Effect of short-term feed restriction on temporal changes in milk components and mammary lipogenic gene expression in mid-lactation Holstein dairy cows.

Investigations of the temporal changes in mammary gene expression that occur during sudden diet change have been limited by the use of mammary tissue as the source of RNA because of the invasive nature of mammary biopsy procedures. However, the cytosolic crescent, present in 1% of the largest milk fat globules, contains mammary epithelial cell RNA that has become trapped between the inner and outer milk fat globule membranes during final formation and secretion of milk fat into the lumen of the mammary alveoli. We hypothesized that cytosolic crescent RNA extracted from milk fat could be used as an alternative source of mammary epithelial cell RNA to measure the immediate temporal changes in gene expression as a result of changes in diet. In this experiment, feed restriction was used to mimic the state of negative energy balance observed in early lactation and induce a rapid change in milk fat yield and lipogenic gene expression. Ten multiparous Holstein dairy were fed a basal diet ad libitum during a 14-d preliminary period followed by a 4-d experimental period where 5 cows remained on ad libitum feeding and 5 cows were fed at 60% of their d 8-14 intakes (restricted) on d 15 to 18 and then returned to ad libitum feeding on d 19 to 21. Milk samples were collected from each milking on d 13 to 20 and the milk fat was immediately isolated, mixed with Trizol LS, and stored at -80°C for subsequent extraction of RNA that was used for measurement of gene expression. Feed restriction tended to increase milk fat percentage. However, total milk and milk fat production were reduced by 21 and 18%, respectively. Consistent with increased use of body fat for milk synthesis, serum nonesterified fatty acids increased 6-fold (0.78 mEq/L in the feed restriction vs. 0.13 mEq/L ad libitum group), whereas the milk fatty acids <C16 decreased and ≥C16 increased by 6.5 percentage units. The lipogenic genes ACACA, FASN, and SCD1, and the transcription factor SREBF1were downregulated by 59, 36, 35, and 43%, respectively, during the feed restriction period. In addition, effects of feed restriction by day within period were detected for the genes AGPAT6, GPAM, BTN1A, and SREBF1, suggesting that temporal (day-to-day) changes in gene expression could also be detected. The results of this experiment were consistent with previous feed restriction studies in which researchers used the mammary biopsy technique to study gene expression in the mammary gland. We concluded that RNA isolated from milk fat could be used as an alternative approach to investigate rapid temporal changes in lipogenic gene expression.