| Imbalances created by excesses or deficiencies of dietary amino acids (AA) reduce the efficiency of dietary protein utilization, limiting milk protein synthesis and litter growth, and increasing nitrogen (N) losses to the environment. The overall goal of the dissertation research described herein was to evaluate the effect of dietary AA availability on AA utilization by the porcine mammary gland during lactation. The main hypothesis was that the efficiency of AA transport by the porcine mammary gland increases in response to dietary crude protein (CP) reduction with subsequent crystalline (C) AA inclusion, and that this increase is mediated via an increase in mRNA abundance of mammary expressed genes encoding for AA transporter proteins. To test this hypothesis, three objectives were addressed: (1) to select suitable reference genes for reverse transcriptase quantitative PCR (RT-qPCR) analysis of AA transporter and milk proteins in porcine mammary gland; (2) to measure mRNA abundance of genes encoding for mammary lysine (Lys) transporters at different stages of mammary physiological activity, and determine the relationship between their expression and that of genes encoding for milk proteins; and (3) to test whether dietary AA availability modulates the efficiency of Lys utilization by the mammary gland and the expression of genes encoding for mammary Lys transporters and milk proteins.;For objective 1, mammary tissue was collected by biopsy from 4 sows on d 110 of gestation, d 5 and d 17 of lactation, and d 5 after weaning. Expression of potential reference genes was measured by RT-qPCR, and their invariance was assessed using a novel analytical model that corrected gene expression for mammary RNA and DNA concentrations. For objective 2, in addition to the mammary tissue collected from sows used in objective 1, samples were taken from 3 additional sows at d 2 and 5 of lactation. Expression of AA transporter genes SLC7A9, SLC7A7, SLC7A6, SLC6A14, SLC7A1 and SLC7A2, and milk protein genes CSN2 and LALBA was measured by RT-qPCR and the relationship between AA transporter and milk protein gene expression was determined. For objective 3, 18 sows were allocated to one of three diets containing 9.5 %, 13.5% and 17.5% CP levels. Mammary tissue and arterial and mammary venous samples were collected on d 7 and 17 of lactation. Expression of SLC7A9, SLC7A6, SLC6A14, SLC7A1, SLC7A2, CSN2 and LALBA was measured by RT-qPCR and AA transport efficiency was measured by AA arterio-venous (AV) difference across the mammary gland relative to the arterial AA concentration. Results of the described studies showed that (1) changes in expression of selected reference genes during lactation are related to variation in RNA and DNA mammary concentration, and therefore such genes can be used as normalization factors for RT-qPCR analysis; (2) mRNA abundance of SLC7A6, SLC6A14, SLC7A1 increases with milk demand and iwas positively related to mRNA abundance of CSN2 and LALBA; and (3) dietary CP reduction with CAA inclusion improves the efficiency of dietary Lys transport across mammary gland and dietary AA utilization for litter growth, but does not change the expression of mammary AA transporter gene during lactation. These results are a novel contribution to our understanding of AA nutrition of the lactating sow and to the critically needed tools for development of mechanistic models of nutrient utilization for milk production. |
