Milk Protein Synthesis Research Articles

Miúda (Nopalea cochenillifera (L.) Salm-Dyck)—The Best Forage Cactus Genotype for Feeding Lactating Dairy Cows in Semiarid Regions

Simple SummaryThe usage of forage cactus is essential for the maintenance of livestock activity in semiarid regions as an alternative to conventional crops. Cactaceae have adaptive characteristics that ensure their development progress under drought conditions. Four genotypes of forage cactus (Gigante, Miúda, IPA Sertânia, and Orelha de Elefante Mexicana) were fed to lactating dairy cows and the diets were then evaluated based on animal performance, milk fatty acid profile, and microbial protein synthesis. Miúda forage cactus led to a higher nutrient intake and milk yield, as well as greater microbial protein synthesis. Higher saturated fatty acids were observed when the Gigante and IPA Sertânia forage cactus genotypes were fed to dairy cows. Orelha de Elefante Mexicana forage cactus caused lower milk yield along with protein yields and content; however, it improved the milk fatty acid profile by promoting a higher ratio of unsaturated to saturated fatty acids and desirable fatty acids. It is concluded that the Miúda forage cactus is the genotype most suitable for the diets of lactating dairy cows.This study aimed to investigate the effects on nutrient intake and digestibility, milk yield (MY) and composition, milk fatty acids profile, and microbial protein synthesis caused by feeding lactating dairy cows four different forage cactus genotypes. Eight Girolando cows (5/8 Holstein × 3/8 Gyr), weighing 490 ± 69.0 kg (means ± standard deviation), and producing 15.5 ± 1.0 kg/d of milk during pretrial were distributed to two contemporaneous 4 × 4 Latin squares. The cows were fed a total mixed ration composed of sorghum silage (385 g/kg of dry matter (DM)), concentrated mix (175 g/kg DM), and forage cactus (440 g/kg DM). The experimental treatments consisted of different cactus genotypes, such as Gigante cactus (GC), Miúda cactus (MC), IPA Sertânia cactus (SC), and Orelha de Elefante Mexicana cactus (OEMC). The feeding of MC provided a higher intake of DM, organic matter (OM), and total digestible nutrients, as well as higher MY, energy-corrected milk, and microbial protein synthesis in comparison with those resulting from the other genotypes tested. The GC promoted lower DM and OM, and the apparent digestibility of neutral detergent fiber. The cows fed with OEMC showed lower MY and milk protein yield and content, and higher unsaturated over saturated fatty acid ratio in milk. Miúda forage cactus increased nutrient intake, digestibility of DM and OM, and microbial synthesis without impairing the milk fatty acid profile.

MiR-204-5p and miR-211 Synergistically Downregulate the αS1-Casein Content and Contribute to the Lower Allergy of Goat Milk.

αS1-Casein (encoded by the CSN1S1 gene) is associated with higher rates of allergy than other milk protein components for humans. microRNAs (miRNAs) as small noncoding RNA molecules regulate gene expression and influence diverse biological processes. However, little is known about the regulation of milk protein synthesis by miRNAs in ruminants. In this study, we aim to investigate the regulatory roles of miR-204 family members (miR-204-5p and miR-211) on αS1-casein in goat mammary epithelial cells (GMEC). Here, we observed that the CSN1S1 mRNA level is upregulated, while miR-204-5p and miR-211 (miR-204-5p/-211) abundance is downregulated during peak lactation compared with middle lactation of dairy goats. We found that miR-204-5p/-211 synergistically inhibit αS1-casein expression via directly binding to the 3'-untranslated region (3'UTR) of CSN1S1 in GMEC. miR-204-5p/-211 increase β-casein mRNA (CSN2) and protein abundance, as well as the signal transducer and activator of transcription 5a (STAT5a) activity. Further, miR-204-5p/-211 enhance β-casein expression via the CSN1S1-STAT5a signaling axis and promote β-casein transcription by activating the STAT5 response element located in the CSN2 promoter. In conclusion, miR-204-5p/-211 regulate αS1-casein and β-casein synthesis via targeting CSN1S1 in GMEC, which provide the strategy for manipulating miR-204 family members to reduce milk allergy potential and improve ruminant milk quality for human consumption.

UFL1 regulates milk protein and fat synthesis–related gene expression of bovine mammary epithelial cells probably via the mTOR signaling pathway

UFL1 is an ufmylation (a novel post-translational modification) E3 ligase, mainly located in the endoplasmic reticulum (ER), that has emerged as a significant regulator of several physiological and pathological processes. Yet its physiological function in milk synthesis in bovine mammary epithelial cells (BMECs) remains unknown. In this study, we investigated the effects of UFL1 in milk protein and fat synthesis-related gene expression, with a particular emphasis on the role of UFL1 in LPS-treated BMECs. Results showed that UFL1 depletion significantly reduced the expression of milk protein and fat synthesis-related gene and mTOR phosphorylation in both normal and LPS-treated BMECs. Overexpression of UFL1 enhanced the activation of the mTOR and milk protein and fat synthesis-related gene expression. Collectively, these above results strongly demonstrate that UFL1 could regulate milk protein and fat synthesis-related gene expression of BMECs probably via the mTOR signaling pathway.

LPS-induced SOCS3 antagonizes the JAK2-STAT5 pathway and inhibits β-casein synthesis in bovine mammary epithelial cells

Bovine mammary epithelial cells (BMECs) are essential for lactation in the dairy cow mammary gland, and are often used as a cellular model to study changes in inflammatory responses and lactation functions with exogenous stimuli. Prolactin (PRL) promotes milk protein synthesis by continuously activating the Janus kinase 2 and signal transducer and activator of transcription 5 (JAK2-STAT5) pathway. Lipopolysaccharides (LPS) activates inflammatory responses in cells and inhibits casein synthesis, but the exact mechanism is still unclear. Suppressor of cytokine signaling-3 (SOCS3) is a negative regulator of the JAK-STATs signaling pathway, and regulates a variety of inflammatory responses by inhibiting STAT3. Previous studies also suggested that SOCS3 plays a role in the development and involution of bovine mammary glands. The purpose of this study was to investigate whether LPS activated SOCS3, and whether SOCS3 resisted the regulation of casein synthesis by PRL in a JAK2-STAT5-dependent manner. We treated in vitro BMECs with 125 ng/mL PRL, 10 μg/mL LPS, SOCS3 siRNA (silencing), a SOCS3-GFP adenovirus overexpression vector, or combinations, to determine β-casein expression. We demonstrated that PRL up-regulated phospho-JAK2, phsopho-STAT5 and β-casein expression, whereas LPS caused the opposite effects, and activated SOCS3. SOCS3 overexpression interrupted the JAK2-STAT5 pathway in BMECs. With SOCS3 was silenced, LPS could not activate the JAK2-STAT5 pathway, and no inhibition of β-casein expression was observed. In conclusion, we showed that LPS activated SOCS3 in BMECs, antagonized the JAK2-STAT5 pathway via SOCS3 regulation, and ultimately reduced β-casein expression in these cells.

Identification of the Potential Role of the Rumen Microbiome in Milk Protein and Fat Synthesis in Dairy Cows Using Metagenomic Sequencing.

Simple SummaryThe rumen is the main digestive and absorption organ of dairy cows. It contains abundant microorganisms and can effectively use human-indigestible plant mass. Therefore, we used metagenomics to explore the role of rumen microbes in the regulation of milk protein and fat in dairy cows. This study showed that Prevotella species and Neocallimastix californiae in the rumen of cows are related to the synthesis of milk components due to their important functions in carbohydrate, amino acid, pyruvate, insulin, and lipid metabolism and transportation metabolic pathways.The rumen contains abundant microorganisms that aid in the digestion of lignocellulosic feed and are associated with host phenotype traits. Cows with extremely high milk protein and fat percentages (HPF; n = 3) and low milk protein and fat percentages (LPF; n = 3) were selected from 4000 lactating Holstein cows under the same nutritional and management conditions. We found that the total concentration of volatile fatty acids, acetate, butyrate, and propionate in the rumen fluid was significantly higher in the HPF group than in the LPF group. Moreover, we identified 38 most abundant species displaying differential richness between the two groups, in which Prevotella accounted for 68.8% of the species, with the highest abundance in the HPF group. Functional annotation based on the Kyoto Encyclopedia of Gene and Genome (KEGG), evolutionary genealogy of genes: Non-supervised Orthologous Groups (eggNOG), and Carbohydrate-Active enzymes (CAZy) databases showed that the significantly more abundant species in the HPF group are enriched in carbohydrate, amino acid, pyruvate, insulin, and lipid metabolism and transportation. Furthermore, Spearman’s rank correlation analysis revealed that specific microbial taxa (mainly the Prevotella species and Neocallimastix californiae) are positively correlated with total volatile fatty acids (VFA). Collectively, we found that the HPF group was enriched with several Prevotella species related to the total VFA, acetate, and amino acid synthesis. Thereby, these fulfilled the host’s needs for energy, fat, and rumen microbial protein, which can be used for increased biosynthesis of milk fat and milk protein. Our findings provide novel information for elucidation of the regulatory mechanism of the rumen in the formation of milk composition.

Kisspeptin-10 Maintains the Activation of the mTOR Signaling Pathway by Inhibiting SIRT6 to Promote the Synthesis of Milk in Bovine Mammary Epithelial Cells.

Kisspeptin-10 (Kp-10) is a peptide hormone that regulates normal physiological processes. The mechanism of Kp-10 in milk synthesis is still unclear. Therefore, bovine mammary epithelial cells (BMECs) were used to study the mechanism by which Kp-10 affects milk synthesis in BMECs. The GPR54 inhibitor and SIRT6 overexpression plasmid and siRNA were used to study the mechanism of regulating milk protein and milk fat synthesis by Kp-10. The results showed that 100 nM Kp-10 increased milk synthesis in BMECs. SIRT6 overexpression could significantly reduce the milk protein and milk fat synthesis in BMECs. Moreover, overexpression of SIRT6 reversed the activation of the Kp-10-induced mTOR signaling pathway. Further analysis suggested that SIRT6 might regulate the signal transduction of mTOR at the transcriptional level. These results strongly suggested that Kp-10/GPR54 activated the mTOR signaling pathway by inhibiting SIRT6 expression and then increased the milk synthesis in BMECs.

Effect of rumen-protected lysine supplementation of diets based on corn protein fed to lactating dairy cows

This trial tested whether rumen-protected Lys (RPL) supplementation would improve the nutritive value of rumen-undegradable protein (RUP) from corn protein. Thirty-two lactating Holstein cows were blocked by days in milk and parity into 8 squares of 4 cows each in replicated 4 × 4 Latin squares. Treatments provided all supplemental crude protein from: (1) soy protein (67% expeller soybean meal plus 33% solvent soybean meal); (2) a blend of soy and corn protein (33% expeller soybean meal, 17% solvent soybean meal, 25% corn gluten meal plus 25% distillers dried grains with solubles); (3) corn protein (50% corn gluten meal plus 50% distillers dried grains with solubles); or (4) corn protein plus RPL [diet 3 top-dressed with RPL (125 g/d of AjiPro-L Generation 1, supplying an estimated 20 g of absorbable Lys/d)]. Diets contained (dry matter basis) 22% alfalfa silage, 43% corn silage, 18% ground high-moisture and dry corn, 2.4% mineral-vitamin premix, 1.5 to 3.9% soy hulls, 15% crude protein, 30 to 32% neutral detergent fiber and predicted to contain equal rumen-degradable protein, RUP, and metabolizable protein. Cows within squares were randomly assigned to treatment sequences and fed diets for 4-wk periods before switching; production data and blood samples were collected during last 2 wk of each period. Data were analyzed using the mixed procedures of SAS. Intake was highest on diet 1, intermediate on diets 2 and 3, and lowest on diet 4; body weight gain was highest on diet 3, intermediate on diets 1 and 2 and lowest on diet 4. Intakes and body weight changes were reflected by differences in milk/dry matter intake, which was highest on diets 2 and 4 and lowest on diet 3. Milk yield was lower on diet 3 (44.3 kg/d) than on diets 1, 2, and 4 (average 45.8 kg/d) and protein yield was highest on diets 1 and 2 (average 1.35 kg/d), intermediate on diet 4 (1.30 kg/d), and lowest on diet 3 (1.25 kg/d). No effects of diet were detected on ruminal metabolites. Free nonessential amino acids and total protein AA were elevated in blood plasma on diet 3, reflecting reduced utilization for milk protein synthesis. These results indicated that 50% dilution of soybean meal RUP with that from corn protein did not reduce yield and that supplementing RPL to the corn protein-based diet increased yield 1.1 kg of milk/d and 50 g of true protein/d.

Supplementing with L-Tryptophan Increases Medium Protein and Alters Expression of Genes and Proteins Involved in Milk Protein Synthesis and Energy Metabolism in Bovine Mammary Cells.

The objective of this study was to investigate the effects of supplementing with L-tryptophan (L-Trp) on milk protein synthesis using an immortalized bovine mammary epithelial (MAC-T) cell line. Cells were treated with 0, 0.3, 0.6, 0.9, 1.2, and 1.5 mM of supplemental L-Trp, and the most efficient time for protein synthesis was determined by measuring cell, medium, and total protein at 0, 24, 48, 72, and 96 h. Time and dose tests showed that the 48 h incubation time and a 0.9 mM dose of L-Trp were the optimal values. The mechanism of milk protein synthesis was elucidated through proteomic analysis to identify the metabolic pathway involved. When L-Trp was supplemented, extracellular protein (medium protein) reached its peak at 48 h, whereas intracellular cell protein reached its peak at 96 h with all L-Trp doses. β-casein mRNA gene expression and genes related to milk protein synthesis, such as mammalian target of rapamycin (mTOR) and ribosomal protein 6 (RPS6) genes, were also stimulated (p < 0.05). Overall, there were 51 upregulated and 59 downregulated proteins, many of which are involved in protein synthesis. The results of protein pathway analysis showed that L-Trp stimulated glycolysis, the pentose phosphate pathway, and ATP synthesis, which are pathways involved in energy metabolism. Together, these results demonstrate that L-Trp supplementation, particularly at 0.9 mM, is an effective stimulus in β-casein synthesis by stimulating genes, proteins, and pathways related to protein and energy metabolism.

Impacts of Heat Stress-Induced Oxidative Stress on the Milk Protein Biosynthesis of Dairy Cows.

Simple SummaryHeat stress (HS) of dairy cows affects milk protein synthesis partially due to a decline in appetite and dry matter intake (DMI). Several published studies indicate that HS causes oxidative stress (OS) with high levels of free radicals in tissues. Furthermore, the involvement of reactive oxygen species (ROS) in inducing apoptosis, in reducing mammary epithelial cell numbers, and in endocrine disruptions are proposed as the main mechanisms by which HS-induced OS modifies milk protein synthesis. However, challenges remain in determining the levels of apoptosis in vivo, as well as the tracking of the sources of ROS formation. Therefore, further investigations are required.Heat stress (HS) is one of the most important factors posing harm to the economic wellbeing of dairy industries, as it reduces milk yield as well as milk protein content. Recent studies suggest that HS participates in the induction of tissue oxidative stress (OS), as elevated levels of reactive oxygen species (ROS) and mitochondrial dysfunction were observed in dairy cows exposed to hot conditions. The OS induced by HS likely contributes to the reduction in milk protein content, since insulin resistance and apoptosis are promoted by OS and are negatively associated with the synthesis of milk proteins. The apoptosis in the mammary gland directly decreases the amount of mammary epithelial cells, while the insulin resistance affects the regulation of insulin on mTOR pathways. To alleviate OS damages, strategies including antioxidants supplementation have been adopted, but caution needs to be applied as an inappropriate supplement with antioxidants can be harmful. Furthermore, the complete mechanisms by which HS induces OS and OS influences milk protein synthesis are still unclear and further investigation is needed.

Structural equation modeling for unraveling the multivariate genomic architecture of milk proteins in dairy cattle

The aims of this study were to investigate potential functional relationships among milk protein fractions in dairy cattle and to carry out a structural equation model (SEM) GWAS to provide a decomposition of total SNP effects into direct effects and effects mediated by traits that are upstream in a phenotypic network. To achieve these aims, we first fitted a mixed Bayesian multitrait genomic model to infer the genomic correlations among 6 milk nitrogen fractions [4 caseins (CN), namely κ-, β-, αS1-, and αS2-CN, and 2 whey proteins, namely β-lactoglobulin (β-LG) and α-lactalbumin (α-LA)], in a population of 989 Italian Brown Swiss cows. Animals were genotyped with the Illumina BovineSNP50 Bead Chip v.2 (Illumina Inc.). A Bayesian network approach using the max-min hill-climbing (MMHC) algorithm was implemented to model the dependencies or independence among traits. Strong and negative genomic correlations were found between β-CN and αS1-CN (-0.706) and between β-CN and κ-CN (-0.735). The application of the MMHC algorithm revealed that κ-CN and β-CN seemed to directly or indirectly influence all other milk protein fractions. By integrating multitrait model GWAS and SEM-GWAS, we identified a total of 127 significant SNP for κ-CN, 89 SNP for β-CN, 30 SNP for αS1-CN, and 14 SNP for αS2-CN (mostly shared among CN and located on Bos taurus autosome 6) and 15 SNP for β-LG (mostly located on Bos taurus autosome 11), whereas no SNP passed the significance threshold for α-LA. For the significant SNP, we assessed and quantified the contribution of direct and indirect paths to total marker effect. Pathway analyses confirmed that common regulatory mechanisms (e.g., energy metabolism and hormonal and neural signals) are involved in the control of milk protein synthesis and metabolism. The information acquired might be leveraged for setting up optimal management and selection strategies aimed at improving milk quality and technological characteristics in dairy cattle.

Interactions of amino acids and hormones regulate the balance between growth and milk protein synthesis in lactating rats fed diets differing in protein content.

Insulin-like growth factor-I (IGF-I), growth hormone (GH), and prolactin (PRL) play important roles in milk protein synthesis, and their plasma concentrations were reported to be affected by dietary protein intake. To investigate the relationship between circulating amino acid (AA) and concentrations of these hormones, 18 Wistar rats aged 14 wk were assigned to a low (LP; 9% protein), standard (SP; 21% protein), or high-protein (HP; 35% protein) diet from parturition through day 15 of lactation. Plasma, liver, pituitary gland, skeletal muscle, and mammary gland samples were collected at the end of treatment. Circulating and hepatic IGF-I concentrations increased linearly with elevated dietary protein concentrations (P < 0.0001). Rats receiving the HP diet had higher circulating GH (P < 0.01) and pituitary PRL concentrations (P < 0.0001) but lower pituitary GH concentration (P < 0.0001) relative to those in rats receiving the LP and SP diets. Pearson correlation test performed on composed data across treatments showed that several circulating AAs were correlated with circulating and tissue concentrations of IGF-I, GH, and PRL. Multiple linear regression analyses identified Leu, Gln, Ala, Gly, and Arg as the main AAs associated with hormone responses (R2 = 0.37 ~ 0.80; P < 0.05). Rats fed the LP and HP diets had greater Igf1 and Ghr gene expression in skeletal muscle than those fed the SP diets (P < 0.01). However, LP treatment decreased Prlr mRNA abundance in mammary glands as compared with the SP and HP treatments (P < 0.05). The HP diets increased AA transporter expression (P < 0.01) but decreased mammalian target of rapamycin (P < 0.05) and 70 kDa ribosomal protein S6 kinase 1 (P < 0.01) phosphorylation in mammary glands as compared with the LP and SP diets. The results of the present study suggested that several circulating AAs mediated the effects of dietary protein supply on concentrations of IGF-I, GH, and PRL, which in turn altered the metabolism status in peripheral tissues including the lactating mammary glands.

Phospho-Tudor-SN coordinates with STAT5 to regulate prolactin-stimulated milk protein synthesis and proliferation of bovine mammary epithelial cells

Tudor staphylococcal nuclease (Tudor‐SN) participates in milk synthesis and cell proliferation in response to prolactin (PRL) and plays a regulatory role on mTOR phosphorylation. However, the complicated molecular mechanism of Tudor‐SN regulating milk protein synthesis and cell proliferation still remains to be illustrated. In present study, we observed that the proteins level of phosphorylated Tudor‐SN and phosphorylated STAT5 were simultaneously enhanced upon PRL treatment in bovine mammary epithelial cells (BMECs). Tudor‐SN overexpression and knockdown experiment showed that Tudor‐SN positively regulated the synthesis of milk protein, cell proliferation and the phosphorylation of STAT5, which was dependent on Tudor‐SN phosphorylation. STAT5 knockdown experiment showed that Tudor‐SN stimulated mTOR pathway through regulating STAT5 activation, which was required for PRL to activate the mTOR pathway. Thus, these results demonstrate the primary mechanism of Tudor‐SN coordinating with STAT5 to regulate milk protein synthesis and cell proliferation under stimulation of PRL in BMECs, which may provide some new perspectives for increasing milk production.

Assessing amino acid uptake and metabolism in mammary glands of lactating dairy cows intravenously infused with methionine, lysine, and histidine or with leucine and isoleucine

The objective of this study was to evaluate the effect of jugular infusions of 2 groups of AA on essential AA (EAA) transport and metabolism by mammary glands. Four Holstein cows in second lactation (66 ± 10 d in milk) were used in 4 × 4 Latin square design with a 2 × 2 factorial arrangement of treatments. Treatments were jugular infusions of saline; Met, Lys, and His (MKH); Ile and Leu (IL); or MKH plus IL (MKH+IL). Each period consisted of 8 d of no infusion followed by 8 d of jugular vein infusion of the treatment solutions. Amino acids were infused at rates of 21 g of Met, 38 g of Lys, 20 g of His, 50 g of Leu, and 22 g of Ile per day. Cows were fed a basal diet consisting of 15.2% crude protein with adequate rumen degradable protein but 15% deficient in MP based on estimates by Cornell Net Carbohydrate and Protein System (v6.5). On the last day of each period, 13C-AA derived from algae was infused into the jugular vein over 6 h, and blood and milk samples were collected before, during, and after infusion. Plasma and milk samples were analyzed for AA isotopic enrichment, and a mammary compartmental model was fitted to the data to derive bidirectional transport and metabolism rates for individual EAA. Influx of Leu increased with IL, whereas influx of other EAA was not different among treatments. Cellular efflux of Met and Lys to venous plasma represented 12 to 34% of influx, whereas cellular efflux of Phe and BCAA represented 29 to 59% of influx. Increased efflux/influx ratios of Ile and Leu with IL but not Met and Lys with MKH demonstrated that increased Ile and Leu influx was mostly returned to plasma resulting in no change in net uptake or efficiency. The isotope results showed that mammary net uptake of Lys and Ile increased during MKH infusion. Net uptake of Met increased with MKH but only in the absence of IL. Catabolism of Lys and Met only increased with MKH alone, resulting in decreased efficiency for milk protein, which demonstrated that Ile and Leu infusion can spare Lys and Met for milk protein synthesis. Total AA uptake to milk output was not different from 1, implying the catabolized Met and Lys contributed nitrogen to nonessential AA. Overall, EAA uptake and metabolism in mammary glands of dairy cows varied across individual EAA and responded differently to respective AA supplements. In addition, uptake, retention, and end use of AA by mammary tissue is variable and dependent on the mix of AA provided. This variability, depending on the mix of AA absorbed, will change the efficiency of utilization of individual AA at the mammary gland level and consequently the whole-body level. Thus, it is inaccurate to use a fixed, constant efficiency within and across AA to represent tissue activity.

Milk Production and Energetic Metabolism of Heat-Stressed Dairy Goats Supplemented with Propylene Glycol.

Simple SummaryHeat stress (HS) causes significant milk production losses and negatively impacts animal health. Previous research indicated that heat-stressed goats eat less and degrade their skeletal muscles to cover shortages in nutrient requirements. Propylene glycol (PG) is a glucogenic precursor and may be a remedy for energy shortage in HS situations. In the current study, dairy goats were exposed to thermoneutral (TN; 15 to 20 °C) or HS (12 h/d at 37 °C and 12 h/d at 30 °C) conditions. In each ambient temperature, goats were fed a control diet (CO) or the same diet supplemented with PG. Goats supplemented with PG gained more body weight, and experienced greater blood glucose and insulin levels compared to CO goats. However, supplementation with PG depressed feed intake and milk fat content. In conclusion, feeding propylene glycol was useful to reduce body weight losses typically observed under HS conditions, but did not improve milk yield or milk composition.Heat-stressed dairy animals increase their reliance on glucose. This elevated glucose demand is partially met by increasing the conversion of glucogenic amino acids (AA) in the liver. Propylene glycol (PG) is a glucogenic precursor and was not tested in dairy goats under thermoneutral (TN) and heat stress (HS) conditions simultaneously. We hypothesize that if HS-goats are fed with PG, they would get more glucose and consequently spare more glucogenic AA for milk protein synthesis rather than gluconeogenesis. Eight multiparous dairy goats (40.8 ± 1.1 kg body weight; 84 ± 1 days in milk) were used in a replicated 4 × 4 Latin square design of 4 periods; 21 d each (14 d adaptation, 5 d for measurements, and 2 d of transition). Goats were allocated to one of 4 treatments in a 2 × 2 factorial arrangement. Factors were control (CO) without PG or 5% of PG, and thermoneutral (TN; 15 to 20 °C) or heat stress (HS; 12 h/d at 37 °C and 12 h/d at 30 °C) conditions. Feed intake, rectal temperature, respiratory rate, milk yield, milk composition, and blood metabolites were measured. Compared to TN, HS goats had lower (p < 0.01) feed intake (–34%), fat-corrected milk (–15%), and milk fat (–15%). Heat-stressed goats also tended (p < 0.10) to produce milk with lower protein (–11%) and lactose (–4%) contents. Propylene glycol increased blood glucose (+7%; p < 0.05), blood insulin (+37%; p < 0.10), and body weight gain (+68%; p < 0.05), but decreased feed intake (–9%; p < 0.10) and milk fat content (–23%; p < 0.01). Furthermore, blood non-esterified fatty acids (–49%) and β-hydroxybutyrate (–32%) decreased (p < 0.05) by PG. In conclusion, supplementation of heat-stressed dairy goats with propylene glycol caused milk fat depression syndrome, but reduced body weight loss that is typically observed under HS conditions. Supplementation with lower doses of PG would avoid the reduced feed intake and milk fat depression, but this should be tested.

A static model to analyze carbon and nitrogen partitioning in the mammary gland of lactating sows

Quantitative estimates of mammary nutrient inputs, outputs and metabolism in sows are scarce, despite being critical elements to identify parameters controlling milk synthesis central for the feeding of lactating sows. The objective of this study was to quantify the mammary gland input and output of nutrients as well as the intramammary partitioning of carbon and nitrogen with the purpose to identify mechanisms controlling mammary nutrient inputs, metabolism and milk production in lactating sows. A data set was assembled by integration of results from four studies. The data set included data on litter performance, mammary arterial-venous concentration differences (AV-difference) of energy metabolites and amino acids, and the contents of lactose, fat and amino acids in milk. Milk yield was estimated based on average litter size and litter gain, and mammary plasma flow (MPF) was estimated using the sum of phenylalanine and tyrosine as internal flow markers. The yield and composition of milk were used to estimate mammary nutrient output in milk, and MPF and AV-difference were used to estimate net mammary input of carbon and nitrogen and output of CO2. Carbon and nitrogen used for the synthesis of lactose, fat and protein in milk and CO2-yielding processes were represented in a static nutrient partitioning model. The origin of mammary CO2 output was calculated using theoretical estimates of carbon released in processes supporting mammary synthesis of de novo fat, protein and lactose in milk, mammary tissue protein turnover and transport of glucose and amino acids. Results indicated that total input of carbon from glucose and lactate was partitioned into lactose (36%), fat (31%) and CO2-yielding processes (34%). Theoretical CO2 estimates indicated that de novo fat synthesis, milk protein synthesis and mammary tissue protein turnover were the main processes related to mammary CO2 production. More than 90% of mammary gland amino acid input was used for milk protein. The quadratic relationship between AV-difference and mammary input of essential amino acids indicated that both changes in AV-difference and MPF contributed to the regulation of mammary input of essential amino acids. The impact of the arterial supply of amino acids on mammary input may be greater for the branched-chain amino acids, arginine and phenylalanine than for other essential amino acids. In conclusion, relationships between input and output parameters indicate that AV-difference and MPF regulate mammary nutrient input to match the supply and demand of nutrients for the mammary gland.

186 Young Scholar Presentation: Immunometabolism during periods of negative nutrient balance or heat stress is altered by dietary methyl donor supply in dairy cows

Abstract Our research examined the effects of enhancing methyl donor supply on immunometabolism during periods of negative nutrient balance (NNB) or heat stress (HS). The first experiment examined the effects of post-ruminal choline supply during NNB on production and pathways of 1-carbon metabolism. Ten primiparous rumen-cannulated cows (158±24 DIM) were used in a replicated 5×5 Latin square design with 4d treatment periods and 10d of recovery. Treatments were unrestricted intake with abomasal infusion of water, restricted intake (R; 60% of net energy for lactation requirements) with abomasal infusion of water or R plus abomasal infusion of 6.25, 12.5, or 25 g/d choline ion. Liver tissue was collected at the end of each treatment period. Enhancing choline supply increased milk yields, but decreased liver triacylglycerol. Activity of betaine homocysteine methyltransferase increased with choline, while methionine synthase tended to increase, and cystathionine β-synthase was decreased. These changes were associated with increased liver and plasma Met. Overall, enhanced supply of choline during NNB increases flux through the Met cycle to regenerate Met and reduce liver triacylglycerol. The second experiment examined the effects of rumen-protected Met (RPM) during HS on mTOR (mechanistic target of rapamycin)-related signaling proteins in the mammary gland. Thirty-two multiparous cows (184±59 DIM) were assigned to an environmental treatment, and a dietary treatment [TMR with RPM (0.105% DM) or without (CON)] in a crossover design. There were 2 periods with 2 phases per period. In phase 1 (9d), all cows were in thermoneutral conditions (TN) and fed ad libitum. During phase 2 (9d), group 1 (n=16) was exposed to HS using electric heat blankets while group 2 (n=16) remained in TN but were pair-fed to HS counterparts. After a washout period (21d), the study was repeated (period 2), with environmental treatments being inverted and dietary treatments remaining the same. Mammary tissue was collected at the end of phase 2. Abundance of phosphorylated mTOR was greater with RPM and tended to be greater with HS. Control cows had a greater decrease in milk protein (%) during phase 2 (difference from phase 1) compared with RPM cows, suggesting that RPM supplementation during HS may support greater milk protein synthesis via mTOR activation. The third experiment investigated the effects of RPM during HS on the response of mammary gland explants to lipopolysaccharide (LPS). Twenty-five mg of tissue obtained from cows in the second experiment was incubated with 0 or 3 μg/mL of LPS for 2h. Incubation with LPS increased abundance of genes associated with inflammation, while HS decreased genes associated with antioxidant responses. Expression of NFKB1was greater in LPS-treated explants from non-HS compared with HS cows. These data indicate that HS reduced immune and antioxidant responses while RPM did not attenuate the inflammatory response induced by LPS in vitro. Overall, data indicated a beneficial effect of choline during NNB and Met during HS on immunometabolism in dairy cows.

Methionyl-Methionine Exerts Anti-Inflammatory Effects through the JAK2-STAT5-NF-κB and MAPK Signaling Pathways in Bovine Mammary Epithelial Cells.

Methionyl-methionine (Met-Met) is a functional dipeptide. Although the role of a dipeptide in milk protein synthesis is clearly established, whether Met-Met has an anti-inflammatory effect and a protective mechanism in bovine mammary epithelial cell (MAC-T) inflammation remains unknown. The purpose of this study was to determine the beneficial effects and underlying mechanisms of Met-Met on lipopolysaccharide (LPS)-induced MAC-T cell inflammation. RNA-seq, siRNA interference, and western blotting were performed to determine the anti-inflammatory mechanisms of Met-Met in the context of LPS exposure. Pretreatment with 2 mM Met-Met could reduce the increase in TNF-α (3.14 ± 0.55 vs 1.54 ± 0.26, P < 0.01), IL-1β (2.30 ± 0.21 vs 1.86 ± 0.11, P < 0.05), and IL-8 (3.49 ± 0.29 vs 0.62 ± 0.20, P < 0.01) after 1 μg/mL LPS exposure. RNA-seq analyses indicated that the overlapping genes were primarily enriched in the nuclear factor kappa-B (NF-κB), mitogen-activated protein kinase (MAPK), and IL-17 pathways. The suppression of NF-κB, P38, and JNK by Met-Met was mediated through the Janus kinase 2-signal transducers and activators of transcription 5 (JAK2-STAT5) pathway. Moreover, the Met-Met-mediated decrease in the LPS-induced activation of p-IκB, NF-κB, and JNK was reversed by knocking down JAK2. Collectively, Met-Met has beneficial effects on MAC-T cell inflammation by activating the JAK2-STAT5 pathway and then inhibiting the NF-κB and MAPK signaling pathways.

Factors that affect heat production in lactating Jersey cows

Heat production (HP) represents a major energy cost in lactating dairy cows. Better understanding of factors that affect HP will improve our understanding of energy metabolism. Our objective was to derive models to explain variation in HP of lactating Jersey cows. Individual animal-period data from 9 studies (n = 293) were used. The data set included cows with a wide range (min to max) in days in milk (44-410) and milk yield (7.8-43.0 kg/d). Diets included corn silage as the predominate forage source, but diets varied (min to max on DM basis) in crude protein (CP; 15.2-19.5%), neutral detergent fiber (NDF; 35.5-43.0%), starch (16.2-31.1%), and crude fat (2.2 to 6.4%) contents. Average HP was (mean ± standard deviation) 22.1 ± 2.86 Mcal/d, or 28.1 ± 3.70% of gross energy intake. Eight models were fit to explain variation in HP: (1) dry matter intake (DMI; INT); (2) milk fat, protein, and lactose yield (MILKCOMP); (3) INT and milk yield (INT+MY); (4) INT and MILKCOMP/DMI (INT+MILKCOMP); (5) mass of digested NDF, CP, and starch (DIG); (6) INT and digested energy (INT+DE); (7) INT and NDF, CP, and starch digestibility (INT+DIG); or (8) INT+MILKCOMP model plus urinary N excretion (INT+MILKCOMP+UN). For all HP models, metabolic body weight was included. All models were derived via a backward elimination approach and included the random effects of study, cow, and period within block within study. The INT models adequately explained variation in HP with a nonrandom effect-adjusted concordance correlation coefficient of 0.84. Similar adjusted concordance correlation coefficients (0.79-0.85) were observed for other HP models. The HP associated with milk protein yield and supply of digestible protein was greater than other milk production and nutrient digestibility variables. The HP associated with urinary N excretion was 5.32. Overall, HP can be adequately predicted from metabolic body weight and DMI. Milk component yield, nutrient digestibility, or urinary N excretion explained similar variation as DMI. Coefficients for milk protein and protein digestion suggest that digestion and metabolism of protein and synthesis of milk protein contribute substantially to HP of a dairy cow.

Effectiveness of feed nitrogen use depending on the level of raw protein and the ratio of protein fractions in the rations of cows

Protein nutrition of ruminants as well as other animal species, should be considered as an amino acid nutrition, since not protein as such, but amino acids are the main material for the synthesis of milk proteins, tissues, enzymes, many hormones, neuropeptides and others that play an important role in ensuring the vital functions of the body. Studying the eff ects of diff erent levels of DPR and ratios UPR:DPR with diff erent content of raw protein in rations for nitrogen metabolism, eff ectiveness of feed protein use and dairy productivity of cows is of great scientifi c and practical importance. The purpose of the researches was to determine the optimal levels of undigested protein in rumen (UPR) and digested protein in rumen (DPR) and to determine the possibility of reducing protein consumption in the rations of high-yielding Holstein cows during the early lactation period. It has been found that in rations with 15,7 % crude protein (CP) the optimal ratio was UPR:DPR = 35:65 (UPR – 5,44 %, DPR – 10,3 % DM), which has been confi rmed by the more active formation of free lysine and methionine in the rumen, the yield of microbial protein, higher coeffi cients of digestibility of dry matter, nitrogen, fi ber, higher yield of lysine and methionine in the duodenal chyme, as well as the lowest losses of protein consumed in the process of rumen digestion. Dairy productivity of cows on this ration was at the level of that of cows on rations with 17,7 % CP – 38,7 kg/day when it contains 3,72 % fat and 3,27 % protein. On the ration with the same CP content (15,7 %), but the ratio of UPR:DPR = 50:50 there was a signifi cant decrease in feed consumption (P &lt; 0,01) and the decrease in dairy productivity of cows, which was probably due to the low level of formation of ammonium nitrogen NH3 -N in the rumen.

Identification of octopus peptide and its promotion of β-casein synthesis in a mouse mammary epithelial cell line.

Octopus protein hydrolysate has been reported to increase milk yield and milk protein production. In this paper, the utilization and underlying mechanisms of bioactive peptide fractions from octopus protein hydrolysate on β-casein expression in mouse mammary epithelial cells (HC11) were investigated. Fraction OPH3-1 significantly stimulated cell proliferation and β-casein synthesis in HC11 cells, which was purified by ultra-filtration and gel-filtration chromatography. The MWs of the peptides from OPH3-1 ranged from 525-2,578Da and consisted of 7-26 amino acid residues. Most of the peptides demonstrated the typical characteristics of milk protein synthesis promotion, especially MGLAGPR, MGDVLNF, EAPLMHV, and TEAPLMHV. Additionally, the mRNA abundances of mTOR, S6K1, 4EBP1, JAK2, and STAT5 were significantly enhanced by OPH3-1, which was consistent with the increased β-casein expression. These results suggest that the OPH3-1 peptides can promote the proliferation of mammary epithelial cells and increase β-casein synthesis. PRACTICAL APPLICATIONS: Breastfeeding mothers are generally recommended to take octopus soup as a daily diet to promote lactation. The peptides fraction OPH3-1 from the enzymatic hydrolysate of Octopus vulgaris which was revealed to significantly stimulate mammary epithelial cell proliferation and β-casein synthesis was obtained. This study suggests that octopus peptides can be used as nutritional supplements to increase the quantity and quality of milk production.