Effects of diet and weight gain on circulating tumour necrosis factor‐α concentrations in Thoroughbred geldings

Low‐grade inflammation precedes the development of obesity‐related metabolic disorders in humans, but whether the same is true in the horse is not known. The objective of this study was to examine the effects of weight gain and diet on the inflammatory state of horses as determined by serum concentrations of tumour necrosis factor‐α (TNF), an inflammatory cytokine. Fifteen mature Thoroughbred geldings with an initial body weight (BW) of 519 ± 12 kg and body condition score (BCS) of 4.3 ± 0.1 were fed a diet of hay plus a concentrate that was either high in non‐structural carbohydrates (NSC) (i.e. starch and sugar), similar to those commercially available (CON) or one that had the energy source replaced with fat and fibre (FAT) for 32 weeks. Weight gain was achieved by feeding an additional 20 Mcal/day in excess of digestible energy maintenance requirements and resulted in a final BW of 608 ± 12 kg and BCS of 6.9 ± 0.1. Horses were exercised twice daily at a walk during the weight gain period. Horses were assessed bi‐weekly for BW and BCS. Serum TNF was analysed from blood samples collected at 4‐week intervals. Although treatment groups began the study with similar mean serum TNF concentrations, 12 weeks of FAT feeding promoted a decrease in circulating TNF that was maintained throughout the study with the exception of weeks 20 and 32. For either diet, there were no linear correlations between serum TNF concentration and BCS when horses increased in BCS from four to seven. The higher level of TNF observed in horses fed the CON diet indicates an increase in some level of systemic inflammation that was independent of their weight gain from a moderately thin to fleshy condition. The influence of diet on serum TNF concentrations should be investigated in horses fed to maintain body condition.     

Both butyrate incubation and hypoxia upregulate genes involved in the ruminal transport of SCFA and their metabolites

Butyrate modulates the differentiation, proliferation and gene expression profiles of various cell types. Ruminal epithelium is exposed to a high intraluminal concentration and inflow of n‐butyrate. We aimed to investigate the influence of n‐butyrate on the mRNA expression of proteins involved in the transmembranal transfer of n‐butyrate metabolites and short‐chain fatty acids in ruminal epithelium. N‐butyrate‐induced changes were compared with the effects of hypoxia because metabolite accumulation after O₂ depletion is at least partly comparable to the accumulation of metabolites after n‐butyrate exposure. Furthermore, in various tissues, O₂ depletion modulates the expression of transport proteins that are also involved in the extrusion of metabolites derived from n‐butyrate breakdown in ruminal epithelium. Sheep ruminal epithelia mounted in Ussing chambers were exposed to 50 mM n‐butyrate or incubated under hypoxic conditions for 6 h. Electrophysiological measurements showed hypoxia‐induced damage in the epithelia. The mRNA expression levels of monocarboxylate transporters (MCT) 1 and 4, anion exchanger (AE) 2, downregulated in adenoma (DRA), putative anion transporter (PAT) 1 and glucose transporter (GLUT) 1 were assessed by RT‐qPCR. We also examined the mRNA expression of nuclear factor (NF) κB, cyclooxygenase (COX) 2, hypoxia‐inducible factor (HIF) 1α and acyl‐CoA oxidase (ACO) to elucidate the possible signalling pathways involved in the modulation of gene expression. The mRNA expression levels of MCT 1, MCT 4, GLUT 1, HIF 1α and COX 2 were upregulated after both n‐butyrate exposure and hypoxia. ACO and PAT 1 were upregulated only after n‐butyrate incubation. Upregulation of both MCT isoforms and NFκB after n‐butyrate incubation could be detected on protein level as well. Our study suggests key roles for MCT 1 and 4 in the adaptation to an increased intracellular load of metabolites, whereas an involvement of PAT 1 in the transport of n‐butyrate also seems possible.     

Duration of feeding linseed diet influences peroxisome proliferator-activated receptor γ and tumor necrosis factor gene expression, and muscle mass of growing–finishing barrows

The aim of the study was to investigate the effect of duration of feeding linseed (rich in n-3 PUFA) on peroxisome proliferator-activated receptor γ (PPARγ) and tumor necrosis factor (TNF-α) gene expression, and muscle mass of growing–finishing barrows. Two isoenergetic and isonitrogenous diets were formulated, and one of which was the basal diet and another one was the linseed diet including linseed at the level of 10%. Twenty-four Landrace × Yorkshire barrows weighing 35 ± 3.7 kg were randomly assigned to four treatments with six individuals per treatment. Pigs in treatment 1 (T1) fed the control diet throughout the experimental period, while pigs in T2, T3 and T4 fed the control diet except for 30, 60, and 90 d prior to slaughter when the linseed diet were fed. The experiment was conducted for 90 days. The longissimus muscle mass and each muscle mass in the hind leg were weighted. PPARγ and TNF-α mRNA expression levels in muscle, spleen and adipose tissue, and plasma concentrations of TNF-α data were measured and analyzed. The results showed that the longissimus muscle mass, quadriceps femoris muscle mass and semitendinosus muscle mass increased linearly (P < 0.01) as prolonged the time of feeding linseed diet. The expression of PPARγ in longissimus muscle and spleen increased (P < 0.01) linearly as prolonged the time of feeding linseed diet, while the expression of PPARγ in adipose tissue were not affected (P = 0.095). Duration of linseed addition linearly decreased (P < 0.01) TNF-α gene expression levels in the longissimus dorsi muscle, adipose and spleen, and serum concentration of TNF-α as well. The expression levels of PPARγ negatively correlated with the expression of TNF-α in muscle (R² = 0.70, P < 0.001) and spleen (R²R² = 0.77, P < 0.001) respectively. Likewise, PPARγ expression level in spleen (R²R² = 0.59, P < 0.01) or muscle (R²R² = 0.52, P < 0.05) negative correlated with serum TNF-α concentration, while there were significant quadratic relation between muscular PPARγ (R²R² = 0.80, P < 0.01) or muscular TNF-α (R²R² = 0.87, P < 0.01) expression and the longissimus dorsi muscle mass. These data suggested that duration of feeding linseed diet lead to a linear decrease of TNF-α gene expression, which may increase the muscle mass in growing–finishing barrows, at least in part, through a PPARγ-dependent mechanism.     

Immune responses of recombinant adenovirus co-expressing VP1 of foot-and-mouth disease virus and porcine interferon alpha in mice and guinea pigs

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating vesicular disease of cloven-hoofed animals. In this study, we constructed and characterized the immune responses and vaccine efficacy conferred by the recombinant adenovirus co-expressing VP1 of FMDV and porcine interferon alpha as fusion protein (rAd-pIFNalpha-VP1). Six groups of female BALB/c mice each with 18 were inoculated subcutaneously twice 2-week intervals with the recombinant adenoviruses. The results showed that the levels of humoral and cell-mediated immune responses in the group inoculated with rAd-pIFNalpha-VP1 were significantly higher than those in the group inoculated with rAd-VP1+rAd-pIFNalpha (P<0.05). Then four groups of guinea pigs each with six were inoculated two times at 2-week intervals intramuscularly with rAd-pIFNalpha-VP1, commercial inactivated FMD vaccine, wild-type adenovirus (wtAd) or PBS, and the protective efficacy of rAd-pIFNalpha-VP1 was determined. The results indicated that all the guinea pigs vaccinated with rAd-pIFNalpha-VP1 as well as inactivated FMD vaccine were protected from FMDV challenge, even though the levels of neutralizing antibodies (1:32-1:40) of the animals vaccinated with rAd-pIFNalpha-VP1 was lower than that in the group inoculated with inactivated FMD vaccine (1:64-1:128). It demonstrated that the newly recombinant adenovirus rAd-pIFNalpha-VP1 might further be an attractive candidate vaccine for preventing FMDV infection in swine.     

Paired box 7 inhibits differentiation in 3T3‐L1 preadipocytes

Myogenesis is precisely proceeded by myogenic regulatory factors. Myogenic stem cells are activated, proliferated and fused into a multinuclear myofiber. Pax7, paired box 7, one of the earliest markers during myogenesis. It has been reported that Pax7 regulates the muscle marker genes, Myf5 and MyoD toward differentiation. The possible roles of Pax7 in myogenic cells have been well researched. However, it has not yet been clarified if Pax7 itself is able to induce myogenic fate in nonmyogenic lineage cells. In this study, we performed experiments using stably expressed Pax7 in 3T3‐L1 preadipocytes to elucidate if Pax7 inhibits adipogenesis. We found that Pax7 represses adipogenic markers and prevents differentiation. These cells showed decreased expression of PDGFRα, PPARγ and Fabp4 and inhibited forming lipid droplets.     

The Effect of Cysteine-Rich Secretory Protein-3 and Lactoferrin on Endometrial Cytokine mRNA Expression After Breeding in the Horse

The equine uterus undergoes a transient innate immune response after breeding, also known as mating-induced endometritis. The deposition of spermatozoa triggers the expression of pro-inflammatory cytokines, which results in the migration of polymorphonuclear neutrophils (PMNs) into the endometrium and the uterine lumen. Select seminal plasma proteins, specifically cysteine-rich secretory protein 3 (CRISP-3) and lactoferrin, have been shown to affect the activity of the PMNs, either by suppressing (CRISP-3) or promoting (lactoferrin) the phagocytosis of spermatozoa based on their viability in vitro. Conjointly, many components of inseminate, including seminal plasma, bacteria, and spermatozoa itself, have shown to have an effect on the expression of endometrial cytokines after breeding. The objective of this study was to determine if select proteins affect the mRNA expression of endometrial cytokines after insemination. Six mares were bred during four consecutive estrous cycles with treatments in randomized order of: 1mg/mL CRISP-3, 150 ug/mL lactoferrin, seminal plasma, or Lactated Ringer’s Solution (LRS) to a total volume of 10 mL combined with 1×10⁹ progressively motile spermatozoa pooled from two stallions. Six hours after treatment, an endometrial biopsy was obtained for qPCR analysis. No treatment effects were found for the mRNA expression of IL-1β, IL-6, IL-8, IL-10, TNFα, and IFNγ, while lactoferrin significantly suppressed the mRNA expression of IL-1RN when compared to LRS. In conclusion, the seminal plasma proteins CRISP-3 and lactoferrin have minimal effect on the expression of select endometrial cytokines at 6 hours post breeding.     

Expression of VEGFR and PDGFR‐α/‐β in 187 canine nasal carcinomas

Radiotherapy represents the standard of care for intranasal carcinomas. Responses to tyrosine kinase inhibitors (TKIs) have been reported but data on expression of target receptor tyrosine kinases (rTKs) is limited. This study characterizes the expression of vascular endothelial growth factor receptor (VEGFR), platelet‐derived growth factor receptor (PDGFR)‐α and PDGFR‐β in canine intranasal carcinomas. Histological samples from 187 dogs were retrieved. Immunohistochemistry was performed using commercially available antibodies. Expression of rTKs was classified into weak, moderate or intense and additionally recorded as cytoplasmic, membranous, cytoplasmic‐membranous, nuclear or stromal. VEGFR was expressed in 158 dogs with predominantly moderate expression (36.9%) and a cytoplasmic‐membranous expression pattern (70.9%). PDGFR‐α was detected in 133 with predominantly weak expression (57.9%) and cytoplasmic pattern (87.9%). PDGFR‐β was identified in 74 patients with a predominantly moderate expression (17.6%) and cytoplasmic expression pattern (63.5%). Co‐expression of rTKs was common. These results confirm expression of VEGFR, PDGFR‐α and PDGFR‐β in canine intranasal carcinomas and support the utility of TKIs.     

Expression of TopBP1 in canine mammary neoplasia in relation to histological type, Ki67, ERalpha and p53

TopBP1 is aberrantly expressed in human and feline mammary carcinomas, but expression of this BRCA1-related protein has not been investigated in canine mammary carcinomas. In this study, 132 canine mammary tumours (46 benign, 86 carcinomas) were examined immunohistochemically for expression of TopBP1, oestrogen receptor α (ERα), Ki67 and p53. Positive staining for TopBP1 was evident in all canine mammary lesions, although five samples had <20% positive cells. The number of samples with high levels of staining increased in different categories from benign mixed tumour to adenoma to carcinoma. Most TopBP1 staining was nuclear, but both nuclear and cytoplasmic staining were observed as the degree of malignancy increased, similar to human and feline mammary carcinomas. Benign mixed tumours, however, had more cytoplasmic staining than adenomas. Expression of p53 and the proliferation marker Ki67 increased from benign mixed tumour to adenoma to carcinoma, but the differences between benign and malignant tumours were more distinct than for TopBP1 expression. ERα expression decreased from malignant to benign tumours, although over half of the benign mixed tumours were negative. TopBP1 was expressed in canine mammary tumours at higher levels than has been reported previously for cats, although the shift in cellular localisation with malignancy was similar.     

Effect of prostaglandin F2alpha on ovarian, adrenal, and pituitary hormones and on luteal blood flow in mares

The effect of a single injection of prostaglandin F2alpha (PGF) during mid-diestrus on systemic concentrations of progesterone, LH, FSH, estradiol, and cortisol and on blood flow to the corpus luteum was studied in 10 controls and 10 PGF-treated mares. Blood flow was assessed by estimating the percentage of corpus luteum with color-Doppler signals of blood flow during real-time scanning of the entire structure and by the diameter of the vascular pedicle near its attachment to the ovary. Treatment was done 8 days after ovulation and 0 h was immediately before the treatment. Examinations and collection of blood samples were done at 0 h, every 5 min until 1h, and then at 1.5, 2, 4, 8, 12, 24, 48, and 72 h. The concentrations of estradiol did not change, but progesterone, LH, FSH, and cortisol increased significantly within 5 min. Concentrations of LH and FSH in the PGF group remained elevated until a temporarily lower concentration at 8 or 4h, respectively, rebounded to 12h, and then slowly decreased. Cortisol remained elevated, until a decrease between 1 and 4h. Progesterone in the PGF group increased significantly until 10 min after 0 h and then decreased by 40 min to below the concentrations in controls. Within the PGF group, progesterone decreased significantly by 45 min to below the concentrations at 0 h. The values for each of the two indicators of blood flow did not differ significantly between the PGF and control groups until a decrease at 24h in the PGF group. Results did not support the hypothesis that the immediate transient post-PGF increase in progesterone was associated with an increase in luteal blood flow. Luteolysis, as indicated by decreasing progesterone, began well before the beginning of a decrease in luteal blood flow.     

Treatment and resolution of zilpaterol hydrochloride toxicity in a Quarter Horse gelding

Zilpaterol hydrochloride is a β‐2 agonist utilised as a repartitioning agent in cattle. In recent years, it has also been used illicitly among human athletes and body builders, and administered to racehorses. While there are numerous anecdotal reports of the effects of zilpaterol in horses, this is the first documented clinical case of equine zilpaterol toxicity. The horse presented with marked tachycardia, profuse sweating and generalised muscle fasciculations. Ultimately, treatment was initiated with propranolol and the horse made a complete recovery.     

The innate immune response against Brucella in humans

Pathogens have developed different strategies to survive and multiply within their host. Among them is the ability to control phagocyte apoptosis while another is to affect the expression of cytokines which is necessary for a normal protective function of the immune response. To establish themselves and cause chronic disease in humans and animals, Brucella spp. invade and proliferate within monocytic phagocytes. We have established that in humans, Brucella suis impairs the apoptosis of monocytes and macrophages, thus preventing its host cell elimination. In mice, which are not naturally colonized by the bacteria, Brucella infection results in Type1 (Th1) cellular immune response which promotes a clearance of the bacterial organism. The development of this response is under the control of major cytokines like TNF-alpha, IFN-gamma and IL-12 produced at the onset of infection. We have observed that in humans, B. suis-infected macrophages which produce IL-1, IL-6, IL-10 and several chemokines including IL-8, do not secrete TNF-alpha. By constructing null mutants, we demonstrated that this inhibition involves the outer membrane protein Omp25 of Brucella, however the mechanism regulating the inhibition has not yet been clearly defined. It is likely that the Omp25-induced effect on TNF-alpha production assists bacterial evasion of antimicrobial defences at different levels. Firstly, by preventing the autocrine activation of macrophages thus inhibiting innate immunity and secondly by impairing the production of IL-12 and the development of a Th1 type specific immunity. In addition to the central role of the macrophage in Brucella infection, others cells of the innate immune response are recruited and influenced by the interactions between bacteria and host. For instance, human Vgamma9Vdelta2 T-cells play an important role in the early response to infection with intracellular pathogens. Evidence has been presented that their number dramatically increased in the peripheral blood of patients with acute brucellosis. We have shown that human Vgamma9Vdelta2 T-cells can be specifically activated by non-peptidic low molecular weight compound(s) from B. suis lysate or by soluble factors produced by B. suis-infected macrophages. Under these conditions, they produce TNF-alpha and IFN-gamma and reduce the bacterial multiplication inside infected autologous macrophages. This impairment of B. suis multiplication is due to both soluble factors released from activated gammadeltaT-cells (including TNF-alpha and IFN-gamma) and to a contact-dependent cytotoxicity directed against the infected cells. The interactions between the bacteria and these cells can counteract the intramacrophagic development of the bacteria and finally influence the further development of the host defense. We hypothesize that the chronicity or the elimination of the infection will depend on the balance between contradictory effects induced by the bacteria which favor either the host or the pathogen. Moreover, the interrelationship between the different cells must be taken into account in the analysis of the virulence of the bacteria and in the development of in vitro models of human macrophage infection.     

Response of whole blood, erythrocyte and plasma vitamin E content to dietary vitamin E intake in the chick

Whole blood, red blood cells (RBC), and plasma vitamin E (VE) levels in chicks fed dietary VE (dl-alpha-tocopheryl acetate, dl-alpha Ta) supplementation in steps of 0.0, 5.0, 10.0, 15.0, 20.0 and 30.0 mg/Kg were determined to examine their usefulness as an index of VE status. The increase in VE level was significant and linear in whole blood (r = 0.90), RBC (r = 0.89) and plasma (r = 0.93) in response to dietary VE intake. There was a close correlation between VE in plasma vs whole blood (r = 0.90), plasma vs RBC (r = 0.91) and whole blood vs RBC (r = 0.95). The plasma VE content was 1.2-1.8 times greater than that of whole blood, and 6.6-12.5 times greater than that of RBC. The plasma total lipids content was not affected by the dietary VE intake, whereas the level of VE in the plasma total lipids was significantly increased with increasing supplementation. Alpha tocopherol was the major isomer (ca 92%) of VE in whole blood, RBC and plasma at hatching. The small proportions of beta-tocopherol (ca 2%), gamma-tocopherol (ca 5%) and alpha-tocotrienol (ca 1%) observed at 1 day of age had decreased or totally disappeared by 7 days of age after feeding the VE-free basal diet. The data showed that in the chick, the whole blood and RBC levels of VE were as sensitive and reliable indexes of dietary VE status as was that of the plasma.     

Effects of hyper‐ and hypothyroidism on the development and proliferation of testicular cells in prepubertal rats

Thyroid hormones are important in the development and regulation of testes. This study was conducted to determine the effects of hyper‐ and hypothyroidism on testicular development in prepubertal rats aged 20–70 days. Weaning male rats (20 days old) until day 70 age were randomly divided into four groups: control, hyperthyroid (hyper‐T), hypothyroid (hypo‐T) and hypothyroid treated with thyroxine (T4) (hypo‐T+T4). The results indicated that thyroid hormones caused a significant effect in body and testis weights, and food and water consumption. In addition there were changes in serum concentrations of tri‐iodothyronine, T4, thyroid stimulating hormone (TSH) and testosterone. Histomorphology showed a significant decrease in seminiferous tubule diameter in hyper‐T compared to the other groups. Leydig cell numbers showed a significant elevation in hyper‐T but not in hypo‐T groups. Immunostaining indicated that TSH receptor (TSHR), thyroid hormone receptors α/β (TRαβ) and proliferating cell nuclear antigen (PCNA) have the roles in testicular development. Our findings suggest that hyper‐ and hypo‐thyroidism regulate testicular cell proliferation and spermatogenesis in prepubertal rats, indicating that expression of TSHR, TRαβ and PCNA may be regulated by thyroid hormones that are involved in testicular development; and that the administration of T4 to the hypo‐T+T4 group leads to an improvement in the testicular condition.     

Detection of anti‐TNFα activity in canine hyperimmune serum using a TNFα inhibition assay

Background: Increased serum tumor necrosis factor‐α (TNFα) activity has been associated with onset of serious inflammatory diseases in dogs. Development of treatment with TNFα‐antagonists has been limited by the unavailability of suitable reagents and potency assays for TNFα. Objectives: The objectives of this study were to optimize a cell‐based assay to measure anti‐TNFα activity in serum and plasma from hyperimmune (vaccinated with an Escherichia coli J5 bacterin) and unvaccinated canine donors; to use the assay to determine whether hyperimmune serum inhibits TNFα activity in vivo; and to determine whether soluble TNF receptor‐1 (sTNFR1, a naturally occurring TNFα antagonist) contributes to anti‐TNFα activity. Methods: Commercial plasma and serum from hyperimmune‐frozen plasma (HFP) donors and unvaccinated fresh‐frozen plasma (FFP) donors were used in the study. An L929‐cell TNFα‐inhibition assay (LTIA) was optimized to measure anti‐TNFα activity. Using a rat subcutaneous pouch model of inflammation, the effects of HFP, FFP, a synthetic TNFα antagonist (Etanercept), and carprofen on TNFα activity were compared in vivo. Immunofluorescence was used to measure soluble sTNFR1 concentration. Results: Using the optimized LTIA, HFP serum but not FFP serum decreased canine TNFα activity (P<.01). HFP plasma and Etanercept (but not FFP plasma or carprofen) significantly decreased TNFα activity in pouch exudates (P<.05). A significantly higher concentration of sTNFR1 was found in HFP than FFP serum. Conclusions: Using the LTIA, anti‐TNFα activity is readily measured in canine serum and inflammatory exudates. sTNFR1 appears to contribute to anti‐TNFα activity in HFP serum. These results suggest HFP should be investigated further as a potential immunotherapeutic agent for controlling canine diseases in which TNFα is implicated.     

β‐hydroxy‐β‐methyl butyrate promotes leucine metabolism and improves muscle fibre composition in growing pigs

The aim of this study was to investigate the effects of excess leucine (Leu) vs. its metabolites α‐ketoisocaproate (KIC) and β‐hydroxy‐β‐methyl butyrate (HMB) on Leu metabolism, muscle fibre composition and muscle growth in growing pigs. Thirty‐two pigs with a similar initial weight (9.55 ± 0.19 kg) were fed 1 of 4 diets for 45 days: basal diet, basal diet + 1.25% L‐Leu, basal diet + 1.25% KIC‐Ca, basal diet + 0.62% HMB‐Ca. Results indicated that relative to the basal diet and HMB groups, Leu and KIC groups exhibited increased Leu concentrations and decreased concentrations of isoleucine, valine and EAAs in selected muscle (p < 0.05) and had lower mRNA levels of MyHC I and higher expression of MyHC IIx/IIb (p < 0.05), and there was no significant difference between the basal and HMB‐supplemented groups. Moreover, the mRNA expression levels of AMPKα and UCP3 were higher but the myostatin mRNA levels were lower in the soleus muscle of the HMB group than those from other groups (p < 0.05). These findings demonstrated that doubling dietary Leu content exerted growth‐depressing effects in growing pigs; dietary KIC supplementation induced muscular branched‐chain amino acid imbalance and promoted muscle toward a more glycolytic phenotype; while dietary HMB supplementation promoted the generation of more oxidative muscle types and increased muscle growth specially in oxidative skeletal muscle, and these effects of HMB might be associated with the AMPKα‐Sirt1‐PGC‐1α axis and mitochondrial biogenesis.     

Expression analysis of an α‐1, 3‐galactosyltransferase,an enzyme that creates xenotransplantation‐related α–Gal epitope,in pig preimplantation embryos

α‐1,3‐Galactosyltransferase (α‐GalT), an enzyme creating Galα1‐3Gal (α‐Gal) epitope on the cell surface in some mammalian species such as pigs, is known to be a key factor that causes hyperacute rejection upon transplantation from pigs to humans. To establish the RNA interference‐based suppression of endogenous α‐GalT messenger RNA (mRNA) synthesis in porcine preimplantation embryos, we determined the suitable embryonic stage at which stage such approach is possible by using the semi‐quantitative RT‐PCR (qRT‐PCR) and the cytochemical method using a fluorescence‐labeled Bandeiraea simplicifolia Isolectin B₄ (BS‐I‐B₄). Staining with BS‐I‐B₄ demonstrated that α‐Gal epitope expression was first recognized at the 8‐cell stage, and increased up to the hatched blastocyst stage. Single embryo‐based qRT‐PCR also confirmed this pattern. These results indicate that creation of α‐Gal epitope is proceeded by de novo synthesis of α‐GalT mRNA in porcine preimplantation embryos with peaking at the blastocyst stage.     

Abnormal synaptic protein expression in two Arabian horses with equine degenerative myeloencephalopathy

Numerous swollen neurons and multiple dystrophic axons were observed in the gracillis and cuneatus nuclei of two male Arabian horses, aged six and 12 months of age, with equine degenerative myeloencephalopathy. Swollen neurons and dystrophic axons showed synaptophysin, synaptosomal-associated protein of 25 kDa, syntaxin-1 and alpha-synuclein immunoreactivity. Moreover, dystrophic axons were strongly immunopositive against the ubiquitin protein and against the anti-phosphorylated 200 kDa neurofilament protein. Abnormal expression of integral synaptic vesicle, synaptic vesicle-associated presynaptic plasma membrane and cytosolic proteins, which participate in the trafficking, docking and fusion of the synaptic vesicle to the plasma membrane, suggest that severe disruption of axonal transport plays a crucial role in the pathogenesis of dystrophic axons in equine degenerative myeloencephalopathy (EDM).     

Effects of acute fasting and age on leptin and peroxisome proliferator‐activated receptor gamma production relative to fat depot in immature and mature pigs

Leptin and peroxisome proliferator‐activated receptor gamma (PPARγ) are adipogenic proteins that are actively involved in metabolic homeostasis of fat. Recently, it was reported that fat tissue in humans and rodents differs in metabolic activity relative to anatomical location of the fat tissue (i.e. depots) and animal age. Hence, we hypothesized that leptin and PPARγ production in various fat depots in female pigs differs in response to acute fasting, and that these responses vary with physiological maturity of the animal. Sixteen intact crossbred immature female pigs [prepubertal (PP); 132.2 ± 4.1 days] and 16 sexually mature female pigs (M; 224 ± 7.4 days) housed in an open‐air, concrete slab, sheltered barn were randomly assigned to either Control or Fasted treatments. Control pigs (PP, n = 8; M, n = 8) had ad libitum access to feed, while Fasted pigs (PP, n = 8; M, n = 8) were denied access to feed from the onset of the study (0 h) to euthanasia at 72 h. Immediately post‐mortem, fat samples were collected from the subcutaneous, pelvic, kidney, and heart (M pigs only) fat depots and analysed for leptin and PPARγ mRNA and protein content. Acute fasting decreased mean leptin mRNA tissue content in a depot specific manner in M pigs (p < 0.01), while mean leptin protein concentrations in fat tissues did not differ with fat depot or age of the pig. Furthermore, acute fasting did not affect mean PPARγ mRNA tissue content in a fat depot or age dependent manner. Mean concentrations of PPARγ protein in fat depots tended to be greater in M vs. PP pigs (p = 0.07). We suggest that these data provide evidence that acute fasting has a greater effect on leptin than PPARγ production in a fat depot dependent manner in M pigs, which may be indicative of changing physiological demands as an animal matures.     

Integrin heterodimer α9β1 is localized on the surface of porcine spermatogonial stem cells in the undifferentiated state

A previous study found that undifferentiated porcine spermatogonial stem cells (SSCs) did not adhere to tenascin C, indicating that the integrin α₉ and β₁ subunits are inactive on the surface of porcine SSCs. However, that study used recombinant tenascin C without FNIII‐like repeats. Therefore, this study re‐evaluated the existence of integrin α₉β₁ actively functioning on the plasma membrane of porcine SSCs using full‐length native tenascin C with FNIII‐like repeats. The localization and function of the integrin heterodimer were confirmed using immunocytochemistry, attachment and antibody inhibition assays. In undifferentiated porcine SSCs with integrin α₉β₁ on the cell surface, adhesion to native tenascin C was significantly higher compared with cells lacking native tenascin C and functional blocking of integrin α₉β₁ significantly inhibited the attachment to native tenascin C compared with no functional blocking. Accordingly, we confirmed that the integrin α₉ and β₁ subunits function as an active heterodimer on the surface of porcine SSCs in the undifferentiated state.