Prediction of apparent total tract digestion of crude protein in adult dogs.

To predict the apparent total tract digestibility (ATTD) of crude protein (CP) in dogs we developed an in vitro system using an in vitro digestion method and a statistical analysis. The experimental diets used chicken meat powder as the protein source, with CP levels of 20% (22.01%, analyzed CP value as dry-based), 30% (31.35%, analyzed CP value as dry-based), and 40% (41.34%, analyzed CP value as dry-based). To simulate in vivo digestive processes a static in vitro digestion was performed in two steps; stomach and small intestine. To analyze ATTD the total fecal samples were collected in eight neutered beagle dogs during the experimental period. CP digestibility was calculated by measuring CP levels in dog food, in vitro undigested fraction, and dog feces. In result, CP digestibility at both in vivo and in vitro was increased with increasing dietary CP levels. To estimate in vivo digestibility the co-relation of in vivo ATTD and in vitro digestibility was investigated statistically and a regression equation was developed to predict the CP ATTD (% = 2.5405 × in vitro CP digestibility (%) + 151.8). The regression equation was evaluated its feasibility by using a commercial diet. The predicted CP digestibility which was calculated by the regression equation showed high index of similarity (100.16%) with that of in vivo in dogs. With that, it would be a feasible non-animal method to predict in vivo CP digestibility by using in vitro digestion method and the proposed linear regression equation in adult dogs.

1-Deoxynojirimycin containing Morus alba leaf-based food modulates the gut microbiome and expression of genes related to obesity.

BACKGROUND: Obesity is a serious disease with an alarmingly high incidence that can lead to other complications in both humans and dogs. Similar to humans, obesity can cause metabolic diseases such as diabetes in dogs. Natural products may be the preferred intervention for metabolic diseases such as obesity. The compound 1-deoxynojirimycin, present in Morus leaves and other sources has antiobesity effects. The possible antiobesity effect of 1-deoxynojirimycin containing Morus alba leaf-based food was studied in healthy companion dogs (n = 46) visiting the veterinary clinic without a history of diseases. Body weight, body condition score (BCS), blood-related parameters, and other vital parameters of the dogs were studied. Whole-transcriptome of blood and gut microbiome analysis was also carried out to investigate the possible mechanisms of action and role of changes in the gut microbiome due to treatment. RESULTS: After 90 days of treatment, a significant antiobesity effect of the treatment food was observed through the reduction of weight, BCS, and blood-related parameters. A whole-transcriptome study revealed differentially expressed target genes important in obesity and diabetes-related pathways such as MLXIPL, CREB3L1, EGR1, ACTA2, SERPINE1, NOTCH3, and CXCL8. Gut microbiome analysis also revealed a significant difference in alpha and beta-diversity parameters in the treatment group. Similarly, the microbiota known for their health-promoting effects such as Lactobacillus ruminis, and Weissella hellenica were abundant (increased) in the treatment group. The predicted functional pathways related to obesity were also differentially abundant between groups. CONCLUSIONS: 1-Deoxynojirimycin-containing treatment food have been shown to significantly improve obesity. The identified genes, pathways, and gut microbiome-related results may be pursued in further studies to develop 1-deoxynojirimycin-based products as candidates against obesity.

Rabbit Meat Extract Induces Browning in 3T3-L1 Adipocytes via the AMP-Activated Protein Kinase Pathway.

The browning of white adipocytes may be an innovative approach to address obesity. This study investigated the effects of rabbit meat extract on 3T3-L1 adipocytes, with a specific emphasis on inducing browning. The browning effects of rabbit meat extract were evaluated by analyzing genes specifically expressed in 3T3-L1 adipocytes using quantitative PCR and immunoblotting. Rabbit meat extract increased the expression of brown adipocyte-specific markers, UCP1 and PGC1α, and mitochondrial biogenesis factors, TFAM and NRF1, without affecting cell viability in fully differentiated 3T3-L1 adipocytes. Moreover, adipocyte differentiation and the triglyceride content were decreased; hormone-sensitive lipase activity was promoted. Rabbit meat extract activated the AMPK pathway in the differentiated 3T3-L1 cells. However, in adipocytes treated with rabbit meat extract, the expression of genes related to browning was reduced by the AMP-activated protein kinase (AMPK) inhibitor, dorsomorphin dihydrochloride. To the best of our knowledge, this is the first study to demonstrate that rabbit meat extract induces the browning of white adipocytes via the activation of the AMPK pathway, thereby demonstrating its therapeutic potential in preventing obesity.

Effects of resistant starch on anti-obesity status and nutrient digestibility in dogs.

This study investigated the effects of corn resistant starch (RS) on the anti-obesity properties, nutrient digestibility, and blood parameters in healthy beagles. Four spayed and six castrated beagle dogs were divided into a control group (CON) with a diet comprising rice and chicken meal and a treatment group (TRT) with a diet comprising corn with an increased RS content by heating-cooling and chicken meal. All dogs in the CON and TRT groups were fed a 1.2-fold higher energy diet than the daily recommended energy requirement for 16 weeks. Throughout the study period, the body weight of dogs in CON increased, whereas no change in body weight was observed in TRT, resulting in a significant difference in body weight between the two groups at the end of the experiment. Moreover, a significant reduction in the apparent total tract digestibility was observed upon the analysis of dry matter, nitrogen-free extract, and organic matter in TRT compared with that in CON. The complete blood cell composition and biochemical parameters were within the reference range in both groups. A significant increase in the concentration of serum adiponectin was found in TRT at the end of the experiment. These results suggest that the corn RS may be beneficial for weight management owing to its reduced nutrient digestibility.

Obese dogs exhibit different fecal microbiome and specific microbial networks compared with normal weight dogs.

Canine obesity is a major health concern that predisposes dogs to various disorders. The fecal microbiome has been attracting attention because of their impact on energy efficiency and metabolic disorders of host. However, little is known about specific microbial interactions, and how these may be affected by obesity in dogs. The objective of this study was to investigate the differences in fecal microbiome and specific microbial networks between obese and normal dogs. A total of 20 beagle dogs (males = 12, body weight [BW]: 10.5 ± 1.08 kg; females = 8, BW: 11.3 ± 1.71 kg; all 2-year-old) were fed to meet the maintenance energy requirements for 18 weeks. Then, 12 beagle dogs were selected based on body condition score (BCS) and divided into two groups: high BCS group (HBCS; BCS range: 7-9, males = 4, females = 2) and normal BCS group (NBCS; BCS range: 4-6, males = 4, females = 2). In the final week of the experiment, fecal samples were collected directly from the rectum, before breakfast, for analyzing the fecal microbiome using 16S rRNA gene amplicon sequencing. The HBCS group had a significantly higher final BW than the NBCS group (P < 0.01). The relative abundances of Faecalibacterium, Phascolarctobacterium, Megamonas, Bacteroides, Mucispirillum, and an unclassified genus within Ruminococcaceae were significantly higher in the HBCS group than those in the NBCS group (P < 0.05). Furthermore, some Kyoto Encyclopedia of Genes and Genomes (KEGG) modules related to amino acid biosynthesis and B vitamins biosynthesis were enriched in the HBCS group (P < 0.10), whereas those related to carbohydrate metabolism were enriched in the NBCS group (P < 0.10). Microbial network analysis revealed distinct co-occurrence and mutually exclusive interactions between the HBCS and NBCS groups. In conclusion, several genera related to short-chain fatty acid production were enriched in the HBCS group. The enriched KEGG modules in the HBCS group enhanced energy efficiency through cross-feeding between auxotrophs and prototrophs. However, further studies are needed to investigate how specific networks can be interpreted in the context of fermentation characteristics in the lower gut and obesity in dogs.

Body Weight Development in Adult Dogs Fed a High Level Resistant Starch Diet.

This study investigated the effect of Dodamssal rice, which has a high content of resistant starch, on obesity and hematologic properties in dogs. In Experiment 1, 24 spayed dogs were divided into three feeding groups: normal-fat basal diet (control), high-fat diet with 12% normal amylose type rice (hNAR), and high-fat diet with 12% high amylose type rice (Dodamssal rice; hHAR). In Experiment 2, 8 spayed dogs were assigned to a normal amylose type rice (NAR) group and a high amylose type rice group (HAR) with a normal-fat basal diet. After 24 weeks, an increase in weight and blood cholesterol was observed in both high-fat diet groups for Experiment 1. Specifically, an increase in serum alanine aminotransferase was observed over time in the hNAR group compared with that of the control; however, no such patterns were present in the hHAR group. Further, a significant weight-loss effect was observed in the HAR group in Experiment 2 at 4 weeks. The effect on body weight was due to the reduced digestibility of amylose and thereby lower dietary ME content. Overall, this confirmed that Dodamssal rice had a positive effect on weight loss in dogs, and these results suggest that Dodamssal rice has potential value as a raw ingredient for preventing obesity in dogs.

Influence of Bile Salts and Pancreatin on Dog Food during Static In Vitro Simulation to Mimic In Vivo Digestion.

The addition of pancreatin and bile salts in different concentrations during in vitro digestion causes changes in the digestibility of crude protein (CP), fat, and dry matter (DM). The effects of bile salts and pancreatin on the digestibility of ether extract (EE), CP, and DM in developing a static in vitro digestion model for dogs were assessed using different concentrations of pancreatin (0, 1, 2.5, 5, and 10 g/L digestive solution) and bile salts (0, 2.5, 6.25, 12.5, and 25 g/L digestive solution). The data were analyzed using one-way analysis of variance. Digestibility of EE increased with the addition of bile salts (p < 0.05), whereas that of CP decreased with ≤0.25 g (1.0 g/L digestive solution) pancreatin. The digestibility of DM decreased significantly in all groups supplemented with ≥3.125 g (12.5 g/L digestive solution) bile salts and 0.25−2.5 g (1−10 g/L digestive solution) pancreatin and was the lowest with 6.25 g (25 g/L digestive solution) of bile salts (p < 0.05). These findings could facilitate the development of effective static in vitro digestion models for dogs.

Evaluating sulfoxaflor residues in pig tissues using animal modeling.

Maximum residue limits (MRL) for pesticides in feed have been set to protect public health and produce safe livestock products. In vivo experiments to establish MRL are essential, as livestock are commonly used to obtain reliable In vivo quantitative information. Here, we aimed to evaluate whether small laboratory animals can replace or reduce monogastric livestock in experiments to quantify pesticide residues In vivo after oral consumption through feed. First, 24 pigs and rats were randomly assigned to four groups and fed 0, 3, 9, or 30 mg/kg of sulfoxaflor. After four weeks, serum, muscle, fat, liver, kidney, and small intestine samples were collected, and sulfoxaflor residues were analyzed using liquid chromatography - tandem mass spectrometry. Sulfoxaflor residues in pig tissues were significantly correlated with those in rat tissues. Model equations were formulated based on the residual sulfoxaflor amount in pig and rat tissues. The calculated and measured sulfoxaflor residues in pigs and rats showed more than 90% similarity. Sulfoxaflor did not affect body weight gain, feed intake, or the feed conversion ratio. Therefore, we concluded that pesticide residue quantification in vivo to establish MRL could be performed using small laboratory animals instead of livestock animals. This would contribute to obtaining In vivo pesticide residue information and reducing large-scale livestock animal experiments.

Gut microbiota profiling in aged dogs after feeding pet food contained Hericium erinaceus.

Health concern of dogs is the most important issue for pet owners. People who have companied the dogs long-term provide the utmost cares for their well-being and healthy life. Recently, it was revealed that the population and types of gut microbiota affect the metabolism and immunity of the host. However, there is little information on the gut microbiome of dogs. Hericium erinaceus (H. erinaceus; HE) is one of the well-known medicinal mushrooms and has multiple bioactive components including polyphenol, ß-glucan, polysaccharides, ergothioneine, hericerin, erinacines, etc. Here we tested a pet food that contained H. erinaceus for improvement in the gut microbiota environment of aged dogs. A total of 18 dogs, each 11 years old, were utilized. For sixteen weeks, the dogs were fed with 0.4 g of H. erinaceus (HE-L), or 0.8 g (HE-H), or without H. erinaceus (CON) per body weight (kg) with daily diets (n = 6 per group). Taxonomic analysis was performed using metagenomics to investigate the difference in the gut microbiome. Resulting from principal coordinates analysis (PCoA) to confirm the distance difference between the groups, there was a significant difference between HE-H and CON due to weighted Unique fraction metric (Unifrac) distance (p = 0.047), but HE-L did not have a statistical difference compared to that of CON. Additionally, the result of Linear discriminate analysis of effect size (LEfSe) showed that phylum Bacteroidetes in HE-H and its order Bacteroidales increased, compared to that of CON, Additionally, phylum Firmicutes in HE-H, and its genera (Streptococcus, Tyzzerella) were reduced. Furthermore, at the family level, Campylobacteraceae and its genus Campylobacter in HE-H was decreased compared to that of CON. Summarily, our data demonstrated that the intake of H. erinaceus can regulate the gut microbial community in aged dogs, and an adequate supply of HE on pet diets would possibly improve immunity and anti-obesity on gut-microbiota in dogs.

Age-related digestibility of nutrients depending on the moisture content in aged dogs.

Digestibility of pet food can affect the health of dog, especially of aged animals. To maintain the health of dogs in an overall good status it is necessary to provide nutritionally balanced food. For example, the digestibility of dogs was known to be decreased along aging. In addition, losing teethes is an often event in aged dogs that could induce a problem to eat a large size dry pet food. Nonetheless, few detailed information is available on the most suited feeding for aged dogs. As part of the nutritional study of food for aged dogs, in this study, we tested whether food type impacts on digestibility on adult versus senior dogs. The methodology to measure the digestibility of nutrients was chosen the index method using chromium oxide. Dogs were fed the same commercial dry or wet diets, which were supplemented with 0.5% chromium oxide. The wet food was prepared by adding twice volume of water in the dry food prior to incubated overnight (14-16 hours) at room temperature. After five days, their feces were collected up to a total weight of > 200 g which was the amount to analyze undigested nutrients in feces as 3 repeats. In the apparent total tract digestibility analysis of the experimental breed, no difference in the digestibility of crude protein, crude fat, crude fiber, ash, and energy was observed regarding the moisture content of the food. Noteworthy, the digestibility of nitrogen free extract was significantly increased in senior dogs fed dry dog food compared with adult dogs fed the same diet, whereas no difference was observed between senior and adult dogs fed wet food. The small breed dogs showed similar results to the experimental breed dogs. However, the digestibility of crude fat was additionally affected by age and food type unlike the experimental breed dogs. This finding suggests that the food moisture content affects the digestibility of nutrients in dogs with aging. Hence, it may be helpful to determine the nutrient contents in foods for senior dogs depending on the food type.

Effects of Dietary Supplementation of Black Soldier Fly (Hermetia illucens) Larvae Oil on Broiler Health.

Insects are a potential source of proteins and fats which can be incorporated into diets of broiler chickens. Accordingly, black soldier fly larvae oil (BSFLO) needs to be tested as an appropriate fat source to produce healthy chickens for consumers. Therefore, the objective of the present study was to evaluate the effects of the replacement of soybean oil (SBO) with BSFLO in broiler diets on intestinal health and blood profiles. A total of 210 one-day-old male broilers were randomly allocated to three dietary treatments (10 replicates of seven birds per group): a control diet and two experimental diets in which SBO was replaced with 50% (50 BSFLO) or 100% (100 BSFLO) BSFLO. At the end of the study (35 days), 18 birds (six broilers per treatment) were slaughtered to determine the intestinal morphology, digestibility, and volatile fatty acid (VFA) profile. Blood samples were collected from 24 randomly selected birds (eight broilers per treatment) to determine the blood profiles. BSFLO supplementation positively affected villus height but did not affect digestibility. BSFLO showed no adverse effects on the VFA and blood profiles. In conclusion, the results of this study suggest that SBO can be replaced by BSFLO without any adverse effects on broiler health.

Effect of black ginseng and silkworm supplementation on obesity, the transcriptome, and the gut microbiome of diet-induced overweight dogs.

Like humans, weight control in overweight dogs is associated with a longer life expectancy and a healthier life. Dietary supplements are one of the best strategies for controlling obesity and obesity-associated diseases. This study was conducted to assess the potential of black ginseng (BG) and silkworm (SW) as supplements for weight control in diet-induced overweight beagle dogs. To investigate the changes that occur in dogs administered the supplements, different obesity-related parameters, such as body condition score (BCS), blood fatty acid profile, transcriptome, and microbiome, were assessed in high energy diet (HD) and HD with BG + SW supplementation (HDT) groups of test animals. After 12 weeks of BG + SW supplementation, total cholesterol and triglyceride levels were reduced in the HDT group. In the transcriptome analysis, nine genes (NUGGC, EFR3B, RTP4, ACAN, HOXC4, IL17RB, SOX13, SLC18A2, and SOX4) that are known to be associated with obesity were found to be differentially expressed between the ND (normal diet) and HD groups as well as the HD and HDT groups. Significant changes in some taxa were observed between the HD and ND groups. These data suggest that the BG + SW supplement could be developed as dietary interventions against diet-induced obesity, and obesity-related differential genes could be important candidates in the mechanism of the anti-obesity effects of the BG + SW supplement.

Effect of commercially purified deoxynivalenol and zearalenone mycotoxins on microbial diversity of pig cecum contents.

OBJECTIVE: Deoxynivalenol (DON) and zearalenone (ZEN) are mycotoxins that frequently contaminate maize and grain cereals, imposing risks to the health of both humans and animals and leading to economic losses. The gut microbiome has been shown to help combat the effects of such toxins, with certain microorganisms reported to contribute significantly to the detoxification process. METHODS: We examined the cecum contents of three different dietary groups of pigs (control, as well as diets contaminated with 8 mg DON/kg feed or 0.8 mg ZEN/kg feed). Bacterial 16S rRNA gene amplicons were acquired from the cecum contents and evaluated by next-generation sequencing. RESULTS: A total of 2,539,288 sequences were generated with ~500 nucleotide read lengths. Firmicutes, Bacteroidetes, and Proteobacteria were the dominant phyla, occupying more than 96% of all three groups. Lactobacillus, Bacteroides, Megasphaera, and Campylobacter showed potential as biomarkers for each group. Particularly, Lactobacillus and Bacteroides were more abundant in the DON and ZEN groups than in the control. Additionally, 52,414 operational taxonomic units were detected in the three groups; those of Bacteroides, Lactobacillus, Campylobacter, and Prevotella were most dominant and significantly varied between groups. Hence, contamination of feed by DON and ZEN affected the cecum microbiota, while Lactobacillus and Bacteroides were highly abundant and positively influenced the host physiology. CONCLUSION: Lactobacillus and Bacteroides play key roles in the process of detoxification and improving the immune response. We, therefore, believe that these results may be useful for determining whether disturbances in the intestinal microflora, such as the toxic effects of DON and ZEN, can be treated by modulating the intestinal bacterial flora.

Evaluation of black soldier fly larvae oil as a dietary fat source in broiler chicken diets.

The present study was conducted to evaluate the effects of black soldier fly larvae oil (BSFLO) from the black soldier fly larvae (BSFL) as a partial or total replacement of soybean oil (SBO) on growth performance, fatty acid (FA) profile, and meat quality of broiler chickens from 1 to 5 wk of age. A total of 210 male broiler chickens (Ross 308) at one-day of age were randomly allotted to 3 dietary treatments (10 replicates and 7 birds/group): a basal control diet (CON), the basal diet in which the SBO was replaced by 50% (50 BSFLO) or 100% (100 BSFLO) of BSFLO. The growth performance, physical measurements and chemical traits of leg meat, and sensory analysis of breast meat were not influenced by diets. However, the relative weight (g/kg) of gizzard of CON was significantly higher (14.85, 12.52, and 13.02 for CON, 50 BSFLO, and 100 BSFLO; p < 0.05) than that of other treatments. As expected, the FA profile of breast meat was affected by BSFLO inclusion. The proportion (%) of saturated fatty acid (SFA) was increased (27.16, 27.58, and 28.72 for CON, 50 BSFLO, and 100 BSFLO; p < 0.05) by BSFLO inclusion and the percentage (%) of MUFA was also increased (43.36, 44.58, and 48.55 for CON, 50 BSFLO, and 100 BSFLO; p < 0.01). On the contrary, the proportion (%) of PUFA was decreased (29.49, 27.84, and 22.74 for CON, 50 BSFLO, and 100 BSFLO; p < 0.01). In conclusion, the present study suggests that the replacement of BSFLO did not show an adverse effect on growth performance and it could be an ingredient as a dietary fat source for a broiler diet.

Difference of gut microbiota composition based on the body condition scores in dogs.

Microorganism residing in the gut has been known to have important roles in the animal body. Microbes and host microenvironment are highly related with host's health including energy metabolism and immune system. Moreover, it reported that gut microbiome is correlated with diseases like obesity in human and dogs. There have been many studies to identify and characterize microbes and their genes in human body. However, there was little information of microbiome in companion animals. Here, we investigated microbiota communities in feaces from twenty - four Beagles (aged 2 years old) and analyzed the taxonomy profile using metagenomics to study the difference among gut microbiome based on body condition score (BCS). gDNA was isolated from feaces, sequenced and clustered. Taxonomy profiling was performed based on the NCBI database. BCS was evaluated once a week according to the description provided by World Small Animal Veterinary Association. Firmicutes phylum was the most abundant followed by Bacteroidetes, Fusobacteria, Proteobacteria and Actinobacteria. That main microbiota in gut were differently distributed based on the BCS. Fusobacteria has been known to be associated with colon cancer in human. Interestingly, Fusobacteria was in the third level from the top in healthy dog's gut microbiome. In addition, Fusobacteria was especially higher in overweight dogs which had 6 scales of BCS. Species Fusobacterium perfoetens was also more abundant when dogs were in BCS 6. It implied that F. perfoetens would be positively related with overweight in dogs. These finding would contribute to further studies of gut microbiome and their functions to improve dog's diets and health condition.

Comparison of sample preparation procedures of inductively coupled plasma to measure elements in dog's hair.

The pre-treatment condition affects on the element analysis of inductively coupled plasma (ICP). In this study the pre-treatment condition of ICP has been studied to quantify elements in dog's hair. The hair samples were collected from twelve female Beagles by clipping them into 1 or 2 cm at the back neck. The qualitative and quantitative analysis of elements in hairs were performed by using ICP. By ICP nine elements were qualitatively detected and quantitatively analyzed (Ca, Cu, Fe, K, Mg, Na, P, Se, Zn). The measured amounts of elements were compared between 3 step and 2 step procedures which were with and without the acetone based washing step. The quantitative analysis showed that the concentrations of K, Na, P, and Se were significantly decreased in hair samples with acetone-based washing (p < 0.005 or 0.001) unlike those without the acetone-based washing. It implied that some minerals are lost by the acetone based washing during the sample preparation step. Therefore, the acetone based washing process is not suitable for quantifying elements in dog's hair. In addition, the results of qualitative and quantitative analysis were compared. Although there was a difference in absolute values of elemental contents in hair, the results of qualitative and quantitative analysis were significantly correlated each other. This finding suggested that the results of qualitative analysis can be used to monitor elemental contents in dog's hair.

A simple method to evaluate body condition score to maintain the optimal body weight in dogs.

Overweight and obesity induce serious health problems that exert negative effects on dog's welfare. Body condition score (BCS) is a common method to evaluate the body fat mass in animals. By palpating and observing fats under the skin it is possible to predict animal's body fat accumulation condition. BCS is also a useful tool to estimate body fat composition in dogs. However, BCS can be subjective when it was performed by non-professionals like pet's owners. To develop a method to avoid the misevaluation of BCS twenty-four Beagles were enrolled and performed BCS evaluation. In addition, the length of chest and abdominal girths were measured. In correlation analysis, the sizes of chest and abdominal girth were significantly correlated with BCS. Especially, the difference and ratio of the chest and abdominal length were highly correlated with the BCS. With that, we suggested that this simple measurement of chest and abdominal girths by a measuring tape would be an effective method to estimate BCS scores in dogs that helps non-professionals to manage their own dog's nutritional condition by monitoring body fat accumulation condition.

Follistatin Rescues Blastocyst Development of Poor Quality Porcine Cumulus-Oocyte Complexes by Delaying Meiotic Resumption With Decreased cGMP.

Mammalian oocytes resume maturation when removed from follicles and cultured in vitro. During folliculogenesis, oocytes are bathed in follicular fluid (FF), which provides an important and specialized microenvironment for oocyte competence. Follistatin (FST) is one component of FF that may play a role in oocyte maturation and embryo development. This study was conducted to examine possible effects of FST on porcine oocyte competence and embryo development. Exogenous FST in oocyte maturation medium for 22 or 44 hours did not improve nuclear maturation and had no effect on good quality cumulus-oocyte complexes (COCs). However, FST improved blastocyst rates in embryos derived from oocytes with less than 2 layers of cumulus. Follistatin treatment of the poor quality COC group increased transcript levels for genes indicative of oocyte quality. Transcript levels were also altered for cumulus expansion-related genes in response to FST when measured during the germinal vesicle breakdown stage. Interestingly, high-quality oocytes remained at germinal vesicle stage much longer than low-quality oocytes, FST treatment induced temporary blockage of spontaneous meiotic resumption when added during culture of both good and poor quality COCs, and levels of cyclic guanosine monophosphate (cGMP) were higher in FST-treated versus untreated groups for both good and poor quality oocytes. In conclusion, FST treatment of porcine oocytes during in vitro maturation can rescue competency of poor quality oocytes to develop to blastocyst stage following in vitro fertilization. Beneficial effects of addition of FST to culture medium may be mediated by inhibiting degradation of cGMP and temporarily delaying nuclear maturation.

Molecular cloning and characterization of porcine ribosomal protein L21.

Ribosomal protein L21 (RPL21) is a structural component of the 60S subunit of the eukaryotic ribosome. This protein has an important role in protein synthesis and the occurrence of hereditary diseases. Pig is a common laboratory model, however, to the best of our knowledge, its RPL21 gene has not been cloned to date. In this study, we cloned and identified the full-length sequence of the pig RPL21 gene for the first time. In addition, we examined its expression pattern and function by using overexpression or knockdown approaches. As a result, we obtained a 604 bp segment that contains a 483 bp open reading frame encoding 160 amino acids. The pig RPL21 gene is located in the "+" strand of chromosome 11, which spans 2167 bp from 4199792 to 4201958. Pig RPL21 protein has nine strands and two helices in its secondary structure. Pig RPL21 is predominantly expressed in ovary and lung, at lower levels in kidney, small intestine, and skin, and at the lowest levels in heart and liver. Furthermore, RPL21 expression is closely connected with cell proliferation and cell cycle arrest. The results are intended to provide useful information for the further study of pig RPL21.

Construction of a Dual-Fluorescence Reporter System to Monitor the Dynamic Progression of Pluripotent Cell Differentiation.

Oct4 is a crucial germ line-specific transcription factor expressed in different pluripotent cells and downregulated in the process of differentiation. There are two conserved enhancers, called the distal enhancer (DE) and proximal enhancer (PE), in the 5' upstream regulatory sequences (URSs) of the mouse Oct4 gene, which were demonstrated to control Oct4 expression independently in embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs). We analyzed the URSs of the pig Oct4 and identified two similar enhancers that were highly consistent with the mouse DE and PE. A dual-fluorescence reporter was later constructed by combining a DE-free-Oct4-promoter-driven EGFP reporter cassette with a PE-free-Oct4-promoter-driven mCherry reporter cassette. Then, it was tested in a mouse ESC-like cell line (F9) and a mouse EpiSC-like cell line (P19) before it is formally used for pig. As a result, a higher red fluorescence was observed in F9 cells, while green fluorescence was primarily detected in P19 cells. This fluorescence expression pattern in the two cell lines was consistent with that in the early naïve pluripotent state and late primed pluripotent state during differentiation of mouse ESCs. Hence, this reporter system will be a convenient tool for screening out ESC-like naïve pluripotent stem cells from other metastable state cells in a heterogenous population.