Sixteen Holstein cows in mid-lactation were used to determine whether alterations of mammary fatty acid metabolism are responsible for the milk fat depression associated with consumption of fish oil. Cows were given a total mixed ration with no added fish oil (control), unprotected fish oil (3.7% of dry matter), or glutaraldehyde-protected microcapsules of fish oil (1.5% or 3.0% of dry matter) for 4 weeks. Milk samples were taken once a week and a mammary biopsy was taken from a rear quarter at the end of the treatment period. Milk fat content was lower in cows given unprotected fish oil (26.0 g/kg), 1.5% protected fish oil (24.6 g/kg) and 3% protected fish oil (20.4 g/kg) than in cows fed the control diet (36.0 g/kg). This was mainly due to a decrease in the synthesis of short-chain fatty acids. Consumption of protected fish oil decreased the abundance of lipogenic enzymes mRNA in the mammary gland. Acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase mRNAs for cows given 3% protected fish oil averaged only 30%, 25% and 25% of control values, respectively. Dietary addition of unprotected fish oil slightly decreased mRNA abundance of these enzymes but markedly reduced the amount of lipoprotein lipase mRNA. Milk fat content was significantly correlated with gene expression of acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase but not lipoprotein lipase. These results suggest that fish oil reduces milk fat percentage by inhibiting gene expression of mammary lipogenic enzymes.

Milk production is a function of the number and activity of mammary epithelial cells, regardless of stage of lactation. Milk yield is generally higher in multiparous cows than in primiparous cows, but persistency is usually greater in the latter group. We compared several measures related to metabolic activity, apoptosis, and endocrine control of mammary cell growth in 8 primiparous and 9 multiparous cows throughout lactation. Mammary gland biopsies were taken in early [10 d in milk (DIM)], peak (50 DIM), and late (250 DIM) lactation to evaluate gene expression and determine DNA and fatty acid synthase (FAS) content. Milk samples taken the day before the biopsies were used to detect protease activities and to determine stanniocalcin-1 (STC) concentrations. Blood samples served to measure insulin-like growth factor-1, prolactin, and STC concentrations. Milk yield was higher in multiparous cows than in primiparous cows at the 10 DIM (32.8 +/- 1.3 and 25.2 +/- 0.8 kg/d) and 50 DIM (38.0 +/- 1.2 and 29.8 +/- 1.1 kg/d), but it was the same for both groups at 250 DIM (23.9 +/- 1.5 and 23.8 +/- 1.1 kg/d). Except for stearoyl-coenzyme A desaturase, expression of genes related to milk synthesis was not affected by stage of lactation. However, gene expression of acetyl-coenzyme A carboxylase, beta-casein, and FAS was lower in early lactation in primiparous cows. Expression of both proapoptotic bax and antiapoptotic bcl-2 genes was higher in primiparous cows, whereas the bax-to-bcl-2 ratio was not changed. Mammary DNA concentration was higher in multiparous cows, as was the amount of FAS protein in early lactation. Two bands of protease activity were found in milk samples, and one of the bands had an apparent molecular weight similar to gelatinase A and was dependent on the stage of lactation. Serum insulin-like growth factor-1 increased with day of lactation and was higher in primiparous cows. Serum prolactin decreased in late lactation, but peak values were observed in early lactation for primiparous cows and peak lactation for multiparous cows. Milk STC content increased with advancing lactation. The results are consistent with a lower degree of differentiation and a greater capacity for cell renewal in the mammary gland of primiparous cows.

Background: In functional genomics, transcript measurement is of fundamental importance. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays are the most popular technology and depend on the initial molecular step, the reverse transcription (RT). This study provides a complex overview of the influence of elements such as RT systems, amount of background RNA, and transcript abundance on the efficiency of qRT-PCR. Using qRT-PCR, we compared the efficiency of some commonly used RT systems and measured the production of PCR-amplifiable products and the influence of PCR inhibitor contents.

Six midlactation Holstein cows were fed a total mixed ration supplemented with either 4.8% canola meal, 3.3% unprotected canola seeds plus 1.5% canola meal, or 4.8% formaldehyde-protected canola seeds, according to a double 3 x 3 Latin square design. Each period lasted 3 wk; experimental analyses were restricted to the last week of each period. Mammary biopsies were taken the last day of each period for gene expression measurements. Milk production and milk protein percentage were reduced by canola seeds, whether protected or unprotected. Protected canola seeds also decreased dry matter intake. Feeding canola seeds reduced the content of C8 to C16 fatty acids in milk and increased the content of oleic acid (C18:1c9). Unprotected canola seeds elevated the concentrations of C18:0. Protected canola seeds increased the C18:2 and C18:3 content, and reduced the C18d:0/C18:1c9 ratio. Similar results were obtained for plasma fatty acids, with some specific features, such as an increased C16:0/C16:1 ratio with protected canola seeds. Canola seeds had no significant effects on insulin, triglycerides, or cholesterol present in serum, but increased the concentration of nonesterified fatty acids; a greater increase was obtained with protected canola seeds. Expression levels of acetyl-CoA carboxylase and delta 9-stearoyl-CoA desaturase genes measured in the mammary gland did not differ significantly between diets. Therefore, the reduced C18s:0/C18:1c9 ratio observed in milk with protected canola seeds was not due to an enhanced expression of the delta-9 desaturase in the mammary gland.

The objective of this study was to evaluate the effect of milking frequency on milk production and composition, mammary cell proliferation, apoptosis, and gene expression. For this investigation, 10 Holstein cows that were being milked twice a day in mid lactation were selected. To study the effect of differential milking, 2 quarters were milked once daily and the other 2 were milked thrice daily for 8wk. After that period, twice-daily milking was resumed for all quarters, and data were collected for an additional 6wk. Mammary gland biopsies were taken 1wk before differential milking (wk -1) and after 4 and 8wk of differential milking. Milk samples were collected weekly throughout the experiment. Once-daily milking resulted in an immediate reduction in milk yield, whereas thrice-daily milking resulted in an increase in milk yield. During differential milking, the daily milk yield of the quarters milked once daily declined by 0.54kg/wk, on average, but remained constant in the quarters milked thrice daily. Part of the difference in milk yield between the glands pairs persisted after twice-daily milking was reinitiated. In the quarters milked once daily, milk BSA concentration increased, indicating an increase in tight junction leakiness, and zymographic analysis of milk enzymes showed increased activity of several proteases. Reducing the milking frequency also increased mammary cell apoptosis and, surprisingly, mammary cell proliferation. Interestingly, milk concentrations of stanniocalcin-1 and insulin-like growth factor-I and mammary gland expression of several genes were also modulated by milking frequency. For example, expression of insulin-like growth factor I receptor was downregulated during once-daily milking. Last, expression of the short and long isoforms of the prolactin receptor and of CSN2 (beta-casein) were upregulated during thrice-daily milking. Taken together, these data suggest that milking frequency not only affects mammary gland remodeling and the expression of paracrine factors but also modulates hormone sensitivity.

Activated neutrophils are able to produce a large quantity of bactericidal molecules such as reactive oxygen species that have been associated with tissue damage in several inflammation models. The protective effects of antioxidants in a context of neutrophil-induced damage to mammary epithelial cells were first evaluated in vitro using a coculture model of activated bovine neutrophils and a bovine mammary epithelial cell line (MAC-T cells). Cell damage was determined by quantifying the release of lactate dehydrogenase by MAC-T cells in culture medium. Morphological observation of cells stained with acridine orange was used to visualize the extent of cell damage. When incubated with neutrophils activated by lipopolysaccharides and phorbol 12-myristate 13-acetate, MAC-T cells released large amounts of lactate dehydrogenase indicating significant cell damage. The addition of dimethylthiourea or bathocuproine disulfonic acid did not reduce the damage whereas catechin, deferoxamine or glutathione ethyl ester significantly reduced neutrophil-induced cytotoxicity in a dose-dependent manner. The effect of deferoxamine, an iron chelator, on the growth of Escherichia coli and the ability of bovine neutrophils to phagocytose these bacteria were then assessed in vitro. Our data showed that deferoxamine did not interfere with the phagocytic activity of neutrophils but inhibited growth of the bacteria. Overall, our results suggest that antioxidants may be effective tools for protecting mammary tissue against neutrophil-induced oxidative stress during bovine mastitis.

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Thirty Holstein cows in mid-lactation (158±20 DIM) were given a total mixed ration based on grass silage, maize silage and rolled barley. After a preliminary period of 1 week, this diet was supplemented with nothing (control), unprotected fish oil (3.7% of dry matter, DM), or two levels of glutaraldehyde-protected microcapsules of fish oil (1.5% and 3.0% of DM, respectively). Unprotected and protected supplements contained, respectively, 74% and 58% of DM as lipids. Cows given the unprotected supplement reduced their feed intake by >25%. Consequently, these cows lost body weight and produced less milk. DM intake, body weight, and milk yield were unaffected by protected fish oil. Fish oil reduced both milk fat and protein percentages, and decreased the proportion of short-chain fatty acids, stearic, and oleic acids in milk fat. Milk trans C18[ratio ]1 fatty acids increased in cows given both unprotected and protected fish oil. Milk fat content of very-long-chain n3 polyunsaturated fatty acids, including C20[ratio ]5 and C22[ratio ]6, increased with fish oil in the diet. Accordingly, the peroxide index increased and a taste panel was able to detect unusual taste in milk from cows consuming the higher level of protected fish oil and disliked the milk from cows given unprotected fish oil. In conclusion, when lactating cows consumed fish oil, milk concentration of long-chain n3 fatty acids increased and mammary de novo synthesis of fatty acids decreased, but milk yield and milk protein content were reduced, and the milk was more susceptible to oxidation and its taste was adversely affected.