Alternative to decrease cholesterol in sheep milk cheeses.

The presence of cholesterol in foods is of nutritional interest because high levels of this molecule in human plasma are associated with an increasing risk of cardiovascular disease and nowadays consumers are demanding healthier products. The goal of this experiment was to diminish the cholesterol content of Manchego, the most popular Spanish cheese manufactured from ewes milk. For this purpose three bulk milks coming from dairy ewe fed with 0 (Control), 3 and 6% of linseed supplement on their diet were used. Nine cheeses (3 per bulk milk) were manufactured and ripened for 3 months. Cholesterol of ewes milk cheese from 6% to 12% linseed supplemented diets decreased by 9.6% and 16.1% respectively, therefore supplying a healthier profile. In a second experiment, different sources of unsaturated fatty acids (rich in oleic, linoleic and α-linolenic acids) were supplemented to dairy ewes and no significant differences were found on cheese cholesterol levels.

Effects of cell culture techniques on gene expression and cholesterol efflux in primary bovine mammary epithelial cells derived from milk and tissue.

Primary bovine mammary epithelial cells (pbMEC) are often used in cell culture to study metabolic and inflammatory processes in the udder of dairy cows. The most common source is udder tissue from biopsy or after slaughter. However, it is also possible to culture them from milk, which is non-invasive, repeatable and yields less contamination with fibroblasts. Generally, not much is known about the influence of cell origin and cell culture techniques such as cryopreservation on pbMEC functionality. Cells were extracted from milk and udder tissue to evaluate if milk-derived pbMEC are a suitable alternative to tissue-derived pbMEC and to test what influence cryopreservation has. The cells were cultivated for three passages and stored in liquid nitrogen. The relative gene expression of the five target genes kappa-casein, lingual antimicrobial peptide (LAP), lactoferrin, lysozyme (LYZ1) and the prolactin receptor normalised with keratin 8 showed a tendency to decrease in the tissue cultures, but not in the milk-derived cultures, suggesting a greater influence of the cultivation process on tissue-derived cells, freezing lowered expression levels in both cultures. Overall expression of LAP and LYZ1 tended to be higher in milk cells. Cholesterol efflux was measured to compare passages one to seven in milk-derived cells. Passage number did not alter the efflux rate (p ≤ 0.05). We showed for the first time that the extraction of pbMEC from milk can be a suitable alternative to tissue extraction.

Effects of continuous milking during the dry period or once daily milking in the first 4 weeks of lactation on metabolism and productivity of dairy cows.

The objective was to compare the effects of 3 management systems in high-yielding dairy cows on metabolic profiles and milk production. Thirty-six multiparous Brown Swiss cows were randomly assigned to 1 of 3 treatment groups (n=12 cows/group): the control (C) group, in which cows were dried off 56 d before calving and milked twice daily throughout next lactation (305 d); the once daily milking (ODM) group, in which cows were dried off 56 d before calving and milked once daily for the first 4 wk of lactation and twice daily for the remaining lactation; and the continuous milking (CM) group, in which cows were milked twice daily until calving and also during the subsequent lactation. Serum glucose concentrations decreased between wk 1 and 4 exclusively in C cows. Serum concentrations of NEFA and BHBA in the first 4 wk of lactation were highest in C cows compared with ODM and CM cows. Decreased backfat thickness during early lactation and reduction of body condition score were markedly more pronounced in C cows compared with ODM and CM cows. Mean lactational milk yield of C cows [11,310+/-601 kg of energy-corrected milk (ECM)/305 d] was approximately 16% higher compared with ODM cows (9,531+/-477 kg of ECM/305 d) and CM cows (9,447+/-310 kg of ECM/305 d). The lactation curve of CM cows compared with C cows was characterized by a similar time of peak yield (wk 3), a reduced peak yield, and no obvious differences in persistency. Mean percentage of milk protein was significantly higher for CM cows (3.91%) compared with C cows (3.52%). In contrast, once daily milking was accompanied by a reduced and significantly delayed peak yield (wk 8) compared with the control treatment, whereas persistency was better and milk protein (3.79%) was higher in ODM cows than in C cows. In conclusion, continuous milking and once daily milking, targeting the interval before or after calving, respectively, substantially reduced the metabolic challenge of fresh cows and improved milk protein percentage. Continuous milking and once daily milking increased milk protein percentage markedly; furthermore, once daily milking during the first 4 wk of lactation improved the lactation curve.

Identification of the bovine cholesterol efflux regulatory protein ABCA1 and its expression in various tissues.

The ATP-binding cassette transporter A1 (ABCA1) is known to play a significant role in cellular export of phospholipids and cholesterol in humans. The ABCA1 transporter might also play a crucial role in cellular cholesterol homeostasis in the cow or in the transfer of cholesterol into the milk, but its presence and tissue distribution in the bovine is unknown. Therefore, we studied the expression and distribution of the bovine ABCA1 transporter using quantitative PCR and sequenced the entire ABCA1 coding region. In addition, the proximal promoter was identified and screened for regulatory elements. Concordant with data from other mammalian species, bovine ABCA1 mRNA was expressed and detected in all tissues tested. The highest expression levels were detected in lung, esophagus, uterus, spleen, and muscle. Sequence analysis revealed that the open reading frame of this gene consists of 6,786 bases and encodes for a protein of 2,261 AA with a predicted molecular weight of 254 kDa. The deduced bovine ABCA1 protein shows the highest AA sequence homology with human (94%), mouse (93%), rat (92%), and chicken (85%). Analysis of the putative ABCA1 promoter region revealed potential transcription factor binding sites associated with ABCA1 transcription and lipid metabolism. This work could open new avenues for elucidating a potential role of ABCA1 in sterol homeostasis in the bovine organism.

Identification, sequence analysis and mRNA tissue distribution of the bovine sterol transporters ABCG5 and ABCG8.

The family of ATP-binding cassette (ABC) transporters consists of several transmembrane proteins that use ATP hydrolysis as an energy source for the transport of a variety of substances through cellular membranes. Two members of this family, ABCG5 and ABCG8, are implicated in the intestinal absorption and biliar excretion of sterols. Cholesterol content in milk is highly variable among species, breeds, and individuals of the same species, but a potential application of these genes in lipid homeostasis in the mammary gland has never been addressed. In the present work, expression of ABCG5 and ABCG8 in the bovine was demonstrated for the first time and characterized by quantitative PCR. The entire coding region and promoter area were sequenced and screened for motifs involved in lipid homeostasis. Both ABCG5 and ABCG8 presented a high level of length and sequence identity with other mammalian species. In the intergenic promoter region, 2 GATA boxes, a liver receptor homolog-1 response element, and a nuclear factor-kappaB response element, important factors in other lipid regulatory processes, were identified. As expected, high expression levels of both ABCG5 and ABCG8 were present in liver and digestive tract samples, and interestingly, in the mammary gland, opening new avenues for further investigation about their potential role in lipid trafficking and excretion during lactation.

Variations in APOE genotype distribution in children from areas with different adult cardiovascular disease mortality in Spain.

We investigate whether a varying distribution of the APOE genotype could help explain regional differences in ischemic heart disease (IHD) mortality in Spain. APOE genotypes were examined by PCR in 1,274 randomly selected healthy children from four Spanish regions with different adult IHD mortality rates (northwest and central Spain with low rates and southeast and southern Spain with high rates). In the population as a whole the prevalence of the higher risk APOE*3/*4 genotype is 16.8% and the prevalence of the APOE*4 allele is 10.1%. In northwest Spain the frequencies of the APOE*3/*4 genotype (12.9%) and of the APOE*4 allele (8.3%) are smaller than in the other regions. The southeast region shows statistically higher frequencies of the APOE*3/*4 genotype (22.5%) and of the APOE*4 allele (13.2%) than in the other regions or in the group as a whole. We can conclude that Spain is not homogeneous in terms of APOE genotype distribution. Although the prevalence of the APOE*4 allele is generally low, there are areas with higher prevalence of the APOE*4 allele and a higher incidence of adult IHD mortality. This allows us to conclude that in Spain this genetic determinant can be associated with IHD mortality in relatively isolated populations.