Porcine Species Research Articles

Safety and efficacy of an essential oil from Origanumvulgare subsp. hirtum (Link) letsw. var. Vulkan when used as a sensory additive in feed for all animal species.

Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on an essential oil from Origanum vulgare subsp. hirtum var Vulkan (DOS 00001) when used as a sensory feed additive for all animal species. Analysis of the oil identified 34 components accounting for > 99% of the oil, with carvacrol being the most prevalent (> 60%). Five tolerance studies in three species (chickens for fattening, weaned piglets and dairy cows) were made to assess the safety for the target species. The recommended use level of 150 mg additive/kg feed was shown to be safe for chickens for fattening and weaned piglets and this conclusion is extended to all poultry and porcine species grown for meat production. A dose of 500 mg additive/head and day (equivalent to ~ 25 mg/kg complete feed) was also demonstrated safe for the dairy cow. The Panel concluded that since the recommended use level differs between the dairy cow and the non‐ruminants tested the lower use level of 25 mg additive/kg feed could be applied to all target animals not included above. Residue studies (meat, liver, fat milk and eggs) showed that the exposure of consumers to products from animals given the additive at the recommended use level did not raise safety concerns. The additive should be considered as an irritant to skin and eyes, and to have a potential for sensitisation of susceptible individuals. Use in animal production of the essential oil extracted from O. vulgare is not expected to pose a risk for the environment. Since oregano and its extracts is recognised to flavour food and its function in feed would be essentially the same as that in food, no further demonstration of efficacy is considered necessary.

FEATURES OF FORMING CORNER SYSTEM OF SORBUS TORMINALIS (L.) CRANTZ ARE IN FOREST PLANTING OF NATURAL ORIGIN MORE SOUTH OF PODOLSK FOREST-STEPPE OF UKRAINE

The method of excavation has investigated the features of the structure of the root system of the bark of the drug of different age categories and the conditions of location in the plantings of natural seed origin SE "Chechelnytsky forestry" of Vinnytsia region. It was discovered that a large, horizontal root system, which is far from the shoots, is characterized by a significant amount of anchor roots that gives the breed a high wind resistance. Consequent, its root system is plastic, with damage or change of edatic conditions, it is easily regenerated, the appearance of strong lateral horizontal or oblique vertical roots under different soil conditions ensures its adaptation in mixed deciduous forests. As a result a specific feature of the root system of the bark is established in different soil conditions: a) the roots penetrate into the ground horizons to a depth of 57 centimetres, but most of them in the upper 10-15 centimetre layer of soil; b) on the slopes of trees there are no rod roots, a powerful root system of horizontal roots in three directions is formed; c) in porcine species, the horizontal roots of 2 to 3 meters are placed at a depth of 5-8 centimetres from the root neck, occupying position above the parent roots. Paying attention to the distribution of the length of the roots by the branches of branching, found that the largest mass of roots of all orientations falls on the first order of branching. Of these, the largest mass is noted for horizontal roots (71-77 %), somewhat lower – for vertical and oblique vertical (29 %). On the 3-4th order of branching there is only 0.1-0.5 % of the mass of roots. Moreover, the patterns have been revealed that rootstock is formed in the seedlings of the bark of the medicinal plant, but its position is different: vertical, horizontal, oblique vertical, which confirms the differences in the formation of the root system in natural plantings. As a result of this study, the roots of oak, maple, and linden are compatible in the rhizosphere with the birch of the initial period of development of root systems of these species. The dynamics, quantity and quality of root extracts and precipitation in ordinary oak and bunch of medicinal varieties, which can have a significant impact on the consumption of nutrients and the growth of trees in their coexistence.

Safety and efficacy of Natuphos® E (6-phytase) as a feed additive for avian and porcine species.

Natuphos® E is a feed additive that contains a 6‐phytase available in powder, granulated and liquid forms which is intended to be used as a feed additive for avian and porcine species. The production strain of the phytase present in the product is a genetically modified strain of Aspergillus niger. The EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) concluded that the genetic modification of the production strain does not give rise to safety concerns. The production strain and its DNA were not detected in the concentrate used to formulate the products. Based on the tolerance studies provided, the Panel concluded that the additive is safe for the target species under the conditions of use with a wide margin of safety. The Panel also concluded that the use of the product as a feed additive does not give rise to concerns for consumers. Evidence was provided showing that the additive is not toxic by inhalation or irritant for skin or eyes, however, it should be regarded as a dermal sensitiser and a potential respiratory sensitiser. The use of the additive as a feed additive poses no risks to the environment. Based on the efficacy studies provided, the Panel concluded that the additive has the potential to be efficacious in chickens for fattening, turkeys for fattening, piglets, pigs for fattening and sows. These conclusions were extended to chickens reared for laying, turkeys reared for breeding and extrapolated to minor poultry species and other avian species for fattening and to the point of lay and to minor porcine species. The Panel considered that there was insufficient information to conclude on the efficacy in laying hens.

Authentication of animal origin of heparin and low molecular weight heparin including ovine, porcine and bovine species using 1D NMR spectroscopy and chemometric tools

High resolution (600MHz) nuclear magnetic resonance (NMR) spectroscopy is used to distinguish heparin and low-molecular weight heparins (LMWHs) produced from porcine, bovine and ovine mucosal tissues as well as their blends. For multivariate analysis several statistical methods such as principal component analysis (PCA), factor discriminant analysis (FDA), partial least squares – discriminant analysis (PLS-DA), linear discriminant analysis (LDA) were utilized for the modeling of NMR data of more than 100 authentic samples. Heparin and LMWH samples from the independent test set (n=15) were 100% correctly classified according to its animal origin. Moreover, by using 1H NMR coupled with chemometrics and several batches of bovine heparins from two producers were differentiated. Thus, NMR spectroscopy combined with chemometrics is an efficient tool for simultaneous identification of animal origin and process based manufacturing difference in heparin products.

Safety and efficacy of VevoVitall® (benzoic acid) as feed additive for minor porcine species.

Following a request from the European Commission, the EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on VevoVitall® (benzoic acid) as a feed additive for minor porcine species, when used as zootechnical additive at a maximum level of 5,000 mg/kg complete feed. The safety and efficacy of the additive had been evaluated by the FEEDAP Panel on weaned piglets (at the maximum supplementation level of 5,000 mg/kg complete feed) and pigs for fattening and pigs for reproduction (at the maximum level of 10,000 mg/kg complete feed). Subsequent to EFSA's assessments, the additive has been authorised for use in feed of weaned piglets, pigs for fattening and sows. To the knowledge of the FEEDAP Panel, there was no new information that might modify its previous assessments on VevoVitall® concerning safety for consumers and the environment; therefore, this opinion focused only on the safety of the target animals and the efficacy of the product. The FEEDAP Panel concluded that VevoVitall® is safe at the supplementation level of 5,000 mg/kg complete feed for minor porcine species for fattening and for reproduction; the FEEDAP could not conclude on the safety of the additive for weaned minor porcine species. VevoVitall® does not represent a risk for the consumer and the environment. VevoVitall® does not pose a risk by inhalation to users and is not skin sensitiser, but is a skin irritant and a severe eye irritant. VevoVitall® has the potential to decrease the urinary pH in minor porcine species at the dose of 5,000 mg/kg complete feed. A remark on the relevance of the claim has been posted by the Panel.

Droplet digital polymerase chain reaction (ddPCR) assays integrated with an internal control for quantification of bovine, porcine, chicken and turkey species in food and feed.

Food adulteration and feed contamination are significant issues in the food/feed industry, especially for meat products. Reliable techniques are needed to monitor these issues. Droplet Digital PCR (ddPCR) assays were developed and evaluated for detection and quantification of bovine, porcine, chicken and turkey DNA in food and feed samples. The ddPCR methods were designed based on mitochondrial DNA sequences and integrated with an artificial recombinant plasmid DNA to control variabilities in PCR procedures. The specificity of the ddPCR assays was confirmed by testing both target species and additional 18 non-target species. Linear regression established a detection range between 79 and 33200 copies of the target molecule from 0.26 to 176 pg of fresh animal tissue DNA with a coefficient of determination (R2) of 0.997–0.999. The quantification ranges of the methods for testing fortified heat-processed food and feed samples were 0.05–3.0% (wt/wt) for the bovine and turkey targets, and 0.01–1.0% (wt/wt) for pork and chicken targets. Our methods demonstrated acceptable repeatability and reproducibility for the analytical process for food and feed samples. Internal validation of the PCR process was monitored using a control chart for 74 consecutive ddPCR runs for quantifying bovine DNA. A matrix effect was observed while establishing calibration curves with the matrix type under testing, and the inclusion of an internal control in DNA extraction provides a useful means to overcome this effect. DNA degradation caused by heating, sonication or Taq I restriction enzyme digestion was found to reduce ddPCR readings by as much as 4.5 fold. The results illustrated the applicability of the methods to quantify meat species in food and feed samples without the need for a standard curve, and to potentially support enforcement activities for food authentication and feed control. Standard reference materials matching typical manufacturing processes are needed for future validation of ddPCR assays for absolute quantification of meat species.

Identification, characterization and purification of porcine Quiescin Q6-Sulfydryl Oxidase 2 protein

BackgroundPost-spermiogenesis membrane surface modifications rely on molecules present in the reproductive tracts. Two isoforms (isoform 1 and 2) from Quiescin Q6-Sulfydryl Oxidase protein family have been identified in the male reproductive tract of rodent species. However, unlike isoform 1, scarce information is available for isoform 2, likely due to its lower expression level and lack of proper purification methods to obtain sufficient protein quantity for further assays.ResultsThis study demonstrated the presence of short and long forms of Quiescin Q6-Sulfydryl Oxidase 2 in boar, likely representing the secretory (short form) and transmembrane (long form) forms of Quiescin Q6-Sulfydryl Oxidase 2. Immunohistochemistry studies revealed the presence of Quiescin Q6-Sulfydryl Oxidase 2 in a broad range of porcine tissues; the pronounced vesicle-contained Quiescin Q6-Sulfydryl Oxidase 2 at the apical region of epididymis and seminal vesicles epithelium suggested its involvement in sperm physiology and its participation in semen formation. The majority of porcine Quiescin Q6-Sulfydryl Oxidase 2 could be purified via either antibody affinity column or be salted out using 10%–40% ammonium sulfate. Higher amount of low molecular weight Quiescin Q6-Sulfydryl Oxidase 2 observed in the seminal vesicle likely represents the secretory form of Quiescin Q6-Sulfydryl Oxidase 2 and reflects an exuberant secretory activity in this organ.ConclusionsWe demonstrated for the first time, the presence of Quiescin Q6-Sulfydryl Oxidase 2 in porcine species; moreover, two forms of Quiescin Q6-Sulfydryl Oxidase 2 were identified and exhibited distinct molecular weights and properties during protein purification processes. This study also provided feasible Quiescin Q6-Sulfydryl Oxidase 2 purification methods from slaughterhouse materials that could potentially allow obtaining sufficient amount of Quiescin Q6-Sulfydryl Oxidase 2 for future functional investigations.

Interleukins and large domestic animals, a bibliometric analysis.

Interleukins have been well described in mice and humans. In large domestic animals the situation is drastically different and there is still a need for further researches aiming at identifying all the homologous interleukins and comparing their functions among species. We performed here a bibliometric analysis of all interleukins described in the literature in various large animal species to identify what is known so far and to underline where there is a need for new studies. Using indicators such as H index but also M quotient, A index, G index, GH ratio, and HG index we ranked 39 interleukins identified so far in bovine, caprine, equine, ovine, and porcine, the main large domestic animals. Indexes and ratio under investigations were higher for IL1, IL2, IL4, IL5, IL6, IL8, IL10, IL12, and IL18 than for other interleukins, particularly in bovine and porcine species and to a certain extent in equine species. Recently discovered interleukins presented low values for the different indexes, quotient, and ratio. Even some “old” interleukins showed low values highlighting the need for further developments in comparative immunology. For instance an interleukin such as IL4 demonstrated variation in its functions between species. In conclusion, this study provides the first bibliometric analysis dedicated to large domestic animal interleukins and underlines the need for more studies to fully determine the structure and the functions of interleukins in other mammal species.

Safety and efficacy of Amylofeed® (endo-1,3(4)-β-glucanase and endo-1,4-β-xylanase and α-amylase) as a feed additive for piglets and minor porcine species.

Amylofeed® is a preparation of endo‐1,3(4)‐β‐glucanase, endo‐1,4‐β‐xylanase and α‐amylase aimed to be used as a feed additive for piglets and young minor porcine species. In a previous assessment, the safety of the additive for the target species, user and environment were established. However, the safety for the consumer and the efficacy of the product could not be established. In that previous assessment, the limitations on the description of the manufacturing process, the characterisation of the additive and on the toxicological studies provided did not allow the Panel to conclude on the safety for the consumer. The applicant has now provided new data/information to assess the safety for the consumer and also new studies in order to support the efficacy of the additive in the target species. The enzymes present in the product are obtained from two different strains and from two different fermentation processes. In the current assessment, the applicant provided complete and detailed information regarding the manufacturing process of the additive, including details on the composition of the intermediate products. New genotoxicity studies were submitted by the applicant and the results showed no genotoxic potential of the test items. The Panel considered that the conclusions drawn in the subchronic oral toxicity study performed and previously submitted were valid for the current assessment; the results showed no evidence for adverse effects. Consequently, the Panel concluded that the additive is safe for the consumers when used as a feed additive. For the evaluation of the efficacy of the additive, the Panel considered four long‐term trials done in weaned piglets. Based on these data, the Panel concluded that the additive has a potential to be efficacious in weaned piglets at the nominal dose of 500 mg/kg feed and extrapolated this conclusion to growing minor porcine species.

Adipogenic differentiation of culture-expanded bone marrow derived porcine mesenchymal stem cells

In order to use Mesenchymal stem cell populations for obesity and related metabolic syndrome studies in cell culture system, as a prerequisite, we evaluated the potency of these stem cells to undergo adipogenic differentiation. Porcine stem cells were chosen to study adipogenesis in due to the fact that pig has a natural tendency to get obese and the species is considered to be the most desired biomedical model for human applications. Porcine MSCs have been exposed to adipogenic induction media following a 21day protocol and observed under microscope for detecting stages of differentiation. At the terminal differentiation stage; morphologically, the cells appeared rounded with numerous large cytosolic lipid spheres. Upon staining with Oil Red O, the lipid spheres stained bright red. Based on this, proprietary medium was found to differentiate MSCs more efficiently than medium formulated on previous reports. Both, the differential morphologic feature corresponding to the adipocyte and positive Oil Red O staining confirmed about successful adipogenic differentiation. We envision that stem cell based culture system from porcine species would aid for studying molecular adipogenesis and subsequent identification of therapeutic targets for obesity and other metabolic diseases.

Safety of Endofeed®DC (endo-1,3(4)-β-glucanase and endo-1,4-β-xylanase) as a feed additive for chickens for fattening, laying hens, pigs for fattening and minor poultry and porcine species.

Endofeed® DC is a preparation of endo‐1,3(4)‐β‐glucanase and endo‐1,4‐β‐xylanase to be used as a feed additive for chickens for fattening, laying hens, pigs for fattening and minor poultry and porcine species. In a previous assessment, the safety of the additive for the target species, user and environment as well as the efficacy were established. In that assessment, the applicant provided in order to address the safety for the consumer, a bacterial reverse mutation assay and an in vitro micronucleus test, and a subchronic oral toxicity study. However, considering the limitations on the description of the manufacturing process, the characterisation of the additive and on the toxicological studies provided, the Panel could not conclude on the safety for the consumer. The applicant provided new data/information on the manufacturing process, characterisation of the additive and new toxicological studies, to support the safety for the consumer, which was the main subject of this opinion. Complete and detailed information regarding the manufacturing process of the additive, including details on the composition of the intermediate products were provided. With the new information on the manufacturing and on the test items used in the toxicological studies evaluated in 2013, the Panel identified the need for new genotoxicity studies, while confirmed the appropriateness of the test item used in the subchronic oral toxicity study. New genotoxicity studies were submitted by the applicant and the results of the tests showed no genotoxic potential of the test items. The Panel considered that the conclusions drawn in the subchronic oral toxicity study previously submitted can be considered valid; the results showed no evidence for adverse effects. Therefore, the Panel concluded that the additive is safe for the consumers when used as a feed additive.

Identification and verification of porcine DNA in commercial gelatin and gelatin containing processed foods

Gelatin, derived from bovine and porcine sources, has been used in many foods and pharmaceutical products. To ensure the compliance of food products with halal regulations, the reliable analytical methods are very much required. In this study, polymerase chain reaction (PCR) assay using species-specific primers was performed to evaluate the halal authenticity of commercial pure gelatin and gelatin-containing processed food products. Based on the specificity and cross-reactivity results of the seven species-specific primers by conventional PCR, the porcine species primer No. 2 was selected and it was able to detect species DNA in 12 out of 36 processed foods. The cloning, sequencing, and blasting at NCBI confirmed the presence of pork DNA in 5 out of 12 porcine DNA positive food samples. The maximum identity (homology) with pork sequence available in NCBI Gene Bank for the five samples ranged from 87% to 97% and the Query Cover ranged from 94% to 100%. The real-time PCR assay detected more positive samples (27 positive amplifications) compared to 12 positive samples with conventional PCR using porcine specific primer No. 2. PCR using species specific primers is a very useful and effective technique for halal authenticity of gelatin and gelatin-containing food products.

Safety and efficacy of Hemicell®HT (endo-1,4-β-d-mannanase) as a feed additive for chickens for fattening, chickens reared for laying, turkey for fattening, turkeys reared for breeding, weaned piglets, pigs for fattening and minor poultry and porcine species.

Hemicell® HT is a feed additive with endo‐1,4‐β‐d‐mannanase as the main enzymatic activity that is available in solid (HT) and liquid (HT‐L) forms. The production strain of the enzyme is a genetically modified strain of Paenibacillus lentus. The recipient strain is considered to be safe, the sequences introduced to obtain the production strain do not raise safety concerns. The absence of the production strain and of recombinant DNA in the product was not proven. The additive is safe for the target species at the corresponding recommended doses. The use of Hemicell® HT as a feed additive does not give rise to concerns for consumers. Hemicell® HT and Hemicell® HT‐L are not irritant to the skin and eyes; Hemicell® HT is a skin sensitiser. No specific data were provided on the effects on the respiratory system; however, considering the nature of the active substance, the additive is considered a potential respiratory sensitiser. The active substance of the additive is a protein, and as such, it will be degraded/inactivated during passage through the digestive tract of the animals or in the environment. However, uncertainty remains on the absence of the production strain and of recombinant DNA in the product, consequently, the EFSA FEEDAP Panel could not conclude on the environmental safety of the product with regard to the genetically modified production strain. The additive has the potential to be efficacious in chickens for fattening at 32,000 U/kg feed and at 48,000 U/kg feed in turkeys for fattening and weaned piglets. These conclusions were extended to chickens reared for laying and turkeys reared for breeding and extrapolated to minor poultry species for fattening or reared for laying/breeding. The Panel could not conclude on the efficacy of the product in pigs for fattening or in minor porcine species.

Overexpression of Porcine Kit Gene in Transgenic Mice Does not Affect Melanogenesis

The Kit gene is proto-oncogene, encoded stem cell factor and involved in melanocyte development. Also, Kit is expressed in epidermis and proliferation of melanoblasts which occur to pigmentation in skin, eyes and other tissues. Here we established porcine Kit genes overexpressed transgenic mice. The role of Kit gene was regulation in melanogensis remains unclear in porcine. Therefore, present study aims to ectopic expression porcine Kit gene in pigmentation of skin tissues. Our results showed that no effect on melanogenesis in ectopic Kit gene overexpressed transgenic mice. Further studies on Kit gene will provide insight into other specific roles in porcine and mice species.

Preattachment Embryos of Domestic Animals: Insights into Development and Paracrine Secretions.

In mammalian species, endometrial receptivity is driven by maternal factors independently of embryo signals. When pregnancy initiates, paracrine secretions of the preattachment embryo are essential both for maternal recognition and endometrium preparation for implantation and for coordinating development of embryonic and extraembryonic tissues of the conceptus. This review mainly focuses on domestic large animal species. We first illustrate the major steps of preattachment embryo development, including elongation in bovine, ovine, porcine, and equine species. We next highlight conceptus secretions that are involved in the communication between extraembryonic and embryonic tissues, as well as between the conceptus and the endometrium. Finally, we introduce experimental data demonstrating the intimate connection between conceptus secretions and endometrial activity and how adverse events perturbing this interplay may affect the progression of implantation that will subsequently impact pregnancy outcome, postnatal health, and expression of production traits in livestock offspring.

89 METHYLATION STATUS OF IGF2/H19 DMR3 REGION AFFECTS IN VITRO BLASTOCYST PRODUCTION IN GOAT (CAPRA HIRCUS)

Parthenogenesis has been observed in lower animals but no known instance has been reported in mammals because both maternal and paternal genomes are a fundamental prerequisite for embryogenesis. A major reason for developmental failure of uniparental zygotes is expression of certain genes in a parent-of-origin-specific manner, i.e. genomic imprinting of genes. Out of many imprinted genes identified so far, IGF2/H19 have been extensively studied and known to play an important role in fetal and placental development. Gene IGF2 is expressed by the paternal allele, H19 is transcribed from the maternal allele, and the reciprocal expression of both genes is regulated by the DMR3 region placed upstream of the H19 gene. In the present study we compared the methylation status of IGF2/H19 DMR in parthenogenetic activated (PA) and IVF goat (Capra hircus) blastocyst through bisulphite sequencing. For this, immature oocytes of usable quality were subjected to in vitro maturation and subsequently used for embryo production through parthenogenesis (n = 993) (by calcium ionophore and 6-DMAP activation) and IVF (n = 1096). It was found that embryo production rate at all the embryonic stages (2-cell, 4-cell, 8–16-cell, morula, and blastocyst) was significantly higher (P < 0.05) in parthenogenesis (74.66 ± 3.35%, 61.90 ± 2.73%, 47.83 ± 2.95%, 38.13 ± 5.28%, and 21.11 ± 2.51%, respectively) as compared with IVF (55.21 ± 2.02%, 38.12 ± 2.48%, 28.53 ± 1.67%, 21.57 ± 1.59%, and 8.23 ± 1.02%, respectively). When blastocysts (n = 6 each) were subjected to TUNEL, it was found that PA blastocyst showed significantly higher (P < 0.05) total cell number (217.83 ± 18.80 v. 159.67 ± 13.94) and significantly low (P < 0.05) apoptotic index (2.04 ± 0.25 v. 4.03 ± 0.29) as compared with IVF blastocysts. For the methylation pattern study, we analysed 17 CpG sites on the DMR3 region of the IGF2/H19 gene. Variable methylation pattern was observed within these CpG sites in different clones (n = 15) of PA and IVF blastocyst. The DMR3 region of the IGF2/H19 gene was significantly hypermethylated (P < 0.05) in PA blastocysts as compared with IVF blastocysts (80.39 ± 2.96, 32.55 ± 4.37, respectively), which suggests higher expression of IGF2 in parthenotes. The result suggests IGF2 might play different roles in different species; the same expression pattern of IGF2 is observed in ovine, but a contrary result is found in porcine species. Our results signify the hypermethylation of IGF2/H19 DMR3, which leads to higher expression of IGF2 to support embryonic development at the blastocyst stage. This work was supported by the NFBSFARA Project on Parthenogenetic Goat (CA-4002), New Delhi, India.

152 SPECIES-SPECIFIC DIFFERENCES IN THE METHYLATION REPROGRAMMING DURING EARLY PRE-IMPLANTATION DEVELOPMENT

Studies in mouse and human have shown extensive DNA methylation reprogramming in pre-implantation development followed by remethylation from implantation. However, the extent to which such reprogramming is conserved in mammals and the timing of demethylation and remethylation are unknown. As part of a major objective to characterise methylation dynamics in the bovine and porcine species from the oocyte to the blastocyst stage, we aimed here to compare the distribution of methylation at single-base resolution in both species at Day 7.5 of development. The DNA methylation profiles were obtained from individual blastocysts at Day 7.5 [pig: 3 in vivo, 3 in vitro; cow: 3 in vivo, 3 in vitro, 3 inner cell mass (ICM) and 3 trophoectoderm (TE) dissected from in vitro blastocysts] using the post-bisulphite adaptor tagging method and Illumina sequencing. For oocytes, data (GEO: GSE63330) from Schroeder et al. 2015 were analysed. Raw sequences were mapped, methylation calls made using Bismark and data analysis and visualisation was done within the SeqMonk platform. Gene expression profiles from individual blastocysts (3 pig, 3 cow) were obtained by RNA-seq. Annotated mRNA features were quantitated in SeqMonk and these were fed into DESeq2 for differential expression analysis (P < 0.05) as previously reported (Love et al. 2014 Genome Biol. 15, 550). Global methylation levels in whole blastocysts differed substantially between porcine and bovine embryos (in vivo: 12.33 ± 3.6 v. 28.33 ± 3.5%; in vitro: 15.02 ± 3.3 v. 24.41 ± 4.1%). In addition, the distribution of methylation differed: the pattern of cytosine methylated seemed random in the porcine genome, but was highly structured in the bovine genome, with methylation predominantly over gene bodies, resembling the profile previously reported in oocytes (Schroeder et al. 2015 PLoS Genet. 11, e1005442). Regarding correlation analysis, gene expression versus methylation were plotted. It suggested that gene body methylation reflected gene expression pattern in oocytes as well as in bovine blastocysts. Pair-wise comparison of isolated ICM and TE was filtered to require 5% change, and replicate set statistics were applied. This revealed very similar total and regional methylation levels in the 2 compartments, indicating that remethylation does not initiate preferentially in one compartment in bovine pre-implantation embryos. This confirms, from a viewpoint of the genome-wide DNA methylation, what has been observed in mouse for specific genes: the trophoblast-specific DNA methylation occurs after the segregation of the TE and ICM (Nakanishi et al. 2012 Epigenetics 7, 173–183). Our study is the first to provide whole genome methylation profiles from single blastocysts of economically important livestock species. Our data demonstrate that methylation reprogramming in early pre-implantation development is species specific. Knowledge of these specific patterns may have high importance when decisions are taken regarding the use of assisted reproductive technologies, cloning, or generation of transgenic animals. This work was funded by AGL2015–66341-R (MINECO-FEDER), PRX14/00348 (MECD), 19595/EE/14 (F. Séneca).

Safety and efficacy of Axtra® PHY 20000 TPT2 (6‐phytase) as a feed additive for poultry and porcine species

Axtra® PHY 20000 TPT2 is a solid preparation that contains a 6-phytase produced with a genetically modified strain of Trichoderma reesei. The production strain and its recombinant DNA were not detected in Axtra® PHY 20000 TPT2. From the results obtained in tolerance studies, the EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) concluded that the additive is safe for the target species at 2,000 FTU/kg feed. The studies provided to address the safety for the consumer were performed with the fermentation product that is used to formulate the additive and the results do not indicate any reason for concern for consumer safety arising from the use of the product as a feed additive. The studies provided to address the safety for the user were performed with the fermentation product that is used to formulate the additive and have been assessed in a previous opinion. Considering the results of those studies and the substances used during the formulation of Axtra® PHY 20000 TPT2, this formulation is not considered a dermal sensitiser. However, it should be considered a potential irritant to skin, eyes and the respiratory tract, and owing to the nature of the active substance, it should be considered a potential respiratory sensitiser. However, the exposure by inhalation is expected to be negligible. No risks to the environment are expected from the use of Axtra® PHY 20000 TPT2 as a feed additive. Based on the results of efficacy studies, the Panel concluded that the additive has the potential to be efficacious at 250 FTU/kg feed.

Whole genome sequences of Japanese porcine species C rotaviruses reveal a high diversity of genotypes of individual genes and will contribute to a comprehensive, generally accepted classification system

Porcine rotavirus C (RVC) is distributed throughout the world and is thought to be a pathogenic agent of diarrhea in piglets. Although, the VP7, VP4, and VP6 gene sequences of Japanese porcine RVCs are currently available, there is no whole-genome sequence data of Japanese RVC. Furthermore, only one to three sequences are available for porcine RVC VP1–VP3 and NSP1–NSP3 genes. Therefore, we determined nearly full-length whole-genome sequences of nine Japanese porcine RVCs from seven piglets with diarrhea and two healthy pigs and compared them with published RVC sequences from a database. The VP7 genes of two Japanese RVCs from healthy pigs were highly divergent from other known RVC strains and were provisionally classified as G12 and G13 based on the 86% nucleotide identity cut-off value. Pairwise sequence identity calculations and phylogenetic analyses revealed that candidate novel genotypes of porcine Japanese RVC were identified in the NSP1, NSP2 and NSP3 encoding genes, respectively. Furthermore, VP3 of Japanese porcine RVCs was shown to be closely related to human RVCs, suggesting a gene reassortment event between porcine and human RVCs and past interspecies transmission. The present study demonstrated that porcine RVCs show greater genetic diversity among strains than human and bovine RVCs.

Editorial: The Physiology and Pharmacology of Leucine-rich Repeat GPCRs.

Editorial: The Physiology and Pharmacology of Leucine-rich Repeat GPCRs.