Effect of source and frequency of rumen-protected protein supplementation on mammary gland amino acid metabolism and nitrogen balance of dairy cattle.

The AA profile of MP affects mammary gland metabolism and milk N efficiency of dairy cattle. Further, the frequency of dietary protein supplementation may influence N partitioning leading to reduced N excretion. This study investigated the effect of source and frequency of rumen-protected (RP) protein supplementation on apparent total-tract digestibility, milk production, mammary gland AA metabolism, and N balance of dairy cattle. Twenty-eight Holstein-Friesian cows (2.3 ± 0.9 lactations; 93 ± 27 DIM; mean ± SD) were used in a randomized complete block design and fed a basal TMR consisting of 41% corn silage, 32% grass silage, and 27% concentrate (DM basis) and formulated to meet 100% and 95% of net energy and MP requirements, respectively. Cows were adapted to the basal TMR in a freestall barn for 7 d, moved to individual tiestalls for 13 d of adaptation to dietary treatments, and then moved into climate respiration chambers for a 4-d measurement period. Treatments consisted of the basal TMR (CON; 159 g CP/kg DM) or the basal TMR including 1 of 3 iso-MP supplements: (1) 315-g mixture of RP soybean meal and RP rapeseed meal fed daily (ST-RPSR), (2) 384-g mixture of RP His, RP Lys, and RP Met fed daily (ST-RPAA), and (3) 768-g mixture of RP His, RP Lys, and RP Met fed every other day (OS-RPAA). The basal TMR with the addition of treatment supplements was designed to deliver 100% of required MP over a 48-h period. The mixture of His, Lys, and Met was formulated to deliver digestible AA in amounts relative to their concentration in casein. Compared with ST-RPSR, ST-RPAA increased milk protein and fat concentration, increased the arterial concentration of total His, Lys, and Met (HLM), decreased mammary clearance of HLM, and increased clearance of Phe, Leu, and Tyr (tendency for Leu and Tyr). Rumen-protected protein source did not affect N balance, but the marginal use efficiency (efficiency of transfer of RP protein supplement into milk protein) of ST-RPAA (67%) was higher than that of ST-RPSR (17%). Milk protein concentration decreased with OS-RPAA compared with ST-RPAA. Arterial concentration of HLM increased on the nonsupplemented day compared with the supplemented day with OS-RPAA, and there was no difference in arterial HLM concentration across days with ST-RPAA. Mammary uptake of HLM tended to increase on the nonsupplemented day compared with the supplemented day with OS-RPAA. Supplementation frequency of RP AA did not affect N balance or overall milk N efficiency, but the marginal use efficiency of OS-RPAA (49%) was lower compared with ST-RPAA. Overall, mammary glands responded to an increased supply of His, Lys, and Met by reducing efflux of other EAA when RP His, RP Lys, and RP Met were supplemented compared with RP plant proteins. Mammary glands increased sequestration of EAA (primarily HLM) on the nonsupplemented day with OS-RPAA, but supplementing RP AA according to a 24-h oscillating pattern did not increase N efficiency over static supplementation.

Dose response to postruminal urea in lactating dairy cattle.

Inclusion of urea in dairy cattle diets is often limited by negative effects of high levels of feed urea on dry matter intake (DMI) and efficiency of rumen N utilization. We hypothesized that supplying urea postruminally would mitigate these limitations and allow greater inclusion of urea in dairy cattle diets. Four rumen-fistulated Holstein-Friesian dairy cows (7 ± 2.1 lactations, 110 ± 30.8 d in milk; mean ± standard deviation) were randomly assigned to a 4 × 4 Latin square design to examine DMI, milk production and composition, digestibility, rumen fermentation, N balance, and plasma constituents in response to 4 levels of urea continuously infused into the abomasum (0, 163, 325, and 488 g/d). Urea doses were targeted to linearly increase the crude protein (CP) content of total DMI (diet plus infusion) by 0%, 2%, 4%, and 6% and equated to 0%, 0.7%, 1.4%, and 2.1% of expected DMI, respectively. Each 28-d infusion period consisted of a 7-d dose step-up period, 14 d of adaptation, and a 7-d measurement period. The diet was fed ad libitum as a total mixed ration [10.9% CP, 42.5% corn silage, 3.5% grass hay, 3.5% wheat straw, and 50.5% concentrate (dry matter basis)] and was formulated to meet 100%, 82%, and 53% of net energy, metabolizable protein, and rumen-degradable protein requirements, respectively. Linear, quadratic, and cubic effects of urea dose were assessed using polynomial regression assuming the fixed effect of treatment and random effects of period and cow. Dry matter intake and energy-corrected milk yield responded quadratically to urea dose, and milk urea content increased linearly with increasing urea dose. Apparent total-tract digestibility of CP increased linearly with increasing urea dose and ruminal NH3-N concentration responded quadratically to urea dose. Mean total VFA concentration was not affected by urea dose. The proportion of N intake excreted in feces decreased linearly and that excreted in urine increased linearly in response to increasing urea dose. The proportion of N intake excreted in milk increased linearly with increasing urea dose. Urinary urea excretion increased linearly with increasing urea dose. Microbial N flow responded cubically to urea dose, but the efficiency of microbial protein synthesis was not affected. Plasma urea concentration increased linearly with increasing urea dose. Regression analysis estimated that when supplemented on top of a low-CP diet, 179 g/d of postruminal urea would maximize DMI at 23.4 kg/d, corresponding to a dietary urea inclusion level of 0.8% of DMI, which is in line with the current recommendations for urea inclusion in dairy cattle diets. Overall, these results indicate that postruminal delivery of urea does not mitigate DMI depression as urea dose increases.

Essential amino acid profile of supplemental metabolizable protein affects mammary gland metabolism and whole-body glucose kinetics in dairy cattle.

This study investigated mammary gland metabolism and whole-body (WB) rate of appearance (Ra) of glucose in dairy cattle in response to a constant supplemental level of metabolizable protein (MP) composed of different essential AA (EAA) profiles. Five multiparous rumen-fistulated Holstein-Friesian dairy cows (2.8 ± 0.4 lactations; 81 ± 11 d in milk; mean ± standard deviation) were abomasally infused according to a 5 × 5 Latin square design with saline (SAL) or 562 g/d of EAA delivered in different profiles where individual AA content corresponded to their relative content in casein. The profiles consisted of (1) a complete EAA mixture (EAAC), (2) Ile, Leu, and Val (ILV), (3) His, Ile, Leu, Met, Phe, Trp, Val (GR1+ILV), and (4) Arg, His, Lys, Met, Phe, Thr, Trp (GR1+ALT). A total mixed ration (58% corn silage, 16% alfalfa hay, and 26% concentrate on a dry matter basis) was formulated to meet 100 and 83% of net energy and MP requirements, respectively, and was fed at 90% of ad libitum intake on an individual cow basis. Each experimental period consisted of 5 d of continuous abomasal infusion followed by 2 d of no infusion. Arterial and venous blood samples were collected on d 4 of each period for determination of mammary gland AA and glucose metabolism. On d 5 of each period, D-[U-13C]glucose (13 mmol priming dose; continuous 3.5 mmol/h for 520 min) was infused into a jugular vein and arterial blood samples were collected before and during infusion to determine WB Ra of glucose. Milk protein yield did not differ between EAAC, GR1+ILV, and GR1+ALT, or between SAL and ILV, and increased over SAL and ILV with EAAC and GR1+ILV. Mammary plasma flow increased with ILV infusion compared with EAAC and GR1+ILV. Infusion of EAAC tended to increase mammary gland net uptake of total EAA and decreased the mammary uptake to milk protein output ratio (U:O) of non-EAA compared with SAL. Infusion of ILV increased mammary net uptake and U:O of Ile, Leu, and Val markedly over all treatments. The U:O of total Ile, Leu, and Val increased numerically (25%) with GR1+ILV infusion compared with EAAC, and the U:O of total Arg, Lys, and Thr tended to decrease, primarily from decreased U:O of Lys. During GR1+ALT infusion, U:O of total Arg, Lys, and Thr was greater than that during EAAC infusion, whereas U:O of Ile, Leu, and Val did not differ from EAAC. Glucose WB Ra increased 16% with GR1+ALT over SAL, and increased numerically 8 and 12% over SAL with EAAC and GR1+ILV, respectively. The average proportion of lactose yield relative to glucose WB Ra did not differ across treatments and averaged 0.53. On average, 28% of milk galactose arose from nonglucose precursors, regardless of treatment. In conclusion, intramammary catabolism of group 2 AA increased to support milk component synthesis when the EAA profile of MP was incomplete with respect to casein. Further, WB and mammary gland glucose metabolism was flexible in support of milk component synthesis, regardless of absorptive EAA profile.

Estimates of daily oxygen consumption, carbon dioxide and methane emissions, and heat production for beef and dairy cattle using spot gas sampling.

A simulation study was conducted to examine accuracy of estimating daily O2 consumption, CO2 and CH4 emissions, and heat production (HP) using a spot sampling technique and to determine optimal spot sampling frequency (FQ). Data were obtained from 3 experiments where daily O2 consumption, emissions of CO2 and CH4, and HP were measured using indirect calorimetry (respiration chamber or headbox system). Experiment 1 used 8 beef heifers (ad libitum feeding; gaseous exchanges measured every 30 min over 3 d in respiration chambers); Experiment 2 used 56 lactating Holstein-Friesian cows (restricted feeding; gaseous exchanges measured every 12 min over 3 d in respiration chambers); Experiment 3 used 12 lactating Jersey cows (ad libitum feeding; gaseous exchanges measured every hour for 1 d using headbox style chambers). Within experiment, averages of all measurements (FQALL) and averages of measurements selected at time points with 12, 8, 6, or 4 spot sampling FQ (i.e., sampling every 2, 3, 4, and 6 h in a 24-h cycle, respectively; FQ12, FQ8, FQ6, and FQ4, respectively) were compared. Within study a mixed model was used to compare gaseous exchanges and HP among FQALL, FQ12, FQ8, FQ6, and FQ4, and an interaction of dietary treatment by FQ was examined. A regression model was used to evaluate accuracy of spot sampling within study [i.e., FQALL (observed) vs. FQ12, FQ8, FQ6, or FQ4 (estimated)]. No interaction of diet by FQ was observed for any variables except for CH4 production in experiment 1. No FQ effect was observed for gaseous exchanges and HP except in experiment 2 where CO2 production was less (5,411 vs. 5,563 L/d) for FQ4 compared with FQALL, FQ12, and FQ8. A regression analysis between FQALL and each FQ within study showed that slopes and intercepts became farther from 1 and 0, respectively, for almost all variables as FQ decreased. Most variables for FQ12 and FQ8 had root mean square prediction error (RMSPE) less than 10% of the mean and concordance correlation coefficient (CCC) greater than 0.80, and RMSPE increased and CCC decreased as FQ decreased. When a regression analysis was conducted with combined data from the 3 experiments (mixed model with study as a random effect), results agreed with those from the analysis for the individual studies. Prediction errors increased and CCC decreased as FQ decreased. Generally, all the estimates from FQ12, FQ8, FQ6, and FQ4 had RMSPE less than 10% of the means and CCC greater than 0.90 except for FQ6 and FQ4 for O2 consumption and CH4 production. In conclusion, the spot sampling simulation with 3 indirect calorimetry experiments indicated that FQ of at least 8 samples (every 3 h in a 24-h cycle) was required to estimate daily O2 consumption, CO2 and CH4 production, and HP and to detect changes in those in response to dietary treatments. This sampling FQ may be considered when using techniques that measure spot gas exchanges such as the GreenFeed and face mask systems.

Energy and nitrogen balance of dairy cattle as affected by provision of different essential amino acid profiles at the same metabolizable protein supply.

Amino acid composition of metabolizable protein (MP) is important in dairy cattle diets, but effects of AA imbalances on energy and N utilization are unclear. This study determined the effect of different AA profiles within a constant supplemental MP level on whole-body energy and N partitioning in dairy cattle. Five rumen-fistulated Holstein-Friesian dairy cows (2.8 ± 0.4 lactations; 81 ± 11 d in milk; mean ± standard deviation) were randomly assigned to a 5 × 5 Latin square design in which each experimental period consisted of 5 d of continuous abomasal infusion followed by 2 d of rest. A total mixed ration consisting of 58% corn silage, 16% alfalfa hay, and 26% concentrate (dry matter basis) was formulated to meet 100 and 83% of net energy and MP requirements, respectively, and was fed at 90% of ad libitum intake by individual cow. Abomasal infusion treatments were saline (SAL) or 562 g/d of essential AA delivered in 4 profiles where individual AA content corresponded to their relative content in casein. The profiles were (1) a complete essential amino acid mixture (EAAC), (2) Ile, Leu, and Val (ILV), (3) His, Ile, Leu, Met, Phe, Trp, Val (GR1+ILV), and (4) Arg, His, Lys, Met, Phe, Thr, Trp (GR1+ALT). The experiment was conducted in climate respiration chambers to determine energy and N balance in conjunction with milk production and composition, digestibility, and plasma constituents. Compared with SAL, infusion of EAAC increased milk, protein, and lactose yield, increased energy retained as body protein, and did not affect milk N efficiency. Total N intake and urine N output was higher with all AA infusions relative to SAL. Compared with EAAC, infusions of GR1+ILV and GR1+ALT produced the same milk yield and the same yield and content of milk fat, protein, and lactose, and had similar energy and N retention. Milk N efficiency was not different between EAAC and GR1+ILV, but was lower with GR1+ALT compared with EAAC, and tended to be lower with GR1+ALT compared with GR1+ILV. Infusion of ILV tended to decrease dry matter intake compared with the other AA infusions. Milk production and composition was not different between ILV and SAL. Compared with EAAC, infusion of ILV decreased or tended to decrease milk, protein, and lactose yields and milk protein content, and increased milk fat and lactose content. Milk N efficiency decreased with ILV compared with SAL, EAAC, and GR1+ILV. Milk urea concentration was not affected by essential amino acid (EAA) infusions. Plasma urea concentration did not differ between EAAC and SAL, tended to increase with ILV and GR1+ILV over SAL, and increased with GR1+ALT compared with EAAC and SAL. In conclusion, removing Arg, Lys, and Thr or removing Ile, Leu, and Val from a complete EAA profile when the total amount of EAA infused remained constant did not impair milk production, but milk N efficiency decreased when Ile, Leu, and Val were absent. Infusion of only Ile, Leu, and Val decreased milk protein yield and content and reduced milk N efficiency compared with a complete EAA profile.

Mammary gland metabolite utilization in response to exogenous glucose or long-chain fatty acids at low and high metabolizable protein levels.

We investigated mammary gland metabolism in lactating dairy cattle in response to energy from glucogenic (glucose; GG) or lipogenic (palm olein; LG) substrates at low (LMP) and high (HMP) metabolizable protein levels. According to a 6 × 6 Latin square design, 6 rumen-fistulated second-lactation Holstein-Friesian dairy cows (97 ± 13 d in milk) were abomasally infused with saline (LMP-C); isoenergetic infusions (digestible energy basis) of 1,319 g/d glucose (LMP-GG), 676 g/d palm olein (LMP-LG), or 844 g/d essential AA (EAA; HMP-C); or isoenergetic infusions of 1,319 g/d glucose + 844 g/d EAA (HMP-GG) or 676 g/d palm olein + 844 g/d EAA (HMP-LG). Each experimental period consisted of 5 d of continuous infusion followed by 2 d of rest. A total mixed ration (42% corn silage, 31% grass silage, and 27% concentrate on a dry matter basis) formulated to meet 100 and 83% of net energy and metabolizable protein requirements, respectively, was fed at 90% of ad libitum intake by individual cow. Arterial and venous blood samples were collected on d 5 of each period. Infusing GG or LG at the HMP level did not affect milk yield or composition differently than at the LMP level. Neither GG nor LG infusion stimulated milk protein or lactose yield, but fat yield tended to decrease with GG and tended to increase with LG. Infusion of GG increased arterial plasma concentrations of glucose and insulin and decreased concentrations of ß-hydroxybutyrate (BHB), nonesterified fatty acids, long-chain fatty acids (LCFA), total AA, EAA, and group 2 AA. Infusion of LG increased arterial triacylglycerides (TAG) and LCFA but did not affect EAA concentrations. Compared with the LMP level, the HMP level increased arterial concentrations of BHB, urea, and all EAA groups and decreased the concentration of total non-EAA. Mammary plasma flow increased with GG and was not affected by LG or protein level. Uptake and clearance of total EAA and group 2 AA were affected or tended to be affected by GG × AA interactions, with their uptakes being lower and their clearances higher with GG, but only at the LMP level. Infusion of LG did not affect uptake or clearance of any AA group. The HMP level increased uptake and decreased clearance of all EAA groups and decreased non-EAA uptake. Infusion of GG tended to increase mammary glucose uptake, and tended to decrease BHB uptake only at the LMP level. Infusion of LG increased mammary uptake of TAG and LCFA and increased or tended to increase clearance of TAG and LCFA. We suspect GG increased mammary plasma flow to maintain intramammary energy and AA balance and stimulated lipogenesis in adipose, accounting for depressed arterial BHB and group 2 AA concentrations. Mammary glucose uptake did not cover estimated requirements for lactose and fat synthesis at the HMP level, except during HMP-GG infusion. Results of this study illustrate flexibility in mammary metabolite utilization when absorptive supply of glucogenic, lipogenic, and aminogenic substrate is increased.

Mammary gland utilization of amino acids and energy metabolites differs when dairy cow rations are isoenergetically supplemented with protein and fat.

Mammary gland utilization of AA and other metabolites in response to supplemental energy from protein (PT) and supplemental energy from fat (FT) was tested in a 2 × 2 factorial arrangement using a randomized complete block design. Fifty-six Holstein-Friesian dairy cows were adapted during a 28-d control period to a basal total mixed ration consisting of 34% grass silage, 33% corn silage, 5% grass hay, and 28% concentrate on a dry matter (DM) basis. Experimental rations were fed for 28 d immediately following the control period and consisted of (1) low protein, low fat (LP/LF), (2) high protein, low fat (HP/LF), (3) low protein, high fat (LP/HF), and (4) high protein, high fat (HP/HF). To obtain the high-protein (HP) and high-fat (HF) diets, intake of the basal ration was restricted and supplemented isoenergetically [net energy (MJ/d) basis] with 2.0 kg/d rumen-protected protein (soybean + rapeseed, 50:50 mixture on a DM basis) and 0.68 kg/d hydrogenated palm fatty acids on a DM basis. Arterial and venous blood samples were collected on d 28 of both periods. Isoenergetic supplements (MJ/d) of protein and fat independently and additively increased milk yield, PT increased protein yield, and FT increased fat yield. A PT × FT interaction affected arterial concentration of all essential AA (EAA) groups, where they increased in response to PT by a greater magnitude at the LF level (on average 35%) compared with the HF level (on average 14%). Mammary gland plasma flow was unaffected by PT or FT. Supplementation with PT tended to decrease mammary clearance of total EAA and decreased group 1 AA clearance by 19%. In response to PT, mammary uptake of total EAA and group 2 AA increased 12 and 14%, respectively, with significantly higher uptake of Arg, Ile, and Leu. Energy from fat had no effect on mammary clearance or uptake of any AA group. The mammary gland uptake:milk protein output ratio was not affected by FT, whereas PT increased this ratio for EAA and group 2 AA. Arterial plasma insulin concentration decreased in response to FT, in particular on the HP/HF diet, as indicated by a PT × FT interaction. Arterial concentrations of nonesterified fatty acids, triacylglycerol, and long-chain fatty acids increased in response to FT, and concentrations of ß-hydroxybutyrate and acetate decreased in response to FT only at the HP level. Mammary clearance and uptake of triacylglycerol and long-chain fatty acids increased in response to FT. Energy from PT and FT increased lactose yield despite no change in arterial glucose concentration or mammary glucose uptake. Mammary-sequestered glucose with PT or FT was used in the same amount for lactose synthesis, and a positive net mammary glucose balance was found across all treatments. Results presented here illustrate metabolic flexibility of the mammary gland in its use of aminogenic versus lipogenic substrates for milk synthesis.

Energy and nitrogen partitioning in dairy cows at low or high metabolizable protein levels is affected differently by postrumen glucogenic and lipogenic substrates.

This study tested the effects of energy from glucogenic (glucose; GG) or lipogenic (palm olein; LG) substrates at low (LMP) and high (HMP) metabolizable protein levels on whole-body energy and N partitioning of dairy cattle. Six rumen-fistulated, second-lactation Holstein-Friesian dairy cows (97 ± 13 d in milk) were randomly assigned to a 6 × 6 Latin square design in which each experimental period consisted of 5 d of continuous abomasal infusion followed by 2 d of rest. A total mixed ration consisting of 42% corn silage, 31% grass silage, and 27% concentrate (dry matter basis) was formulated to meet 100 and 83% of net energy and metabolizable protein requirements, respectively, and was fed at 90% of ad libitum intake by individual cow. Abomasal infusion treatments were saline (LMP-C), isoenergetic infusions (digestible energy basis) of 1,319 g/d of glucose (LMP-GG), 676 g/d of palm olein (LMP-LG; major fatty acid constituents are palmitic, oleic, and linoleic acid), or 844 g/d of essential AA (HMP-C), or isoenergetic infusions of 1,319 g/d of glucose + 844 g/d of essential AA (HMP-GG) or 676 g/d of palm olein + 844 g/d of essential AA (HMP-LG). The experiment was conducted in climate respiration chambers to determine energy and N balance in conjunction with milk production and composition, nutrient digestibility, and plasma constituents. Infusion of GG and LG decreased dry matter intake, but total gross energy intake from the diet plus infusions was not affected by GG or LG. Furthermore, GG or LG did not affect total milk, protein, or lactose yields. Infusing GG or LG at the HMP level did not affect milk production differently than at the LMP level. Infusion of GG stimulated energy retention in body tissue, increased plasma glucose and insulin concentrations, decreased lipogenic metabolites in plasma, and decreased milk fat yield and milk energy output. Nitrogen intake decreased and milk N efficiency increased in response to GG, and N retention was not affected. Infusion of LG tended to increase metabolizable energy intake, increased milk fat yield and milk energy output, increased plasma triacylglycerides and long-chain fatty acid concentrations, and had no effect on energy retention. Infusion of LG decreased N intake but did not affect milk N efficiency or N retention. Compared with the LMP level, the HMP level increased dry matter intake, gross and metabolizable energy intake, and total milk, fat, protein, and lactose yields. Milk energy output increased at the HMP level, and protein level did not affect total energy retention. Heat production increased at the HMP level, but only when GG and LG were infused. The HMP level increased N intake, milk N output, and plasma urea concentration, tended to increase N retention, and decreased milk N efficiency. Regardless of protein level, GG promoted energy retention and improved milk N efficiency, but not through increased milk protein yield. Infusion of LG partitioned extra energy intake into milk and had no effect on milk N efficiency.

Expression of genes related to energy metabolism and the unfolded protein response in dairy cow mammary cells is affected differently during dietary supplementation with energy from protein and fat.

Secretory capacity of bovine mammary glands is enabled by a high number of secretory cells and their ability to use a range of metabolites to produce milk components. We isolated RNA from milk fat to measure expression of genes involved in energy-yielding pathways and the unfolded protein response in mammary glands of lactating cows given supplemental energy from protein (PT) and fat (FT) tested in a 2 × 2 factorial arrangement. We hypothesized that PT and FT would affect expression of genes in the branched-chain AA catabolic pathway and tricarboxylic acid (TCA) cycle based on the different energy types (aminogenic versus lipogenic) used to synthesize milk components. We also hypothesized that the response of genes related to endoplasmic reticulum (ER) homeostasis via the unfolded protein response would reflect the increase in milk production stimulated by PT and FT. Fifty-six multiparous Holstein-Friesian dairy cows were fed a basal total mixed ration (34% grass silage, 33% corn silage, 5% grass hay, and 28% concentrate on a dry matter basis) for a 28-d control period. Experimental rations were then fed for 28 d, consisting of (1) low protein, low fat (LP/LF); (2) high protein, low fat (HP/LF); (3) low protein, high fat (LP/HF); or (4) high protein and high fat (HP/HF). To obtain the high-protein (HP) and high-fat (HF) diets, intake of the basal ration was restricted and supplemented isoenergetically (net energy basis) with 2.0 kg/d rumen-protected protein (soybean + rapeseed, 50:50 mixture on dry matter basis) and 0.68 kg/d hydrogenated palm fatty acids on a dry matter basis. RNA from milk fat samples collected on d 27 of each period underwent real-time quantitative PCR. Energy from protein increased expression of BCAT1 (branched-chain amino acid transferase 1) mRNA, but only at the LF level, and tended to decrease expression of mRNA encoding the main subunit of the branched-chain keto-acid dehydrogenase complex. mRNA expression of malic enzyme, a proposed channeling route for AA though the TCA cycle, was decreased by PT, but only at the LF level. Expression of genes associated with de novo fatty acid synthesis was not affected by PT or FT. Energy from fat had no independent effect on genes related to ER homeostasis. At the LF level, PT activated XBP1 (X-box binding protein 1) mRNA. At the HF level, PT increased mRNA expression of the gene encoding GADD34 (growth arrest and DNA damage-inducible 34). These findings support our hypothesis that mammary cells use aminogenic and lipogenic precursors differently for milk component production when dietary intervention alters AA and fatty acid supply. They also suggest that mammary cells respond to increased AA supply through mechanisms of ER homeostasis, dependent on the presence of FT.

Reporter bacteriophage T7NLC utilizes a novel NanoLuc::CBM fusion for the ultrasensitive detection of Escherichia coli in water.

Rapid detection of bacteria responsible for foodborne diseases is a growing necessity for public health. Reporter bacteriophages (phages) are robust biorecognition elements uniquely suited for the rapid and sensitive detection of bacterial species. The advantages of phages include their host specificity, ability to distinguish viable and non-viable cells, low cost, and ease of genetic engineering. Upon infection with reporter phages, target bacteria express reporter enzymes encoded within the phage genome. In this study, the T7 coliphage was genetically engineered to express the newly developed luceriferase, NanoLuc (NLuc), as an indicator of bacterial contamination. While several genetic approaches were employed to optimize reporter enzyme expression, the novel achievement of this work was the successful fusion of the NanoLuc reporter to a carbohydrate binding module (CBM) with specificity to crystalline cellulose. This novel chimeric reporter (nluc::cbm) bestows the specific and irreversible immobilization of NanoLuc onto a low-cost, widely available crystalline cellulosic substrate. We have shown the possibility of detecting the immobilized fusion protein in a filter plate which resulted from a single CFU of E. coli. We then demonstrated that microcrystalline cellulose can be used to concentrate the fusion reporter from 100 mL water samples allowing a limit of detection of <10 CFU mL-1E. coli in 3 hours. Therefore, we conclude that our phage-based detection assay displays significant aptitude as a proof-of-concept drinking water diagnostic assay for the low-cost, rapid and sensitive detection of E. coli. Additional improvements in the capture efficiency of the phage-based fusion reporter should allow a limit of detection of <10 CFU per 100 mL.

African born women living with HIV in the United States: unmet needs and opportunities for intervention.

Africa born (immigrant) women comprise a disproportionate number of Black women living with HIV in the United States. Though they are at risk for mental health disorders, including psychological distress and depression, little is known about their experience with these important predictors of quality of life, retention in care and adherence to antiretroviral therapy. In this qualitative study, we used constructivist grounded theory to explore the psychosocial and mental health challenges of African born women living with HIV in Boston and New York City. We conducted one-on-one semi-structured interviews with 45 women. Major themes contributing to psychological distress and depressive symptoms included (1) pre-immigration HIV-related stigma; (2) persistent HIV-related stigma post-immigration, (3) undocumented immigration status, (4) economic insecurity, and (5) intimate partner violence (IPV). Many participants described ongoing depressive symptoms or histories of depressive episodes. Yet, most had not been formally diagnosed or treated for depression. Prayer, consultation with faith leadership, and support groups were described most frequently as useful interventions. Future research should explore these thematic areas among a larger, more representative sample of African born women living with HIV to determine differences by country of origin across thematic areas. These data would be useful to inform development of innovative and culturally appropriate interventions.

Feed and nitrogen efficiency are affected differently but milk lactose production is stimulated equally when isoenergetic protein and fat is supplemented in lactating dairy cow diets.

Fifty-six Holstein-Friesian cows were used in a randomized complete block design to test the effects of supplemental energy from protein (PT) and fat (FT) on lactation performance and nutrient digestibility in a 2 × 2 factorial arrangement. During the control period, cows were adapted for 28 d to a basal total mixed ration consisting of 34% grass silage, 33% corn silage, 5% grass hay, and 28% concentrate on a dry matter (DM) basis. Experimental rations were fed for 28 d immediately following the control period and consisted of (1) low protein, low fat (LP/LF), (2) high protein, low fat (HP/LF), (3) low protein, high fat (LP/HF), or (4) high protein and high fat (HP/HF). To obtain the HP and HF diets, intake of the basal ration was restricted and supplemented isoenergetically (net energy basis) with 2.0 kg/d of rumen-protected protein (soybean + rapeseed, 50:50 mixture on DM basis) and 0.68 kg/d of hydrogenated palm fatty acids (FA) on a DM basis. Milk production and composition, nutrient intake, and apparent digestibility were measured during the final 7 d of the control and experimental periods. No interaction was found between PT and FT on milk production and composition. Yields of milk, fat- and protein-corrected milk, and lactose increased in response to PT and FT and lactose concentration was unaffected by treatment. Milk protein concentration and yield increased in response to PT, and protein yield tended to increase in response to FT. Milk fat concentration and yield increased in response to FT and were unaffected by PT. Milk urea concentration increased and nitrogen efficiency decreased in response to PT. Feed and nitrogen efficiency were highest on the LP/HF diet and both parameters increased in response to FT, whereas milk urea concentration was not affected by FT. Energy from fat increased the concentration and yield of ≥16-carbon FA in milk and decreased the concentration of FA synthesized de novo, but had no effect on their yield. Concentration and yield of de novo-synthesized FA increased in response to PT. Concentration and yield of polyunsaturated FA increased and decreased in response to PT and FT, respectively. Apparent total-tract digestibility of crude fat decreased in response to PT, and FT increased crude protein digestibility. Energy supplementation through rumen-inert hydrogenated palm FA appears to be an efficient feeding strategy to stimulate milk production with regard to feed and nitrogen efficiency compared with supplementing an isoenergetic level of rumen-protected protein.

Exogenous essential amino acids stimulate an adaptive unfolded protein response in the mammary glands of lactating cows.

The phosphorylation of mammalian target of rapamycin complex 1 (mTORC1) components and integrated stress response networks in the mammary glands of lactating cows have not accounted for the stimulation of milk protein yield by chronic supplementation with AA or glucose. Faster milk protein synthesis could be a consequence of increased milk protein mRNA per cell, the number of ribosomes per cell, the secretory capacity of cells, or the mammary cell number. To investigate these 4 possibilities using a translational and transcriptional approach, we performed protein and gene expression analyses of mammary and longissimus dorsi tissue collected from lactating dairy cows after 5 d of abomasal infusion with saline or 844 or 1,126 g/d of an essential AA (EAA) mixture, with and without 1,000 g/d glucose. Infusion with EAA increased milk protein yield but did not affect the phosphorylation of mTORC1-related proteins in the mammary gland. In skeletal muscle, phosphorylation of 4EBP1 (eIF4E-binding protein 1) increased in response to both EAA and glucose, and phosphorylated S6K1 (70-kDa ribosomal protein S6 kinase) increased with glucose. In response to EAA, mammary mRNA expression of the marker genes for milk proteins, ribosome biogenesis, and cell proliferation were not upregulated. Instead, reciprocal regulation of 2 arms of the unfolded protein response occurred. Infusion of EAA for 5 d activated XBP1 (X-box binding protein 1) mRNA, encoding a transcription factor for endoplasmic reticulum biogenesis, and it decreased the mRNA expression of genes encoding pro-apoptotic protein CHOP (C/EBP homologous protein) and downstream GADD34 (growth arrest and DNA damage-inducible 34). These findings implicate non-stress-related, adaptive capabilities of the unfolded protein response in the long-term nutritional regulation of milk protein yield in lactating dairy cows.

Glucose supplementation stimulates peripheral branched-chain amino acid catabolism in lactating dairy cows during essential amino acid infusions.

To determine how glucose modulates protein synthesis when essential AA are in abundant supply, 5 early-lactation, rumen-fistulated Holstein dairy cows were fed a diet containing 6.95 MJ/kg of net energy for lactation and 12.4% crude protein and abomasally infused for 5 d with saline, 844 or 1,126 g/d of a complete essential AA mix, with and without the inclusion of 1,000 g/d of glucose, in a 5×5 Latin square design. Infusion of essential AA increased milk yield by 4.1 kg/d, milk protein by 256 g/d, milk fat by 95 g/d, and milk urea nitrogen by 70% compared with saline, with no differences between the level of essential AA infusion. The addition of glucose to essential AA infusate did not stimulate milk protein yield or concentration, but reduced milk urea nitrogen by 17% and decreased milk fat yield. Arterial concentrations of total essential AA increased 3- to 4-fold, mammary clearance decreased 61%, and mammary uptake of essential AA increased 65% in response to essential AA infusion. Arterial branched-chain AA concentrations declined 29% in response to glucose and mammary clearance increased 48%, but mammary AA uptake was unchanged. Essential AA infusion increased plasma 3-methylhistidine by 50% and reduced muscle branched-chain α-keto acid dehydrogenase kinase abundance by 14%, indicating stimulation of muscle protein turnover and branched-chain AA catabolism, respectively. Glucose had no further effect on muscle branched-chain α-keto acid dehydrogenase kinase abundance but decreased mRNA expression of branched chain aminotransferase 1. Lack of further increases in plasma 3-methylhistidine or greater stimulation of muscle branched-chain AA catabolism indicates that muscle protein degradation was unchanged with glucose but that accretion may have been stimulated. The decrease in circulating branched-chain AA concentrations and nitrogen excretion in response to glucose suggests that surplus essential AA were redirected to peripheral, extra-mammary tissues.

The effect of dysmenorrhea severity and interference on reactions to experimentally-induced pain.

Introduction: Dysmenorrhea is associated with increased risk of chronic pain and hyperalgesia. Menstruating individuals with dysmenorrhea are more likely to have elevated pain reactivity when experiencing experimental pain, than those without. However, no study has examined intragroup differences in reactions to experimentally induced pain for individuals with dysmenorrhea. The main aim of this study was to examine the relative roles of dysmenorrhea severity and interference in the experience of experimentally-induced pain. Methods: Participants were 120 menstruating individuals involved in a larger research study examining the influence of expectations on experimentally-induced pain. As part of the study, participants completed an online questionnaire regarding demographic and menstrual information and participated in a cold pressor task. Participants were randomized into four groups based on the manipulation of two independent variables: (1) high vs. low expectations about pain severity (pain-expectations); (2) and high vs. low expectations about one's pain tolerance (self-expectations). Participants verbally rated their pain severity throughout the cold pressor task using a 0-10 scale. Regression analyses were conducted examining the relationships between dysmenorrhea experience (i.e., average severity and interference) and cold pressor data [pain severity ratings and pain tolerance (i.e., total time in the cold pressor)], controlling for the manipulated expectations and age. Then, moderation analyses were conducted examining expectation group differences. Results: When controlling for manipulated expectations and age, dysmenorrhea severity significantly predicted initial pain severity rating (p = 0.022) but did not predict final pain severity rating (p = 0.263) or pain tolerance (p = 0.120). Dysmenorrhea interference did not predict initial pain severity rating (p = 0.106), final pain severity rating (p = 0.134), or pain tolerance (p = 0.360). A moderation analysis indicated that the relationship between dysmenorrhea severity and initial pain severity rating was not moderated by pain-expectations, χ 2(1) = 0.412, p = 0.521. Discussion: During an experimentally-induced pain task, dysmenorrhea severity but not interference predicted initial pain severity rating, such that higher levels of dysmenorrhea severity predicted greater initial pain severity rating. This suggests individuals with more severe dysmenorrhea pain may experience greater initial sensitivity to pain and be at risk for increased sensitivity to acute pain and potentially the development of chronic pain.

Fitness consequences of dispersal: is leaving home the best of a bad lot?

Using 20 years of demographic and genetic data from four populations of banner-tailed kangaroo rats (Dipodomys spectabilis), we asked whether dispersing individuals gain benefits during adulthood that might compensate for the substantial survival costs they experience as juveniles. Compared to philopatric animals, within- and between-population dispersers gained no measureable advantages in adult survival, fecundity, or probability of recruiting offspring to adulthood. Restricting analyses to members of two central populations living more than 15 times the median dispersal distance from the study site edge, and using peripheral populations only to detect dispersal or offspring recruitment "offsite," did not change this result. Population density during year of birth had small negative effects on adult survival and fecundity, but there were no interactions with dispersal phenotype. We found no evidence that dispersers gained access to superior habitat or that their offspring suffered less inbreeding depression. Our results are consistent with fitness advantages being indirect; by leaving, dispersers release their kin from competition. Our results are also consistent with the possibility that dispersal is the "best of a bad lot." If dispersal is a conditional strategy, then the benefits may be obscured in observational data that compare dispersing individuals to philopatric individuals rather than to individuals whose dispersal phenotype is experimentally manipulated.

Mapping rainbow trout immune genes involved in inflammation reveals conserved blocks of immune genes in teleosts.

We report the genetic map location of 14 genes involved in the inflammatory response to salmonid bacterial and viral pathogens, which brings the total number of immune genes mapped in rainbow trout (RT, Oncorhynchus mykiss) to 61. These genes were mapped as candidate genes that may be involved in resistance to bacterial kidney disease, as well as candidates for known QTL for resistance to infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus and Ceratomyxa shasta. These QTL map to one or more of the linkage groups containing immune genes. The combined analysis of these linkage results and those of previously mapped immune genes in RT shows that many immune genes are found in syntenic blocks of genes that have been retained in teleosts despite species divergence and genome duplication events.