Dairy herds are major contributors to N pollution because 70% of the N intake is lost to the environment and 30% or less is retained in milk protein. Plasma amino acids (AA) that are not used for protein synthesis in mammary glands (MG) are catabolized in post splanchnic tissues (liver plus gastrointestinal tract, pancreas, spleen, portal system, and associated adipose tissue) and two thirds of the net supply of essential AA (EAA) are cleared in splanchnic tissues. Thus, increasing AA capture in MG would be expected to reduce AA catabolism and thereby increase efficiency of AA utilization. The objectives of the work presented in this dissertation were to test the effect of energy and N intake on cell regulatory mechanisms, nutrient kinetics, milk, milk protein yield, and N efficiency in dairy cows.

The aim of the first study was to test whether metabolizable protein (MP) and dietary energy exerted independent effects on milk protein synthesis and postabsorptive N efficiency. Forty mid-lactation cows (32 multiparous Holstein and 8 primiparous Holstein x Jersey cross-breds) were used in a complete randomized design with a 2 x 2 factorial arrangement of diets. Cows were assigned to one of four dietary treatments: high-energy, high-protein (HE/HP); high-energy, low-protein (HE/LP); low-energy, high-protein (LE/HP); and low-energy, low-protein (LE/LP). Energy concentrations were 1.55 (HE/HP and HE/LP) or 1.44 (LE/HP and LE/LP) Mcal NEL/kg DM according to the NRC model. Changes in predicted MP were achieved by feeding diets with 6.6 (HE/HP and LE/HP) or 4.6% (HE/LP and LE/LP) ruminally undegradable protein (DM basis). Ruminally degradable protein was held constant at 10.1% of DM. All cows were fed HE/HP diet from day 1 to 21 followed by the respective treatments from day 22 to 43 (n=10). Milk protein yield was reduced as dietary energy was reduced. There were no interactions between dietary energy and protein for either milk or protein yield. Milk urea N was significantly affected by energy and protein with an interaction (HE/HP=17.2, HE/LP=12.2, LE/HP=21.0, LE/LP=12.2 mg/dl). Nitrogen efficiency was affected by energy and protein supplies with no interaction and ranged from a low of 31% (LE/HP) to a high of 43% (HE/LP). Although energy and protein independently affected milk and protein yield the tissue and cellular mechanisms that regulate milk production were not studied.

The second experiment studied cellular mechanisms in MG that contributed to the regulation of protein synthesis in the presence of energy or protein supply. We hypothesized that metabolism of AA in the MG is controlled by systemic and local tissue adaptations and when combined with altered mammary cell function controlled milk protein yield. Six primiparous mid-lactation Holstein cows with rumen cannulas were randomly assigned to abomasal infusions of casein and starch using a 2 x 2 factorial arrangement. The design was a replicated incomplete 4 x 4 Latin-square. All animals received the same basal diet (17.6% CP and 1.58 Mcal NEL/kg DM) throughout the study. Cows were restricted to 70% of ad libitum intake and infused abomasally for 36 h with water, starch (2 kg/d), casein (0.86 kg/d), or the combination (2 kg/d starch + 0.86 kg/d casein) using peristaltic pumps. Milk weights, milk samples, and arterial and venous blood samples were collected during the last 8 h of infusions. Mammary biopsy samples were collected and tissue protein prepared to evaluate cell signaling. Animals infused with casein had increased arterial concentrations of NEAA and EAA, as well as net uptake and clearance; however, milk protein yield did not increase. Animals infused with starch however, exhibited reduced arterial concentrations of NEAA and EAA but increased clearance and net uptake of most AA. Additionally, infusions of starch increased circulating concentration of insulin, IGF-I, and glucose as well as the rate of mammary plasma flow. Abomasal infusions of starch activated mammary activity of ribosomal protein S6 irrespective of other treatments. However, mammary tissue mTOR increased activity in response to casein only when starch was present during the infusions. These results suggest that cell signaling activation responded to different nutritional stimuli. Milk and protein yield increased in animals infused with starch. Therefore, MG positively responded to energy supply and engaged local and intracellular regulatory mechanisms to achieve that response. Understanding these adaptations could be beneficial in the development of mathematical representations for nutrients utilization in lactating animals. These two studies supported our hypotheses that regulatory mechanism are activated during limiting supply of AA to sustain protein synthesis in MG. The accuracy of mathematical models for lactating animals would increase if effects of energy on AA metabolism and cell signaling related to protein synthesis were included in the representation of milk protein synthesis.