Worldwide research on the health effects of bovine milk containing A1 and A2 ß-casein: Unraveling the current scenario and future trends through bibliometrics and text mining.

The possible adverse effect of consuming bovine milk with A1 ß-casein (but not with A2 ß-casein) on health aspects due to the release of ß-casomorphin-7 (BCM-7) is currently under debate. The aim of this study was to perform a bibliometric analysis of studies extracted from Scopus to explore the relationship between BCM-7, A1 or A2 bovine milk with different aspects of health. Over time, several research groups were formed that are no longer active and although some authors have returned to the field of study, they have focused their efforts mainly on conducting reviews that show the same imprecise conclusions due to the few original articles. Research is concentrated in Europe and Asia, where New Zealand, China and Germany are the countries with the most publications, records and citations on the subject, respectively. On the other hand, no country in Africa or South America has scientific production, which opens the possibility of building collaborations between countries and exploring areas that lack scientific studies. Based on conflicting information from primarily in vitro and animal studies, and limited clinical trials with poor designs, A1 milk presents pro-inflammatory and oxidative activity, but the evidence is insufficient to associate its consumption with negative health effects. However, A2 milk may be better tolerated by the digestive system of some individuals, suggesting its possible modulating role in the intestinal microbiota. Stronger scientific evidence is needed to reach a consensus on whether the presence of ß-casein A1 can significantly negatively affect health. The information shown will allow a better understanding of the subject and consumers will be able to make their own decisions regarding A1 or A2 milk.

Estructura, propiedades y genética de las caseínas de la leche: una revisión / Structure, properties and genetic of milk caseins: a review / Estrutura, propriedades e genética das caseínas do leite: uma revisão

Resumen La leche es un alimento esencial para los humanos y una de sus importancias radica en el contenido de proteínas lácteas. Las proteínas más frecuentes en este preciado líquido son las caseínas (αS1-caseína, αS2-caseína, β-caseína y κ-caseína), las cuales son fuente de aminoácidos para la dieta de los mamíferos en sus primeros días de vida. En la leche, las caseínas, están formadas por agregados moleculares de proteínas de tamaños variables denominados micelas. El objetivo de esta revisión es presentar un panorama general de la estructura, propiedades y genética de las caseínas lácteas y su relación con la salud humana. A partir de esta revisión, se pudo establecer, que las αs1 y αs2 caseínas se encuentran en conjunto con la β-caseína, formando el núcleo micelar, interactuando con los iones de calcio, para formar y mantener la micela estable. Animales caracterizados genéticamente con algunas variantes de estas proteínas, se asocian con un rendimiento en el volumen de leche. La κ-caseína, por su parte, está asociada con un aumento en el rendimiento y calidad de los quesos, de ahí su importancia económica, mientras que las formas más comunes de β-caseína en razas de ganado lechero son A1 y A2. La β-caseína A2 no presenta efectos negativos a la salud humana, por el contrario, ha sido asociada con propiedades reductoras de colesterol y triacilglicéridos. Sin embargo, la variante A1 de la β-caseína produce un péptido bioactivo denominado β-casomorfina-7 (BCM-7), que puede desempeñar un papel etiológico poco claro en el desarrollo de algunas enfermedades en humanos, tales como: enfermedad isquémica del corazón, diabetes mellitus tipo 1, síndrome de muerte súbita infantil (SIDS), desórdenes neurológicos, como autismo y esquizofrenia.

Abstract Milk is an essential food for humans and one of the reasons of its importance lies in the content of milk proteins. The most frequent proteins in this precious liquid are caseins (αS1-casein, αS2-casein, β-casein and κ-casein), which are a source of amino acids for the diet of mammals in their first days of life. In milk, caseins are made up of molecular aggregates of proteins of varying sizes called micelles. The objective of this review is to present an overview of the structure, properties and genetics of dairy caseins and their relation with human health. From this review, it was established that αs1 and αs2 caseins are found together with β-casein, forming the micellar nucleus and interacting with calcium ions, to form and maintain stable the micelle. Animals genetically characterized with some variants of these proteins are associated with a yield in milk volume. For its part, κ-casein is associated with an increase in the yield and quality of cheeses, hence its economic importance, while the most common forms of β-casein in dairy cattle are A1 and A2. β-casein A2 does not have negative effects on human health; on the contrary, it has been associated with lowering properties of cholesterol and triacylglycerides. However, the A1 variant of β-casein produces a bioactive peptide called β-casomorphin-7 (BCM-7), which may play an unclear etiological role in the development of some diseases in humans, such as: ischemic heart disease, type 1 diabetes mellitus, sudden infant death syndrome (SIDS), neurological disorders, such as autism and schizophrenia.

Resumo O leite é um alimento essencial para o ser humano e uma de suas principais característica é o teor de proteínas do leite. As proteínas mais frequentes neste líquido são as caseínas (αS1-caseína, αS2-caseína, β-caseína e κ-caseína), que são uma fonte de aminoácidos para a dieta dos mamíferos nos primeiros dias de vida. As caseínas no leite são constituídas por agregados moleculares de proteínas de variados tamanhos, chamados micelas. O objetivo desta revisão é apresentar uma visão geral da estrutura, propriedades e genética das caseínas lácteas e sua relação com a saúde humana. A partir desta revisão, foi estabelecido que as caseínas αs1 e αs2 são encontradas em conjunto com a β-caseína, formando o núcleo micelar, interagindo com os íons cálcio, para formar e manter a micela estável. Animais geneticamente caracterizados com algumas variantes dessas proteínas estão associados com o rendimento da produção de leite. Por sua vez, a κ-caseína está associada ao aumento do rendimento da produção e da qualidade dos queijos, por isso sua importância econômica, enquanto as formas mais comuns de β-caseína em bovinos leiteiros são A1 e A2. A Β-caseína A2 não tem efeitos negativos na saúde humana, pelo contrário, tem sido associada a propriedades redutoras do colesterol e dos triacilglicéridos. No entanto, a variante A1 da β-caseína produz um peptídeo bioativo denominado β-casomorfina-7 (BCM-7), que pode desempenhar uma função etiológico ainda desconhecida no desenvolvimento de algumas doenças em humanos, tais como: doença isquêmica do coração, diabetes mellitus tipo 1, síndrome da morte súbita infantil (SMSL), distúrbios neurológicos, como autismo e esquizofrenia.

Análisis genético de cinco polimorfismos de nucleótido simple de caseínas lácteas obtenidos con chips genómicos en ganado Holstein de Antioquia, Colombia / Genetic analysis of five single nucleotide polymorphisms of dairy caseins obtained with genomic chips in Holstein cattle from Antioquia, Colombia

RESUMEN Los polimorfismos genéticos asociados con las caseínas de la leche son de gran importancia, ya que pueden ser usados como marcadores genéticos para mejorar el rendimiento productivo en los hatos lecheros. El objetivo de este estudio fue evaluar la diversidad y estructura genética de 5 SNP de caseínas de la leche, obtenidos con chips genómicos en vacas y toros de raza Holstein en Antioquia (Colombia). Fueron muestreados 113 animales de raza Holstein en 3 regiones del departamento de Antioquia (norte, centro y oriente) y un cuarto grupo de sementales comerciales. Los animales fueron genotipificados con chips genómicos de alta densidad (Illumina BovineHD e Illumina SNP50 v2), a partir de los cuales se identificaron 5 SNP (ARS-BFGL-NGS-8140, BTA-77380-no-rs, BTA-32346-no-rs, BTB-00821654 y ARS-BFGL-NGS-15809). Para cada SNP se realizó un análisis genético mediante un análisis de varianza molecular (amova) usando el software GenAIEx 6.501. Los SNP con mayor heterocigosidad total (HT) fueron ARS-BFGL-NGS-8140 y BTA-32346-no-rs, con resultados cercanos al 45%; sin embargo, la Ht para ARS-BFGL-NGS-15809, BTA-77380-no-rs y BTB-00821654 estuvo por debajo del 15%. El SNP con mayor diversidad genética fue BTA-32346-no-rs (Ho-He = 0,06; p < 0,05). En esta investigación se evaluó una subpoblación de toros comerciales extranjeros, en la cual se obtuvieron frecuencias alélicas y genotípicas similares a las obtenidas para las subpoblaciones locales, sugiriendo que los alelos de los toros muy posiblemente están fijados en dichas subpoblaciones, por lo que la estructura y diversidad genética tienden a ser bajas en la muestra de estudio.

ABSTRACT Genetic polymorphisms associated with milk caseins have a great importance since they can be used as genetic markers to improve productive performance in dairy herds. The main goal of the present study was to evaluate the diversity and genetic structure of 5 SNPs of milk caseins, obtained with genomic chip in Holstein cows and bulls from Antioquia (Colombia). 113 Holstein animals were sampled in 3 regions of Antioquia (north, center, and east), and a fourth group of commercial sires. Animals were geno-typed with high-density SNP chips (Illumina BovineHD and Illumina SNP50 v2), from which 5 SNPs were identified (ARS-BFGL-NGS-8140, BTA-77380-no-rs, BTA-32346-no-rs, BTB-00821654 and ARS-BFGL-NGS-15809). For each SNP, a genetic analysis was performed by means of an analysis of molecular variance (AMOVA) using the GenAIEx 6.501 software. The SNPs with the highest total heterozygosity (Ht) were ARS-BFGL-NGS-8140 and BTA-32346-no-rs, with results close to 45%; however, the HT for ARS-BFGL-NGS-15809, BTA-77380-no-rs, and BTB-00821654 were below 15%. The SNP with the highest genetic diversity was BTA-32346-no-rs (Ho-He = 0,06; p < 0,05). In this research a subpopulation of foreign commercial bulls was evaluated, in which similar allelic and genotypic frequencies to those for local subpopulations were obtained, suggesting that the alleles of the bulls are very possibly fixed in these subpopulations, so that the structure and genetic diversity tend to be low in the study sample.

Water buffaloes (Bubalus bubalis) only have A2A2 genotype for beta-casein.

Beta-casein is a milk protein that has two variants: A1 and A2. Some individuals have difficulties digesting beta-casein A1, which can cause gastrointestinal disorders. A2 milk has emerged as an alternative. This milk only contains beta-casein A2 and is obtained from females carrying the A2A2 genotype of the gene. In cattle, allele and genotype frequencies vary according to breed and marker-assisted selection is performed to obtain A2A2 animals and the consequent production of A2 milk that is easier to digest. This study aimed to evaluate the alleles of beta-casein in buffaloes. A total of 657 buffaloes of four different breeds were genotyped and all animals carried the A2A2 genotype, i.e., allele A1 does not exist in the buffalo species. Thus, all milk products of buffaloes are naturally A2. This result adds value to products derived from buffalo milk.

Allele and genotype frequency for milk beta-casein in dairy cattle in the northern region of Tocantins State, Brazil / Frequências alélicas e genotípicas para beta-caseína do leite em bovinos leiteiros da microrregião de Araguaína, Tocantins

At present, there is a concern about the quality of milk and diseases related to its consumption, as it can generate discomfort and allergic reactions in some individuals due to its protein components. Thus, the present study was developed to identify the allele and genotype frequencies of genes for ß-casein, A1 and A2, in dairy herds in the region of Araguaína-TO, Brazil. Genetic material from 421 animals (crossbred dairy cattle in lactation) was used. All animals were numbered for identification, and DNA samples were extracted from hair bulbs. Samples for two markers from the polymorphic regions were characterized and confirmed by real-time PCR using the ABI Prism® 7500 Sequence Detection System (Applied Biosystems). Allele and genotype frequencies were determined using the TaqMan™ detection system, where the primer and probe release different fluorescence signals for each allele of the polymorphism. The sampled herd showed frequencies of 28.27% for the A1 allele and 71.73% for the A2 allele. Genotype frequencies were 52.96% (223/421) for A2A2; 37.53% (158/421) for the A1A2 genotype; and 9.50% (40/421) for the A1A1 genotype. The frequency of the A1 allele for ß-casein in dairy herds from the northern region of Tocantins was low and is per the results of previous studies. Although the A2A2 genotype of ß-casein had a high relative frequency, the A1A2 genotype is still rather frequent, warranting greater selection pressure.(AU)

Atualmente existe uma preocupação em relação à qualidade e doenças que estão relacionadas ao consumo de leite, pois o mesmo pode gerar desconfortos e reações alergicas em alguns indivíduos devido aos seus constituintes protéicos. Assim, o presente estudo teve como objetivo identificar a frequência alélica e genotípica de genes para beta caseína, A1 e A2, em rebanhos leiteiros da região de Araguaína-TO. Foram utilizados material genético de 421 animais (bovinos leiteiros mestiços em lactação), e todos os animais foram numerados para identificação e amostras de DNA foram extraídas de bulbo de folículos pilosos. As amostras para dois marcadores das regiões polimórficas foram caracterizadas e confirmadas por PCR em tempo real, usando um sistema de detecção de sequências ABI Prism® 7500 (Applied Biosystems). As frequências alélicas e genotípicas foram determinadas utilizando o sistema de detecção TaqMan ™, no qual o primer e a sonda emitem diferentes sinais de fluorescência para cada alelo do polimorfismo. Observou-se frequência do alelo A1 de 28,27%, e do alelo A2 de 71,73% no rebanho amostral. A frequência genotípica de A2A2 foi de 52,96% (223/421), com genótipo A1A2 de 37,53% (158/421), e de 9,50% (40/421) animais com genótipo A1A1. A frequência do alelo A1 para beta-caseína em rebanhos leiteiros da região norte do Tocantins foi baixa e seguiu a mesma tendência já observada em estudos anteriores. Os genótipos A2A2 da beta-caseína apresentaram frequência relativa alta, entretanto o genótipo A1A2 ainda é bastante frequente, necessitando de maior pressão de seleção.(AU)

Bisphenol A and benzophenone-3 exposure alters milk protein expression and its transcriptional regulation during functional differentiation of the mammary gland in vitro.

The plastic monomer and plasticizer bisphenol A (BPA), and the UV-filter benzophenone-3 (BP3) have been shown to have estrogenic activities that could alter mammary gland development. Our aim was to analyze whether BPA or BP3 direct exposure affects the functional differentiation of the mammary gland using an in vitro model. Mammary organoids were obtained and isolated from 8 week-old virgin female C57BL/6 mice and were differentiated on Matrigel with medium containing lactogenic hormones and exposed to: a) vehicle (0.01% ethanol); b) 1 × 10-9 M or 1 × 10-6 M BPA; or c) 1 × 10-12 M, 1 × 10-9 M or 1 × 10-6 M BP3 for 72 h. The mRNA and protein expression of estrogen receptor alpha (ESR1) and progesterone receptor (PR) were assessed. In addition, mRNA levels of PR-B isoform, glucocorticoid receptor (GR), prolactin receptor (PRLR) and Stat5a, and protein expression of pStat5a/b were evaluated at 72 h. The mRNA and protein expression of milk proteins and their DNA methylation status were also analyzed. Although mRNA level of PRLR and GR was similar between treatments, mRNA expression of ESR1, total PR, PR-B and Stat5a was increased in organoids exposed to 1 × 10-9 M BPA and 1 × 10-12 M BP3. Total PR expression was also increased with 1 × 10-6 M BPA. Nuclear ESR1 and PR expression was observed in all treated organoids; whereas nuclear pStat5a/b alveolar cells was observed only in organoids exposed to 1 × 10-9 M BPA and 1 × 10-12 M BP3. The beta-casein mRNA level was increased in both BPA concentrations and 1 × 10-12 M BP3, which was associated with hypomethylation of its promoter. The beta-casein protein expression was only increased with 1 × 10-9 M BPA or 1 × 10-12 M BP3. In contrast, BPA exposure decreased alpha-lactalbumin mRNA expression and increased DNA methylation level in different methylation-sensitive sites of the gene. Also, 1 × 10-9 M BPA decreased alpha-lactalbumin protein expression. Our results demonstrate that BPA or BP3 exposure alters milk protein synthesis and its transcriptional regulation during mammary gland differentiation in vitro.

Production of Cow's Milk Free from Beta-Casein A1 and Its Application in the Manufacturing of Specialized Foods for Early Infant Nutrition.

Beta-casein (BC) is frequently expressed as BC A2 and BC A1 in cow's milk. Gastrointestinal digestion of BC A1 results in the release of the opioid peptide beta-casomorphin 7 (BCM7) which is less likely to occur from BC A2. This work was aimed to produce milk containing BC A2 with no BC A1 (BC A2 milk) using genetically selected CSN2 A2A2 Jersey cows. Additionally, we aimed to develop an infant formula (IF) suitable for healthy full-term infants during the first six months of life based on BC A2 milk. The concentration of BCM7 released from BC A2 IF, from commercially available IFs as well as from human milk and raw cow's milk was evaluated after simulated gastrointestinal digestion (SGID). BC A2 IF presented the lowest mean relative abundance of BC A1 (IF 1 = 0.136 ± 0.010), compared with three commercially available IFs (IF 2 = 0.597 ± 0.020; IF 3 = 0.441 ± 0.014; IF 4 = 0.503 ± 0.011). Accordingly, SGID of whole casein fraction from BC A2 IF resulted in a significantly lower release of BCM7 (IF 1 = 0.860 ± 0.014 µg/100 mL) compared to commercially available IFs (IF 2 = 2.625 ± 0.042 µg/100 mL; IF 3 = 1.693 ± 0.012 µg/100 mL; IF 4 = 1.962 ± 0.067 µg/100 mL). Nevertheless, BCM7 levels from BC A2 IF were significantly higher than those found in SGID hydrolysates of BC A2 raw milk (0.742 ± 0.008 µg/100 mL). Interestingly, results showed that BCM7 was also present in human milk in significantly lower amounts (0.697 ± 0.007 µg/100 mL) than those observed in IF 1 and BC A2 milk. This work demonstrates that using BC A2 milk in IF formulation significantly reduces BCM7 formation during SGID. Clinical implications of BC A2 IF on early infant health and development need further investigations.

In vitro culture of ovine mammary gland cells expressing beta-lactoglobulin and beta-casein / Células da glândula mamária ovina cultivadas in vitro expressam beta-lactoglobulina e beta-caseina

The expression of milk proteins in vitro is essential to exploit the mammary gland cells as a biological model. Enzymatic tissue disaggregation has been widely used to establish mammary cell culture, but its effect in long-term ovine mammary cell culture is not completely elucidated. This study aimed at comparing mechanical/enzymatic and mechanical dissociation methods to establish ovine mammary cell culture. We compared cellular differentiation induced by lactating ewe serum or fetal bovine serum based on the gene expression levels of milk proteins (beta-lactoglobulin, alpha s1-casein, and betacasein). Mechanically dissociated cells were positive immunostaining for cytokeratin 8.13, such as mammary epithelial cells. These cells are responsible for milk protein expression and they are low immunostaining for vimentin, mesenchymal marker. Mechanical/enzymatic dissociation cells were positive for vimentin. The fastest cell growth (cell/hour) was observed in the mechanical dissociation group cultured with 10% fetal bovine serum medium. Mechanically and mechanically/enzymatically derived cells were able to express beta-casein and beta-lactoglobulin, but not alpha s1-casein. The relative expression of beta-lactoglobulin was not affected by the tissue dissociation method or culture media, beta-casein relative expression was down regulated in mechanically dissociated cells cultured in the presence of lactating ewe serum, (P = 0.019). Beta-casein relative expression was also down regulated in mechanically/enzymatically dissociated cells cultured with fetal bovine serum (P = 0.021). In the present conditions, we conclude that mechanical dissociation followed by culture with 10% of fetal bovine serum was the most efficient method to induce milk proteins' mRNA expression by ovine mammary epithelial cells in vitro.(AU)

A expressão in vitro de proteínas do leite é essencial para explorar as células da glândula mamária como um modelo biológico. A desagregação tecidual via enzimática é amplamente utilizada para o estabelecimento cultivo de células mamárias. No entanto, seu efeito a longo prazo no cultivo de células da glândula mamária ovina ainda não é bem elucidado. Este estudo tem como objetivo comparar dois métodos de dissociação tecidual, mecânico/enzimático e mecânico, para estabelecer cultivo celular de glândula mamária ovina. A indução da diferenciação celular, por adição de soro de ovelha lactante ou soro fetal bovino, foi avaliada pelos níveis de expressão de proteínas do leite (beta-lactoglobulina, alpha s1-caseína e beta-caseína). Células mecanicamente dissociadas foram positivamente marcadas para a presença de citoqueratina 8.13, marcador para células epiteliais mamárias. Essas células são as responsáveis pela produção das proteínas do leite e são pouco marcadas para a presença de vimentina, marcador para células de origem mesenquimal. Já as células obtidas da dissociação mecânica/ enzimática foram positivamente marcadas para presença de vimentina. A maior velocidade de crescimento (células/hora) foi observado para o grupo com dissociação mecânica cultivado em meio com 10% de soro fetal bovino. As células obtidas tanto da dissociação mecânica quanto mecânica/enzimática foram capazes de expressar beta-lactoglobulina e beta-caseína, mas não alfa s1-caseína. A expressão relativa de beta-lactoglobulina não foi afetada pelo método de dissociação ou meio de cultivo. A expressão relativa da beta-caseína foi negativamente regulada para células mecanicamente dissociadas e cultivadas na presença de soro de ovelha lactante (P = 0,019). A expressão relativa da beta-caseína também foi negativamente regulada para células dissociadas de forma mecânica/enzimática e cultivadas com soro fetal bovino (P = 0,021). Nas condições do presente estudo, concluímos que [...](AU)

In vitro culture of ovine mammary gland cells expressing beta-lactoglobulin and beta-casein / Células da glândula mamária ovina cultivadas in vitro expressam beta-lactoglobulina e beta-caseina

The expression of milk proteins in vitro is essential to exploit the mammary gland cells as a biological model. Enzymatic tissue disaggregation has been widely used to establish mammary cell culture, but its effect in long-term ovine mammary cell culture is not completely elucidated. This study aimed at comparing mechanical/enzymatic and mechanical dissociation methods to establish ovine mammary cell culture. We compared cellular differentiation induced by lactating ewe serum or fetal bovine serum based on the gene expression levels of milk proteins (beta-lactoglobulin, alpha s1-casein, and betacasein). Mechanically dissociated cells were positive immunostaining for cytokeratin 8.13, such as mammary epithelial cells. These cells are responsible for milk protein expression and they are low immunostaining for vimentin, mesenchymal marker. Mechanical/enzymatic dissociation cells were positive for vimentin. The fastest cell growth (cell/hour) was observed in the mechanical dissociation group cultured with 10% fetal bovine serum medium. Mechanically and mechanically/enzymatically derived cells were able to express beta-casein and beta-lactoglobulin, but not alpha s1-casein. The relative expression of beta-lactoglobulin was not affected by the tissue dissociation method or culture media, beta-casein relative expression was down regulated in mechanically dissociated cells cultured in the presence of lactating ewe serum, (P = 0.019). Beta-casein relative expression was also down regulated in mechanically/enzymatically dissociated cells cultured with fetal bovine serum (P = 0.021). In the present conditions, we conclude that mechanical dissociation followed by culture with 10% of fetal bovine serum was the most efficient method to induce milk proteins' mRNA expression by ovine mammary epithelial cells in vitro.(AU)

A expressão in vitro de proteínas do leite é essencial para explorar as células da glândula mamária como um modelo biológico. A desagregação tecidual via enzimática é amplamente utilizada para o estabelecimento cultivo de células mamárias. No entanto, seu efeito a longo prazo no cultivo de células da glândula mamária ovina ainda não é bem elucidado. Este estudo tem como objetivo comparar dois métodos de dissociação tecidual, mecânico/enzimático e mecânico, para estabelecer cultivo celular de glândula mamária ovina. A indução da diferenciação celular, por adição de soro de ovelha lactante ou soro fetal bovino, foi avaliada pelos níveis de expressão de proteínas do leite (beta-lactoglobulina, alpha s1-caseína e beta-caseína). Células mecanicamente dissociadas foram positivamente marcadas para a presença de citoqueratina 8.13, marcador para células epiteliais mamárias. Essas células são as responsáveis pela produção das proteínas do leite e são pouco marcadas para a presença de vimentina, marcador para células de origem mesenquimal. Já as células obtidas da dissociação mecânica/ enzimática foram positivamente marcadas para presença de vimentina. A maior velocidade de crescimento (células/hora) foi observado para o grupo com dissociação mecânica cultivado em meio com 10% de soro fetal bovino. As células obtidas tanto da dissociação mecânica quanto mecânica/enzimática foram capazes de expressar beta-lactoglobulina e beta-caseína, mas não alfa s1-caseína. A expressão relativa de beta-lactoglobulina não foi afetada pelo método de dissociação ou meio de cultivo. A expressão relativa da beta-caseína foi negativamente regulada para células mecanicamente dissociadas e cultivadas na presença de soro de ovelha lactante (P = 0,019). A expressão relativa da beta-caseína também foi negativamente regulada para células dissociadas de forma mecânica/enzimática e cultivadas com soro fetal bovino (P = 0,021). Nas condições do presente estudo, concluímos que o método de dissociação mecânica seguido pelo cultivo em meio com 10% de soro fetal bovino foi o método mais eficiente para induzir a expressão mRNA de proteínas do leite por células epiteliais mamárias ovinas in vitro.(AU)