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In addition to fulfilling many breeders’ curiosity, equine embryonic sex determination can have a profound commercial impact. However, the application of currently described assays for equine embryonic sexing has rendered variable diagnosis and validation rates, with sensitivity being the main problem. In addition, while pregnancy results of in vivo-flushed equine embryos following a needle aspiration biopsy equal those of non-biopsied embryos, the effect on in vitro-produced embryos is unknown. Here, we aimed to develop a highly sensitive and specific assay for equine sex determination that can be directly performed on few embryonic cells, and to test the effect of a needle aspiration biopsy on the viability of the in vitro-produced embryo. To this end, a multiplex quantitative real-time PCR (qPCR) assay with dual-labelled probes was designed to allow the simultaneous generation of both male-specific and control fragments in a single closed-tube reaction, avoiding potential sample loss or contamination. To improve sensitivity, multicopy and polymeric genes were chosen to be specifically amplified, i.e., eight copies of Y-chromosomal ETSTY5 as male-specific and four autosomal UBC monomers as control fragment. Specificity was enhanced by the equine-specific character of ETSTY5 and by using dual-labelled probes. The assay was optimised with equine male and female genomic DNA and demonstrated a 100% accuracy and a >95% qPCR efficiency down to 10 pg of DNA. The assay was subsequently applied to determine the sex of 44 in vitro-produced embryos, collecting trophectoderm biopsies by means of a needle aspiration biopsy and herniating cells. Of all trophectoderm biopsies and herniating cell samples (n = 54), 87% could be diagnosed. Assay results were validated on a second sample obtained from the biopsied embryo (n = 18) or, by ultrasound-based sex determination of the foetus (n = 7) following the transfer of the biopsied embryo to a recipient mare, with about half of the embryos being fillies and colts. The needle aspiration biopsy procedure did not impair initial pregnancy rate or early pregnancy losses as compared to non-biopsied embryos. In conclusion, we report a safe, reliable, fast, and cost-effective assay for equine sex determination which was validated for the sex determination of in vitro-produced embryos based on few embryonic cells, and needle aspiration biopsy did not impair the embryo's viability. The assay and safe biopsy strategy hold potential for other applications.

Sustainable food provision for a continuously growing human population is one of the major challenges for the next decades. Cultured meat represents one of the alternatives which is currently extensively explored. Yet, the most appropriate cell type, capable of long-term proliferation and myogenic differentiation, remains to be identified. Bovine mesenchymal stromal cells (MSCs) are considered as a promising cell source. Within the context of cultured meat production, it is mandatory to maximize cell yield per tissue source. Although many enzymatic methods to isolate MSCs from adipose tissue (AT) have been described, cell yield has never been compared. In this study, we evaluate 32 isolation conditions including four enzyme mixtures (Collagenase type I, Collagenase type I + Trypsin, Liberase TM and Collagenase type IV) at varying concentrations and incubation times, regarding their efficiency to isolate MSCs from bovine subcutaneous AT. The highest cell yield in combination with a low population doubling time was obtained using Liberase TM at a concentration of 0.1% for 3 h. MSC identity of the cells was confirmed by tri-lineage differentiation potential and cell surface marker expression. Subsequently, isolated cells were myogenically differentiated using 5-aza-2'-deoxycytidine and galectin-1. mRNA levels of the myogenic regulatory factors (MRF) myogenic factor 5 (MYF5), myogenic differentiation 1 (MYOD1), MYF6, and myogenin (MYOG) were increased, while less paired box 3 (PAX3) mRNA expression was observed when compared to undifferentiated MSCs. The presence of desmin (DES), tropomyosin (TM), and myosin heavy chain (MyHC) in myogenically differentiated bovine AT-MSCs was confirmed using immunofluorescence stainings. When considering MSCs from bovine AT as potential cell source to produce cultured meat, it is recommended to use 0.1% Liberase TM for 3 h to ensure a high cell yield., (© 2024. The Author(s).)

X-linked recessive dystrophinopathies are the most common muscular dystrophies (MDs) in humans and dogs. To date, 20 breed-specific MD-associated variants are described in the canine dystrophin gene (DMD), including one associated with dystrophin-deficient MD in the Border Collie mixed breed. Here, we report the diagnosis and follow-up of mild dystrophin-deficient MD in a 5-month-old male Border Collie, associated with a novel DMD variant. Diagnosis was based on neurological examination and laboratory evaluations including creatine kinase activity, electromyography and muscle biopsies with immunofluorescent staining. Inspection of the Sashimi plots of the RNA-seq data from the affected muscle biopsy led to the discovery of a 162-bp L1 pseudoexon in DMD intron 63, introducing a frameshift and a premature stop codon (NM_001003343.1: c.9271_9272insN[162] p.(Ala3091fs*21)). Reduced DMD mRNA levels were detected for both the non-pseudoexon (50× less) and pseudoexon (3× less) containing transcripts in the affected muscle, compared with the level of the non-pseudoexon containing transcript in a control muscle, resulting in very low dystrophin protein levels and the upregulation of utrophin. Because the variant was only found in the affected dog, not in the healthy mother and grandmother, or in 108 unrelated Border Collies from the Belgian population (46 males and 62 females), it was considered a de novo variant. Although the prognosis for dystrophinopathy is generally regarded as poor, the dog stabilised at the age of 6 months and is still clinically stable at the age of 2 years., (© 2024 Stichting International Foundation for Animal Genetics.)

Background: KCNJ10 and CAPN1 variants cause "spinocerebellar" ataxia in dogs, but their association with generalized myokymia and neuromyotonia remains unclear., Objective: To investigate the association between KCNJ10 and CAPN1 and myokymia or neuromyotonia, with or without concurrent spinocerebellar ataxia., Animals: Thirty-three client-owned dogs with spinocerebellar ataxia, myokymia neuromytonia, or a combination of these signs., Methods: Genetic analysis of a cohort of dogs clinically diagnosed with spinocerebellar ataxia, myokymia or neuromyotonia. KCNJ10 c.627C>G and CAPN1 c.344G>A variants and the coding sequence of KCNA1, KCNA2, KCNA6, KCNJ10 and HINT1 were sequenced using DNA extracted from blood samples., Results: Twenty-four Jack Russell terriers, 1 Jack Russell terrier cross, 1 Dachshund and 1 mixed breed with spinocerebellar ataxia were biallelic (homozygous) for the KCNJ10 c.627C>G variant. Twenty-one of those dogs had myokymia, neuromyotonia, or both. One Parson Russell terrier with spinocerebellar ataxia alone was biallelic for the CAPN1 c.344G>A variant. Neither variant was found in 1 Jack Russell terrier with ataxia alone, nor in 3 Jack Russell terriers and 1 Yorkshire terrier with myokymia and neuromyotonia alone. No other causal variants were found in the coding sequence of the investigated candidate genes in these latter 5 dogs., Conclusion: The KCNJ10 c.627C>G variant, or rarely the CAPN1 c.344G>A variant, was confirmed to be the causal variant of spinocerebellar ataxia. We also report the presence of the KCNJ10 c.627C>G variant in the Dachshund breed. In dogs with myokymia and neuromyotonia alone the reported gene variants were not found. Other genetic or immune-mediated causes should be investigated to explain the clinical signs of these cases., (© 2023 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

In addition to fulfilling many breeders' curiosity, equine embryonic sex determination can have a profound commercial impact. However, the application of currently described assays for equine embryonic sexing has rendered variable diagnosis and validation rates, with sensitivity being the main problem. In addition, while pregnancy results of in vivo-flushed equine embryos following a needle aspiration biopsy equal those of non-biopsied embryos, the effect on in vitro-produced embryos is unknown. Here, we aimed to develop a highly sensitive and specific assay for equine sex determination that can be directly performed on few embryonic cells, and to test the effect of a needle aspiration biopsy on the viability of the in vitro-produced embryo. To this end, a multiplex quantitative real-time PCR (qPCR) assay with dual-labelled probes was designed to allow the simultaneous generation of both male-specific and control fragments in a single closed-tube reaction, avoiding potential sample loss or contamination. To improve sensitivity, multicopy and polymeric genes were chosen to be specifically amplified, i.e., eight copies of Y-chromosomal ETSTY5 as male-specific and four autosomal UBC monomers as control fragment. Specificity was enhanced by the equine-specific character of ETSTY5 and by using dual-labelled probes. The assay was optimised with equine male and female genomic DNA and demonstrated a 100% accuracy and a >95% qPCR efficiency down to 10 pg of DNA. The assay was subsequently applied to determine the sex of 44 in vitro-produced embryos, collecting trophectoderm biopsies by means of a needle aspiration biopsy and herniating cells. Of all trophectoderm biopsies and herniating cell samples (n = 54), 87% could be diagnosed. Assay results were validated on a second sample obtained from the biopsied embryo (n = 18) or, by ultrasound-based sex determination of the foetus (n = 7) following the transfer of the biopsied embryo to a recipient mare, with about half of the embryos being fillies and colts. The needle aspiration biopsy procedure did not impair initial pregnancy rate or early pregnancy losses as compared to non-biopsied embryos. In conclusion, we report a safe, reliable, fast, and cost-effective assay for equine sex determination which was validated for the sex determination of in vitro-produced embryos based on few embryonic cells, and needle aspiration biopsy did not impair the embryo's viability. The assay and safe biopsy strategy hold potential for other applications., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Bovine mesenchymal stromal cells (MSCs) display important features that render them valuable for cell therapy and tissue engineering strategies, such as self-renewal, multi-lineage differentiation, as well as immunomodulatory properties. These cells are also promising candidates to produce cultured meat. For all these applications, it is imperative to unequivocally identify this cell population. The isolation and in vitro tri-lineage differentiation of bovine MSCs is already described, but data on their immunophenotypic characterization is not yet complete. The currently limited availability of monoclonal antibodies (mAbs) specific for bovine MSC markers strongly hampers this research. Following the minimal criteria defined for human MSCs, bovine MSCs should express CD73, CD90, and CD105 and lack expression of CD14 or CD11b, CD34, CD45, CD79α, or CD19, and MHC-II. Additional surface proteins which have been reported to be expressed include CD29, CD44, and CD106. In this study, we aimed to immunophenotype bovine adipose tissue (AT)-derived MSCs using multi-color flow cytometry. To this end, 13 commercial Abs were screened for recognizing bovine epitopes using the appropriate positive controls. Using flow cytometry and immunofluorescence microscopy, cross-reactivity was confirmed for CD34, CD73, CD79α, and CD90. Unfortunately, none of the evaluated CD105 and CD106 Abs cross-reacted with bovine cells. Subsequently, AT-derived bovine MSCs were characterized using multi-color flow cytometry based on their expression of nine markers. Bovine MSCs clearly expressed CD29 and CD44, and lacked expression of CD14, CD45, CD73, CD79α, and MHCII, while a variable expression was observed for CD34 and CD90. In addition, the mRNA transcription level of different markers was analyzed using reverse transcription quantitative polymerase chain reaction. Using these panels, bovine MSCs can be properly immunophenotyped which allows a better characterization of this heterogenous cell population., (© 2023 International Society for Advancement of Cytometry.)

Paradoxical pseudomyotonia has previously been described in the English Cocker Spaniel (ECS) and English Springer Spaniel (ESS) breeds, without the identification of potentially causative variants. This disease is characterised by episodes of exercise-induced generalised myotonic-like muscle stiffness, phenotypically similar to congenital pseudomyotonia in cattle, and paramyotonia congenita and Brody disease in people. Four additional affected ESS dogs with paradoxical pseudomyotonia are described in this report, together with the identification of the autosomal recessive c.126C>A(p.(Cys42Ter)) SLC7A10 nonsense variant as candidate disease-causing variant in both ECS and ESS. The variant has an estimated prevalence of 2.5% in both breeds in the British study samples, but was not identified in the Belgian study samples. Genetic testing-based breeding should be a useful tool to eliminate this disease in the future, although an effective treatment option is available for severely affected dogs., (© 2023 Stichting International Foundation for Animal Genetics.)

Hereditary ataxias are a large group of neurodegenerative diseases that have cerebellar or spinocerebellar dysfunction as core feature, occurring as an isolated sign or as part of a syndrome. Based on neuropathology, this group of diseases has so far been classified into cerebellar cortical degenerations, spinocerebellar degenerations, cerebellar ataxias without substantial neurodegeneration, canine multiple system degeneration, and episodic ataxia. Several new hereditary ataxia syndromes are described, but most of these diseases have similar clinical signs and unspecific diagnostic findings, wherefore achieving a definitive diagnosis in these dogs is challenging. Eighteen new genetic variants associated with these diseases have been discovered in the last decade, allowing clinicians to reach a definitive diagnosis for most of these conditions, and allowing breeding schemes to adapt to prevent breeding of affected puppies. This review summarizes the current knowledge about hereditary ataxias in dogs, and proposes to add a "multifocal degenerations with predominant (spino)cerebellar component" category regrouping canine multiple system degeneration, new hereditary ataxia syndromes that do not fit in 1 of the previous categories, as well as specific neuroaxonal dystrophies and lysosomal storage diseases that cause major (spino)cerebellar dysfunction., (© 2023 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC. on behalf of the American College of Veterinary Internal Medicine.)

Background: Weaning is a critical phase in the pigs' life and gut health might be compromised. Gluconic acid was shown to be poorly absorbed but readily fermented to butyrate in the gut which in turn can improve gut function. Hence, a total of 144 weaning pigs were fed the experimental diets for 42 days. Three treatments were replicated in 8 pens with 6 piglets each: control; low dietary dose of gluconic acid, 9 g/kg; and high dietary dose of gluconic acid, 18 g/kg. After 21 days, one piglet from each pen was sampled for blood haematology and biochemistry, fore- and hindgut digesta characteristics and microbiota, and distal small intestinal histo-morphological indices and gene expression., Results: Feeding gluconic acid enhanced performance in period d 0-14 post-weaning, in particular feed intake was increased (P = 0.028), though the high dose did not show benefits over the low dose. Regarding d 0-42, feed intake was elevated (P = 0.026). At d 21, piglets fed 18 g/kg gluconic acid showed a trend for lower number of total white blood cells (P = 0.060), caused by particularly lower numbers of lymphocytes as compared to control (P = 0.028). Highly reduced plasma urea was found for groups fed gluconic acid, it amounted to 2.6 and 2.6 mmol/L for the 9 and 18 g/kg level, respectively, as compared to 3.8 mmol/L in control (P = 0.003). Feeding gluconic acid promoted the relative abundance of lactic-acid-producing and acid-utilizing bacteria. In distal small intestine, Lactobacillus amylovorus increased substantially from 11.3 to 82.6% for control and gluconic acid 18 g/kg, respectively (P < 0.05). In mid-colon, the butyrate producers Faecalibacterium prausnitzii (P > 0.05) and Megasphaera elsdenii (P < 0.05) showed highest abundance in gluconic acid 18 g/kg. Consequently, in caecum and mid-colon, increased relative molar percentage of butyrate were found, e.g., 10.0, 12.9 et 14.7% in caecum for gluconic acid at 0, 9, and 18 g/kg, respectively (P = 0.046). Elevated mRNA anti-inflammatory cytokine and survival signalling levels in distal small intestinal mucosa were found by feeding gluconic acid which might be mediated by butyrate., Conclusions: Gluconic acid may have potential to alleviate the postweaning growth-check in pigs by altering microbiota composition and fermentation in the gut., (© 2023. The Author(s).)

(1) Idiopathic epilepsy (IE) is thought to have a genetic cause in several dog breeds. However, only two causal variants have been identified to date, and few risk loci are known. No genetic studies have been conducted on IE in the Dutch partridge dog (DPD), and little has been reported on the epileptic phenotype in this breed. (2) Owner-filled questionnaires and diagnostic investigations were used to characterize IE in the DPD. A genome-wide association study (GWAS) involving 16 cases and 43 controls was performed, followed by sequencing of the coding sequence and splice site regions of a candidate gene within the associated region. Subsequent whole-exome sequencing (WES) of one family (including one IE-affected dog, both parents, and an IE-free sibling) was performed. (3) IE in the DPD has a broad range in terms of age at onset, frequency, and duration of epileptic seizures. Most dogs showed focal epileptic seizures evolving into generalized seizures. A new risk locus on chromosome 12 (BICF2G630119560; p raw = 4.4 × 10 -7 ; p adj = 0.043) was identified through GWAS. Sequencing of the GRIK2 candidate gene revealed no variants of interest. No WES variants were located within the associated GWAS region. However, a variant in CCDC85A (chromosome 10; XM&#95;038680630.1: c.689C > T) was discovered, and dogs homozygous for the variant (T/T) had an increased risk of developing IE (OR: 6.0; 95% CI: 1.6-22.6). This variant was identified as likely pathogenic according to ACMG guidelines. (4) Further research is necessary before the risk locus or CCDC85A variant can be used for breeding decisions.

Background: In Belgian Malinois, a KCNJ10 variant causes progressive spinocerebellar degeneration., Hypothesis/objectives: Describe the clinical, diagnostic, pathological and genetic features of spinocerebellar degeneration in the Bouvier des Ardennes breed., Animals: Five affected Bouvier des Ardennes puppies with spinocerebellar ataxia (SCA), 8 healthy related dogs, and 63 healthy unrelated Bouvier des Ardennes., Methods: Sequential case study., Results: Clinical signs started at 6 weeks of age in 1 puppy with severe signs of cerebellar disease, and at 7 to 10 weeks of age in the 4 remaining puppies with milder signs of spinocerebellar disease. The first puppy displayed severe intention tremors and rapidly progressive generalized hypermetric ataxia, whereas the 4 others developed a milder progressive SCA. Euthanasia after progression to nonambulatory status was performed by 8 weeks of age in the first puppy, and before 11 months of age in the 4 remaining puppies. Histopathology revealed cerebellar spongy degeneration and a focal symmetrical demyelinating myelopathy. All cases were homozygous for KCNJ10 XM&#95;545752.6:c.986T>C(p.(Leu329Pro)), which is pathogenic for SCA with (or without) myokymia, seizures or both (SAMS) and spongy degeneration and cerebellar ataxia (SDCA) 1 in Belgian Malinois dogs. All sampled parents were heterozygous and none of the healthy dogs were homozygous for this recessive variant. This variant has an allele frequency of 15% in the 63 healthy dogs studied., Conclusions and Clinical Importance: Inherited spinocerebellar degeneration also affects the Bouvier des Ardennes breed and is caused by a KCNJ10 variant. It can present with a spectrum of severity grades, ranging from severe cerebellar to milder spinocerebellar signs., (© 2022 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

Background: Primary congenital hypothyroidism (CH) is a rare endocrine disorder in cats with a largely unknown genetic cause., Objectives: Describe the clinical presentation of CH in 11 affected cats and identify the causal genetic variant., Animals: Eleven CH-cats from 10 unrelated families, 11 CH-free family members, 21 unrelated CH-free cats, and 155 unrelated nondiagnosed cats from different breeds., Methods: Case control study of CH-cats and their siblings (2019-2021). Diagnosis was based on low to low-normal serum thyroxine (T4) concentrations, high thyroid-stimulating hormone (TSH) concentrations and clinical signs compatible with CH. We identified the causal variant using Sanger sequencing, genotyping via PCR-RFLP and variant interpretation using ACMG/AMP guidelines., Results: All CH-cats (5 weeks-8 years) had disproportionate dwarfism. A goiter was not palpable in all. Thyroid scintigraphy with radiopertechnetate showed abnormally high uptake by thyroid glands, whereas scintigraphy with radioiodine showed abnormally low uptake, compatible with a defect in iodine organification by thyroid peroxidase (TPO). All cases were homozygous for TPO variant XM&#95;006930524.4:c.430G>A(p.(Gly144Arg)), while none of the CH-free cats were. All sampled parents were heterozygous for this recessive variant. This variant was found in 15 cat breeds with an estimated allele frequency of 9%., Conclusions and Clinical Importance: Disproportionate dwarfism, abnormally high TSH and abnormally low to low-normal T4 concentrations are diagnostic for CH in cats. All cases had dyshormonogenesis demonstrated by thyroid scintigraphy. This novel TPO missense variant (not described in humans) causes CH in cats and awareness of it can assist in diagnosis and breeding., (© 2022 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

Overconditioning is a risk factor for upregulated pre- and postpartum fat mobilization. Therefore, we hypothesized that overconditioning at the end of pregnancy leads to the accumulation of lipids in the liver and modifications of the hepatic gene expression pattern. The aim of this study was to evaluate the effect of normal- versus overconditioning on the hepatic transcriptomic profile of dairy cows at the end of pregnancy. Ten dry multiparous Holstein cows were killed 2 wk before expected calving. Body condition score (BCS) and backfat thickness (BFT) were evaluated, and blood samples for nonesterified fatty acids (NEFA) were taken before cows were killed. After cows were killed, liver biopsy samples were collected for further assessment of total lipids and RNA sequencing. Five cows were classified as normal-conditioned (median BCS = 3, range 2.75-3.5) and 5 as overconditioned (median BCS = 4, range 4-5). Regression models confirmed that normal-conditioned cows had lower BFT (1.29 ± 0.29 cm; least squares means ± standard error) and serum NEFA (0.16 ± 0.04 mmol/L) in comparison to overconditioned cows (3.14 ± 0.43 cm and 0.38 ± 0.07 mmol/L for BFT and NEFA, respectively). Total liver lipid percentage tended to be lower in normal- versus overconditioned cows (4.63 ± 0.40% and 6.06 ± 0.44%, respectively). In comparison to the mean liver lipid percentage of the normal- and overconditioned cows, 1 overconditioned cow had a relatively low (5.21%) and 1 normal-conditioned cow had a relatively high (6.07%) liver lipid percentage. Differentially expressed genes analysis (edgeR quasi-likelihood method) showed that normal-conditioned cows presented 11 upregulated and 12 downregulated genes in comparison to overconditioned cows. Linear discriminant analysis effects size revealed 133 differentially expressed genes between normal- versus overconditioned cows. Notably, the liver of normal-conditioned cows had upregulated genes associated with liver functionality (ALB, SELENOP, IGF1, and IGF2). On the other hand, overconditioned cows had upregulated genes associated with the acute-phase response (C3, HPX, and, LBP). High basal lipolysis in overconditioned cows at the end of pregnancy increased liver lipid content, and this may alter the hepatic gene expression pattern to a pro-inflammatory state., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Background: The varroa mite is one of the main causes of honey bee mortality. An important mechanism by which honey bees increase their resistance against this mite is the expression of suppressed mite reproduction. This trait describes the physiological inability of mites to produce viable offspring and was found associated with eight genomic variants in previous research., Results: This paper presents the development and validation of high-throughput qPCR assays with dual-labeled probes for discriminating these eight single-nucleotide variants. Amplicon sequences used for assay validation revealed additional variants in the primer/probe binding sites in four out of the eight assays. As for two of these the additional variants interfered with the genotyping outcome supplementary primers and/or probes were developed. Inclusion of these primers and probes in the assay mixes allowed for the correct genotyping of all eight variants of interest within our bee population., Conclusion: These outcomes underline the importance of checking for interfering variants in designing qPCR assays. Ultimately, the availability of this assay allows genotyping for the suppressed mite reproduction trait and paves the way for marker assisted selection in breeding programs.

Transient receptor potential (TRP) channels contribute to sensory transduction in the body, agonized by a variety of stimuli, such as phytochemicals, and they are predominantly distributed in afferent neurons. Evidence indicates their expression in non-neuronal cells, demonstrating their ability to modulate gastrointestinal function. Targeting TRP channels could potentially be used to regulate gastrointestinal secretion and motility, yet their expression in the pig is unknown. This study investigated TRPA1 and TRPV1 expression in different gut locations of piglets of varying age. Colocalization with enteroendocrine cells was established by immunohistochemistry. Both channels were expressed in the gut mucosa. TRPV1 mRNA abundance increased gradually in the stomach and small intestine with age, most notably in the distal small intestine. In contrast, TRPA1 exhibited sustained expression across ages and locations, with the exception of higher expression in the pylorus at weaning. Immunohistochemistry confirmed the endocrine nature of both channels, showing the highest frequency of colocalization in enteroendocrine cells for TRPA1. Specific co-localization on GLP-1 immunoreactive cells indicated their possible role in GLP-1 release and the concomitant intestinal feedback mechanism. Our results indicate that TRPA1 and TRPV1 could play a role in gut enteroendocrine activity. Moreover, age and location in the gut significantly affected gene expression.

Background: Early-life antibiotic administration is known to affect gut microbiota and host adiposity, but the effects of antibiotic exposure on skeletal muscle properties remain unknown. The present study evaluated the changes in skeletal muscle properties including myofiber characteristics and composition, as well as intramuscular fat (IMF) content in skeletal muscle of piglets when exposed to a tylosin-containing diet., Results: A total of 18 piglets (28 days of age) were randomly allocated into two groups: control basal diet (Control) and Control + 100 mg tylosin phosphate/kg of feed (Antibiotic). The trial lasted for 39 days. High-throughput amplicon sequencing revealed that no significant difference in initial gut microbiota composition was existed between Control and Antibiotic groups. Antibiotic administration increased body weight and growth rate and decreased feed to gain ratio of pigs (P < 0.05). The carcass lean and fat volumes of pigs were increased by the tylosin administration (P < 0.05). Antibiotic treatment increased myofiber density and the expression of genes related to type I and type IIb myofibers in longissimus muscle (P < 0.05). The IMF content in longissimus muscle was increased by antibiotic exposure (P < 0.05). Antibiotic administration increased expression of genes related to fatty acid uptake and de novo synthesis, and decreased expression of genes related to triglyceride hydrolysis (P < 0.05). Tylosin administration affected taxonomic distribution and beta diversity of the caecal and colonic microbiota of piglets., Conclusion: These results confirm that the growth performance, myofiber composition and muscle lipid metabolism are affected by antibiotic administration, which may be associated with an altered gut microbiota, suggesting that the gut microbiota could be served as a potential target for modulating skeletal muscle properties of host.

Farmers face difficulties in redeeming their investment in larger litter sizes since this comes with larger litter heterogenicity, lower litter resilience and risk of higher mortality. Dietary oligosaccharides, given to the sow, proved beneficial for the offspring's performance. However, giving oligosaccharides to the suckling piglet is poorly explored. Therefore, this field trial studied the effect of dietary short-chain fructo-oligosaccharides (scFOS; 1g/day; drenched) supplementation to low (LBW, lower quartile), normal (NBW, two intermediate quartiles) and high (HBW, upper quartile) birth weight piglets from birth until 7 or 21 days of age. Performance parameters, gut microbiome and short-chain fatty acids profile of feces and digesta were assessed at birth (d 0), d 7, weaning (d 21.5) and 2 weeks post-weaning (d 36.5). Additional parameters reflecting gut health (intestinal integrity and morphology, mucosal immune system) were analysed at d 36.5. Most parameters changed with age or differed with the piglet's birth weight. Drenching with scFOS increased body weight by 1 kg in NBW suckling piglets and reduced the post-weaning mortality rate by a 100%. No clear difference in the IgG level, the microbiota composition and fermentative activity between the treatment groups was observed. Additionnally, intestinal integrity, determined by measuring intestinal permeability and regenerative capacity, was similar between the treatment groups. Also, intestinal architecture (villus lenght, crypt depth) was not affected by scFOS supplementation. The density of intra-epithelial lymphocytes and the expression profiles (real-time qPCR) for immune system-related genes (IL-10, IL-1ß, IL-6, TNFα and IFNγ) were used to assess mucosal immunity. Only IFNγ expression, was upregulated in piglets that received scFOS for 7 days. The improved body weight and the reduced post-weaning mortality seen in piglets supplemented with scFOS support the view that scFOS positively impact piglet's health and resilience. However, the modes of action for these effects are not yet fully elucidated and its potential to improve other performance parameters needs further investigation., Competing Interests: The funder provided salaries for two authors, who collaborated in the conceptualisation but not in the design of the experiment, data collection and analysis or in the decision to publish. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: Paramyotonia congenita and Brody disease are well-described conditions in humans, characterized by exercise-induced myotonic-like muscle stiffness. A syndrome similar to Brody disease has been reported in cattle. Reports of a similar syndrome in dogs are scarce., Objectives: To define and describe the clinical, diagnostic, and genetic features and disease course of paradoxical pseudomyotonia in Spaniel dogs., Animals: Seven client-owned dogs (4 English Springer Spaniels and 3 English Cocker Spaniels) with clinically confirmed episodes of exercise-induced generalized myotonic-like muscle stiffness., Methods: Sequential case study., Results: All dogs were <24 months of age at onset. The episodes of myotonic-like generalized muscle stiffness always occurred with exercise, and spontaneously resolved with rest in <45 seconds in all but 1 dog. Extreme outside temperatures seemed to considerably worsen episode frequency and severity in most dogs. Complete blood count, serum biochemistry including electrolytes, urinalysis, brain magnetic resonance imaging, cerebrospinal fluid analysis, electromyography, motor nerve conduction velocity, ECG, and echocardiography were unremarkable. Muscle biopsy samples showed moderate but nonspecific muscle atrophy. The episodes seemed to remain stable or decrease in severity and frequency in 6/7 dogs, and often could be decreased or prevented by avoiding the episode triggers. The underlying genetic cause is not identified yet, because no disease-causing variants could be found in the coding sequence or splice sites of the 2 major candidate genes, SCN4A and ATP2A1., Conclusions and Clinical Importance: Paradoxical pseudomyotonia is a disease affecting Spaniels. It is of variable severity but benign in most cases., (© 2019 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

The possible impact of natural heat stress on animal fertility is currently a major concern for breeding companies. Here, we aimed to address this concern by determining the effects of natural heat stress on the fertility of Holstein bulls located in the Netherlands. Semen samples were collected from six bulls at two locations in March 2016 (low temperature-humidity index (THI) group; maximum THI of 51.8 and 55 at their respective locations) or August (high THI group; maximum THI of 77.9 and 80.5 during meiotic and spermiogenic stages of spermatogenesis, 42 to 14 days prior to semen collection). The effect of heat stress on semen quality was assessed by sperm morphology, motility, reactive oxygen species production, lipid peroxidation, viability, and DNA fragmentation. Moreover, we evaluated the development of embryos generated in vitro by low and high THI semen, and determined inner cell mass/trophectoderm ratio, apoptotic cell ratio, and embryonic gene expression in day-8 blastocysts. An increase in cell death (propidium iodide-positive cells; P = 0.039) was observed in the high THI group (31.5%) compared to the low THI group (27.6%). Moreover, a decrease (P < 0.001) was observed in the total blastocyst rates at day 7 post-insemination (15.3 vs 20.9%) and day 8 (23.2 vs 29.6%) in the high THI compared to the low THI group, respectively. There were no differences in the relative abundance of candidate transcripts examined. In conclusion, sperm samples from dairy bulls obtained during a period with higher THI had reduced viability and led to a decrease in blastocyst development and delayed hatching, compared to semen collected during a period with low THI., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Hypertrophic cardiomyopathy (HCM) is the most common inherited human heart disease. The same disease has a high prevalence in cats, where it is also suspected to be inherited. More than 1500 variants in MYBPC3, MYH7 and other sarcomeric genes are associated with human HCM, while in cats, only two causative variants in MYBPC3 are currently known. Here, we describe an adult Domestic Shorthair cat with arterial thromboembolism and heart failure that was diagnosed with HCM on necropsy. Sequencing of the coding regions of MYBPC3 and MYH7 revealed 21 variants, of which the MYH7 c.5647G>A (p.(Glu1883Lys)) variant was further analysed, because its orthologous variant had already been reported in a human patient with HCM, but with limited causal evidence. This variant affects the highly conserved assembly competence domain, is predicted in silico to be damaging and was found only once in population databases. Recently, functional studies have confirmed its predicted damaging effect and a paralogous variant in MYH6 has been associated with cardiac disease in humans as well. This report of an orthologous variant in a cat with HCM and its absence in 200 additional cats provides further evidence for its disease-causing nature. As the first report of feline HCM caused by a variant in MYH7, this study also emphasises this gene as a candidate gene for future studies in cats and highlights the similarity between human and feline HCM.

Clinical, pathological, and genetic findings of a primary hereditary ataxia found in a Malinois dog family are described and compared with its human counterpart. Based on the family history and the phenotype/genotype relationships already described in humans and dogs, a causal variant was expected to be found in KCNJ10. Rather surprisingly, whole-exome sequencing identified the SLC12A6 NC&#95;006612.3(XM&#95;014109414.2): c.178&#95;181delinsCATCTCACTCAT (p.(Met60Hisfs*14)) truncating variant. This loss-of-function variant perfectly segregated within the affected Malinois family in an autosomal recessive way and was not found in 562 additional reference dogs from 18 different breeds, including Malinois. In humans, SLC12A6 variants cause "agenesis of the corpus callosum with peripheral neuropathy" (ACCPN, alias Andermann syndrome), owing to a dysfunction of this K + -Cl - cotransporter. However, depending on the variant (including truncating variants), different clinical features are observed within ACCPN. The variant in dogs encodes the shortest isoform described so far and its resultant phenotype is quite different from humans, as no signs of peripheral neuropathy, agenesis of the corpus callosum nor obvious mental retardation have been observed in dogs. On the other hand, progressive spinocerebellar ataxia, which is the most important feature of the canine phenotype, hindlimb paresis, and myokymia-like muscle contractions have not been described in humans with ACCPN so far. As this is the first report of a naturally occurring disease-causing SLC12A6 variant in a non-human species, the canine model will be highly valuable to better understand the complex molecular pathophysiology of SLC12A6-related neurological disorders and to evaluate novel treatment strategies.

It is generally accepted that intracellular killing of microorganisms by production of reactive oxygen species (ROS) in the phagosome of the neutrophil is an important arm of innate defense. High-producing dairy cows are prone to periparturient metabolic and infectious diseases. Both myeloperoxidase (MPO) activity and ROS production decrease the day of parturition. Several studies have demonstrated changes in the expression of genes involved in, for example, metabolism and defense in the circulating neutrophil during peripartum. In this study, we wanted to further characterize the periparturient neutrophil in terms of its oxidative killing capacity by analyzing the oxidative burst at 3 levels. First, the ROS phenotype was evaluated using chemiluminescence. The cows (sampled within 24 h after parturition and at 135 d in milk) showed a significantly slower production of ROS at parturition. Both primiparous (n = 13) and multiparous (n = 12) cows were included in this study, but parity did not affect the kinetics of ROS production. Second, the expression of 11 genes involved in ROS production was measured in the same cows: cytochrome b-245 α and β chain (CYBA, CYBB; coding for membrane-bound constituents of NADPH oxidase); neutrophil cytosolic factors 1, 2, and 4 (NCF1, NCF2, and NCF4); Rac family small GTPase 1 and 2 (RAC1 and RAC2; coding for regulatory proteins of NADPH oxidase); superoxide dismutase 2 (SOD2); catalase (CAT); myeloperoxidase (MPO; coding for enzymes involved in metabolizing downstream ROS); and spleen-associated tyrosine kinase (SYK; involved in signaling). During peripartum, a shift in expression in the oxidative killing pathway was observed, characterized by a downregulation of MPO and a simultaneous upregulation of the genes coding for NADPH oxidase. Third, as total DNA methylation is known to change during pregnancy, we investigated whether the observed differences were due to different methylation patterns. Promotor regions initiate transcription of particular genes; therefore, we analyzed the methylation status in annotated CpG islands of MPO and SOD2, 2 genes with a significant difference in expression between both lactation stages. The differences in methylation of these CpG islands were nonsignificant. High-throughput techniques may be necessary to obtain more detailed information on the total DNA methylation dynamics in bovine neutrophils and increase our understanding of how gene expression is controlled in neutrophils., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Host-parasite co-evolution history is lacking when parasites switch to novel hosts. This was the case for Western honey bees (Apis mellifera) when the ectoparasitic mite, Varroa destructor, switched hosts from Eastern honey bees (Apis cerana). This mite has since become the most severe biological threat to A. mellifera worldwide. However, some A. mellifera populations are known to survive infestations, largely by suppressing mite population growth. One known mechanism is suppressed mite reproduction (SMR), but the underlying genetics are poorly understood. Here, we take advantage of haploid drones, originating from one queen from the Netherlands that developed Varroa-resistance, whole exome sequencing and elastic-net regression to identify genetic variants associated with SMR in resistant honeybees. An eight variants model predicted 88% of the phenotypes correctly and identified six risk and two protective variants. Reproducing and non-reproducing mites could not be distinguished using DNA microsatellites, which is in agreement with the hypothesis that it is not the parasite but the host that adapted itself. Our results suggest that the brood pheromone-dependent mite oogenesis is disrupted in resistant hosts. The identified genetic markers have a considerable potential to contribute to a sustainable global apiculture.

In neutrophils, toll-like receptor and complement component 5a (C5a) signaling are critical pathways regulating innate immunity. In cows, not much is known about the second C5a receptor, complement component 5a receptor 2 (C5AR2). It is an interesting player in sepsis treatment because it is considered to have an anti-inflammatory effect during normal inflammation. Periparturient cows are prone to severe infections, and the objectives of this study were to investigate the expression and functionality of C5AR2 during peripartum. We investigated the effect of 2 major inflammatory stimuli, C5a and lipopolysaccharide (LPS), on the expression of a selected number of genes (C5AR1, C5AR2, TLR4, ITGAM, COX2, and CXCL8) and functions linked to these receptors. Overall, TLR4, ITGAM, and C5AR2, all of which are involved in early inflammation, showed a lower expression in periparturient cows. However, an overall lower expression seems not to be the only explanation for the increased risk of sepsis in periparturient cows. Normally, in response to inflammation and as seen in the mid-lactation group, the expression of these genes increases after stimulation with LPS. However, in periparturient cows, stimulation with LPS led to a decrease in expression of these receptors, indicating a different response of neutrophils in response to LPS during this period. A decrease in ITGAM (coding for CD11b) expression complicates correct neutrophil localization and phagocytosis. Its downregulation upon stimulation might be detrimental for adequate eradication of the pathogen and might increase the risk of an imbalanced inflammation; C5AR2 seems to play a central role in this altered response. In addition, myeloperoxidase (MPO) activity in periparturient cows is lower in response to C5a stimulation. It has been suggested that MPO plays an important role in neutrophil shutdown and, thereby, timely resolution of inflammation. A decreased MPO activity might thus prolong the inflammatory reaction of the neutrophils. This finding was supported by the increased viability of the neutrophils obtained from periparturient cows. Even after stimulation, we found a lower caspase-3 activity in this group, indicating that they might be activated for a longer time compared with the neutrophils from mid-lactation cows. Accordingly, these alterations might contribute to a temporal mismatch in inflammatory responses, as often seen in severe periparturient infections., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The aim of this study was to determine the number of adipose tissue macrophages (ATM) and the mRNA expression of adipokines [adiponectin (ADIPOQ), leptin (LEP), interleukin 6 (IL6), tumor necrosis factor (TNF), and interleukin 10 (IL10)] in different adipose depots from cows with a variable body condition score (BCS) at the end of the dry period. We hypothesized that the number of ATM and the expression of these adipokines depend on adipocyte size and the anatomical location of the adipose depot. Subcutaneous, omental, mesenteric, perirenal, and intrapelvic adipose tissue samples were taken immediately after euthanasia of 10 Holstein Friesian dairy cows (upcoming parity 2 to 5, age 3.9 ± 1.4 yr; mean ± standard deviation) at the end of pregnancy (actual days of pregnancy at the moment of euthanasia: 269 ± 5 d). During the dry period, all animals received similar diets to meet but not exceed requirements. Five animals were considered to have a normal BCS (2.5-3.5) and 5 animals were considered to be over-conditioned (BCS = 3.75-5). Body weight of the animals at the moment of euthanasia was 717 ± 77 kg. Expression of the different genes was determined by reverse transcription quantitative real-time PCR. Adipocyte size was determined by measuring the area of 100 adipocytes on histological sections. Average adipocyte area was 10,475 ± 1,019, 8,500 ± 780, 10,383 ± 1,227, 11,466 ± 1,039, and 11,087 ± 1,632 µm 2 for the subcutaneous, mesenteric, omental, intrapelvic, and perirenal adipose depot, respectively. Immunohistochemistry using anti-bovine CD172a antibodies was performed to determine the proportion of ATM (the number of CD172a-positive cells per 100 adipocytes, given as a percentage). Expression of LEP, IL6, and TNF was positively associated with adipocyte size, whereas no association could be detected between ADIPOQ and IL10 with the size of the adipocytes. The omental adipose depot was especially infiltrated with ATM (1.92 ± 0.55, 1.10 ± 0.33, and 8.28 ± 2.24% for the subcutaneous, mesenteric, and omental adipose depot, respectively). The proportion of ATM was positively associated with the size of the adipocytes in the omental and mesenteric adipose depot. Expression of ADIPOQ, LEP, IL6, TNF, and IL10 differed among depots, which suggests differences in inflammatory characteristics depending on the anatomical location of depots. In conclusion, the results of the present study confirm the adipose tissue as a potential source of inflammatory mediators and demonstrate ATM infiltration, especially in the omental adipose depot., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Background: Deoxynivalenol (DON) is a mycotoxin produced by Fusarium species in the field, commonly found in cereal grains, which negatively affects performances and health of animals. Mycotoxin binders are supposed to reduce the toxicity of mycotoxins., Method: The effect of a mycotoxin binder (containing acid-activated bentonite, clinoptilolite, yeast cell walls and organic acids) on growth performance and gut health was studied. Hundred and twenty weaning piglets were allocated to 4 treatments, with 5 pens of 6 piglets each, arranged in a 2 × 2 factorial design: control diet; control diet with 1 kg/t binder; control diet with DON; and control diet with DON and 1 kg/t binder. From d0-14, the diet of DON-challenged groups was artificially contaminated with a mixture of DON (2.6 mg/kg), 3-acetyl-deoxynivalenol (0.1 mg/kg) and 15-acetyl-deoxynivalenol (0.3 mg/kg), after which the total contamination level was reduced to 1 mg/kg, until d37. On d14, one pig from each pen was euthanized and distal small intestinal mucosa samples were collected for the assessment of intestinal permeability, and gene expression of tight junction proteins, toll-like receptor 4, inflammatory cytokines and intestinal alkaline phosphatase., Results: After 37 d, there were no differences in growth performance between control and DON-challenged groups ( P  > 0.05). Nevertheless, groups that received diets with binder had a significantly higher average daily gain (ADG) and average daily feed intake (ADFI) for the first 14 d as well as for the whole period, compared to groups without binder ( P  ≤ 0.05). Groups with binder in the diet also exhibited lower expression of toll-like receptor 4 in distal small intestinal mucosa at d14, compared to groups without binder ( P  ≤ 0.05). Interestingly, comparing the two DON treatments, piglets fed DON and binder had significantly higher ADFI and ADG compared to those with only DON for the first 14-d ( P  ≤ 0.05). Addition of binder to DON contaminated diets, also down-regulated the gene expression of toll-like receptor 4 ( P  ≤ 0.05) and increased mRNA level zona occludens 1 ( P  ≤ 0.10) as compared to DON., Conclusions: The present data provide evidence that the binder improves growth rate in piglets associated with reduction of toll-like receptor-4 and increase of tight junction protein gene expression. However, the current study does not allow to assess whether the effects of the binder are mediated by alterations in the toxicokinetics of the mycotoxin.

Connexins (Cxs) are required for normal embryo development and implantation. They form gap junctions (GJs) connecting the cytoplasm of adjacent cells and hemichannels (HCs), which are normally closed but open in response to stress conditions. Excessive HC opening is detrimental for cell function and may lead to cell death. We found that hatching of in vitro-produced bovine embryos, matured in serum-containing conditions, was significantly improved when vitrification/warming was done in the presence of Gap26 that targets GJA1 (Cx43) and GJA4 (Cx37). Further work showed that HCs from blastocysts produced after oocyte maturation in the presence of serum were open shortly after vitrification/warming, and this was prevented by Gap26. Gap26, applied for the exposure times used, inhibited Cx43 and Cx37 HCs while it did not have an effect on GJs. Interestingly, Gap26 had no effect on blastocyst degeneration or cell death. We conclude that blocking HCs protects embryos during vitrification and warming by a functional effect not linked to cell death.

SeSAME/EAST syndrome is a multisystemic disorder in humans, characterised by seizures, sensorineural deafness, ataxia, developmental delay and electrolyte imbalance. It is exclusively caused by homozygous or compound heterozygous variations in the KCNJ10 gene. Here we describe a similar syndrome in two families belonging to the Malinois dog breed, based on clinical, neurological, electrodiagnostic and histopathological examination. Genetic analysis detected a novel pathogenic KCNJ10 c.986T>C (p.(Leu329Pro)) variant that is inherited in an autosomal recessive way. This variant has an allele frequency of 2.9% in the Belgian Malinois population, but is not found in closely related dog breeds or in dog breeds where similar symptoms have been already described. The canine phenotype is remarkably similar to humans, including ataxia and seizures. In addition, in half of the dogs clinical and electrophysiological signs of neuromyotonia were observed. Because there is currently no cure and treatment is nonspecific and unsatisfactory, this canine translational model could be used for further elucidating the genotype/phenotype correlation of this monogenic multisystem disorder and as an excellent intermediate step for drug safety testing and efficacy evaluations before initiating human studies.

Alexander disease (AxD) is a fatal neurodegenerative disorder of astrocyte dysfunction in man, for which already a number of causal variants are described, mostly de novo dominant missense variants in the glial fibrillary acidic protein (GFAP). A similar disorder was already phenotypically described in animals but without the identification of causal variants. We diagnosed a Labrador retriever with a juvenile form of AxD based on clinical (tetraparesis with spastic front limbs mimicking 'swimming puppy syndrome') and pathological (the detection of GFAP containing Rosenthal fibers in astrocytes) features. In order to identify a causal variant, the coding sequences of the four detected GFAP transcript variants (orthologues from human transcript variants α, γ, δ/ɛ and κ) were sequenced. From the five detected variants, a heterozygous c.719G>A nucleotide substitution resulting in a p.Arg240His substitution was considered to be causal, because it is orthologous to the heterozygous de novo dominant c.716G>A (p.Arg239His) hotspot variant in man, proven to cause a severe phenotype. In addition, the variant was not found in 50 unrelated healthy Labrador retrievers. Because the condition in dogs is morphologically similar to man, it could be a promising animal model for further elucidating the genotype/phenotype correlation in order to treat or prevent this disease.

Retrotransposons are transposable elements that insert extra copies of themselves throughout the genome via an RNA intermediate using a 'copy and paste' mechanism. They account for more than 44% of the bovine genome and have been reported to be functional, especially during preimplantation embryo development. In the present study, we tested whether high oxygen tension (20% O 2 ) influences global DNA methylation analysed by immunofluorescence staining of developing bovine embryos and whether this has an effect on the expression of some selected retrotransposon families. High oxygen tension significantly increased global DNA methylation in 4-cell embryos and blastocysts. A significant expression difference was observed for ERV1-1-I&#95;BT in female blastocysts, but no significant changes were observed for the other retrotransposon families tested. Therefore, the study indicates that global DNA methylation is not necessarily correlated with retrotransposon expression in bovine preimplantation embryos.

Neonates with intrauterine growth restriction (IUGR) show lower efficiency of nutrient utilization compared to normal birth weight (NBW) newborns. This study was conducted using neonatal piglets as a model to test the hypothesis that IUGR affects the intestinal barrier function, intestinal structure, and antioxidant system development during the suckling period. The small intestinal mucosae were obtained from IUGR and NBW littermates in the suckling period (d 0, 3, 8, and 19 postnatal). The epithelial barrier function was assessed by FITC-dextran 4 (FD4) and horseradish peroxidase (HRP) fluxes across the epithelium, histomorphologic measurements, and expression of tight-junction proteins. Redox status represented by the glutathione disulfide/glutathione ratio and malondialdehyde concentrations was determined, whereas mRNA expressions of some redox-sensitive proteins were quantified. Results showed that IUGR piglets exhibited a 2-fold higher intestinal permeability in the proximal small intestine on d 0 (P < 0.05), and this difference between IUGR and NBW piglets was widened to 3 and 4 times for FD4 and HRP, respectively (P < 0.05), on d 3. In accordance, expression of occludin was down-regulated at the transcriptional level in IUGR piglets at d 0 and 19 (P < 0.01). Furthermore, the transcription of heme oxygenase 1, catalase, and thioredoxin reductase genes was down-regulated in IUGR piglets, mainly on postnatal d 0 and 19 (P < 0.01). It appears that IUGR subjects have a lower capacity to mount an antioxidant response in the early postnatal period. Collectively, these results add to our understanding of the mechanisms responsible for intestinal dysfunction in IUGR neonates., (© FASEB.)

Although the equine oviduct clearly affects early embryo development and the selective transport of equine embryos through the oviduct indicates a reciprocal interaction, the influence of the embryo on gene expression in the oviduct remains to be determined in the horse. The aim of this study was to examine this by means of RNA sequencing. Four days after ovulation, epithelial cells ipsilateral and contralateral to the ovulation side from five cyclic and five pregnant mares were collected from the oviduct. RNA was extracted, samples were sequenced, and data analysis was performed to determine differentially expressed genes (DEGs) (P value ≤0.05 and absolute fold change ≥2) and to provide functional interpretation. A total of 10 743 transcripts were identified and 253 genes were found to be upregulated and 108 to be downregulated in the pregnant ipsilateral oviduct when compared to the cyclic ipsilateral oviduct. Comparison of the ipsilateral and the contralateral oviduct indicated 164 DEGs in pregnant mares and 77 DEGs in cyclic mares. Enriched functional categories were detected only in the comparison of pregnant and cyclic ipsilateral oviducts and showed that the equine embryo affects the expression of immune response-related genes in the oviduct, with marked upregulation of interferon-associated genes. This research represents the foundation for further assessment of the role of specific genes in the early embryo-maternal dialogue of the horse., (© 2016 by the Society for the Study of Reproduction, Inc.)

Background: Since the development of in vitro embryo production in cattle, different supplements have been added to culture media to support embryo development, with serum being the most popular. However, the addition of serum during embryo culture can induce high birthweights and low viability in calves (Large Offspring Syndrome). Analysis of global gene expression in bovine embryos produced under different conditions can provide valuable information to optimize culture media for in vitro embryo production., Results: We used RNA sequencing to examine the effect of in vitro embryo production, in either serum-containing or serum-free media, on the global gene expression pattern of individual bovine blastocysts. Compared to in vivo derived embryos, embryos produced in serum-containing medium had five times more differentially expressed genes than embryos produced in serum-free conditions (1109 vs. 207). Importantly, in vitro production in the presence of serum appeared to have a different impact on the embryos according to their sex, with male embryos having three times more genes differentially expressed than their female counterparts (1283 vs. 456). On the contrary, male and female embryos produced in serum-free conditions showed the same number (191 vs. 192) of genes expressed differentially; however, only 44 of those genes were common in both comparisons. The pathways affected by in vitro production differed depending on the type of supplementation. For example, embryos produced in serum-containing conditions had a lower expression of genes related to metabolism while embryos produced in serum-free conditions showed aberrations in genes involved in lipid metabolism., Conclusions: Serum supplementation had a major impact on the gene expression pattern of embryos, with male embryos being the most affected. The transcriptome of embryos produced in serum-free conditions showed a greater resemblance to that of in vivo derived embryos, although genes involved in lipid metabolism were altered. Male embryos appeared to be most affected by suboptimal in vitro culture, i.e. in the presence of serum.

Eukaryotic translation elongation factor 1 alpha (EEF1A) plays a key role in protein synthesis. In higher vertebrates EEF1A occurs in two isoforms, EEF1A1 and EEF1A2, encoded by distinct genes. The purpose of this study was to compare the two porcine genes as for the genomic sequence, gene organization and mRNA expression in different tissues, as well as to search for polymorphism and chromosomal assignment. Standard methods of DNA and mRNA analysis were used. We determined the complete genomic sequence of the porcine EEF1A1 and EEF1A2 genes. The two genes differ in the lengths of transcription units (3102 and 8588 bp, respectively), but have similar genomic organization and their coding sequences are highly similar (78% identity of coding sequences and 92.4% identity of amino acid sequences). Several polymorphisms in the two genes were detected. EEF1A1 and EEF1A2 were mapped to SSC1p11.1 and SSC17q23.3, respectively. mRNA of EEF1A1 was expressed in all studied tissues (the highest expression was in 44-day fetal muscle and low expression in adult liver and brain), while EEF1A2 was expressed only in skeletal-muscle, tongue, heart, diaphragm and brain tissues. EEF1A2 was not expressed in fetal muscle tissue (44 days). In this paper results are provided on genomic sequences, genomic organization, polymorphism, chromosomal assignment and spatial and temporal expressions of the porcine EEF1A1 and EEF1A2 genes. Novel polymorphisms were described in both genes. Porcine EEF1A2 was studied for the first time.

F4 enterotoxigenic Escherichia coli (ETEC) cause diarrhoea and mortality in piglets leading to severe economic losses. Oral immunization of piglets with F4 fimbriae induces a protective intestinal immune response evidenced by an F4-specific serum and intestinal IgA response. However, successful oral immunization of pigs with F4 fimbriae in the presence of maternal immunity has not been demonstrated yet. In the present study we aimed to evaluate the effect of maternal immunity on the induction of a systemic immune response upon oral immunization of piglets. Whereas F4-specific IgG and IgA could be induced by oral immunization of pigs without maternal antibodies and by intramuscular immunization of pigs with maternal antibodies, no such response was seen in the orally immunized animals with maternal antibodies. Since maternal antibodies can mask an antibody response, we also looked by ELIspot assays for circulating F4-specific antibody secreting cells (ASCs). Enumerating the F4-specific ASCs within the circulating peripheral blood mononuclear cells, and the number of F4-specific IgA ASCs within the circulating IgA(+) B-cells revealed an F4-specific immune response in the orally immunized animals with maternal antibodies. Interestingly, results suggest a more robust IgA booster response by oral immunization of pigs with than without maternal antibodies. These results demonstrate that oral immunization of piglets with F4-specific maternal antibodies is feasible and that these maternal antibodies seem to enhance the secondary systemic immune response. Furthermore, our ELIspot assay on enriched IgA(+) B-cells could be used as a screening procedure to optimize mucosal immunization protocols in pigs with maternal immunity.

Previous observational studies suggest an association between polymorphism c.980A>G in the CXCR1 gene, encoding the chemokine (C-X-C motif) receptor 1, and the innate immunity and infection status of the mammary gland. Mammary glands of eight Holstein heifers were experimentally infected with a Staphylococcus chromogenes isolate originating from a chronic intramammary infection (IMI) to study differences between CXCR1 genotypes c.980AG and c.980GG. Quarters from heifers with genotypes c.980AG and c.980GG developed subclinical mastitis but showed differences in the early response at 6-18 h post challenge. Bacterial count at 18 h post challenge tended to be higher in quarters from c.980AG heifers compared to c.980GG heifers. Somatic cell count (SCC) was higher at 6 h post challenge and tended to be higher at 9 h post challenge in c.980AG heifers compared to c.980GG heifers. Milk production decreased similarly. Milk neutrophils of c.980AG heifers showed more apoptosis at 9 h post challenge and tended to show more necrosis at 6, 9 and 12 h post challenge than c.980GG heifers. Differences were less pronounced in the later stage (>18 h) of infection. The results demonstrate that CXCR1 polymorphism can influence SCC and milk neutrophil viability following experimental IMI., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Developmental toxicity testing could greatly benefit from the availability of an in vitro alternative model based on the use of animal embryos that have better human-like physiology than the currently-used alternative models. These current models are insufficient, as extrapolation of the results can be challenging. Therefore, an in vitro bovine embryo culture system was used to expose individual morulae to test substances, and to study developmental characteristics up to the blastocyst stage. Cadmium was chosen as the reference toxicant to investigate the sensitivity of the bovine morulae to various concentrations and exposure times. Oocytes from slaughterhouse-obtained bovine ovaries, were maturated, fertilised and cultured up until the morula stage. Morulae were exposed to different cadmium concentrations for 18 or 70 hours, and developmental competence, embryo quality and the expression of cadmium exposure-related genes were evaluated. Cadmium exposure hampered embryonic developmental competence and quality. Compared with the 18-hour exposure, the 70-hour exposure induced a 20-fold higher toxic response with regard to developmental competence and a more 'cadmium-typical' transcript expression. The bovine morula might be a promising tool for toxicity testing as, following exposure, the embryos reacted in a sensitive and 'cadmium-typical' manner to our reference toxicant., (2015 FRAME.)

Background: Chemokine (C-X-C motif) receptor 1 (CXCR1 or IL-8RA) plays an important role in the bovine mammary gland immunity. Previous research indicated polymorphism c.980A > G in the CXCR1 gene to influence milk neutrophils and mastitis resistance. In the present study, four c.980AG heifers and four c.980GG heifers were experimentally infected with Staphylococcus chromogenes. RNA was isolated from milk somatic cells one hour before and 12 hours after the experimental intramammary challenge. Expression of CXCR1 and eight candidate reference genes (ACTB, B2M, H2A, HPRT1, RPS15A, SDHA, UBC and YWHAZ) was measured by reverse transcription quantitative real-time PCR (RT-qPCR). Differences in relative CXCR1 expression between c.980AG heifers and c.980GG heifers were studied and the effect of the experimental intramammary challenge on relative expression of CXCR1 and the candidate reference genes was analyzed., Results: Relative expression of CXCR1 was not associated with polymorphism c.980A > G but was significantly upregulated following the experimental intramammary challenge. Additionally, differential expression was detected for B2M, H2A, HPRT1, SDHA and YWHAZ., Conclusions: This study reinforces the importance of CXCR1 in mammary gland immunity and demonstrates the potential effect of experimental intramammary challenge on expression of candidate reference genes in milk somatic cells.

The CXCR1 gene plays an important role in the innate immunity of the bovine mammary gland. Associations between single nucleotide polymorphisms (SNP) CXCR1c.735C>G and c.980A>G and udder health have been identified before in small populations. A fluorescent multiprobe PCR assay was designed specifically and validated to genotype both SNP simultaneously in a reliable and cost-effective manner. In total, 3,106 cows from 50 commercial Flemish dairy herds were genotyped using this assay. Associations between genotype and detailed phenotypic data, including pathogen-specific incidence rate of clinical mastitis (IRCM), test-day somatic cell count, and test-day milk yield (MY) were analyzed. Staphylococcus aureus IRCM tended to associate with SNP c.735C>G. Cows with genotype c.735GG had lower Staph. aureus IRCM compared with cows with genotype c.735CC (rate ratio = 0.35, 95% confidence interval = 0.14–0.90). Additionally, a parity-specific association between Staph. aureus IRCM and SNP c.980A>G was detected. Heifers with genotype c.980GG had a lower Staph. aureus IRCM compared with heifers with genotype c.980AG (rate ratio = 0.15, 95% confidence interval = 0.04–0.56). Differences were less pronounced in multiparous cows. Associations between CXCR1 genotype and somatic cell count were not detected. However, MY was associated with SNP c.735C>G. Cows with genotype c.735GG out-produced cows with genotype c.735CC by 0.8 kg of milk/d. Results provide a basis for further research on the relation between CXCR1 polymorphism and pathogen-specific mastitis resistance and MY.