Effect of L‐carnitine on maturation,cryo‐tolerance and embryo developmental competence of bovine oocytes

In this study, the effects of the addition of L‐carnitine in in vitro maturation (IVM) medium for bovine oocytes on their nuclear maturation and cryopreservation were investigated; they were matured in IVM medium supplemented with 0.0, 0.3, 0.6 and 1.2 mg/mL of L‐carnitine (control, 0.3, 0.6 and 1.2 groups, respectively) and some of them were vitrified by Cryotop. Moreover, the effects of L‐carnitine during in vitro fertilization (IVF) and in vitro culture (IVC) on the developmental potential and quality of IVF embryos were also examined. A significantly higher maturation rate of oocytes was obtained for 0.3 and 0.6 mg/mL groups compared with the control (P < 0.05). The blastocyst formation rate in the 0.6 group was significantly improved, whereas the rate in the 1.2 group was significantly decreased when compared with the control group (P < 0.05). No significant difference was found in embryo development between the control and the L‐carnitine group after oocyte vitrification. Supplementation of IVF and IVC media with L‐carnitine had no effect on development to the blastocyst stage of IVM oocytes treated with 0.6 mg/mL L‐carnitine. In conclusion, the supplementation of L‐carnitine during IVM of bovine oocytes improved their nuclear maturation and subsequent embryo development after IVF, but when they were vitrified the improving effects were neutralized.     

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.     

Effect of L‐carnitine on proliferative response and mRNA expression of some of its associated factors in splenic mononuclear cells of male broiler chicks

The effect of L‐carnitine supplementation on mitogen (concanavalin A, Con A) induced proliferation of mononuclear cells (MNC) in the spleen was investigated in broiler chickens at different ages. Day‐old chickens were fed a diet supplemented with or without L‐carnitine (100 ppm) for 24 days. The carnitine‐supplemented group showed greater proliferation of MNC in the spleen in response to Con A than that of the control group at 24 days of age. In addition, at 24 days of age the carnitine‐supplemented group showed higher expression of interleukin (IL)‐2 and interferon (IFN)‐γ mRNA, but lower expression of inducible nitric oxide synthase (iNOS) in the Con A‐stimulated splenic MNC than the control group. The enhancement effect of L‐carnitine on MNC proliferation and IL‐2 mRNA expression was not found in chicks at 14 days of age. Addition of L‐carnitine (50 nmol/mL) to the culture medium enhanced proliferation and IL‐2 mRNA expression of splenic MNC obtained from 24‐day‐old but not from 14‐day‐old broiler chickens. The results suggest that L‐carnitine is capable of enhancing MNC proliferation in broiler chickens at 24 days of age partly through increasing IL‐2 and IFN‐γ production and decreasing NO production.     

Effects of in ovo feeding of L‐arginine on the development of digestive organs,intestinal function and post‐hatch performance of broiler embryos and hatchlings

This study was to investigate the effects of in ovo feeding (IOF) L‐arginine (Arg) solution on the development of digestive organs, the duodenal mucosa of broiler embryos and hatchlings, and the growth performance of chicks during the first week post‐hatch. A total of 720 fertilized eggs with similar weight were randomly allocated to three groups, consisting of eight replicates of 30 eggs each. Three treatments were arranged as non‐injected control, diluent‐injected (0.75% NaCl solution) group and Arg solution‐injected group containing 1% Arg, dissolved in diluent. At 17.5 days of incubation, 0.6 ml of IOF solution was injected into amniotic fluid of each egg of injected groups. Results showed IOF of Arg solution increased (p < .05) the chick embryo weight at 19 days of incubation; the body weight gain of post‐hatch broilers during 1–7 days; the weights of liver, pancreas, proventriculus and gizzard; the concentrations of duodenal ghrelin, vasoactive intestinal peptide and glucagon‐like peptide 2; and the duodenum mucosal enzyme activities of alkaline phosphatase, maltase, sucrase and inducible nitric oxide synthase of 7‐day‐old post‐hatch broilers compared with other groups. The IOF of Arg solution also increased (p < .05) the villus height (VH) and the ratio of VH to crypt depth (CD) and decreased (p < .05) the CD in duodenum of broiler embryos and post‐hatch hatchlings, except for the CD at 19 days of incubation. In conclusion, IOF of 1% Arg solution into the amnion at 17.5 days of incubation could improve the development of digestive organs, the duodenal morphology, the releasing of gastrointestinal hormones and mucosal enzyme activities of broiler embryos and hatchlings and finally the growth performance of chicks during the first week post‐hatch. Therefore, IOF of appropriate Arg solution could be an effective technology for regulating early nutrition supply and subsequent growth development in poultry industry.     

Effects of exposure to artificial long days on milk yield,maternal insulin‐like growth factor 1 levels and kid growth rate in subtropical goats

This study was designed to determine whether any relationship exists between exposure to artificial long days, milk yield, maternal plasma insulin‐like growth factor 1 (IGF‐1) levels, and kid growth rate in goats. One group of lactating goats was maintained under naturally decreasing day length (control group; n = 19), while in another one, they were kept under artificial long days (LD group; n = 19). Milk yield was higher in goats from the LD group than that in the control group (P < 0.05). Maternal IGF‐1 levels at day 57 of lactation were higher (P < 0.05) in goats from the LD group than the levels in the control group and were positively correlated with the total milk yields per goat at days 43 and 57 of lactation (r = 0.77 and r = 0.84, respectively; P < 0.01). Daily weight gain at week 4 was higher (P < 0.01) in kids from the LD group than that in kids from the control group and was correlated with total and average IGF‐1 maternal levels (r = 0.60 and r = 0.60, P < 0.05). It was concluded that submitting lactating goats to artificial long days increases milk yield, plasma IGF‐1 maternal levels and the growth rate of the kids.     

Serum α‐1‐acid glycoprotein concentration in clinically healthy puppies and adult dogs and in dogs with various diseases

Background: α‐1‐acid glycoprotein (AGP) is an acute‐phase protein and a serum marker of inflammation and neoplasia in humans. AGP concentrations in diseased dogs and the potential effects of age, breed, and sex have not been elucidated. Objective: The purpose of this study was to examine differences in AGP concentration based on age, sex, and breed in a large population of clinically healthy dogs and to compare AGP concentrations in dogs with various diseases. Methods: Serum was obtained from clinically healthy puppies (n=74) and adults (n=172) of both sexes, and included mongrels (n=205) and Beagles (n=41). Serum also was obtained from 192 dogs with various diseases, including 8 with pyometra that were sampled before, and 1, 2, 3, and 10 days after surgery. AGP concentration was measured by single radial immunodiffusion. Statistical comparisons were made among age, sex, breed, and disease groups. Results: Serum AGP in healthy adult mongrels was 364±106 mg/L (reference interval, 152–576 mg/L). AGP was lowest in newborns (n=11, 122±54 mg/L) and gradually increased to adult levels by 3 months of age. Median AGP concentration was highest in dogs with parvovirus (n=17, 2100 mg/L), distemper (n=7, 1250 mg/L), and pyometra (n=18, 2480 mg/L) and was also significantly higher in dogs with acute filariasis, renal failure, urolithiasis, pancreatitis, hepatitis, trauma, hyperadrenocorticism, and immune‐mediated hemolytic anemia. Dogs with acute filariasis and acute hepatopathy had significantly higher AGP concentrations than dogs with chronic filariasis and chronic hepatopathy. Serum AGP concentration decreased gradually following surgery for pyometra but remained increased after 10 days (896±175 mg/L). Conclusions: Because of significantly lower AGP in puppies, the age of dogs should be considered when using AGP as a marker of disease. Serum AGP may be a useful marker of inflammatory disease in dogs and may help differentiate acute and chronic stages of disease.     

Effect of L‐theanine on meat quality,muscle amino acid profiles,and antioxidant status of broilers

This study investigated the effect of L ‐theanine on carcass traits, meat quality, muscle antioxidant capacity, and amino acid (AA) profiles of broilers. Three hundred 1‐day‐old Ross 308 male broilers were randomly allotted to five groups with six replicates. Birds were fed the basal diet or basal diet with 300, 600, 900, or 1,500 mg/kg L ‐theanine for 42 consecutive days. The results showed that L ‐theanine quadratically increased dressing percentage, eviscerated percentage, and leg muscle yield (p < .05). Meanwhile, drip loss, cooking loss, shear force, L*_24h, and muscle lactate content decreased quadratically in response to dietary L ‐theanine supplementation (p < .05), while pH_24h and muscle glycogen content were quadratically improved by L ‐theanine (p < .05). Notably, the contents of muscle malondialdehyde and protein carbonyl, and the activities of muscle total antioxidant capacity, catalase, and glutathione peroxidase decreased quadratically in response to dietary L ‐theanine supplementation (p < .05), suggesting that the oxidative stress level of muscle was decreased quadratically. Moreover, L ‐theanine quadratically increased the concentrations of most of muscle essential AA, nonessential AA, and flavor AA (p < .05). In conclusion, L ‐theanine can be used as a valuable feed additive to modulate carcass traits, meat quality, muscle antioxidant status, and AA profiles of boilers, and its optimum addition level is 600 mg/kg based on the present study.     

Haematological and febrile response to Escherichia coli lipopolysaccharide in 12‐week‐old cockerels of genetically diverse layer lines fed diets with increasing L‐arginine levels

Due to its decisive function in the avian metabolic, endocrine and immune system L‐arginine (Arg) is dietary indispensable for chickens. In 12‐week‐old cockerels of two high‐ and two low‐performing purebred layer lines, the effects of increasing dietary Arg on the haematological and febrile response were studied over 48 h after single lipopolysaccharide (LPS) injection. The offered diets contained Arg equivalent to 70%, 100% and 200% of recommended supply. Pathophysiological alterations in weight gain, feed intake, body temperature and differential blood count were examined in comparison with their physiological initial values. Within the first 24 h after LPS injection, cockerels reduced feed intake and lost body weight subsequently. Thereby, low‐performing genotypes lost body weight to a lesser extent than high‐performing ones. The loss of body weight was further intensified by deficient dietary Arg. Within the following 24 h, cockerels recovered by improving feed intake and weight gain. Furthermore, LPS induced genotype‐specific fever response: both brown genotypes showed initial hypothermia followed by longer lasting moderate hyperthermia, whereas the white genotypes exhibited biphasic hyperthermia. Fever response was accompanied by significant changes in differential blood counts. Characterized by lymphopenia and heterophilia, a severe leucopenia was observed from 4 to 8 h after LPS injection and replaced by a marked leucocytosis with longer lasting monocytosis up to 48 h after LPS injection. Under given pathophysiological conditions, deficiently Arg‐supplied cockerels showed higher total leucocyte counts than adequately and excessively Arg‐supplied cockerels. However, deficient and surplus dietary Arg tended to cause higher ratios between heterophils and lymphocytes. To conclude, present results confirmed that LPS induced numerous immunological changes in 12‐week‐old cockerels and emphasized that chicken's genotype is a source of variation to be considered for immunological studies. Deficient dietary Arg intensified acute changes in differential blood counts and weight gain during LPS‐induced inflammation.     

Effects of nutritional level of concentrate‐based diets on meat quality and expression levels of genes related to meat quality in Hainan black goats

The present study investigated the effects of the nutritional levels of diets on meat quality and related gene expression in Hainan black goat. Twenty‐four goats were divided into six dietary treatments and were fed a concentrate‐based diet with two levels of crude protein (CP) (15% or 17%) and three levels of digestive energy (DE) (11.72, 12.55 or 13.39 MJ/kg DM) for 90 days. Goats fed the concentrate‐based diet with 17% CP had significantly (P < 0.05) higher average daily gains (ADG) and better feed conversion rates (FCR). The pH _24h value tended to decrease (P < 0.05) with increasing DE levels. The tenderness of Longissimus dorsi muscle (LD) and Semimembranosus muscle (SM) reduced with increasing CP levels (P < 0.05). With increasing DE levels, tenderness was increased (P < 0.05). The heart fatty acid‐binding protein (H‐FABP) mRNA expression levels in LD and SM increased with increasing DE levels (P < 0.05), but decreased with increasing CP levels (P < 0.05). The calpastatin (CAST) and μ‐calpain mRNA expressions levels in LD and SM were affected significantly (P < 0.05) by CP and DE levels in the diet. Therefore, the nutritional levels of diets affect meat quality and expression levels of genes associated with meat quality in Hainan black goats.     

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.     

Effect of genistein on IGF‐1 and IGFBP‐1 in young and aged female rat ovary

The objective of this study was to assess the effects of genistein (GEN) on expression of insulin‐like growth factor 1 (IGF‐1) and insulin‐like growth factor binding protein 1 (IGFBP‐1) in young and aged rat ovary. Forty young female Sprague Dawley (SD) rats (200 ± 20 g) and forty aged female SD rats (490 ± 20 g) were selected and according to weight, they were divided into the following five groups with eight animals in each: negative control group (NC), low‐dose group (L), middle‐dose group (M), high‐dose group (H) and positive control group (PC). GEN group received GEN of 15, 30, 60 mg/kg respectively. It lasted 30 days. Concentrations of serum hormones, IGF‐1 and IGFBP‐1 were determined by enzyme‐linked immunosorbent assay (ELISA). Gene and protein expressions of IGF‐1 and IGFBP‐1 were determined by real‐time PCR and Western blot respectively. Compared with NC, GEN significantly increased oestradiol‐17β(E₂) level in aged rat, reduced luteinizing hormone (LH) level in young and aged rat. Serum levels of IGFBP‐1 in young rats were significantly higher in GEN groups (p < 0.05). mRNA and protein expression levels of IGF‐1 and IGFBP‐1 were positively correlated with GEN dose. GEN could significantly reduce the ratio of IGF‐1/IGFBP‐1 of aged rats. Multivariate Cox regression analysis result showed IGF‐1 and IGFBP‐1 levels significantly correlated with GEN dose. We speculate that there is an association between the addition of GEN and expression of IGF‐1 and IGFBP‐1, and the relationship between them is different in young and aged rat.     

Polymorphism in promoter region of growth hormone receptor is associated with potential production capacity of insulin‐like growth factor‐1 in pre‐pubertal Holstein heifers

Insulin‐like growth factor‐1 (IGF‐1) is one of the important factors for growth, milk production and reproductive functions and mainly released from the liver in response to growth hormone (GH) via GH receptor (GHR) in cattle. Recently, some single nucleotide polymorphisms (SNPs) were identified in the bovine GHR gene. Some GHR‐SNPs were shown to be related to plasma IGF‐1 concentration in cattle. Hence, the capacity to IGF‐1 production in the liver might be affected by GHR‐SNP and associated with performance in the future. This study examined whether GHR‐SNP is associated with IGF‐1 production in the liver of pre‐pubertal heifers. In 71 Holstein calves, blood samples for genomic DNA extraction were obtained immediately after birth. To genotype the GHR‐SNPs in the promoter region, polymerase chain reaction (PCR) products were digested with restriction enzyme NsiI (cutting sites: AA, AG and GG). All heifers at 4 months of age were intramuscularly injected with 0.4 mg oestradiol benzoate. Blood samples were obtained from the jugular vein just before (0 h) and 24 h after injection. The number of AA, AG and GG at the NsiI site was 0, 17 and 54 respectively. In AG and GG, plasma GH concentrations were higher pre‐injection than 24 h post‐injection (p < 0.01). Moreover, plasma GH concentrations in AG post‐injection were higher than in GG (p < 0.05). In contrast, the GG genotype exhibited higher plasma IGF‐1 concentrations in pre‐injection than post‐injection (p < 0.01), although oestradiol did not change IGF‐1 concentration in the AG genotype. We conclude that the GG polymorphism in the promoter region of GHR is associated with a higher potential capacity of IGF‐1 production in the liver of cattle.     

COX‐2, mPGES‐1 and EP2 receptor immunohistochemical expression in canine and feline malignant mammary tumours

Prostaglandin (PG) signalling is involved in human and animal cancer development. PG E₂ (PGE₂) tumour‐promoting activity has been confirmed and its production is controlled by Cyclooxygenase‐2 (COX‐2) and microsomal PGE synthase‐1 (mPGES‐1). Evidence suggests that mPGES‐1 and COX‐2 contribute to carcinogenesis through the EP2 receptor. The aim of our study was to detect by immunohistochemistry COX‐2, mPGES‐1 and EP2 receptor expression in canine (n = 46) and feline (n = 50) mammary tumours and in mammary non‐neoplastic tissues. COX‐2 positivity was observed in 83% canine and 81% feline mammary carcinomas, mPGES‐1 in 75% canine and 66% feline mammary carcinomas and the EP2 receptor expression was observed in 89% canine and 54% feline carcinomas. The frequency of COX‐2, EP2 receptor and mPGES‐1 expression was significantly higher in carcinomas than in non‐neoplastic tissues and adenomas. COX‐2, mPGES‐1 and EP2 receptor expression was strongly associated. These findings support a role of the COX‐2/PGE2 pathway in the pathogenesis of these tumours.     

Effects of L‐methionine on performance,gut morphology and antioxidant status in gut and liver of piglets in relation to DL‐methionine

This study investigated the hypothesis that dietary supplementation of L‐methionine (L‐Met) in weaned piglets in relation to DL‐methionine (DL‐Met) results in a higher antioxidant status and lower need for antioxidant enzyme activation in intestinal epithelium and body tissues, and improves gut morphology and gut barrier function as well as performance. A total of 99 early‐weaned 21‐day old piglets were allotted to six groups and fed a semi‐synthetic wheat–barley‐based basal diet supplemented with 0.067%, 0.107% and 0.147% of either DL‐Met (MetAmino; Evonik, Hanau, Germany) or L‐Met (L‐Met100; CJ Europe, Schwalbach am Taunus, Germany) to reach dietary Met concentrations of 0.16%, 0.20% and 0.24%, of which the latter met the requirements for maintenance and growth based on a pre‐experiment. Feed intake and body weights were recorded weekly, and samples of plasma, liver and duodenum and jejunum mucosa were collected after 3 weeks at slaughter. Plasma concentrations of L‐Met were similar, and those of D‐Met and total Met were higher in piglets fed DL‐Met in relation to those fed L‐Met. Feed intake, daily gains and feed:gain ratio, and the relative bio‐efficacy based on gains and feed:gain ratio were similar for both groups. Likewise, villi length, crypt depth, the villi length:crypt depth ratio in duodenum and jejunum and gene expression of tight junction proteins in the jejunum did not differ. Concentrations of antioxidants like glutathione and tocopherol, the total antioxidant capacity, the mRNA abundance or activity of antioxidant enzymes like superoxide dismutase and glutathione peroxidase, concentrations of thiobarbituric acid reactive substances, markers for oxidative damage of lipids and the expression of inflammatory genes were similar in liver and jejunum mucosa. These data indicate that the effects of L‐Met and DL‐Met supplementation are comparable considering both piglet performance and parameters of gut health and function like gut morphology and the intestinal antioxidant status.     

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.