéria seca de alfafa em condições secas.

Determining the performance of alfalfa population collected from a narrow agroeceological zone of Turkey / Determinação da população de alfafa coletada em uma estreita zona agroecológica da Turquia

ABSTRACT: This study was carried out to determine the yield and quality characteristics of some alfalfa village populations cultivated by producers in Bingol province of Turkey. Alfalfa seeds were obtained from 23 different locations in 2015. A three replicated, randomised complete block designed field trial was established in 2016 including these genotypes and four registered varieties. As a result of three year trials, it was determined that the populations obtained from the villages of Servi, Mutluca, Garip, Sarıçiçek-1 and Bagliisa produced highest green herbage and hay yields. When we compare the quality results, Kumgecit, Küçüktekören and Şenköy-1 populations stands out in terms of crude protein ratio. Highest crude protein yield was obtained from Servi population. In terms of relative feed value, it was observed that the varieties were better qualfied than the populations. It is concluded that the populations of Servi, Mutluca, Garip, Sarıçiçek-1 and Bagliisa can be used in breeding studies especially to obtain genetic progress in yield of alfalfa for feed and bioenergy production. For the genetic improvement of current genotypes with higher crude protein ratio, Kumgecit, Küçüktekören and Şenköy-1 populations can be used as a source. This study showed that, East Anatolia region is not just live-conserving highly diversified species but also covering special intra-species genetic diversity in microclimatic zones of Turkey to be used to improve the global forage and bioenergy cropproduction.

RESUMO: Este estudo foi realizado para determinar as características de rendimento e qualidade de algumas populações de alfafa cultivadas por produtores na província de Bingol, na Turquia. As sementes de alfafa foram obtidas de 23 locais diferentes em 2015. Em 2016, foi estabelecido um ensaio de campo com três repetições, aleatórias e desenvolvido em bloco, incluindo esses genótipos e quatro variedades registradas. Com o resultado de testes de três anos, determinou-se que as populações obtidas nas aldeias de Servi, Mutluca, Garip, Sarıçiçek-1 e Bagliisa produziam maiores colheitas de forragem verde e feno. Quando comparamos os resultados de qualidade, as populações Kumgecit, Küçüktekören e Şenköy-1 se destacam em termos de proporção de proteína bruta. O maior rendimento de proteína bruta foi obtido da população de Servi. Em termos de valor relativo de ração, observou-se que as variedades eram mais qualificadas do que as populações. Conclui-se que as populações de Servi, Mutluca, Garip, Sarıçiçek-1 e Bagliisa podem ser utilizadas em estudos de melhoramento, especialmente para obter progresso genético no rendimento de alfafa para produção de ração e bioenergia. Para o aprimoramento genético dos genótipos atuais com maior proporção de proteína bruta, as populações Kumgecit, Küçüktekören e Şenköy-1 podem ser usadas como fonte. Este estudo mostrou que, a região da Anatólia Oriental não é apenas espécies altamente diversificadas conservadoras de vida, mas também abrange diversidade genética intraespécie especial em zonas microclimáticas da Turquia, a ser usada para melhorar a produção global de forragem e bioenergia.

Efecto del consumo del extracto de alfalfa (medicago sativa) en anemia ferropénica inducida, en ratones (mus musculus) / Effect of consumption of alfalfa extract (Medicago sativa) on induced iron deficiency anemia in mice (Mus musculus)

RESUMEN Objetivos Determinar el efecto del consumo del extracto de alfalfa en anemia ferropé-nica inducida, en ratones. Materiales y Métodos Se utilizaron treinta ratones albinos M. musculus de la cepa Balb/c, machos de peso promedio 23±33,7 g. Se formaron tres grupos de diez ratones cada uno: a) grupo control negativo hierro suficiente (HS), recibió 40g/d de alimento balanceado durante siete semanas; b) grupo control positivo hierro deficiente (HD), recibió 40g/d de dieta ferropénica durante siete semanas y; c) grupo experimental hierro deficiente (HD), recibió 40g/d de dieta ferropénica durante siete semanas y a partir de la semana cinco se agregó 20g/d de extracto de alfalfa (EA). Resultados Al finalizar el tratamiento se observó diferencia significativa en los niveles de hemoglobina entre los grupos control positivo (8.41±3.9 g/dL) y experimental (13.4±3.3 g/dL) (t student, p<0,05). No se encontró diferencia significativa en los niveles de hemoglobina, al término del periodo de inducción entre los grupos control positivo (8.76±3.9 g/dL) y experimental (8.59± 3.1 g/dL) (t student, p>0,05). Conclusiones En condiciones experimentales, la alfalfa presenta efecto antianémico, sustentado en los resultados de los niveles de hemoglobina.(AU)

ABSTRACT Objective To determine the effect of alfalfa extract consumption on induced iron deficiency anemia in mice. Materials and Methods Thirty M. musculus albino mice of the BALB/c strain were used for this study. All of them were males, with a mean weight of 23±33.7 g. Three groups of 10 mice each were formed: a) negative control group with sufficient iron (HS), which received 40g/d of balanced feed for seven weeks; b) positive control group with iron deficiency (HD), which received 40g/d of a diet plan for anemia for seven weeks; and c) experimental group with iron deficiency (HD), which received 40g/d of a diet plan for anemia for seven weeks and 20g/d of alfalfa extract (EA) from week five. Hemoglobin levels were measured. Results At the end of the treatment, a significant difference was observed in hemoglobin levels between the positive (8.41±3.9 g/dL) and experimental (13.4±3.3 g/dL) control groups (T student, p<0.05). There was no significant difference in hemoglobin levels at the end of the induction period between the positive (8.76±3.9 g /dL) and experimental (8.59±3.1 g/dL) groups (T student, p>0.05). Conclusions Under experimental conditions, alfalfa has an antianemic effect based on the results of hemoglobin levels.(AU)

Efecto del consumo de extracto de alfalfa (Medicago sativa L) sobre el recuento de leucocitos, en ratones (Mus musculus) / Effect of the consumption of alfalfa extract (Medicago sativa L.) on the leukocyte count, in mice (Mus musculus)

La alfalfa, Medicago sativa L, es una leguminosa altamente ecológica, de fácil cultivo y comercialización. En Perú, se cultiva como forraje para animales. Objetivos: Determinar el efecto del consumo del extracto de alfalfa sobre el recuento de leucocitos, en ratón albino. Material y métodos: Estudio experimental, realizado en el Bioterio de la Facultad de Farmacia y Bioquímica de la Universidad Nacional Mayor de San Marcos. Se utilizaron veinte ratones albinos de la cepa balb/c, machos, de peso promedio 23,2 ± 32,8 g. Se formaron dos grupos: experimental y control, cada uno, de diez ratones. Al grupo experimental se le administró, vía oral por canulación extracto de alfalfa, en dosis de 250 mg/kg, volumen de 2 ml, y al grupo control, también, vía oral por canulación, placebo, volumen de 2 ml, cada cinco horas, durante una semana, alternando con nutrientes estándar. Se realizó recuento de leucocitos. Resultados: Dentro del periodo de acondicionamiento, se observó un ligero incremento en la media de leucocitos del grupo control (3940 ± 1549,3) frente al grupo experimental (3840 ± 1468,3), cuya diferencia no fue significativa. Luego de administrar al grupo experimental el extracto de Medicago sativa L., alternando con sus nutrientes respectivos, se observó diferencia significativa de la media (7800 ± 2885,2) frente al grupo control (3930± 1518,1) (p<0,05). Conclusiones: En condiciones experimentales, el consumo del extracto de Medicago sativa L. incrementó significativamente el número de leucocitos. (AU)

Alfalfa, Medicago sativa L, is a highly organic legume, easy to grow and commercialize. In Peru, it is cultivated as fodder for animals. Objectives: To determine the effect of alfalfa extract consumption on the white blood cell count in albino mouse. Methods: Experimental study. carried out in Bioterio of the Facultad de Farmacia y Bioquímica of Universidad Nacional Mayor de San Marcos. Twenty albino mice of the balb / c strain, male, of average weight 23.2 ± 32.8 g were used. Two groups were formed: experimental and control, each of ten mice. The experimental group was administered, orally by cannulation alfalfa extract, in a dose of 250 mg / kg, volume of 2 ml, and the control group, also, orally by cannulation, placebo, volume of 2 ml, every five hours, for a week, alternating with standard nutrients. Leukocyte count was performed. Results: Within the conditioning period, there was a slight increase in leukocytes mean of the control group (3940 ± 1549.3) compared to the experimental group (3840 ± 1468.3), whose difference was not significant. After administering the extract of Medicago sativa L to the experimental group, alternating with their respective nutrients, a significant difference was observed between the mean (7800 ± 2885.2) and the control group (3930 ± 1518.1) (p <0.05). Conclusions: Under experimental conditions, the consumption of Medicago sativa extract significantly increased the number of leukocytes. (AU)

Transcriptome sequencing analysis of alfalfa reveals CBF genes potentially playing important roles in response to freezing stress

Abstract Alfalfa (Medicago sativa L.) is an important perennial forage, with high nutritional value, which is widely grown in the world. Because of low freezing tolerance, its distribution and production are threatened and limited by winter weather. To understand the complex regulation mechanisms of freezing tolerance in alfalfa, we performed transcriptome sequencing analysis under cold (4 °C) and freezing (-8 °C) stresses. More than 66 million reads were generated, and we identified 5767 transcripts differentially expressed in response to cold and/or freezing stresses. These results showed that these genes were mainly classified as response to stress, transcription regulation, hormone signaling pathway, antioxidant, nodule morphogenesis, etc., implying their important roles in response to cold and freezing stresses. Furthermore, nine CBF transcripts differentially expressed were homologous to CBF genes of Mt-FTQTL6 site, conferring freezing tolerance in M. truncatula, which indicated that a genetic mechanism controlling freezing tolerance was conservative between M. truncatula and M. sativa. In summary, this transcriptome dataset highlighted the gene regulation response to cold and/or freezing stresses in alfalfa, which provides a valuable resource for future identification and functional analysis of candidate genes in determining freezing tolerance.

Ammonia-N concentration in alfalfa silage and its effects on dairy cow performance: A meta-analysis / Concentración del nitrógeno amoniacal en el ensilaje de alfalfa y sus efectos sobre el desempeño de vacas lecheras: Meta-análisis / Concentração de nitrogênio amoniacal em silagem de alfafa e seus efeitos sobre o desempenho de vacas leiteiras: Meta-análise

Abstract Background: Ammonia nitrogen (N) in alfalfa silage is used to evaluate silage quality, but its effect on dairy cows production is unknown. Objective: A meta-analysis was conducted to assess the effects of ammonia-N concentration in alfalfa silage on dairy cow performance. Methods: Thirty-four comparisons from 10 studies were analyzed with linear and quadratic models by the mixed-effect models of the R program using study as a random effect. The variance explained by the models was evaluated calculating marginal R2 (m) and conditional R2 (c) . Results: Dry matter intake declined (DMI; p = 0.007), and milk production and milk fat yield tended to decrease linearly in response to increasing ammonia-N concentration (p = 0.08). Milk fat and milk protein percentages were unaffected. Milk protein yield, however, showed a positive linear trend effect (p = 0.07) along with a significant quadratic effect (p = 0.01) in response to increasing ammonia-N concentration. Values of R2 (m) and R2 (c) indicated less variation for significant models. Conclusion: Increased ammonia-N in alfalfa silage reduced DMI, negatively impacting milk production and milk fat yield. The quadratic response in milk protein yield might be explained by the initial reduction in milk product ion, and the subsequent utilization of ammonia-N to produce more rumen microbial protein, which results in more milk protein.

Resumen Antecedentes: El nitrógeno (N) amoniacal en el ensilaje de alfalfa es utilizado para evaluar la calidad del ensilaje, pero se desconoce su efecto sobre la producción en vacas lecheras. Objetivo: Se realizó un meta-análisis para evaluar los efectos de la concentración del N amoniacal en el ensilaje de alfalfa sobre el desempeño de vacas lecheras. Métodos: Treinta y cuatro comparaciones de 10 estudios fueron analizadas utilizando modelos lineales y cuadráticos mediante modelos de efectos mixtos de R, incluyendo el estudio como efecto aleatorio. La varianza explicada por los modelos se evaluó calculando la R2 (m) marginal y la R2 (c) condicional. Resultados: El consumo de materia seca se redujo (DMI; p = 0,007), y la producción de leche y grasa en leche tendieron a disminuir linealmente (p = 0,08) en respuesta al incremento en la concentración de N amoniacal en el ensilaje. Los porcentajes de grasa y proteína en leche no fueron afectados. La producción de la proteína en leche, sin embargo, mostró una tendencia de efecto lineal positivo (p = 0,07) junto a un efecto cuadrático significativo (p = 0,01) al aumentar el N amoniacal del ensilaje. Los valores de R2 (m) y R2 (c) indicaron menor variación en los modelos significativos. Conclusión: El incremento del N amoniacal en el ensilaje de alfalfa redujo el DMI, la producción de leche y la producción de grasa en leche. La respuesta cuadrática en la producción de proteína en leche puede ser explicada por la reducción inicial en producción de leche, con el uso subsecuente a nivel ruminal del N amoniacal para producir más proteína microbiana, lo cual resulta en mayor proteína en leche.

Resumo Antecedentes: O nitrogênio amoniacal na silagem de alfafa é utilizado para avaliar a qualidade da silagem, porém o seu efeito sobre a produção de vacas leiteiras é desconhecido. Objetivo: Realizar uma meta-análise para avaliar os efeitos da concentração de nitrogênio amoniacal na silagem de alfafa no desempenho de vacas leiteiras. Métodos: Trinta e quatro comparações compiladas a partir de dez trabalhos de pesquisa foram analisadas usando modelos mistos com coefi ciente linear e quadrático do programa R, e considerando o estudo como efeito aleatório. A variância explicada do modelo foi avaliada calculando a distribuição marginal R2 (m) e a condicional R2 (c) . Resultados: Houve redução no consumo de matéria seca (DMI; p = 0,007), e uma tendência em reduzir linearmente a produção de leite e de gordura em resposta ao aumento da concentração de nitrogênio amoniacal na silagem (p = 0,08). As porcentagens de gordura e proteína do leite não foram afetadas. No entanto, houve uma tendência linear (p = 0,07), acompanhada de um signifi cativo efeito quadrático (p = 0,01) para o rendimento de proteínas do leite em resposta ao aumento do nitrogênio amoniacal na silagem de alfafa. Os valores de R2 (m) e R2 (c) indicaram menor variação para os modelos signifi cativos. Conclusaõ: O aumento do nitrogênio amoniacal na silagem de alfafa resultou na redução do DMI, afetando negativamente a produção de leite e o teor de gordura no leite. A resposta quadrática para o rendimento de proteínas do leite pode ser explicada pela redução inicial da produção de leite e à subsequente utilização do nitrogênio amoniacal presente na silagem para produzir mais proteína microbiana sintetizada a nível ruminal e consequentemente, mais proteínas do leite.

Estado nutricional e produção de massa seca da alfafa em função de doses de potássio em dois solos / Nutritional status and dry mass production of alfalfa in function of potassium rates in two soils

A adubação potássica é de fundamental importância para cultura da alfafa em termos de rendimento, qualidade e persistência da forrageira, sobretudo em solos naturalmente pobres em K, como Latossolos e Argissolos. Assim, para avaliar os efeitos da aplicação de K na produção e estado nutricional da alfafa, conduziu-se um experimento em casa de vegetação utilizando-se amostras de um Latossolo Vermelho distrófico textura média (LV) (0,6 mmolc dm-3 de K) e um Argissolo Vermelho-Amarelo distrófico textura arenosa/média (PVA) (2,2 mmolc dm-3 de K). Adotou-se delineamento experimental inteiramente casualizado, em esquema fatorial 6 x 2 (seis doses de K e dois solos), com quatro repetições. As doses de K utilizadas foram: 0, 25, 50, 100, 150 e 200 mg kg-1 de K. Adubação potássica aumentou o teor de K no solo e na parte aérea das plantas. Com a concentração de K na parte aérea por volta de 10 g kg-1, as plantas apresentaram sintomas típicos de deficiência deste nutriente. O fornecimento de K elevou também a concentração de N na parte aérea das plantas cultivadas no LV. Com exceção do primeiro corte das plantas cultivadas no PVA, a adição de K incrementou a produção de massa seca da alfafa. Os níveis críticos de K no solo e na parte aérea de alfafa foram, 1,8 mmolc dm-3 e 16,7 g kg-1, respectivamente.

Potassium fertilization is very important to alfalfa crop in terms of yield, quality and persistence of forage, especially on soils naturally poor K. Thus, to assess the effects of K fertilization in alfalfa production and nutritional status, was carried out an experiment in a greenhouse using samples of a Dystrophic Oxisol medium texture (LV) (0.6 mmolc dm-3 K) and a Dystrophic Ultisol sandy/medium texture (PVA) (2.2 mmolc dm-3 K). A completely randomized design in a factorial arrangement 6 x 2 (six K rates and two soils) was used, with four replications. The K rates used were: 0, 25, 50, 100, 150 and 200 mg kg-1 K. Potassium fertilization increased K content in soil and shoots. Dry matter production was increased with the K addition. However, in the PVA, this occurred only in the second cut. In LV, potassium fertilization increased N concentration in alfalfa shoots in both cuts. Plants with K concentration around 10 g kg-1 had typical symptoms of this nutrient deficiency. The K critical levels of K in soil and shoots were 1.8 mmolc dm-3 and 16.7 g kg-1, respectively.

Efecto estrogénico del extracto hidroalcóholico de alfalfa (Medicago sativa) en ratas albinas ovariectomizadas / Estrogen effect of hydroalcoholic extract of alfalfa (Medicago sativa) in ovariectomized albino rats

Objetivo: Demostrar las propiedades estrogénicas del extracto hidroalcohólico de la especie Medicago sativa L (alfalfa) en ratas albinas ovariectomizadas (OVX). Método: Se emplearon 48 ratas albinas Sprague Dawley hembras de 200 a 250 g. de 8 semanas de edad de las cuales 40 fueron sometidas a extirpación quirúrgica de ambos ovarios (OVX) siguiendo la técnica de ovariectomía bilateral por el flanco. Después del post-operatorio las ratas se dividieron en seis grupos y se suministró el extracto hidroalcohólico de Medicago sativa (alfalfa) bajo el siguiente esquema de trabajo: Grupo 1:Control Negativo (OVX), vehículo, VO,2mL/kg; Grupo2: Control Positivo (OVX) ,Estradiol, SC, 3ug/kg; Grupo 3: (OVX)Extracto Alfalfa ,VO, 100 mg/kg; Grupo 4: (OVX),Extracto Alfalfa, VO, 500 mg/kg; Grupo 5: (OVX), Extracto Alfalfa, VO, 1000 mg/kg; Grupo 6: (No OVX),Control del procedimiento quirúrgico. El tratamiento duró 14 días. Los parámetros evaluados fueron peso del útero, peso corporal, cambios del ciclo estral mediante frotis vaginal y análisis del perfil hormonal. Resultados: Se observó aumento en el peso de útero en las dosis de 500mg/kg y 1000mg/kg.; además de la presencia de alcaloides, flavonoides y saponinas en el extracto hidroalcohólico de alfalfa en cantidad regular. En la técnica quirúrgica empleada la combinación anestésica xilazina (2mg/kg), ketamina (40mg/kg) indujo un plano quirúrgico óptimo (plano 2), sin complicaciones en el post-operatorio ni la muerte de los animales. Conclusiones: El extracto hidroalcohólico de Medicago sativa L (alfalfa) causa un efecto estrogénico al incrementar el peso de útero en ratas OVX en las dosis de 500mg/kg y 1000mg/kg. Además contiene saponinas en cantidad regular. La técnica quirúrgica ovariectomía bilateral por el flanco es un método práctico, confiable y menos traumático para los animales que permite realizar investigaciones sobre defiencia de estrógenos.

The change of splenocyte subsets in Balb/c mice by immunization with the transgenic alfalfa(Medicago sativa) containing Eg95-EgA31 fusion gene of Echinococcus granulosus / 中国地方病学杂志

Objective To investigate the change of splenocyte subsets in Balb/c mice immunized with transgenic alfalfa(Medicago sativa)containing Eg95-EgA31 fusion gene of Echinococcus granulosus(Eg) and challenged with Eg protoscoleces.Methods Leaf protein was extracted from transgenic alfalfa containing Eg95-EgA31 fusion gene by heat-coagulation method,and concentration of 20 g/L was used in the study.Meanwhile,leaf protein extracted from the transgenic alfalfa containing blank vector(pBI121)and the normal alfalfa was served as control.Thirty-two female Balb/c mice were randomly divided into 4 groups,8 mice in each group.Oral group was immunized with the leaf protein containing Eg95-EgA31 fusion antigen intragastrically(100μl per mouse);intranasal group was immunized with the leaf protein containing Eg95-EgA31 fusion antigen intranasally(10 μl per mouse);blank vector group was vaccinated intranasally with 10μl leaf protein with blank vector(pBI121);and normal control group was given 100μl normal leaf protein intragastrically.All mice in the above mentioned groups were immunized every 3 days for 2 months.Then,the mice were challenged intraperitoneally with Eg protoscoleces(50 protoscoleces per mouse)8 weeks after last vaccination and sacrified 24 weeks pest infection to separate the splenocytes.Flow cytometry was used to measure the percentages of CD4+ and CD8+ T ceils subsets.Resuits Compared with the normal control group(0.166±0.018,0.083±0.006,2.019 ±0.369),the percentages of CD4+(0.286±0.009)and CD8+(0.102±0.004)T cell subsets and the ratio of CD4+/CD8+(2.814±0.014)in oral group increased significantly (P<0.01 or<0.05).The percentage of CD4+ subset(0.269±0.016)and the ratio of CD4+/CD8+(2.955±0.986) in intranasal group was significantly higher than that ofthe normal control group(all P<0.01).The percentage of CD4+ subset in oral group was significantly higher than that of the intranasal group(P<0.05).No significant difference was found in the percentages of CD4+ and CD8+ T cell subsets and the ratio of CD4+/CD8+ between the blank vector group(0.169±0.018,0.093±0.019,1.852±0.188)and the normal control group(all P>0.05).Conclusions CD4+ T cell may play an important role in the protection induced by transgenic alfalfa vaccine against the challenge of Eg protoscoleces.Intragastrical immunization may be a good route.

The dynamic observation on splenocyte cytokines in mice immunized with the transgenic alfalfa containing Eg95-EgA31 fusion gene of Echinococcus granulosus / 中国地方病学杂志

Objective To investigate the dynamic changes of splenocyte cytokines in mice immunized with the transgenic alfalfa containing Eg95-EgA31 fusion gene of Echinococcus granulosus (Eg). Methods Eighty-eight Balb/c mice were divided into 2 groups randomly according to body weights,and immunized orally or intranasally with 100μl or 10μl extracted leaf protein from the transgenic alfalfa(20 g/L) respectively once per 3 days for 2 months. Four mice randomized from each group were killed to get splenocyte on week 0(control),2,4,6,8,10,12,14,16,18 and 20 after the last immunization. The splenocyte were cultured in medium for 48 hours with EgAg or concanavalin A (ConA) stimulation to induce the interleukin (IL)-12,interferon γ(IFN-γ) and IL-10,and cultured for 72 hours with EgAg or lipopolysaccharide (LPS) stimulus to induce the tumor necrosis factor α (TNF-α). Then the supernatant was collected to measure the level of IL-12,IFN-γ,TNF-α and IL-10 by ELISA. Results In the oral immunization group,the level of IL-12,IFN-γ,TNF-α and IL-10 increased significantly from week 4 to week 6,week 2 to week 8,week 2 to week 6 and week 4 to week 12,respectively,reaching the highest level(25.0±5.8)ng/L on week 4,(575.0±28.9)ng/L on week 2,(50.0±11.5)ng/L on week 2 and (42.5±2.9)ng/L on week 8,respectively,as compared with the values on week 0[(11.3±2.5),(125.0±28.9),(11.3±2.5),(12.5±2.9)ng/L,all P < 0.01]; in the intranasal immunization group,it was similar about the values of IL-12,IFN-γ,TNF-α and IL-10 could be seen from week 4 to week 6,week 2 to week 10,week 4 to week 10 and week 6 to week 16,respectively,reaching the highest level(25.0±5.8)ng/L on week 6,(725.0±28.9)ng/L on week 4,(27.5±2.9)ng/L on week 6 and (60.0±11.5)ng/L on week 6,respectively,as compared with the values on week 0[(11.3±2.5),(125.0±28.9),(11.3±2.5),(12.5±2.9)ng/L,all P < 0.01]. The cytokine levels in the groups with EgAg,ConA or LPS stimulus were significantly higher than those in the corresponding splenocyte suspension groups(P < 0.05 or < 0.01),and the cytokine levels in the groups with ConA or LPS stimulus were obviously higher than those in the corresponding groups with EgAg stimulation (P < 0.05 or < 0.01). Conclusion The mixed responses of Th1 and Th2 types can be induced in mice immunized with the transgenic alfalfa in the early period post immunization(2-10 weeks).

Dynamic observation on splenocytes subsets in mice immunized with the transgenic alfalfa vaccine containing Eg95-EgA31 fusion gene of Echinococcus granulosus / 中国人兽共患病学报

To observe changes on subsets of splenocytes in mice immunized with the transgenic alfalfa (Medicago sativa) vaccine containing Eg95-EgA31 fusion gene of Echinococcus granulosus dynamically,the leaf protein was extracted from the transgenic alfalfa by heat-coagulation method and prepared for a solution with a concentration of 20 g/ L.The 132 BALB/c mice were divided into 3 groups randomly and immunized intranasally or orally with the leaf protein solution once per 3 days for 2 months.At the same time,the group of intranasal immunization with leaf protein from the non-transgenic alfalfa was set as control group.4 mice randomized from each group were killed to get the spleens at Week 0,2,4,6,8,10,12,14,16,18 and 20 after last immunization,and the splenocytes were separated to measure CD_4~+and CD_8~+T cell subsets by FCM.In the oral group,CD_4~+subset increased significantly from Week 6 to Week 10 after the last immunization and reached the peak at Week 6.While CD+ 8 subset increased obviously from Week 4 to Week 12 and reached the highest level at Week 8.In the intranasal group,the significant increase of the CD_4~+subset was observed from Week 4 to Week 6 and also reached the peak at Week 6.The similar trend of CD_8~+ subset was observed from Week 4 to Week 10 and reached the highest level at Week 8.It was suggested that CD+ 4and CD+ 8subsets played an important role in the protection induced in mice immunized with the transgenic alfalfa vaccine.

Cultivation and identification of the transgenic alfalfa containing Echinococcus granulosus Eg95 gene / 中国地方病学杂志

Objective To cultivate and identify the transgenic affalfa containing Echinococcus granulosus Eg95 gene. Methods The alfalfa plants were transformed by co-cultivating alfalfa cotyledons via recombinant Agrobacterium tumefaciens LBA4404 harboring pBI-Eg95. The transgenic alfalfa explants were selected by kanamyein after calli formation, shoots and roots regeneration in the selective medium, the seedlings of transgenic plants were obtained which were finally transplanted into pots containing nutrient soil. After 2-3 months growth, the complete transgenic alfalfa plants containing Echinococcus granulosus Eg95 gene were obtained. To identify the transgenic alfalfa plants, the total DNA, RNA and leaf protein were extracted from fresh leaf tissue of the transgenic alfalfa plants and confirmed by PCR, RT-PCR, SDS-PAGE and Western blot assay. Results A specific band around 471 bp was amplified by PCR with total DNA, and the same band was obtained by RT-PCR with total RNA, which confirmed that the Eg95 gene was stably integrated into the transformed alfalfa genome. SDS-PAGE analysis showed that the relative molecular mass(Mr) of the expressed protein was about 16.5×103, consistent with the Eg95 protein, and the level of Eg95 expression was up to 0.06% of total soluble leaf protein by Bio-Rad Quantity one assay. Western blot verified the expressed protein was reactive with the sera of mice infected with Echinococcus granulosus. Conclusion The transgenic alfalfa plants containing Echinococcus granulosus Eg95 gene are successfully cultivated.

Alelopatia intra-específica de extratos aquosos de folhas e raízes de alfafa na germinação e no crescimento inicial de plântulas de dois materiais de alfafa: crioulo e melhorado / Intra-specific allelopathy of leaves and roots aqueous extracts on germination and early seedling growth of two alfalfa materials: crioulo and improved

A alfafa é uma das culturas conhecidas que apresentam um tipo específico de alelopatia denominado autotoxicidade. Dessa forma, o objetivo deste trabalho foi avaliar os efeitos da concentração de extratos aquosos de partes (folhas e raízes) da planta de alfafa (material Crioulo) na germinação e no crescimento inicial de plântulas de dois materiais de alfafa (Crioulo e P5454). Placas de Petri contendo 50 sementes de cada variedade foram umedecidas com 10ml dos extratos em duas concentrações: 50 por cento e 100 por cento (extrato puro). Água deionizada foi usada como controle. O delineamento experimental foi inteiramente casualizado, com cinco repetições para cada variedade. Após seis dias da germinação, procedeu-se à contagem do número de sementes germinadas, bem como à medição do comprimento do hipocótilo e da radícula de cada plântula. Os extratos aquosos das folhas inibiram a germinação e o comprimento da radícula e do hipocótilo das duas variedades. No entanto, para o material Crioulo, a inibição na germinação e no desenvolvimento inicial só ocorreu com o uso do extrato puro das folhas, enquanto que na P5454 o extrato diluído a 50 por cento já reduziu tanto a germinação quanto o comprimento das plântulas. Os extratos aquosos das raízes não causaram inibição na germinação nem no comprimento das plântulas. Os resultados sugerem variações intra-específicas na tolerância aos compostos aleloquímicos e uma produção preferencial dos mesmos na parte aérea da planta de alfafa, material Crioulo.

Alfalfa is one of the known crops that present a specific type of allelopathy called autotoxicity. Hence, this research was aimed at evaluating the concentration effects of aqueous extracts of plant parts (leaves and roots) of alfalfa (var. Crioula) on germination and early development of seedlings of two alfalfa (Crioula and P5454) varieties. Fifty seed of each variety were placed in Petri dishes with moistened filter paper. Aliquots (ten ml) from each part extracts at two concentrations, 50 or 100 percent (pure extract), were added to petri dishes, and distilled water was used as a control. Treatments placed in Petri dishes were randomly arranged in a completely randomized design with five replicates. Measurements included: germination, and radicle and hypocotyl lengths taken six days after germination. The aqueous extracts of leaves inhibited germination and radicle and hypocotyl length of the two alfalfa varieties. However, on the var. Crioula germination and early development was inhibited only when it was used pure extracts of leaves, while in var. P5454 leaves extracts diluted to 50 percent already had some reduction on germination and seedlings length. The aqueous extracts of the alfalfa roots did not cause inhibition on germination and seedlings lengths. The results suggest intra-specific variations on tolerance to the allelochemicals and a preferential production of them in the aerial part of the alfalfa plant, var. Crioula.