Evaluation of milk somatic cells as a source of mRNA for study of lipogenesis in the mammary gland of lactating beef cows supplemented with dietary high-linoleate safflower seeds1

Generally, once-daily milking (ODM) decreases milk yield. This effect may be the consequence of a decrease in mammary epithelial cell (MEC) activity or a reduction in their number. The aim of this study was to determine the effect of ODM on the synthetic activity and rate of apoptosis of MEC using a non-invasive method. Eight Alpine goats were subjected to ODM or twice-daily milking for two 5-week periods. MECs were purified by centrifugation and immunocytochemical binding in milk after 1 and 5 weeks of each period. mRNA levels of some proteins involved in lactose and milk protein synthesis and in apoptosis were evaluated using real-time PCR. Isolation of MEC from milk was a useful method to investigate transcriptional regulation in a timeline study. ODM induced greater decreases in milk, lactose and protein yields after 1 week than after 5 weeks. This suggests an adaptation of the mammary gland to ODM, which reduces the inhibitory effect of this practice. Reductions in milk component yields were associated with lower a-lactalbumin transcripts, suggesting a transcriptional decrease of lactose synthesis during ODM. Glucose transporter GLUT1 transcripts were downregulated under ODM, suggesting that lactose precursor uptake by MEC might be involved in the regulation of lactose synthesis. k-Casein mRNA levels tended to be lower during ODM. ODM increased levels of the pro-apoptotic transcript Bax after both 1 and 5 weeks, but no variation was observed in the Bax/Bcl-2 ratio. ODM affected cell synthetic activity through transcriptional regulation and may have induced apoptosis. The reduction of the negative effect of ODM on milk yield suggests that Alpine goats are able to adapt to ODM. Further studies are needed to investigate the effect of ODM on MEC turnover.

Section: Discussionmentioning

confidence: 89%

Section: Introductionmentioning

confidence: 99%

Use of milk epithelial cells to study regulation of cell activity and apoptosis during once-daily milking in goats

Chedly

Lacasse

Marnet

et al. 2011

“…FO is more effective than plant oils in elevating milk fat cis-9,trans-11-CLA content (Offer et al, 1999) because of the inhibitory effect of docosahexaenoic acid (DHA) in FO on VA reduction to stearic acid in the rumen (Boeckaert et al, 2008). These increases in milk cis-9,trans-11-CLA and trans-11-18:1 can be further enhanced when FO is provided in combination with plant oils or oilseeds rich in 18:2n-6 (Whitlock et al, 2002;AbuGhazaleh et al, 2004).…”

Section: Discussionmentioning

confidence: 99%

“…Most of the studies on the effect of nutrition on mammary lipid metabolism have been carried out using mammary tissue collected at the time of slaughtering or by biopsy, while only a few studies have been reported using milk cells from ruminants as a non-invasive method (Boutinaud et al, 2002 andMurrieta et al, 2006, Feng et al, 2007. The second aim of the present study was to investigate the effect of lipid-supplemented diets on the amount of milk purified mammary epithelial cells (MEC) and on the gene expression of lipogenic enzymes from MEC.…”

Section: Discussionmentioning

confidence: 99%

Effects of polyunsaturated fatty acids from plant oils and algae on milk fat yield and composition are associated with mammary lipogenic and SREBF1 gene expression

Angulo

Mahecha

Nuernberg

et al. 2012

The main aim of the present study was to examine the effects of long-term supplementing diets with saturated or unprotected polyunsaturated fatty acids from two different plant oils rich in either n-3 or n-6 fatty acids (FAs) plus docosahexaenoic acid (DHA)-rich algae on mammary gene expression and milk fat composition in lactating dairy cows. Gene expression was determined from mammary tissue and milk epithelial cells. Eighteen primiparous German Holstein dairy cows in mid-lactation were randomly assigned into three dietary treatments that consist of silage-based diets supplemented with rumen-stable fractionated palm fat (SAT; 3.1% of the basal diet dry matter, DM), or a mixture of linseed oil (2.7% of the basal diet DM) plus DHA-rich algae (LINA; 0.4% of the basal diet DM) or a mixture of sunflower oil (2.7% of the basal diet DM) plus DHA-rich algae (SUNA; 0.4% of the basal diet DM), for a period of 10 weeks. At the end of the experimental period, the cows were slaughtered and mammary tissues were collected to study the gene expression of lipogenic enzymes. During the last week, the milk yield and composition were determined, and milk was collected for FA measurements and the isolation of milk purified mammary epithelial cells (MECs). Supplementation with plant oils and DHA-rich algae resulted in milk fat depression (MFD; yield and percentage). The secretion of de novo FAs in the milk was reduced, whereas the secretion of trans-10,cis-12-CLA and DHA were increased. These changes in FA secretions were associated in mammary tissue with a joint down-regulation of mammary lipogenic enzyme gene expression (stearoyl-CoA desaturase, SCD1; FA synthase, FASN) and expression of the regulatory element binding transcription factor (SREBF1), whereas no effect was observed on lipoprotein lipase (LPL) and glycerol-3-phosphate acyltransferase 1, mitochondrial (GPAM). A positive relationship between mammary SCD1 and SREBF1 mRNA abundances was observed, suggesting a similar regulation for these genes. Such data on mammary gene expression in lactating cows presenting MFD contribute to strengthen the molecular mechanisms that govern milk fat synthesis in the mammary glands. In purified MEC, the dietary treatments had no effect on gene expressions. Differences between mammary tissue and milk purified MEC gene expression were attributed to the effect of lipid supplements on the number of milk purified MEC and its RNA quality, which are determinant factors for the analysis of gene expression using milk cells.

“…In order to reduce the invasive effect of the study we should minimise the number of mammary biopsy collection and chose other techniques especially for kinetic studies. The collection of MEC from milk is a non-invasive method that has been used previously to study mammary gene expression in cows (Hayashi et al, 2004;Murrieta et al, 2006;Boutinaud et al, 2008) and in goats (Boutinaud et al, 2002;Ben Chedly et al, 2011). The use of this technique avoids the carryover effect of sampling that may be observed in mammary biopsies.…”

Section: Introductionmentioning

confidence: 99%

The decrease in milk yield during once daily milking is due to regulation of synthetic activity rather than apoptosis of mammary epithelial cells in goats

Chedly

Lacasse

Marnet

et al. 2013

Once daily milking (ODM) is a management practice that can improve working conditions and reduce production costs in dairy farming compared with twice daily milking (TDM). However, ODM is associated with a decrease in milk yield. Previous research indicated that disruption of tight junctions in the mammary gland may be one of the regulatory factors involved in the milk yield decrease observed during ODM. The aim of this study was to investigate the involvement of mammary epithelium disruption in the regulation of the activity and dynamics of mammary epithelial cells (MEC) during 5 weeks of ODM in goats. Twelve alpine goats (producing 3.67 ± 0.64 kg/day and 47 ± 1.6 days in milk) were assigned to two groups that were milked once or twice a day during 5 weeks and then switched back to TDM. Mammary biopsies were collected before and on days 2 and 16 of both ODM and TDM switchback periods. Milk purified epithelial cells were collected before and on days 1, 7, 21 and 28 during ODM as well on days 1 and 7 of the TDM switchback period. The mRNA levels of genes involved in the regulation of synthetic activity and apoptosis were analysed by RT-PCR in milk MEC and mammary biopsies. ODM decreased yields of milk (−23%), lactose (−23%) and casein (−16%). Lactose synthesis was regulated at the transcriptional level by downregulation of α-lactalbumin mRNA levels in both biopsy samples (−30%) and milk MEC (−74%). TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling) staining of mammary gland biopsies did not show any increase in cell apoptosis after 2 and 16 days of ODM (0.8% and 1%, respectively) despite upregulation of Bax mRNA levels in milk MEC. This suggests that the decrease in milk yield observed during ODM is attributable to a decrease in synthetic activity rather than to induction of MEC cell death. ODM induced the disruption of tight junctions in the mammary gland only on the first day of the treatment as indicated by increased blood lactose concentration. This indicates that the decrease in MEC activity observed over the 5 weeks of ODM was not due to disruption of the mammary gland tight junctions. There was no carryover effect of 5 weeks of ODM on milk production. Therefore, it appears that the decrease in milk yield that occurs during ODM in goats is due to regulation of synthetic activity rather than to apoptosis of MEC.