Enqin Zheng

Genome-wide association analysis reveals genetic loci and candidate genes for feeding behavior and eating efficiency in Duroc boars

Efficient use of feed resources is a challenge in the pork industry because the largest variability in expenditure is attributed to the cost of fodder. Efficiency of feeding is directly related to feeding behavior. In order to identify genomic regions controlling feeding behavior and eating efficiency traits, 338 Duroc boars were used in this study. The Illumina Porcine SNP60K BeadChip was used for genotyping. Data pertaining to individual daily feed intake (DFI), total daily time spent in feeder (TPD), number of daily visits to feeder (NVD), average duration of each visit (TPV), mean feed intake per visit (FPV), mean feed intake rate (FR), and feed conversion ratio (FCR) were collected for these pigs. Despite the limited sample size, the genome-wide association study was acceptable to detect candidate regions association with feeding behavior and eating efficiency traits in pigs. We detected three genome-wide (P < 1.40E-06) and 11 suggestive (P < 2.79E-05) single nucleotide polymorphism (SNP)-trait associations. Six SNPs were located in genomic regions where quantitative trait loci (QTLs) have previously been reported for feeding behavior and eating efficiency traits in pigs. Five candidate genes (SERPINA3, MYC, LEF1, PITX2, and MAP3K14) with biochemical and physiological roles that were relevant to feeding behavior and eating efficiency were discovered proximal to significant or suggestive markers. Gene ontology analysis indicated that most of the candidate genes were involved in the development of the hypothalamus (GO:0021854, P < 0.0398). Our results provide new insights into the genetic basis of feeding behavior and eating efficiency in pigs. Furthermore, some significant SNPs identified in this study could be incorporated into artificial selection programs for Duroc-related pigs to select for increased feeding efficiency.

Novel transgenic pigs with enhanced growth and reduced environmental impact

In pig production, inefficient feed digestion causes excessive nutrients such as phosphorus and nitrogen to be released to the environment. To address the issue of environmental emissions, we established transgenic pigs harboring a single-copy quad-cistronic transgene and simultaneously expressing three microbial enzymes, β-glucanase, xylanase, and phytase in the salivary glands. All the transgenic enzymes were successfully expressed, and the digestion of non-starch polysaccharides (NSPs) and phytate in the feedstuff was enhanced. Fecal nitrogen and phosphorus outputs in the transgenic pigs were reduced by 23.2–45.8%, and growth rate improved by 23.0% (gilts) and 24.4% (boars) compared with that of age-matched wild-type littermates under the same dietary treatment. The transgenic pigs showed an 11.5–14.5% improvement in feed conversion rate compared with the wild-type pigs. These findings indicate that the transgenic pigs are promising resources for improving feed efficiency and reducing environmental impact.

Birth weight, umbilical and placental traits in relation to neonatal loss in cloned pigs

Cloned piglets generated through somatic cell nuclear transfer (SCNT) have a high rate of neonatal death. Postnatal loss is associated with low birth weight, umbilical status and placental parameters in fertilisation-derived piglets. To investigate whether or not this relationship also exists in cloned piglets, birth weight, umbilical status, placental parameters, placental morphology and gene expression pattern were compared among four piglet groups, namely, SCNT-derived male piglets that died within 4 days (SCNT-DW4), SCNT-derived male piglets that survived over 4 days (SCNT-SO4), artificial insemination (AI)-generated male piglets that died within 4 days (AI-DW4) and AI-generated male piglets that survived over 4 days (AI-SO4). Results showed that the occurring frequency of abnormal umbilical cord in SCNT-DW4 piglets was significantly higher than that in AI-SO4 piglets but was similar to that in SCNT-SO4 and AI-DW4 piglets. The birth weight, placental surface area and placental weight of AI-SO4, AI-DW4 and SCNT-SO4 groups were similar but were significantly higher than those in SCNT-DW4 group. SCNT-SO4 placentas exhibited mild but SCNT-DW4 placentas showed severe morphological abnormalities compared with AI-SO4 placentas. The expression profiles of imprinting, angiopoiesis, nutrient transport, apoptosis and oxidative stress-related genes in SCNT-DW4 placentas were erroneous compared with those in SCNT-SO4 and AI-SO4 placentas, which both had similar gene expression patterns. These results indicate that birth weight, umbilical status, placental parameters, placental morphology and gene expression were associated with neonatal death of cloned piglets. The high loss of cloned piglets during neonatal age may be caused by severe deficiency of extra-embryonic development during prenatal stage.

A global comparison of the microbiome compositions of three gut locations in commercial pigs with extreme feed conversion ratios

In an attempt to increase profits and sustainability in the swine industry, the gut microbiome has become a focus of much research. In this study, we performed a comparative analysis of the gut microbiome in the ileum, cecum, and colon of Duroc × (Landrace × Yorkshire) (DLY) pigs showing two extreme feed conversion ratios (FCRs) using 16S rRNA gene sequencing. The results revealed that the microbial community in the cecum and colon had significantly higher alpha diversity than the ileum. We further identified 11, 55, and 55 operational taxonomic units (OTUs) with significantly different relative abundances between the high and low FCR pigs among the three gut locations, respectively. These OTUs were mainly associated with bacteria that participate in the metabolism of dietary polysaccharides and proteins. We then identified two and nine metabolic pathways that were enriched in the cecum and colon of the high FCR pigs, respectively. The results suggested that the short chain fatty acids and indolic compounds produced by microbial fermentation might influence porcine feed efficiency. These results should improve our understanding of microbiota compositions in the different gut locations of commercial pigs and provide important insights into the effect of gut microbiota on porcine FCRs.

Production of functional human nerve growth factor from the saliva of transgenic mice by using salivary glands as bioreactors

The salivary glands of animals have great potential to act as powerful bioreactors to produce human therapeutic proteins. Human nerve growth factor (hNGF) is an important pharmaceutical protein that is clinically effective in the treatment of many human neuronal and non-neuronal diseases. In this study, we generated 18 transgenic (TG) founder mice each carrying a salivary gland specific promoter-driven hNGF transgene. A TG mouse line secreting high levels of hNGF protein in its saliva (1.36 μg/mL) was selected. hNGF protein was successfully purified from the saliva of these TG mice and its identity was verified. The purified hNGF was highly functional as it displayed the ability to induce neuronal differentiation of PC12 cells. Furthermore, it strongly promoted proliferation of TF1 cells, above the levels observed with mouse NGF. Additionally, saliva collected from TG mice and containing unpurified hNGF was able to significantly enhance the growth of TF1 cells. This study not only provides a new and efficient approach for the synthesis of therapeutic hNGF but also supports the concept that salivary gland from TG animals is an efficient system for production of valuable foreign proteins.

Genome-wide association study reveals genetic loci and candidate genes for average daily gain in Duroc pigs

Objective Average daily gain (ADG) is an important target trait of pig breeding programs. We aimed to identify single nucleotide polymorphisms (SNPs) and genomic regions that are associated with ADG in the Duroc pig population. Methods We performed a genome-wide association study involving 390 Duroc boars and by using the PorcineSNP60K Beadchip and two linear models. Results After quality control, we detected 3,5971 SNPs, which included seven SNPs that are significantly associated with the ADG of pigs. We identified six quantitative trait loci (QTL) regions for ADG. These QTLs included four previously reported QTLs on Sus scrofa chromosome (SSC) 1, SSC5, SSC9, and SSC13, as well as two novel QTLs on SSC6 and SSC16. In addition, we selected six candidate genes (general transcription factor 3C polypeptide 5, high mobility group AT-hook 2, nicotinamide phosphoribosyltransferase, oligodendrocyte transcription factor 1, pleckstrin homology and RhoGEF domain containing G4B, and ENSSSCG00000031548) associated with ADG on the basis of their physiological roles and positional information. These candidate genes are involved in skeletal muscle cell differentiation, diet-induced obesity, and nervous system development. Conclusion This study contributes to the identification of the casual mutation that underlies QTLs associated with ADG and to future pig breeding programs based on marker-assisted selection. Further studies are needed to elucidate the role of the identified candidate genes in the physiological processes involved in ADG regulation.

Generation of a novel growth-enhanced and reduced environmental impact transgenic pig strain

In pig production, insufficient feed digestion causes excessive nutrients such as phosphorus and nitrogen, which are then released to the environment. To address the issue of environmental emissions, we have established transgenic pigs harboring a single-copy quad-cistronic transgene and simultaneously expressing three microbial enzymes, β-glucanase, xylanase, and phytase in the salivary glands. All the transgenic enzymes were successfully expressed, and the digestion of non-starch polysaccharides (NSPs) and phytate in the feedstuff was enhanced. Fecal nitrogen and phosphate outputs were reduced by 23%–46%, and growth rate improved by 23.4% (gilts) and 24.4% (boars) when the pigs were fed on a corn and soybean-based diet and high-NSP diet. The transgenic pigs showed a 11.5%– 14.5% improvement in feed conversion rate compared to the age-matched wild-type littermates. These findings indicate that transgenic pigs are promising resources for improving feed efficiency and reducing nutrient emissions to the environment.

Constitutive expression of antimicrobial peptide PR-39 in transgenic mice significantly enhances resistance to bacterial infection and promotes growth

Use of huge amounts of antibiotics in farm animal production has promoted the prevalence of antibiotic-resistant bacteria, which poses a serious threat to public health. Therefore, alternative approaches are needed to reduce or replace antibiotic usage in the food animal industry. PR-39 is a pig-derived proline-rich antimicrobial peptide that has a broad spectrum of antibacterial activity and a low propensity for development of resistance by microorganisms. To test whether ubiquitous expression of PR-39 in transgenic (TG) mice can increase resistance against bacterial infection, we generated TG mice that ubiquitously express a pig-derived antimicrobial peptide PR-39 and analyzed their growth and resistance to infection of the highly pathogenic Actinobacillus pleuropneumoniae (APP) isolated from swine. The growth performance was significantly increased in TG mice compared with their wild-type (WT) littermates. After the APP challenge, TG mice exhibited a significantly higher survival rate and significantly lower tissue bacterial load than WT littermates. Furthermore, the tissue lesion severity that resulted from APP infection was milder in TG mice than that in their WT littermates. This study provides a good foundation for the development of PR-39-expressing TG animals, which could reduce the use of antibiotics in the farm animal industry.

Suppressing Ku70/Ku80 expression elevates homology-directed repair efficiency in primary fibroblasts

The main DNA repair pathways, nonhomologous end joining (NHEJ) and homology-directed repair (HDR), are complementary to each other; hence, interruptions of the NHEJ pathway can favor HDR. Improving HDR efficiency in animal primary fibroblasts can facilitate the generation of gene knock-in animals with agricultural and biomedical values by somatic cell nuclear transfer. In this study, we used siRNA to suppress the expression of Ku70 and Ku80, which are the key factors in NHEJ pathway, to investigate the effect of Ku silencing on the HDR efficiency in pig fetal fibroblasts. Down-regulation of Ku70 and Ku80 resulted in the promotion of the frequencies of multiple HDR pathways, including homologous recombination, single strand annealing, and single-stranded oligonucleotide-mediated DNA repair. We further evaluated the effects of Ku70 and Ku80 silencing on promoting HR-mediated knock-in efficiency in two porcine endogenous genes and found a significant increase in the amount of knock-in cells in Ku-silenced fibroblasts compared with control. The RNA interference strategy will benefit the generation of cell lines and organisms with precise genetic modifications.

Genetic Architecture of Feeding Behavior and Feed Efficiency in a Duroc Pig Population

Increasing feed efficiency is a major goal of breeders as it can reduce production cost and energy consumption. However, the genetic architecture of feeding behavior and feed efficiency traits remains elusive. To investigate the genetic architecture of feed efficiency in pigs, three feeding behavior traits (daily feed intake, number of daily visits to feeder, and duration of each visit) and two feed efficiency traits (feed conversion ratio and residual feed intake) were considered. We performed genome-wide association studies (GWASs) of the five traits using a population of 1,008 Duroc pigs genotyped with an Illumina Porcine SNP50K BeadChip. A total of 9 genome-wide (P < 1.54E-06) and 35 suggestive (P < 3.08E-05) single nucleotide polymorphisms (SNPs) were detected. Two pleiotropic quantitative trait loci (QTLs) on SSC 1 and SSC 7 were found to affect more than one trait. Markers WU_10.2_7_18377044 and DRGA0001676 are two key SNPs for these two pleiotropic QTLs. Marker WU_10.2_7_18377044 on SSC 7 contributed 2.16 and 2.37% of the observed phenotypic variance for DFI and RFI, respectively. The other SNP DRGA0001676 on SSC 1 explained 3.22 and 5.46% of the observed phenotypic variance for FCR and RFI, respectively. Finally, functions of candidate genes and gene set enrichment analysis indicate that most of the significant pathways are associated with hormonal and digestive gland secretion during feeding. This study advances our understanding of the genetic mechanisms of feeding behavior and feed efficiency traits and provide an opportunity for increasing feeding efficiency using marker-assisted selection or genomic selection in pigs.