In vitro gastrointestinal digestion study of two wheat cultivars and evaluation of xylanase supplementation

Background:The filamentous fungus Talaromyces versatilis is known to improve the metabolizable energy of wheatbased poultry diets thanks to its ability to produce a pool of CAZymes and particularly endo-β(1,4)-xylanases.In order to appreciate their in vivo mode of action,the supplementation effect of two of its xylanases,XynD and XynB from families GH10 and GH11 respectively,have been evaluated on two different wheat cultivars Caphorn and Isengrain,which were chosen amongst 6 varieties for their difference in non starch polysaccharides content and arabinoxylan composition.Results:Polysaccharides digestion was followed during 6 h along the digestive tract using the TNO gastrointestinal model-1,to mimic monogastric metabolism.Polysaccharide degradation appeared to occur mainly at the jejunal level and was higher with Isengrain than with Caphorn.For both cultivars,XynD and XynB supplementation increased notably the amount of reducing end sugars into the jejuno-ileal dialysates,which has been confirmed by a valuable increase of the soluble glucose into the jejunal dialysates.Conclusions:The amounts of arabinose and xylose into the dialysates and ileal deliveries increased consequently mainly for Caphorn,suggesting that XynD and XynB supplementation in wheat-based diet could alleviate the anti-nutritional effects of arabinoxylans by limiting the physical entrapment of starch and could increase the available metabolizable energy.     

In vitro development of canine somatic cell nuclear transfer embryos in different culture media

The objective of the present study was to investigate the effects of three different culture media on the development of canine somatic cell nuclear transfer (SCNT) embryos. Canine cloned embryos were cultured in modified synthetic oviductal fluid (mSOF), porcine zygote medium-3 (PZM-3), or G1/G2 sequential media. Our results showed that the G1/G2 media yielded significantly higher morula and blastocyst development in canine SCNT embryos (26.1% and 7.8%, respectively) compared to PZM-3 (8.5% and 0%) or mSOF (2.3% and 0%) media. In conclusion, this study suggests that blastocysts can be produced more efficiently using G1/G2 media to culture canine SCNT embryos.     

Time-dependent low-field MRI characteristics of canine blood: an in vitro study

This study was conducted to assess time-sensitive magnetic resonance (MR) changes in canine blood using low-field MR. Arterial and venous blood samples were collected from eight healthy beagle dogs. Samples were placed in 5-mL tubes and imaged within 3 hours of collection at 1 day intervals from day 1 to day 30. The following sequences were used: T1-weighted (T1W), T2-weighted (T2W), fluid-attenuated inversion recovery (FLAIR), short tau inversion recovery (STIR), and T2-star gradient-echo (T2^*-GRE). Visual comparison of the images revealed that four relatively homogenous blood clots and twelve heterogeneous blood clots developed. The margination of the clot and plasma changed significantly on day 2 and day 13. On day 2, heterogeneous blood clots were differentiated into 2 to 3 signal layers in the T2W, T1W, and especially the STIR images. Hypointense signal layers were also detected in the blood clots in STIR images, which have T2 hypo, FLAIR hypo, and T1 hyper intense signals. In all images, these signal layers remained relatively unchanged until day 13. Overall, the results suggest that hematomas are complex on low-field MRI. Accordingly, it may not be feasible to accurately characterize hemorrhages and predict clot age based on low-field MRI.     

The effects analysis of two neonicotinoid insecticides on in vitro maturation of porcine oocytes using hanging drop monoculture method

Acetamiprid (ACE) and imidacroprid (IMI) are known neonicotinoid insecticides with strong affinities for the insect-selective nicotinic acetylcholine receptor. These provide insect control by hyperstimulating insect nerves and are used for agricultural pest management. However, it has also been reported that ACE and IMI affect mammalian reproductive function. We determined the effects of ACE and IMI on the in vitro maturation of porcine oocytes. Significant decreases in nuclear maturation rates were observed in the ACE or IMI-exposed groups. Also, in matured oocytes from the ACE or IMI-exposed groups, irregular chromosomes were observed. Our results suggest that ACE and IMI exposure was detrimental to porcine oocytes and the extent of the effects depends on the concentration of exposure.     

Development of in vitro produced porcine embryos according to serum types as macromolecule

This study was conducted to establish an in vitro maturation (IVM) system by selection of efficient porcine serum during porcine in vitro production. To investigate the efficient porcine serum (PS), different types of PS [newborn pig serum, prepubertal gilt serum (PGS), estrus sow serum, and pregnancy sow serum] were used to supplement IVM media with or without gonadotrophin (GTH) and development rates of parthenogenetic activation (PA) and in vitro fertilization (IVF) embryos were then compared. The maturation rates of the PGS group was significantly higher when GTH was not added. Additionally, during development of PA embryos without GTH, the PGS group showed significantly higher cleavage and blastocyst formation rates. Moreover, the cleavage rates of IVF embryos were significantly higher in the PGS group, with no significant differences in the blastocyst formation. However, when GTH was supplemented into the IVM media, there were no significant differences among the four groups in the cleavage rates, development rates of the blastocyst, and cell number of the blastocyst after PA and IVF. In conclusion, PGS is an efficient macromolecule in porcine IVM, and GTH supplementation of the IVM media is beneficial when PS is used as macromolecule, regardless of its origin.     

Seasonal patterns of gastrointestinal nematode infection in goats on two Lithuanian farms

Background

This study investigated seasonal changes in naturally acquired gastrointestinal nematode (GIN) infections on two Lithuanian goat farms with different parasite control practices.

Findings

On both farms, nematode faecal egg counts (FEC) and larval cultures were obtained from 15 adult and 10 young goats at bi-weekly intervals from April 2012 to April 2013. Goats on farm A were dewormed with ivermectin (0.3 mg/kg body weight) in October/November 2012, whereas the animals on farm B were left untreated. Thirteen young goats were slaughtered in August/November 2012 and April 2013 and worm burdens in the gastrointestinal tract were enumerated. In goats from both farms, Teladorsagia, Trichostrongylus, Oesophagostomum, Chabertia and Haemonchus were the dominant GIN genera. Herbage contamination with infective third-stage larvae (L₃) peaked in July/August and resulted in high FEC in September/October. Parasitological examination at slaughter showed that Teladorsagia spp. and Haemonchus contortus survived the winter, both in the abomasal mucosa as adults and as early fourth-stage larvae (EL₄). Deworming on farm A significantly reduced FEC, especially of H. contortus, at the start of the grazing period compared with the untreated farm B (P < 0.05).

Conclusions

Goats were heavily infected with several GIN throughout the year. Strategic anthelmintic treatment during housing significantly reduced nematode egg output, in particular by H. contortus, at the start of the grazing season.     

In vitro study of Pasteurella multocida adhesion to trachea, lung and aorta of rabbits

In vitro experiments were undertaken to study the adhesion and colonization to tracheal mucosa, lung and aorta explants from freshly killed rabbits of two different strains of Pasteurella multocida. Serotype A:3 (capsulated, fimbriae +, haemagglutination -, dermonecrotic toxin -) isolated from a rabbit with rhinitis, and serotype D:1 (non-capsulated, fimbriae +, haemagglutination +, dermonecrotic toxin +) isolated from a dead rabbit with septicaemia, were used. When the explants were observed under the scanning electron microscope, the type D strain was highly adherent to trachea and aorta explants compared to the type A strain. Adhesion to lung explants was best achieved by the type A strain after 45 min incubation, but after 2 h incubation no significant difference was observed between the strains. Our data indicate that the presence of fimbriae and the absence of capsule seem to enhance the adherence of P. multocida type D strain to tracheal tissue. The capsular material of P. multocida type A strain and the toxin of the type D strain seem to influence the adherence to lung tissue in rabbit. Adhesion of strain D to aorta may indicate the expression of receptors on the endothelium to that strain and may also explain the ability of certain strains to cause septicaemia.     

Microdroplet In Vitro Fertilization Can Reduce the Number of Spermatozoa Necessary for Fertilizing Oocytes

Successful in vitro fertilization (IVF) in mice has been achieved using spermatozoa at concentrations specifically optimized for the experimental conditions, such as species and source of spermatozoa. Although IVF in mice is mostly performed using about 80–500 µl drops, it is expected that the number of spermatozoa used for insemination can be reduced by decreasing the size of the IVF drops. The present study was undertaken to examine the extent to which the number of spermatozoa used for IVF could be reduced by using small droplets (1 µl). We devised the experimental parameters using frozen–thawed spermatozoa from C57BL/6 mice in anticipation of broader applications to other mouse facilities. We found that as few as 5 spermatozoa per droplet could fertilize oocytes (1 or 3 oocytes per droplet), although the fertilization rates were low (13–15%). Practical fertilization rates (> 40%) could be achieved with frozen-thawed C57BL/6J spermatozoa, which are sensitive to cryopreservation, when 20 sperm per droplet were used to inseminate 3 oocytes. Even with spermatozoa from a very poor quality suspension (10% motility), about 25% of oocytes were fertilized. Our calculations indicate that the number of inseminated spermatozoa per oocyte can be reduced to 1/96–1/240 by this method. In two separate embryo transfer experiments, 60% and 47%, respectively, of embryos developed to term. Our microdroplet IVF method may be particularly advantageous when only a limited number of motile spermatozoa are available because of inadequate freezing-thawing or genetic reasons.     

麻叶荨麻醇提液对大鼠离体子宫运动的影响

为探究荨麻对子宫运动的影响及其可能机制,本试验用生物信号处理系统测定了浓度递增(0.5%、1.0%、2.0%、4.0%、8.0%和16.0%)的麻叶荨麻(Urtica cannabina,UC)醇提液对未孕大鼠离体子宫收缩频率、收缩张力和收缩幅度的影响,并用特异性M-受体阻断剂硫酸阿托品(atropine,A,2.5 μg/mL)和2.0% UC联合用药,分为3个处理: UC单独处理、UC+A和A+UC,观察UC对子宫收缩的影响是否与M-受体有关.结果显示,在UC浓度递增到4.0%时收缩频率较用药前显著增加(P <0.05);浓度递增到4.0%、8.0%时,收缩幅度较用药前极显著降低(P <0.01);但浓度递增UC对子宫收缩张力影响不显著(P >0.05).一次性加入2.0% UC后子宫收缩张力迅速增加并接近僵直,之后逐渐降低.无论UC+A或A+UC处理都使子宫收缩频率较用药前明显加快,收缩张力极显著增加(P <0.01),但2个联合用药处理组之间差异不显著(P >0.05);UC+A和A+UC处理组收缩张力较UC单独用药组均极显著降低(P <0.01).结果表明,UC可兴奋未孕大鼠离体子宫,A可缓解UC引起的子宫僵直或张力增加,但不能阻断UC引起的子宫收缩频率增加.提示UC对未孕大鼠离体子宫的兴奋作用和激动与M-受体有一定关系,但可能还存在其他机制.     

Evaluation of nutritive value and in vitro rumen fermentation gas accumulation of de-oiled algal residues

Background

Algae are widely recognized for their high oil content and for exponentially accumulating biomass with particular potential to provide single cell protein for human consumption or animal feed. It is believed that along with biodiesel from algae, the high protein de-oiled algal residue may become an alternative feed supplement option in the future. This study was conducted to investigate de-oiled algal residue obtained from the common Chlorella species, Thalassiosira weissflogii, Selenarstrum capricornutum, Scenedesmus sp., and Scenedesmus dimorphus for assessment as potential feed supplements for ruminants by comparing with soybean (Glycine max) meal and alfalfa (Medicago sativa) hay.

Results

With the exception of T. weissflogii, algal residue had higher concentrations of Cu, Zn, and Mn and lower concentration of Ca, Mg, and K than soybean meal and alfalfa hay. The algal residue CP (crude protein) concentrations ranged from 140 to 445 g/kg DM and varied among the de-oiled residues. In vitro rumen fermentation gas accumulation curves indicated that algal biomass degradation potential was less than that of soybean meal or alfalfa hay by up to 41.7%.The gas production curve, interpreted with a dual pool logistic model, confirmed that the fraction sizes for fast fermenting and slow fermenting of de-oiled algal residues were smaller than those in soybean meal and alfalfa hay, and the fermenting rate of the fractions was also low.

Conclusions

Inferior in vitro rumen gas accumulation from the five de-oiled algal residues suggests that these algal byproducts are less degradable in the rumen.     

In vitro susceptibilities of Leptospira spp. and Borrelia burgdorferi isolates to amoxicillin, tilmicosin, and enrofloxacin

Antimicrobial susceptibility testing was conducted with 6 different spirochetal strains (4 strains of Leptospira spp. and 2 strains of Borrelia burgdorferi) against 3 antimicrobial agents, commonly used in equine and bovine practice. The ranges of MIC and MBC of amoxicillin against Leptospira spp. were 0.05-6.25 µg/ml and 6.25-25.0 µg/ml, respectively. And the ranges of minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of amoxicillin against B. burgdorferi were 0.05-0.39 µg/ml and 0.20-0.78 µg/ml, respectively. The ranges of MIC and MBC of enrofloxacin against Leptospira spp. were 0.05-0.39 µg/ml and 0.05-0.39 µg/ml, respectively. Two strains of B. burgdorferi were resistant to enrofloxacin at the highest concentration tested for MBC (≥100 µg/ml). Therefore, the potential role of tilmicosin in the treatment of leptospirosis and borreliosis should be further evaluated in animal models to understand whether the in vivo studies will confirm in vitro results. All spirochetal isolates were inhibited (MIC) and were killed (MBC) by tilmicosin at concentrations below the limit of testing (≤0.01 µg/ml).     

In vitro toxicity of melamine against Tetrahymena pyriformis cells

This study assessed the toxicity of melamine against the unicellular eukaryotic system of Tetrahymena (T.) pyriformis exposed to 0, 0.05, 0.25, 0.5, 2.5, and 5 mg/mL of melamine. Cell growth curves of different cultures, the half maximum inhibition concentration (IC₅₀) value of melamine, and morphological changes in cells were obtained via optical and transmission electron microscopic observation. The effects of eleven melamine concentrations, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 and 5 mg/mL, on protein expression levels of T. pyriformis were examined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The results showed an obvious inhibitory effect of melamine on the growth of eukaryotic cells. Cell growth dynamics indicated that the IC₅₀ value of melamine on T. pyriformis was 0.82 mg/mL. The cellular morphology was also affected in a concentration-dependent manner, with characteristics of atrophy or cell damage developing in the presence of melamine. The relative contents of the top four main proteins corresponding to peak mass-to-charge ratios (m/z) of 4466, m/z 6455, m/z 6514, and m/z 7772 in the MALDI-TOF-MS spectra were all found to be closely correlated with the melamine concentrations. In conclusion, exposure of eukaryotic cells to melamine could inhibit cell growth, cause changes in cytomorphology and even disturb the expression of proteins in a concentration-dependent manner. The described method of examining four sensitive proteins affected by melamine was also proposed to be used in a preliminary study to identify protein biomarkers in T. pyriformis.     

In Vitro Fertilization and Development of Porcine Oocytes Matured in Follicular Fluid

This study was conducted to assess the fertilization and development of porcine oocytes matured in a solo follicular fluid (pFF) using different in vitro culture systems and insemination periods. Cumulus-oocyte complexes (COCs), follicular cells (FCs), and pFF were collected from the follicles of ovaries. The pFF was used as a maturation medium (MpFF) after supplementation with follicle stimulating hormone (FSH) and antibiotics. The COCs were matured in a 15 ml test tube containing 3.5 ml of MpFF with FCs (5.2 × 10⁶ cells/ml; rotating culture system) or 2 ml of MpFF without FCs in a 35-mm petri dish (static culture system) for 44 to 48 h. After maturation culture, oocytes were co-incubated with frozen-thawed spermatozoa for 5 h and then cultured for 7 days. The total mean rates of sperm penetration, normal fertilization, male pronucleus (MPN) formation, cleavage, and development to the blastocyst stage of oocytes after insemination were significantly higher (P<0.01) in the rotating culture system than in the static culture system. In conclusion, compared with the static culture system, the rotating culture system is adequate for the production of developmentally competent porcine oocytes when MpFF is used as a maturation medium.     

A field evaluation of two vaccines against Mycoplasma hyopneumoniae infection in pigs

Background

A field trial was carried out with two Mycoplasma hyopneumoniae vaccines in order to investigate the benefit of vaccination under field conditions in modern Danish pig production facilities with pigs being positive for M. hyopneumoniae. The M. hyopneumoniae infection of the herd was confirmed through blood samples that were positive for antibodies against M. hyopneumoniae combined with gross lesions of the lungs related to M. hyopneumoniae at slaughter and detection of M. hyopneumoniae by polymerace chain reaction in these lesions.

Results

A total of 2,256 pigs from two herds were randomly divided into three groups. Group 1 received 2 mL ThoroVAX^®VET, Group 2 received 1 mL Ingelvac^®MycoFLEX, and Group 3 was a non-vaccinated control group. The vaccination was performed by a person who was not involved in the rest of the trial and vaccination status thereby blinded to the evaluators.The prevalence of lung lesions related to M. hyopneumoniae were significantly lower for pigs vaccinated with ThoroVAX^®VET but not for pigs vaccinated with Ingelvac^®MycoFLEX^®, when compared to non-vaccinated pigs. There was no significant effect of vaccination on growth rate, antibiotic consumption or mortality.

Conclusion

This trial demonstrated that vaccination with Thoro^®VAX VET was effective in reducing the prevalence of lung lesion in pig units infected with M. hyopneumoniae.     

卵丘细胞对猪卵母细胞体外成熟及孤雌胚胎体外发育的影响

为探讨表皮生长因子(epidermal growth factor,EGF)的添加浓度及脱卵丘细胞时间对猪卵母细胞体外成熟及孤雌胚胎体外发育的影响.试验通过在体外成熟液中添加不同浓度(0、10、15、20、30、40 ng/mL)的EGF来研究其对培养44 h的卵母细胞成熟率以及孤雌胚胎发育的影响;在培养开始后的不同时间(18、24、38、44 h)进行脱卵丘细胞处理来研究不同时间脱卵丘处理对培养44 h的卵母细胞成熟率以及孤雌胚胎发育的影响.结果表明,成熟培养基中添加10 ng/mL EGF能显著提高卵母细胞的卵裂率和囊胚率(P <0.05).共培组和独培组卵母细胞培养18 h后脱卵丘细胞成熟率均低于44 h,但差异不显著(P >0.05);共培组卵母细胞培养18 h后脱卵丘细胞的卵裂率和囊胚率显著高于培养44 h(P <0.05);独培组卵母细胞培养18 h后脱卵丘细胞的卵裂率与44 h无显著差异(P >0.05),但囊胚率显著高于培养44 h后脱卵丘细胞(P <0.05).添加10 ng/mL EGF对猪卵母细胞体外成熟及孤雌胚胎体外发育较好;卵母细胞培养18 h后脱卵丘细胞可提高孤雌胚胎早期发育能力.     

Experimental in vitro transmission of Babesia sp. (EU1) by Ixodes ricinus

Babesia sp. (EU1), first characterized in 2003, has been implicated in human cases of babesiosis in Italy, Austria and Germany. It has been identified in roe deer and in its suspected tick vector, Ixodes ricinus, in several European countries. The aim of the present study was to validate the competence of I. ricinus as a vector of Babesia sp. (EU1) via experimental infections. For this purpose, a parasite strain isolated from roe deer was cloned in sheep erythrocytes. After experimental infections, parasite DNA was successfully amplified by PCR in both eggs and larvae originating from infected I. ricinus females and in the salivary glands of females exposed to Babesia sp. (EU1) as nymphs. We also demonstrate that infected females were able to transmit parasite DNA during a new blood meal. Together with previous epidemiological studies, these results validate I. ricinus as a competent vector for Babesia sp. (EU1).     

Production of Diabetic Offspring Using Cryopreserved Epididymal Sperm by In Vitro Fertilization and Intrafallopian Insemination Techniques in Transgenic Pigs

Somatic cell nuclear transfer (SCNT) is a useful technique for creating pig strains that model human diseases. However, production of numerous cloned disease model pigs by SCNT for large-scale experiments is impractical due to its complexity and inefficiency. In the present study, we aimed to establish an efficient procedure for proliferating the diabetes model pig carrying the mutant human hepatocyte nuclear factor-1α gene. A founder diabetes transgenic cloned pig was generated by SCNT and treated with insulin to allow for normal growth to maturity, at which point epididymal sperm could be collected for cryopreservation. In vitro fertilization and intrafallopian insemination using the cryopreserved epididymal sperm resulted in diabetes model transgenic offspring. These results suggest that artificial reproductive technology using cryopreserved epididymal sperm could be a practical option for proliferation of genetically modified disease model pigs.