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Osteoarthritis (OA) is the most common degenerative joint disease characterized by articular cartilage degradation and joint degeneration. The articular cartilage is mainly formed by chondrocytes and a collagen-proteoglycan extracellular matrix that contains high levels of glycosylated proteins. It was reported that the shift from glycoproteins containing &alpha, 2,6-linked sialic acids to those that contain &alpha, 2,3 was associated with the onset of common types of arthritis. However, the pathophysiology of &alpha, 2,3-sialylation in cartilage has not been yet elucidated. We show that cartilage from osteoarthritic patients expresses high levels of the &alpha, 2,3-sialylated transmembrane mucin receptor, known as podoplanin (PDPN). Additionally, the Maackia amurensis seed lectin (MASL), that can be utilized to target PDPN, attenuates the inflammatory response mediated by NF-kB activation in primary chondrocytes and protects human cartilage breakdown ex vivo and in an animal model of arthritis. These findings reveal that specific lectins targeting &alpha, 2,3-sialylated receptors on chondrocytes might effectively inhibit cartilage breakdown. We also present a computational 3D molecular model for this interaction. These findings provide mechanistic information on how a specific lectin could be used as a novel therapy to treat degenerative joint diseases such as osteoarthritis.

The study was aimed at assessing heat load-related risk of clinical mastitis (CM) in dairy cows. Records of CM for the years 2014 and 2015 were obtained from a large conventional dairy farm milking about 1,200 Holstein cows in central Italy. A case of CM was defined by the presence of clinical signs and veterinary confirmation. Quarter milk samples were collected and bacteriological investigated for each CM. Etiological agents were identified and classified as environmental or contagious pathogens. Hourly weather data from the nearest weather station were used to calculate heat load index (HLI). Upper and lower thresholds of HLI, at which the animal accumulates or dissipates heat, were settled and used to measure heat load balance through the accumulated heat load (AHL) model. Zero and positive values of AHL indicate periods of thermo-neutral and heat accumulation, respectively. Each case of CM was associated with HLI-AHL values recorded 5 d before the event. The risk of CM was evaluated using a case-crossover design. A conditional logistic regression model was used to calculate the odds ratio and 95% confidence intervals of CM recorded in thermo-neutral (AHL = 0) or heat load (AHL0) days, pooled or stratified for pathogen type (environmental or contagious). Classes of AHL as low (6.5), medium (6.6-34.9), and high (35) were included in the model. Other variables included in the model were milk yield as liters (20, 20-30, and30), days in milk (60, 60-150, and150), and parity (1, 2-3, and3). A total of 1,086 CM cases were identified from 677 cows. Escherichia coli, Streptococcus spp., and Streptococcus uberis were the environmental pathogens isolated with the highest frequency; Staphylococcus aureus prevailed within contagious species. The analysis of pooled data indicated a significant effect of heat load on the occurrence of CM in the contagious pathogen stratum. Higher milk yield, middle and late stage of lactation, and older parity increased the risk of CM under heat load conditions. However, the association between pathogen type and these factors was not clear because the model provided significant odds ratios within all pathogen categories. The present study provided the first evidence of an association between HLI and CM in dairy cattle and suggested the ability of the AHL model to assess the risk of mastitis associated with heat load.

The aim of this study was to determine the anti-inflammatory potential of pomegranate peel extracts (PPE) prepared from waste material of pomegranate juice production both in vitro on Caco-2 cells and ex vivo using porcine colonic tissue explants. Caco-2 cells were stimulated in vitro by TNF and colonic tissue explants were stimulated ex vivo with lipopolysaccharide (LPS). Both tissues were co-treated with PPE at 0, 1.0, 2.5, 5.0, 10 and 25 μg/mL. The secretion of CXCL8 in the supernatant of both experiments was determined by enzyme linked immunosorbent assay (ELISA) and the relative expression of inflammatory cytokines were evaluated in the colonic tissue by quantitative polymerase chain reaction (QPCR). The 2.5 to 25 μg/mL of PPE suppressed CXCL8 (p &lt, 0.001) in the Caco-2 cells, whereas CXCL8 production was suppressed by only 5 and 25 μg/mL (p &lt, 0.01) of PPE in the colonic explants. The 5 μg/mL of PPE also suppressed the expression of IL1A (p &lt, 0.05), IL6 (p &lt, 0.01) and CXCL8 (p &lt, 0.05) in LPS challenged colonic tissues compared to controls. In conclusion, the 5 μg/mL of PPE consistently elicits strong anti-inflammatory activity. These results support the potential of bioactive compounds from the waste peel of pomegranate in terms of their anti-inflammatory activity in cells and tissues of the gastrointestinal tract.

(-)-Epigallocatechin-3-gallate and hydroxytyrosol improved antioxidative and anti-inflammatory responses in bovine mammary epithelial cells L. Basiricò1, P. Morera1, D. Dipasquale1, R. Bernini1, L. Santi1, A. Romani2, N. Lacetera1 and U. Bernabucci1† 1Dipartimento di Scienze Agrarie e Forestali (DAFNE), Università degli Studi della Tuscia, via San Camillo de Lellis, 01100, Viterbo, Italy; 2Dipartimento di Statistica, Informatica, Applicazioni (DiSiA) “Giuseppe Parenti”, Università degli Studi di Firenze, via Morgagni 59, 50134, Firenze, Italy (-)-Epigallocatechin-3-gallate (EGCG), the major phenolic compound of green tea, and hydroxytyrosol (HTyr), a phenol found in olive oil, have received attention due to their wide-ranging health benefits. To date, there are no studies that report their effect in bovine mammary gland. Therefore, the aim of this study was to evaluate the anti-oxidative and anti-inflammatory effects of EGCG and HTyr in bovine mammary epithelial cell line (BME-UV1) and to compare their antioxidant and anti-inflammatory in vitro efficacy. Sample of EGCG was obtained from a commercially available green tea extract while pure HTyr was synthetized in our laboratories. The mammary oxidative stress and inflammatory responses were assessed by measuring the oxidative stress biomarkers and the gene expression of inflammatory cytokines. To evaluate the cellular antioxidant response, glutathione (GSH/ GSSH), γ-glutamylcysteine ligase activity, reactive oxygen species and malondialdehyde (MDA) production were measured after 48-h incubation of 50 μM EGCG or 50 μM of HTyr. Reactive oxygen species production after 3 h of hydrogen peroxide (50 μM H2O2) or lipopolysaccharide (20 μM LPS) exposure was quantified to evaluate and to compare the potential protection of EGCG and HTyr against H2O2-induced oxidative stress and LPS-induced inflammation. The anti-inflammatory activity of EGCG and HTyr was investigated by the evaluation of pro and anti-inflammatory interleukins (tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and IL-10) messenger RNA abundance after treatment of cells for 3 h with 20 μM of LPS. Data were analyzed by one-way ANOVA. (-)-Epigallocatechin-3-gallate or HTyr treatments induced higher concentrations of intracellular GSH compared to control cells, matched by an increase of γ-glutamylcysteine ligase activity mainly in cells treated with HTyr. Interestingly, EGCG and HTyr prevented oxidative lipid damage in the BME-UV1 cells by a reduction of intracellular MDA levels. (-)-Epigallocatechin-3-gallate and HTyr were able to enhance cell resistance against H2O2-induced oxidative stress. It was found that EGCG and HTyr elicited a reduction of the three inflammatory cytokines TNF-α, IL-1β, IL-6 and an increase of the anti-inflammatory cytokine IL-10. Hydroxytyrosol has proved to be a strong antioxidant compound, and EGCG has shown mainly an anti-inflammatory profile. These results indicated that EGCG and HTyr may provide dual protection because they were able to attenuate oxidative stress and inflammatory responses, suggesting that these phenolic compounds are potential natural alternatives to be used in dairy cattle as feed supplement for reducing the development of oxidative and inflammatory processes related to parturition or as topical treatments for the control of bovine intramammary inflammation. Animal (2019), 13:12, pp 2847–2856

Climate change has the potential to impair livestock health, with consequences for animal welfare, productivity, greenhouse gas emissions, and human livelihoods and health. Modelling has an important role in assessing the impacts of climate change on livestock systems and the efficacy of potential adaptation strategies, to support decision making for more efficient, resilient and sustainable production. However, a coherent set of challenges and research priorities for modelling livestock health and pathogens under climate change has not previously been available. To identify such challenges and priorities, researchers from across Europe were engaged in a horizon-scanning study, involving workshop and questionnaire based exercises and focussed literature reviews. Eighteen key challenges were identified and grouped into six categories based on subject-specific and capacity building requirements. Across a number of challenges, the need for inventories relating model types to different applications (e.g. the pathogen species, region, scale of focus and purpose to which they can be applied) was identified, in order to identify gaps in capability in relation to the impacts of climate change on animal health. The need for collaboration and learning across disciplines was highlighted in several challenges, e.g. to better understand and model complex ecological interactions between pathogens, vectors, wildlife hosts and livestock in the context of climate change. Collaboration between socio-economic and biophysical disciplines was seen as important for better engagement with stakeholders and for improved modelling of the costs and benefits of poor livestock health. The need for more comprehensive validation of empirical relationships, for harmonising terminology and measurements, and for building capacity for under-researched nations, systems and health problems indicated the importance of joined up approaches across nations. The challenges and priorities identified can help focus the development of modelling capacity and future research structures in this vital field. Well-funded networks capable of managing the long-term development of shared resources are required in order to create a cohesive modelling community equipped to tackle the complex challenges of climate change.

Heat stress (HS) has a negative impact on dairy cows' health, milk production, reproductive performance and immune defenses. Cellular and molecular responses to high temperatures in bovine polymorphonuclear cells and peripheral blood mononuclear cells (PBMCs) have been investigated so far. On the contrary, the effects of high temperatures on isolated monocytes remain almost undisclosed. The aim of this study was to unravel the in vitro effects of high temperatures, simulating a severe HS related body hyperthermia, on bovine lifespan and M1/M2 polarisation. The PBMCs were isolated from whole blood of 9 healthy dairy cattle. Monocytes were sorted by magnetic activated cell sorting and cultured over night at 39 °C (normothermia) or 41 °C (HS). Apoptotic rate and viability were assessed and mRNA abundance for heat shock proteins (HSPs), heat transcription factors (HSFs) and genes involved in monocyte/macrophage polarization (STAT1, STAT2, STAT3, STAT6, IL1β, TGF1β, IL-10, COX2) were quantified by qPCR. We found that apoptosis increased in monocytes exposed to 41 °C, as compared to control, while viability conversely decreased. HS increased the abundance of HSF1 and HSP70. The concomitant decrease of STAT1 and STAT2 and the increase of STAT6 genes abundance at 41 °C suggest, at transcriptional factors level, a polarization of monocytes from a classical activated M1 to a non-classically activated M2 monocytes. In conclusion, the exposure of bovine monocytes to high temperatures affects their lifespan as well as the abundance of genes involved in HS response and in monocyte/macrophages polarization phenotype, confirming that bovine immune response may be significantly affected by hyperthermia.

Natural Product Research, DOI: 10.1080/14786419.2018.1508149 Antioxidant and anti-inflammatory effects of pomegranate peel extracts on bovine mammary epithelial cells BME-UV1 Fabio Mastrogiovanni a, Roberta Bernini a, Loredana Basiricò a,Umberto Bernabucci a , Margherita Campo b, Annalisa Romani b, Luca Santi a and Nicola Lacetera a aDepartment of Agricultural and Forestry Sciences (DAFNE), University of Tuscia, Viterbo, Italy; bDepartment of Statistics, Computing, Applications “Giuseppe Parenti” DiSIA, Phytolab, University of Florence, Florence, Italy Pomegranate peel extracts (PPE) were tested for the first time on BME-UV1, a valid cellular model to study the bovine mammary epithelial metabolism, to evaluate the effects on the oxidative stress and inflammatory status. Based on the statistical analysis of MTT data, PPE at 0.1, 1.0 and 10 lg/mL resulted not cytotoxic after 24 h, 48 h and 7 days of treatment. At the same concentrations, PPE induced a reduction of ROS production elicited by the addition of hydrogen peroxide or lipopolysaccharide evidencing an antioxidant effect confirmed also by a decrease of malondialdehyde. At 10 lg/mL, PPE reduced pro-inflammatory cytokines expressions showing an anti-inflammatory effect on BME-UV1 treated with lipopolysaccharide. Although in vivo experiments are necessary, the results of this study are promising for future applications of PPE as feed supplement for dairy cattle, in particular around calving, when the animals are more subject to oxidative stress and inflammatory diseases.

Livestock plays an important role in the global economy. Climate change effects are not only limited to crop production, but also affect livestock production, for example reduced milk yields and milk quality, reduced meat production and reduced fertility. Therefore, livestock-based food security is threatened in many parts of the world. Furthermore, multiple stressors are a common phenomenon in many environments, and are likely to increase due to climate change. Among these stresses, heat stress appears to be the major factor which negatively influences livestock production. Hence, it is critical to identify agro-ecological zone-specific climate resilient thermo-tolerant animals to sustain livestock production. Livestock responds to the changing environments by altering their phenotypic and physiological characters. Therefore, survivability of the animal often depends on its ability to cope with or adapt to the existing conditions. So to sustain livestock production in an environment challenged by climate change, the animals must be genetically suitable and have the ability to survive in diversified environments. Biological markers or biomarkers indicate the biological states or alterations in expression pattern of genes or state of protein that serve as a reference point in breeding for the genetic improvement of livestock. Conventionally, identification of animals with superior genetic traits that were economically beneficial was the fundamental reason for identifying biomarkers in animals. Furthermore, compared with the behavioural, morphological or physiological responses in animals, the genetic markers are important because of the possibility of finding a solution to animal adaptability to climate change.

Sustainability of food systems is one of the big challenges facing humanity. Local food networks, especially those using organic methods, are proliferating worldwide, and little is known about their carbon footprints. This study aims to assess greenhouse gas (GHG) emissions associated with a local organic beef supply chain using a cradle-to-grave approach.The study determined an overall burden of 24.46 kg COThe identification of the major sources of GHG emissions associated with organic beef produced and consumed in a local food network may stimulate debate on environmental issues among those in the network and direct them toward processes, choices and habits that reduce carbon pollution. © 2018 Society of Chemical Industry.

This study investigated the mortality of dairy cows during heat waves. Mortality data (46,610 cases) referred to dairy cows older than 24mo that died on a farm from all causes from May 1 to September 30 during a 6-yr period (2002-2007). Weather data were obtained from 12 weather stations located in different areas of Italy. Heat waves were defined for each weather station as a period of at least 3 consecutive days, from May 1 to September 30 (2002-2007), when the daily maximum temperature exceeded the 90th percentile of the reference distribution (1971-2000). Summer days were classified as days in heat wave (HW) or not in heat wave (nHW). Days in HW were numbered to evaluate the relationship between mortality and length of the wave. Finally, the first 3 nHW days after the end of a heat wave were also considered to account for potential prolonged effects. The mortality risk was evaluated using a case-crossover design. A conditional logistic regression model was used to calculate odds ratio and 95% confidence interval for mortality recorded in HW compared with that recorded in nHW days pooled and stratified by duration of exposure, age of cows, and month of occurrence. Dairy cows mortality was greater during HW compared with nHW days. Furthermore, compared with nHW days, the risk of mortality continued to be higher during the 3 d after the end of HW. Mortality increased with the length of the HW. Considering deaths stratified by age, cows up to 28mo were not affected by HW, whereas all the other age categories of older cows (29-60, 61-96, and >96mo) showed a greater mortality when exposed to HW. The risk of death during HW was higher in early summer months. In particular, the highest risk of mortality was observed during June HW. Present results strongly support the implementation of adaptation strategies which may limit heat stress-related impairment of animal welfare and economic losses in dairy cow farm during HW.

Animal welfare during transportation has been investigated in several studies, as opposed to post-transportation phases. In this study, we evaluated the effect of a novel environment after transportation on 26 Friesian bulls, 242 ± 42 day-old, from ten different dairy farms. Animals were shipped to a breeding center in different seasons, and selected parameters of innate immunity (serum bactericidal activity, hemolytic complement, serum albumin, α, β, and γ-globulins, interleukin-6, TNF-α) were monitored before and after the arrival at days--4/0/4/15/30. Our results showed significant differences of IL-6 and TNF-α protein levels at destination in December (94 ± 1.3 pg/ml) and June (+788 pg/ml), respectively. Moreover, the serum levels of these cytokines increased between days 0 and 15 after the arrival, the modulation of IL-6 being in agreement with established models of physical and/or psychological stress. Concerning the modulation of albumin, alpha and beta-globulins, the highest levels were detected in April, whereas a significant decrease was observed between day 15 and 30 after arrival; on the contrary, γ-globulin levels significantly increased after day 15. The results of this study highlight the occurrence of innate immune responses of young bulls to the combined effects of climate (season) and novel farming conditions.

Heat stress represents a key factor that negatively affects the productive and reproductive performance of farm animals. In the present work, a new measure of tolerance to heat stress for dairy cattle was developed using principal component analysis. Data were from 590,174 test-day records for milk yield, fat and protein percentages, and somatic cell score of 39,261 Italian Holstein cows. Test-day records adjusted for main systematic factors were grouped into 11 temperature-humidity index (THI) classes. Daughter trait deviations (DTD) were calculated for 1,540 bulls as means of the adjusted test-day records for each THI class. Principal component analysis was performed on the DTD for each bull. The first 2 principal components (PC) explained 42 to 51% of the total variance of the system across the 4 traits. The first PC, a measure of the level at which the curve is located, was interpreted as a measure of the level at which the DTD curve was located. The second PC, which shows the slope of increasing or decreases DTD curves, synthesized the behavior of the DTD pattern. Heritability of the 2 component scores was moderate to high for level across all traits (range = 0.23-0.82) and low to moderate for slope (range = 0.16-0.28). For each trait, phenotypic and genetic correlations between level and slope were equal to zero. A genome-wide association analysis was carried out on a subsample of 423 bulls genotyped with the Illumina 50K bovine bead chip (Illumina, San Diego, CA). Two single nucleotide polymorphisms were significantly associated with slope for milk yield, 4 with level for fat percentage, and 2 with level and slope of protein percentage, respectively. The gene discovery was carried out considering windows of 0.5 Mb surrounding the significant markers and highlighted some interesting candidate genes. Some of them have been already associated with the mechanism of heat tolerance as the heat shock transcription factor (HSF1) and the malonyl-CoA-acyl carrier protein transacylase (MCAT). The 2 PC were able to describe the overall level and the slope of response of milk production traits across increasing levels of THI index. Moreover, they exhibited genetic variability and were genetically uncorrelated. These features suggest their use as measures of thermotolerance in dairy cattle breeding schemes.

Heat stress exerts a direct negative effect on farm animal health, triggering physiological responses. Environmental high temperature induces immunosuppression in dairy cows, increasing the risk of mastitis and milk somatic cell counts. The influence of heat stress on leukocytes activities has not been fully elucidated. The present in vitro study was aimed at assessing whether the exposure to temperature simulating conditions of severe whole body hyperthermia affects defensive functions of bovine blood polymorphonuclear cells. Blood was collected from seven clinically healthy, multiparous, late lactating Holstein cows. After isolation, PMN were incubated at either 39 or 41°C. Phagocytosis, respiratory burst and apoptosis were then investigated. The selected temperatures of 39°C or 41°C mimicked conditions of normothermia or severe heat stress, respectively. Phagocytosis assay was carried out by measuring the fluorescence of phagocyted fluorescein-labelled E. coli bioparticles. The modulation of oxidative burst activity was studied by the cytochrome C reduction method. Apoptosis was determined by measuring the activities of two enzymes that play an effector role in the process, namely Caspase-3 and Caspase-7. Statistical analyses were performed using SPSS 22.0. A Student t-test for paired samples and a Generalised Estimating Equation were used based on data distribution. The phagocytosis rate was reduced (-37%, P

BACKGROUND A hydatid cyst is a parasitic disease caused by the tapeworm Echinococcus granulosus. Kidney involvement represents 4% of all cases, and is rare compared to that in the liver or lung, even more as an isolated site of infection. We present a case report of a woman with septic status, cutaneous fistula and a renal cystic mass revealed to be a solitary hydatid cyst of the kidney. METHODS A 60-year-old woman was referred acutely by another hospital to our department because of septic fever, cutaneous lumbar fistula and a left kidney cystic mass of 10 x 8 cm. We suspected a renal abscess and the patient underwent immediate left nephrectomy. RESULTS We performed an extraperitoneal nephrectomy with a lumbar access under the 12th rib with complete resection of the fistula. The histopathological examination revealed it to be a hydatid cyst involving 2/3 of the kidney. After surgery a medical therapy with albendazol was administrated for 6 months, and the patient did not have any other localization 24 months after surgery. CONCLUSIONS An isolated renal hydatid cyst presenting as cutaneous fistula with a septic status is a very rare condition. A pre-surgical diagnosis is not always possible as in this case. The surgical therapy (nephron-sparing or radical) is the key of the success and a medical therapy after surgery is recommended to prevent other localizations of this parasitic disease.

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 B(1) (AFB(1)) and fumonisin B(1) (FB(1)), on the oxidative status of bovine peripheral blood mononuclear cells (PBMC) incubated for 2 and 7 days at different levels of AFB(1) (0, 5 and 20 μg/ml) and FB(1) (0, 35 and 70 μg/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 AFB(1) and all concentrations of FB(1) caused an increase (p

Mitogens are diverse compounds of plant and microbial origin, widely employed to test immunocompetence in animals. The blastogenic response of bovine Peripheral Blood Mononuclear Cells (PBMC) to lypopolysaccharides (LPS) has been investigated in our laboratories for a long time. In particular, a possible correlation between blastogenic response to LPS and disease resistance of periparturient dairy cows had been observed in previous studies. Most important, low responder cows presented a higher frequency of disease cases after calving, compared with high responder animals. Owing to the above, different aspects of the blastogenic response to LPS were investigated on PBMC of healthy Friesian cows, using a 72-hour Bromodeoxyuridin (BrDU) cell proliferation assay. Stimulation with LPS induced little if any replication of bovine PBMC over 72 hours despite consistent BrDU detection in all the PBMC samples under study. Poor replication of LPS-stimulated PBMC was confirmed by cell cycle and cell growth flow cytometry analyses. In particular, LPS stimulation gave rise to very low percentages of S phase cells, sometimes lower than in control, unstimulated cells, as opposed to Concanavalin A-stimulated PBMC. Magnetic separation and analysis of BrDU-treated bovine PBMC after exposure to LPS showed that both B and CD4 T cells are involved in the blastogenic response to LPS, in contrast with current data based on human and murine models. Finally, LPS caused an early, specific up-regulation of TNF-α and TLR4 genes in bovine PBMC, and significant correlations were shown between the expression of inflammatory cytokine and Indoleamine-pyrrole 2,3-dioxygenase (IDO1) genes. On the whole, our data indicate that differences in the blastogenic response to LPS could be partly accounted for by heterogenicity of responding cells (B and T lymphocytes), which might also have an impact on induction and regulation of inflammatory responses and endotoxin tolerance.

The 2 studies described investigated seasonal variations of mortality and temperature-humidity index (THI)-mortality relationships in dairy cows. Mortality data were extracted from the Italian Bovine Spongiform Encephalopathy databases, which contain records on cows older than 24 mo that died on a farm from all causes (98% of total records), were slaughtered in an emergency state, or were sent for normal slaughter but were sick in the preslaughter inspection (2% of total records). Both studies evaluated mortality data during a 6-yr period (2002 to 2007). The seasonal pattern study was conducted throughout Italy and was based on 320,120 deaths. An association between season and deaths was found for all 6 yr. Summer and spring were the seasons with the highest and lowest frequency of deaths (15,773.3 +/- 2,861 and 11,619.3 +/- 792.3), respectively, and within summer months, the number of deaths in July and August (5,435 +/- 284 and 5,756 +/- 676.2, respectively) was higher than in June (4,839 +/- 344.8). The THI-mortality relationships study was carried out only for deaths (51,240) reported for the Lombardia and Emilia Romagna regions. For this study, the mortality databases were integrated with THI data, which were calculated by using data from 73 weather stations. Each farm where deaths were recorded was assigned the THI values (maximum and minimum) calculated at the closest weather station for each day the events (deaths) were reported. Analysis of data indicated that approximate THI values of 80 and 70 were the maximum and minimum THI, respectively, above which the number of deaths in dairy farms starts to increase. Maximum and minimum THI values of 87 and 77 were the upper critical THI above which the risk of death for dairy cows becomes maximum. This study defined quantitative relationships between mortality risk and THI in dairy cows and may help to provide emergency interventions and mitigation measures, which may ensure survival of dairy cows and reduce replacement costs associated with heat stress-related mortality.

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 Pparγ 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. Pparγ 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.

The objective of the study was to assess the effects of intravenous infusion of triacylglycerol (TAG) emulsions derived from different lipid sources on responses to mitogens of peripheral blood mononuclear cells (PBMC) isolated from fasted dairy cows. Six multiparous, non-pregnant, non-lactating Holstein cows were used in a replicated 3×3 Latin Square design. For 4 d, cows were fasted and infused intravenously with a 20% TAG emulsions derived from tallow (TA), linseed oil (LO) or fish oil (FO). Fasting was employed to induce energy deficit and lipid mobilization. Emulsions were administered for 20 to 30 min every 4 h throughout the 4 d fast at a rate of 0·54 g TAG/kg BW/d. Blood samples were taken before the first infusion, and then every 24 h during the fast. Blood was utilized to assess DNA synthesis, IgM and interferon-gamma (IFN-γ) secretion by PBMC stimulated with mitogens. In TA infused cows there was a decline of PBMC ability to respond to mitogens, which was significant 48 h after initiation of the infusion period for DNA synthesis and IFN-γ secretion. In LO or FO infused cows, PBMC responses to mitogens were not altered during the infusion period, and in some cases PBMC responses to mitogen was improved at 72 and 96 h after initiation of treatments. Effects of TAG infusion on PBMC responses to mitogens depended on the lipid source suggesting that LO or FO can attenuate the negative effects of fasting on immune functions.

The data set consisted of 1,016,856 inseminations of 191,012 first, second, and third parity Holstein cows from 484 farms. Data were collected from year 2001 through 2007 and included meteorological data from 35 weather stations. Nonreturn rate at 56 d after first insemination (NR56) was considered. A logit model was used to estimate the effect of temperature-humidity index (THI) on reproduction across parities. Then, least squares means were used to detect the THI breakpoints using a 2-phase linear regression procedure. Finally, a multiple-trait threshold model was used to estimate variance components for NR56 in first and second parity cows. A dummy regression variable (t) was used to estimate NR56 decline due to heat stress. The NR56, both for first and second parity cows, was significantly (unfavorable) affected by THI from 4 d before 5 d after the insemination date. Additive genetic variances for NR56 increased from first to second parity both for general and heat stress effect. Genetic correlations between general and heat stress effects were -0.31 for first parity and -0.45 for second parity cows.

Objective—To evaluate vaccine safety, antibody response, and nonspecific lymphocyte blastogenesis following inoculation of a commercial monovalent live attenuated bluetongue virus (BTV) serotype 2 vaccine in goats. Animals—12 nonpregnant and nonlactating Saanen goats. Procedure—6 goats were inoculated with the monovalent live attenuated BTV serotype 2 vaccine, which has been widely used in Italy during the proceding 2 years. The other 6 goats were unvaccinated and represented negative controls. Nonspecific lymphocyte blastogenesis was evaluated 14 and 7 days before and 7, 21, and 49 days after vaccination by measuring DNA synthesis in peripheral blood mononuclear cells (PBMCs) stimulated with phytohemagglutinin, concanavalin-A, and pokeweed mitogen. On the same days as lymphocyte blastogenesis, blood samples were taken to determine serum concentrations of anti-BTV antibodies. Results—During the 7 weeks following vaccination, PBMCs obtained from vaccinated goats had a significantly decreased response to mitogens in terms of DNA synthesis, compared with PBMCs from the same goats before vaccination. Conversely during the experiment, no significant change was found in the response of the PBMCs obtained from unvaccinated goats. Starting from 21 days after vaccination, serum from vaccinated goats had anti-BTV antibodies. No anti-BTV antibodies were detected in the serum from unvaccinated goats. Conclusions and Clinical Relevance—Inoculation of goats with the monovalent live attenuated BTV serotype 2 vaccine described herein resulted in a profound depression of nonspecific lymphocyte blastogenesis, which might compromise the resistance of vaccinated goats to pathogens. (Am J Vet Res 2004;65:1331–1334)

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 B(100) (ApoB(100)), apolipoprotein E (ApoE), and microsomal triglyceride transfer protein (MTP) play in that VLDL limitation. To our knowledge, no studies have simultaneously measured ApoB(100), 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 ApoB(100), ApoE, and MTP were 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 ApoB(100) mRNA synthesis was lower, whereas MTP and ApoE mRNA abundance were higher. Negative correlation (r = -0.57) between plasma NEFA concentration and ApoB(100) mRNA abundance, and positive correlation between ApoB(100) 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 ApoB(100) may be consistent with decreased synthesis and/or secretion of VLDL from liver during the periparturient period.

Objective—To assess the effects of nonesterified fatty acids (NEFA) and β-hydroxybutyrate (BHBA) on functions of mononuclear cells obtained from ewes.Animals—6 Sardinian ewes.Procedure—Mononuclear cells were cultured with concentrations of NEFA (0, 15.6, 31.2, 62.5, 125, 250, 500, 1,000, or 2,000 µmol/L) and BHBA (0, 0.45, 0.9, 1.8, or 3.6 mmol/L). Concentrations of NEFA and BHBA were intended to mimic those of ketotic or healthy ewes, and NEFA and BHBA were tested alone and in combination. Synthesis of DNA was stimulated by use of concanavalin A (Con A) or pokeweed-mitogen (PWM). Secretion of IgM was stimulated by use of PWM.Results—Synthesis of DNA stimulated by Con A and PWM was significantly inhibited by high concentrations of NEFA (≥ 250 µmol/L) or by a combination of high concentrations of NEFA (≥ 250 µmol/L) and all concentrations of BHBA (≥ 0.45 mmol/L). In contrast, DNA synthesis was not inhibited by low concentrations of NEFA (≤ 125 µmol/L) or by a combination of low concentrations of NEFA (≤ 125 µmol/L) and the lowest concentration of BHBA (0.45 mmol/L). Secretion of IgM was significantly inhibited by all concentrations of NEFA and by all combinations of NEFA and BHBA concentrations. When used alone, none of the concentrations of BHBA inhibited DNA synthesis or IgM secretion.Conclusions and Clinical Relevance—Reduced immunoresponsiveness during ketosis is likely to be associated with an increase in plasma concentration of NEFA and not with an increase in plasma concentration of BHBA. (Am J Vet Res2002;63:414–418)

The study was based on data collected during 5 yr (2003-2007) and was aimed at assessing the effects of the month, slaughter house of destination (differing for stocking density, openings, brightness, and cooling device types), length of the journey, and temperature-humidity index (THI) on mortality of heavy slaughter pigs (approximately 160 kg live weight) during transport and lairage. Data were obtained from 24,098 journeys and 3,676,153 pigs transported from 1,618 farms to 3 slaughter houses. Individual shipments were the unit of observation. The terms dead on arrival (DOA) and dead in pen (DIP) refer to pigs that died during transport and in lairage at the abattoir before slaughtering, respectively. These 2 variables were assessed as the dependent counts in separate univariate Poisson regressions. The independent variables assessed univariately in each set of regressions were month of shipment, slaughter house of destination, time traveled, and each combination of the month with the time traveled. Two separate piecewise regressions were done. One used DOA counts within THI levels over pigs transported as a dependent ratio and the second used DIP counts within THI levels over pigs from a transport kept in lairage as a dependent ratio. The THI was the sole independent variable in each case. The month with the greatest frequency of deaths was July with a risk ratio of 1.22 (confidence interval: 1.06-1.36; P0.05) and 1.27 (confidence interval: 1.06-1.51; P0.05) for DOA and DIP, respectively. The lower mortality risk ratios for DOA and DIP were recorded for January and March (P0.05). The aggregated data of the summer (June, July, and August) versus non-summer (January, March, September, and November) months showed a greater risk of pigs dying during the hot season when considering both transport and lairage (P0.05). The mortality risk ratio of DIP was lower at the slaughter house with the lowest stocking density (0.64 m(2)/100 kg live weight), large open windows on the roof and sidewalls, low brightness (40 lx) lights, and high-pressure sprinklers as cooling devices. The mortality risk ratio of DOA increased significantly for journeys longer than 2 h, whereas no relationship was found between length of transport and DIP. The piecewise analysis pointed out that 78.5 and 73.6 THI were the thresholds above which the mortality rate increased significantly for DOA and DIP, respectively. These results may help the pig industry to improve the welfare of heavy slaughter pigs during transport and lairage.

The data set for this study comprised 1,488,474 test-day records for milk, fat, and protein yields and fat and protein percentages from 191,012 first-, second-, and third-parity Holstein cows from 484 farms. Data were collected from 2001 through 2007 and merged with meteorological data from 35 weather stations. A linear model (M1) was used to estimate the effects of the temperature-humidity index (THI) on production traits. Least squares means from M1 were used to detect the THI thresholds for milk production in all parities by using a 2-phase linear regression procedure (M2). A multiple-trait repeatability test-model (M3) was used to estimate variance components for all traits and a dummy regression variable (t) was defined to estimate the production decline caused by heat stress. Additionally, the estimated variance components and M3 were used to estimate traditional and heat-tolerance breeding values (estimated breeding values, EBV) for milk yield and protein percentages at parity 1. An analysis of data (M2) indicated that the daily THI at which milk production started to decline for the 3 parities and traits ranged from 65 to 76. These THI values can be achieved with different temperature/humidity combinations with a range of temperatures from 21 to 36°C and relative humidity values from 5 to 95%. The highest negative effect of THI was observed 4 d before test day over the 3 parities for all traits. The negative effect of THI on production traits indicates that first-parity cows are less sensitive to heat stress than multiparous cows. Over the parities, the general additive genetic variance decreased for protein content and increased for milk yield and fat and protein yield. Additive genetic variance for heat tolerance showed an increase from the first to third parity for milk, protein, and fat yield, and for protein percentage. Genetic correlations between general and heat stress effects were all unfavorable (from -0.24 to -0.56). Three EBV per trait were calculated for each cow and bull (traditional EBV, traditional EBV estimated with the inclusion of THI covariate effect, and heat tolerance EBV) and the rankings of EBV for 283 bulls born after 1985 with at least 50 daughters were compared. When THI was included in the model, the ranking for 17 and 32 bulls changed for milk yield and protein percentage, respectively. The heat tolerance genetic component is not negligible, suggesting that heat tolerance selection should be included in the selection objectives. © 2014 American Dairy Science Association.

A retrospective study on seasonal variations in the characteristics of cow’s milk and temperature–humidity index (THI) relationship was conducted on bulk milk data collected from 2003 to 2009. The THI relationship study was carried out on 508 613 bulk milk data items recorded in 3328 dairy farms form the Lombardy region, Italy. Temperature and relative humidity data from 40 weather stations were used to calculate THI. Milk characteristics data referred to somatic cell count (SCC), total bacterial count (TBC), fat percentage (FA%) and protein percentage (PR%). Annual, seasonal and monthly variations in milk composition were evaluated on 656 064 data items recorded in 3727 dairy farms. The model highlighted a significant association between the year, season and month, and the parameters analysed (SCC, TBC, FA%, PR%). The summer season emerged as the most critical season. Of the summer months, July presented the most critical conditions for TBC, FA% and PR%, (52 054±183 655, 3.73%±0.35% and 3.30%±0.15%, respectively), and August presented higher values of SCC (369 503±228 377). Each milk record was linked to THI data calculated at the nearest weather station. The analysis demonstrated a positive correlation between THI and SCC and TBC, and indicated a significant change in the slope at 57.3 and 72.8 maximum THI, respectively. The model demonstrated a negative correlation between THI and FA% and PR% and provided breakpoints in the pattern at 50.2 and 65.2 maximum THI, respectively. The results of this study indicate the presence of critical climatic thresholds for bulk tank milk composition in dairy cows. Such indications could facilitate the adoption of heat management strategies, which may ensure the health and production of dairy cows and limit related economic losses.

Objective—To determine the effects of moderate feed restriction, single or twin pregnancy, and subclinical pregnancy toxemia (PT) on immune responses of ewes. Animals—16 Sardinian ewes. Procedure—Six weeks before lambing, ewes were assigned to 1 of 2 groups (n = 8/group) matched for number of fetuses, body condition score, and plasma glucose, non-esterified fatty acids (NEFA), and β- hydroxybutyrate (BHBA) concentrations, and feed intake was restricted for 1 of the groups. Cell-mediated immunity was evaluated in vivo and in vitro. Humoral immunity was evaluated in vivo by determining production of IgG antibodies against keyhole limpet hemocyanin (KLH). Results—Four ewes developed subclinical PT (plasma BHBA concentration > 0.86 mmol/L without any clinical signs of disease). Whether feed was restricted and type of pregnancy (single vs twin) did not have any significant effects on cell-mediated and humoral immune responses. Ewes with subclinical PT had significantly lower in vitro proliferation of phytohemagglutinin- stimulated peripheral blood mononuclear cells and significantly lower values for KLH-specific IgG than did healthy ewes. Plasma BHBA and NEFA concentrations were negatively correlated with in vitro proliferation of peripheral blood mononuclear cells; plasma NEFA concentration was negatively correlated with values for KLH-specific IgG. Conclusions and Clinical Relevance—Results suggest that subclinical PT may be associated with impairments in cell-mediated and humoral immune responses in sheep. Am J Vet Res 2001;62:1020–1024)

This study was performed to ascertain whether a standardized extract from Echinacea angustifolia (Polinacea) affects proliferation and interferon gamma (IFN-gamma) secretion in bovine peripheral blood mononuclear cells (PBMC). PBMC from six Holstein heifers were incubated with 0, 6.3, 20, 60, or 180 microg/ml of the tested compound. Proliferation was stimulated by concanavalin A (ConA) or pokeweed-mitogen (PWM). Secretion of IFN-gamma was stimulated by ConA. All concentrations of Polinacea exerted a mitogenic effect. With respect to control PBMC (0 microg/ml), the lowest and highest increase of proliferation were observed with Polinacea at 6.3 (2-fold increase) or 180 (10-fold increase) microg/ml, respectively. Polinacea at 180 microg/ml reduced ConA-driven proliferation, whereas at 20 and 60 microg/ml improved proliferation of PWM-stimulated PBMC. IFN-gamma secretion was not affected. In conclusion, Polinacea modulates bovine PBMC proliferation, and deserves to be tested in vivo to define conditions that may benefit from its utilization.

This preliminary study aimed at assessing whether the in vitro proliferation of peripheral blood mononuclear cells in response to lipopolysaccharide permits individual characterization of periparturient dairy cows, and whether this parameter may be associated with incidence of infections and with some of the single nucleotide polymorphisms located on the toll-like receptor 4 ( TLR4 ) gene. Based on the average response of peripheral blood mononuclear cells to lipopolysaccharide over 7 time points during the transition period, 31 cows were categorized as low (LO), medium (MED), and high (HI) responders. This categorization identified 7 HI, 19 MED, and 5 LO cows, respectively. Genomic DNA was genotyped for P-226 C>G and E3+2021 C>T TLR4 single nucleotide polymorphisms. Monitoring of the health status revealed that 8 of the 31 cows suffered from clinical mastitis, metritis, or interdigital dermatitis during the first 60d in milk. The association study pointed out that none of the HI cows and all of the LO cows developed an infection; cows with the CCGT haplotype remained healthy and none of them belonged to the LO responder category.

The study was undertaken to describe the temperature humidity index (THI) dynamics over the Mediterranean basin for the period 1971-2050. The THI combines temperature and humidity into a single value, and has been widely used to predict the effects of environmental warmth in farm animals. The analysis was based on daily outputs of the temperature and relative humidity from the Max Planck Institute data using the Intergovernmental Panel on Climate Change Special Report Emission Scenario A1B. Data revealed a gradual increase of both annual and seasonal THI during the period under investigation and a strong heterogeneity of the Mediterranean area. In particular, the analysis indicated that Spain, southern France and Italy should be expected to undergo the highest THI increase, which in the last decade under study (2041-2050) will range between 3 and 4 units. However, only during summer months the area presents characteristics indicating risk of thermal (heat) stress for farm animals. In this regard, scenario maps relative to the summer season suggested an enlargement of the areas in the basin where summer THI values will likely cause thermal discomfort in farm animals. In conclusion, the study indicated that the Mediterranean basin is likely to undergo THI changes, which may aggravate the consequences of hot weather on animal welfare, performances, health and survival and may help farmers, nutritionists, veterinarians, and policy-makers to develop appropriate adaptation strategies to limit consequences of climate change for the livestock sector in the Mediterranean countries.

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 transcription-polymerase 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

The study was aimed at assessing whether the peri-parturient period is associated with changes of intracellular and plasma inducible heat shock proteins (Hsp) 72 kDa molecular weight in dairy cows, and to establish possible relationships between Hsp72, metabolic, and immunological parameters subjected to changes around calving. The study was carried out on 35 healthy periparturient Holstein cows. Three, two, and one week before the expected calving, and 1, 2, 3, 4, and 5 weeks after calving, body conditions score (BCS) was measured and blood samples were collected to separate plasma and peripheral blood mononuclear cells (PBMC). Concentrations of Hsp72 in PBMC and plasma increased sharply after calving. In the post-calving period, BCS and plasma glucose declined, whereas plasma nonesterified fatty acids (NEFA) and tumor necrosis factor-alpha increased. The proliferative responses of PBMC to lipopolysaccharide (LPS) declined progressively after calving. The percentage of PBMC expressing CD14 receptors and Toll-like receptors (TLR)-4 increased and decreased in the early postpartum period, respectively. Correlation analysis revealed significant positive relationships between Hsp72 and NEFA, and between PBMC proliferation in response to LPS and the percentage of PBMC expressing TLR-4. Conversely, significant negative relationships were found between LPS-triggered proliferation of PBMC and both intracellular and plasma Hsp72. Literature data and changes of metabolic and immunological parameters reported herein authorize a few interpretative hypotheses and encourage further studies aimed at assessing possible cause and effect relationships between changes of PBMC and circulating Hsp72, metabolic, and immune parameters in dairy cows. L'articolo è disponibile sul sito dell'editore http://link.springer.com/journal/12192

Effects of different periods of exposure to hot environments on rumen function, diet digestibility and digesta passage rate were studied in four adult notpregnant Sardinian ewes housed in a climatic chamber. The ewes were kept in individual metabolic cages. The trial lasted 83 days; 17 days were spent under thermal comfort conditions (TC) [temperature-humidity index (THI)=65.0±2.0], followed by 49 days under elevated THI (ETHI: THI=82.0±2.5) and 17 days under thermal comfort (TC; THI=65.0±1.0). Five digestibility and passage rate trials were carried out during the 83 days. Trials 1 and 5 were carried out under TC; trials 2, 3 and 4 were carried out under ETHI. Values of rectal temperatures (39.7±0.3°C) and respiratory rate (118.4±31.8 breaths/min) indicated that sheep under ETHI were heat-stressed. Heat stress caused an increase (P

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. L’articolo è disponibile sul sito dell’editore http://www.sciencedirect.com/science/journal/01652427

Twenty-four dairy cows were monitored during the transition period. We observed changes of oxidative status and relationships between oxidative and metabolic status. Body condition score (BCS) of the 24 animals at the beginning of the trial (30.4 +/- 2 d before expected calving) was between 2.0 and 3.6. The BCS was recorded and blood samples were collected weekly during the last 30 d of pregnancy and the first 30 DIM. Plasma samples were analyzed to determine indices of oxidative status: reactive oxygen metabolites (ROM); thiobarbituric acid-reactive substances (TBARS); thiol groups (SH); glutathione peroxidase (GSH-Px), and indices of energy metabolism: glucose, beta-hydroxybutyrate, and nonesterified fatty acids. In erythrocytes we determined indices of oxidative status: GSH-Px, superoxide dismutase (SOD), and intracellular SH. Before calving, cows showed an increase of plasma SH, SOD, and GSH-Px, a decrease of erythrocyte GSH-Px and plasma ROM, and no changes in erythrocyte SH. After calving, cows showed a decrease of plasma and erythrocyte SH and SOD, and an increase of ROM, TBARS, and plasma GSH-Px. Cows with higher BCS at the beginning of the trial and greater loss of BCS after calving, had higher plasma ROM, TBARS, and SH, and lower SOD and erythrocyte SH in the postpartum period. Oxidative status of dairy cows was related to energy status. Cows with higher BHBA and NEFA showed higher ROM and TBARS and lower levels of antioxidants. Results of the present study demonstrated that cows can experience oxidative stress during the peripartum period, and cows with higher BCS and greater BCS losses are more sensitive to oxidative stress.

This study was carried out to ascertain the effects of intense high environmental temperatures (HET) on lymphocyte functions in periparturient dairy cows. The study was undertaken from the beginning of March through the end of July 2003 in a commercial dairy unit located approximately 40 km north of Rome. Thirty-four Holstein cows were utilised in the study. Twenty-two of these cows gave birth in spring (SP cows), from 28 March to 30 April. The remaining 12 cows gave birth in summer (SU cows), between 15 June and 2 July. The two groups of cows were balanced for parity and were fed the same rations. Blood samples were taken 4, 3, 2 and 1 week before calving, and 1, 2 and 4 weeks after calving, in order to evaluate peripheral blood mononuclear cell (PBMC) function in vitro, and to determine plasma cortisol concentrations. After isolation, the PBMC were stimulated with mitogens and their response in terms of DNA synthesis and IgM secretion was measured. During spring, either the day (9– 20 h) or the night (21–8 h) temperature humidity index (THI) was below the upper critical THI (72) established for dairy cows. During summer, the mean daily THI values were 79.5±2.9 during the day and 70.1±4.7 during the night. Furthermore, during summer, three heat waves (a period of at least 3 consecutive days during which there were less than 10 recovery hours) occurred. Recovery hours were intended hours with a THI below 72. The first heat wave lasted 5 days, the second 6 days, and the third 15 days. Compared to the SP cows, over the entire periparturient period the extent of DNA synthesis and IgM secretion levels were lower (P ranging from

The study was undertaken during spring and summer months in a territory representative of the Mediterranean climate to assess the effects of season on some immunological parameters of dairy cows. Twenty Holstein cows were used. Eleven of those cows gave birth during spring; the remaining nine cows gave birth in summer. The two groups of cows were homogeneous for parity. Values of air temperatures and relative humidity were recorded both during spring and summer, and were utilized to calculate the temperature humidity index (THI). One week before the expected calving, rectal temperatures and respiratory rates of the cows were recorded (1500 hours), and cell-mediated immunity was assessed by measuring the proliferation of mitogen-stimulated peripheral blood mononuclear cells (PBMC). Within 3 h of calving, one colostrum sample was taken from each cow and analysed to determine content of immunoglobulin (Ig) G1, IgG2, IgM and IgA. At 48 h after birth, passive immunization of the calves was assessed by measuring total serum IgG. During summer, daytime (0900–2000 hours) THI values were above the upper critical value of 72 [75.2, (SD 2.6)] indicating conditions that could represent moderate heat stress. That THI values were able to predict heat stress was confirmed by the values of rectal temperatures and respiratory rates, which were higher (P < 0.05 and P < 0.001 respectively) during summer. Proliferation of PBMC, the colostral concentration of Ig fractions and serum levels of IgG in their respective offspring did not differ between spring and summer cows. Results indicated that moderate heat stress due to the hot Mediterranean summer does not modify cell-mediated immunity, the protective value of colostrum and passive immunization of the offspring in dairy cows.

Water-in-oil-type adjuvants prepared with natural or synthetic vitamin E as the oil phase, Arlacel A or Montanide 103 as emulsifier and 1.0% aqueous solution of bovine serum albumin (BSA) as antigen were tested in mice. An emulsion of 0.42 ml vitamin E, 0.42 ml special mineral oil, 0.15 ml Arlacel A and 1.0 ml BSA gave maximal humoral response, but 0.85 ml special mineral oil, 0.15 ml Arlacel A and 1.0 ml BSA (standard Freund's incomplete adjuvant formulation) gave the greatest delayed-type hypersensitivity response.

The aim of this preliminary study was to determine the effects of selenium (Se) injection on Se status, cell-mediated immunity (CMI), milk yield and milk somatic cell count (MSCC) of ewes fed adequate amounts of Se, and on Se status, passive immunization and CMI of their offspring. Thirty days before lambing, 36 Sardinian ewes were assigned to one of three groups. One group (NT) was not treated; a second group (BL) was given 5 mg of Se on day 30 before lambing; a third group (BLL) was given 2.5 mg of Se on day 30 before lambing and at lambing. Selenium was given intramuscularly as sodium selenite. Selenium status was assessed by measuring glutathione peroxidase activity of erythrocytes (GSHpx-E). The CMI was measured by determining the increases in double skinfold thickness after intradermal injection of phytohaemagglutinin (PHA). Compared to their NT counterparts, ewes belonging to BL and BLL groups and their offspring had significantly higher GSHpx-E (P0.01). The GSHpx-E values of lambs were positively related to those of their mothers (P0.0005). Ewes of group BL had a greater (P0.01) response to PHA 6 h after injection than ewes of the NT group. Lambs born to BL and BLL ewes had a greater (P0.0001) response to PHA 24 h after injection. Responses of ewes and lambs to PHA 24 h after injection were positively related (P0.05). Serum immunoglobulin at 10 days of age did not differ significantly among the three groups of lambs. Compared to the NT group, milk yield on day 70 of lactation was significantly higher in BL ewes (P0.05). The MSCC was not affected significantly by Se injection. Immunoresponsiveness and milk yield might represent additional and appropriate criteria to consider when re-evaluating Se requirements of dairy sheep.

To determine effects of selenium (Se) and vitamin E (VE) administration in late pregnancy on Se status, plasma immunoglobulin concentrations, and colostrum and milk production of dairy cows, and on Se status, passive immunity, and growth of their offspring.25 Holstein cows and their offspring.3 and 1.5 weeks before calving, sodium selenite (5 mg/100 kg of body weight) and d,l-alpha-tocopheryl acetate (25 IU/100 kg) were administered to 13 cows. The other 12 cows were not treated. Se status was assessed by measurement of glutathione peroxidase activity of erythrocytes (GSH-Px-E).The 13 treated cows had higher (P0.01) GSH-Px-E values at calving and during the first 12 weeks of lactation. Changes in plasma immunoglobulin concentrations before or after calving did not differ between the 2 groups of cows. During the first 36 hours after calving (4 milkings), treated cows produced 22% more colostrum than did their nontreated counterparts (P0.005). Percentages of colostral immunoglobulins did not differ between the 2 groups. During the first 12 weeks of lactation, treated cows produced 10% more milk than did nontreated cows (P0.005). GSH-Px-E values at birth and 28 days of life were significantly higher in calves from treated cows. Plasma immunoglobulin concentrations and body weight during the first 56 days after birth did not differ between calves born to treated or nontreated cows.Cows given Se and VE in late pregnancy produce large quantities of colostrum and milk. Colostrum produced from cows given Se and VE is suitable to feed newborn calves and to be stored for later use. Improvement of Se status in calves born to cows given Se and VE in late pregnancy is not beneficial to passive immunity and growth.

The effect of beta carotene on disease protection and humoral immunity in chickens was investigated in comparison with the effect of other lipid-soluble antioxidant vitamins, vitamin E and A, which are both proven immunoenhancers and contributors to disease protection. Beta carotene alone was not as effective as either vitamin in protecting chickens from Escherichia coli infection, nor did it significantly enhance humoral immunity. In combination with vitamin E, however, beta carotene significantly increased disease protection and reduced hepatomegaly caused by E. coli infection.

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, beta-OH-butyrate, and cholesterol were determined in plasma samples. Abundance of leptin mRNA, and Ob-Ra and Ob-Rb leptin receptor mRNA 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, beta-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/microg 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/microg 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/microg 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 prolactin-mediated effect of photoperiod on AT leptin modulation may be proposed in lactating dairy cows.

Effects of short- and long-term exposure to a hot environment on diet digestibility and rumen passage rate were studied in four, 10-mo-old Friesian heifers housed in a climatic chamber. The trial lasted 65 d. Twenty-five days were spent under thermal comfort (temperature-humidity index = 64), and 40 d were spent under hot conditions (temperature-humidity index = 84). Three digestibility and rumen passage rate trials were performed during the 65 d. Chromium oxide was used as an external marker. The first digestibility and rumen passage rate trial (trial 1) was performed under thermal comfort; trials 2 and 3 were performed under hot conditions. Exposure to the hot environment reduced dry matter intake and increased water intake and rectal temperature compared with those during the thermal comfort period. Digestibility coefficients for dry matter, organic matter, neutral detergent fiber, and acid detergent fiber were higher in trial 2 than in trials 1 and 3. No statistical differences were found between trials 1 and 3 for these variables. Rumen passage rate was more rapid in trial 1 than in trials 2 and 3. No difference was observed between trials 2 and 3. These results indicated that exposure to a hot environment can affect digestibility in a time-dependent fashion, suggesting an adaptation of the digestive tract to hot environments.

The study was carried out to assess whether hot seasons affect the oxidative status of transition dairy cows. Twenty Holstein cows were utilized. Eleven of those cows gave birth in spring; the remaining nine cows gave birth in summer. Oxidative status was evaluated from 21 d before to 35 d after calving, determining oxidative markers in plasma [glutathione peroxidase activity, thiol groups, reactive oxygen metabolites, and thiobarbituric acid reactive substances (TBARS)] and in erythrocytes [glutathione peroxidase activity (GSH-Px-E), intracellular thiols (SH), superoxide dismutase activity (SOD) and TBARS]. The season did not modify plasma oxidative markers. In contrast, cows exposed to moderate heat stress (39.5 +/- 0.2 degrees C rectal temperatures) due to summer temperature-humidity index (73.2 +/- 2.5 mean daily THI), showed higher erythrocyte SOD, GSH-PxE, SH, and TBARS compared with spring cows, indicating a condition of oxidative stress in summer transition dairy cows. Some changes (the increase of SOD, for example), represent compensatory changes the cow is undergoing in response to increased oxidative stress. Under the conditions compared, the effect of heat stress on oxidative status in transition dairy cows by use of plasma markers does not give enough information to reach definitive conclusions. Erythrocytes are an appropriate and sensitive model to study the oxidative status of transition dairy cows exposed to hot environments.

The objective of the study was to evaluate the relationships between body condition and lymphocyte functions in periparturient dairy cows. Thirty days before expected calving, 21 Holstein cows were categorized as thin (n = 6), medium (n = 8), or overconditioned (n = 7) based on body condition score (BCS). Blood samples were collected on 21, 14, 7, and 3 d before calving and on d 3, 7, 14, 21, 28, and 35 after parturition. An aliquot of blood was used to determine plasma nonesterified fatty acids (NEFA) and glucose. At 14 and 7 d before, and 14 and 35 d after calving, a second aliquot of blood was used to assess peripheral blood mononuclear cell (PBMC) functions: DNA synthesis, immunoglobulin (Ig) M, and interferon-gamma (IFN-gamma) secretion after mitogen stimulation. During the experiment, all 21 cows showed a decline in BCS. Overconditioned cows lost significantly more BCS than thin cows. After calving, overconditioned cows had higher plasma NEFA compared with thin and medium cows. Conversely, plasma glucose never differed between the 3 categories of cows. Regardless of BCS, DNA synthesis and IgM secretions were significantly lower in PBMC isolated on 7 d before calving compared with those recorded 14 and 35 d after parturition. Conversely, PBMC from the 21 cows did not show any change of IFN-gamma secretion during the experimental period. Taking into consideration the BCS categories, PBMC isolated from overconditioned cows presented lower IgM secretion compared with thin cows on d 14 and 35 after calving. Furthermore, PBMC isolated from overconditioned cows secreted less IFN-gamma compared with thin and medium cows on d 7 before calving. The DNA synthesis of PBMC stimulated with the 3 mitogens did not differ between the 3 categories of cows. In conclusion, immunodepression occurring in cows around calving would be particularly evident in overconditioned cows.

An in vitro study was conducted to examine the influence of nonesterified fatty acids (NEFA) on bovine polymorphonuclear leukocytes (PMN). Eight healthy, midlactating Holstein cows were used as blood donors. Blood PMN were isolated and incubated with a mixture of NEFA, reflecting composition of bovine plasma NEFA at concentrations that were intended to mimic those found in blood of cows undergoing high, moderate, or low lipomobilization intensity (2, 1, 0.5, 0.25, 0.125, and 0.0625 mM). Control samples were incubated in absence of NEFA. Phagocytosis and oxidative burst activities were assessed by a 2-color flow cytometric method, which was based on oxidation of intracellular dihydrorhodamine 123 to green fluorescent rhodamine 123. Oxidative burst products were generated by incubating PMN with Staphylococcus aureus labeled with propidium iodide. A flow cytometric technique was used to detect PMN viability, necrosis, and apoptosis using fluorescein isothiocyanate-labeled annexin-V and propidium iodide. Phagocytic activity was not affected by NEFA. The highest concentration of NEFA (2 mM) was associated with a dramatic increase of phagocytosis-associated oxidative burst activities with a reduction in cell viability (48.0 vs. 97.5% in control samples) and with a marked increase of necrosis (49.4 vs. 0.5% in control samples). Conversely, the mixture of NEFA did not affect the occurrence of apoptosis. Enhancement of the oxidative burst associated with the highest concentration of NEFA might explain the reduced viability and higher percentage of necrosis observed under the same conditions. This study demonstrated a substantial resistance of bovine PMN to an overload of fatty acids. However, observation that the highest concentration of NEFA regulated some PMN functions encourages the possibility of in vivo studies to assess the relationships between intensity of lipomobilization, plasma NEFA, and bovine PMN functions.

This study examined the effects of heat stress on composition of colostrum from primiparous cows during late pregnancy and the early postpartum period. Two groups of 6 Holstein heifers were utilized. During the last 3 wk of pregnancy and during the first 36 h after calving, one group was exposed to thermal comfort (temperature-humidity index = 65); the other group was exposed to high air temperatures (temperature-humidity index = 82 from 0900 to 2000 h and temperature-humidity index = 76 from 2100 to 0800 h). Heifers under heat stress had higher rectal temperatures and respiratory rates; lower plasma triiodothyronine and glucose; higher plasma nonesterified fatty acids and beta-hydroxybutyrate; lower intakes of dry matter, net energy for lactation, and crude protein; higher water intakes; and lower body condition scores. The decline of plasma immunoglobulins (Ig) over the final 2 wk of pregnancy was less pronounced for heifers under heat stress. For the first four milkings, colostrum of cows exposed to high air temperatures had lower mean concentrations of IgG and IgA; lower mean percentages of total protein, casein, lactalbumin, fat, and lactose; lower contents (grams per liter) of short- and medium-chain fatty acids; lower energy; lower titratable acidity; and higher pH. Thus, high air temperatures during late pregnancy and the early postpartum period markedly affected the composition of colostrum from primiparous dairy cows.

This in vitro study was performed to assess the effects of various concentrations of nonesterified fatty acids (NEFA) on lymphocyte function of heifers. Nine Holstein heifers were studied. Peripheral blood mononuclear cells were incubated with various concentrations of NEFA (0, 0.0625, 0.125, 0.25, 0.5, 1, and 2 mmol/L). The mixture of NEFA was represented by C16:0 (30%), C16:1 (5%), C18:0 (15%), C18:1 (45%), and C18:2 (5%). The DNA synthesis was diminished at concentrations of NEFA of 2, 1, and 0.5 mmol/L. The IgM secretion was inhibited at concentrations of NEFA of 2, 1, 0.5, and 0.25 mmol/L. Secretion of IFN-gamma was depressed at concentrations of NEFA of 2, 1, 0.5, 0.25, and 0.125 mmol/L. Increases of plasma NEFA might contribute to explain the higher incidence of infections observed in cows suffering from energy deficit.

Objective—To assess effects on functions of peripheral blood mononuclear cells (PBMC) obtained from ewes for each of several fatty acids represented in ovine plasma at concentrations mimicking those of ketotic or healthy ewes.Sample Population—Blood samples obtained from 6 Sardinian ewes.Procedure—The PBMC were cultured in media that contained oleic (OA), palmitic (PA), stearic (SA), linoleic (LA), or palmitoleic (POA) acid at concentrations similar to those of ketotic or healthy ewes. Synthesis of DNA was stimulated by use of concanavalin A or pokeweed mitogen (PWM). Secretion of IgM was stimulated by use of PWM.Results—High concentrations (900, 450, and 225 µmol/L) of OA significantly inhibited DNA synthesis and IgM secretion of PBMC. Conversely, low concentrations (56 or 28 µmol/L) of OA significantly enhanced DNA synthesis of PBMC. High concentrations of PA (600, 300, 150, 75, 37.5, or 18.7 µmol/L) and SA (300, 150, or 75 µmol/L) significantly inhibited DNA synthesis of PBMC. High concentrations of PA (600, 300, 150, 75, 37.5, or 18.7 µmol/L) and SA (300, 150, 75, or 38 µmol/L) also significantly inhibited IgM secretion of PBMC. None of the concentrations of LA and POA affected PBMC functions.Conclusion and Clinical Relevance—Impaired immunoresponsiveness of ketotic ewes is likely associated with an increase of plasma concentrations of OA, PA, or SA and not with that of LA or POA. At physiologic concentrations, single fatty acids are likely to participate in modulation of immunoresponsiveness by exerting suppressive or stimulatory effects on immune cells. (Am J Vet Res2002;63:958–962)