Generation of genetically modified pigs is an efficient strategy for improving various indicators of pig performance, such as feed utilisation, piglet survival rate and pork nutritional composition. In our previous study, we generated a transgenic pigs expressing four microbial enzymes, namely, bg17A, eg1314, xynB and eappA, in salivary glands specifically[1]. During the feeding process, we found that the N and P emissions of the TG pigs were substantially reduced, whereas their nutrient intake and absorption from the feed increased. However, the TG pigs carry the EGFP gene. This gene has potential animal and human health hazards [12]. In the present study, we developed TG pigs without the EGFP marker. The MF-TG pigs were found to efficiently secrete the microbial enzymes phytase, β-glucanase and xylanase as the previous TG pigs. We compared the MF-TG pigs with WT pigs fed with complete diets and noted that these genetically modified pigs fed with nutrition-deficient experimental diets considerably reduced their N, P and Ca emissions in the manure without compromising production performance. However, when compared with WT pigs fed with the same nutrition-deficient experimental diets, these genetically modified pigs had substantially improved ADG and feed utilisation efficiency but only reduced faecal P emissions. This result was not consistent with that of our previous trial in metabolic cage under restrictive feeding and movement[1]. The main difference between the previous study and the present work was that all pigs herein were measured in cages without feeding and movement restrictions. Other prior studies reported that supplementary enzymes in the feed have a positive effect on the digestibility of feed nutrients[3, 13]. Phytase has the ability to liberate P from phytate by step-wise dephosphorylation of phytate. β-glucanase and xylanase can effectively degrade glucan and xylan, respectively. Hence, β-glucanase, xylanase and phytaseare are both nutritionally and ecologically beneficial because they enhance P/N absorption while reducing P/N excretion[1].
The issue of whether the digestive enzymes secreted by genetically modified pigs would affect their intestinal microbiotas remains unclear. In recent years, metagenomics methods based on high-throughput sequencing technology have rapidly promoted the study of the composition and function of intestinal microorganism floras[14]. In the present study, all experimental pigs were selected from populations with a similar genetic background, of the same gender and raised under the same environmental, nutritional and management conditions to minimise the variability caused by genetic, gender and external factors. Nevertheless, results showed that the gut microbiotas of MF-TG and WT pigs were different, consistent with those of previous studies[15–17]. The MF-TG pigs had higher levels of probiotics, such as L. reuteri and Streptococcus, in the caecum than WT pigs. Lactobacillus reuteri strongly adheres to the intestinal mucosa; thus, this bacterium can improve the distribution of intestinal microbes, antagonise the colonisation of other harmful bacteria and prevent the development of intestinal diseases [18]. In addition, Lactobacillus reuteri can produce reuterin, a non-protein broad-spectrum antibacterial substance that can greatly inhibit the growth of Gram-positive/negative bacteria, yeast, fungi and pathogens [18]. Streptococcus is generally considered a health-promoting microorganism because of its role in regulating human health. Numerous Streptococcus species are involved in carbohydrate fermentation, starch hydrolysis and glucan production from sucrose[17]. Streptococcus gallolyticus can ferment mannitol, trehalose and inulin and produce acids from starch and glycogen. Therefore, the presence of these bacteria suggested that the MTF-TG pigs were healthier than the WT pigs because they have more probiotics to promote gut health or degrade carbohydrates in their diet. By contrast, the levels of Campylobacter and Chlamydia in WT pigs were higher than those in MF-TG pigs. Campylobacter and Chlamydia are common parasites in the digestive tract of numerous livestock, such as cattle, sheep, pig and poultry, and often cause diarrhoea and enteritis[9–11]. The presence of these parasites indicated that the WT pigs were more susceptible to diarrhoea and enteritis than the MF-TG pigs. Other studies also suggested that the levels of NSP-degrading enzymes tend to increase the population of beneficial bacteria, thereby enhancing gut physiology, for example, reducing the relative weight of organs in the digestive system and increasing villus height[19].
We also compared the functions and abundance of microbial genes of MF-TG and WT pigs. Results showed that the abundance of K07258, K00610 and K06142 in the caecum of MF-TG pigs was more active than that of WT pigs. These genes are associated with cysteine and methionine metabolism, peptidoglycan biosynthesis and nucleotide metabolism. Prior studies reported that pigs with high feed utilisation have high abundance of methionine metabolism, peptidoglycan biosynthesis and nucleotide metabolism pathways. These features seem to verify that the gut microorganisms in MF-TG pigs can adapt to multiple digestive enzymes and evolve new mechanisms to proliferate despite altered metabolic conditions[17]. In addition, the abundance of 50 carbohydrate enzymes in the caecal microbes of MF-TG pigs was high as expected. These results on the functions of microbial genes indicated that the microorganisms promoted the adaptability of transgenic enzymes and increased the feed efficiency of the MF-TG pigs. Moreover, common veterinary drugs were associated with their corresponding resistance genes in the pig intestine, suggesting that the intestinal microbes acquired resistance under the pressure of antibiotic selection. Nevertheless, the evolution of antibiotic resistance seemed to have no relationship with the MF-TG pigs expressing exogenous digestive enzymes.