Valine and nonessential amino acids affect bidirectional transport rates of leucine and isoleucine in bovine mammary epithelial cells.

A more complete understanding of the mechanisms controlling AA transport in mammary glands of dairy cattle will help identify solutions to increase nitrogen feeding efficiency on farms. It was hypothesized that Ala, Gln, and Gly (NEAAG), which are actively transported into cells and exchanged for all branched-chain AA (BCAA), may stimulate transport of BCAA, and that Val may antagonize transport of the other BCAA due to transporter competition. Thus, we evaluated the effects of varying concentrations of NEAAG and Val on transport and metabolism of the BCAA Ala, Met, Phe, and Thr by bovine mammary epithelial cells. Primary cultures of bovine mammary epithelial cells were assigned to treatments of low (70% of mean in vivo plasma concentrations of lactating dairy cows) and high (200%) concentrations of Val and NEAAG (LVal and LNEAAG, HVal and HNEAAG, respectively) in a 2 × 2 factorial design. Cells were preloaded with treatment media containing [15N]-labeled AA for 24 h. The [15N]-labeled media were replaced with treatment media containing [13C]-labeled AA. Media and cells were harvested from plates at 0, 0.5, 1, 5, 15, 30, 60, and 240 min after application of the [13C]-labeled AA and assessed for [15N]- and [13C]-AA label concentrations. The data were used to derive transport, transamination, irreversible loss, and protein-synthesis fluxes. All Val fluxes, except synthesis of rapidly exchanging tissue protein, increased with the HVal treatment. Interestingly, the rapidly exchanging tissue protein, transamination, and irreversible-loss rate constants decreased with HVal, indicating that the significant flux increases were primarily driven by mass action with the cells resisting the flux increases by downregulating activity. However, the decreases could also reflect saturation of processes that would drive down the mass-action rate constants. This is supported by decreases in the same rate constants for Ile and Leu with HVal. This could be due to either competition for shared transamination and oxidation reactions or a reduction in enzymatic activity. Also, NEAAG did not affect Val fluxes, but influx and efflux rate constants increased for both Val and Leu with HNEAAG, indicating an activating substrate effect. Overall, AA transport rates generally responded concordantly with extracellular concentrations, indicating the transporters are not substrate-saturated within the in vivo range. However, BCAA transamination and oxidation enzymes may be approaching saturation within in vivo ranges. In addition, System L transport activity appeared to be stimulated by as much as 75% with high intracellular concentrations of Ala, Gln, and Gly. High concentrations of Val antagonized transport activity of Ile and Leu by 68% and 15%, respectively, indicating competitive inhibition, but this was only observable at HNEAAG concentrations. The exchange transporters of System L transport 8 of the essential AA that make up approximately 40% of milk protein, so better understanding this transporter is an important step for increased efficiency.

Effects of varying extracellular amino acid concentrations on bidirectional amino acid transport and intracellular fluxes in mammary epithelial cells.

Understanding the regulation of cellular AA uptake as protein supply changes is critical for predicting milk component yields because intracellular supplies partly regulate protein synthesis. Our objective was to evaluate cellular uptake and kinetic behavior of individual AA when cells are presented with varying extracellular AA supplies. Bovine primary mammary epithelial cells were grown to confluency and transferred to medium with an AA profile and concentration similar to that of plasma from dairy cows for 24 h. Treatments were 4 AA concentrations, 0.36, 2.30, 4.28, and 6.24 mM, which represented 16, 100, 186, and 271% of typical plasma AA concentrations, respectively, in lactating dairy cows. Twenty-four plates of cells (89.4 × 19.2 mm) were assigned to each treatment. Cells were first subjected to treatment medium enriched with 15N-labeled AA for 24 h and then incubated with treatment medium enriched with 13C-labeled AA for 0, 15, 60, 300, 900, 1,800, and 3,600 s. Intracellular free AA, intracellular protein-bound AA, and extracellular medium free AA were analyzed for concentrations and isotopic enrichment using gas chromatography-mass spectrometry. A dynamic, 12-pool model was fitted to the data for 14 AA to derive unidirectional uptake and efflux, protein turnover, transamination, oxidation, and synthesis. The derived concentration for half the maximal uptake (km) indicated no saturation of AA uptake at typical in vivo concentrations for 11 of the 14 AA. Arginine, Pro, and Val appeared to exhibit saturation kinetics. Net uptake of all essential AA except Phe was positive across treatments. Most nonessential AA exhibited negative net uptake values. Efflux of AA was quite high, with several AA exhibiting greater than 100% efflux of the respective influx. Intracellular pool turnover was rapid for most AA (e.g., 2 min for Arg), demonstrating plasticity in matching needs for protein translation to supplies. Intracellular AA concentrations increased linearly in response to treatment for most AA, whereas 9 AA exhibited quadratic responses. Amino acid uptake is responsive to varying extracellular supplies to maintain homeostasis. No saturation of uptake was evident for most AA, indicating that transporter capacity is likely not a limitation for most AA except possibly Arg, Val, and Pro in mammary epithelial cells.

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.

A method of assessing essential amino acid availability from microbial and ruminally undegraded protein in lactating dairy cows.

The objective of this study was to extend a stable isotope-based assessment of AA absorption from rumen-degradable protein (RDP) sources to include determination of essential AA (EAA) availability from microbial protein (MCP). To demonstrate the technique, a study using a 2 × 2 factorial arrangement of treatments applied in a repeated 4 × 4 Latin square design was undertaken. Factors were high and low rumen-degradable protein and high and low starch. Twelve lactating cows were blocked into 3 groups according to days in milk and randomly assigned to the 4 treatment sequences. Each period was 14 d in length with 10 d of adaption followed by 4 d of ruminal infusions of 15N-labeled ammonium sulfate. On the last day of each period, a 13C-labeled AA mixture was infused into the jugular vein over a 6-h period to assess total AA entry. Rumen, blood, urine, and milk samples were collected during the infusions. Ruminal bacteria and blood samples were assessed for AA enrichment. Total plasma AA absorption rates were derived for 6 EAA from plasma 13C AA enrichment. Absorption of 6 EAA from MCP was calculated from total AA absorption based on 15N enrichment in blood and rumen bacteria. Essential AA absorption rates from total protein, MCP, and rumen-undegradable protein were derived with standard errors of the mean of 6, 14, and 14%, respectively. An average of 45% of absorbed EAA were from MCP, which varied among 6 EAA and was interactively affected by starch and RDP in diets. Microbial AA availability measured by isotope dilution method increased with the high RDP diets and was unaffected by starch level, except for Met, which decreased with high starch. Microbial protein outflow, estimated from urinary purine derivatives, increased with RDP and was not significantly affected by starch. This was consistent with measurements from the isotope dilution method. Total AA absorption rates measured from isotope dilution were similar to estimates from CNCPS (v. 6.55), but a lower proportion of absorbed AA was derived from MCP for the former method. Compared with the isotope and CNCPS estimates, the Fleming model underestimated microbial EAA and total EAA availability. An average of 58% of the absorbed EAA was converted into milk, which varied among individual AA and was interactively affected by starch and RDP in diets. The isotope dilution approach is advantageous because it provides estimates of EAA availability for individual EAA from rumen-undegradable protein and MCP directly with fewer errors of measurement than can be achieved with intestinal disappearance methods.

An in vitro method for assessment of amino acid bidirectional transport and intracellular metabolic fluxes in mammary epithelial cells.

Understanding uptake of AA by mammary tissue as supply varies is critical for predicting milk component production. Our objective was to develop an in vitro method to quantify cellular uptake, efflux, and intracellular metabolism of individual AA that could be implemented for evaluating these factors when AA supply and profile are varied. Bovine primary mammary epithelial cells were grown to confluency and exposed to medium with an AA profile and concentration similar to lactating dairy cow plasma for 24 h. Cells were then preloaded in medium enriched with 15N-labeled AA for 24 h followed by removal of the 15N-labeled medium and incubation with medium enriched with 13C-labeled AA for 0, 15, 60, 300, 900, 1,800, and 3,600 s. Extracellular free AA and intracellular free and protein-bound AA were analyzed for concentrations and isotopic enrichment by gas chromatography-mass spectrometry. A dynamic, 12-pool model was constructed representing extracellular and intracellular free and protein-bound pools of an AA, and their respective 15N and 13C isotopes. Markov chain Monte Carlo simulation (n = 5,000) was conducted to evaluate prediction errors by deriving standard errors and posterior distributions for rate constants, fluxes, and pools. Cellular Ala influx and efflux were higher than Leu, reflecting Ala role in driving system L transport and the high capacity of sodium-dependent transport. The Ala and Leu turnover rates were 181 and 95, 580 and 857, and 74 and 157% per hour for extracellular, intracellular, and fast protein-bound pools, respectively. The intracellular and extracellular Ala to Leu ratios were quite different, meaning the blood AA profile is not the AA profile provided for protein translation. The high level of exchange and rapid turnover of pools provide a mechanism for matching the AA supplies to the precision necessary for translation. This also understates the importance of using experimental medium similar to what is observed in vivo given that some AA depend on other AA for influx (exchange driven). The average root mean squared prediction error across the isotope enrichments, pools, and concentrations was 9.7 and 14.1% for Ala and Leu, respectively, and collinearity among parameters was low, indicating adequate fit and identifiability. The described model provides insight on individual AA transport kinetics and a method for future evaluation of AA transport and intracellular metabolism when subjected to varying AA supplies.

Effects of jugular infused methionine, lysine, and histidine as a group or leucine and isoleucine as a group on production and metabolism in lactating dairy cows.

Essential AA (EAA), particularly leucine, isoleucine, methionine, and histidine, possess signaling properties for promoting cellular anabolic metabolism, whereas methionine, lysine, and histidine are considered also to be substrate limiting AA. The objective of this study was to evaluate production responses to supplementation of 2 AA groups in a 2 × 2 factorial design. Eight cows (99 ± 18 days in milk) were assigned to 4 jugular infusion treatments consisting of saline (CON), methionine plus lysine plus histidine (MKH), isoleucine plus leucine (IL), or MKH plus IL, in a replicated 4 × 4 Latin square design. Periods were 18 d in length, comprising 8 d of rest followed by 10 d of jugular infusion. Daily infusion amounts were 21 g of methionine, 38 g of lysine, 20 g of histidine, 50 g of leucine, and 22 g of isoleucine. Cows were ad libitum fed a common diet consisting of 15.2% crude protein and 1.61 Mcal/kg NEL on a dry matter basis that was predicted to meet rumen degradable protein requirements but was 15% deficient in metabolizable protein. Milk and energy-corrected milk yields increased by 2.3 kg/d and 1.9 kg/d, respectively, with infused IL, and no change was observed for MKH. Milk protein concentration increased by 0.13 percentage units for MKH, whereas milk protein yield increased for both MKH and IL by 84 g/d and 64 g/d, respectively. The milk protein yield increase for MKH+IL was 145 g/d versus CON. Gross feed efficiency tended to increase with IL infusion, and N efficiency tended to increase with MKH infusion. Aggregate arterial EAA concentrations less Met, Lys, and His declined by 7.2% in response to MKH infusion. Arterial EAA less Ile and Leu also declined by 6.2% in response to IL infusion. Net total AA (TAA) and EAA uptake by the udder tended to increase in response to MKH infusion, whereas mammary blood flow increased in response to IL infusion, but TAA and EAA net uptakes were unaffected. Apparent udder affinity increased for TAA and EAA less Met, Lys, and His in response to MKH infusion, whereas affinity for EAA less Ile and Leu increased for IL infusion. Venous Met and Leu concentrations increased by 192% and 35% from the MKH and IL infusions, respectively, compared with CON, which indicates that intracellular concentration of these EAA changed substantially. Increases in milk protein yield were observed from 2 groups of amino acids independently and additively, which contradicts the single limiting amino acid theory that a single EAA will limit milk protein yield.

Assessing availability of amino acids from various feedstuffs in dairy cattle using a stable isotope-based approach.

Nitrogen efficiency in dairy cows can be improved by more precisely supplying essential amino acids (EAA) relative to animal needs, which requires accurate estimates of the availability of individual EAA from feedstuffs. The objective of this study was to determine EAA availability for 7 feed ingredients. Seven heifers (258 ± 28 kg BW) were randomly chosen and assigned to 8 treatment sequences in a 7 × 8 incomplete Latin square design. Treatments were a basal diet (BD), and 10% (on a dry matter basis) of BD replaced by corn silage (CS), grass hay (GH), alfalfa hay (AH), dried distillers grain (DDGS), soybean hulls (SH), wet brewers grain (BG), or corn grain (CG). Total plasma AA entry rates were estimated for each EAA within each diet by fitting a 4-pool dynamic model to observed plasma, 13C AA enrichment resulting from a 2-h constant infusion of a 13C algal AA mixture. Individual EAA availability from each test ingredient was determined by regression of entry rates for that AA on crude protein intake for each ingredient. The derived plasma total EAA entry rates for corn silage, grass hay, alfalfa hay, dried distillers grain, soyhulls, brewers grain, and corn grain were 30.6 ± 3.4, 27.4 ± 3.2, 31.3 ± 3.4, 37.2 ± 3.2, 26.4 ± 3.2, 37.8 ± 3.2, and 33.5 ± 3.2% (±standard error) of EAA from each ingredient, respectively. Using the previous estimate of 8.27% EAA utilization by splanchnic tissues during first pass, total rumen-undegradable protein EAA absorbed from the gut lumen was 33.4, 29.9, 34.1, 40.6, 28.8, 41.2, and 36.5% of the EAA in each ingredient respectively.

Effects of varying extracellular amino acid profile on intracellular free amino acid concentrations and cell signaling in primary mammary epithelial cells.

Extracellular amino acid profiles affect intracellular AA concentrations and profile as well as signaling proteins that regulate protein translation rates. The objective of this study was to assess whether various extracellular AA profiles and varied ratios of Lys to Met would increase the phosphorylation of signaling proteins related to protein metabolism. Six AA profiles, reflecting Dulbecco's modified Eagle's medium (DMEM), blood meal (BM), corn gluten meal (CGM), casein (CAS), plasma of lactating cows (PLA), and a negative control (NEG) represented the first factor (F1), and the ratio of Lys to Met (unaltered or set to 3:1) was the second factor (F2). Treatments were arranged in a 6 × 2 factorial manner, resulting in 12 treatments that were replicated 4 times. The total AA masses for all treatments were set to 659 mg/L (63% of DMEM) except NEG (0 mg/L). Confluent mammary epithelial cells were exposed to treatment media for 80 min (SD = 7.4). Intracellular concentrations of 17 AA were changed according to F1. The Met and Leu percent of total intracellular AA mass, as an example, varied from 0.58 (PLA) to 6.94 (NEG, +F2) for Met and 0.05 (NEG, -F2) to 4.63 (CGM, +F2) for Leu. Overall, balancing for Lys and Met at a 3:1 ratio increased intracellular concentrations of Lys and Met by 54 and 71%, respectively. Within the mechanistic target of rapamycin (mTOR) pathway, phosphorylation of mTOR (Ser2448), ribosomal protein S6 (Ser235/236), and eukaryotic initiation factor 4E binding protein 1 (Thr37/46) (4EBP1) were increased by all 5 AA profiles compared with the NEG control. We found no differences in phosphorylation state among the 5 AA profiles, indicating lack of sensitivity to various AA profiles. This lack of sensitivity between AA profiles might also be due to assay imprecision or other experimental limitations. Only phosphorylation of 4EBP1 was increased for F2. Phosphorylation of eukaryotic initiation factor 2 α subunit (Ser51) was unaffected by either F1 or F2 factors. Regression analyses indicated that intracellular concentrations of Met, Thr, Ile, and Leu predicted phosphorylation of mTOR-related proteins with adequate precision and accuracy, suggesting that multiple EAA dictate regulation, regardless of AA ratios. Changes in extracellular AA profiles translated to modified intracellular AA profiles, and no single profile uniquely stimulated phosphorylation of the mTOR pathway-related proteins.

An in vivo stable isotope-based approach for assessment of absorbed amino acids from individual feed ingredients within complete diets.

Accurate assessment of the nutritional content of feed ingredients is required for precise diet formulation. Characterizing ingredients in terms of absorption and digestibility of individual AA is challenging, and this information often relies on indirect methods. The purpose of this research was to evaluate an in vivo stable isotope-based method of determining plasma entry rates of individual AA from feather meal (FM), blood meal (BM), and a rumen-protected AA (RPMet). Abomasal infusions of unprotected Ile, Leu, Met, and sodium caseinate were used as control treatments to assess technique reliability and accuracy. Isotopic enrichment of plasma AA in response to a 2-h constant jugular infusion of a mixture of 13C labeled AA was measured and modeled using a dynamic 4-pool model, which was fitted to each AA by infusion to derive diet entry rates. The resulting entry rate matrix was used to derive plasma entry rates of individual AA from each ingredient by regression. The mean of plasma AA entry for abomasally infused Ile, Leu, and Met was 93.4 ± 7.35% of that infused, indicating that 6.6% was used by splanchnic tissues during first pass. The mean of the plasma essential AA entry for abomasally infused casein was 86.7 ± 4.81% of that present in the source protein, which represents a mean of 8.7% first-pass use assuming 95% digestibility. Individual AA appearances ranged from 86 to 93% of the source content except Ile, which was 73%. These fractional appearance percentages were similar to those previously reported when using a dietary regression approach. The mean plasma essential AA entry rate for FM was 52.7% of the AA in the source ingredient, with a range across AA of 48 to 58%. The mean plasma essential AA entry rate for BM was 47.5%, with a range of 30 to 61%. However, estimated Met availability from the RPMet was lower (9.9%) than expected (42%). This may be due to the relatively larger errors of measurement for Met entry rates and a small change in RPMet inclusion. Assuming that rumen-undegraded protein absorption is reflective of aggregated essential AA entry rates after correction for first-pass use, 52.6 and 61.2% of dietary FM and BM CP was absorbed from the intestine, respectively, which yielded an estimated intestinal digestibility of 70 and 66%, respectively. This method appears to provide an accurate and precise in vivo assessment of individual AA plasma entry rates that can be used to better characterize individual feed ingredients in ruminants. Such information will result in more robust economic assessments of feeds and increased precision of diet formulation.

Consonant differentiation mediates the discrepancy between non-verbal and verbal abilities in children with ASD.

BACKGROUND: Many children with autism spectrum disorder (ASD) demonstrate verbal communication disorders reflected in lower verbal than non-verbal abilities. The present study examined the extent to which this discrepancy is associated with atypical speech sound differentiation. METHODS: Differences in the amplitude of auditory event-related potentials elicited by contrasting consonant-vowel syllables during a passive listening paradigm were used to assess speech sound differentiation in 24 children with ASD and 18 chronological age-matched children with typical development (TD), M age 6.90 years (SD = 1.39). RESULTS: Results revealed that compared with TD peers, children with ASD showed reduced consonant differentiation in the 84- to 308-ms period. Brain responses indexing consonant differentiation were negatively related to the degree of discrepancy in non-verbal and verbal abilities and mediated the relationship between diagnostic group membership and the greater discrepancy. CONCLUSIONS: We discuss the theoretical and clinical implications of the brain's response to speech sound contrasts possibly explaining the greater non-verbal versus language ability in children with ASD compared with that in typically developing children.

Perspectives of health care providers on the provision of intermittent preventive treatment in pregnancy in health facilities in Malawi.

BACKGROUND: Nearly 20 years after the adoption by the government of Malawi of the provision of intermittent preventive treatment in pregnancy (IPTp) for malaria, only 55% of pregnant women received at least two doses of sulfadoxine-pyrimethamine (SP) in 2010. Although several reasons for the low coverage have been suggested, few studies have examined the views of health care providers. This study examined the experiences of the nurses and midwives in providing antenatal care (ANC) services. METHODS: This study was conducted in health facilities in Malawi that provide routine ANC services. Providers of ANC in Malawi were selected from in eight health care facilities of Malawi. Selected providers were interviewed using a semi-structured interview guide designed to address a series of themes related to their working conditions and their delivery of IPTp. RESULTS: Nurses displayed detailed knowledge of ANC services and the rationale behind them. Nurses understood that they should provide two doses of IPTp during a pregnancy, but they did not agree on the timing of the doses. Nurses gave SP as directly observed therapy (DOT) at the clinic. Nurses did not give SP pills to women to take home with them because they did not trust that women would take the pills. Women who resisted taking SP explained they do not take drugs if they had not eaten, or they feared side effects, or they were not sick. Reasons for not giving the first or second dose of SP included a delay in the first ANC visit, testing positive for HIV, and presenting with malaria. None of the nurses were able to show any specific written guidelines on when to give SP. The challenges faced by the nurses include being overworked and persuading women to take SP under observation. CONCLUSION: The findings show that the nurses had gained the knowledge and technical skills to provide appropriate ANC services. With regard to IPTp, nurses need guidelines that would be available at the health facility about how and when to give SP. The adoption of the WHO guidelines and their diffusion to health care facilities could help increase the coverage of IPTp2 (at least two doses of sulfadoxine-pyrimethamine) in Malawi.

A statistical filtering procedure to improve the accuracy of estimating population parameters in feed composition databases.

Accurate estimates of mean nutrient composition of feeds, nutrient variance (i.e., standard deviation), and covariance (i.e., correlation) are needed to develop a more quantitative approach of formulating diets to reduce risk and optimize safety factors. Commercial feed-testing laboratories have large databases of composition values for many feeds, but because of potentially misidentified feeds or poorly defined feed names, these databases are possibly contaminated by incorrect results and could generate inaccurate statistics. The objectives of this research were to (1) design a procedure (also known as a mathematical filter) that generates accurate estimates of the first 2 moments [i.e., the mean and (co)variance] of the nutrient distributions for the largest subpopulation within a feed in the presence of outliers and multiple subpopulations, and (2) use the procedure to generate feed composition tables with accurate means, variances, and correlations. Feed composition data (>1,300,000 samples) were collected from 2 major US commercial laboratories. A combination of a univariate step and 2 multivariate steps (principal components analysis and cluster analysis) were used to filter the data. On average, 13.5% of the total samples of a particular feed population were removed, of which the multivariate steps removed the majority (66% of removed samples). For some feeds, inaccurate identification (e.g., corn gluten feed samples included in the corn gluten meal population) was a primary reason for outliers, whereas for other feeds, subpopulations of a broader population were identified (e.g., immature alfalfa silage within a broad population of alfalfa silage). Application of the procedure did not usually affect the mean concentration of nutrients but greatly reduced the standard deviation and often changed the correlation estimates among nutrients. More accurate estimates of the variation of feeds and how they tend to vary will improve the economic evaluation of feeds and risk assessment of diets, and provide the ability to implement stochastic programming.

Effects of short-term variation in forage quality and forage to concentrate ratio on lactating dairy cows.

Within-farm variation in forage composition can be substantial and potentially costly, and it presents challenges for sampling the forage accurately. We hypothesized that day-to-day variation in forage neutral detergent fiber (FNDF) concentrations and diet variation caused by sampling error would have negative effects on production measures in lactating dairy cows. Twenty-four Holstein cows (73 d in milk) were used in 8 replicated 3 × 3 Latin squares with 21-d periods. Treatments were (1) control (CON), (2) variable (VAR), and (3) overreacting (ORR). On average, over the 21-d period, all 3 treatments were the same [24.7% FNDF and 48.2% forage dry matter (DM) composed of 67% alfalfa silage and 33% grass silage]. The CON treatment was essentially consistent day-to-day in total forage and FNDF concentrations and proportion of alfalfa and grass silages. The VAR treatment changed daily (in a random pattern) in proportion of alfalfa and grass silages fed, which resulted in day-to-day changes in FNDF (range was 21.5 to 28%). The ORR treatment varied in a 5-d cyclic pattern in total forage and FNDF concentrations (26, 24, 28, and 21.5% FNDF). Over the 21 d, ORR (25.1 kg/d) had higher DM intake compared with CON (24.5 kg/d) and VAR (24.3 kg/d). Milk production (42.8 kg/d), milk fat (3.5%), and milk protein (2.8%) were not affected by treatment; however, a treatment × day interaction was observed for milk production. Lower daily milk yields for VAR and ORR compared with CON were rare; they only followed sustained 4- and 5-d periods of feeding higher FNDF diets compared with CON. In contrast, increased daily milk yields for VAR and ORR versus CON were more frequent and followed sustained diet changes of only 2 or 3d. Lipolytic and lipogenic-related enzyme mRNA abundances in subcutaneous adipose tissue were not affected by treatment. Treatment × day interactions were observed for milk fatty acid markers of cellulolytic bacteria (iso-14:0, iso-15:0, iso-16:0) and lipolysis (18:0) and generally followed the expected response to changes in daily rations. Overall, extreme daily fluctuations in FNDF had no cumulative negative effect on production measures over a 21-d period, and daily responses to transient increases in FNDF were less than expected.

Measuring coverage in MNCH: indicators for global tracking of newborn care.

Neonatal mortality accounts for 43% of under-five mortality. Consequently, improving newborn survival is a global priority. However, although there is increasing consensus on the packages and specific interventions that need to be scaled up to reduce neonatal mortality, there is a lack of clarity on the indicators needed to measure progress. In 2008, in an effort to improve newborn survival, the Newborn Indicators Technical Working Group (TWG) was convened by the Saving Newborn Lives program at Save the Children to provide a forum to develop the indicators and standard measurement tools that are needed to measure coverage of key newborn interventions. The TWG, which included evaluation and measurement experts, researchers, individuals from United Nations agencies and non-governmental organizations, and donors, prioritized improved consistency of measurement of postnatal care for women and newborns and of immediate care behaviors and practices for newborns. In addition, the TWG promoted increased data availability through inclusion of additional questions in nationally representative surveys, such as the United States Agency for International Development-supported Demographic and Health Surveys and the United Nations Children's Fund-supported Multiple Indicator Cluster Surveys. Several studies have been undertaken that have informed revisions of indicators and survey tools, and global postnatal care coverage indicators have been finalized. Consensus has been achieved on three additional indicators for care of the newborn after birth (drying, delayed bathing, and cutting the cord with a clean instrument), and on testing two further indicators (immediate skin-to-skin care and applications to the umbilical cord). Finally, important measurement gaps have been identified regarding coverage data for evidence-based interventions, such as Kangaroo Mother Care and care seeking for newborn infection.

Estimates of female genital mutilation/cutting in 27 African countries and Yemen.

The practice of female genital mutilation/cutting (FGM/C) has been documented in many countries in Africa and in several countries in Asia and the Middle East, yet producing reliable data concerning its prevalence and the numbers of girls and women affected has proved a major challenge. This study provides estimates of the total number of women aged 15 years and older who have undergone FGM/C in 27 African countries and Yemen. Drawing on national population-based survey data regarding FGM/C prevalence and census data regarding the number of women in each country, we find that almost 87 million girls and women aged 15 and older have been cut in these 28 countries. Producing reliable figures for the number of women affected by FGM/C in these countries allows researchers and program directors to better comprehend the impact of the practice and to mobilize resources for advocacy against it.

Comparing sequential associations within a single dyad.

We present a new application of sampled permutation testing to examine whether two sequential associations are different within a single dyad (e.g., a teacher and a student). A Monte Carlo simulation with the same (i.e., 100 vs. 100) or a different (100 vs. 400) number of event pairs was used to simulate designs that use time-based (typically producing equal-length comparisons) and event-based (typically producing different-length comparisons) data, respectively. For these pairs of simulated data streams, we compared the Type I error rates and the kappa for agreement on significance decisions, using the sampled permutation tests and the more traditional asymptotic log linear analysis. The results provide the first evidence relevant to evaluating the accuracy of log linear analysis and sampled permutation testing for the purpose of comparing sequential associations within a single dyad.

Intentional communication elicits language-facilitating maternal responses in dyads with children who have developmental disabilities.

An experimental test of whether intentional communication elicits maternal responses purported to facilitate language development is presented. Fifty-eight prelinguistic children with disabilities and their mothers participated. Children were randomly assigned to two staff-implemented treatments that were designed to experimentally increase intentional communication ability. We predicted that treatment effects on maternal responses would vary by maternal education level and maternal responsive interaction style at the pretreatment period. We predicted any treatment effect on maternal responses would be mediated by posttreatment intentional communication. Results indicated that treatment effects on maternal responses varied by pretreatment maternal education level. A pattern of results also supports the conclusion that posttreatment intentional communication to mother was, at least in part, responsible for the effect on posttreatment maternal responses.

Relative treatment effects of two prelinguistic communication interventions on language development in toddlers with developmental delays vary by maternal characteristics.

This paper tests whether two prelinguistic communication interventions have a differential effect on productive and receptive language development 6 and 12 months after the end of treatment. We predicted that treatment effects on language development would vary as a function of pretreatment maternal responsivity or amount of mothers' formal education. Fifty-eight prelinguistic children with developmental delays and their mothers participated in the study. Children were randomly assigned to one of two staff-implemented treatments that were designed to increase intentional communication ability. Results confirmed the prediction that treatment effects on children's receptive and expressive language 6 and 12 months after the end of interventions vary as a function of pretreatment maternal responsivity and education level.

Predicting spoken language level in children with autism spectrum disorders.

Thirty-five children who received an autism spectrum diagnosis at the age of 2 years (24 with autism, 11 with PDD-NOS) were re-evaluated 2 years later to examine factors related to the development of spoken language. Child variables (play level, motor imitation ability and joint attention) and environmental variables (socioeconomic status and hours of speech/language therapy between ages 2 and 3) were used to predict an aggregate measure of language outcome at age 4. After controlling for age 2 language skills, the only significant predictors were motor imitation and number of hours of speech/language therapy. Implications of these results for understanding the early developmental course of autism spectrum disorders and the effects of intervention are discussed.

Histological comparison of corneal ablation with Er:YAG laser, Nd:YAG optical parametric oscillator, and excimer laser.

PURPOSE: To use histological techniques to assess and compare the ablation depth, local damage, and surface quality of corneal ablations by a Q-switched Er:YAG laser, an optical parametric oscillator laser at 2.94 microm, a long pulse Er:YAG laser, and a 193-nm excimer laser. METHODS: Human cadaver eyes and in vivo cat eyes were treated with a 6.0-mm diameter, 30-microm-deep phototherapeutic keratectomy ablation and a 6.0-mm diameter, -5.00-D photorefractive keratectomy ablation. Human cadaver eyes were also treated with a 5.0-mm diameter, -5.00-D laser in situ keratomileusis (LASIK) ablation. Fluences and pulse widths used were 200 mJ/cm2 and 70 ns for the Q-switched Er:YAG, 150 mJ/cm2 and 7 ns for the optical parametric oscillator laser (OPO), 500 mJ/cm2 and 50 microseconds for the long pulse Er:YAG, and 160 mj/cm2 and 20 ns for the excimer laser. In the ablation rate study, 12 porcine eyes were ablated by the OPO laser with a range of layers and at different fluences ranging from 60 to 150 mJ/cm2, all using a 1.5-mm spot on the eye. The ablation depth of these acute ablations was evaluated by light microscopy examination. RESULTS: In the acute damage study, light microscopy showed a thin surface layer in all samples with minimal thermal damage except on the long pulse Er:YAG corneas. Transmission electron microscopy revealed less than 0.3-microm surface damage for all specimens of both the optical parametric oscillator and the excimer laser samples with no evidence of collagen shrinkage. Transmission electron microscopy showed damage layers of 0.5 to 3 microm for Q-switched Er:YAG and 3 to 10 microm for long pulse Er:YAG. Scanning electron microscopy showed smooth surfaces in all eyes, although the excimer was the roughest. In the porcine eye study, ablations were produced in both PTK and PRK modes with the ablation rate per layer increasing with the fluence. At 120 mJ/cm2, the average ablation rate was 1.9 microm per layer. CONCLUSIONS: The histology from the short pulse mid-infrared optical parametric oscillator laser at 2.94 microm was comparable to the 193-nm excimer with a smooth, damage-free, ablation zone when performing PRK and LASIK.