A new approach of immunotherapy against Crotalus snakes envenoming: ostrich (Struthio camelus) egg yolk antibodies (IgY-technology) / Un nuevo enfoque de inmunoterapia contra el envenenamiento de serpientes Crotalus: anticuerpos de yema de huevo de avestruz (Struthio camelus) (tecnología IgY)

Abstract Crotalid envenomation is a neglected collective health problem involving many countries in America, which need secure and inexpensive snake anti-venom treatments. Here, high antibody titers (IgY) were raised in the Ostrich (Struthio camelus) egg yolk by immunizing with the venom of Venezuelan venomous Crotalus snakes. Ostriches were immunized with a pool of venoms from common rattlesnake (Crotalus durissus cumanensis), Uracoan rattlesnake (Crotalus vegrandis), Guayana rattlesnake (Crotalus durissus ruruima) and black rattlesnake (Crotalus pifanorum). The anti-snake venom antibodies were prepared from egg yolk by the water dilution method, enriched by the addition of caprylic acid (CA) and precipitation with ammonium sulfate at 30% (W/V). The purity and molecular mass of the final product was satisfactory, yielding a single ∼ 175 kDa band in SDS-PAGE gels ran under non-reducing conditions. In the immunoblot analysis, specific binding of the antivenom was observed with most venom proteins. The LD50 was 16.5 g/mouse (825 μg/kg body weight). High titers of IgY against Crot/pool venom were shown by ELISA. The median effective dose (ED50) was 19.66 mg/2LD50. IgY antibodies neutralized efficiently the Crot/pool venom lethality. As far as we know, this is the first anti-snake venom produced in ostriches, which could make this technology an affordable alternative for low-income countries, since it is likely to produce manteniabout 2-4 g of IgY per ostrich egg. Hence, almost 400 g of IgY can be purified from only one ostrich during a year. In addition, there are enormous differences in the cost of investment in the maintenance of horses, from the points of view of infrastructure, feeding and veterinary care, in which the cost can reach USD 100 per animal per day, compared to a maintenance cost of USD 146 per month per producing bird. These results are encouraging and could easily be extrapolated to the manufacturing of other antivenoms and antitoxins as well, as they could be applied to the manufacturing of potential diagnostic tools.

Resumen El envenenamiento por crotálidos es un problema de salud colectiva desatendido, que involucra a muchos países del continente americano, los cuales necesitan tratamientos seguros y económicos. En este trabajo, se obtuvieron títulos altos de anticuerpos (IgY) producidos en yema de huevo de avestruz (Struthio camelus) mediante la inmunización con el veneno de serpientes venezolanas del genero Crotalus. Se inmunizaron avestruces con una colección de veneno de serpientes de cascabel común (Crotalus durissus cumanensis), cascabel de Uracoa (Crotalus vegrandis), cascabel de Guayana (Crotalus durissus ruruima) y cascabel negra (Crotalus pifanorum). Los anticuerpos anti-veneno de serpiente se prepararon a partir de yema de huevo por el método de dilución en agua, enriquecidos mediante la adición de ácido caprílico (CA), seguido de una precipitación con sulfato de amonio al 30% (P/V). La pureza y masa molecular de los anticuerpos (IgY) se definieron mediante ensayos de SDS-PAGE nativos y las masas moleculares se establecieron electroforéticamente, obteniéndose una única banda de IgY de ∼ 175 kDa. El análisis de inmunotransferencia mostró la unión específica del antiveneno con la mayoría de las proteínas del veneno. La DL50 fue de 16,5 μg/ratón (825 μg / kg de peso corporal); Se mostraron títulos altos de IgY contra el veneno de Crot / pool mediante ELISA. La dosis mediana efectiva (DE50) fue de 19,66 mg/2 LD50. Los anticuerpos IgY neutralizaron eficazmente la letalidad del veneno de Crot / pool. Hasta donde sabemos, se trata del primer antídoto de serpiente producido en avestruces, lo que podría abaratar la producción de este tratamiento en países del tercer mundo. Ya que es probable que se obtengan alrededor de 2-4 g de IgY por huevo de avestruz. Por lo tanto, se podrían purificar casi 400 g de IgY de un solo avestruz durante un año. Asimismo, debido a las enormes diferencias en el costo de inversión en el mantenimiento de los caballos desde el punto de vista de infraestructura, alimentación y atención veterinaria, en los que el costo puede llegar a los 100 USD por día, frente a los 146 USD por mes de mantenimiento de la producción de aves. Estos resultados abren un campo terapéutico, para la fabricación de otros antivenenos contra un amplio espectro de toxinas y también como probables herramientas de diagnóstico.

Study on bacterial endotoxin detection of egg yolk lecithin of Chinese Pharmacopoeia 2020 Edition / 中国药理学通报

Abstract; Aim To solve the problems in the appliea- tion of egg yolk leeithin endotoxin test method, and and to establish the baeterial endotoxin examination method for egg yolk lecithin (for injeetion). Methods The ethanol solution of Tween 80 ( the volume ratio of tween 80 to anhydrous ethanol was 2. 5 • 2. 7, mixed for 4 min) was used to prepare lecithin solution of egg yolk at 0. 1 kg • L 1 , and 10 test water was added to 1 mL lecithin solution of egg yolk (500 EU • mL 1 standard solution of endotoxin IOjxL was added for pos¬itive control). After diluted 20 times with endotoxin test water, the standard curve range was 10 ~0. 01 EU • mL 1 by kinetic-turbidimetrie assay. Methodology of endotoxin test was studied using limulus lysate from two manufacturers and eight hatches of samples. Results The recoveries of eight hatches of samples all met the requirement of interference test between 50% and 200% stipulated in the pharmacopoeia, which solved the problems of the current endotoxin test method in practical application. Conclusions The bacterial en¬dotoxin test method of egg yolk lecithin with good dura-bility is established to provide the basis for the revision of pharmacopoeia.

Effect of low-density lipoproteins and trehalose on the quality of cryopreserved bovine semen / Efecto de las lipoproteínas de baja densidad y la trehalosa en la calidad del semen bovino criopreservado / Efeito das lipoproteínas de baixa densidade e trealose na qualidade do sêmen bovino criopreservado

Abstract Background: In artificial insemination, chicken egg yolk is added to bovine semen to protect it during the cryopreservation process, although it contains substances that can affect the microbiological quality and metabolism of sperm. Objective: To evaluate post-thaw quality of bovine cryopreserved semen added with centrifuged and non-centrifuged egg yolk, low-density lipoproteins (LDL), and trehalose (T). Methods: Ten ejaculates from five bulls were cryopreserved under the treatments T1: pure egg yolk (PEY) at 20% v/v, T2: centrifuged egg yolk (CEY) at 20% v/v, T3: LDL at 8% v/v, T4: T at 100 mM, and T5: T at 100 mM plus LDL at 8% v/v (TLDL). Spermatic motility and kinetics, functional membrane integrity (FMI), structural membrane integrity (SMI), sperm vitality (SV) and abnormal morphology (AM) were assessed using the Sperm Class Analyzer (SCA®) system, hypoosmotic test (HOST), SYBR/PI probes, and eosin-nigrosin staining, respectively. A completely randomized design was used. Normal distribution of the variables was validated through the Kolmogórov- Smirnov test. A generalized linear model was used to determine sources of variation. Means were compared using the Tukey test. Results: Inclusion of CEY or LDL had a similar effect on sperm protection, and were superior for motility, kinetics and membrane integrity compared to the other treatments (p<0.05). CEY was superior for progressive motility (p<0.05). The cryoprotective action of LDL was similar to TLDL for motility and kinetics, SMI, SV, and AM (p<0.05). Inclusion of PEY and T resulted in the lowest semen quality (p<0.05). The use of T resulted in a reduction in FMI and SMI (p<0.05). No differences in AM between treatments were found (p>0.05). Conclusions: Egg yolk can be replaced by centrifuged egg yolk or low-density lipoproteins in the freezing extender used for bovine semen used in artificial insemination.

Resumen Antecedentes: la yema de huevo de gallina se agrega al semen bovino usado en inseminación artificial para protegerlo durante el proceso de criopreservación, aunque ésta tiene sustancias que pueden afectar el metabolismo espermatico y la calidad microbiológica del semen. Objetivo: evaluar la calidad post-descongelación del semen bovino criopreservado agregado con yema de huevo centrifugada y no centrifugada, lipoproteínas de baja densidad (LDL) y trehalosa (T). Métodos: diez eyaculados de cinco toros se criopreservaron bajo los tratamientos, T1: yema de huevo pura (PEY) al 20% v/v, T2: yema de huevo centrifugada (CEY) al 20% v/v, T3: LDL al 8% v/v, T4: T a 100 mM, y T5: T a 100 mM más LDL al 8% v/v (TLDL). La movilidad y la cinética espermática, la integridad funcional de la membrana (FMI), la integridad estructural de la membrana (SMI), la vitalidad espermática (SV) y la morfología anormal (AM), se determinaron mediante el sistema Sperm Class Analyzer (SCA®), la prueba hipoosmótica (HOST), las sondas SYBR/PI y la tinción con eosina-nigrosina, respectivamente. Se utilizó un diseño completamente al azar. La normalidad de las variables se validó mediante la prueba de Kolmogórov-Smirnov. Se utilizó un modelo lineal generalizado para determinar las fuentes de variación. Las medias de los tratamientos se compararon mediante la prueba de Tukey. Resultados: CEY y LDL tuvieron un efecto similar en la protección de los espermatozoides, siendo superiores a los demás tratamientos respecto a movilidad, cinética e integridad de la membrana (p<0,05). CEY fue superior para la movilidad progresiva (p<0,05). La acción crioprotectora de LDL fue similar a TLDL para movilidad y cinética, SMI, AM y SV (p<0,05). PEY y T resultaron en la más baja calidad seminal (p<0,05). El uso de T redujo la FMI y la SMI (p<0,05). No se encontraron diferencias en AM entre los tratamientos (p>0,05). Conclusiones: la yema de huevo puede reemplazarse por yema de huevo centrifugada o por lipoproteinas de baja densidad en el diluyente de congelación usado para semen bovino destinado a inseminacion artificial.

Resumo Antecedentes: a gema de ovo de galinha tem sido utilizada com a finalidade de proteger o sêmen bovino durante o processo de criopreservação, embora tenha substâncias que possam afetar o metabolismo dos espermatozóides e a qualidade microbiológica do sêmen utilizado para a inseminação artificial. Objetivo: avaliar a qualidade pós-descongelamento do sêmen bovino criopreservado com gema de ovo centrifugada e não centrifugada, lipoproteínas de baixa densidade (LDL) e trealose (T). Métodos: dez ejaculados de cinco touros foram criopreservados sob os tratamentos, T1: gema de ovo pura (PEY) 20% v/v, T2: gema de ovo centrifugada (CEY) 20% v/v, T3: LDL 8% v/v, T4: T 100 mM e T5: T 100 mM mais LDL 8% v/v (TLDL). Mobilidade e cinética espermática, integridade funcional da membrana (FMI), integridade estrutural da membrana (SMI), vitalidade espermática (SV) e morfologia anormal (AM) foram determinadas por o sistema Sperm Class Analyzer (SCA®), teste hiposmótico (HOST), coloração com SYBR/PI e eosina-nigrosina, respectivamente. Um design completamente aleatoriedade foi usado. A normalidade das variáveis foi validada pelo teste de Kolmogorov-Smirnov. Um modelo linear generalizado foi utilizado para determinar as fontes de variação. As médias dos tratamentos foram comparadas pelo teste de Tukey. Resultados: T2 (CEY) e T3 (LDL) tiveram efeito similar na proteção espermática, sendo superior aos demais tratamentos para mobilidade, cinética e integridade da membrana (p<0,05). T2 (CEY) foi superior para mobilidade progressiva (p<0,05). A ação crioprotetora de T3 (LDL) foi semelhante à T5 (TLDL) para motilidade e cinética, SMI, SV e AM (p<0,05). T1 (PEY) e T4 (T) tiveram a menor qualidade seminal (p<0,05). O uso de T4 (T) produziu uma redução na SMI e FMI (p<0,05). Não foram encontradas diferenças na AM entre os tratamentos (p>0,05). Conclusões: a gema de ovo pode ser substituída por gema de ovo centrifugada ou lipoproteínas de baixa densidade no diluente de congelamento de sêmen bovino.

Effect of egg yolk-based extender and seminal plasma removal on sperm viability of cooled donkey semen / Efeito do diluente à base de gema de ovo e remoção do plasma seminal na viabilidade espermática do sêmen refrigerado de jumento

Developing effective cooled semen protocols is essential to increase pregnancy rates and reproductive efficiency in donkeys. This study aimed to evaluate the effect on sperm kinetic parameters and membrane integrity in cooled donkey semen diluted with defined milk proteins extender with 1% or 2% of egg yolk and the removal of seminal plasma. Twenty-four ejaculates from six jackasses were collected. Each ejaculate was divided into four aliquots that were diluted in extender with 1% (EY1) or 2% (EY2) egg yolk. One sample from each group was centrifuged, seminal plasma was removed (CEY1, CEY2 groups, respectively), and the samples were then refrigerated at 5 °C for 24 h. Fresh and cooled semen samples were assessed for sperm motility, morphology, and plasma membrane integrity. Total motility, progressive motility, sperm kinetic parameters, or live sperm cells were not statistically different when semen was cooled with an extender supplemented with 1% or 2% of egg yolk. Seminal plasma removal does not affect total motility or sperm kinetic parameters. However, progressive motility decreased (P<0.05) when semen was extended with 2% of egg yolk and seminal plasma was removed. Membrane integrity was affected (P<0.05) in centrifuged samples. In conclusion, the obtained results suggest that there is no difference in sperm kinetics and membrane integrity when 1% or 2% of egg yolk was added to the Equiplus extender. Also, the removal of seminal plasma by centrifugation did not have any beneficial effect on cooled donkey semen. Further studies are needed to relate these results with in vivo fertility tests with cooled donkey semen.(AU)

O desenvolvimento de protocolos de sêmen resfriado eficazes é essencial para aumentar as taxas de prenhez e eficiência reprodutiva em jumentos. O objetivo desse estudo foi avaliar o efeito do diluente à base de proteínas do leite com 1 ou 2% de gema de ovo sobre os parâmetros cinéticos do sêmen e integridade da membrana em sêmen resfriado de jumento, com ou sem a remoção do plasma seminal. Vinte e quatro ejaculados de seis jumentos foram coletados. Cada ejaculado foi dividido em quatro alíquotas e diluído em diluente com 1% (EY1) ou 2% (EY2) de gema de ovo. Uma amostra por grupo foi centrifugada e o plasma seminal removido (grupos CEY1 e CEY2, respectivamente). Os pellets foram novamente ressuspendidos nas mesmas concentrações e diluentes. Em seguida, as quatro alíquotas foram refrigeradas a 5°C por 24 horas. Amostras de sêmen fresco e refrigerado foram avaliadas quanto à motilidade espermática e integridade da membrana plasmática. Motilidade total, motilidade progressiva, parâmetros de cinética espermática ou células espermáticas vivas não apresentaram diferença significativa quando o sêmen foi resfriado com diluente suplementado com 1% ou 2% de gema de ovo. A remoção do plasma seminal não afetou a motilidade total ou os parâmetros de cinética espermática; entretanto, a motilidade progressiva diminuiu (P<0,05) quando o sêmen foi diluído com 2% de gema de ovo e o plasma seminal removido. Nas amostras centrifugadas, a integridade da membrana foi afetada (P<0,05). Em conclusão, os resultados sugerem que não há diferença na cinética espermática e na integridade da membrana quando 1% ou 2% de gema de ovo são adicionados ao diluente Equiplus e a remoção do plasma seminal por centrifugação não teve nenhum efeito benéfico no resfriamento de sêmen de jumento. Mais estudos são necessários para relacionar esses resultados com testes de fertilidade in vivo com sêmen resfriado em jumentos.(AU)

Hyperresponsiveness to Boiled Egg Yolk in Early Life Leads to Prolonged Egg Allergy

Hen's egg is the most common allergen in IgE-mediated food allergy among children in Japan. Although the majority of patients with egg allergy can eat heated egg yolk safely because of its low allergenicity, severely allergic patients show an immediate-type reaction to heated egg yolk. We hypothesized that patients with hyperresponsiveness to boiled egg yolk may have difficulty in acquiring tolerance to egg. The purpose of this study was to examine the prognosis of patients with hyperresponsiveness to boiled egg yolk. Data from 121 patients with egg allergy who underwent oral food challenge (OFC) with boiled egg yolk between January 2012 and December 2013 were analyzed retrospectively. The proportion of patients who could consume heated whole egg 3 years after OFC was 15.4% in the OFC-positive group and 75.8% in the OFC-negative group. Hyperresponsiveness to boiled egg yolk in early life might lead to prolonged egg allergy in children. This finding might aid in the selection of an appropriate population requiring practical immunotherapy.

IgY antialérgenos específicos del grupo 1 de ácaros del polvo doméstico inducidos por oligopéptidos sintéticos no glicosilados / Specific IgY anti-group 1 dust mite allergens induced by unglycosylated synthetic oligopeptides

Resumen Introducción. La obtención de anticuerpos específicos capaces de detectar alérgenos del grupo 1 de ácaros del polvo doméstico representa una estrategia potencial de salud pública para reducir la exposición y la sintomatología clínica asociada con el asma y la rinitis alérgica. Objetivo. Producir y purificar anticuerpos aviares antialérgenos específicos del grupo 1 de los ácaros Dermatophagoides sp.y Blomia tropicalis utilizando la tecnología IgY. Materiales y métodos. Se diseñaron y sintetizaron oligopéptidos que evidenciaran epítopes inmunogénicos de los alérgenos Der p1, Der f1 y Blo t1 empleados posteriormente para producir anticuerpos IgY policlonales en gallinas Hy Line Brown. Las IgY presentes en las yemas de los huevos se purificaron mediante cromatografía tiofílica. Su inmunorreactividad y especificidad se determinaron mediante un inmunoensayo ELISA indirecto y Dot Blot. Resultados. Se obtuvo una reactividad elevada de las IgY contra epítopes de alérgenos presentes en extractos de cuerpo entero de D. farinae, D. pteronyssinus y B. tropicalis. Los niveles más altos de IgY se produjeron entre los días 32 y 40 de inmunización. Los anticuerpos mostraron mayor inmunorreactividad y especificidad en el reconocimiento de proteínas de D. farinae, con un límite de detección mayor de 0,03 µg de proteína total delcaroajo las condiciones experimentales analizadas. Las IgY purificadas no mostraron reactividad significativa frente al extracto de Periplaneta americana. Conclusión. La tecnología IgY permitió la producción de anticuerpos específicos contra alérgenos del grupo 1 de los ácaros del polvo al utilizar oligopéptidos sintéticos no glicosilados. Hasta donde se sabe, esta es la primera vez que se usan estos reactivos inmunológicos para la detección de ácaros de importancia médica.

Abstract Introduction: The use of specific antibodies capable of detecting allergens of the group 1 of house dust mites represents a potential strategy to reduce exposure and clinical symptomatology associated with asthma and allergic rhinitis. Objective: To produce and purify chicken antibodies specific for the dust mites Dermatophagoides sp. and B. tropicalis using the IgY technology. Materials and methods: We designed and synthesized oligopeptides showing immunogenic epitopes of Der p1, Der f1, and Blo t1. These were used to produce IgY antibodies in Hy Line Brown chickens. IgY were extracted from egg yolk using thiophilic chromatography. The immunogenicity and specificity were assayed by indirect ELISA and Dot Blot. Results: We obtained high reactivity of IgY antibodies against epitopes of allergens present in whole body mites extracts of D. farinae, D. pteronyssinus, and B. tropicalis. The highest IgY levels were registered between days 32 and 40 after immunization. The antibodies showed high immunoreactivity and specificity towards D. farinae proteins with detection limits above 0.03 µg of mite proteins under the experimental conditions used. Purified IgY did not show significant reactivity when binding to Periplaneta americana extract. Conclusion: The IgY technology allowed the production of specific antibodies against house dust mites group 1 allergens using non-glycosylated synthetic peptides. To our knowledge, this is the first time that this immunochemicals are used in the detection of mites of medical relevance.

Advances in structure, function and application of egg yolk antibody in human and animal diseases / 中国人兽共患病学报

Egg yolk immunoglobulin(IgY),also called egg yolk antibody,is a specific antibody secreted by birds after stimulation with certain antigens.For that it exhibits good resistance to heat,acid and proteinase-mediated degradation,its early-stage products were developed as food or food additives,and they have gained great social and economic benefits in both food safety and ecological agriculture fields.Due to the specific antibodies contained in these products,the products can help to resist pathogen infection.Therefore egg yolk antibody shows great value for development and application in the prevention and treat ment of animal and human diseases,and also shows great potential in human health care area.This review focuses on the development and applications of IgY biologics related with antiviral,antibacterial,antiparasitic and antitumor activities.

Preparation of egg yolk antibodies against human isomaltase and determination of their biological activities / 医学研究生学报

Objective Antibody drugs are one of the hot topics in biomedical research.This study aims to develop egg yolk antibodies (IgYs) against human isomaltase and determine their biological activities.Methods The purified recombinant isomaltase protein was used as an antigen to immunize egg-laying hens in combination with complete Freund adjuvant (CFA).Anti-isomahase IgYs were extracted by water dilution-sodium sulfate extraction assay and further analyzed for their purity,specificity,titer and stability by SDS-PAGE,Western blot and ELISA respectively,and their inhibitory effect on human alpha-glycosidase enzymes was evaluated by the PNPG method.Results Anti-isomaltase IgYs were obtained,with a titer of 1 ∶ 12800,capable of specifically binding human isomaltase,and with a good thermal stability,acid/alkali stability and pepsin resistance.Conclusion Anti-human isomaltase IgYs were successfully prepared,which may provide an experimental ground for further investigation of oral antihyperglycemic agents for type Ⅱ diabetes mellitus.

Preparation of high immunity yolk antibody against Vibrio parahemolyticus and comparison of effectiveness between different extraction methods / 吉林大学学报(医学版)

Objective:To compare the effectiveness between three methods for purifying the immunoglobulin of egg yolk(IgY) which are polyethylene glycol (PEG) method, chloroform extraction method and chloroform / PEG method, and to provide basis for obtaining the batch of IgY.Methods:The inactivated vaccine of Vibrio parahemolyticus (V. parahemolyticus) was prepared and the hens were immunized by multi-point intramuscular injection.The eggs were collected and the IgY was purified by PEG method, chloroform extraction method and chloroform/PEG method.The protein extraction rate, the IgY titer and the purity of the antibody which purified by different methods were detected.Furthermore, the operation process, cost and safety of the three methods were analyzed.Results:The protein contents of the extraction belonging three methods from high to low in turn were chloroform extraction method, chloroform/PEG method, and PEG method.There was no significant difference in the antibody titer between three methods, and the tiler of chloroform extraction method was slightly high.The purities of purified antibody from high to low in turn were PEG method, chloroform/PEG method and chloroform method.The PEG method had better security but relatively lower extraction efficiency and higher cost.The chloroform/PEG method had high extraction efficiency and good antibody purity.Conclusion:The PEG method is suitable for a small amount of extraction in the laboratory.The chloroform/PEG method is appropriate for extracting the high quality IgY in a batch as it has high extraction efficiency and good antibody purity.

Sperm-binding to the perivitelline membrane of chicken egg yolk as a functional test for sperm evaluation in dogs / Teste de ligação espermática à membrana perivitelínica da gema de ovo de galinha como avaliação funcional do sêmen de cães

During fertilization, spermatozoa interact with the zona pellucida (ZP) through the binding between the acrosome and proteins 2 and 3 (ZP2 and ZP3). The perivitelline membrane of chicken egg yolk is homologous to the mammalian ZP3, which allows the binding of sperm of several species. The aim of this study was to standardize and evaluate the efficiency of sperm-binding to the perivitelline membrane of chicken eggs as a functional method for canine semen evaluation. For this purpose, nine post-thaw sperm samples were used, which were divided into two aliquots: the first was kept in water bath at 37ºC (live sample) and the second was submitted to cold shock to induce cellular damage (dead sample). The two aliquots were mixed on five proportions, corresponding to 0%, 25%, 50%, 75%, and 100% of viable cells, and the binding test was performed by analyzing the number of spermatozoa bonded to the perivitelline membrane by means of computerized assessment of sperm motility (CASA) or conventional microscopy. Additionally, samples were submitted to sperm motility analysis, evaluation of plasmatic and acrosomal membrane integrity, and sperm mitochondrial activity. The sperm-binding test to the perivitelline membrane of chicken egg yolk was considered a feasible sperm analysis test for both fertilizing capacity and overall sperm attributes evaluation, mainly when the analysis is performed by a conventional microscope, which expands its practicality to the majority of canine reproduction laboratories.(AU)

Durante a fecundação, os espermatozoides interagem com a zona pelúcida (ZP) por meio da ligação entre o acrossomo e as proteínas 2 e 3 (ZP2 e ZP3). A membrana perivitelínica da gema de ovo de galinhas é homóloga à ZP3 de mamíferos, possibilitando a ligação espermática de diversas espécies. Este trabalho padronizou e avaliou a eficiência do teste de ligação espermática à membrana perivitelínica da gema de ovo de galinhas como avaliação funcional do sêmen de cães. Para tal, foram utilizadas nove amostras seminais previamente criopreservadas. Cada amostra foi dividida em duas alíquotas: a primeira foi mantida em banho-maria à 37ºC (vivos) e a segunda submetida a choque térmico com o intuito de induzir dano celular (mortos). As duas alíquotas foram misturadas, correspondendo a 0, 25, 50, 75 e 100% de células viáveis. As amostras foram avaliadas quanto ao número de espermatozoides ligados à membrana perivitelínica por meio da análise computadorizada da motilidade (CASA) ou microscopia convencional. Ademais, as amostras foram avaliadas quanto à motilidade espermática, integridade das membranas acrossomal e plasmática e atividade mitocondrial espermática. O teste de ligação espermática à membrana perivitelínica de ovos de galinha foi considerado um teste de análise seminal exequível tanto para avaliar a capacidade fecundante dos espermatozoides como atributos seminais gerais, especialmente quando realizado em microscopia convencional, expandindo sua praticidade para a maioria dos laboratórios de análise de sêmen canino.(AU)

Preparation and neutralization efficacy of IgY antibodies raised against Deinagkistrodon acutus venom

Background The five-paced pit viper (Deinagkistrodon acutus), endemic to China and northern Vietnam, is responsible for most snakebites in the Chinese territory. Antivenom produced from horses is the main treatment for snakebites, but it may cause numerous clinical side effects and have other disadvantages involved in their production such as the welfare of animals. The present study was conducted aiming to develop an alternative antibody (IgY) from the egg yolk of leghorn chickens immunized with snake venom. Methods IgY from the egg yolk of white leghorn chickens previously immunized intramuscularly with D. acutus venom was extracted by water, precipitated by ammonium sulfate and purified by affinity chromatographic system. IgY was identified by SDS-PAGE, ELISA and Western blot. Finally, IgY neutralization assays to test its efficacy against hemorrhagic, edema-forming and myotoxic activities of D. acutus venom were conducted on mice. Results For the first time, IgY antibodies against D. acutus venom were raised successfully in egg yolk of chickens injected with D. acutus venom multiple times. By three steps, including caprylic acid extraction, ammonium sulfate precipitation and affinity chromatography, IgY antibodies were isolated and purified from egg yolk, which exhibited a single protein band on SDS-PAGE and two bands (about 65 kDa and 35 kDa, respectively) under reducing conditions, and presented a high titer (1:40,000) tested by ELISA. Immunoblot analysis confirmed that these IgY were polyclonal antibodies since they bound to components of D. acutus venom. Furthermore, immunodiffusion assay showed that anti-D. acutus venom IgY cross-reacted with the venoms of Trimeresurus albolabris and D. saxatilis Emelianov, but did not react to the venoms of Bungarus multicinctus and Naja atra. In the neutralizing lethal assay, the median effective dose of anti-D. acutus venom IgY was 14.14 mg/kg of mouse body weight under the challenge dose (3 LD50 of D. acutus venom). In neutralizing the hemorrhagic, edema-forming and myotoxic activities of D. acutus venom, IgY showed the characteristic dose-dependent neutralization effects against all these toxic activities of D. acutus venom. Conclusion Anti-D. acutus venom IgY antibodies with high purity and titer were for the first time raised successfully in egg yolk of chickens immunized with D. acutus venom. They were effective in neutralizing the lethal effects, and the hemorrhagic, edema-forming and myotoxic acitivities of D. acutus venom. IgY could be an effective source to develop a treatment against snake bites in humans or animals in the future.(AU)

Preparation and neutralization efficacy of IgY antibodies raised against Deinagkistrodon acutus venom

Abstract Background The five-paced pit viper (Deinagkistrodon acutus), endemic to China and northern Vietnam, is responsible for most snakebites in the Chinese territory. Antivenom produced from horses is the main treatment for snakebites, but it may cause numerous clinical side effects and have other disadvantages involved in their production such as the welfare of animals. The present study was conducted aiming to develop an alternative antibody (IgY) from the egg yolk of leghorn chickens immunized with snake venom. Methods IgY from the egg yolk of white leghorn chickens previously immunized intramuscularly with D. acutus venom was extracted by water, precipitated by ammonium sulfate and purified by affinity chromatographic system. IgY was identified by SDS-PAGE, ELISA and Western blot. Finally, IgY neutralization assays to test its efficacy against hemorrhagic, edema-forming and myotoxic activities of D. acutus venom were conducted on mice. Results For the first time, IgY antibodies against D. acutus venom were raised successfully in egg yolk of chickens injected with D. acutus venom multiple times. By three steps, including caprylic acid extraction, ammonium sulfate precipitation and affinity chromatography, IgY antibodies were isolated and purified from egg yolk, which exhibited a single protein band on SDS-PAGE and two bands (about 65 kDa and 35 kDa, respectively) under reducing conditions, and presented a high titer (1:40,000) tested by ELISA. Immunoblot analysis confirmed that these IgY were polyclonal antibodies since they bound to components of D. acutus venom. Furthermore, immunodiffusion assay showed that anti-D. acutus venom IgY cross-reacted with the venoms of Trimeresurus albolabris and D. saxatilis Emelianov, but did not react to the venoms of Bungarus multicinctus and Naja atra. In the neutralizing lethal assay, the median effective dose of anti-D. acutus venom IgY was 14.14 mg/kg of mouse body weight under the challenge dose (3 LD50 of D. acutus venom). In neutralizing the hemorrhagic, edema-forming and myotoxic activities of D. acutus venom, IgY showed the characteristic dose-dependent neutralization effects against all these toxic activities of D. acutus venom. Conclusion Anti-D. acutus venom IgY antibodies with high purity and titer were for the first time raised successfully in egg yolk of chickens immunized with D. acutus venom. They were effective in neutralizing the lethal effects, and the hemorrhagic, edema-forming and myotoxic acitivities of D. acutus venom. IgY could be an effective source to develop a treatment against snake bites in humans or animals in the future.

Anti-Trimeresurus albolabris venom IgY antibodies: preparation, purification and neutralization efficacy

Background Snakebite incidence in southwestern China is mainly attributed to one of the several venomous snakes found in the country, the white-lipped green pit viper Trimeresurus albolabris. Since antivenom produced from horses may cause numerous clinical side effects, the present study was conducted aiming to develop an alternative antivenom antibody (immunoglobulin Y - IgY) from leghorn chickens. Methods IgY in egg yolk from white leghorn chicken previously injected with T. albolabris venom was extracted by water, precipitated by ammonium sulfate and purified by affinity chromatographic system. IgY was identified by SDS-PAGE, ELISA and Western blot, and its neutralizing assay was conducted on mice. Results Chickens injected multiple times with T. albolabris venom elicited strong antibody responses, and from their egg yolk IgY was isolated and purified, which exhibited a single protein band on SDS-PAGE and two bands (about 65 and 35 kDa, respectively) under reduced conditions. Immunoblot analysis revealed that these IgY are polyclonal antibodies since they bind with most venom components. In the neutralizing assay, all mice survived while the ratios of IgY/venom reached up to 3.79 (50.0 mg/13.2 mg). Conclusions IgY antibody response was successfully conducted in white leghorn chicken injected with T. albolabrisvenom. IgY against T. albolabris venom was obtained for the first time, and it exhibited strong neutralizing potency on mice. These results may lay a foundation for the development of IgY antivenom with clinical applications in the future.(AU)

Anti-Trimeresurus albolabris venom IgY antibodies: preparation, purification and neutralization efficacy

Snakebite incidence in southwestern China is mainly attributed to one of the several venomous snakes found in the country, the white-lipped green pit viper Trimeresurus albolabris. Since antivenom produced from horses may cause numerous clinical side effects, the present study was conducted aiming to develop an alternative antivenom antibody (immunoglobulin Y - IgY) from leghorn chickens. Methods IgY in egg yolk from white leghorn chicken previously injected with T. albolabris venom was extracted by water, precipitated by ammonium sulfate and purified by affinity chromatographic system. IgY was identified by SDS-PAGE, ELISA and Western blot, and its neutralizing assay was conducted on mice. Results Chickens injected multiple times with T. albolabris venom elicited strong antibody responses, and from their egg yolk IgY was isolated and purified, which exhibited a single protein band on SDS-PAGE and two bands (about 65 and 35 kDa, respectively) under reduced conditions. Immunoblot analysis revealed that these IgY are polyclonal antibodies since they bind with most venom components. In the neutralizing assay, all mice survived while the ratios of IgY/venom reached up to 3.79 (50.0 mg/13.2 mg). Conclusions IgY antibody response was successfully conducted in white leghorn chicken injected with T. albolabrisvenom. IgY against T. albolabris venom was obtained for the first time, and it exhibited strong neutralizing potency on mice. These results may lay a foundation for the development of IgY antivenom with clinical applications in the future.

Exigência nutricional de proteína bruta para codornas japonesas (Coturnix coturnix japonica) na fase de postura / Nutritional requirement of crude protein for japanese quail (Coturnix coturnix japonica) in the production phase

O objetivo do trabalho foi determinar o melhor nível de proteína bruta na alimentação de codornas japonesas em fase de produção. Foram utilizadas 300 codornas com 16 semanas de idade, alojadas em gaiolas, em um delineamento inteiramente casualizado, com cinco tratamentos, seis repetições e dez aves por unidade experimental. Os níveis avaliados foram 14, 17, 20, 23 e 26 por cento de proteína bruta, e as dietas foram formuladas para serem isoenergéticas. Foi verificado efeito quadrático para consumo de ração, peso de ovo, massa de ovo, ingestão de energia, eficiência energética por dúzia de ovo, coeficiente de digestibilidade de nitrogênio, retenção de nitrogênio, peso de gema, casca e albúmen. Efeito linear foi verificado para produção de ovos, ingestão de proteína, conversão alimentar por massa de ovos, eficiência energética por massa de ovo em kg, peso final e gravidade específica. Não houve efeito significativo para conversão alimentar por dúzia e concentração sérica de ácido úrico. Recomenda-se nível de 20 por cento de proteína bruta para codornas japonesas em fase de postura...

The aim of this study was to determine the best level of crude protein in the diet of Japanese quails in the production phase. A total of 300 quails at 16 weeks of age in cages, in a completely randomized design with five treatments and six replicates of ten birds per experimental unit was used. The levels evaluated were 14, 17, 20, 23 and 26 percent crude protein and diets were formulated to be isoenergetic. A quadratic effect was observed for feed intake, egg weight, egg mass, energy intake, energy efficiency per dozen eggs, nitrogen digestibility, shell percentage, nitrogen retention, weight of yolk, albumen and shell. A linear effect was observed for egg production, protein intake, feed conversion by egg mass, energy efficiency per egg mass in kg, final weight and yolk percentage and specific gravity. There was no significant effect on feed conversion per dozen, percentage of albumen, shell thickness and serum uric acid. The recommendation is of a 20 percent crude protein level for Japanese quail during the laying phase...

Cryoprotection effectiveness of low concentrations of natural and lyophilized LDL (low density lipoproteins) on canine spermatozoa / Eficiência de baixas concentrações de lipoproteínas de baixa densidade natural e liofilizada na crioproteto de espermatozoides caninos

The aim of this study was to evaluate the use of low concentrations of natural and lyophilized low density lipoprotein (LDL) from hen's egg yolk for cryopreservation of canine semen. Different ammonium sulphate concentrations were tested to extract LDL from egg yolk. The yolk was centrifuged, and LDL was isolated using 10, 20, 40, 45, or 50 percent ammonium sulphate solution (ASS). The LDL-rich floating fraction was collected for chemical characterization. Dry matter content was lowest (P<0.05) in the LDL extracted with the 50 percent ASS. The purification of LDL increased in association with increasing ammonium sulphate concentrations. SDS-PAGE showed that the 50 percent ASS solution yielded a purer fraction of LDL from egg yolk. For semen cryopreservation, TRIS extender was used replacing 20 percent egg yolk (control) by natural or lyophilized LDL using 1, 2, and 3 percent (w/v). Semen was centrifuged (755Xg for 7 min), diluted with one of the extenders, packed into 0.5mL straws (100x106 sperm/mL), and placed in a programmable cryopreservation machine. Thawed semen (37°C/ 30s) was analyzed for sperm motility, morphology, and by the hypoosmotic and epifluorescence tests (CFDA/ PI). Natural LDL extracted with 50 percent ASS was as effective as whole egg yolk to preserve canine frozen sperm when using low concentrations. The lyophilized LDL, mainly in the two higher concentrations tested (2 and 3 percent), was unsuitable to maintain the effectiveness of the LDL cryoprotective effect on dog sperm...

O objetivo deste estudo foi avaliar o uso de baixas concentrações da lipoproteína de baixa densidade (LBD) extraída da gema do ovo de galinha, nas formas natural e liofilizada, na criopreservado do sêmen canino. Diferentes concentrações de sulfato de amônio também foram testadas na extrato da LBD da gema do ovo. A gema foi centrifugada, sendo a LBD isolada usando-se soluto saturada de sulfato de amônio (SSA) nas concentrações de 10, 20, 40, 45 e 50 por cento. A frado rica em LBD foi coletada para caracterizado química. O conteœdo de matéria seca foi menor (P<0,05) na LBD extraída com SSA 50 por cento. A pureza da LBD melhorou medida que se aumentou a concentrado de SSA utilizada. SDS-PAGE mostrou que a SSA 50 por cento produziu uma frado mais pura de LBD oriunda da gema do ovo. Para o congelamento de sêmen, o meio diluidor TRIS teve a gema do ovo a 20 por cento (controle) substituída pela LBD a 1, 2 e 3 por cento (p/v), nas formas natural e liofilizada. O sêmen foi centrifugado (755xg por 7min), diluído em um dos meios diluidores em teste e envasado em palhetas de 0,5mL (100x106 sptz/mL), sendo congelado em máquina de congelamento programável. O sêmen descongelado (37°C/30s) foi analisado quanto motilidade e morfologia espermática e nos testes hiposm-tico e de epifluorescência (DACF/IP). A LBD natural extraída com SSA 50 por cento foi tão eficiente quanto a gema do ovo na preservado do espermatozoide canino congelado nas baixas concentrações testadas. A LBD liofilizada, principalmente as duas maiores concentrações (2 e 3 por cento), não foi adequada para manter o efeito crioprotetor da LBD sobre o espermatozoide canino...

Avian IgY antibodies: characteristics and applications in immunodiagnostic / Anticorpos IgY aviário: características e aplicações em imunodiagnóstico

Immunoglobulin Y (IgY) is the major antibody isotype in birds, reptiles, amphibia, and lungfish, playing a similar biological role as mammal IgG. Due to its phylogenetic distance, immune diversification and presence in the egg yolk, IgY provide a number of advantages in immunodiagnostic compared to IgG from mammals. Moreover, IgY production is in agreement with international efforts to reduce, refine and if possible, to replace animals in experimentation, contributing substantially in favor of animal welfare. This article presents an overview about structural and functional features, production and applications of IgY in immunodiagnostic, as well as the advantages of chicken antibodies use.

A imunoglobulina Y (IgY) é a classe de anticorpos de maior importância em aves, répteis, anfíbios e peixes pulmonados, desempenhando um papel semelhante a IgG de mamíferos. Devido a sua distância filogenética, mecanismos de diversificação imune e presença na gema do ovo, IgY proporciona uma série de vantagens em imunodiagnóstico, quando comparada a IgG de mamíferos. Além disso, esse método alternativo de produção de anticorpo está de acordo com os esforços internacionais para reduzir, refinar e, se possível, substituir animais em experimentação, contribuindo substancialmente a favor do bem-estar animal. Este artigo apresenta uma revisão sobre as características estruturais e funcionais da IgY, bem como os métodos de produção, vantagens e aplicações em imunodiagnóstico, além das vantagens da sua utilização.

Effect of chicken egg yolk antibody on canine parvoviral enteritis in pups / 대한수의학회지

Preventive and therapeutic effects of egg yolk antibody, immunoglobulin Y (IgY), against canine parvovirus (CPV) was evaluated in 25 pups orally challenged with CPV-2a. Oral administration of IgY using powder, paste and coated paste delivery systems was compared. Each type of IgY was administered orally for 17 days from 3 days before challenge. The group of pups administered coated IgY showed mild symptoms such as a moderate decrease in total white blood cell count, no depression, vomiting and diarrhea when compared with other groups. The overall clinical score of the group of pups administered coated IgY was significantly lower than that of the challenge control group. However, mortality did not differ among groups because not all pups received symptomatic treatment. These results implied that oral treatment of coated IgY could improve therapeutic effects against CPV challenge if pups received symptomatic treatment.

Isolation, purification and characterization of the egg-yolk proteins from the oocytes of the Indian freshwater murrel, Channa punctatus (Bloch).

In oviparous organisms, yolk accumulation in the oocytes is critical and indispensable for the development of the newly hatched young ones. In fish and many other oviparous vertebrates, the major constituents of the egg-yolk are synthesized as a precursor in the liver. The precursor is transported to the oocyte for uptake and cleaved into major yolk proteins lipovitellin, phosvitin and β’-components. The eggs of Channa punctatus are pelagic, have large oil globule and exceptionally high lipid content. Lipovitellin was isolated by single step gel filtration chromatography on Sepharose 6B. Purified native lipovitellin showed immunological reactivity with vitellogenin antiserum. Phosvitin isolated by phenol extraction method could not be visualized with routine protein staining methods, whereas incorporation of trivalent ions in the coomassie brilliant blue stained phosvitin. It was characterized by in vivo labeling of egg-yolk proteins with 32P. The molecular mass of murrel phosvitin was less than 14,000 kDa.

Efeito da utilização de diferentes diluidores para a produção in vitro de embriões bovinos / Effect of different bovine semen extenders on in vitro development of bovine embryos

Objetivou-se avaliar as características morfológica e funcional do sêmen bovino congelado comparando-se a eficácia de dois diferentes diluidores. O ejaculado de quatro touros foi dividido em duas partes iguais, uma submetida ao diluidor Tris e gema de ovo (A) e outra ao diluidor à base de lecitina de soja (Andromed®) (B). No experimento I, cinco palhetas dos diluidores A e B de cada touro foram descongeladas e avaliadas quanto à motilidade, vigor, concentração, morfologia espermática e teste de termor-resistência lento. Foram feitas, ainda, avaliação da integridade de membranas, por meio da associação das sondas iodeto de propídio, isotiocionato de fluoresceína - Pisum sativum e carbocianina catiônica lipofílica, e avaliação funcional da membrana plasmática com teste hiposmótico. A avaliação da integridade da cromatina foi realizada pelo método de coloração com laranja de acridina. No experimento II, o sêmen com os diferentes diluidores foi utilizado na fecundação in vitro, sendo observadas taxas de clivagem e desenvolvimento embrionário in vitro. Em relação aos resultados obtidos, apenas a porcentagem de espermatozoides no sêmen congelado foi discretamente maior com o diluidor A, concluindo-se que o diluidor composto por lecitina de soja pode substituir o composto por Tris e gema de ovo, respeitando-se as variações individuais de cada touro utilizado no presente experimento.

The goal of this study was to evaluate the protective effect of egg yolk compared with a soybean lecithin-based extender on morphologic and functional characteristics of frozen bovine semen. The ejaculate of four bulls was divided into two equal parts, one diluted with egg-yolk-Tris extender (A) and the other with a soy-lecithin based extender (Andromed®) (B). In experiment I, five straws of extender A and B from each bull were thawed and assessed regarding motility (subjective and computerized analysis), vigor, concentration, sperm morphology and slow thermoresistance (STR), evaluation of membrane integrity through association of propidium iodite probes (PI), fluorescein isotiocianate - Pisum sativum (FITC-PSA) and lipophilic cationic carbocyanine (JC-1) and functional evaluation of the plasmatic membrane through Hyposmotic Swelling Test (HOST). An evaluation of chromatin integrity was performed with the acridine orange staining procedure. In experiment II, the frozen semen with different extenders was used for in vitro fertilization (IVF), analyzing cleavage rates and in vitro embryo development. No statistical difference between extenders was identified, suggesting that the soy lecithin extender may substitute egg-yolk-Tris extender, considering the individual variations of each bull used in this experiment.