Composition, functional properties and sensory characteristics of Mozzarella cheese manufactured from different somatic cell counts in milk

In the present study, composition, functional properties and sensory characteristics of Mozzarella cheese produced from milk with somatic cell counts (SCC) at low (<200,000 cells/mL), intermediate (≈400,000 cells/mL) and high (>800,000 cells/mL) levels were investigated. Three batches of cheese were produced for each SCC category. The cheeses were vacuum packed in plastic bags and analysed after 2, 9, 16, 23 and 30 days of storage at 4ºC. SCC level did not affect the moisture, fat, total protein and ash content, mesophilic and psychrotrophic bacteria, and sensory parameters of Mozzarella cheese. However, meltability increased in cheese manufactured from high SCC milk. Results indicated that raw milk used to produce Mozzarella cheese should not contain high SCC (>800,000 cells/mL) in order to avoid changes in the functional properties of the Mozzarella cheese.

Short communication: Evaluation of an on-farm test to estimate somatic cell count

The objective of this study was to compare the results of an on-farm test, named Somaticell, with results of electronic cell counting and for milk somatic cell count (SCC) among readers. The Somaticell test correctly determined the SCC in fresh quarter milk samples. Correlation between Somaticell and electronic enumeration of somatic cells was 0.92 and. coefficient 0.82. Using a threshold of 205,000 cells/mL, the sensitivity and specificity for determination of intramammary infections were 91.3 and 96.0%, respectively. The SCC was greater for milk samples from which major mastitis pathogens were recovered. Minor variation among readers was observed and most likely associated with the mixing procedure. However, the final analysis indicated that this variation was not significant and did not affect the amount of samples classified as having subclinical mastitis. The on-farm test evaluated in this study showed adequate capacity of determining SCC on quarter milk samples and may be considered as an alternative for on-farm detection of subclinical mastitis.

Effects of immunosuppressants on hepatocyte cell mitosis during liver regeneration in growing animal models of partial hepatectomy

Objective. We sought to evaluate the effects of immunosuppressant drugs (corticosteroid, cyclosporine [CsA], and tacrolimus [Tac]) on liver regeneration in growing animals submitted to 70% hepatectomy. Materials and Methods. Newborn and weaning rats were submitted to 70% hepatectomy receiving separately methylprednisolone, CsA, or Tac. All animals were sacrificed 24 hours after the procedure. The remnant liver lobes were subjected to histomorphometric analyses with determination of hepatocyte mitotic index. Results., Administration of immunosuppressants did not change the mitotic index of the regenerating liver in newborn animals. In weaning rats, methylprednisolone reduced the mitotic index (P = .01) and Tac caused a greater increase in this rate (P = .001). CsA had no effect on mitotic index. The number of hepatocyte mitoses in newborn animal livers was greater than that in weaning animal livers (P = .001). Conclusion. In situations in which intense, fast processes of liver regeneration are crucial, the advantages of the use of Tac must be considered, such as in pediatric transplant patients.

Apoptosis and necrosis of polymorphonuclear leukocytes in goat milk with high and low somatic cell counts

The purpose of the present trial was to compare the percentages of necrotic and apoptotic polymorphonuclear leukocytes (PMNL) in goat milk with low and high somatic cell count (SCC). Twenty eight milk samples were collected from 20 lactating goats, determined to be negative in bacteriological examination, and divided in three groups, according to their SCC: samples with SCC lower than 500 x 10(3) cells/mL; between 500 and 1500 x 10(3) cells/mL; and higher than 1500 x 10(3) cells/m L. SCC was performed in an automatic somatic cell counter. Apoptosis and necrosis were quantified using dual-color flow cytometry with fluorescein labeled annexin-V and propidium iodide (PI). Results of the present study showed a significant positive correlation between the percentage of the viable PMNL and milk SCC(r = 0.495, P=0.008), as well as a significant negative correlation between apoptotic PMNL and milk SCC(r = -0.486, P = 0.009). Results also pointed out lower PMNL viability rates due to higher apoptosis rates in milk samples with SCC lower than 5 x 10(5) cells/mL. (C) 2011 Elsevier B.V. All rights reserved.

Evaluation of somatic cell count thresholds to detect subclinical mastitis in Gyr cows

The objectives of this study were (1) to determine the sensitivity (Se) and specificity (Sp) of somatic cell count (SCC) thresholds to identify subclinical mastitis in Gyr cows caused by major and minor pathogens; (2) to study the effects of month of sampling, rear or front mammary quarters, herd, intramammary infection (IMI), and bacterial species on SCC at quarter level; and (3) to describe the prevalence of IMI in Gyr cows in commercial dairy herds. In total, 221 lactating Gyr cows from 3 commercial dairy farms were selected. Milk samples were collected from individual quarters once a month for 1 yr from all lactating cows for SCC and bacteriological analysis. Mammary quarters were considered the experimental units and the SCC results were log(10)-transformed. Four SCC thresholds (100, 200, 300 and 400 x 10(3) cells/mL) were used to determine Se and Sp to identify infected mammary quarters. The overall prevalence of IMI in quarter milk samples of Gyr cows was 49.8%, and the prevalence of minor pathogens was higher (31.9%) than that of major pathogens (17.8%). Quarter samples with microbial isolation presented higher SCC compared with negative samples. Sensitivity and Sp of selected SCC thresholds varied according to the group of pathogen (major and minor) involved in the IMI definition. Sensitivity increased and Sp decreased when mammary quarters with only major pathogens isolation were considered positive. The use of a single SCC analysis to classify quarters as uninfected or infected in Gyr cows may not be a useful test for this breed because Se and Sp of SCC at the studied thresholds were low. The occurrence of IMI and the bacterial species are the main factors responsible for SCC variation in mammary quarters of Gyr cows. Milk samples with major pathogens isolation elicited higher SCC than those with minor pathogens.

Mechanism of mitosis-specific activation of MEK1

Activation of cyclin B-Cdc2 is an absolute requirement for entry into mitosis, but other protein kinase pathways that also have mitotic functions are activated during G(2)/M progression. The MAPK cascade has well established roles in entry and exit from mitosis in Xenopus, but relatively little is known about the regulation and function of this pathway in mammalian mitosis. Here we report a detailed analysis of the activity of all components of the Ras/Raf/MEK/ERK pathway in HeLa cells during normal G(2)/M. The focus of this pathway is the dramatic activation of an endomembrane-associated MEK1 without the corresponding activation of the MEK substrate ERK. This is because of the uncoupling of MEK1 activation from ERK activation. The mechanism of this uncoupling involves the cyclin B-Cdc2-dependent proteolytic cleavage of the N-terminal ERK-binding domain of MEK1 and the phosphorylation of Thr(286). These results demonstrate that cyclin B-Cdc2 activity regulates signaling through the MAPK pathway in mitosis.

Evaluation of somatic cell variants deficient in glycosylphosphatidyl-inositol anchoring as candidates for genetic correction.

Many cell surface glycoproteins are anchored in the lipid bilayer by a glycosylphosphatidyl-inositol (GPI) structure. Recently, a number of cell lines which are deficient in the biosynthesis and/or addition of this anchor have been described. In this report, we summarize the current knowledge on these lines and discuss their potential use to isolate the genes involved in the GPI anchor biosynthetic pathway with a specific emphasis on L cell fibroblasts.

Casein fractions of ultra high temperature milk with different somatic cell counts

The objective of this work was to evaluate the effect of somatic cell counts (SCC) in casein fractions of ultra high temperature (UHT) milk. Raw milks were categorized in SCC groups of low (200,000-320,000 cells mL-1), intermediate (380,000-560,000 cells mL-1) and high cells (600,000-800,000 cells mL-1). Five replicates of UHT milks within each SCC category were analyzed for casein fractions after 8, 30, 60, 90 and 120 days of storage through high performance liquid chromatography. SCC showed effect only on beta-casein reduction. SCC in raw milk increases the proteolysis of UHT milk, as a consequence of beta-casein degradation.

Expression of steroidogenic proteins and genes in bovine placenta from conventional and somatic cell nuclear transfer (SCNT) gestations

Pendant la grossesse, les hormones stéroïdes jouent un rôle indispensable dans la régulation des principales manifestations physiologiques telles que la reconnaissance maternelle de la gestation, la réceptivité de l'endomètre, le début du développement embryonnaire ainsi que le maintien de la gestation. Cependant, on sait très peu sur la production de ces hormones et les principaux facteurs des voies intracellulaires impliqués dans le processus de stéroïdogenèse dans le placenta bovin pendant les stades initiaux et plus avancés de la gestation. Par ailleurs, certaines anomalies du placenta chez les bovins suite à une mauvaise production de stéroïdes n'ont pas encore été démontrées. Les objectifs de cette thèse étaient donc de : 1) déterminer la présence et la localisation des principales protéines stéroïdiennes dans le placenta de bovins provenant de gestations de 50 à 120 jours, 2) comparer l'expression placentaire d'une série de gènes et de protéines stéroïdiennes entre une gestation impliquant un transfert de noyaux de cellules somatiques (SCNT) et une gestation non-clonale; 3) étudier l'impact des hormones trophiques et des seconds messagers sur la stéroïdogenèse dans le placenta bovin à 140 +10 jours de gestation. L’utilisation de techniques d’immunohistochimie, d’immunobuvardage et de PCR quantitatif nous a permis d’évaluer la présence d'un large éventail de gènes stéroïdiens (STAR, CYP11A1, HSD3B1, CYP17A1 et SCARB1) qui participent au transport du cholestérol et dans la production de différents types de stéroïdes. Dans cette thèse, nous avons démontré la capacité du placenta bovin d’initier la stéroïdogenèse au début de la gestation et nous avons également déterminé les principales cellules impliquées dans ce processus. Nous avons constaté que les tissus maternels expriment les principaux marqueurs de stéroïdogenèse suggérant une plus grande capacité stéroïdogénique que les tissus fœtaux. En outre, un modèle d'expression des protéines complémentaires stéroïdogéniques entre la caroncule et le cotylédon a été observé, indiquant que la stéroïdogenèse placentaire exige une communication cellule à cellule entre les cellules de la mère et du fœtus. Après avoir démontré les principales cellules impliquées dans la synthèse des hormones stéroïdiennes dans le placenta bovin en début de gestation, nous avons ensuite étudié les modifications possibles de la stéroïdogenèse dans les tissus SCNT cotylédonaires à 40 jours de gestation. Nous avons identifié d'importantes modifications dans l'expression des gènes STAR, CYP11A1, HSD3B1, CYP17A1, et SULT1E1. Conséquemment, nous postulons que l'expression réduite des gènes stéroïdiens peut provoquer une insuffisance de la biosynthèse des hormones stéroïdiennes, ce qui pourrait contribuer à un développement anormal du placenta et du fœtus dans les gestations SCNT à court ou long terme. Finalement, nous avons développé un modèle efficace de culture d’explants de placentome qui nous a permis d'explorer les mécanismes sous-jacents spécifiques à la stéroïdogenèse placentaire. Nous avons exploré l'effet stimulant des hormones trophiques et différents messagers secondaires sur l'expression de différentes protéines stéroïdogéniques ainsi que le taux de progestérone (P4) dans les explants de placentome. En utilisant les techniques de RIA et de PCR quantitatif, nous avons constaté que même si les analogues de l'hormone lutéinisante (hCG) ont un effet stimulant sur plusieurs gènes stéroïdiens, le calcium ionophore est le principal modulateur dans la synthèse de la P4. Ces résultats suggèrent que dans le placenta bovin, la synthèse de la P4 est modulée principalement par l'afflux de calcium intracellulaire, et apparemment les nucléotides cycliques ne semblent pas contrôler ce processus. En conclusion, cette étude contribue de manière significative à une meilleure compréhension des mécanismes d'entraînement de la synthèse des stéroïdes placentaires au début de la gestation et permet aussi d’apporter de nouveaux éclairages sur l'importance des stéroïdes placentaires dans la régulation du développement du placenta et du fœtus.

Somatic Cell Therapy: A Genetic Rescue for a Tattered Immune System?

Étude de cas / Case study

Relationship between the somatic cell count in raw milk and the casein fractions of UHT milk

The relationship between somatic cell count (SCC) in raw milk and casein fractions of 15 batches of the corresponding ultra-high-temperature (UHT) milk was examined. Raw milk was collected, pasteurised and submitted to UHT treatment. Samples of the UHT milk were taken on days 8, 30, 60, 90 and 120 of storage at room temperature and their casein fractions analysed by high performance liquid chromatography. SCC ranged from 197,000 to 800,000 cells/mL. No correlation (p>0.05) was found between SCC and K-casein concentrations in raw or UHT milks. The alpha(s2) and P-casein concentrations in raw milk were negatively correlated with SCC (p<0.05). In UHT milk, negative correlations were observed for a,1-casein (p<0.05) and beta-casein (p<0.05) on the 8th day, and for alpha S-2-casein (p<0.01) on the 60th day of storage. Results indicate that higher SSC in raw milk is associated with substantial degradation of beta-casein and alpha(s)-casein, which may lead to quality defects in UHT milk during storage. Aust. J. Dairy Technol. 63, 45-49

Loss of Methylation at H19 DMD Is Associated with Biallelic Expression and Reduced Development in Cattle Derived by Somatic Cell Nuclear Transfer

Although cloning of mammals has been achieved successfully, the percentage of live offspring is very low because of reduced fetal size and fewer implantation sites. Recent studies have attributed such pathological conditions to abnormal reprogramming of the donor cell used for cloning. The inability of the oocyte to fully restore the differentiated status of a somatic cell to its pluripotent and undifferentiated state is normally evidenced by aberrant DNA methylation patterns established throughout the genome during development to blastocyst. These aberrant methylation patterns are associated with abnormal expression of imprinted genes, which among other genes are essential for normal embryo development and gestation. We hypothesized that embryo loss and low implantation rates in cattle derived by somatic cell nuclear transfer (SCNT) are caused by abnormal epigenetic reprogramming of imprinted genes. To verify our hypothesis, we analyzed the parental expression and the differentially methylated domain (DMD) methylation status of the H19 gene. Using a parental-specific analysis, we confirmed for the first time that H19 biallelic expression is tightly associated with a severe demethylation of the paternal H19 DMD in SCNT embryos, suggesting that these epigenetic anomalies to the H19 locus could be directly responsible for the reduced size and low implantation rates of cloned embryos in cattle.

Effect of somatic cell counts on lipolysis, proteolysis and apparent viscosity of UHT milk during storage

In this work, lipolysis, proteolysis and viscosity of ultra-high temperature (UHT) milk containing different somatic cell counts (SCC) were investigated. UHT milks were analysed on days 8, 30, 60, 90 and 120 of storage. Lipolysis as measured by free fatty acids increase, casein degradation and viscosity of UHT milk were not affected by SCC but increased during storage. A negative relationship was observed between SCC and casein as a percentage of true protein on the 120th day of storage, hence indicating that high SCC increases the proteolysis of UHT milk by the end of its shelf life.

Viable Calves Produced by Somatic Cell Nuclear Transfer Using Meiotic-Blocked Oocytes

Somatic cell nuclear transfer (SCNT) has had an enormous impact on our understanding of biology and remains a unique tool for multiplying valuable laboratory and domestic animals. However, the complexity of the procedure and its poor efficiency are factors that limit a wider application of SCNT. In this context, oocyte meiotic arrest is an important option to make SCNT more flexible and increase the number of cloned embryos produced. Herein, we show that the use of butyrolactone I in association with brain-derived neurotrophic factor (BDNF) to arrest the meiotic division for 24 h prior to in vitro maturation provides bovine (Bos indicus) oocytes capable of supporting development of blastocysts and full-term cloned calves at least as efficiently as nonarrested oocytes. Furthermore, the procedure resulted in cloned blastocysts with an 1.5- and twofold increase of POU5F1 and IFNT2 expression, respectively, which are well-known markers of embryonic viability. Mitochondrial DNA (mtDNA) copy number was diminished by prematuration in immature oocytes (718,585 +/- 34,775 vs. 595,579 +/- 31,922, respectively, control and treated groups) but was unchanged in mature oocytes (522,179 +/- 45,617 vs. 498,771 +/- 33,231) and blastocysts (816,627 +/- 40,235 vs. 765,332 +/- 51,104). To our knowledge, this is the first report of cloned offspring born to prematured oocytes, indicating that meiotic arrest could have significant implications for laboratories working with SCNT and in vitro embryo production.