, Stoughton, MA, USA). Data were collected from three independent experiments and the results are presented as means �� standard deviations. Lysozyme Activity Assays Micrococcus selleckchem Pacritinib lysodeikticus cells (China General Microbiological Culture Collection Center, Beijing, China) were revived and prepared for the gel diffusion and turbidimetric assays, which were used to evaluate rHLZ activity in the milk of transgenic pigs. A suspension of M. lysodeikticus with mixed solid culture medium containing 1.5% nutrient broth agar (Sigma-Aldrich) was used as the medium for the gel diffusion assay. Quantitative filter paper discs (6-mm diameter) were placed on the agar plates, which were loaded with milk samples and then incubated at 28��C for 24 h. The results were assessed by measuring the inhibition zones around the filter paper discs.

The turbidimetric assay, as described by Shugar [29], was used to monitor the reduction in turbidity of a suspension of M. lysodeikticus cells at an absorbance of 450 nm (A450). Briefly, 2.5 mL of M. lysodeikticus cell suspension (A450, 0.60�C0.7) as the substrate was prepared at 25��C in 66 mM potassium phosphate buffer (pH 6.24) and then placed in a 4-mL cuvette at room temperature. The reaction was initiated by adding 100 ��L of 1100 dilutions of pig milk samples (test group) or 100 ��L of ddH2O (blank group). A450 was recorded at 30-s intervals over a 5-min period. The ��A450 per minute was used as the maximum linear rate for all groups. One unit will produce a ��A450 of 0.001 nm/min at pH 6.24 at 25��C in a 2.6-mL reaction mixture.

All samples were measured in triplicate. Composition Analysis of Milk Up to 50 mL of colostrum (3�C6 h) and mature milk (14 days) were collected from six transgenic pigs and three non-transgenic pigs for analysis of gross milk composition, including fat, protein, and lactose. All testing was completed by the Beijing Research Institute for Nutritional Resources, which has established the criteria and methods used for analysis of large quantities of biochemical and nutritional components. Colostrum and milk composition was measured according to the national food safety standard of raw milk issued by the Chinese Ministry of Health (GB19301-2010). Animals and Feeding Experiment As female offspring of the founder animals attained puberty, transgenic and sibling non-transgenic gilts (F1) were bred to non-transgenic Large White boars.

Of the gilts bred, four transgenic and four non-transgenic (control) gilts that conceived and farrowed successfully were used in this study. On day 107 of pregnancy, the gilts were moved into a farrowing house and monitored for signs of parturition. At parturition, gilts Dacomitinib and their newborns were monitored during farrowing to ensure safe delivery and successful nursing afterward.