http://http://dspace.unitus.it:80 The DSpace digital repository system captures, stores, indexes, preserves, and distributes digital research material. 2013-06-18T04:34:02Z http://hdl.handle.net/2067/141 Title: Abundance of mRNA of apolipoprotein B100, apolipoprotein E, and microsomal triglyceride transfer protein in liver from periparturient dairy cows Authors: Bernabucci, Umberto; Ronchi, Bruno; Basiricò, Loredana; Pirazzi, Daniela; Rueca, Fabrizio; Lacetera, Nicola; Nardone, Alessandro Abstract: Limited secretion of very low density lipoproteins (VLDL) in dairy cows is strongly related to fatty liver and other metabolic disorders in the early postpartum. Currently, there is limited information on which roles apolipoprotein B100 (ApoB100), apolipoprotein E (ApoE), and microsomal triglyceride transfer protein (MTP)play in that VLDL limitation. To our knowledge, no studies have simultaneously measured ApoB100, ApoE, and MTP mRNA in periparturient dairy cows. Therefore, a trial was conducted to assess liver gene expression of these proteins in transition dairy cows and to evaluate the relationships between their expression and metabolic status. Eight multiparous Holstein cows were monitored during the transition period. To evaluate metabolic and nutritional status, body condition score was registered, and plasma indexes of energy metabolism and VLDL were determined from 35 d before to 35 d after calving. Liver biopsies were performed on d −35, 3, and 35 relative to day of calving, and gene expression of ApoB100, ApoE, andMTPwere determined on liver tissue. Body condition, plasma glucose and VLDL decreased, and plasma NEFA and BHBA increased after calving. Compared with values of d −35, on d 3 after calving the ApoB100 mRNA synthesis was lower, whereas MTP and ApoE mRNA abundance were higher. Negative correlation (r = −0.57) between plasma NEFA concentration and ApoB100 mRNA abundance, and positive correlation between ApoB100 mRNA abundance and plasma cholesterol (r = 0.65) and plasma albumins (r = 0.52) were detected at 3 d postpartum. Data on changes of gene expression of the 3 main proteins involved in the regulation of synthesis and secretion of VLDL in the liver suggest that decreased mRNA for ApoB100 may be consistent with decreased synthesis and/or secretion ofVLDL from liver during the periparturient period. 2003-12-31T23:00:00Z http://hdl.handle.net/2067/148 Title: Heat Stress Elicits Different Responses in Peripheral Blood Mononuclear Cells from Brown Swiss and Holstein Cows Authors: Lacetera, Nicola; Bernabucci, Umberto; Scalia, Daniela; Basiricò, Loredana; Morera, Patrizia; Nardone, Alessandro Abstract: This study was undertaken to assess whether peripheral blood mononuclear cells (PBMC) isolated from Brown Swiss (Br) and Holstein (Ho) cows and stimulated with concanavalin A differ in response to chronic exposure to incubation temperatures simulating conditions of hyperthermia. Five multiparous Br and 5 Ho cows were utilized as blood donors. Peripheral blood mononuclear cells were subjected for 65 h to each of 5 treatments (T). Cells were exposed to 39°Ccontinuously (T39) and three 13-h cycles at 40 (T40), 41 (T41), 42 (T42) or 43°C (T43), respectively, which were interspersed with two 13-h cycles at 39°C. Treatment T39 was adopted to mimic normothermia; T40, T41, T42, and T43 mimicked conditions of more severe hyperthermia alternating with normothermia. Measures evaluated at the end of the incubation period were proliferative response (DNA synthesis), intracellular reactive oxygen species (ROS) concentrations, and mRNA abundance of the 72-kDa heat-shock protein (Hsp72). In Br cows,DNAsynthesis began to decline whenPBMCwere repeatedly exposed to 41°C (−22%), whereas DNA synthesis in cells isolated from Ho cows did not begin to decline until 42°C (−40%). Furthermore, under T41 and T42, DNA synthesis from Br cows was lower than in Ho (−24 and −54%, respectively). In both breeds, increased incubation temperatures caused a reduction of intracellular ROS (from −39.6 and −69.7%). Increase in incubation temperatures enhanced Hsp72 mRNA levels only in PBMC isolated from Br cows. The Hsp72 mRNA in Br cows increased significantly under T41 and T43 compared with T39. In both breeds, DNA synthesis was positively and negatively correlated with intracellular ROS and Hsp72 mRNA abundance, respectively (r = 0.85 and r = −0.70, respectively). Results indicated that PBMC from Br cows are less tolerant to chronic heat exposure than those from Ho cows, and that the lower tolerance is associated with higher expression of Hsp72, suggesting that the same level of hyperthermia may be associated with a differential decline of immune function in the 2 breeds. 2005-12-31T23:00:00Z http://hdl.handle.net/2067/149 Title: Photoperiod Affects Gene Expression of Leptin and Leptin Receptors in Adipose Tissue from Lactating Dairy Cows Authors: Bernabucci, Umberto; Basiricò, Loredana; Lacetera, Nicola; Morera, Patrizia; Ronchi, Bruno; Accorsi, Pier Attilio; Seren, Eraldo; Nardone, Alessandro Abstract: Leptin is mainly secreted by adipocytes and is implicated in the regulation of metabolic status, feed intake, and body condition. Day length (DL) can affect leptin gene expression and secretion. The aim of the study was to evaluate the effect of DL on gene expression of leptin and leptin receptors in adipose tissue (AT). Four lactating and pregnant Holstein cows were housed in a climate-controlled chamber for 51 d. The first 30 d were used to adapt animals to the new housing conditions. During that period the DL adopted was 12 h light:12 h dark (12:12). The experimental period included 3 different and consecutive phases: 7 d of neutral DL (12:12); 7 d of long DL (18 h light:6 h dark); and 7 d of short DL (6 h light:18 h dark). Subcutaneous AT biopsies were performed at the end of each phase. Prolactin, growth hormone, cortisol, leptin, glucose, nonesterified fatty acids, β-OH-butyrate, and cholesterol were determined in plasma samples. Abundance of leptin mRNA, and Ob-Ra and Ob-Rb leptin receptormRNA were determined in AT samples by ribonuclease protection assay. Day length did not affect feed intake or body condition score. Exposure to short DL significantly reduced milk yield (13.1 ± 2.2 vs. 15.8 ± 1.7 and 16.0 ± 2.0 kg/d for short vs. neutral and long DL, respectively). Plasma leptin, growth hormone, cortisol, nonesterified fatty acids, β-OH-butyrate, and glucose were not affected by DL; cholesterol was lowest under short DL (3.93 ± 0.38 vs. 4.36 ± 0.39 and 4.07 ± 0.38 mmol/L for short vs. neutral and long DL, respectively). Prolactin increased under long DL (134.82 ± 16.94 vs. 81.98 ± 20.25 and 96.16 ± 0.38 ng/mL for long vs. neutral and short DL, respectively). Gene expression of leptin and its receptors was affected by DL. Leptin mRNA increased under long DL (11.91 ± 0.84 vs. 7.82 ± 0.84 and 7.56 ± 0.84 pg of mRNA/g of total RNA for long vs. neutral and short DL, respectively). Leptin receptors Ob-Ra and Ob-Rb mRNA were higher under long DL, whereas Ob-Ra and Ob-Rb mRNA were lower under short DL (Ob-Ra: 1.91 ± 0.41, 2.49 ± 0.41, and 0.65 ± 0.41 pg of mRNA/g of total RNA for neutral, long, and short DL, respectively; Ob-Rb: 5.29 ± 0.79, 5.98 ± 0.68, and 2.02 ± 0.70 pg of mRNA/g of total RNA for neutral, long, and short DL, respectively). Results of the present study appear to exclude an effect of feed intake and metabolic status on leptin gene expression. A prolactinmediated effect of photoperiod on AT leptin modulation may be proposed in lactating dairy cows. 2005-12-31T23:00:00Z http://hdl.handle.net/2067/1331 Title: Cellular thermotolerance is associated with heat shock protein 70.1 genetic polymorphisms in Holstein lactating cows Authors: Basiricò, Loredana; Morera, Patrizia; Primi, Valentina; Lacetera, Nicola; Nardone, Alessandro; Bernabucci, Umberto Abstract: The present study aimed to investigate the association between inducible Hsp70.1 single nucleotide polymorphisms (SNPs) and heat shock (HS) response of peripheral blood mononuclear cells (PBMC) in dairy cows. Exposure to HS differently affected cell viability and gene and protein expression of Hsp70.1 in the selected genotypes. These results indicate that the presence of SNPs (C/- and G/T) in the 5′-UTR region of inducible Hsp70.1 ameliorates HS response and tolerance to heat of bovine PBMC. These mutation sites may be useful as molecular genetic markers to assist selection for heat tolerance. Il presente studio ha lo scopo di studiare l’associazione tra il polimorfismo genetico al locus delle Hsp70.1 e la risposta allo shock termico (ST) da caldo in linfociti ottenuti da vacche da latte. L’Esposizione allo ST ha influenzato in modo differente la vitalità cellulare e la espressione genetica e proteica delle Hsp70.1 nei diversi polimorfismi testati. I risultati indicano che la presenza di SNP (C/- e G/T) nella regione 5’-UTR delle Hsp70.1 migliora la risposta e la tolleranza allo ST. Queste mutazioni potrebbero essere utilizzate come marcatori genetici molecolari per assistere la selezione genetica verso la termo tolleranza al caldo. 2010-12-31T23:00:00Z http://hdl.handle.net/2067/1332 Title: Aflatoxin B1 and fumonisin B1 affect the oxidative status of bovine peripheral blood mononuclear cells Authors: Bernabucci, Umberto; Colavecchia, Luciana; Danieli, Pier Paolo; Basiricò, Loredana; Lacetera, Nicola; Nardone, Alessandro; Ronchi, Bruno Abstract: This in vitro study was focused on the effect of two widely occurring mycotoxins, aflatoxin B1 (AFB1) and fumonisin B1 (FB1), on the oxidative status of bovine peripheral blood mononuclear cells (PBMC) incubated for 2 and 7 days at different levels of AFB1 (0, 5 and 20 lg/ml) and FB1 (0, 35 and 70 lg/ml). Results of the present study demonstrate that, even though by different mechanisms, AFB1 and FB1 may induce cytotoxicity through an impairment of the oxidative status of PBMC. Lo studio in vitro ha lo scopo di verificare l’effetto della esposizione ad aflatossina B1 (AFB1) e fumonisina B1 (FB1) di sullo stato ossidativo di linfociti bovini incubati per 2 o 7 giorni con differenti concentrazioni di tossine (AFB1: 0, 5 and 20 mcg/ml and FB1: 0, 35 and 70 mcg/ml). I risultati del presente studio hanno dimostrato, anche se attraverso meccanismi differenti, che entrambe le tossine inducono citotossicità attraverso il peggioramento dello stato ossidativo. 2010-12-31T23:00:00Z http://hdl.handle.net/2067/1801 Title: Aflatoxin B1 and fumonisin B1 affect the oxidative status of bovine peripheralblood mononuclear cells Authors: Bernabucci, Umberto; Colavecchia, Luciana; Danieli, Pier Paolo; Basiricò, Loredana; Lacetera, Nicola; Nardone, Alessandro; Ronchi, Bruno Abstract: Mycotoxins are secondary metabolites having a high cytotoxic potential. They are produced by molds and released in food and feed. To date, the mechanisms underlying the mycotoxin-induced cytotoxicity have not been fully clarified. The induction of oxidative stress, as a possible mechanism, has been postulated. This in vitro study was focused on the effect of two widely occurring mycotoxins, aflatoxin B1 (AFB1) and fumonisin B1 (FB1), on the oxidative status of bovine peripheral blood mononuclear cells (PBMC) incubated for 2 and 7 days at different levels of AFB1 (0, 5 and 20 lg/ml) and FB1 (0, 35 and 70 lg/ml). Reactive oxygen metabolites (ROM), intracellular thiols (SH), malondialdehyde (MDA) and gene expression of cytoplasmic superoxide dismutase (SOD) and glutathione peroxidase (GSHPX-1) were measured on PBMC after incubation. The highest concentration of AFB1 and all concentrations of FB1 caused an increase (p < 0.05) of intracellular ROM without any time dependent effect. Intracellular SH decreased with 20 lgAFB1/ml (p < 0.05) and the effect was particularly marked after 7 days of exposure. Intracellular SH were not affected by FB1 even though a lower (p < 0.05) SH level after 2 days exposure than after 7 days was observed. MDA increased (p < 0.05) in AFB1 or FB1 treated PBMC. The exposure to FB1 for 7 days increased MDA (p < 0.05) only in cells treated with 70 lg/ml. Exposure of PBMC to AFB1 reduced SOD mRNA while FB1 decreased both SOD and GSHPX-1 mRNA abundance. These results demonstrate that, even though by different mechanisms, AFB1 and FB1 may induce cytotoxicity through an impairment of the oxidative status of PBMC. 2010-12-31T23:00:00Z http://hdl.handle.net/2067/1827 Title: Heat shock modulates adipokines expression in 3T3-L1 adipocytes Authors: Bernabucci, Umberto; Basiricò, Loredana; Morera, Patrizia; Lacetera, Nicola; Ronchi, Bruno; Nardone, Alessandro Abstract: Studies have demonstrated that heat shock is associated with alteration in energy metabolism. In this study, we investigated the effect of heat shock on gene expression and secretion of adiponectin and leptin, and gene expression of Hspa2 and Pparg in 3T3-L1 adipocytes. Compared with 37 8C, adiponectin mRNA was higher at 39 8C, and lower at 41 8C. Leptin mRNA was higher when adipocytes were exposed to 41 8C compared with 37 and 39 8C. Secretion of adiponectin increased at 39 8C, and when cells were exposed to 41 8C it was not detectable. Leptin secretion increased significantly at 41 8C, compared with 37 and 39 8C. Hspa2 mRNA was increased at 39 8C, and the highest level was reached at 41 8C. Pparg mRNA exhibited a substantial increase in a temperature-dependent manner. The study provides the first evidence of a possible direct effect of heat shock on adiponectin and leptin gene expression and secretion, and demonstrates that the expression of the two adipokines is differentially regulated at the temperatures tested. 2008-12-31T23:00:00Z http://hdl.handle.net/2067/1802 Title: Heat shock impairs DNA synthesis and down-regulates gene expression for leptin and Ob-Rb receptor in concanavalin A-stimulated bovine peripheral blood mononuclear cells Authors: Lacetera, Nicola; Bernabucci, Umberto; Basiricò, Loredana; Morera, Patrizia; Nardone, Alessandro Abstract: This study verified whether leptin or its long isoform receptor (Ob-Rb) genes are expressed in proliferating lymphocytes from bovine species, and whether their expression changes with increased temperatures. Peripheral blood mononuclear cells (PBMC) from five Holstein cows were incubated in the presence of concanavalin A, and alternatively subjected for 65 h to each of the following treatments (T): 39 8C continuously (T39) or three 13-h cycles at 40 (T40), 41 (T41) or 42 8C (T42), respectively, which were alternated with two 13-h cycles at 39 8C. T39 mimicked normothermia; T40, 41 and 42 mimicked conditions of hyperthermia alternated with normothermia. PBMC proliferation declined under T42. Compared with T39, levels of mRNA for leptin was lower under T42, whereas mRNA for Ob-Rb was lower in lymphocytes cultured both under T41 and T42. DNA synthesis was positively correlated with leptinmRNA. This study supports the concept that severe heat stress impairs proliferation of bovine PBMC, confirms that bovine lymphocytes express Ob-Rb gene, and provides the first experimental evidence that bovine lymphocytes express gene for leptin, and that increased temperatures are associated with altered gene expression for leptin and Ob-Rb. 2008-12-31T23:00:00Z http://hdl.handle.net/2067/1779 Title: Cellular thermotolerance is associated with heat shockprotein 70.1 genetic polymorphisms in Holstein lactating cow Authors: Basiricò, Loredana; Morera, Patrizia; Primi, Valentina; Lacetera, Nicola; Nardone, Alessandro; Bernabucci, Umberto Abstract: Heat shock proteins (Hsp) are known to protect cells from several stressors. Nucleotide changes in the flanking regions [5′- and 3′-untranslated region (UTR)] of Hsp gene might affect inducibility, degree of expression, or stability of Hsp70 mRNA. The present study aimed to investigate the association between inducible Hsp70.1 single nucleotide polymorphisms (SNPs) and heat shock (HS) response of peripheral blood mononuclear cells (PBMC) in dairy cows. Four hundred forty-six Italian Holstein cows were genotyped for four Hsp70.1 SNPs: g895 C/- and g1128 G/T in 5′-UTR, and g2154 G/A and g64 G/T in 3′-UTR. Genetic polymorphisms in 3′-UTR of bovine Hsp70.1 gene resulted monomorphic. Distribution of alleles of the nucleotide sequence polymorphism within the 5′-UTR of the bovine Hsp70.1 gene were 81.2% and 18.8% for C and -, respectively, and 77.8% and 22.2% for G and T, respectively. Among the 446 genotyped animals, a group of cows balanced for days in milk and parity was selected to be representative of the following genotypes: CC (n=8), C- (n=7), and – (n=7) and GG (n=8), GT (n=11), and TT (n=3) in 5′-UTR. PBMC were isolated from blood samples and heated at 43°C in thermal bath for 1 h and then incubated at 39°C in atmosphere of 5% CO2 for 1,2, 4, 8, 16, and 24 h (recovery times). Cell viability was determined by XTT assay. Gene and protein expression of Hsp70.1 was determined by real-time reverse transcriptionpolymerase chain reaction and by ELISA assay, respectively.For the two SNPs detected, one allele was the most frequent (C, 66.8% and G, 56.8%). Genotypes – and TG showed higher (P<0.05) viability compared with CC and GG, respectively. Genotypes C- and TT had intermediate viability. Gene expression of Hsp70.1 showed higher (P<0.001) levels in – and TG genotype compared with their counterparts. Genotypes – and TG showed the higher level of inducible Hsp70.1 protein in respect to C-, TT and CC, GG. In conclusion, exposure to HS differently affected cell viability and gene and protein expression of Hsp70.1 in the selected genotypes. These results indicate that the presence of SNPs (C/- and G/T) in the 5′-UTR region of inducible Hsp70.1 ameliorates HS response and tolerance to heat of bovine PBMC. These mutation sites may be useful as molecular genetic markers to assist selection for heat tolerance. 2010-12-31T23:00:00Z http://hdl.handle.net/2067/1781 Title: Down-regulation of hepatic ApoB100 expression during hot season in transition dairy cows Authors: Basiricò, Loredana; Morera, Patrizia; Lacetera, Nicola; Ronchi, Bruno; Nardone, Alessandro; Bernabucci, Umberto Abstract: Heat stress is recognized as one of the main factors adversely affecting yield, reproduction and health of dairy ruminants. The peripartum period is the most critical phase for dairy cows. The interaction between heat stress and physiological stress imposed by the peripartum, might worsen the metabolic status of transition dairy cows. Therefore, the objective of the present study was to investigate the effect of hot season on metabolic status and on hepatic apolipoprotein B100 (ApoB100) expression during the peripartum period in Holstein dairy cows. Twenty-four multiparous Holstein cows were utilized in the study. Twelve cows gave birth in spring (SP: 28 March to 30 April). The remaining 12 cows gave birth in summer (SU: 15 June to 2 July). The two groups were balanced for parity and body condition score (BCS) and monitored from −30 to 35 d relative to calving. Values of air temperature (°C) and relative humidity (%) were recorded at 1 h intervals and were utilized to calculate the temperature–humidity index (THI). Milk production and BCS were recorded weekly. Blood samples were taken at 0800 h from the jugular vein −30, −18, and −4 d prepartum and 3, 17, and 35 d postpartum. Plasma was analyzed for glucose, urea, cholesterol, β-OH butyrate (BHBA), non-esterified fatty acids (NEFA) and ApoB100. Liver biopsies were performed on d −30, 3, and 35 relative to day of calving, and tissue samples were used for histopathological examination, and for ApoB100 mRNA and protein determination. Milk yield in SU cows was lower than in their SP counterparts. During summer, transition cows showed lower glucose and cholesterol, higher NEFA and BHBA, had lower liver ApoB100 gene and protein expression and higher lipid accumulation in liver compared with transition cows during spring. The current study provides the first evidence about the negative effects of hot season on ApoB100 gene and protein expression in transition dairy cows. The down-regulation of ApoB100 expression together with higher circulating NEFA and altered lipid metabolism are likely to be responsible for the higher risk of liver lipidosis in transition SU cows. These metabolic conditions make transition cows more susceptible to metabolic diseases during hot season. 2010-12-31T23:00:00Z http://hdl.handle.net/2067/1867 Title: Heat shock induced changes of adipokines gene expression in 3T3-L1 adipocytes Authors: Bernabucci, Umberto; Basiricò, Loredana; Morera, Patrizia; Lacetera, Nicola; Ronchi, Bruno; Nardone, Alessandro Abstract: To study the effects of heat shock on adipokines gene expression 3T3-L1 adipocytes were used. Heat shock differently affected gene expression of leptin, adiponectin and acylation stimulating protein (ASP): exposure of cells to temperature higher than 39°C caused upregulation of leptin and downregulation of adiponectin and ASP genes. The present study provides the first evidence about the effects of heat shock on adipokines gene expression. Changes in gene expression of the three adipokines may help to explain the alteration of lipid metabolism and liver functionality occurring in animals exposed to hot conditions. 2006-12-31T23:00:00Z http://hdl.handle.net/2067/1868 Title: Fatty acids affect proliferation of peripheral blood mononuclear cells in dairy cows Authors: Lacetera, Nicola; Kuzminsky, Giorgina; Morera, Patrizia; Basiricò, Loredana Abstract: In vitro studies were performed to assess the effects of bovine plasma fatty acids on proliferation of peripheral blood mononuclear cells (PBMC). PBMC from 6 Holstein heifers were cultured in media containing oleic (OA), palmitic (PA), stearic (SA), linoleic (LA), palmitoleic (POA), or linolenic (LNA) acid at concentrations mimicking different degree of lipomobilisation. Proliferation of PBMC was stimulated by concanavalin A or pokeweed mitogen. Concentrations of OA, PA, SA and LA mimicking moderate-intense lipomobilisation impaired PBMC proliferation. Concentrations of OA or LA mimicking low degree of lipomobilisation enhanced PBMC proliferation. None of the POA, and LNA concentrations affected proliferation of PBMC. 2006-12-31T23:00:00Z http://hdl.handle.net/2067/1869 Title: Gene expression and protein secretion of apolipoprotein B100 (ApoB100) in transition dairy cows under hot or thermoneutral environments Authors: Basiricò, Loredana; Bernabucci, Umberto; Morera, Patrizia; Lacetera, Nicola; Nardone, Alessandro Abstract: The aim of the study was to investigate the effects of hot season on gene expression and protein secretion of ApoB100 in transition dairy cows. Hot season strongly down-regulated ApoB100 gene and protein expression. This condition and the higher circulating NEFA were responsible for the higher lipid accumulation in liver of heat-stressed transition cows. 2008-12-31T23:00:00Z http://hdl.handle.net/2067/1834 Title: Heat shock induced changes of adipokines gene expression in 3T3-L1 adipocytes Authors: Bernabucci, Umberto; Basiricò, Loredana; Morera, Patrizia; Lacetera, Nicola; Ronchi, Bruno; Nardone, Alessandro Abstract: To study the effects of heat shock on adipokines gene expression 3T3-L1 adipocytes were used. Heat shock differently affected gene expression of leptin, adiponectin and acylation stimulating protein (ASP):exposure of cells to temperature higher than 39°C caused upregulation of leptin and downregulation of adiponectin and ASP genes. The present study provides the first evidence about the effects of heat shock on adipokines gene expression. Changes in gene expression of the three adipokines may help to explain the alteration of lipid metabolism and liver functionality occurring in animals exposed to hot conditions. 2008-12-31T23:00:00Z http://hdl.handle.net/2067/1656 Title: Heat shock modulates adipokines expression in 3T3-L1 adipocytes Authors: Bernabucci, Umberto; Basiricò, Loredana; Morera, Patrizia; Lacetera, Nicola; Ronchi, Bruno; Nardone, Alessandro Abstract: Studies have demonstrated that heat shock is associated with alteration in energy metabolism. In this study, we investigated the effect of heat shock on gene expression and secretion of adiponectin and leptin, and gene expression of Hspa2 and Pparg in 3T3-L1 adipocytes. Compared with 37 8C, adiponectin mRNA was higher at 39 8C, and lower at 41 8C. Leptin mRNA was higher when adipocytes were exposed to 41 8C compared with 37 and 39 8C. Secretion of adiponectin increased at 39 8C, and when cells were exposed to 41 8C it was not detectable. Leptin secretion increased significantly at 41 8C, compared with 37 and 39 8C. Hspa2 mRNA was increased at 39 8C, and the highest level was reached at 41 8C. Pparg mRNA exhibited a substantial increase in a temperature-dependent manner. The study provides the first evidence of a possible direct effect of heat shock on adiponectin and leptin gene expression and secretion, and demonstrates that the expression of the two adipokines is differentially regulated at the temperatures tested. 2008-12-31T23:00:00Z http://hdl.handle.net/2067/1658 Title: Liver apolipoprotein B100 expression and secretion are down-regulated early postpartum in dairy cows Authors: Bernabucci, Umberto; Basiricò, Loredana; Pirazzi, Daniela; Rueca, Fabrizio; Lacetera, Nicola; Lepri, Enrica; Nardone, Alessandro Abstract: Fatty liver is a typical condition early postpartum in dairy cows. The aim of the present study was to evaluate gene expression, synthesis and secretion of liver apolipoprotein B100 (Apo-B100) in periparturient dairy cows. The relationships between Apo-B100 with liver lipid accumulation, and metabolic and oxidative status were also investigated. The trial was carried out in a commercial dairy herd. Sixteen multiparous Holstein cows due to calve in Aprilwere monitored from 30 d prepartum to 35 d postpartum. Milk yield was recorded, and body condition (BCS) of cows was scored. Blood samples were taken at − 30±3, − 18±2, and − 4±1 d prepartum (before expected calving) and at 3, 17, and 35 d postpartum. Plasma and erythrocyte samples were obtained and analyzed. Glucose, NEFA, BHBA, cholesterol, albumin, Apo-B100, reactive oxygen metabolites, thiol groups, and glutathione peroxidase were determined in plasma. Reactive oxygen metabolites, thiol groups, glutathione peroxidase and superoxide dismutase were determined in erythrocytes. Liver biopsies were performed on d−30, 3, and 35 relative to day of calving. Liver samples were analyzed to determine fat accumulation, Apo-B100 mRNA and Apo-B100 protein. Milk yield, BCS, and plasma indices of metabolic status showed expected changes for that physiological phase. Oxidative status indices indicated an oxidative stress condition early postpartum. Fat accumulation in liver increased after calving and no significant difference was found between values observed at 3 and 35 DIM. Gene expression as well as cellular and plasma concentration of Apo-B100 were higher prepartum compared with postpartum. Apo-B100 mRNA was not different between 3 and 35 DIM, whereas both cellular and plasma Apo-B100 increased from 3 to 35 DIM. The study demonstrates that Apo-B100 is regulated at the level of transcription and post-transcription. Changes of gene and protein Apo-B100 expression may contribute to the accumulation of triglyceride in liver taking place early postpartum in dairy cows. 2008-12-31T23:00:00Z http://hdl.handle.net/2067/1859 Title: Down-regulation of hepatic ApoB100 expression during hot season in transition dairy cows. Authors: Basiricò, Loredana; Morera, Patrizia; Lacetera, Nicola; Ronchi, Bruno; Nardone, Alessandro; Bernabucci, Umberto Abstract: Heat stress is recognized as one of the main factors adversely affecting yield, reproduction and health of dairy ruminants. The peripartum period is the most critical phase for dairy cows. The interaction between heat stress and physiological stress imposed by the peripartum, might worsen the metabolic status of transition dairy cows. Therefore, the objective of the present study was to investigate the effect of hot season on metabolic status and on hepatic apolipoprotein B100 (ApoB100) expression during the peripartum period in Holstein dairy cows. Twenty-four multiparous Holstein cows were utilized in the study. Twelve cows gave birth in spring (SP: 28 March to 30 April). The remaining 12 cows gave birth in summer (SU: 15 June to 2 July). The two groups were balanced for parity and body condition score (BCS) and monitored from −30 to 35 d relative to calving. Values of air temperature (°C) and relative humidity (%) were recorded at 1 h intervals and were utilized to calculate the temperature–humidity index (THI). Milk production and BCS were recorded weekly. Blood samples were taken at 0800 h from the jugular vein −30, −18, and −4 d prepartum and 3, 17, and 35 d postpartum. Plasma was analyzed for glucose, urea, cholesterol, β-OH butyrate (BHBA), non-esterified fatty acids (NEFA) and ApoB100. Liver biopsies were performed on d −30, 3, and 35 relative to day of calving, and tissue samples were used for histopathological examination, and for ApoB100 mRNA and protein determination. Milk yield in SU cows was lower than in their SP counterparts. During summer, transition cows showed lower glucose and cholesterol, higher NEFA and BHBA, had lower liver ApoB100 gene and protein expression and higher lipid accumulation in liver compared with transition cows during spring. The current study provides the first evidence about the negative effects of hot season on ApoB100 gene and protein expression in transition dairy cows. The down-regulation of ApoB100 expression together with higher circulating NEFA and altered lipid metabolism are likely to be responsible for the higher risk of liver lipidosis in transition SU cows. These metabolic conditions make transition cows more susceptible to metabolic diseases during hot season. 2010-12-31T23:00:00Z http://hdl.handle.net/2067/1872 Title: Heat shock impairs DNA synthesis for leptin and Ob-Rb receptor in concanavalin A-stimulated peripheral blood mononuclear cells Authors: Lacetera, Nicola; Bernabucci, Umberto; Basiricò, Loredana; Morera, Patrizia; Nardone, Alessandro Abstract: This study verified whether leptin or its long isoform receptor (Ob-Rb) genes are expressed in proliferating lymphocytes from bovine species, and whether their expression changes with increased temperatures. Peripheral blood mononuclear cells (PBMC) from five Holstein cows were incubated in the presence of concanavalin A, and alternatively subjected for 65 h to each of the following treatments (T): 39 8C continuously (T39) or three 13-h cycles at 40 (T40), 41 (T41) or 42 8C (T42), respectively, which were alternated with two 13-h cycles at 39 8C. T39 mimicked normothermia; T40, 41 and 42 mimicked conditions of hyperthermia alternated with normothermia. PBMC proliferation declined under T42. Compared with T39, levels of mRNA for leptin was lower under T42, whereas mRNA for Ob-Rb was lower in lymphocytes cultured both under T41 and T42. DNA synthesis was positively correlated with leptinmRNA. This study supports the concept that severe heat stress impairs proliferation of bovine PBMC, confirms that bovine lymphocytes express Ob-Rb gene, and provides the first experimental evidence that bovine lymphocytes express gene for leptin, and that increased temperatures are associated with altered gene expression for leptin and Ob-Rb. 2008-12-31T23:00:00Z http://hdl.handle.net/2067/1860 Title: Cellular. thermotolerance is associated with heat shock protein 70.1 genetic polymorphisms in Holstein lactating cows. Authors: Basiricò, Loredana; Morera, Patrizia; Primi, Valentina; Lacetera, Nicola; Nardone, Alessandro; Bernabucci, Umberto Abstract: Heat shock proteins (Hsp) are known to protect cells from several stressors. Nucleotide changes in the flanking regions [5′- and 3′-untranslated region (UTR)] of Hsp gene might affect inducibility, degree of expression, or stability of Hsp70 mRNA. The present study aimed to investigate the association between inducible Hsp70.1 single nucleotide polymorphisms (SNPs) and heat shock (HS) response of peripheral blood mononuclear cells (PBMC) in dairy cows. Four hundred forty-six Italian Holstein cows were genotyped for four Hsp70.1 SNPs: g895 C/- and g1128 G/T in 5′-UTR, and g2154 G/A and g64 G/T in 3′-UTR. Genetic polymorphisms in 3′-UTR of bovine Hsp70.1 gene resulted monomorphic. Distribution of alleles of the nucleotide sequence polymorphism within the 5′-UTR of the bovine Hsp70.1 gene were 81.2% and 18.8% for C and -, respectively, and 77.8% and 22.2% for G and T, respectively. Among the 446 genotyped animals, a group of cows balanced for days in milk and parity was selected to be representative of the following genotypes: CC (n=8), C- (n=7), and – (n=7) and GG (n=8), GT (n=11), and TT (n=3) in 5′-UTR. PBMC were isolated from blood samples and heated at 43°C in thermal bath for 1 h and then incubated at 39°C in atmosphere of 5% CO2 for 1, 2, 4, 8, 16, and 24 h (recovery times). Cell viability was determined by XTT assay. Gene and protein expression of Hsp70.1 was determined by real-time reverse transcriptionpolymerase chain reaction and by ELISA assay, ì respectively. For the two SNPs detected, one allele was the most frequent (C, 66.8% and G, 56.8%). Genotypes – and TG showed higher (P<0.05) viability compared with CC and GG, respectively. Genotypes C- and TT had intermediate viability. Gene expression of Hsp70.1 showed higher (P<0.001) levels in – and TG genotype compared with their counterparts. Genotypes – and TG showed the higher level of inducible Hsp70.1 protein in respect to C-, TT and CC, GG. In conclusion, exposure to HS differently affected cell viability and gene and protein expression of Hsp70.1 in the selected genotypes. These results indicate that the presence of SNPs (C/- and G/T) in the 5′-UTR region of inducible Hsp70.1 ameliorates HS response and tolerance to heat of bovine PBMC. These mutation sites may be useful as molecular genetic markers to assist selection for heat tolerance. 2010-12-31T23:00:00Z http://hdl.handle.net/2067/1711 Title: Down-regulation of hepatic ApoB100 expression during hot season in transition dairy cows Authors: Basiricò, Loredana; Morera, Patrizia; Lacetera, Nicola; Ronchi, Bruno; Nardone, Alessandro; Bernabucci, Umberto 2010-12-31T23:00:00Z http://hdl.handle.net/2067/1856 Title: Fatty acids affect proliferation of peripheral blood mononuclear cells in dairy cows. Authors: Lacetera, Nicola; Kuzminsky, Giorgina; Morera, Patrizia; Basiricò, Loredana Abstract: In vitro studies were performed to assess the effects of bovine plasma fatty acids on proliferation of peripheral blood mononuclear cells (PBMC). PBMC from 6 Holstein heifers were cultured in media containing oleic(OA), palmitic (PA), stearic (SA), linoleic (LA), palmitoleic (POA), or linolenic (LNA) acid at concentrations mimicking different degree of lipomobilisation. Proliferation of PBMC was stimulated by concanavalin A or pokeweed mitogen. Concentrations of OA, PA, SA and LA mimicking moderate-intense lipomobilisation impaired PBMC proliferation. Concentrations of OA or LA mimicking low degree of lipomobilisation enhanced PBMC proliferation. None of the POA, and LNA concentrations affected proliferation of PBMC. 2006-12-31T23:00:00Z http://hdl.handle.net/2067/1838 Title: Heat shock impairs DNA synthesis and down-regulates gene expression for leptin and Ob-Rb receptor in concanavalin A-stimulated bovine peripheral blood mononuclear cells. Authors: Lacetera, Nicola; Bernabucci, Umberto; Basiricò, Loredana; Morera, Patrizia; Nardone, Alessandro Abstract: This study verified whether leptin or its long isoform receptor (Ob-Rb) genes are expressed in proliferating lymphocytes from bovine species, and whether their expression changes with increased temperatures. Peripheral blood mononuclear cells (PBMC) from five Holstein cows were incubated in the presence of concanavalin A, and alternatively subjected for 65 h to each of the following treatments (T): 39 8C continuously (T39) or three 13-h cycles at 40 (T40), 41 (T41) or 42 8C (T42), respectively, which were alternated with two 13-h cycles at 39 8C. T39 mimicked normothermia; T40, 41 and 42 mimicked conditions of hyperthermia alternated with normothermia. PBMC proliferation declined under T42. Compared with T39, levels of mRNA for leptin was lower under T42, whereas mRNA for Ob-Rb was lower in lymphocytes cultured both under T41 and T42. DNA synthesis was positively correlated with leptinmRNA. This study supports the concept that severe heat stress impairs proliferation of bovine PBMC, confirms that bovine lymphocytes express Ob-Rb gene, and provides the first experimental evidence that bovine lymphocytes express gene for leptin, and that increased temperatures are associated with altered gene expression for leptin and Ob-Rb. 2008-12-31T23:00:00Z http://hdl.handle.net/2067/1857 Title: Heat shock modulates adipokines expression in 3T3-L1 adipocytes. Authors: Bernabucci, Umberto; Basiricò, Loredana; Morera, Patrizia; Lacetera, Nicola; Ronchi, Bruno; Nardone, Alessandro Abstract: Studies have demonstrated that heat shock is associated with alteration in energy metabolism. In this study, we investigated the effect of heat shock on gene expression and secretion of adiponectin and leptin, and gene expression of Hspa2 and Ppar in 3T3-L1 adipocytes. Compared with 37 °C, adiponectin mRNA was higher at 39 °C, and lower at 41 °C. Leptin mRNA was higher when adipocytes were exposed to 41 °C compared with 37 and 39 °C. Secretion of adiponectin increased at 39 °C, and when cells were exposed to 41 °C it was not detectable. Leptin secretion increased significantly at 41 °C, compared with 37 and 39 °C. Hspa2 mRNA was increased at 39 °C, and the highest level was reached at 41 °C. Ppar mRNA exhibited a substantial increase in a temperature-dependent manner. The study provides the first evidence of a possible direct effect of heat shock on adiponectin and leptin gene expression and secretion, and demonstrates that the expression of the two adipokines is differentially regulated at the temperatures tested. 2008-12-31T23:00:00Z http://hdl.handle.net/2067/1858 Title: Gene expression and protein secretion of apolipoprotein B100 (ApoB100) in transition dairy cows under hot or thermoneutral environments. Authors: Basiricò, Loredana; Bernabucci, Umberto; Morera, Patrizia; Lacetera, Nicola; Nardone, Alessandro Abstract: The aim of the study was to investigate the effects of hot season on gene expression and protein secretion of ApoB100 in transition dairy cows. Hot season strongly down-regulated ApoB100 gene and protein expression. This condition and the higher circulating NEFA were responsible for the higher lipid accumulation in liver of heat-stressed transition cows 2008-12-31T23:00:00Z http://hdl.handle.net/2067/1861 Title: Photoperiod Affects Gene Expression of Leptin and Leptin Receptors in Adipose Tissue from Lactating Dairy Cows Authors: Bernabucci, Umberto; Basiricò, Loredana; Lacetera, Nicola; Morera, Patrizia; Ronchi, Bruno; Accorsi, Pier Attilio; Seren, Eraldo; Nardone, Alessandro Abstract: Leptin is mainly secreted by adipocytes and is implicated in the regulation of metabolic status, feed intake, and body condition. Day length (DL) can affect leptin gene expression and secretion. The aim of the study was to evaluate the effect of DL on gene expression of leptin and leptin receptors in adipose tissue (AT). Four lactating and pregnant Holstein cows were housed in a climate-controlled chamber for 51 d. The first 30 d were used to adapt animals to the new housing conditions. During that period the DL adopted was 12 h light:12 h dark (12:12). The experimental period included 3 different and consecutive phases: 7 d of neutral DL (12:12); 7 d of long DL (18 h light:6 h dark); and 7 d of short DL (6 h light:18 h dark). Subcutaneous AT biopsies were performed at the end of each phase. Prolactin, growth hormone, cortisol, leptin, glucose, nonesterified fatty acids, β-OH-butyrate, and cholesterol were determined in plasma samples. Abundance of leptin mRNA, and Ob-Ra and Ob-Rb leptin receptormRNA were determined in AT samples by ribonuclease protection assay. Day length did not affect feed intake or body condition score. Exposure to short DL significantly reduced milk yield (13.1 ± 2.2 vs. 15.8 ± 1.7 and 16.0 ± 2.0 kg/d for short vs. neutral and long DL, respectively). Plasma leptin, growth hormone, cortisol, nonesterified fatty acids, β-OH-butyrate, and glucose were not affected by DL; cholesterol was lowest under short DL (3.93 ± 0.38 vs. 4.36 ± 0.39 and 4.07 ± 0.38 mmol/L for short vs. neutral and long DL, respectively). Prolactin increased under long DL (134.82 ± 16.94 vs. 81.98 ± 20.25 and 96.16 ± 0.38 ng/mL for long vs. neutral and short DL, respectively). Gene expression of leptin and its receptors was affected by DL. Leptin mRNA increased under long DL (11.91 ± 0.84 vs. 7.82 ± 0.84 and 7.56 ± 0.84 pg of mRNA/g of total RNA for long vs. neutral and short DL, respectively). Leptin receptors Ob-Ra and Ob-Rb mRNA were higher under long DL, whereas Ob-Ra and Ob-Rb mRNA were lower under short DL (Ob-Ra: 1.91 ± 0.41, 2.49 ± 0.41, and 0.65 ± 0.41 pg of mRNA/g of total RNA for neutral, long, and short DL, respectively; Ob-Rb: 5.29 ± 0.79, 5.98 ± 0.68, and 2.02 ± 0.70 pg of mRNA/g of total RNA for neutral, long, and short DL, respectively). Results of the present study appear to exclude an effect of feed intake and metabolic status on leptin gene expression. A prolactinmediated effect of photoperiod on AT leptin modulation may be proposed in lactating dairy cows. 2005-12-31T23:00:00Z http://hdl.handle.net/2067/1862 Title: Heat Stress Elicits Different Responses in Peripheral Blood Mononuclear Cells from Brown Swiss and Holstein Cows Authors: Lacetera, Nicola; Bernabucci, Umberto; Scalia, Daniela; Basiricò, Loredana; Morera, Patrizia; Nardone, Alessandro Abstract: This study was undertaken to assess whether peripheral blood mononuclear cells (PBMC) isolated from Brown Swiss (Br) and Holstein (Ho) cows and stimulated with concanavalin A differ in response to chronic exposure to incubation temperatures simulating conditions of hyperthermia. Five multiparous Br and 5 Ho cows were utilized as blood donors. Peripheral blood mononuclear cells were subjected for 65 h to each of 5 treatments (T). Cells were exposed to 39°Ccontinuously (T39) and three 13-h cycles at 40 (T40), 41 (T41), 42 (T42) or 43°C (T43), respectively, which were interspersed with two 13-h cycles at 39°C. Treatment T39 was adopted to mimic normothermia; T40, T41, T42, and T43 mimicked conditions of more severe hyperthermia alternating with normothermia. Measures evaluated at the end of the incubation period were proliferative response (DNA synthesis), intracellular reactive oxygen species (ROS) concentrations, and mRNA abundance of the 72-kDa heat-shock protein (Hsp72). In Br cows,DNAsynthesis began to decline whenPBMCwere repeatedly exposed to 41°C (−22%), whereas DNA synthesis in cells isolated from Ho cows did not begin to decline until 42°C (−40%). Furthermore, under T41 and T42, DNA synthesis from Br cows was lower than in Ho (−24 and −54%, respectively). In both breeds, increased incubation temperatures caused a reduction of intracellular ROS (from −39.6 and −69.7%). Increase in incubation temperatures enhanced Hsp72 mRNA levels only in PBMC isolated from Br cows. The Hsp72 mRNA in Br cows increased significantly under T41 and T43 compared with T39. In both breeds, DNA synthesis was positively and negatively correlated with intracellular ROS and Hsp72 mRNA abundance, respectively (r = 0.85 and r = −0.70, respectively). Results indicated that PBMC from Br cows are less tolerant to chronic heat exposure than those from Ho cows, and that the lower tolerance is associated with higher expression of Hsp72, suggesting that the same level of hyperthermia may be associated with a differential decline of immune function in the 2 breeds 2005-12-31T23:00:00Z