Abomasal infusion of essential fatty acids and conjugated linoleic acid during late pregnancy and early lactation affects immunohematological and oxidative stress markers in dairy cows.

Oxidative stress and inflammation, as natural parts of metabolic adaptations during the transition from late gestation to early lactation, are critical indicators of dairy cows' metabolic health. This study was designed to investigate the effects of abomasal infusion of essential fatty acids (EFA), particularly α-linolenic acid, and conjugated linoleic acid (CLA) on plasma, erythrocyte, and liver markers of oxidative stress in dairy cows during the transition period. Rumen-cannulated German Holstein cows (n = 38) in their second lactation (11,101 ± 1,118 kg milk/305 d, mean ± standard deviation) were abomasally infused with one of the following treatments from d -63 antepartum until d 63 postpartum (PP): CTRL (n = 9; 76 g/d coconut oil); EFA (n = 9; 78 g/d linseed plus 4 g/d safflower oil); CLA (n = 10; isomers cis-9,trans-11 and trans-10,cis-12 CLA; 38 g/d); and EFA+CLA (n = 10; 120 g/d). Hematological parameters as well as markers of oxidative status were measured in plasma, erythrocytes, and liver before and after calving. Immunohematological parameters, including erythrocyte number, hematocrit, hemoglobin, mean corpuscular hemoglobin, leukocytes, and basophils, were affected by time, and their peak levels were observed on the day after calving. The oxidative stress markers glutathione peroxidase 1 and reactive oxygen metabolites in plasma and erythrocytes were both affected by time, exhibiting the highest levels on d 1 PP, whereas ß-carotene, retinol, and tocopherol were at their lowest levels at the same time. Immunohematological parameters were only marginally affected by fatty acid treatment in a time-dependent manner. As such, lymphocyte and atypical lymphocyte counts were both significantly highest in the groups that received EFA at d 1 PP. Moreover, EFA supplementation increased the mean corpuscular volume and showed a trend for induction of mean corpuscular hemoglobin compared with the CLA group during the transition period. The PP mean thrombocyte volume was higher in the EFA than in the CLA group (except for d 28) and both EFA and CLA reduced number of thrombocytes and thrombocrit at distinct time points. Hepatic mRNA abundance of markers related to oxidative status, including glutathione peroxidase (GPX-1) and catalase (CAT), was lower (P < 0.05) in EFA-treated than non-EFA-treated cows at d 28 PP. Dairy cows at the onset of lactation were characterized by induced markers of both oxidative stress and inflammation. Supplementing EFA and CLA had minor and time-dependent effects on markers of oxidative stress in plasma, erythrocytes, and liver. A comparison of EFA supplementation with CLA or CTRL showed higher immunohematological response at d 1 PP and lower hepatic antioxidant levels by d 28 PP. Supplementation with EFA+CLA had only a minor effect on oxidative markers, which were more similar to those with the EFA treatment. Altogether, despite the time-dependent differences, the current findings show only minor effects of EFA and CLA supplementation in the prevention of early lactation-induced oxidative stress.

Genotype by environment interaction due to heat stress in Brown Swiss cattle.

Due to its geographical position and a highly variable orography, Italy is characterized by several climatic areas and thus, by many different dairy cow farming systems. Brown Swiss cattle, in this context, are a very appreciated genetic resource for their adaptability and low metabolic requirement. The significant heterogeneity in farming systems may consist of genotype by environment (G × E) interactions with neglected changes in animals' rank position. The objective of this study was to investigate G × E for heat tolerance in Brown Swiss cattle for several production traits (milk, fat, and protein yield in kilograms; fat, protein, and cheese yield in percentage) and 2 derivate traits (fat-corrected milk and energy-corrected milk). We used the daily maximum temperature-humidity index (THI) range, calculated according to weather stations' data from 2008 to 2018 in Italy, and 202,776 test-day records from 23,396 Brown Swiss cows from 639 herds. Two different methodologies were applied to estimate the effect of the environmental variable (THI) on genetic parameters: (1) the reaction norm model, which uses a continuous random covariate to estimate the animal additive effect, and (2) the multitrait model, which splits each production pattern as a distinct and correlated trait according to the first (a thermal comfort condition), third (a moderate heat stress condition), and fifth (a severe heat stress condition) mean THI value quintile. The results from the reaction norm model showed a descending trend of the additive genetic effect until THI reached the value of 80. Then we recorded an increase with high extreme THI values (THI 90). Permanent environmental variance at increasing THI values revealed an opposite trend: The plot of heritability and the ratio of animal permanent environmental variance to phenotypic variance showed that when the environmental condition worsens, the additive genetic and permanent environmental component for production traits play a growing role. The negative additive genetic correlation between slope and linear random coefficient indicates no linear relationship between the production traits or under heat stress conditions, except for milk yield and protein yield. In tridimensional wireframe plots, the extreme margin decreases until a minimum of ∼0.90 of genetic correlation in the ECM trait, showing that the magnitude of G × E interaction is greater than the other traits. Genetic correlation values in Brown Swiss suggest the possibility of moderate changes in animals' estimated breeding value in heat stress conditions. Results indicated a moderate G × E interaction but significant variability in sire response related to their production level.

Estimation of maximum thermo-hygrometric index thresholds affecting milk production in Italian Brown Swiss cattle.

It is known that heat stress affects dairy cow performance in multiple ways: physiological, behavioral, reproductive, and productive. The aim of the present study was to determine if a threshold of temperature-humidity index (THI) exists for multiple milk production traits (milk yield, fat-corrected milk, protein and fat yield and percentage, energy-corrected milk, cheese production, and cheese yield) in Italian Brown Swiss dairy cows from the period 15 d before the day of the Italian Breeders Association test-day sampling. A 10-yr data set (2009-2018) containing 202,776 test-day records of 23,296 Brown Swiss cows was matched with the maximum THI. In all parities considered, no THI thresholds were observed for milk yield in Brown Swiss. In contrast, a THI threshold of 75 was identified for fat-corrected milk. No THI threshold was found for fat percentage, but fat yield showed the highest THI thresholds in cows of first and second parity. Protein yield and cheese production were affected by heat stress with average THI threshold of 74. The THI thresholds identified indicate that the Brown Swiss breed has higher thermal tolerance versus literature values reported for Holstein cows. As THI rises, Brown Swiss cows tend to produce the same volume of milk, but with a decreasing quality with regard to components. Further study is necessary to estimate the genetic component of heat tolerance, in Brown Swiss cattle, considering that the correct estimation of THI thresholds represents the first step to identify components that could be included in selection procedures.

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.

Effects of abomasal infusion of essential fatty acids and conjugated linoleic acid on performance and fatty acid, antioxidative, and inflammatory status in dairy cows.

The objective of this study was to test the effects of essential fatty acids (EFA), particularly α-linolenic acid, and conjugated linoleic acid (CLA) supplementation on fatty acid (FA) composition, performance, and systemic and hepatic antioxidative and inflammatory responses in dairy cows. Four cows (126 ± 4 d in milk) were investigated in a 4 × 4 Latin square and were abomasally infused with 1 of the following for 6 wk: (1) coconut oil (control treatment, CTRL; 38.3 g/d; providing saturated FA), (2) linseed and safflower oil (EFA treatment; 39.1 and 1.6 g/d, respectively; providing mainly α-linolenic acid), (3) Lutalin (BASF, Ludwigshafen, Germany; CLA treatment; cis-9,trans-11 and trans-10,cis-12 CLA, 4.6 g/d each), (4) or EFA+CLA. The initial dosage was doubled every 2 wk, resulting in 3 dosages (dosage 1, 2, and 3). Cows were fed a corn silage-based total mixed ration with a high n-6/n-3 FA ratio. Dry matter intake and milk yield were recorded daily, and milk composition was measured weekly. The FA compositions of milk fat and blood plasma were analyzed at wk 0, 2, 4, and 6. The plasma concentration and hepatic mRNA abundance of parameters linked to the antioxidative and inflammatory response were analyzed at wk 0 and 6 of each treatment period. Infused FA increased in blood plasma and milk of the respective treatment groups in a dose-dependent manner. The n-6/n-3 FA ratio in milk fat was higher in CTRL and CLA than in EFA and EFA+CLA. The sum of FA

(-)-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.

Effect of hot season on blood parameters, fecal fermentative parameters, and occurrence of Clostridium tyrobutyricum spores in feces of lactating dairy cows.

High temperature influences rumen and gut health, passage rate, and diet digestibility, with effects on fermentative processes. The main aim of the study was to investigate the effect of hot season on hindgut fermentation, the occurrence of Clostridium tyrobutyricum spores in bovine feces, and on their relationship with metabolic conditions in dairy cows producing milk used for Grana Padano cheese. The study was carried out on 7 dairy farms located in the Po Valley (Italy), involving 1,950 Italian Friesian dairy cows. The study was carried out from November 2013 till the end of July 2014. Temperature and relative humidity were recorded daily by weather stations. Constant management conditions were maintained during the experimental period. Feed and diet characteristics, metabolic conditions, and fecal characteristics were recorded in winter (from late November 2013 to the end of January 2014), spring (from April to May 2014), and summer (July 2014) season. In each season, blood samples were collected from 14 multiparous lactating dairy cows per herd to measure biochemical indices related to energy, protein, and mineral metabolism, as well as markers of inflammation and some enzyme activities. Fecal samples were also collected and measurements of moisture, pH and volatile fatty acids (VFA) were performed. The DNA extracted and purified from fecal samples was used to detect Clostridium tyrobutyricum spores in a quantitative real-time PCR assay. The daily mean temperature-humidity index was 40.7 ± 4.6 (range 25 to 55), 61.2 ± 3.7 (range 39 to 77), and 70.8 ± 3.2 (range 54 to 83) in winter, spring, and summer, respectively. Total VFA concentration in feces progressively decreased from winter to summer. The seasonal changes of acetate and propionate followed the same trend of total VFA; conversely, butyrate did not show any difference between seasons, and its molar proportion was greater in summer compared with winter. A greater occurrence of Cl. tyrobutyricum spores in summer compared with the other seasons was observed. The plasma concentrations of glucose, urea, albumin, Ca, Mg, Cl, Zn, and alkaline phosphatase activity were lower in summer compared with winter, whereas the opposite occurred for bilirubin and Na. Our results show that summer season, through direct and indirect effect of heat stress, affected fecal fermentative parameters and hindgut buffering capacity, and was responsible for the increasing occurrence of Cl. tyrobutyricum spores in feces.

Anti-inflammatory effects of conjugated linoleic acid isomers and essential fatty acids in bovine mammary epithelial cells.

Fatty acids are important modulators of inflammatory responses, in particular, n-3 and n-6 essential fatty acids and CLA have received particular attention for their ability to modulate inflammation. The objectives of this study were to compare the effects of CLA and essential fatty acids on the expression of pro and anti- inflammatory cytokines and their protective efficacy against inflammatory status in mammary gland by an in vitro model based on bovine mammary epithelial cells (BME-UV1). Bovine mammary epithelial cells were treated with complete medium containing either 50 µM of cis-9, trans-11 CLA (c9,t11 CLA) or trans-10, cis-12 CLA (t10,c12 CLA) or (α)-linolenic acid (aLnA) or (γ)-linolenic acid (gLnA) or linoleic acid (LA). After 48 h by fatty acids administration the cells were treated for 3 h with 20 µM of lipopolysaccharide (LPS) to induce inflammatory stimulus. Reactive oxygen species (ROS) production after treatments was assessed to verify and to compare the potential protection of different fatty acids against LPS-induced oxidative stress. The messenger RNA abundance of bovine pro and anti-inflammatory cytokines (tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and interleukine-10 (IL-10)) and peroxisome proliferator receptor-α/γ (PPARγ/α) were determined in BME-UV1 by real-time PCR. The results showed that cells treated with fatty acids and LPS increased ROS production compared with control cells. Among treatments, cells treated with c9,t11 CLA and t10,c12 CLA isomers revealed significant lower levels of ROS production compared with other fatty acids. All fatty acids reduced the gene expression of pro- and anti-inflammatory cytokines. Among fatty acids, t10,c12 CLA, LA and gLnA showed an homogeneous reduction of the three pro-inflammatory cytokines and this may correspond to more balanced and efficient physiological activity and may trigger a better protective effect. The PPARγ gene expression was significantly greater in cells treated with t10,c12 CLA, aLnA and LA, whereas the PPARα gene expression levels were significantly lower in cells treated with all different fatty acids, compared with the control. These results suggest that fatty acids inhibited the transcription of pro-inflammatory cytokines by the upregulation of PPARγ expression.

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.

Comparison between conjugated linoleic acid and essential fatty acids in preventing oxidative stress in bovine mammary epithelial cells.

Some in vitro and in vivo studies have demonstrated protective effects of conjugated linoleic acid (CLA) isomers against oxidative stress and lipid peroxidation. However, only a few and conflicting studies have been conducted showing the antioxidant potential of essential fatty acids. The objectives of the study were to compare the effects of CLA to other essential fatty acids on the thiol redox status of bovine mammary epithelia cells (BME-UV1) and their protective role against oxidative damage on the mammary gland by an in vitro study. The BME-UV1 cells were treated with complete medium containing 50 µM of cis-9,trans-11 CLA, trans-10,cis-12 CLA, α-linolenic acid, γ-linolenic acid, and linoleic acid. To assess the cellular antioxidant response, glutathione, NADPH, and γ-glutamyl-cysteine ligase activity were measured 48 h after addition of fatty acids (FA). Intracellular reactive oxygen species and malondialdehyde production were also assessed in cells supplemented with FA. Reactive oxygen species production after 3 h of H2O2 exposure was assessed to evaluate and to compare the potential protection of different FA against H2O2-induced oxidative stress. All FA treatments induced an intracellular GSH increase, matched by high concentrations of NADPH and an increase of γ-glutamyl-cysteine ligase activity. Cells supplemented with FA showed a reduction in intracellular malondialdehyde levels. In particular, CLA isomers and linoleic acid supplementation showed a better antioxidant cellular response against oxidative damage induced by H2O2 compared with other FA.

Fate of transgenic deoxyribonucleic acid fragments in digesta and tissues of rabbits fed genetically modified soybean meal.

Numerous animal feeding studies have investigated the presence of DNA from transgenic plants in tissues from different animal species, but the data reported are sometimes controversial. The aim of this study was to investigate the presence of transgenic DNA (tDNA) in the digesta and tissues of a meat rabbit breed fed genetically modified (GM) soybean meal. Fifteen male New Zealand White rabbits were used for the experimental trial. Ten rabbits (treated group [TG]) were fed a mixed feed containing 10% GM soybean meal and 5 rabbits (control group [CG]) received a mixed feed containing conventional soybean meal, both from weaning (28 d of age) to slaughter (80 ± 3 d). Samples of blood, liver, kidney, heart, stomach, intestine (jejunum), lateral quadricep muscle, longissimus muscle, and perirenal adipose tissue were collected to assess the possible DNA transfer from GM feed to animal tissues. Samples of stomach contents and feces were also taken to study the degradability of ingested tDNA from feed in the digestive tract of rabbit. Moreover, samples of hair were collected to determine the possible environmental contamination from feed powders present on the farm. The DNA extraction was performed using specific genomic DNA kits. All samples were monitored, by using real-time PCR, for oligonucleotide primers and probes specific for the transgenic Roundup Ready soybean 40-3-2 and for the endogenous () gene. As an internal control of rabbit tissues, the presence of the () gene was used. In this study, no fragments of tDNA were detectable in tissue DNA samples of rabbits except in the extracted DNA from stomach digesta, feces, and hair of rabbits fed with GM soybean. Similar results were found for the reference gene, whereas the presence of the gene was detected in all rabbit tissues. The lack of tDNA of soybean in rabbit tissues represents an important result, which demonstrates that meat from rabbits fed a diet containing GM feed is as that derived from rabbits fed conventional crops. The recombinant DNA recovered in the stomach digesta and in feces indicates an incomplete digestion of the soybean DNA in the gastrointestinal tract of the rabbit, whereas the presence of trace soybean transgene in the hair of the TG rabbits is suggestive of an environmental contamination.

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.

Release of angiotensin converting enzyme-inhibitor peptides during in vitro gastrointestinal digestion of Parmigiano Reggiano PDO cheese and their absorption through an in vitro model of intestinal epithelium.

The occurrence of 8 bovine casein-derived peptides (VPP, IPP, RYLGY, RYLG, AYFYPEL, AYFYPE, LHLPLP, and HLPLP) reported as angiotensin converting enzyme-inhibitors (ACE-I) was investigated in the 3-kDa ultrafiltered water-soluble extract (WSE) of Parmigiano Reggiano (PR) cheese samples by ultra-performance liquid chromatography coupled to high-resolution mass spectrometry via an electrospray ionization source. Only VPP, IPP, LHLPLP, and HLPLP were revealed in the WSE, and their total amount was in the range of 8.46 to 21.55 mg/kg of cheese. Following in vitro static gastrointestinal digestion, the same ACE-I peptides along with the newly formed AYFYPEL and AYFYPE were found in the 3 kDa WSE of PR digestates. Digestates presented high amounts (1,880-3,053 mg/kg) of LHLPLP, whereas the remaining peptides accounted for 69.24 to 82.82 mg/kg. The half-maximal inhibitory concentration (IC50) values decreased from 7.92 ± 2.08 in undigested cheese to 3.20 ± 1.69 after in vitro gastrointestinal digestion. The 3-kDa WSE of digested cheeses were used to study the transport of the 8 ACE-I peptides across the monolayers of the Caco-2 cell culture grown on a semipermeable membrane of the transwells. After 1h of incubation, 649.20 ± 148.85 mg/kg of LHLPLP remained in the apical compartment, whereas VPP, IPP, AYFYPEL, AYFYPE, and HLPLP accounted in total for less than 36.78 mg/kg. On average, 0.6% of LHLPLP initially present in the digestates added to the apical compartment were transported intact to the basolateral chamber after the same incubation time. Higher transport rate (2.9%) was ascertained for the peptide HLPLP. No other intact ACE-I peptides were revealed in the basolateral compartment. For the first time, these results demonstrated that the ACE-I peptides HLPLP and LHLPLP present in the in vitro digestates of PR cheese are partially absorbed through an in vitro model of human intestinal epithelium.

Conjugated linoleic acid isomers strongly improve the redox status of bovine mammary epithelial cells (BME-UV1).

Some studies have shown the protective effects of conjugated linoleic acid (CLA) isomers against oxidative stress and lipid peroxidation in animal models, but no information is available about CLA and changes in oxidative status of the bovine mammary gland. The objectives of the study were to assess in vitro the effect of CLA on the cellular antioxidant response of bovine mammary cells, to examine whether CLA isomers could play a role in cell protection against the oxidative stress, and to study the molecular mechanism involved. For the study, BME-UV1 cells, a bovine mammary epithelial cell line, were used as the experimental model. The BME-UV1 cells were treated with complete medium containing 50 µM cis-9,trans-11 CLA (c9,t11 CLA), trans-10,cis-12 CLA (t10,c12 CLA), and CLA mixture (1:1, cis-9,trans-11: trans-10,cis-12 CLA). To monitor cellular uptake of CLA isomers, cells and culture medium were collected at 0, 3, and 48 h from CLA addition for lipid extraction and fatty acid analyses. To assess the cellular antioxidant response, glutathione (GSH/GSSH), NADPH, and γ-glutamyl-cysteine ligase activity was measured after 48 h from addition of CLA. Cytoplasmic superoxide dismutase, glutathione peroxidase, glutathione S-transferase, and glutathione reductase activities and mRNA were also determined. Intracellular reactive oxygen species and thiobarbituric acid reactive substance production were assessed in cells supplemented with CLA isomers. Cell viability after 3h to H2O2 exposure was assessed to evaluate and to compare the potential protection of different CLA isomers against H2O2-induced oxidative stress. Mammary cells readily picked up all CLA isomers, their accumulation was time dependent, and main metabolites at 48 h are two 18:3 isomers. The CLA treatment induced an intracellular GSH increase, matched by high concentration of NADPH, and an increase of γ-glutamyl-cysteine ligase activity mainly in cells treated with the t10,c12 CLA isomer. The CLA isomer treatment of bovine mammary cells increased superoxide dismutase, glutathione peroxidase, and glutathione S-transferase activity and decreased glutathione reductase activity, but no changes in gene expression of these antioxidant enzymes were observed. Cells supplemented with CLA isomers showed a reduction in intracellular reactive oxygen species and thiobarbituric acid reactive substance levels. All CLA isomers were able to enhance cell resistance against H2O2-induced oxidative stress. These suggest an antioxidant role of CLA, in particular of t10,c12 CLA, by developing a significantly high redox status in cells.

The effect of calving in the summer on the hepatic transcriptome of Holstein cows during the peripartal period.

The liver is the main metabolic organ coordinating the adaptations that take place during the peripartal period of dairy cows. A successful transition into lactation, rather than management practices alone, depends on environmental factors such as temperature, season of parturition, and photoperiod. Therefore, we analyzed the effect of calving season on the hepatic transcriptome of dairy cows during the transition period. A total of 12 Holstein dairy cows were assigned into 2 groups based on calving season (6 cows March-April, spring; 6 cows June-July, summer, SU). The RNA was extracted from liver samples obtained at -30, 3, and 35 DIM via percutaneous biopsy and hybridized to the Agilent 44K Bovine (V2) Gene Expression Microarray (Agilent Technologies Inc., Santa Clara, CA). A quantitative PCR on 22 target genes was performed to verify and expand the analyses. A total of 4,307 differentially expressed genes were detected (false discovery rate ≤0.05) in SU compared with spring. Furthermore, 73 unique differentially expressed genes were detected in SU compared with spring cows after applying a fold-change threshold ≥3 and ≤-3. For Kyoto Encyclopedia of Genes and Genomes pathways analysis of differentially expressed genes, we used the dynamic impact approach. Ingenuity Pathway Analysis software was used to analyze upstream transcription regulators and perform gene network analysis. Among metabolic pathways, energy metabolism from lipids, carbohydrates, and amino acids was strongly affected by calving in SU, with a reduced level of fatty acid synthesis, oxidation, re-esterification, and synthesis of lipoproteins, leading to hepatic lipidosis. Glycan-synthesis was downregulated in SU cows probably as a mechanism to counteract the progression of this lipidosis. In contrast, calving in the SU resulted in upregulation of gluconeogenesis but also greater use of glucose as an energy source. Among nonmetabolic pathways, the heat-shock response was obviously activated in SU cows but was also associated with inflammatory and intracellular stress response. Furthermore, data support a recent finding that cows experience endoplasmic reticulum stress around parturition. Transcription regulator analysis revealed how metabolic changes are related to important regulatory mechanisms, including epigenetic modification. The holistic analyses of the liver transcriptome response to calving in the summer at high environmental temperatures underscore how transition cows should be carefully managed during this period, as they experience alterations in liver energy metabolism and inflammatory state increasing susceptibility to health disorders in early postpartum.

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.

Effect of summer season on milk protein fractions in Holstein cows.

Milk characteristics are affected by heat stress, but very little information is available on changes of milk protein fractions and their relationship with cheesemaking properties of milk. The main objective of the study was to evaluate the effect of hot season on milk protein fractions and cheesemaking properties of milk for Grana Padano cheese production. The study was carried out in a dairy farm with a cheese factory for transforming the milk to Grana Padano cheese. The study was carried out from June 2012 to May 2013. Temperature and relative humidity of the inside barn were recorded daily during the study period using 8 electronic data loggers programmed to record every 30 min. Constant managerial conditions were maintained during the experimental periods. During the experimental period, feed and diet characteristics, milk yield, and milk characteristics were recorded in summer (from June 29 to July 27, 2012), winter (from January 25 to March 8, 2013), and spring (from May 17 to May 31, 2013). Milk yield was recorded and individual milk samples were taken from 25 cows selected in each season during the p.m. milking. Content of fat, proteins, caseins (CN), lactose and somatic cell count (SCC), titratable acidity, and milk rennet coagulation properties were determined on fresh samples. Milk protein fraction concentrations were determined by the sodium dodecyl sulfate-PAGE. Data were tested for nonnormality by the Shapiro-Wilk test. In case of nonnormality, parameters were normalized by log or exponential transformation. The data were analyzed with repeated measures ANOVA using a mixed model procedure. For all the main milk components (fat, protein, total solids, and solids-not-fat), the lowest values were observed in the summer and the greatest values were observed in the winter. Casein fractions, with the exception of γ-CN, showed the lowest values in the summer and the greatest values in the winter. The content of IgG and serum albumin was greater in summer than in the winter and spring. A mild effect of season was observed for milk SCC, with greater values in summer than in the winter and spring. A worsening of milk coagulation properties was observed in summer season. The alteration of cheesemaking properties during hot season seems strictly linked with changes of milk protein fractions mainly with the decrease of αS-CN and ß-CN and the increase of undefined proteins.

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.