During the 21-day lactation period, the sow��s milk was the only source of nutrition for the piglets. All 40 piglets had free access to water and were weighed every 2 days from the third day after farrowing. Necropsy and Sample Collection All piglets were slaughtered for necropsy at 22 days of age. Blood samples selleck inhibitor were obtained by anterior vena cava puncture and captured in 6-mL anticoagulant-free Vacutainer tubes (Becton Dickinson and Company, Franklin Lakes, NJ, USA) and then centrifuged at 3000��g for 10 min to obtain serum. The serum samples were stored at ?80��C. The contents of the duodenum, jejunum, and ileum were collected separately for microbial analysis. The mid sections of the duodenum, jejunum, and ileum tissues were collected, flushed with a 0.

9% physiological saline, immersed in 4% paraformaldehyde, and stored at 4��C for future microscopic assessment of mucosal morphology. Microbial Analysis In vitro survival of Salmonella, E. coli, Bifidobacterium, Lactobacillus, total aerobes, and total anaerobes was assessed using methods previously described [29]. Briefly, 1.0 g of digesta was taken from each sample and serially diluted with 9 mL of sterile physiological saline, resulting in dilutions ranging from 10?1 to 10?6 for enumeration. Then, 0.1 mL of two or three consecutive dilutions of each sample were plated on appropriate medium. The morphology of the flora and individual cells, Gram staining, and oxygen consumption were used to identify the bacteria. Two replicates and one blank control were produced from each sample plate.

The number of microbes was expressed as log10 colony-forming units (CFU)/g. Histology Intestinal samples were fixed in 4% paraformaldehyde, embedded in paraffin, and sliced to 4-��m thick sections, which were stained with hematoxylin and eosin. Villus height, crypt depth, and the villus height to crypt depth ratio were measured at 40�� magnification using Image-Pro Plus software (Image-Pro Plus 6.0; Media Cybernetics, Silver Spring, MD, USA). Ten, well-oriented, intact villi were selected and measured in triplicate for each pig. Statistical Analysis All experimental data were analyzed using SPSS software (ver. 19.0; SPSS Inc., Chicago, IL, USA). A probability (p) value <0.05 was considered statistically significant.

Results Generation of Cloned Transgenic Pigs and Transgene Transmission The transgene vector, pBC2-HLY-NEOR, contained a 5,238-bp genomic DNA fragment of hLZ, a bovine ��-casein peptide DNA sequence signal, and the neomycin resistance gene (Neor) as a selection marker (Figure 1A), which was previously used successfully to generate transgenic cattle. After somatic cell nuclear transfer, 380 embryos were transferred into two recipient gilts (one with 200 and the other with 180). Thirteen male pigs were born. By PCR analysis, hLZ was AV-951 integrated into the genome of seven transgenic pigs (Figure 1B).