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Effects of micronutrient supplementation on performance and epigenetic status in dairy cows
- M. Gasselin, M. Boutinaud, A. Prézelin, P. Debournoux, M. Fargetton, E. Mariani, J. Zawadzki, H. Kiefer, H. Jammes
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The postpartum period is crucial in dairy cows and is marked by major physiological and metabolic changes that affect milk production, immune response and fertility. Nutrition remains the most important lever for limiting the negative energy balance and its consequences on general health status in highly selected dairy cows. In order to analyze the effect of a commercial micronutrient on intrinsic parameters, performances and the epigenome of dairy cows, 2 groups of 12 Holstein cows were used: 1 fed a standard diet (mainly composed of corn silage, soybean meal and non-mineral supplement) and the other 1 fed the same diet supplemented with the commercial micronutrient (µ-nutrient supplementation) for 4 weeks before calving and 8 weeks thereafter. Milk production and composition, BW, body condition score (BCS), DM intake (DMI) and health (calving score, metritis and mastitis) were recorded over the study period. Milk samples were collected on D15 and D60 post-calving for analyses of casein, Na+ and K+ contents and metalloprotease activity. Milk leukocytes and milk mammary epithelial cells (mMECs) were purified and counted. The viability of mMECs was assessed, together with their activity, through an analysis of gene expression. At the same time points, peripheral blood mononuclear cells (PBMCs) were purified and counted. Using genomic DNA extracted from PBMCs, mMECs and milk leukocytes, we assessed global DNA methylation (Me-CCGG) to evaluate the epigenetic imprinting associated with the µ-nutrient-supplemented diet. The µ-nutrient supplementation increased BCS and BW without modifying DMI or milk yield and composition. It also improved calving condition, reducing the time interval between calving and first service. Each easily collectable cell type displayed a specific pattern of Me-CCGG with only subtle changes associated with lactation stages in PBMCs. In conclusion, the response to the µ-nutrient supplementation improved the body condition without alteration of global epigenetic status in dairy cows.
Review: the cellular mechanisms underlying mammary tissue plasticity during lactation in ruminants
- M. Boutinaud, L. Herve, H. Quesnel, V. Lollivier, L. Finot, F. Dessauge, E. Chanat, P. Lacasse, C. Charton, J. Guinard-Flament
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The mammary tissue is characterized by its capacity to adapt in response to a wide variety of changing conditions. This adaptation capacity is referred to as the plasticity of mammary tissue. In dairy ruminants, lactation is challenged by modifications that can either be induced on purpose, such as by modifying management practices, or occur involuntarily, when adverse environmental constraints arise. These modifications can elicit both immediate changes in milk yield and composition and carryover effects that persist after the end of the challenge. This review focuses on the current knowledge concerning the cellular mechanisms underlying mammary tissue plasticity. The main mechanisms contributing to this phenomenon are changes in the activity and number of mammary epithelial cells (MECs). Changes in the number of these cells result from variations in the rates of cell proliferation and death as well as changes in the rate MEC exfoliation. The number of MECs also depends on the number of resident adult mammary stem cells and their progenitors, which can regenerate the pools of the various mammary cells. Several challenges, including changes in milking frequency, changes in level of feed supply and hormonal manipulations, have been shown to modulate milk yield together with changes in mammary cell activity, turnover and exfoliation. Epigenetic changes may be an additional mechanism of adaptation. Indeed, changes in DNA methylation and reductions in milk yield have been observed during once-daily milking and during mastitis in dairy cows and may affect cell activity persistently. In contrast to what has been assumed for a long time, no carryover effect on milk yield were observed after feed supply challenges in dairy cows and modification of milking frequency in dairy goats, even though the number of mammary cells was affected. In addition, mammary tissue plasticity has been shown to be influenced by the stage of lactation, health status and genetic factors. In conclusion, the cellular mechanisms underlying mammary tissue plasticity are diverse, and the mammary tissue either does or does not show elastic properties (with no permanent deformation), in response to environmental changes.
Review: Inhibition of prolactin as a management tool in dairy husbandry
- P. Lacasse, X. Zhao, N. Vanacker, M. Boutinaud
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Accumulating evidence supports that the hormone prolactin (PRL) is galactopoietic in dairy ruminants. Accordingly, the inhibition of PRL secretion by the dopamine agonists quinagolide and cabergoline causes a sharp decline in milk production and could be useful in several critical periods. First, PRL inhibition may reduce the incidence during the periparturient period of metabolic disorders caused by the abrupt increase in energy demand for milk production. Metabolic disturbances can be lessened by reducing milk output by milking once a day or incompletely in the first few days of lactation. The injection of cows with quinagolide for the first 4 days of lactation reduced milk production during the first week of lactation without any residual effects. Blood glucose and calcium concentrations were higher and β-hydroxybutyric acid concentration was lower in the quinagolide-treated cows. Second, PRL inhibition may help sick or injured lactating cows, considering that they can fall into severe negative energy balance when they are unable to consume enough feed to support their milk production. This leads to a weakened immune system and increased susceptibility to diseases. When cows were subjected to feed restriction and were treated with quinagolide, the decrease in milk production was accelerated without any residual effects. The quinagolide-treated cows had higher glucose and lower β-hydroxybutyric acid and non-esterified fatty acid concentrations than the control cows did. Third, PRL inhibition may facilitate drying-off in high-yielding cows, because they are often dried off while still producing significant quantities of milk, which delays mammary involution and increases risk of mastitis. Therefore, strategies that reduce milk production before drying-off and accelerate mammary gland involution could be an important management tool. In this context, inhibition of PRL was utilised to accelerate mammary gland dry-off. Quinagolide decreased milk production within the first day of treatment, and both quinagolide and cabergoline induced more rapid changes in several markers of mammary gland involution after drying-off. In addition, quinagolide improved the animals’ resistance to intramammary infection. These results suggest that the inhibition of PRL could be a strategy for facilitating drying-off, reducing metabolic stress during the postpartum period, and alleviating acute nutritional stress during illness without compromising the overall productivity of dairy ruminants.
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
- H. Ben Chedly, P. Lacasse, P-G. Marnet, M. Boutinaud
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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.
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
- J. Angulo, L. Mahecha, K. Nuernberg, G. Nuernberg, D. Dannenberger, M. Olivera, M. Boutinaud, C. Leroux, E. Albrecht, L. Bernard
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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.
Use of milk epithelial cells to study regulation of cell activity and apoptosis during once-daily milking in goats
- H. Ben Chedly, P. Lacasse, P.-G. Marnet, M. Komara, S. Marion, M. Boutinaud
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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 α-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. κ-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.