Abstract
Lysozyme is often used as a feed additive and acts as an antimicrobial protein that enhances immune function and defends against pathogenic bacteria in pigs. In this study, we genetically added recombinant human lysozyme (rhLZ) to sow milk by somatic cell nuclear transfer and investigated whether the presence of recombinant human lysozyme can influence intestinal microbiota and mophology in sucking pigs. We generated transgenic cloned pigs and the first-generation hybrids (F1) produced high levels of rhLZ in milk. The average concentration of rhLZ was 116.34±24.46 mg/L in the milk of F1 sows, which was 1500-fold higher than that of the native pig lysozyme. In vitro, it was demonstrated that rhLZ in milk of transgenic pigs had enzyme levels at 92,272±26,413 U/mL. In a feeding experiment, a total of 40 newborn piglets were nursed by four transgenic sows and four sibling non-transgenic sows (F1), with five piglets per gilt. The piglets were allowed to nurse for 21 days and the sow milk was the only source of nutrition for the piglets. All piglets were slaughtered on postnatal day 22. Six types of bacteria were cultured and analyzed to detect the impact of rhLZ on gut microbiota. The number of Escherichia coli in the duodenum of piglets reared by transgenic sows was significantly decreased (p<0.001) and their villus height to crypt depth ratio in the intestine were increased due to the significant decrease of crypt depth in the duodenum, jejunum, and ileum (p<0.001). Together, we successfully generated rhLZ transgenic cloned pigs and elevated lysozyme level in nuring piglets. The results of the feeding experiments demonstrated that rhLZ-enhanced milk can inhibit the growth of E. coli in the duodenum and positively influence intestinal morphology without adversely affecting weight gain or piglet growth.
Highlights
One of the greatest aspirations in modern biology is the ability to utilize the ever expanding knowledge of the genetic basis of the enormous phenotypic diversity that exists in contemporary livestock and other organisms
Two founder boars (0303 and 0309) and samples from the bred F1 pigs were subjected to Southern blot analysis (Figure 1C). Quantitative Real-time PCR (qPCR) analysis showed that the average copy number of inserted hLZ gene sequences in the genome of the F0 and F1 generations was 1.9860.21
We have sucessfully produced transgenic pigs with human lysozyme expressing in milk at 1500-fold higher than that of the native pig lysozyme and greatly elevated lysozyme level in nursing piglets
Summary
One of the greatest aspirations in modern biology is the ability to utilize the ever expanding knowledge of the genetic basis of the enormous phenotypic diversity that exists in contemporary livestock and other organisms. Owing to the development of transgenic technologies, researchers are able to produce transgenic animals, including livestock species, as specific biomedical research models for various human afflictions, including Alzheimer’s disease, cystic fibrosis, diabetes, and providing tissues for xenotransplantation [1,2,3,4]. The work reported here is an initial step in breeding pigs for diarrhea resistance by expressing human lysozyme in mammary gland tissue to benefit piglet health and improve their ability to resist bacterial infections. Both suckling and weaning piglets are more susceptible to diarrheal diseases. Lysozyme displays anti-inflammatory capabilities by decreasing the expression of pro-inflammatory cytokines and increasing the expression of anti-inflammatory cytokines [16], and immune regulatory function by directly and indirectly modulating the complement system [17]
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