Vaginal temperature of lactating cows during heat waves or normal summer day and effect of additional daily cooling treatments as heat load mitigation strategy.

The vaginal temperature (VT) of lactating Holstein cows was monitored in not heat wave (NHW) and in heat wave (HW) summer days. Temperature humidity index (THI) was monitored and assigned to four classes of heat load (HL): THI < 68 null; 68 < THI < 74 low; 74 < THI < 80 moderate; and THI > 80 high.Five daily treatments consisting of continuous forced ventilation and sprayed water (1' on follow by 5' off) were assumed as control cooling protocol (CC) and compared with two experimental cooling protocols (EC) applied in the feed bunk and based on the CC plus two additional cooling treatments which lasted a total of 90' (EC90) or 150' (EC150) in the day.Sixty lactating cows were enrolled in two summer trials carried out in NHW or HW. In each trial, 10 cows were cooled by CC, 10 by EC90 and 10 by EC150. Twenty additional cows were monitored in a fall trail to have reference value of THI and VT under thermoneutral conditions (TN). Each trial lasted 72 h, and measurements of VT were carried out by intra-vaginal data loggers.The 33% of observed THI was within the high class of HL during HW, whereas THI never exceeded the upper threshold of moderate or low class of HL in NHW and TN, respectively.Multiparous and high yielding cows were more sensitive to HL, and the increased daily cooling treatments reduced heat load during hot conditions. However, during heat waves a certain degree of hyperthermia occurred even with intensive cooling management based on seven daily treatments.

Heat load increases the risk of clinical mastitis in dairy cattle.

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 (AHL > 0) 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, and >30), days in milk (<60, 60-150, and >150), and parity (1, 2-3, and >3). 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.

(-)-Epigallocatechin-3-gallate and hydroxytyrosol improved antioxidative and anti-inflammatory responses in bovine mammary epithelial cells.

(-)-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)-1ß, 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-1ß, 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.

Review: Adaptation of animals to heat stress.

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.

Derivation and genome-wide association study of a principal component-based measure of heat tolerance in dairy cattle.

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.

Short communication: Effect of heat stress on nonreturn rate of Italian Holstein cows.

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.

The effect of heat waves on dairy cow mortality.

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.

Analysis of factors associated with mortality of heavy slaughter pigs during transport and lairage.

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; P < 0.05) and 1.27 (confidence interval: 1.06-1.51; P < 0.05) for DOA and DIP, respectively. The lower mortality risk ratios for DOA and DIP were recorded for January and March (P < 0.05). 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 (P < 0.05). 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.

Seasonal variations in the composition of Holstein cow's milk and temperature-humidity index relationship.

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.

The effects of heat stress in Italian Holstein dairy cattle.

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.

Temperature humidity index scenarios in the Mediterranean basin.

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.

Metabolic and hormonal acclimation to heat stress in domesticated ruminants.

Environmentally induced periods of heat stress decrease productivity with devastating economic consequences to global animal agriculture. Heat stress can be defined as a physiological condition when the core body temperature of a given species exceeds its range specified for normal activity, which results from a total heat load (internal production and environment) exceeding the capacity for heat dissipation and this prompts physiological and behavioral responses to reduce the strain. The ability of ruminants to regulate body temperature is species- and breed-dependent. Dairy breeds are typically more sensitive to heat stress than meat breeds, and higher-producing animals are more susceptible to heat stress because they generate more metabolic heat. During heat stress, ruminants, like other homeothermic animals, increase avenues of heat loss and reduce heat production in an attempt to maintain euthermia. The immediate responses to heat load are increased respiration rates, decreased feed intake and increased water intake. Acclimatization is a process by which animals adapt to environmental conditions and engage behavioral, hormonal and metabolic changes that are characteristics of either acclimatory homeostasis or homeorhetic mechanisms used by the animals to survive in a new 'physiological state'. For example, alterations in the hormonal profile are mainly characterized by a decline and increase in anabolic and catabolic hormones, respectively. The response to heat load and the heat-induced change in homeorhetic modifiers alters post-absorptive energy, lipid and protein metabolism, impairs liver function, causes oxidative stress, jeopardizes the immune response and decreases reproductive performance. These physiological modifications alter nutrient partitioning and may prevent heat-stressed lactating cows from recruiting glucose-sparing mechanisms (despite the reduced nutrient intake). This might explain, in large part, why decreased feed intake only accounts for a minor portion of the reduced milk yield from environmentally induced hyperthermic cows. How these metabolic changes are initiated and regulated is not known. It also remains unclear how these changes differ between short-term v. long-term heat acclimation to impact animal productivity and well-being. A better understanding of the adaptations enlisted by ruminants during heat stress is necessary to enhance the likelihood of developing strategies to simultaneously improve heat tolerance and increase productivity.

Seasonal pattern of mortality and relationships between mortality and temperature-humidity index in dairy cows.

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.

Influence of different periods of exposure to hot environment on rumen function and diet digestibility in sheep.

Effects of different periods of exposure to hot environments on rumen function, diet digestibility and digesta passage rate were studied in four adult not-pregnant 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 degrees C) and respiratory rate (118.4 +/- 31.8 breaths/min) indicated that sheep under ETHI were heat-stressed. Heat stress caused an increase (P < 0.01) in water intake, and reductions (P < 0.05) in dry matter intake, rumen pH, rumen cellulolytic and amylolytic bacteria count, rumen osmolarity, organic matter, dry matter, neutral detergent fibre, acid detergent fibre and non-structural carbohydrates digestibility coefficients, and a reduction of digesta passage rates. Under ETHI, diet digestibility and passage rate of digesta were reduced in a time-dependent fashion. Variation of diet digestibility under ETHI was not related to passage rate of digesta and feed intake. Reduction of cellulolytic and amylolytic bacteria and the adaptive response to hot environment seem to be related to alteration of digestibility observed in ewes chronically exposed to hot environment.

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.

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 degrees C continuously (T39) or three 13-h cycles at 40 (T40), 41 (T41) or 42 degrees C (T42), respectively, which were alternated with two 13-h cycles at 39 degrees C. 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 leptin mRNA. 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.

Photoperiod affects gene expression of leptin and leptin receptors in adipose tissue from lactating dairy cows.

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.

Heat stress elicits different responses in peripheral blood mononuclear cells from Brown Swiss and Holstein cows.

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 degrees C continuously (T39) and three 13-h cycles at 40 (T40), 41 (T41), 42 (T42) or 43 degrees C (T43), respectively, which were interspersed with two 13-h cycles at 39 degrees 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, DNA synthesis began to decline when PBMC were repeatedly exposed to 41 degrees C (-22%), whereas DNA synthesis in cells isolated from Ho cows did not begin to decline until 42 degrees 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.

In vitro effects of nonesterified fatty acids on bovine neutrophils oxidative burst and viability.

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.

Lymphocyte functions in overconditioned cows around parturition.

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.

Influence of body condition score on relationships between metabolic status and oxidative stress in periparturient dairy cows.

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.