Xueyan Lin

Bovine mammary gene expression profiling during the onset of lactation.

Background Lactogenesis includes two stages. Stage I begins a few weeks before parturition. Stage II is initiated around the time of parturition and extends for several days afterwards. Methodology/Principal Findings To better understand the molecular events underlying these changes, genome-wide gene expression profiling was conducted using digital gene expression (DGE) on bovine mammary tissue at three time points (on approximately day 35 before parturition (−35 d), day 7 before parturition (−7 d) and day 3 after parturition (+3 d)). Approximately 6.2 million (M), 5.8 million (M) and 6.1 million (M) 21-nt cDNA tags were sequenced in the three cDNA libraries (−35 d, −7 d and +3 d), respectively. After aligning to the reference sequences, the three cDNA libraries included 8,662, 8,363 and 8,359 genes, respectively. With a fold change cutoff criteria of ≥2 or ≤−2 and a false discovery rate (FDR) of ≤0.001, a total of 812 genes were significantly differentially expressed at −7 d compared with −35 d (stage I). Gene ontology analysis showed that those significantly differentially expressed genes were mainly associated with cell cycle, lipid metabolism, immune response and biological adhesion. A total of 1,189 genes were significantly differentially expressed at +3 d compared with −7 d (stage II), and these genes were mainly associated with the immune response and cell cycle. Moreover, there were 1,672 genes significantly differentially expressed at +3 d compared with −35 d. Gene ontology analysis showed that the main differentially expressed genes were those associated with metabolic processes. Conclusions The results suggest that the mammary gland begins to lactate not only by a gain of function but also by a broad suppression of function to effectively push most of the cells resources towards lactation.

Methionine, leucine, isoleucine, or threonine effects on mammary cell signaling and pup growth in lactating mice

Two studies were undertaken to assess the effects of individual essential AA supplementation of a protein-deficient diet on lactational performance in mice using litter growth rates as a response variable. The first study was designed to establish a dietary protein response curve, and the second to determine the effects of Leu, Ile, Met, and Thr supplementation of a protein-deficient diet on lactational performance. In both studies, dams were fed test diets from parturition through d 17 of lactation, when the studies ended. Mammary tissue was collected on d 17 from mice on the second experiment and analyzed for mammalian target of rapamycin (mTOR) pathway signaling. Supplementation with Ile, Leu, or Met independently increased litter weight gain by 11, 9, and 10%, respectively, as compared with the protein-deficient diet. These responses were supported by independent phosphorylation responses for mTOR and eIF4E binding protein 1 (4eBP1). Supplementation of Ile, Leu, and Met increased phosphorylation of mTOR by 55, 34, and 47%, respectively, as compared with the protein-deficient diet. Phosphorylation of 4eBP1 increased in response to Ile and Met supplementation by 60 and 40%, respectively. Supplementation of Ile and Met increased phosphorylation of Akt/protein kinase B (Akt) by 41 and 59%, respectively. This work demonstrated that milk production responds nonlinearly to protein supply, and milk production and the mTOR pathway responded independently to supplementation of individual AA. The former demonstrates that a linear breakpoint model is an inappropriate description of the responses, and the latter demonstrates that no single factor limits AA for lactation. Incorporation of a multiple-limiting AA concept and nonlinear responses into milk protein response models will help improve milk yield predictions and allow derivation of diets that will increase postabsorptive N efficiency and reduce N excretion by lactating animals.

Effect of dietary roughage level on chewing activity, ruminal pH, and saliva secretion in lactating Holstein cows

Increasing dietary roughage level is a commonly used strategy to prevent subacute ruminal acidosis. We hypothesized that high-roughage diets could promote chewing activity, saliva secretion, and hence more alkaline to buffer rumen pH. To verify the hypothesis, 12 multiparous Holstein cows in mid lactation were randomly allocated to 4 treatments in a triplicated 4 × 4 Latin square experiment with one cow in each treatment surgically fitted with a ruminal cannula. Treatments were diets containing 40, 50, 60, or 70% of roughage on a DM basis. Increasing dietary roughage level decreased DM, CP, OM, starch, and NEL intake, increased ADF intake, and decreased milk yield linearly. Intake of NDF was quite stable across treatments and ranged from 7.8 to 8.1 kg/d per cow. Daily eating time increased linearly with increased roughage level. The increase in eating time was due to increased eating time per meal but not number of meals per day, which was stable and ranged from 8.3 to 8.5 meals per day across treatments. Increasing dietary roughage level had no effect on ruminating time (min/d), the number of ruminating periods (rumination periods per d), and chewing time per ruminating period (min/ruminating period). Ruminating time per kilogram of NDF intake and total chewing time per kilogram of ADF intake were similar across treatments (57.4 and 183.8 min/kg, respectively). Increasing dietary roughage level linearly increased daily total chewing time; linearly elevated the mean, maximum, and minimum ruminal pH; and linearly decreased total VFA concentration and molar proportion of propionate in ruminal fluid. Saliva secretion during eating was increased, the secretion during rumination was unaffected, but the secretion during resting tended to decrease with increased dietary roughage level. As a result, total saliva secretion was not affected by treatments. In conclusion, the results of the present study did not support the concept that high-roughage diets elevated ruminal pH through increased salivary recycling of buffering substrates.

MEN1 /Menin regulates milk protein synthesis through mTOR signaling in mammary epithelial cells

The MEN1 gene, which encodes the protein Menin, was investigated for its regulatory role in milk protein synthesis in mammary glands. Menin responds to nutrient and hormone levels via the PI3K/Akt/mTOR pathway. Bovine mammary epithelial cells and tissues were used as experimental models in this study. The results revealed that the milk protein synthesis capacity of mammary epithelial cells could be regulated by MEN1/Menin. The overexpression of Menin caused significant suppression of factors involved in the mTOR pathway, as well as milk protein κ-casein (CSNK). In contrast, a significant increase in these factors and CSNK was observed upon MEN1/Menin knockdown. The repression of MEN1/Menin on the mTOR pathway was also observed in mammary gland tissues. Additionally, MEN1/Menin was found to elicit a negative response on prolactin (PRL) and/or insulin (INS), which caused a similar downstream impact on mTOR pathway factors and milk proteins. Collectively, our data indicate that MEN1/Menin could play a regulatory role in milk protein synthesis through mTOR signaling in the mammary gland by mediating the effects of hormones and nutrient status. The discovery of Menin’s role in mammary glands suggests Menin could be potential new target for the improvement of milk performance and adjustment of lactation period of dairy cows.

Effects of graded removal of lysine from an intravenously infused amino acid mixture on lactation performance and mammary amino acid metabolism in lactating goats

To investigate responses of milk protein synthesis and mammary AA metabolism to a graded decrease of postruminal Lys supply, 4 lactating goats fitted with jugular vein, mammary vein, and carotid artery catheters and transonic blood flow detectors on the external pudic artery were used in a 4 × 4 Latin square experiment. Goats were fasted for 24 h and then received a 9-h intravenous infusion of an AA mixture plus glucose. Milk yield was recorded and samples were taken in h 2 to 8 of the infusion period; a mammary biopsy was performed in the last hour. Treatments were graded decrease of lysine content in the infusate to 100 (complete), 60, 30, or 0% as in casein. Lysine-removed infusions linearly decreased milk yield, tended to decrease lactose yield, and tended to increase milk fat to protein ratio. Milk protein content and yield were linearly decreased by graded Lys deficiency. Mammary Lys uptake was concomitantly decreased, but linear regression analysis found no significant relationship between mammary Lys uptake and milk protein yield. Treatments had no effects on phosphorylation levels of the downstream proteins measured in the mammalian target or rapamycin pathway except for a tended quadratic effect on that of eukaryotic initiation factor 2, which was increased and then decreased by graded Lys deficiency. Removal of Lys from the infusate linearly increased circulating glucagon and glucose. Removal of Lys from the infusate linearly decreased arterial and venous concentrations of Lys. Treatments also had a significant quadratic effect on venous Lys, suggesting mechanisms to stabilize circulating Lys at a certain range. The 2 infusions partially removing Lys resulted in a similar 20% decrease, whereas the 0% Lys infusion resulted in an abrupt 70% decrease in mammary Lys uptake compared with that of the full-AA mixture infusion. Consistent with the abrupt decrease, mammary Lys uptake-to-output ratio decreased from 2.2 to 0.92, suggesting catabolism of Lys in the mammary gland could be completely prevented when the animal faced severe Lys deficiency. Mammary blood flow was linearly increased, consistent with the linearly increased circulating nitric oxide by graded Lys deficiency, indicating mechanisms to ensure the priority of the mammary gland in acquiring AA for milk protein synthesis. Infusions with Lys removed increased mammary clearance rate of Lys numerically by 2 to 3 fold. In conclusion, the decreased milk protein yield by graded Lys deficiency was mainly a result of the varied physiological status, as indicated by the elevated circulating glucagon and glucose, rather than a result of the decreased mammary Lys uptake or depressed signals in the mTOR pathway. Mechanisms of Lys deficiency to promote glucagon secretion and mammary blood flow and glucagon to depress milk protein synthesis need to be clarified by future studies.

Menin Modulates Mammary Epithelial Cell Numbers in Bovine Mammary Glands Through Cyclin D1

Menin, the protein encoded by the MEN1 gene, is abundantly expressed in the epithelial cells of mammary glands. Here, we found MEN1/menin expression slowly decreased with advancing lactation but increased by the end of lactation. It happened that the number of bovine mammary epithelial cells decreases since lactation, suggesting a role of menin in the control of mammary epithelial cell growth. Indeed, reduction of menin expression through MEN1-specific siRNA transfection in the bovine mammary epithelial cells caused cell growth arrest in G1/S phase. Decreased mRNA and protein expression of Cyclin D1 was observed upon MEN1 knockdown. Furthermore, menin was confirmed to physically bind to the promoter region of Cyclin D1 through a ChIP assay, indicating that menin plays a regulatory role in mammary epithelial cell cycle progression. Moreover, lower expression of MEN1/menin induced increased epithelial cell apoptosis and caused extracellular matrix remodeling by down-regulating its associated genes, such as DSG2 and KRT5, suggesting that menin’s role may also be involved in the control of cell–cell adhesion in normal mammary glands. Taken together, our data revealed an unknown molecular function of menin in epithelial cell proliferation, which may be important in the regulation of lactation behavior of mammary glands.

High-production dairy cattle exhibit different rumen and fecal bacterial community and rumen metabolite profile than low-production cattle

Our aim was to simultaneously investigate the gut bacteria typical characteristic and conduct rumen metabolites profiling of high production dairy cows when compared to low‐production dairy cows. The bacterial differences in rumen fluid and feces were identified by 16S rDNA gene sequencing. The metabolite differences were identified by metabolomics profiling with liquid chromatography mass spectrometry (LC‐MS). The results indicated that the high‐production dairy cows presented a lower rumen bacterial richness and species evenness when compared to low‐production dairy cows. At the phylum level, the high‐production cows increased the abundance of Proteobacteria and decreased the abundance of Bacteroidetes, SR1, Verrucomicrobia, Euryarchaeota, Planctomycetes, Synergistetes, and Chloroflexi significantly (p < 0.05). At the genus level, the rumen fluid of the high‐production group was significantly enriched for Butyrivibrio, Lachnospira, and Dialister (p < 0.05). Meanwhile, rumen fluid of high‐production group was depleted for Prevotella, Succiniclasticum, Ruminococcu, Coprococcus,YRC22, CF231, 02d06, Anaeroplasma, Selenomonas, and Ruminobacter significantly (p < 0.05). A total of 92 discriminant metabolites were identified between high‐production cows and low‐production cows. Compared to rumen fluid of low‐production dairy cows, 10 differential metabolites were found up‐regulated in rumen fluid of high‐production dairy cows, including 6alpha‐Fluoropregn‐4‐ene‐3,20‐dione, 3‐Octaprenyl‐4‐hydroxybenzoate, disopyramide, compound III(S), 1,2‐Dimyristyl‐sn‐glycerol, 7,10,13,16‐Docosatetraenoic acid, ferrous lactate, 6‐Deoxyerythronolide B, vitamin D2, L‐Olivosyl‐oleandolide. The remaining differential metabolites were found down‐regulated obviously in high‐production cows. Metabolic pathway analyses indicated that most increased abundances of rumen fluid metabolites of high‐yield cows were related to metabolic pathways involving biosynthesis of unsaturated fatty acids, steroid biosynthesis, ubiquinone and other terpenoid‐quinone biosynthesis. Most down‐regulated metabolic pathways were relevant to nucleotide metabolism, energy metabolism, lipid metabolism and biosynthesis of some antibiotics.

Effect of hay supplementation timing on rumen microbiota in suckling calves

An animal feeding trial was conducted on 18 seven‐day‐old Holstein dairy bull calves weighing 42 ± 3 kg each. Calves were randomly assigned into three groups (n = 6 each). The dietary treatments were as follows: (1) milk and starter for the control group (MS), (2) supplementation of oat hay from week 2 on the basis of milk and starter (MSO2), and (3) supplementation of oat hay from week 6 on the basis of milk and starter (MSO6). All animals were fed starter and oat hay ad libitum. The major phyla in the different groups of rumen fluid included Firmicutes, Actinobacteria, Bacteroidetes, Proteobacteria, and Euryarchaeota. The major genera were identified, and major genera proportions in the three groups were as follows: Methanobrevibacter (Euryarchaeota), 2.1%, 1.7%, and 2.1%; Olsenella (Actinobacteria), 23.9%, 17.7%, and 12.8%; Prevotella (Bacteroidetes), 10.5%, 16.5%, and 19.2%; Dialister (Firmicutes), 3.3%, 4.1%, and 2.8%; Succiniclasticum (Firmicutes), 3.8%, 4.7%, and 9.2%; and Sharpea (Firmicutes), 0.4%, 2.5%, and 0.2%, respectively. There were no significant differences in the various phyla among the three groups (p > .05). The results showed that calves hay supplementation time did not affect the diversity of the rumen microbiota in the suckling calves. However, the hay supplementation altered the proportion of the various microbial populations, supplementation of oat hay from week 2 on the basis of milk and starter could improve calves rumen pH.

Gastrointestinal Tract Development in Unweaned Calves Feeding Different Amounts of Milk and Different Starters

This study was conducted to determine the effect of different starter diets and different amounts of milk on growth performance and gastrointestinal tract development in unweaned calves. 16 calves were assigned to 4 groups, 4 calves in each group. These four groups received the following treatments respectively: 1) high milk (6 L) + low starch (21%), high NDF (28%), high molasses (10%) starter (HMLS group); 2) high milk (6L) + high starch (40%), low NDF (14%), low molasses (5%) starter (HMHS group); 3) low milk (3 L) + low starch (21%), high NDF (28%), high molasses (10%) starter (LMLS group); 4) low milk (3 L) + high starch (40%), low NDF (14%), low molasses (5%) starter (LMHS group). The trial was of 2 × 2 factorial design. All calves had free access to hay and water. Results showed that the low milk allowance increased calf concentrate dry matter intake (DMI) and total DMI, reduced body height at 4 weeks of age, reduced heart girth at 6 weeks of age (P < 0.05). The low milk allowance increased complex stomach full weight, reticulorumen full weight, and the percentages of stomach full weight and reticulorumen full weight over body weight (P < 0.05). The low starch, high fiber, high molasses starters reduced the complex stomach full weight, the proportion of the complex stomach empty weight over body weight, reticulorumen empty weight, abomasum full weight, the proportion of abomasum empty weight over body weight and reticulorumen volume (P < 0.05). The low milk allowance increased calf intestine length, small intestine full weight, the proportion of small intestine full weight over body weight, but reduced the jejunum villus width (P < 0.05), increased ruminal pH (P < 0.05). The low milk allowance reduced papilla length in rumen anterior ventral blind sac, but increased papilla length in the posterior ventral sac (P < 0.05).

Effect of initial time of forage supply on growth and rumen development in preweaning calves

To determine the effects of the initial timing of forage supply on growth, ruminal fermentation parameters and rumen development in preweaning calves, 18 7-day-old Holstein calves of 42 ± 3 kg were randomly divided into three treatment groups. The dietary treatments were (1) milk and commercial starter diet (MS) control, (2) milk and starter diet supplemented with oat hay at 2 weeks (MSO2), and (3) milk and starter diet supplemented with oat hay at 6 weeks (MSO6). Starter feed and oat hay were provided ad libitum, and 2 L of milk was provided twice daily. Samples were collected at 64 days of age. Supplementing with hay increased the dry-matter intake; P 0.05). The concentrations of serum glucose (P = 0.07) and β-hydroxybutyric acid; P < 0.05) were lower in the MSO6 group than in the MS and MSO2 groups. Rumination time was longer, and time spent on non-nutritive oral behaviour (such as e.g. licking surfaces, tongue rolling, wood-shaving consumption) was lower for hay-supplemented calves than for the control (P < 0.05). Although ruminal pH of hay-supplemented calves was significantly higher than that of the control, total rumen fluid volatile fatty acid concentrations were not significantly different among treatments. Calves in the MSO2 group had a smaller ratio of empty weight to slaughter weight (P < 0.05) and a larger total digestive tract weight (P < 0.05); the empty gastrointestinal tract weights were similar among the three treatments, suggesting that MSO2 calf weight gain may have resulted from intestinal chyme accumulation. Compared with the control, hay-supplemented calves had reduced rumen papilla width and epithelium thickness (P < 0.05), and no discernable plaque formation. Hay supplementation in the diet of preweaning calves improved the overall dry-matter intake, improved rumination, reduced non-nutritive oral behaviours, improved rumen pH, and ensured healthy rumen development; furthermore, productivity and rumen development were better in calves supplemented with hay from the second week.