Progesterone and Androstenedione Are Important Follicular Fluid Factors Regulating Porcine Oocyte Maturation Quality.

Oocytes matured in vitro are useful for assisted human and farm animal reproduction. However, the quality of in vitro matured oocytes is usually lower than that of in vivo matured oocytes, possibly due to the absence of some important signal regulators in vitro. In this study, untargeted metabolomics was used to detect the changes in the metabolites in the follicular fluid (FF) during in vivo pig oocyte maturation and in the culture medium during in vitro maturation. Our results showed that the total metabolite changing profile of the in vivo FF was different from that of the in vitro maturation medium, but the levels of 23 differentially expressed metabolites (DEMs) changed by following the same trend during both in vivo and in vitro pig oocyte maturation. These 23 metabolites may be important regulators of porcine oocyte maturation. We found that progesterone and androstenedione, two factors in the ovarian steroidogenesis pathway enriched from the DEMs, were upregulated in the FF during in vivo pig oocyte maturation. The levels of these two factors were 31 and 20 fold, respectively, and they were higher in the FF than in the culture medium at the oocyte mature stage. The supplementation of progesterone and androstenedione during in vitro maturation significantly improved the pig oocyte maturation rate and subsequent embryo developmental competence. Our finding suggests that a metabolic abnormality during in vitro pig oocyte maturation affects the quality of the matured oocytes. This study identified some important metabolites that regulate oocyte maturation and their developmental potential, which will be helpful to improve assisted animal and human reproduction.

Digestion and utilization of plant-based diets by transgenic pigs secreting ß-glucanase, xylanase, and phytase in their salivary glands.

Novel transgenic (TG) pigs co-expressing three microbial enzymes, ß-glucanase, xylanase, and phytase, in their salivary glands were previously generated, which exhibited reduced phosphorus and nitrogen emissions and improved growth performances. In the present study, we attempted to explore the age-related change of the TG enzymic activity, the residual activity of the enzymes in the simulated gastrointestinal tract, and the effect of the transgenes on the digestion of nitrogen and phosphorus content in the fiber-rich, plant-based diets. Results showed that all the three enzymes were stably expressed over the growing and finishing periods in the F2 generation TG pigs. In simulated gastric juice, all the three enzymes exhibited excellent gastrointestinal environment adaptability. The apparent total tract digestibility of phosphorus was increased by 69.05% and 499.64%, while fecal phosphate outputs were decreased by 56.66% and 37.32%, in the TG pigs compared with the wild-type littermates fed with low non-starch polysaccharides diets and high fiber diets, respectively. Over half of available phosphorus and water-soluble phosphorus in fecal phosphorus were reduced. We also found the performance of phosphorus, calcium, and nitrogen retention rates were significantly improved, resulting in faster growth performance in TG pigs. The results indicate that TG pigs can effectively digest the high-fiber diets and exhibit good growth performance compared with wild type pigs.

Knockdown of YY1 Inhibits XIST Expression and Enhances Cloned Pig Embryo Development.

The technique of cloning has wide applications in animal husbandry and human biomedicine. However, the very low developmental efficiency of cloned embryos limits the application of cloning. Ectopic XIST-expression-induced abnormal X chromosome inactivation (XCI) is a primary cause of the low developmental competence of cloned mouse and pig embryos. Knockout or knockdown of XIST improves cloning efficiency in both pigs and mice. The transcription factor Yin yang 1(YY1) plays a critical role in XCI by triggering the transcription of X-inactive specific transcript (XIST) and facilitating the localization of XIST RNA on the X chromosome. This study aimed to investigate whether RNA interference to suppress the expression of YY1 can inhibit erroneous XIST expression, rescue abnormal XCI, and improve the developmental ability of cloned pig embryos. The results showed that YY1 binds to the 5' regulatory region of the porcine XIST gene in pig cells. The microinjection of YY1 siRNA into cloned pig embryos reduced the transcript abundance of XIST and upregulated the mRNA level of X-linked genes at the 4-cell and blastocyst stages. The siRNA-mediated knockdown of YY1 altered the transcriptome and enhanced the in vitro and in vivo developmental efficiency of cloned porcine embryos. These results suggested that YY1 participates in regulating XIST expression and XCI in cloned pig embryos and that the suppression of YY1 expression can increase the developmental rate of cloned pig embryos. The present study established a new method for improving the efficiency of pig cloning.

Supplementation of SDF1 during Pig Oocyte In Vitro Maturation Improves Subsequent Embryo Development.

The quality of in vitro matured oocytes is inferior to that of in vivo matured oocytes, which translates to low developmental capacity of embryos derived from in vitro matured oocytes. The developmental potential of in vitro matured oocytes is usually impaired due to oxidative stress. Stromal cell-derived factor-l (SDF1) can reduce oxidative stress and inhibit apoptosis. The aim of this study was to investigate the effects of SDF1 supplementation during pig oocyte in vitro maturation (IVM) on subsequent embryo development, and to explore the acting mechanisms of SDF1 in pig oocytes. We found that the IVM medium containing 20 ng/mL SDF1 improved the maturation rate of pig oocytes, as well as the cleavage rate and blastocyst rate of embryos generated by somatic cell nuclear transfer, in vitro fertilization, and parthenogenesis. Supplementation of 20 ng/mL SDF1 during IVM decreased the ROS level, increased the mitochondrial membrane potential, and altered the expression of apoptosis-related genes in the pig oocytes. The porcine oocyte transcriptomic data showed that SDF1 addition during IVM altered the expression of genes enriched in the purine metabolism and TNF signaling pathways. SDF1 supplementation during pig oocyte IVM also upregulated the mRNA and protein levels of YY1 and TET1, two critical factors for oocyte development. In conclusion, supplementation of SDF1 during pig oocyte IVM reduces oxidative stress, changes expression of genes involved in regulating apoptosis and oocyte growth, and enhances the ability of in vitro matured pig oocytes to support subsequent embryo development. Our findings provide a theoretical basis and a new method for improving the developmental potential of pig in vitro matured oocytes.

Amphiregulin Supplementation During Pig Oocyte In Vitro Maturation Enhances Subsequent Development of Cloned Embryos by Promoting Cumulus Cell Proliferation.

The oocyte in vitro maturation (IVM) technique is important in animal husbandry, biomedicine, and human-assisted reproduction. However, the developmental potential of in vitro matured oocytes is usually lower than that of in vivo matured (IVVM) oocytes. Amphiregulin (AREG) is an EGF-like growth factor that plays critical roles in the maturation and development of mammalian oocytes. This study investigated the effects of AREG supplementation during pig oocyte IVM on the subsequent development of cloned embryos. The addition of AREG to pig oocyte IVM medium improved the developmental competence of treated oocyte-derived cloned embryos by enhancing the expansion and proliferation of cumulus cells (CCs) during IVM. The positive effect of AREG on enhancing the quality of IVVM pig oocytes might be due to the activation of proliferation-related pathways in CCs by acting on the AREG receptor. The present study provides an AREG treatment-based method to improve the developmental competence of cloned pig embryos.

Maternal dietary supplementation of arginine increases the ratio of total cloned piglets born to total transferred cloned embryos by improving the pregnancy rate of recipient sows.

The extremely low full-term developmental efficiency of cloned pig embryos limits the practical application of pig cloning techniques. Maternal dietary supplementation of the nutritionally important amino acid, arginine, can enhance prenatal developmental rate of in vivo fertilization-derived pig embryos. It was hypothesized that maternal dietary addition of arginine can also improve the developmental capacity of cloned pig embryos. To test this hypothesis, there was a comparison of the reproductive performance between recipient sows fed an L-arginine-supplemented diet (L-Arg group) and those fed the control diet (control group). There was a subsequent comparison of the developmental indexes of cloned piglets farrowed in the L-Arg and control groups of surrogate sows. Dietary supplementation of L-arginine during gestation days 14-75 increased the plasma concentrations of arginine and arginine metabolites, including nitric oxide, spermidine, and putrescine in recipient sows of transferred cloned pig embryos. Although maternal arginine addition did not affect the birth weight and placental development indexes of newborn cloned piglets, it significantly increased the ratio of total cloned piglets born to total transferred cloned pig embryos by increasing the pregnancy rate of recipient sows. The results of this study suggest that nutritional management of recipient sows is an effective approach to improve the developmental rate of cloned pig embryos.

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, ß-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.

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.

Improvement of anti-nutritional effect resulting from ß-glucanase specific expression in the parotid gland of transgenic pigs.

ß-Glucan is the predominant anti-nutritional factors in monogastric animal feed. Although ß-glucanase supplementation in diet can help to eliminate the adverse effects, enzyme stability is substantially modified during the feed manufacturing process. To determine whether the expression of endogenous ß-glucanase gene (GLU) in vivo can improve digestibility of dietary ß-glucan and absorption of nutrients, we successfully produced transgenic pigs via nuclear transfer which express the GLU from Paenibacillus polymyxa CP7 in the parotid gland. In three live transgenic founders, ß-glucanase activities in the saliva were 3.2, 0.07 and 0.03 U/mL, respectively, and interestingly the enzyme activities increased in the pigs from 178 days old to 789 days old. From the feed the amount of gross energy, crude protein and crude fat absorbed by the transgenic pigs was significantly higher than the non-transgenic pigs. Meanwhile the moisture content of the feces was significantly reduced in transgenic pigs compared with the non-transgenic pigs. Furthermore, in all positive G1 pigs, ß-glucanase activity was detectable and the highest enzyme activity reached 3.5 U/mL in saliva. Also, crude protein digestion was significantly higher in G1 transgenic pigs than in control pigs. Taken together, our data showed that the transgenic ß-glucanase exerted its biological catalytic function in vivo in the saliva, and the improved performance of the transgenic pigs could be accurately passed on to the offspring, indicating a promising alternative approach to improving nutrient availability was established to improve utilization of livestock feed through transgenic animals.