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Studies in other species indicate high protein diets increase urinary calcium (Ca) excretion and may lead to negative calcium balance and reduced bone density. As overfeeding of protein is commonplace in the horse industry, this study was undertaken to determine the effects of excess dietary protein on growth, physiologic response, mineral balance, bone density, and bone geometry in immature horses. Sixteen 10-month-old American Quarter Horses were blocked by age and sex into two dietary treatments. The control diet was formulated to provide the NRC (1989) recommended concentration of crude protein, while the high protein diet provided 130% of NRC (1989) recommendations. All other nutrients were formulated at or slightly above NRC (1989) recommendations. Blood samples, feces, and urine were collected during the 116-day study to determine any diet effect on pH and mineral balance. Radiographs were made of the left third metacarpal (MCIII) to determine bone density via radiographic bone aluminum equivalence (RBAE), and bone geometry was determined metrically from the radiographs. Urine pH decreased over time (p < 0.001), but there were no diet effects on blood pH or urine pH. Conversely, when normalized to day 0 values, fecal pH was reduced by feeding the high protein treatment (p < 0.02). Density of dorsal and palmar cortices increased over time (p < 0.001), but no differences were observed between diets. But, normalized total medial-lateral (ML) width of the MCIII was higher in the control diet (p < 0.05). Fecal Ca loss was greater in horses fed the high protein diet (p < 0.005), while Ca absorption and retention were lower for horses on the high protein treatment (p < 0.02). Phosphorus (P) balance was not different between diets, although feeding the high protein diet resulted in higher P intake overall (p < 0.001). While excess dietary protein may decrease fecal pH, increase fecal Ca excretion, and decrease Ca absorption and retention, there was no consistent effect of the high protein diet on bone density over the course of this study. Further research is necessary to determine if feeding high-protein diets is detrimental to bone quality in the growing horse.

Dietary protein is required to support recovery and adaptation following exercise training. While prior research demonstrates that many athletes meet total daily protein needs, intake seems to be predominantly skewed toward the evening meal. An even distribution of protein doses throughout the course of a day is theorized to confer greater skeletal muscle anabolism compared to a skewed pattern of intake. Protein quality is also an important dietary consideration for athletes, with the amino acid leucine being the primary driver of the postprandial anabolic response. The present thesis investigates the protein consumption characteristics among a selected cohort of NCAA Division 1 collegiate soccer players and evaluates differences between male and female athletes. Athletes were instructed to complete 3-day food diaries, which were subsequently analyzed and compared to nutrition guidelines for soccer players. Breakfast, lunch, and dinner explained 81.4% of total daily dietary protein intake. Most athletes (77.8%) ingested optimum amounts of protein at dinner, but not at breakfast (11.1%) or lunch (47.2%). In addition, statistically significant sex-based differences in daily dietary protein intake, meal-specific protein amounts, and protein quality measures were detected. Findings indicate suboptimal dietary protein intake practices among the collegiate athletes.

Exercise and diet may preserve muscle mass and bone health, thereby preventing age-related functional decline among older community-dwelling subjects. We used data from two studies to examine effects of exercise administered through a home-based program for hip fracture patients and the effects of dietary protein, vitamin D, and calcium on physical functioning in older adults. In the Health and Independence Post-Rehabilitation (HIP) study, the effects of a home-based exercise program (vs. attention-based control) (n=232) on functional recovery following hip fracture was assessed using two standardized physical performance batteries. In a sub-analysis (n=56), we evaluated the effects of food frequency questionnaire-derived protein, calcium, and vitamin D intakes on these outcomes. In the Framingham Offspring Study (FOS), dietary protein, calcium, and vitamin D intakes were measured using two sets of 3-day diet records. Physical functioning over 12 years was measured using the Rosow-Breslau and Nagi scales. In the HIP study, the exercise intervention led to significant improvement in physical performance at 6 months for all primary endpoints [(SPPB (P<0.001), AMPAC Mobility Function (P=0.01) and Daily Activity (P=0.01). However, we found no beneficial effects of dietary protein, calcium, or vitamin D on physical functioning. In the FOS, Cox proportional hazard’s models were used to estimate risk of functional decline over 12 years, adjusting for potential confounding by age, sex, education, physical activity, cigarettes per day, height, and energy intake. Subjects consuming ≥ 1.2 gm/kg protein intake (vs <0.8 gm/kg) had a lower risk of becoming dependent the following tasks: heavy work at home; walking one-half mile; climbing stairs; stooping, kneeling, crouching; lifting >10 lbs, lifting <10 lbs. Subjects consuming ≥ 1.2 (vs <0.8) gm/kg/day of protein had a 41% lower risk of becoming dependent in ≥1 functional task over 12 years. The beneficial effect of higher protein intake was strengthened among subjects who were more active and had higher SMM. There was also evidence of a beneficial interaction of both vitamin D and calcium with physical activity and SMM. These results suggest that participation in home-based functional exercise in older post-hip fracture adults and higher intakes of dietary protein help to preserve functional capacity among community-dwelling older adults.

High levels of dietary protein may be detrimental to reproductive performance in cattle. The objective of Exp. 1 was to determine the effects of differences in dietary protein on the production and quality of bovine embryos collected from superovulated donors. Angus cows were randomly assigned to receive one of three experimental diets: a daily ration of 5.7 kg poultry litter, 2.0 kg hay, 3.1 kg corn, and 0.5 kg peanut hulls (LITTER; n = 15); a daily ration of 6.2 kg peanut hulls, 2.2 kg soybean meal, 2.0 kg hay, 0.5 kg corn, and 0.4 kg dicalcium phosphate (SBM; n = 15); or a daily ration of 6.2 kg peanut hulls, 2.0 kg hay, and 3.1 kg corn (CON; n = 19). Diets differed in the amount of total, soluble and degradable protein, but were comparable in energy. After 30 d on the diets, all cows were treated to induce superovulation (28.8 mg FSH/cow, Folltropin) and synchronize estrus. After the detection of estrus each cow was inseminated with semen from one of four Holstein bulls. Embryos were collected 7 d after estrus and evaluated for quality (according to the International Embryo Transfer Society (IETS) standards) and stage of development. Prior to treatment to induce superovulation, blood samples were collected 6 h after feeding. Samples were analyzed to assess dietary effects on plasma urea nitrogen (PUN). Mean levels of PUN were higher (P < 0.01) in cows fed the LITTER or SBM diet (16.3 mg/dL, LITTER; 21.8 mg/dL, SBM; 9.7 mg/dL, CON) than in cows fed the CON diet. Additionally, concentration of PUN was higher in cows fed SBM than in those fed LITTER (P < 0.01). An average of 9.2 transferable embryos (Grade 1, 2 and 3) was collected from each cow and there were no significant differences in the number of transferable embryos collected among groups (9.2, LITTER; 9.3, SBM; 9.1, CON). The number of degenerate embryos or unfertilized ova did not differ among dietary groups. High-protein diets elevated PUN, but did not affect the number or quality of embryos collected from superovulated donors. Cryopreservation of bovine embryos is an important aspect of a successful embryo transfer program. The objective of Exp. 2 was to evaluate the post-thaw viability of bovine embryos collected in Exp. 1 in an in vitro culture system after the embryos had been held at room temperature or refrigerated for 2 to 12 h prior to freezing. Upon embryo recovery, each embryo was randomly assigned to be placed in holding media for 2, 6 or 12 h prior to freezing. During this interval, one-half of the embryos were maintained in a refrigerated environment (5 °C), while the remaining half of the embryos were held at room temperature (20.5 to 22 °C) until freezing. Immediately prior to freezing, embryos were removed from the holding media, transferred to a well containing ethylene glycol (10%) in ovum culture media and loaded individually into a 0.25-mL plastic straw. Straws were then placed in a freezer unit (-6 °C) and seeded to induce ice crystal formation through all columns of the straw. The temperature of the freezer was then decreased 0.6 °C/min to -32 °C, and straws were loaded into canes and plunged into a liquid nitrogen tank (-196 °C). After storage, each straw was exposed to a 5-s air thaw and placed in a water bath at 35 °C for 20 s. Each embryo was then washed to remove excess ethylene glycol prior to in vitro culture. Embryos were individually cultured in Ham's F-10 media supplemented with 4% fetal bovine serum for 72 h. Embryos were evaluated at 24 h intervals throughout the culture period and assigned a stage of development and quality grade score (according to IETS standards). The percentage of embryos that developed to the expanded blastocyst stage and hatched from the zona pellucida was greater for embryos held 2 or 6 h prior to freezing (P < 0.05) than for embryos held for 12 h after collection before being frozen (62.9, 52.0 and 31.1%, respectively). The percentage of embryos that degenerated during in vitro culture was lower for embryos held 2 or 6 h prior to freezing (20.4 and 26.6%; P < 0.05) than for embryos held for 12 h before freezing (50.8%). Furthermore, embryo quality grade was more desirable for embryos held for 2 or 6 h (1.5 and 1.7; P < 0.05) than for those held for 12 h before freezing (2.1). The semen used to inseminate donors and the diet fed to donors for 4 wk prior to embryo collection did not influence the proportion of embryos that hatched or degenerated during the 72 h of in vitro culture. Additionally, holding embryos in a refrigerated environment from the time of collection until freezing did not enhance embryonic development during post-thaw culture. Thus, embryonic viability may be impaired when embryos are held longer than 6 h following embryo recovery before being frozen; however, the storage temperature during the interval from collection to freezing does not influence embryonic development post-thaw. / Master of Science

Background Maintenance of lean muscle mass and related strength is associated with lower risk for numerous chronic diseases of aging in women. Objective Our aim was to evaluate whether the association between dietary protein and lean mass differs by physical activity level, amino acid composition, and body mass index categories. Design We performed a cross-sectional analysis of a prospective cohort. Participants/setting Participants were postmenopausal women from the Womens Health Initiative with body composition measurements by dual-energy x-ray absorptiometry (n=8,298). Main outcome measures Our study measured percent lean mass, percent fat mass, and lean body mass index. Statistical analyses performed Linear regression models adjusted for scanner serial number, age, calibrated energy intake, race/ethnicity, neighborhood socioeconomic status, and recreational physical activity were used to determine the relationship between protein intake and body composition measures. Likelihood ratio tests and stratified analysis were used to investigate physical activity and body mass index as potential effect modifiers. Results Biomarker-calibrated protein intake was positively associated with percent lean mass; women in the highest protein quintile had 6.3 percentage points higher lean mass than the lowest quintile (P<0.001). This difference rose to 8.5 percentage points for physically active women in the highest protein quintile (P-interaction=0.023). Percent fat mass and lean body mass index were both inversely related to protein intake (both P<0.001). Physical activity further reduced percent fat mass (P-interaction=0.022) and lean body mass index (P-interaction=0.011). Leucine intake was associated with lean mass, as were branched chain amino acids combined (both P<0.001), but not independent of total protein. All associations were observed for normal-weight, overweight, and obese women. Conclusions Protein consumption up to 2.02 g/kg body weight daily is positively associated with lean mass in postmenopausal women. Importantly, those that also engage in physical activity have the highest lean mass across body mass index categories.

A study of the adaptive changes of some enzyme activities to the dietary protein intake was made in the liver, kidney, and pancreas of rats and serum of humans. The rats fed the 40 per cent casein diet had a higher rate of weight gain and the weights of the liver and kidney were higher than in the rats fed the 10 per cent casein diet. Three enzymes involved in the elimination of excess nitrogen from the body were found to show a similar response to increased dietary protein intake. These enzymes were: D-amino acid oxidase in the kidneys, arginase in the liver and kidneys, and glutamic-pyruvic transaminase in the liver of rats. The rate of enzyme induction was detectable the second day, being high during the first four days and reaching the maximum value on the fourth day. Thereafter, the enzymatic activity did not change much. The alkaline phosphatase activities in the liver and kidneys of the rats did not show significant change during consumption of the diets containing 10 per cent or 40 per cent casein. Samples of pancreatic trypsinogen and chymotrypsinogen of the rats were not collected during the appropriate time to note any significant changes caused by the diets . A more appropriate experimental design, including proper timing for feedings and dissections, for the study of the adaptation of the proteolytic enzymes in the pancreas of the rats is desirable and recommended. The values for the circulating enzymes of the human serum, 0-amino acid oxidase, glutamic-pyruvic transaminase, and alkaline phosphatase were not found to be affected significantly by the diets containing 12 per cent and 30 per cent protein calories.

The long-term effects of total dietary protein and individual food sources of dietary protein on cardiometabolic risk are not clearly understood. The effects of the amount consumed and the timing of dietary protein intake as well as the contribution of overall dietary patterns on various cardiometabolic outcomes are largely unknown, particularly in children. The objective of this dissertation is to estimate the effects of patterns of dietary protein intake and egg consumption on cardiometabolic risk in adolescents and adults. Prospective data from two studies were used: the National Growth and Health Study (NGHS) with 2105 pre-adolescent girls followed for 10 years and the Framingham Offspring Study (FOS) with 2054 middle-aged adults followed for 12 years. Diet was assessed in both cohorts via 3-day diet records. NGHS outcomes included %body fat, %truncal fat, waist circumference, body mass index, skeletal muscle mass (SMM), fasting glucose (FG), insulin resistance, blood pressure (BP), and lipids. FOS outcomes included FG and BP. Multivariable models including analysis of covariance, logistic regression, and Cox proportional hazards models were used. Girls consuming ≥75 g/day of total protein (vs. less) had less body fat (p<0.0001) and more SMM (p<0.0001) by late adolescence. Girls consuming more morning protein had the highest total protein intakes, perhaps contributing to the observed beneficial effects of morning protein on body composition. Consumption of ≥3.5 eggs/week in 9-17 year-old girls was associated with lower %body fat (p=0.019) and higher %SMM (p=0.026) by later adolescence. There was no evidence that higher egg intake was detrimental to any cardiometabolic outcomes. Girls who consumed more eggs (ages 9-17 years) in combination with more fiber, fruits/vegetables, or physical activity led to statistically significant 43-58% reduced risks of becoming overweight by late adolescence. Adults who consumed ≥5 eggs/week had lower FG (p=0.0004) and systolic BP (p=0.0284) over time. Higher egg intakes were associated with a 27% lower risk of IFG or T2D (95% CI:0.51-1.04) and 30% lower risk of HBP (95% CI:0.52-0.96). In summary, total protein intake, including regular egg consumption, has no adverse effects on cardiometabolic risk in adolescence or adults and may benefit body composition over time.