Nucleotide sequence polymorphisms of beta1‐, beta2‐, and beta3‐adrenergic receptor genes on Jinhua,Meishan, Duroc and Landrace pigs

The full amino acid coding sequences of adrenergic receptor genes beta1, beta2, and beta3 (ADRB1, ADRB2, and ADRB3)were determined for Jinhua, Meishan, Duroc and Landrace pigs. Non‐synonymous substitution of Arg458Pro was found in the porcine ADRB1 gene, resulting in a 469 amino acid sequence. Continuous substitutions of Asn29Asp and Glu30Gln were found in the porcine ADRB2 gene, resulting in a 418 amino acid sequence. Additionally, a Lys30 polymorphism of the ADRB2 gene was found in the Jinhua pigs. There were three non‐synonymous substitutions of Asn24Thr, Arg264Gln and Asn398Asp on the porcine ADRB3 gene. A thymine insertion in the ADRB3 gene, resulting in a protein with two fewer amino acids, was found in the Jinhua and Meishan pigs. To assess the effect of ADRB polymorphisms on porcine subcutaneous fat layer thickness, we calculated the genetic frequency of the variants in fatty and lean groups, each consisting of 24 pigs that were crossbreds of Duroc and Jinhua pigs. The effect of the ADRB3 gene polymorphism was not evaluated, because there was insufficient variation on the ADRB3 gene in the examined groups. Although Fisher's exact test showed no significant difference in the frequency of ADRB1 and ADBR2 variants between the two groups, the Arg458 variant of ADRB1 was higher (P = 0.11) in the lean group, and pigs in that group had a thinner fat layer than did those with the Pro458 variant. These results imply a possibility of ADRB1 polymorphism as a minor factor in porcine fat layer thickness. The Asp29 variant of ADRB2 was higher in the lean group (P = 0.11), and the Glu30 variant was higher in the fatty group (P = 0.15), but the Asp29 variant was found only in the Chinese pigs. Thus, the effect of ADRB2 polymorphisms was not clear in this study.     

The novel polymorphism of the beta 3-adrenergic receptor gene and its distribution in domestic pigs and wild boars in Asia

The beta 3‐adrenergic receptor (ADRB3) is a G protein‐coupled receptor that is involved in regulating energy homeostasis. We have studied DNA sequences of porcine ADRB3 to find candidate genetic polymorphisms for economically important growth and performance traits in pigs. Five novel haplotypes derived from the three In/dels and 44 SNPs were identified among domestic pigs and wild boars. Three of them encode non‐synonymous amino acid sequences by five missense polymorphisms and a frameshift by a thymine insertion. The amino acid polymorphic sites were distributed as follows: one substitution was in extracellular loop 1, three substitutions were in intracellular loop3 and one substitution and the deletion of two amino acids were at the carboxyl‐terminal. There was no polymorphism in the transmembrane domains. In addition, we surveyed the allelic frequency of the thymine insertion that cause frameshift in South‐east Asian local pigs, including some commercial breeds and wild boars. This thymine insertion was distributed widely in the domestic pigs and wild boars. The frequencies of this allele were relatively low in Western improved breeds, while they were very common in Asian breeds and wild boars in Asia. This result indicates that this insertion originally occurred in ancient Asian wild boars and then circulated among Asian domestic pigs. This allele also spread over Western breeds, probably through the introgression of Asian pigs into European stocks during the 18th and 19th centuries.     

Association of an SNP marker in exon 24 of a class 3 phosphoinositide‐3‐kinase (PIK3C3) gene with production traits in Duroc pigs

A C?T single nucleotide polymorphism (SNP) on exon 24 of the porcine class 3 phosphoinositide‐3‐kinase (PIK3C3) gene is considered a possible genetic marker for selecting backfat (BF) thickness and carcass fat, although only one study has published results on its effects by performing experiments on a single resource family. We analyzed the association of this PIK3C3 polymorphism with production traits in 739 Duroc pigs. The C allele frequency was 67.9% in our study population. PIK3C3 polymorphism showed significant effects on average daily weight gain (ADG), BF thickness, intermuscular fat content (IMF), and the size of the loin eye muscle area (EMA). The C alleles increased ADG, BF and IMF, and decreased EMA. The predicted differences in traits between the homozygous pigs of the C and T alleles were 40 g/day for DG, 1.2 mm for BF, 0.44% for IMF, and 1.6 cm² for EMA. Furthermore, the statistical models for estimating the breeding values of each trait had lower Akaike's information criterion values when adding PIK3C3 genotype information. We therefore confirmed that the polymorphism in PIK3C3 (C2604T) has the potential to be a genetic marker for production traits in Duroc pigs.     

Identification and examination of a novel 9‐bp insert/deletion polymorphism on porcine SFTPA1 exon 2 associated with acute lung injury using an oleic acid‐acute lung injury model

The pulmonary surfactant‐associated protein (SFTPA1, SP‐A) gene has been studied as a candidate gene for lung disease resistance in humans and livestock. The objective of the present study was to identify polymorphisms of the porcine SFTPA1 gene coding region and its association with acute lung injury (ALI). Through DNA sequencing and the PCR‐single‐strand conformation polymorphism method, a novel 9‐bp nucleotide insertion (+) or deletion (‐) was detected on exon 2 of SFTPA1, which causes a change in three amino acids, namely, alanine (Ala), glycine (Gly) and proline (Pro). Individuals of three genotypes (?/?, +/? and +/+) were divided into equal groups from 60 Rongchang pigs that were genotyped. These pigs were selected for participation in the oleic acid (OA)‐ALI model by 1‐h and 3‐h injections of OA, and there were equal numbers of pigs in the control and injection groups. The lung water content, a marker for acute lung injury, was measured in this study; there is a significant correlation between high lung water content and the presence of the 9‐bp indel polymorphism (P < 0.01). The lung water content of the OA injection group was markedly higher than that of the control group and lung water content for the +/+ genotype was significantly higher than that of the others in the 1‐h group (P < 0.01). No differences in the expression of the SFTPA1 gene were found among individuals with different SFTPA1 genotypes, indicating that the trait is not caused by a linked polymorphism causing altered expression of the gene. The individuals with the ?/? genotype showed lower lung water content than the +/+ genotype pigs, which suggests that polymorphism could be a potential marker for lung disease‐resistant pig breeding and that pig can be a potential animal model for human lung disease resistance in future studies.     

Effects of transforming growth factor‐β1 treatment on muscle regeneration and adipogenesis in glycerol‐injured muscle

Transforming growth factor (TGF)‐β1 is associated with fibrosis in many organs. Recent studies demonstrated that delivery of TGF‐β1 into chemically injured muscle enhances fibrosis. In this study, we investigated the effects of exogenous TGF‐β1 on muscle regeneration and adipogenesis in glycerol‐injured muscle of normal mice. Tibialis anterior (TA) muscles were injured by glycerol injection. TGF‐β1 was either co‐injected with glycerol, as an ‘early treatment’ group, or injected at day 4 after glycerol, as a ‘late treatment’ group and the TA muscles were collected at day 7 after initial injury. Myotube density was significantly lower in the early treatment group than in the glycerol‐injured group (without TGF‐β1 treatment). Moreover, the Oil red O‐positive area was significantly smaller in the early treatment group than in the late treatment group and glycerol‐injured group. Furthermore, TGF‐β1 treatment increased endomysial fibrosis and induced immunostaining of α‐smooth muscle actin. The greater inhibitory effects of early TGF‐β1 treatment than that of late TGF‐β1 treatment during regeneration in glycerol‐injured muscle suggest a more potent effect of TGF‐β1 on the initial stage of muscle regeneration and adipogenesis. Combination of TGF‐β1 with glycerol might be an alternative to enhance muscle fibrosis for future studies.     

Time‐coordinated prevalence of extracellular HGF,FGF2 and TGF‐β3 in crush‐injured skeletal muscle

Successful regeneration and remodeling of neuromuscular junctions are critical for restoring functional capacities and properties of skeletal muscle after damage, and axon‐guidance molecules may be involved in the signaling that regulates such restoration. Recently, we found that early‐differentiated satellite cells up‐regulate a secreted neural chemorepellent Sema3A upon in vivo muscle‐crush injury. The study also revealed that Sema3A expression is up‐regulated in primary satellite‐cell cultures in response to hepatocyte growth factor (HGF) and basic fibroblast growth factor (FGF2) and is prevented by transforming growth factor (TGF)‐β2, 3. In order to verify the physiological significance of this regulation in vitro, the present study was designed to estimate the time‐course of extracellular HGF, FGF2 and TGF‐β3 concentrations after crush‐injury of Gastrocnemius muscle in the rat lower hind‐limb, using a combination of a non‐homogenization/non‐spin extraction of extracellular wound fluids and enhanced chemiluminescence–Western blotting analyses. Results clearly demonstrated that active HGF and FGF2 are prevalent in 2–8 days post‐crush, whereas active TGF‐β3 increases after 12 days, providing a better understanding of the time‐coordinated levels of HGF, FGF2 and TGF‐β3 that drive regulation of Sema3A expression during regenerative intramuscular moto‐neuritogenesis.     

Sudden death in the presence of overt β‐adrenergic receptor activation in guinea pigs immediately following isoflurane anesthesia

Observations A case series of sudden death is reported in five consecutive guinea pigs following anesthesia with inhalational isoflurane during β‐adrenergic receptor stimulation with isoproterenol. Sustained‐release isoproterenol pellets or mini‐osmotic pumps were implanted subcutaneously in male Dunkin–Hartley guinea pigs as part of a research study to assess the interplay of adrenergic receptor activation and the development of atrial arrhythmias. The continuous exposure to isoproterenol resulted in a similar presentation and eventual sudden death in all guinea pigs exposed to inhalational isoflurane between 15 to 40 minutes after discontinuation of anesthesia. Death occurred in guinea pigs in this case series despite the fact that doses of isoproterenol used were more than 10‐fold lower than previously reported in guinea pigs in the absence of isoflurane anesthesia. The cause of death was suspected to be due to an interaction of isoproterenol with isoflurane anesthesia, as placebo implantation or anesthesia alone did not result in cardiac arrest. Of four subsequent guinea pigs anesthetized with the combination of xylazine and ketamine (X/K), three survived isoproterenol implantation for the full 21‐day study period while one died perioperatively. Conclusions There was an increased rate of post‐anesthetic mortality associated with isoproterenol pellet implantation in guinea pigs anesthetized with isoflurane compared to X/K. This may be due to the detrimental effects of the combination of isoflurane during overt β‐adrenergic receptor activation or cardioprotective effects of X/K anesthesia during β‐adrenergic receptor hyperactivity.     

Acute stimulation of a smooth muscle constrictor by oestradiol‐17β via GPER1 in bovine oviducts

Oviducts play roles in reproductive processes, including gametes transport, fertilization and early embryo development. Oviductal transport is controlled by various factors such as endothelins (EDNs) and nitric oxide (NO), smooth muscle contracting and relaxing factor, respectively. EDNs and NO production depend on an ovarian steroid hormone, oestradiol‐17β (E2) and E2 quickly exerts their biological functions through G protein‐coupled oestrogen receptor 1 (GPER1), which mediates rapid intracellular signalling. Because follicular fluid which contains a high concentration of E2 enters the oviduct, we hypothesized that E2 in the follicular fluid participates via GPER1 in producing EDNs and NO. To test this hypothesis, we investigated 1) the expression and localization of GPER1 in bovine oviductal tissues and 2) rapid effects of E2 via GPER1 on EDN1, EDN2 and inducible NO synthase (iNOS) expression in cultured bovine oviductal isthmic epithelial cells. GPER1 was observed in the oviductal epithelium, stroma and smooth muscle, and its expression was highest in the isthmus. Short‐term treatments (≤1 hr) of E2 increased EDN2 mRNA expression in the isthmic epithelial cells, although E2 did not affect EDN1 and iNOS mRNA expressions. Results of GPER1‐selective agonist G‐1 and GPER1‐selective antagonist G‐15 treatments revealed acute stimulation by E2, which is mediated via GPER1. The overall findings suggested that E2 in follicular fluid rapidly stimulates EDN2 expression via GPER1 in the isthmic epithelial cells. Follicular fluid may play a role in retention of the ovulated oocyte in the end of ampulla by contracting the isthmus for successful fertilization.     

A possible solution to model nonlinearity in elimination and distributional clearances with α2‐adrenergic receptor agonists: Example of the intravenous detomidine and methadone combination in sedated horses

The alpha(α)₂‐agonist detomidine is used for equine sedation with opioids such as methadone. We retrieved the data from two randomized, crossover studies where detomidine and methadone were given intravenously alone or combined as boli (STUDY 1) (Gozalo‐Marcilla et al., 2017, Veterinary Anaesthesia and Analgesia, 2017, 44 , 1116) or as 2‐hr constant rate infusions (STUDY 2) (Gozalo‐Marcilla et al., 2019, Equine Veterinary Journal, 51 , 530). Plasma drug concentrations were measured with a validated tandem Mass Spectrometry assay. We used nonlinear mixed effect modelling and took pharmacokinetic (PK) data from both studies to fit simultaneously both drugs and explore their nonlinear kinetics. Two significant improvements over the classical mammillary two‐compartment model were identified. First, the inclusion of an effect of detomidine plasma concentration on the elimination clearances (Cls) of both drugs improved the fit of detomidine (Objective Function Value [OFV]: ?160) and methadone (OFV: ?132) submodels. Second, a detomidine concentration‐dependent reduction of distributional Cls of each drug further improved detomidine (OFV: ?60) and methadone (OFV: ?52) submodel fits. Using the PK data from both studies (a) helped exploring hypotheses on the nonlinearity of the elimination and distributional Cls and (b) allowed inclusion of dynamic effects of detomidine plasma concentration in the model which are compatible with the pharmacology of detomidine (vasoconstriction and reduction in cardiac output).